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Aug 6, 2007 - 2Department of Botany, Faculty of Science, Charles University in Prague, ...... Heywood, N. A. Burges, D. M. Moore, D. H. Valentine, S. M..
American Journal of Botany 94(8): 1391–1401. 2007.

COMPLEX

DISTRIBUTION PATTERNS OF DI-, TETRA-, AND

EUROPEAN HIGH MOUNTAIN CARNIOLICUS (ASTERACEAE)1

HEXAPLOID CYTOTYPES IN THE PLANT

SENECIO

JAN SUDA,2,3,7 HANNA WEISS-SCHNEEWEISS,4 ANDREAS TRIBSCH,5 GERALD M. SCHNEEWEISS,6 PAVEL TRA´VNI´CˇEK,3,2 AND PETER SCHO¨NSWETTER6 2

Department of Botany, Faculty of Science, Charles University in Prague, Bena´tska´ 2, CZ-128 01 Prague, Czech Republic; 3 Institute of Botany, Academy of Sciences of the Czech Republic, Pru˚honice 1, CZ-252 43, Czech Republic; 4 Department of Systematic and Evolutionary Botany, Faculty Centre Botany, University of Vienna, Rennweg 14, A-1030 Vienna, Austria; 5Department of Organismic Biology, AG Ecology and Diversity of Plants, University of Salzburg, Hellbrunnerstraße 34, A-5020 Salzburg, Austria; and 6Department of Biogeography and Botanical Garden, Faculty Centre Botany, University of Vienna, Rennweg 14, A-1030 Vienna, Austria

DNA ploidy levels were estimated using DAPI-flow cytometry of silica-dried specimens of the European mountain plant Senecio carniolicus (Asteraceae), covering its entire distribution area in the Eastern Alps (77 populations, 380 individuals) and the Carpathians (five populations, 22 individuals). A complex pattern of ploidy level variation (2x, 4x, 5x, 6x, and 7x cytotypes) was found in this species, which has been considered uniformly hexaploid. Hexaploids predominated in the Eastern Alps and was the only cytotype found in the Carpathians, while odd ploidy levels (5x, 7x) constituted a small fraction of the samples (,1.3%). Tetraploids occurred in two disjunct areas, which correspond with putative Pleistocene refugia for silicicolous alpine plants. Diploids occurred in large portions of the Alps but were absent from areas most extensively glaciated in the past. Intrapopulational cytotype mixture was detected in 22 populations—the majority involving diploids and hexaploids—with intermediate ploidy levels mostly lacking, suggesting limited gene flow and the evolution of reproductive isolation. Significant and reproducible intracytotype variation in nuclear DNA content was observed. Higher genome size in western diploids might be due to ancient introgression with the closely related S. incanus or to different evolutionary pathways in the geographically separated diploids. Key words: Alps; Asteraceae; cytogeography; cytotype mixture; DNA ploidy; flow cytometry; genome size variation; Senecio carniolicus.

In recent years, a wealth of data on the occurrence and ubiquity of polyploidy in angiosperms has been produced, and it is currently believed that the vast majority of angiosperms are at least anciently polyploid (Soltis, 2005). In many plant groups, allopolyploids have been identified, their parentage established, and genomic changes after polyploidization analyzed (e.g., Tragopogon [Asteraceae]: Ownbey, 1950; Pires et al., 2004; Soltis et al., 2004; Gossypium [Malvaceae]: Brubaker et al., 1999; Cronn and Wendel, 2004). A recent paradigm shift concerns autopolyploids, which are no longer considered ‘‘dead ends of evolution’’ (Stebbins, 1971) but to have great evolutionary potential (Hancock, 1992; Soltis and Soltis, 1995). Although they are much less commonly discovered than allopolyploids (Soltis, 2005), the number of well-investigated autopolyploid complexes is increasing (e.g., Qu et al., 1998; Weiss et al., 2003; Stuessy et al., 2004; Li, 2005). In many cases, both allo- and autopolyploids have been shown to originate recurrently and to be successfully maintained (e.g., Brochmann et al., 1992; Segraves et al., 1999; Soltis and Soltis, 2000; Sharbel and Mitchell-Olds, 2001; Guo et al., 2005), emphasizing their evolutionary significance. Ploidy differences are not restricted to comparisons between 1 Manuscript received 21 October 2006; revision accepted 31 May 2007. The authors thank A.-M. Csergo, T. Englisch, and M. Ronikier, who collected plant material. This study was partly supported by the Austrian Science Fund (FWF P13874-BIO and T218-BIO); Czech Science Foundation (206/06/0598); Ministry of Education, Youth and Sport of the Czech Republic (MSM 0021620828); and Academy of Sciences of the Czech Republic (AV0Z60050516).

