13(3)_05 raycheva, tz. & al. rumex pulcher in the - bio.bas.bg
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13(3)_05 Raycheva, Tz. & al. Rumex pulcher in the Bulgarian
flora.inddRumex pulcher (Polygonaceae) in the Bulgarian flora:
distribution, morphology, and karyology
Tzvetanka Raycheva1, Eva M. Temsch2 & Dessislava Dimitrova3
1 Department of Botany, Agricultural University of Plovdiv, 12 Mendeleev St., 4000 Plovdiv, Bulgaria, e-mail: [email protected]
2 Faculty Centre of Botany, University of Vienna, 14 Rennweg St., A-1030, Vienna, Austria, e-mail: [email protected]
3 Institute of Botany, Bulgarian Academy of Sciences, Acad. Georgi. Bonchev S., bl. 23, 1113 Sofia, Bulgaria, e-mail: [email protected]
Received: April 05, 2007 Accepted: April 24, 2007
Abstract. Of the four subspecies of Rumex pulcher distributed in Europe, three occur in Bulgaria. The type subspecies pulcher is widely distributed in the country, while R. pulcher subspp. woodsii and raulinii are registered in the more southern parts of the country with Mediterranean climatic conditions. We have applied a set of classical (morphological and karyological) and modern (genome size measurements and scanning microscopy) methods to identify the taxonomically most reliable features of the three taxa. An identification key for the three subspecies is presented. The chromosome number of the three subspecies is 2n = 20. Their genome size (1C-values) varies between 0.76 and 0.88 pg.
Key words: Bulgaria, genome size, karyology, morphology, Rumex pulcher, scanning microscopy
Introduction
Rumex pulcher L. is a polymorphic species repre- sented in Europe by four subspecies (Akeroyd 1993). According to this author, R. pulcher L. subsp. pulcher and R. pulcher subsp. woodsii (De Not) Archang. are distributed in Bulgaria. Prior to Vulev (1966), no sub- specific taxa have been recognized in R. pulcher for the Bulgarian flora (Stojanov & Stefanov 1924, 1933, 1948; Stojanov & al. 1966). In the latest Bulgarian flo- ristic literature (Delipavlov 2003), R. pulcher subsp. raulinii (Boiss.) Rech. f. is also reported for the nation- al flora. Our field studies confirm the three subspecies for the territory of Bulgaria.
Rumex pulcher subsp. pulcher is widely distributed across the country and often behaves as a weed. The remaining two subspecies are of more restricted distri-
bution in areas with clearly expressed Mediterranean climatic conditions.
Different authors have treated the morpholo- gical variation of R. pulcher at different taxono mic ranks (Hayek 1924; Rechinger 1932; Cullen 1967). We present here the results of a comparative mor- phological study of the three subspecies distributed in Bulgaria, including scanning microscopy of the valves and fruits.
The karyology of R. pulcher s.l. has been studied by different authors from various geographic regions (Table 1). Most of the counts, including earlier ones from Bulgaria (Stoeva 1987; Raycheva 2005), show a diploid chromosome number 2n = 20. Only Dahlgren & al. (1971) and Garcia & al. (1989) have reported a tetraploid number for R. pulcher subsp. woodsii from Spain.
322 Raycheva, Tz. & al. • Rumex pulcher in the Bulgarian flora
Table 1. References on the chromosome counts for the subspecies of R. pulcher distributed in Bulgaria. Taxon 2n Origin Reference
R. pulcher subsp. pulcher
20 Turkey Degraeve (1975) 20 Bulgaria Stoeva (1987) 20 Central Europe Löve & Löve (1961) 20 Spain Garcia & al. (1989) 20 Sweden Löve (1967) 20 Denmark Ichikawa & al. (1971) 20 California Löve (1986) 20 Japan Himi & al. (1999) 20 Greece Baltisberger (1991) 20 Bulgaria Raycheva (2005)
R. pulcher subsp. woodsii
20 Albania Strid (1971) 40 Spain Dahlgren & al. (1971) 40 Balearic Islands Garcia & al. (1989) 20 Bulgaria Raycheva (2005) 20 Italy Löve (1967)
R. pulcher subsp. raulinii 20 Greece Löve (1967) 20 Bulgaria Raycheva (2005)
In the last decades, estimation of the amount of DNA contained in an organism‘s chromosome complement (C-value) and also, in polyploids, in the constituent chromosome sets (Cx-value), has proven to be a relia- ble taxonomic tool, as the closely related species or sub- specific entities can measurably differ in their genome size (Bennett & al. 2000; Gregory 2005). Genome size is measured with flow cytometry and Feulgen densitome- try. Some plant species pose significant technical prob- lems caused by protein- and DNA-binding endogenous metabolites (Greilhuber 1986). The representatives of subgenus Rumex are known to have such compounds in their cells. So far the DNA content of R. pulcher has not been measured (Bennett & Leitch 2004).