7

Author for correspondence (e-mail: [email protected])

species but also occur frequently within species (e.g., Ehrendorfer, 1958; Miller, 1978; Burton and Husband, 1999; Weiss et al., 2003; Baack, 2004). Assessment of ploidy level distribution within populations, greatly enhanced by the use of flow cytometric (FCM) techniques, revealed that cytotype mixture, i.e., the presence of more than one ploidy level within a population, is much more frequent than anticipated (e.g., Nicholson, 1983; van Dijk et al., 1992; Husband and Schemske, 1998; Keeler and Davis, 1999; Suda, 2003; Weiss et al., 2002; Schneeweiss et al., 2004; Stuessy et al., 2004). Two alternative explanations for the sympatric occurrence of various cytotypes are directional selection and balanced selection. The first hypothesis assumes that cytotype mixture is a transitional stage and that eventually one cytotype will outcompete the other as a result of, for example, frequencydependent mating success in out-crossers (minority cytotype exclusion principle: Levin, 1975). Because of its temporary nature, this mode is difficult to observe but is expected to be particularly important in the case of primary hybrid zones, i.e., higher polyploids emerging within lower-ploid populations (Petit et al., 1999). The second hypothesis assumes that different cytotypes can be maintained by isolating mechanisms, such as ecological sorting (Fowler and Levin, 1984; Lumaret et al., 1987; Felber-Girard et al., 1996; Husband and Schemske, 1998; Johnson et al., 2003) or divergence in flowering time (van Dijk et al., 1992; Petit et al., 1997; Bretagnolle and Thompson, 2001; Nuismer and Cunningham, 2005), which might lead to reproductive isolation and eventual speciation (e.g., Otto and Whitton, 2000; Husband and Sabara, 2004). This mode can occur in both primary and secondary hybrid

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zones (Baack, 2004; Nuismer and Cunningham, 2005), the latter defined as hybrid zones where different cytotypes re-gain contact after phases of geographic separation (Petit et al., 1999). One of the prerequisites for polyploid research in natural systems is knowledge of the geographical distribution of cytotypes. Distributional data complement phylogenetic and experimental data by, for example, revealing the spatial proximity of polyploids with diploid parental taxa and thus the possibility of gene flow between different ploidy levels (Baack, 2004, 2005a; Baack and Stanton, 2005). Furthermore, distributional data serve as a foundation for exploring questions of niche separation and potential barriers to coexistence and might give insights into the frequency of polyploid formation (Segraves et al., 1999) as well as into the historical development of modern distribution patterns of polyploid alliances (e.g., Ehrendorfer, 1958). Recent technical and methodological advancements in cytological research, in particular genome size estimation from dehydrated plant vouchers (Suda and Tra´vnı´cˇek, 2006b), make the efficient and detailed assessment of cytotype distribution feasible. Our initial chromosome counts revealed the presence of three cytotypes (di-, tetra-, and hexaploid) in several populations of the European high mountain plant Senecio carniolicus Willd. (Asteraceae), which was previously thought to be uniformly hexaploid (Favarger, 1964) because occasional diploid counts were considered dubious (Dobesˇ and Gutermann, 2002). Senecio carniolicus is member of sect. Jacobaea and belongs to a group of closely related southern European mountain plants informally called the Incani s.l. (Pelser et al., 2003, 2004). It is a common and abundant acidophilic species of alpine grasslands, moraines, and stable screes (Ellenberg, 1996) occurring up to 3300 m above sea level (Reisigl and Pitschmann, 1958) and is endemic to the Eastern Alps (eastern Switzerland, Italy, Austria, Slovenia) and the Western and Southern Carpathians (Poland, Slovakia, Romania). Although often treated as a subspecies of the diploid S. incanus L. from the Western Alps (France, Italy, western and central Switzerland) and the northern Apennines (Italy), it is clearly distinct on the species level based on morphological, cytological, and molecular (DNA sequences and AFLPs) data (Hess et al., 1980; A. Tribsch et al., unpublished data). Typical plants of S. incanus have pinnatifid leaves with a dense sericeous-lanate indumentum and partly hairy achenes, while those of S. carniolicus have shallowly lobed leaves with a rather sparse indumentum and glabrous achenes (Chater and Walters, 1976). It has been suggested that populations at the southwestern edge of the distribution area (the southern Alpi Lepontine and western Alpi Bergamasche in Switzerland and Italy) are morphologically intermediate between S. carniolicus and S. incanus (Chenevard, 1906; Braun-Blanquet, 1913; Chater and Walters, 1976). They were initially described as S. carniolicus var. insubricus Chenevard but were later transferred as subsp. insubricus (Chenevard) Br.-Bl. to S. incanus. Experimental information on the breeding system as well as data on the longevity of S. carniolicus are currently lacking. The high pollen to ovule ratio (H. Weiss-Schneeweiss, unpublished data) as well as the pronounced protandry, however, suggest the prevalence of allogamy. We determined the geographic pattern of cytotype distribution in S. carniolicus, applying the recently developed method of estimating DNA ploidy level using flow cytometry of desiccated plant tissue (Suda and Tra´vnı´cˇek, 2006b). The

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following questions were addressed: (1) How are the cytotypes distributed, in particular in relation to presumed Pleistocene refugia? (2) Does the distribution of the cytotypes allow the inference of the mode of origin (primary and/or secondary hybrid zones) of the polyploids? (3) Do populations with different ploidy levels exist (cytotype mixture)? If so, which cytotypes are involved, and are they connected by individuals with intermediate ploidy levels, suggesting inter-cytotype gene flow? MATERIALS AND METHODS Plant material—Eighty-five Senecio populations were sampled between 1999 and 2005 in Austria, Italy, France, Poland, Romania, Slovakia, and Switzerland (44.138 N–49.228 N and 7.148 E–24.928 E) (Table 1, Fig. 1). For S. carniolicus, 77 populations were sampled in the Eastern Alps, with a further five populations originating from the Carpathians. Three populations of the closely related Western Alpine S. incanus were also included. Typically, leaf material of five plants per population separated by up to a few hundred meters was collected and desiccated using silica gel. Herbarium vouchers are deposited in WU, and living plants of some populations are being cultivated in the Botanical Garden of the University of Vienna (HBV; Table 1). Chromosome counts—Actively growing root meristems were pretreated with 0.1% colchicine for 2 h at room temperature and 2 h at 48C, fixed in 3 : 1 ethanol : acetic acid for 12 h at room temperature, and stored at 208C until use. Feulgen staining with Schiff’s reagent was performed according to a standard protocol (Weiss et al., 2002). Preparations with at least 10 complete well-spread chromosome plates were chosen for analysis using a Reichert Jung Polyvar light microscope (Leitz Optical Systems, Vienna, Austria). Photographs were taken on black and white T-Max film (Eastman Kodak, Rochester, New York, USA), scanned, and manipulated using Corel Photo-Paint 10.0 (Corel Corp., Ottawa, Quebec, Canada) only using options that apply uniformly to the whole image. Flow cytometry—Flow cytometry (FCM) of 4 0 ,6-diamidino-2-phenylindole (DAPI)-stained nuclei was used to estimate DNA ploidy levels of silica gel-dried Senecio samples (Suda and Tra´vnı´cˇek, 2006a). The prefix ‘‘DNA’’ indicates that ploidy levels were mostly inferred from nuclear DNA content without knowledge of the exact chromosome numbers (Suda et al., 2006). Desiccated green leaf tissue (c. 0.5 cm2) of the analyzed plant was chopped along with an appropriate amount of fresh intact leaf of internal reference standard in 0.5 mL of ice-cold Otto I buffer (0.1 M citric acid, 0.5% Tween 20). The nuclear suspension was filtered through a nylon mesh (42 lm) to remove large debris and incubated at room temperature for 5 min, after which 1 mL of staining solution was added. The staining solution consisted of Otto II buffer (0.4 M Na2HPO412 H2O) supplemented with DAPI (Sigma, St. Louis, Missouri, USA) at a final concentration of 4 lg/mL and 2-mercaptoethanol (2 ll/mL). After incubation for 5 min at room temperature, the relative fluorescence intensity of 5000 particles was recorded using a Partec PA II flow cytometer (Partec GmbH, Mu¨nster, Germany) equipped with an HBO mercury arc lamp. Generally, only histograms with both peaks (sample and standard) of approximately the same height were considered. If the coefficient of variation (CV) of the G0/G1 peak of the sample exceeded the 5% threshold, the analysis was discarded and the sample remeasured. Pisum sativum cv. Ctirad (2C ¼ 9.09 pg) was selected as a primary reference standard (Dolezˇel et al., 1998). The genome size of pea is optimal for estimating relative fluorescence intensity of diploid and low-polyploid (2x–5x) Senecio plants. Vicia faba cv. Inovec served as an internal standard in FCM assays of high polyploids (6x, 7x) to minimize the standard : sample peak ratio and thus to avoid potential nonlinearity of measurements. The relative nuclear DNA amount of Vicia was calibrated against Pisum (average ratio 3.14, based on seven replications on different days). Several Senecio samples were reanalyzed (up to four times) on different days to assess between-run fluctuation and guarantee result reliability. FCM analyses using fresh individuals were also performed to evaluate any potential fluorescence shift after tissue desiccation. DNA ploidy levels of the Senecio samples were determined based on FCM profiles of individuals with karyologically determined ploidy levels (diploid: 2n ¼ 2x ¼ 40; tetraploid: 2n ¼

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Fig. 1. Distribution of cytotypes of Senecio carniolicus in the Eastern Alps. (A) Overall distribution of cytotypes. The inset shows the study area within Europe and gives the localities of the Carpathian populations. Even ploidy levels are represented by dots (white, diploid; gray, tetraploid; black, hexaploid). Cytotype mixture is illustrated by mixed shading. The odd ploidy levels, pentaploid and heptaploid, are indicated with the numbers 5 and 7, respectively. To aid legibility, the distributions of di-, tetra-, and hexaploids are given separately in (B), (C), and (D), respectively. (E) Shows the position of toponyms mentioned in the text and gives the maximal extent of the ice sheet during the last glaciation (modified from van Husen, 1987).

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TABLE 1. Geographic origin, DNA ploidy levels with corresponding number of plants, total number of analyzed individuals, relative fluorescence intensities of DAPI-stained nuclei (relative to Pisum sativum cv. Ctirad), type of analyzed tissue, chromosome counts, and voucher information (identifier for the Herbarium Scho¨nswetter & Tribsch stored in the herbarium WU) for 85 populations of Senecio incanus L. and S. carniolicus Willd. from the Alps and the Carpathians. Geographic coordinates

Country

Col Lombard Ormea Nufenenpass

F I CH

44.20 44.13 46.48

7.14 7.78 8.39

1 3 4

1 3 4

0.824 0.914 6 0.011 0.885 6 0.004

Silica Silica Fresh

S. carniolicus Alps C-1 C-2 C-3 C-4 C-5 C-6

Cima dell’Uomo Pizzo di Gino Monte Legnone Monte Spluga Valle Vicime Cima Cadelle