To our knowledge, no data on the microscopic char- acteristics (SEM electron microscopy) of the valves of R. pulcher have been published so far. Taking into consider- ation that valves provide a number of taxonomically re- liable morphological features, we present data about the surface of the valves and the tuberculae.
The present study of R. pulcher s.l. provides data for a clear delimitation of subspp. pulcher, woodsii and raulinii in the Bulgarian flora.
Material and methods
The herbarium collections of R. pulcher s.l. in the three national herbaria (SOM, SOA and SO) have been stu- died. For the karyological studies, genome size esti-
mation and scanning microscopy, plant material has been collected from 15 natural accessions in Bulgaria, in the period 2003–2005 (Table 2). Voucher speci- mens were deposited in the herbarium SOA (Agrarian University, Plovdiv). The chorological information has been processed according to Kozhuharov & al. (1983), mapped and included in a data base with the help of dSOA software (Stoyanov 2003). The floristic regions follow the standard accepted in Flora R Bulgaria.
Plant material collected by the authors has been used for scanning microscopy (Table 2). The ob- jects have been observed directly, without any physi- cal or chemical treatment. The valves have been pre- pared for observation prior to scanning, following the methodology of Terziisky & Atanasov (1977) and Terziisky (1983). The observations have been con- ducted with a JEOL electron scanning microscope, with scanning adaptor JSM-5500.
A classical squash technique has been applied for the karyological studies. Root tips were pretreated with 0.05 % colchicine for 2 hours, fixed in ethanol:glacial acetic acid (3:1) for at least 2 h at room temperature or for 24 h in the refrigerator, and stored in 96 % ethanol until required. Hydrolysis was conducted in 1N HCl at 60 °C for 20–40 min. Then the root tips were trans- ferred into HCl:di-ethyl ether (1:1) for 9 min at 60 °C, washed thoroughly in distilled water and stained with haematoxylin after Gomori (Melander & Wingstrand 1953) for 20–25 min at 60 °C. Finally, the root tips were squashed in 45 % acetic acid and mounted in Canadian balsam.
Nuclear C-values of R. pulcher were investi ga ted by the two available methods: flow cytometry and Feulgen densitometry.
The nuclei isolation procedure for flow cytometry was based on Galbraith & al. (1983). Young leaves of Hordeum vulgare 'Ditta' (1C = 5.02 pg DNA, Doleel & al. 1998) served as internal standard. After prepa- ration of the nuclei suspension and its incubation with propidium iodide (PI) as described in Baranyi & Greilhuber (1996), the measurement was conducted on a Ploidy Analyser II (PAII, Partec, Münster, Germany) equipped with a mercury lamp and the appropriate fil- ter combination (TK560, EM520). Depending on the availability of the plant material, one to three indivi- duals were measured from every accession. For each individual and isolate, three runs of 5000 counts each were measured. Coefficients of variation of G1 peaks of R. pulcher and H. vulgare mostly did not exceed 3 %.