CH I I I I I

46.23 46.12 46.10 46.18 46.10 46.05

8.95 9.14 9.42 9.55 9.72 9.74

5 5 8

5 5 8 5 1 5

Fresh Silica Fresh Silica Silica Silica

C-7 C-8 C-9 C-10

Piz Julier Bocchetta Forbici Schwarzhorn Pizzo di Coca

CH I CH I

46.48 46.32 46.73 46.08

9.75 9.90 9.93 10.00

C-11 C-12 C-13 C-14 C-15 C-16

Monte Vago Hohes Rad 1 Hohes Rad 2 Valluga Monte Verva Monte Colombine

I A A A I I

46.44 46.88 46.91 47.15 46.42 45.85

10.07 10.10 10.11 10.20 10.22 10.36

C-17 C-18

Passo Crocedomini Stilfser Joch

I I

45.93 46.52

10.43 10.46

C-19 C-20 C-21 C-22 C-23 C-24 C-25 C-26 C-27

Piz Lad Paso di Gavia 1 Paso di Gavia 2 Passo Tonale Pfisser Joch Val Folgorida Weißseejoch Verpeilspitze Pfossental

I I I I A I A A I

46.83 46.34 46.35 46.23 47.07 46.17 46.87 47.03 46.75

10.47 10.47 10.50 10.57 10.58 10.60 10.68 10.80 11.02

C-28

Gaisbergtal

A

46.85

C-29 C-30

Schrankogel Monte Ziolera

A I

C-31 C-32

Glungezer Schrotthorn

C-33

0.837 6 0.001 0.834 6 0.011 0.840 6 0.004 2.208 6 0.028 1.583 2.161 6 0.026, 2.520 2.164 6 0.032 2.259 6 0.020 2.134 6 0.031 1.529 6 0.004, 1.890 2.221 6 0.030 2.155 6 0.021 2.159 6 0.029 2.215 6 0.014 2.139 6 0.049 0.793 6 0.016, 2.181 6 0.033 1.507 6 0.019 1.495 6 0.012, 1.826 6 0.004, 2.095 2.179 6 0.024 1.484 6 0.018 1.485 6 0.011 1.481 6 0.011 2.199 6 0.018 1.505 6 0.008 2.177 6 0.031 2.150 6 0.028 0.792, 2.206 6 0.036 0.797 6 0.020, 2.204 6 0.029 2.148 6 0.038 0.808 6 0.004, 2.197 2.174 6 0.026 0.803, 2.170 6 0.020 0.793 6 0.011, 1.542 6 0.018, 2.120 0.801 6 0.005, 2.158 0.817 6 0.007, 2.134 6 0.007 2.182 6 0.062 0.810, 2.181 6 0.044 0.802 6 0.003 2.138 6 0.035 0.780 6 0.006, 2.138 6 0.020 0.813 6 0.007, 2.106 6 0.011

Population

Locality

S. incanus I-1 I-2 I-3

Longitude (8 E)

DNA ploidy level

Latitude (8 N)

2x

4x

5x

6x

7x

5 1 4 5 5 5 6

1

2 5 2

2

1

N

Relative fluorescence intensity (mean 6 SD)

Tissue

5 5 5 7

5 5 5 5 5 6

5 5 5 5 5 8

1

5 5

5

1

5 5 4

5 2 5 5 5 3 5 5 5

11.05

3

2

5

47.03 46.17

11.10 11.45

4

5 1

5 5

A I

47.20 46.73

11.52 11.55

1

3 4

3 5

Cima d’Asta

I

46.18

11.60

4

1

10

C-34

Plose

I

46.68

11.70

4

1

5

C-35

Cavalazza Piccola

I

46.29

11.78

3

2

5

C-36 C-37

Cima Margerita Passo Pordoi

I I

46.37 46.42

11.80 11.82

1

5 5

5 6

C-38 C-39 C-40

Tristenspitz Staller Sattel Toblacher Pfannhorn

A A A

46.95 46.88 46.78

11.82 12.20 12.28

2

5 3

5 5 5

C-41

Ka¨rlsspitze

A

46.82

12.28

3

2

5

2 5 5 5 3

5

5

Silica Silica Silica Fresh Silica Silica Silica Silica Silica Silica

Chromosome count, 2n (no. of individuals counted)

40 (4)

40 40 40 120

(5) (1) (5) (2)

Voucher no.

4696 4677 Cult. in HBV

120 (2)

5114 4984 4969 4957 — 4996

80 (6)

5083 4948 5064 4975

120 (2)

5043 5196 — 5191 5100 3662

Silica Silica

5025 3677

Silica Silica Silica Silica Silica Silica Silica Silica Silica

5068 5017 3664 5005 4935 5032 5208 — 4922

120 (1) 120 (2)

Silica

3690

Silica Silica

— 3650

Silica Silica

5187 4919

Silica

4661

Silica

4907

Silica

4650

Silica Silica

4647 3638

Silica Silica Silica

4898 4894 4594

Silica

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Continued. Geographic coordinates

Population

Locality

Country

Latitude (8 N)

Longitude (8 E)

DNA ploidy level

2x

4x

5x

6x

7x

N

C-42 C-43 C-44 C-45 C-46 C-47 C-48

Col Quaterna` Kalser Ho¨he Golzentipp Monte Peralba Schleinitz Monte Crostis Hoher Sadnig

I A A I A I A

46.67 47.00 46.73 46.63 46.89 46.57 46.93

12.47 12.60 12.60 12.72 12.75 12.88 12.98

C-49 C-50

Kalkbretterkopf Scharnik

A A

47.13 46.80

13.03 13.03

C-51 C-52 C-53 C-54 C-55 C-56 C-57

Dechantalm Ankogel Hocheck Grosser Hafner Goldeck Bartelmann Wandspitze

A A A A A A A

46.83 47.05 46.88 47.07 46.75 46.94 47.02

13.15 13.25 13.40 13.40 13.45 13.45 13.53

3 1

C-58 C-59

Seekarspitze Rosennock

A A

47.27 46.88

13.54 13.72

1

C-60

Samspitze

A

47.27

13.73

2

C-61

Hochgolling

A

47.27

13.76

9

9

C-62 C-63 C-64 C-65

Greifenberg Klafferscharte Hochwildstelle Eisenhut

A A A A

47.29 47.30 47.34 46.95

13.79 13.80 13.83 13.93

3 4 7 4

1

3 4 7 5

C-66 C-67 C-68 C-69 C-70

Deneck Rettlkirchspitze Belsˇcˇica Hohenwart Schiesseck

A A A/SLO A A

47.28 47.26 46.45 47.33 47.24

14.06 14.13 14.14 14.23 14.33

7 3 1

7 3 5 4 5

C-71 C-72 C-73 C-74 C-75

Bo¨senstein Zirbitzkogel 2 Zirbitzkogel 1 Saualpe Seckauer Zinken

A A A A A

47.44 47.10 47.07 46.85 47.34

14.42 14.55 14.57 14.65 14.75

2 5 5 2

5 2 5 5 3

C-76 C-77

Ameringkogel Gleinalm

A A

47.07 47.22

14.82 15.05

5 5

5 5

PL

49.24

19.90

5

PL

49.22

20.00

SK

49.19

RO RO

Carpathians C-78 Western Tatras: Czerwone Wierchy C-79 High Tatras: Kasprowy Wierch C-80 High Tatras: Sedielko (Lodowa Przelecz) Pass C-81 Southern Carpathians: Peleaga C-82 Southern Carpathians: Iezer

2

5 5 5 5 5 5 3

5 5 5 5 5 5 5

5 4

1

5 5

5

4

5 5 5 5 4 3 5

4

5 5

5 5 5 4

5 1

3

Relative fluorescence intensity (mean 6 SD)

2.138 6 0.018 2.111 6 0.021 2.098 6 0.041 2.169 6 0.020 2.125 6 0.015 2.107 6 0.022 0.766 6 0.007, 2.152 6 0.010 0.756 6 0.006 0.755 6 0.006, 2.122 2.078 6 0.008 0.775 6 0.005 2.072 6 0.025 0.758 6 0.005 2.068 6 0.017 0.743 6 0.007 0.763, 2.112 6 0.019 1.485 6 0.015 external (2x), 2.105 6 0.036 1.451 6 0.038, 1.794 1.517 6 0.016 1.510 6 0.009 1.504 6 0.010 1.437 6 0.014 1.469 6 0.012, 2.170 1.482 6 0.016 1.502 6 0.005 0.821 6 0.006 1.496 6 0.007 0.778 6 0.004, 1.527, 2.285 1.509 6 0.005 2.104 6 0.012 2.111 6 0.023 2.153 6 0.029 external (4x), 2.158 6 0.018 2.116 6 0.019 2.143 6 0.022

Tissue

Chromosome count, 2n (no. of individuals counted)

Voucher no.

Silica Silica Silica Silica Silica Silica Silica

4589 4881 4602 4576 4610 4574 5597

Silica Silica

3711 4613

Silica Silica Silica Silica Silica Silica Silica

— 3770 — 3759 — — 4633

Silica Silica

5168 3747

Silica



7 Silica, 2 Fresh Silica Silica Silica Silica

80 (2)

3798 Cult. in HBV Cult. in HBV 3804 4557

Silica Silica Silica Silica Silica

— 7302 9679 Cult. in HBV —

Silica Silica Silica Silica Silica

— — 3735 4564 5218

Silica Silica

5220 5229

5 2.133 6 0.048

Silica

7011

5

5 2.097 6 0.031

Silica

7005

20.18

2

2 external (6x)

Silica

Cult. in HBV

45.37

22.88

5

5 2.232 6 0.022

Silica

10 562

45.50

24.92

5

5 2.154 6 0.030

Silica

9688

5 3

4 1 5

1

Note: A ¼ Austria; CH ¼ Switzerland; F ¼ France; I ¼ Italy; PL ¼ Poland; SK ¼ Slovakia; SLO ¼ Slovenia; RO ¼ Romania; Cult. ¼ cultivated.

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Fig. 2. Mitotic metaphase chromosomes of (A, B) diploid Senecio carniolicus var. insubricus (C-2, C-3: both 2n ¼ 2x ¼ 40); (C) tetraploid (C-61: 2n ¼ 4x ¼ 80); and (D) hexaploid S. carniolicus var. carniolicus (C27: 2n ¼ 6x ¼ 120). Scale bar ¼ 5 lm.

4x ¼ 80; and hexaploid: 2n ¼ 6x ¼ 120). The total number of FCM acquisitions was 683.

Chromosomes of polyploid cytotypes are slightly smaller than those of diploids (1–2 lm in hexaploids; Fig. 2D).

Statistical analyses—Data were analyzed with the SAS 8.1 statistical package using CORR and NPAR1WAY procedures (SAS Institute, Cary, North Carolina, USA). The Spearman-rank correlation coefficient was used to test whether mean fluorescence intensity was related to the geographic location of a population. Differences in relative fluorescence intensity between two disjunct groups of tetraploid populations were analyzed using the Kruskal– Wallis test.