323Phytol. Balcan. 13(3) • Sofia • 2007
Table 2. List of accessions studied and methods applied to R. pulcher s.l. in Bulgaria. No Taxon, locality, UTM, voucher number Methods applied R. pulcher subsp. pulcher 1 Black Sea Coast (Southern): grassy places around Sozopol, 20 m, 03.07.2004; NG-59 (Raycheva) K, Map, M
2 Black Sea Coast (Southern): Kraimorie village, 20 m, 23.06.2003, NG-67 (Raycheva) Fl, Fe, Map, M
3 Danubian Plain: Somovit village, Nikopol district, 50 m, 25.06.2003, LJ-24 (Raycheva) SOA 56363 K, Map, M
4 Forebalkan (Western): hay meadows along the left bank of Bela Reka River, near Dolna Bela Rechka village, Montana district, 400 m, 27.6.2002, FN-98 43,1218 N, 23,1931 E, (Dimitrova)
Fl, Fe, Map, M
5 Rhodopi Mts (Eastern): near Ivailovgrad – Doupkata Protected Area, 104 m, 14.07.2005, MF-29 (Raycheva) SEM, Map, M
6 Rhodopi Mts (Eastern): village Odrintsi, 194 m, 15.07.2005, MF-28 (Raycheva) Fe, Map, M
7 Thracian Lowland: near Pyasuchnik dam, 300 m, 22.06.2003, KG-99 (Raycheva) SOA 56407 Fl, Fe, K, Map, M
R. pulcher subsp. woodsii 8. Black Sea Coast (Southern): light oak forests near Sinemorets village, 40 m, 03.07.2004, NG-85 (Raycheva) SOA 57068 Fl, Fe, K, Map, M
9* Rhodopi Mts (Eastern): betwen Siv Kladenets and Odrintsi villages, 194 m, 15.07.2005, MF-28 (Raycheva) SOA 56931 Fl, Fe, Map, M
10* Rhodopi Mts (Eastern): near Belopolyane village, in sunflower fields, 170 m, 15.07.2005; MF-38 (Raycheva) SOA 56929 SEM, Fe, Map, M
11* Rhodopi Mts (Eastern): abandoned lands and pastures near Ivailovgrad, 160 m, 15.07.2005, MF-29 (Raycheva) SOA 56930 Fe, Map, M
R. pulcher subsp. raulinii 12* Black Sea Coast (Southern): rare oak forests near Sinemorets village, 40 m, 03.07.2004, NG-85 (Raycheva) SOA 57067 Fl, Fe, K, Map, M
13* The Valley of Strouma River: Kresna town, 180 m, 18.06.2005, FM-72 (Raycheva) SOA 56926 K, Map, M
14* Belasitsa Mts: between Skrut and Klyuch villages, 300 m, 18.06.2005, F-L68 (Raycheva) SOA 56927 SEM, Fl, Fe, Map, M
15* Rila Mts: Dupnitsa town, above the road tunnels, 510 m, 19.06.2005, FM-78 (Raycheva) SOA 56928 K, Map, M
Fl – flow cytometry; Fe – Feulgen densitometry; SEM – scanning electron microscopy; K – karyological studies with classical haema- toxylin method; Map – mapped locality; M – gross-morphological studies; * – plants from a new floristic region.
For quantitative Feulgen staining (Feulgen & Rossenbeck 1924), seedlings of R. pulcher were co- fixed in 4 % neutral formaldehyde solution, togeth- er with primary root tips of the standard organism Pisum sativum 'Kleine Rheinländerin' (1C = 4.42 pg DNA, Greil hu ber & Ebert 1994). These samples were post-fixed with methanol acetic acid (3:1) and after- wards stored in 96 % ethanol in the freezer until use. Following the preparation method of Greilhuber & Ebert (1994), rehydrated material was hydrolyzed in 5N HCl for 75 min at 20.0 °C (Greilhuber & Baranyi 1999; Greilhuber & Temsch 2001) and stained in Schiff´s reagent for 1.5 hours at room temperature. After washing out in SO2-water, the plant tissue was squashed in 45 % acetic acid, briefly rinsed in 96 % ethanol, and finally air-dried. Three slide pairs of R. pulcher and the standard tissue were produced in par- allel per accession. For each slide, 20 G1/0 or 10 te- lophase plus 10 prophase nuclei were measured. We have used the Cell Image Retrieval and Evaluation System (CIRES, Kontron, Munich, Germany) for measurement.
Results and discussion
Chorology and ecology
Rumex pulcher subsp. pulcher occurs in the Medi te- r ra nean region, Balkan Peninsula, Asia Minor, Hun- garian lowlands, Atlantic West Europe. It has spread into Asia and got naturalized in America (Akeroyd 1993). In Bulgaria, the subspecies grows in open grassy habitats, along roads, and as a weed in agri- cultural phytocoenoses across the country, from 0 up to 1500 m a.s.l. (Fig. 1, Table 3).