Flow cytometry—DNA ploidy levels were estimated in 402 plants of S. carniolicus from 82 populations (380 individuals from 77 Eastern Alpine populations and 22 individuals from five Carpathian populations) and in eight plants from three populations of the closely related S. incanus. FCM analyses mostly yielded high-resolution histograms, with average sample CV of 2.95% (range 1.29–4.80%) and average standard CV of 2.01% (range 0.91–3.72%). The arbitrary threshold of 3.0% was achieved in 58.5% and 95.3% of sample and standard runs, respectively. Importantly, living and desiccated samples gave virtually identical fluorescence values. In addition, only negligible between-day variation (less than 3.2%) was observed when the same silica gel-dried sample was re-analyzed. Collectively, these measures of quality indicate that the recorded fluorescence values are stable and reliable. Three distinct groups of fluorescence intensities were obtained when 28 karyologically-counted diploid (2n ¼ 2x ¼ 40; 11 plants), tetraploid (2n ¼ 4x ¼ 80; eight plants), and hexaploid (2n ¼ 6x ¼ 120; nine plants) individuals of S. carniolicus were analyzed cytometrically. In addition, four

RESULTS Chromosome counts—Chromosome numbers were estimated for one to six individuals from each of 11 populations (32 individuals in total), confirming the presence of di- (2n ¼ 2x ¼ 40; Fig. 2A, B), tetra- (2n ¼ 4x ¼ 80; Fig. 2C), and hexaploid (2n ¼ 6x ¼ 120; Fig. 2D) cytotypes (Table 1). The chromosomes are mostly submeta- to metacentric and relatively small (2.5–5.0 lm in prometaphase and 1.0–2.5 lm in fully condensed metaphase; Fig. 2A and B, respectively), although they vary in size within chromosome complement, the smallest being less than half the size of the largest (Fig. 2A).

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Fig. 3. Fluorescence histograms of individual DNA ploidy levels of DAPI-stained nuclei isolated from silica-dried material of Senecio carniolicus and from fresh tissue of internal reference standards (A–C, Pisum sativum cv. Ctirad as primary reference standard; D–E, Vicia faba cv. Inovec as secondary reference standard) analyzed simultaneously using flow cytometry. The reference peak is marked with an asterisk. (A) 2x: population C-32; (B) 4x: population C-61; (C) 5x: population C-18; (D) 6x: population C-80; (E) 7x: population C-6.

plants of S. incanus with diploid number of chromosomes (2n ¼ 2x ¼ 40) formed a group of their own, with slightly but significantly higher relative fluorescence values than diploids of S. carniolicus. FCM investigation of 378 plants lacking any chromosome count further confirmed this differentiation and revealed two additional groups, corresponding to previously unknown DNA-pentaploids and DNA-heptaploids of S. carniolicus (Fig. 3). The majority of S. carniolicus plants belonged to DNAhexaploids (55.5%), while DNA-diploids and DNA-tetraploids made up 21.1% and 22.1% of the samples, respectively. In addition, rare odd-ploidy level cytotypes were detected, with about 1.0% DNA-pentaploids and 0.3% DNA-heptaploids (Fig. 1). Sympatric occurrence of two or more cytotypes was

encountered in more than one quarter of populations from the Alps (22 of 77), despite the rather limited number of analyzed individuals per population (on average fewer than five). The most frequent cytotype combination, 2x þ 6x, recorded in 14 populations, outnumbered other ploidy combinations: 4x þ 5x (two populations), 4x þ 6x (one population), and 6x þ 7x (one population). A few populations with more than two cytotypes were also detected: 2x þ 4x þ 6x plants co-occurred in populations C-33 (Italy, Cima d’Asta) and C-70 (Austria, Schiesseck), and 4x þ 5x þ 6x plants co-occurred in population C-18 (Italy, Stilfser Joch). Table 2 summarizes relative fluorescence intensities for S. carniolicus cytotypes. The nuclear DNA amount per monoploid genome (Cx-value according to Greilhuber et al., 2005)

TABLE 2. Summary of relative fluorescence intensities for cytotypes of Senecio carniolicus based on individual plant measurements (DAPI staining), using Pisum sativum cv. Ctirad (2C ¼ 9.09 pg) as a unit value. Ploidy level

Mean fluorescence intensity per cytotype 6 SD

Min. relative fluorescence intensity per cytotype

Max. relative fluorescence intensity per cytotype

Intra-cytotype variation (Max./Min.; %)

Mean fluorescence per monoploid genome

2x 4x 5x 6x 7x

0.790 6 0.027 1.498 6 0.029 1.834 6 0.035 2.151 6 0.051 2.520

0.734 1.413 1.794 2.044 —

0.849 1.583 1.890 2.285 —

15.7 12.0 5.4 11.8 —

0.395 0.375 0.367 0.359 0.360

Note: Min. ¼ minimum; Max. ¼ maximum.

N

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Fig. 4. Flow cytometric histograms documenting intraspecific genome size variation within diploid, tetraploid, and hexaploid cytotypes of Senecio carniolicus. Nuclei from dehydrated tissues of plants of the same ploidy level with different fluorescence intensities were isolated, stained with DAPI, and measured simultaneously. (A) 2x; (B) 4x; and (C) 6x cytotypes. Differences between mean peak positions are 3.5%, 5.3%, and 8.3%, respectively.

in even cytotypes decreased with increasing ploidy level. DNA-diploids had basic genomes that were on average 5.5% larger than the DNA-tetraploids and 10.0% larger than the DNA-hexaploids. Monoploid genome size in the DNApentaploids was almost exactly midway between 4x and 6x cytotypes, while DNA-heptaploids had the same Cx-value as the 6x plants. Variation in fluorescence intensity per cytotype within populations was mostly low, averaging 2.7%. Nevertheless, differences above the level of artifactual fluctuation caused by instrumental drift and/or nonidentical sample preparation were observed in several populations (an arbitrary threshold of 4% was passed in 14 populations; the actual resolution, however, largely depends on CV values and even smaller differences may be discriminated in case of very low CVs, see Fig. 4A). The most prominent examples were diploid plants from population C-28 (Austria, Gaisbergtal) with 6.1% fluorescence divergence, tetraploids from population C-60 (Austria, Samspitze) with 5.4% divergence, and hexaploids from populations C-36 (Italy, Cima Margerita) and C-78 (Poland, Czerwone Wierchy) with 7.1% and 7.3% divergence, respectively. Simultaneous FCM analyses confirmed the presumed differences in nuclear DNA amount and yielded histograms with clearly separated or at least bifurcated peaks. Relative fluorescence intensities differed not only within populations but also among populations. This variation amounted to 13.1% (2x populations), 10.2% (4x), 5.4% (5x), and 10.5% (6x) (see also Table 2 for overall variation between plants). Once again, the difference was reproducible and repeatedly confirmed in simultaneous FCM runs (Fig. 4). The relationship between geographic location of even ploidy level populations in the Alps and their mean fluorescence intensity was also tested. The relative fluorescence in DNAdiploids decreased significantly from both west to east (Spearman r ¼ 0.614, P ¼ 0.001, N ¼ 24) and south to north (r ¼ 0.707, P , 0.001, N ¼ 24). The same trend was also observed in other cytotypes, although only the correlation between fluorescence values of hexaploid populations and longitude was statistically significant (r ¼ 0.504, P , 0.001, N ¼ 51). The disjunct tetraploid populations did not significantly differ in fluorescence intensity based on the Kruskal–Wallis test (P ¼ 0.302, N ¼ 21).

DISCUSSION Large cytotype diversity—Flow cytometric screening of DNA ploidy levels in 402 individuals from 82 populations together with 28 confirmatory chromosome counts revealed the presence of di-, tetra-, penta-, hexa-, and heptaploid cytotypes in S. carniolicus, three of which (4x, 5x, 7x) were previously unrecorded. The even ploidy levels 2x, 4x, and 6x were found in 25, 22, and 56 populations, respectively, while pentaploids were restricted to three populations and heptaploids to one population. The large cytotype diversity in S. carniolicus contrasts sharply with previous ideas that assumed little ploidy variation and the prevalence of hexaploids (2n ¼ 6x ¼ 120; reports from the Alps and the Carpathians: Favarger, 1964, 1991; Murı´n and Ma´jovsky´, 1976; Dobesˇ and Vitek, 2000). Single reports of diploid S. carniolicus (Mattick-Ehrensberger in Tischler, 1950) from the Austrian Stubaier Alpen are not supported by voucher specimens and have been considered doubtful because of the notorious unreliability of the source (Dobesˇ and Gutermann, 2002), but they might be correct in light of the current results (see next section). The degree of intraspecific ploidy variation in S. carniolicus is remarkable, and there are few reports of comparable cytotype differentiation in other sexually reproducing plant species. Six different ploidy levels (2x to 7x, plus some aneuploids) are found in Cardamine pratensis s.s. (Brassicaceae) in the Iberian Peninsula (Lihova´ et al., 2003), and the same number (3x to 8x) occurs in Ixeris nakazonei (Asteraceae) in the Ryukyu Archipelago and Taiwan (Denda and Yokota, 2004). To gain unbiased insights into intraspecific ploidy diversity, large-scale population sampling is necessary, and in this respect, flow cytometry has already revolutionized the field of cytogeography. Distribution of cytotypes—While only hexaploid cytotypes seem to occur in the Carpathians (five populations with 22 individuals were analyzed; Table 1), the cytotype distribution in the Alps is complex. Diploids are widespread in the Eastern Alps (Fig. 1). In the majority of populations (16 out of 25), diploids occur together with polyploids (mostly hexaploids; see next section), and exclusively diploid populations are restricted to three main areas: (1) the southwestern part of the distribution area (Alpi Lepontine, westernmost Alpi Bergamasche; popu-