Rumex pulcher subsp. woodsii is abundant and often occurs as a ruderal plant in the Aegean region, SW Asia and the central part of East Mediterranean. The subspe- cies has been introduced in Central and West Europe and different parts of America and South Africa. In Bulgaria, the taxon has been reported in literature from the Black Sea Coast (Southern), Balkan Range (Eastern) and Toundzha Hilly Country. In the course of the cur- rent study, the subspecies was confirmed for the Black Sea Coast (Southern), and registered for the first time
324 Raycheva, Tz. & al. • Rumex pulcher in the Bulgarian flora
Table 3. Chorological data on Rumex pulcher s.l. in Bulgaria. Literature sources Herbarium specimens Floristic regions Altitude Floristic regions Altitude R. pulcher L. subsp. pulcher 1.1 (Urumov 1908b); 1.2 (Urumov 1901; Davidov 1905; Rechinger 1932, 1933); 2 (Urumov 1901; 1905a; Kovachev 1903; Rechinger 1932; Stoeva 1987); 3 (Urumov 1898, 1901, 1902, 1910, 1917, 1925, 1926, 1928, 1935a); 4.1 (Velenovský 1898; Urumov 1898, 1901, 1905a, 1917, 1925, 1926, 1928, 1935a; Rechinger 1932, 1933); 4.2 (Urumov 1897, 1926, 1928); 5.1 (Urumov 1902, 1905b, 1935a); 5.2 (Urumov 1897, 1898, 1917, 1929b; Neichev 1908; Baev 1947); 5.3 (Velenovský 1891; Urumov 1909); 6 (Velenovský 1891; Urumov 1905b, 1906, 1910, 1929a, 1930, 1935b); 7 (Urumov 1905b, 1913, 1935b); 8 (Urumov 1929a, 1930); 9 (Urumov 1904, 1913, 1935b); 11 (Urumov 1905a); 14.1 (Urumov 1923); 15 (Urumov 1904, 1906); 16.1 (Urumov 1902, 1905a, 1908a, 1935b); 16.2 (Urumov 1904; 1910, 1929b); 17.1 (Urumov 1906, Rechinger 1932); 18 (Velenovský 1891; Urumov 1906, 1908b, 1917, 1929b; Rechinger 1932); 19 (Urumov 1909, 1910). Vulev (1966), Andreev (1992), and Delipavlov (2003) report the taxon for whole territory of the country
up to 2000 m 1.1; 1.2; 2; 3; 4; 4.2; 5.2; 5.3; 6; 7; 8; 9; 10*; 11; 12*;
13*; 14.1; 15; 16.1; 16.2; 17.1; 17.2*; 17.3*; 18; 19; 20*
up to 1100 m
R. pulcher subsp. woodsii 1.1 (Panov 1987); 5.3 (Panov 1987); 19 (Panov 1987). Andreev (1992) and Delipavlov (2003) report the taxon for the Black Sea Coast, Balkan Range (Eastern) and Toundzha Hilly Country.
– 1.1; 17.3* up to 200 m
R. pulcher subsp. raulinii Vulev (1966), Andreev (1992), and Delipavlov (2003) report the taxon for Black Sea Coast, Forebalkan (Eastern) and Thracian Llowland.
– 1.1; 10*; 11*; 15* up to 500 m
1 – Black Sea Coast (1.1 – Southern, 1.2 – Northern); 2 – NE Bulgaria; 3 – Danubian Plain; 4 – Forebalkan (4.1 – Western, 4.2 – East- ern); 5 – Balkan Range (5.1 – Western, 5.2 – Central, 5.3 – Eastern); 6 – Sofia region; 7 – Znepole region; 8 – Vitosha region; 9 – West Frontier Mts; 10 – The Valley of Strouma River (10.1 – Southern, 10.2 – Northern); 11 – Mt Belasitsa; 12 – Mt Slavyanka; 13 – The Val- ley of Mesta River; 14 – Pirin Mts (14.1 – Southern, 14.2 – Northern); 15 – Rila Mts; 16 – Mt Sredna Gora (16.1 – Western, 16.2 – East- ern); 17 – Rhodopi Mts (17.1 – Western, 17.2 – Central, 17.3 – Eastern); 18 – Thracian Lowland; 19 – Toundzha Hilly Country; 20 – Mt Strandzha; * – new data.
Fig. 1. Distribution of R. pulcher subsp. pulcher in Bulgaria. Note: Th is fi gure includes also data on R. pulcher s.l. from the Bulgarian fl oristic literature prior to Vulev (1966), where the subspecifi c variation has not been considered.
– new and unpublished data
325Phytol. Balcan. 13(3) • Sofia • 2007
in the Rhodopi Mts (Eastern) (Fig. 2, Table 3). The sub- species occurs in secondary herbaceous plant commu- nities, with Lolium perenne L., Dactylis glomerata L., Aegilops triuncialis L., Alopecurus myosuroides Huds., Cirsium sp., Filago vulgaris Lam., Plantago lanceolata L., Eryngium campestre L., Hypericum perforatum L., Daucus guttatus Sm., and Potentilla pedata Willd.