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lations C-1, C-2, and C-3); (2) the eastern parts of the Hohe Tauern (populations C-49, C-52, C-54, and C-56); and (3) the Karawanken in the southeastern part of the Alpine distribution area (population C-68), the last of which corresponds to the type locality of S. carniolicus. The diploid populations in the Alpi Lepontine and the western Alpi Bergamasche correspond to the distribution range of S. carniolicus var. insubricus (Hess et al., 1980). The only published chromosome count for this taxon is n ¼ c. 60 (under the name S. incanus subsp. insubricus: Favarger, 1965); however, the material originated from the central Alpi Bergamasche, where only polyploids occur (Fig. 1) and where morphological variability prevents the reliable discrimination of var. insubricus (Caccianiga et al., 2000). The hexaploid is the most widespread cytotype and occurs throughout most of the distribution area of S. carniolicus, often mixed with diploids. It is apparently absent from the most southwestern and most northeastern parts of the Eastern Alps (Fig. 1), where it is replaced by diploids or tetraploids. The latter cytotype is found in two disjunct areas in the southwest and the northeast of the distribution of S. carniolicus (Fig. 1). All three cytotypes occur in putative Pleistocene refugia for silicicolous alpine plants suggested by Tribsch and Scho¨nswetter (2003) and Scho¨nswetter et al. (2005), but while diploids and hexaploids successfully colonized terrain covered by glaciers during the last glacial maximum (van Husen, 1987), the tetraploid cytotype’s distribution is largely associated with formerly unglaciated areas (Fig. 1). With the current data, it is not possible to determine whether the current distribution of tetraploids is due to independent origins or, instead, the restriction of a once continuous distribution during Pleistocene glaciations and a subsequent failure to re-colonize. The most surprising result, however, is the wide distribution of diploids. In other polyploid complexes from the Alps, such as Biscutella laevigata s.l. (Brassicaceae) or Galium pumilum agg. (Rubiaceae), diploid taxa and/or cytotypes are confined to presumed peripheral refugia (Ehrendorfer, 1958; Tremetsberger et al., 2002). The hexaploid cytotype is expected to be the most successful colonizer of formerly glaciated areas (Levin, 1983; Otto and Whitton, 2000). This hypothesis is corroborated by the sole occurrence of hexaploids in the northwestern part of the Alpine distribution area (Fig. 1), which was most strongly affected by the Pleistocene glaciations (van Husen, 1987). Cytotype-mixture in populations is frequent—In 22 of 77 populations (28.6%) of S. carniolicus from the Alps, more than one cytotype was present, although, on average, only 4.94 individuals per population were investigated. This represents a very high proportion of mixed populations compared to other well-investigated species, such as Chamerion angustifolium (Onagraceae: Husband and Schemske, 1998) or Ranunculus adoneus (Ranunculaceae: Baack, 2004), where single-cytotype populations largely predominated. The majority of mixed populations contained di- and hexaploid cytotypes (16 populations; Table 1), and only in two (populations C-33 and C-70) were tetraploids also present. Five individuals each were collected from different altitudes from the former locality, the lower (sub)population being tetraploid and the higher one being mixed with di- and hexaploids. With the exception of population C-18 (Italy, Stilfser Joch), the mixed populations of tetra- and hexaploids and of diploids and tetraploids also lack intermediate ploidy levels. These results indicate that gene flow between different cytotypes is quite limited due to balanced selection (e.g., ecological sorting on a microscale or flowering

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time divergence: Felber-Girard et al., 1996; Husband and Schemske, 1998; Johnson et al., 2003) or that hybrids are not viable or have reduced viability, imposing reduction of fitness because of loss of gametes in intercytotype crosses. Alternatively, but less likely, S. carniolicus might be (predominantly) autogamous, which would also allow the establishment of autopolyploids within diploid populations (Baack, 2005b). More data on phenology, niche differentiation, and crossing behavior as well as more representative sampling in each population are obviously needed to test these hypotheses. Mode of origins of higher ploidy levels—The distribution pattern of the three main cytotypes is too complex to allow inferences about the modes of polyploid origin, given our fairly low sample size within populations. The interpretation of S. carniolicus var. insubricus as morphologically intermediate between S. incanus and S. carniolicus s.s. (Chenevard, 1906; Braun-Blanquet, 1913; Chater and Walters, 1976) might suggest a hybridogenous origin of (allopolyploid) S. carniolicus with S. incanus as one parent. However, the presence of diploid S. carniolicus (including var. insubricus) makes autopolyploidy a more likely hypothesis. This does not preclude the possibility of polyploidization after crosses of already differentiated diploid lineages, as suggested by variation in nuclear DNA content (see following section) and phylogenetic patterns inferred from nuclear ITS sequences (A. Tribsch et al., unpublished data). Nuclear DNA content variation—Significant intracytotype variation in relative fluorescence intensity, as high as 15.7% (Table 2), was detected in all cytotypes in which multiple individuals were analyzed cytometrically. Although the differences were recorded in silica-dried material, they can be considered reliable and unbiased for several reasons: (1) low CVs were achieved, which are not compatible with the presence of interfering metabolites; (2) reinvestigation of the same sample gave virtually identical fluorescence values even when single FCM analyses were performed over a time span of 5 mo; and more importantly, (3) simultaneous analysis of samples with different fluorescence intensities yielded two distinct peaks or, at least, a clearly bifurcated one (Fig. 4). The appearance of two separate peaks in co-processed runs is the most convincing test for true differences in DNA content (Greilhuber, 2005). Aneuploidy may explain part of the variation observed, although only euploid chromosome numbers are reported in the literature and have also been found in the 32 individuals analyzed here. Another possible explanation is the introgression of a closely related taxon with different genome size, S. incanus from the Western Alps being an ideal candidate (see Table 1). However, molecular fingerprint data (A. Tribsch and P. Scho¨nswetter, unpublished) do not support the introgression hypothesis but rather indicate distinct evolutionary pathways for the geographically separated diploids. The correlation of genome size with proximity to putative refugial areas, which has also been observed in the grass Festuca pallens (Sˇmarda and Buresˇ, 2006), suggests that a reduction in genome size due to, for instance, activities of transposable elements or sequence elimination (Bennetzen, 2000; Bennetzen et al., 2005) might be advantageous for (re-)colonization. Further FCM research, including more samples of S. incanus s.s. and the use of molecular techniques, are required to elucidate causes of this variation in clinal genome size.

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