Rumex pulcher subsp. raulinii is distributed in the Aegean region. The subspecies has been reported in the Bulgarian literature from the Black Sea Coast, Forebalkan, Balkan Range (Central), and Thracian Lowland. Our revision of the Bulgarian herbaria has revealed one specimen belonging to R. pulcher sub- sp. raulinii (SV: coll. B. Davidov, SOM 17832, in hu- midis Deli Orman, July 1903) that had been first de- termined as R. palustris, and subsequently revised by B. Achta rov as R. pulcher. We have confirmed the taxon for the Black Sea Coast (Southern), and disco- vered it for the first time in the Valley of River Strou- ma, Mt Belasi tsa, and Rila Mts (Fig. 3, Table 3). It occurs seldom in thin oak forests (Quercus cerris L., Q. frainetto Ten.), accom panied by Briza maxima L., Poa bulbosa L., P. sylvicola Guss., Bromus tectorum L., Lolium perenne L., Festuca pratensis Huds., Cistus in- canus L., and Cynosurus cristatus L.
Morphological characteristics
Rumex pulcher s.l. shows a typical tendency to xero- phytization, e.g, formation of fleshy tubers on the main root, reduction in the leaf size, distinctly fiddle- shaped leaves, and indumentum on stems and leaves. These features appear in all three subspecies but are most clearly expressed in the typical one.
Rumex pulcher is a perennial plant. In R. pulcher subsp. woodsii, tubers can be frequently found on the main root. Typical for this taxon is the fiddle- like shape of the basal leaves, but our observations have shown that this cannot be used as a diagnostic feature on suspecific level, since it is regularly found in the three subspecies distributed in Bulgaria. This is contrary to Rechinger (1932), according to whom the fiddle-shaped leaves distinguish R. pulcher subsp. woodsii from R. pulcher subspp. pulcher and raulinii. The leaf and stem indumentum varies considerably in the three taxa. It is mostly papillose, and seldom (in subsp. woodsii) consists of unicellular trichomes. The stem height also varies between the subspecies but in ge neral the typical subspecies has shorter, flexuose, often intricate stems, while R. pulcher subsp. woodsii has high stems with long lateral divaricate branches.
– new and unpublished data
– herbarium specimens
– literature data Fig. 2. Distribution of R. pulcher subsp. woodsii in Bulgaria.
326 Raycheva, Tz. & al. • Rumex pulcher in the Bulgarian flora
Our observations have shown that most of the taxo nomically reliable morphological features for sub ge nus Rumex can be found in the valves of the mature plants (length/width of valves, tuberculae and ochreae, size and number of spines) (Table 4).
Table 4. Morphological parameters of the valves of R. pulcher s.l.
Character
±S (mm)
±S (mm)
±S (mm)
Valve length 4.73±0.02 5.46±0.03 5.34±0.03 Valve width 3.00±0.03 4.65±0.03 2.96±0.02 Tubercule length 2.38±0.03 2.81±0.02 2.48±0.03 Tubercule width 1.18±0.01 1.55±0.01 1.13±0.02 Ochrea length 2.33±0.03 2.84±0.02 2.00±0.02 Ochrea width 1.46±0.02 1.84±0.02 1.50±0.02
The identification key given below is based on morphological features that have proven to be most discrete and reliable for identification of the three sub- species. The data related to plant habitus, morphology of basal leaves and indumentum type are considered reliable for the purpose and are not used, in contrast to Akeroyd (1993).
Identification key
1 Valves clearly dentate, oblong to narrowly ovate, 4.5–5.5 mm long, 3(–3.4) mm wide, with 4–6 irregular spines longer than 1 mm; branches short, flexuose, often intricate…………………………………………2
1* Valves slightly dentate, rounded in shape, 5.2–6 mm long, 4.2–5.2 mm wide, with 6–8 almost regular 0.5–0.8 mm long spines; branches long, divari- cate……………………………………subsp. woodsii
2 Some spines 3–3.5 mm long ………subsp. raulinii
2* Some spines 1.5–2 mm long ………subsp. pulcher
Scanning microscopy supports the significance of the valves morphology for identification of the three subspecies. Furthermore, the surface of the tubercu- lae also shows some differences that correspond to the subspecific taxonomic concept. In R. pulcher subsp. raulinii their structure is more robust and papillose as compared to the other two taxa.
The surface of the valves is similar in all three sub- species, but when compared to other species in subge- nus Rumex, their thick and robust venation outlines
– new and unpublished data
Fig. 3. Distribution of R. pulcher subsp. raulinii in Bulgaria.
327Phytol. Balcan. 13(3) • Sofia • 2007
a clear tendency towards xerophytization. In R. pul- cher subsp. woodsii the surface of the valves is larger and spines are shorter (0.4–0.8 mm). In the remaining two subspecies the valves are narrower and elongated, while the spines are longer and more robust, reaching 2.8–3.2 mm and exceeding the valve width.
The tuberculae are usually three in number, only in R. pulcher subsp. woodsii two of them are reduced to nodes. The surface of the valve tuberculae is irregular- ly concave-undulate, but distinctly differs in the three taxa. Most clearly differentiated are the concave struc- tures in R. pulcher subsp. raulinii, where the pits are strongly undulated (Plate I, 3b), while in the remain- ing two subspecies the pit surface is smooth. The dif- ference in valve architecture correlates with the gross- morphology of the three taxa and their ecological characteristics. Rumex pulcher subsp. pulcher, which shows the strongest tendency to xerophytization, has the smallest concave structures with irregular shape as compared to the remaining two subspecies.
Karyology and genome size variation
Further studies (in addition to Raycheva 2005) on the karyology of R. pulcher in Bulgaria have shown stable chromosome numbers 2n = 2x = 20 for the three sub- species (Fig. 4).
Apart from classical karyology, we have applied flow cytometry and Feulgen densitometry to test for nuclear C-value variation among the Bulgari- an subspecies of R. pulcher. An earlier comparison with the Feulgen stained samples fixed in formalde- hyde or methanol acetic acid (3:1) revealed the pres- ence of secondary metabolites in the present materi- al (Fig. 5). Such substances are known as inhibitors of DNA staining (Greilhuber 1986, 1988), but sus-
metry diverge widely. However, in our investigation we found good data agreement, when formaldehyde fixed material was used for Feulgen densitometry.
The mean 1C-values were 0.76 pg DNA in R. pul- cher subsp. woodsii, 0.83 pg DNA in R. pulcher subsp. raulinii, and 0.82 pg DNA in R. pulcher subsp. pulcher. The C-value of R. pulcher subsp. woodsii was approx. 7 % lower than the values of the other two subspecies (Table 5). This difference underlines the more specific morphological syndrome of R. pulcher subsp. woodsii and its more divergent position within R. pulcher s.l.
Table 5. Comparative DNA data obtained by flow cytometry and Feulgen densitometry. Locality numbers follow the sequence in Table 2.
Locality No Flow cytometry pg/1C±S.D
Feulgen densitometry pg/1C±S.D
R. pulcher L. subsp. pulcher 2 0.80±0.02 0.83±0.05 4 0.79±0.02 0.81±0.04 6 – 0.81±0.03 7 0.79±0.02 0.88±0.08 R. pulcher subsp. wodsii 8 0.76±0.01 0.76±0.06 9 – 0.70±0.06 10 – 0.81±0.04 11 – 0.81±0.09 R. pulcher subsp. raulinii 12 0.80±0.02 0.82±0.03 14 – 0.85±0.004
Fig. 4. Metaphase plate of R. pulcher subsp. woodsii. Scale bar = 5 μm.
ceptibility of the two approaches (flow cy- tometry and Feulgen densitometry) to that problem is different. A strong influence of in- hibitors can often be demonstrated, when re- sults with flow cytomet- ry and Feulgen densito-
Fig. 5. Root-tip cells of R. pulcher; fi xation in 4 % formaldehyde, Feulgen staining. Arrowhead shows polymerized secondary compounds (probably condensed tannins) in many cells and (n) shows regular staining of nuclei.
7• Phytol. Balcan. 13(3) • 2007
328 Raycheva, Tz. & al. • Rumex pulcher in the Bulgarian flora
SEM of valves and ochreae surface of the three subspecies of R. pulcher: 1a, 1b, subsp. pulcher; 2a, 2b, subsp. woodsii; 3a, 3b, subsp. raulinii.
Plate I
1a 1b
2a 2b
3a 3b
Conclusions
The biosystematic study of R. pulcher in Bulgaria has confirmed the existence of three subspecies in the na- tional flora: R. pulcher subspp. pulcher, woodsii and raulinii. Morphological studies and genome size esti- mations have confirmed the more divergent position of R. pulcher subsp. woodsii in the plant group.
Acknowledgements. The study has been supported financially by Bulgarian National Science Fund (Project VUB 10/5) and FWF Austrian Science Fund (P14607 B03). The authors are grateful to Prof. Johann Greilhuber for his support and useful comments on the paper and to the anonymous reviewer for the constructive and valuable suggestions.
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Tzvetanka Raycheva1, Eva M. Temsch2 & Dessislava Dimitrova3
1 Department of Botany, Agricultural University of Plovdiv, 12 Mendeleev St., 4000 Plovdiv, Bulgaria, e-mail: [email protected]
2 Faculty Centre of Botany, University of Vienna, 14 Rennweg St., A-1030, Vienna, Austria, e-mail: [email protected]
3 Institute of Botany, Bulgarian Academy of Sciences, Acad. Georgi. Bonchev S., bl. 23, 1113 Sofia, Bulgaria, e-mail: [email protected]
Received: April 05, 2007 Accepted: April 24, 2007
Abstract. Of the four subspecies of Rumex pulcher distributed in Europe, three occur in Bulgaria. The type subspecies pulcher is widely distributed in the country, while R. pulcher subspp. woodsii and raulinii are registered in the more southern parts of the country with Mediterranean climatic conditions. We have applied a set of classical (morphological and karyological) and modern (genome size measurements and scanning microscopy) methods to identify the taxonomically most reliable features of the three taxa. An identification key for the three subspecies is presented. The chromosome number of the three subspecies is 2n = 20. Their genome size (1C-values) varies between 0.76 and 0.88 pg.
Key words: Bulgaria, genome size, karyology, morphology, Rumex pulcher, scanning microscopy
Introduction
Rumex pulcher L. is a polymorphic species repre- sented in Europe by four subspecies (Akeroyd 1993). According to this author, R. pulcher L. subsp. pulcher and R. pulcher subsp. woodsii (De Not) Archang. are distributed in Bulgaria. Prior to Vulev (1966), no sub- specific taxa have been recognized in R. pulcher for the Bulgarian flora (Stojanov & Stefanov 1924, 1933, 1948; Stojanov & al. 1966). In the latest Bulgarian flo- ristic literature (Delipavlov 2003), R. pulcher subsp. raulinii (Boiss.) Rech. f. is also reported for the nation- al flora. Our field studies confirm the three subspecies for the territory of Bulgaria.
Rumex pulcher subsp. pulcher is widely distributed across the country and often behaves as a weed. The remaining two subspecies are of more restricted distri-
bution in areas with clearly expressed Mediterranean climatic conditions.
Different authors have treated the morpholo- gical variation of R. pulcher at different taxono mic ranks (Hayek 1924; Rechinger 1932; Cullen 1967). We present here the results of a comparative mor- phological study of the three subspecies distributed in Bulgaria, including scanning microscopy of the valves and fruits.
The karyology of R. pulcher s.l. has been studied by different authors from various geographic regions (Table 1). Most of the counts, including earlier ones from Bulgaria (Stoeva 1987; Raycheva 2005), show a diploid chromosome number 2n = 20. Only Dahlgren & al. (1971) and Garcia & al. (1989) have reported a tetraploid number for R. pulcher subsp. woodsii from Spain.
322 Raycheva, Tz. & al. • Rumex pulcher in the Bulgarian flora
Table 1. References on the chromosome counts for the subspecies of R. pulcher distributed in Bulgaria. Taxon 2n Origin Reference
R. pulcher subsp. pulcher
20 Turkey Degraeve (1975) 20 Bulgaria Stoeva (1987) 20 Central Europe Löve & Löve (1961) 20 Spain Garcia & al. (1989) 20 Sweden Löve (1967) 20 Denmark Ichikawa & al. (1971) 20 California Löve (1986) 20 Japan Himi & al. (1999) 20 Greece Baltisberger (1991) 20 Bulgaria Raycheva (2005)
R. pulcher subsp. woodsii
20 Albania Strid (1971) 40 Spain Dahlgren & al. (1971) 40 Balearic Islands Garcia & al. (1989) 20 Bulgaria Raycheva (2005) 20 Italy Löve (1967)
R. pulcher subsp. raulinii 20 Greece Löve (1967) 20 Bulgaria Raycheva (2005)
In the last decades, estimation of the amount of DNA contained in an organism‘s chromosome complement (C-value) and also, in polyploids, in the constituent chromosome sets (Cx-value), has proven to be a relia- ble taxonomic tool, as the closely related species or sub- specific entities can measurably differ in their genome size (Bennett & al. 2000; Gregory 2005). Genome size is measured with flow cytometry and Feulgen densitome- try. Some plant species pose significant technical prob- lems caused by protein- and DNA-binding endogenous metabolites (Greilhuber 1986). The representatives of subgenus Rumex are known to have such compounds in their cells. So far the DNA content of R. pulcher has not been measured (Bennett & Leitch 2004).
To our knowledge, no data on the microscopic char- acteristics (SEM electron microscopy) of the valves of R. pulcher have been published so far. Taking into consider- ation that valves provide a number of taxonomically re- liable morphological features, we present data about the surface of the valves and the tuberculae.
The present study of R. pulcher s.l. provides data for a clear delimitation of subspp. pulcher, woodsii and raulinii in the Bulgarian flora.
Material and methods
The herbarium collections of R. pulcher s.l. in the three national herbaria (SOM, SOA and SO) have been stu- died. For the karyological studies, genome size esti-
mation and scanning microscopy, plant material has been collected from 15 natural accessions in Bulgaria, in the period 2003–2005 (Table 2). Voucher speci- mens were deposited in the herbarium SOA (Agrarian University, Plovdiv). The chorological information has been processed according to Kozhuharov & al. (1983), mapped and included in a data base with the help of dSOA software (Stoyanov 2003). The floristic regions follow the standard accepted in Flora R Bulgaria.
Plant material collected by the authors has been used for scanning microscopy (Table 2). The ob- jects have been observed directly, without any physi- cal or chemical treatment. The valves have been pre- pared for observation prior to scanning, following the methodology of Terziisky & Atanasov (1977) and Terziisky (1983). The observations have been con- ducted with a JEOL electron scanning microscope, with scanning adaptor JSM-5500.
A classical squash technique has been applied for the karyological studies. Root tips were pretreated with 0.05 % colchicine for 2 hours, fixed in ethanol:glacial acetic acid (3:1) for at least 2 h at room temperature or for 24 h in the refrigerator, and stored in 96 % ethanol until required. Hydrolysis was conducted in 1N HCl at 60 °C for 20–40 min. Then the root tips were trans- ferred into HCl:di-ethyl ether (1:1) for 9 min at 60 °C, washed thoroughly in distilled water and stained with haematoxylin after Gomori (Melander & Wingstrand 1953) for 20–25 min at 60 °C. Finally, the root tips were squashed in 45 % acetic acid and mounted in Canadian balsam.
Nuclear C-values of R. pulcher were investi ga ted by the two available methods: flow cytometry and Feulgen densitometry.
The nuclei isolation procedure for flow cytometry was based on Galbraith & al. (1983). Young leaves of Hordeum vulgare 'Ditta' (1C = 5.02 pg DNA, Doleel & al. 1998) served as internal standard. After prepa- ration of the nuclei suspension and its incubation with propidium iodide (PI) as described in Baranyi & Greilhuber (1996), the measurement was conducted on a Ploidy Analyser II (PAII, Partec, Münster, Germany) equipped with a mercury lamp and the appropriate fil- ter combination (TK560, EM520). Depending on the availability of the plant material, one to three indivi- duals were measured from every accession. For each individual and isolate, three runs of 5000 counts each were measured. Coefficients of variation of G1 peaks of R. pulcher and H. vulgare mostly did not exceed 3 %.
323Phytol. Balcan. 13(3) • Sofia • 2007
Table 2. List of accessions studied and methods applied to R. pulcher s.l. in Bulgaria. No Taxon, locality, UTM, voucher number Methods applied R. pulcher subsp. pulcher 1 Black Sea Coast (Southern): grassy places around Sozopol, 20 m, 03.07.2004; NG-59 (Raycheva) K, Map, M
2 Black Sea Coast (Southern): Kraimorie village, 20 m, 23.06.2003, NG-67 (Raycheva) Fl, Fe, Map, M
3 Danubian Plain: Somovit village, Nikopol district, 50 m, 25.06.2003, LJ-24 (Raycheva) SOA 56363 K, Map, M
4 Forebalkan (Western): hay meadows along the left bank of Bela Reka River, near Dolna Bela Rechka village, Montana district, 400 m, 27.6.2002, FN-98 43,1218 N, 23,1931 E, (Dimitrova)
Fl, Fe, Map, M
5 Rhodopi Mts (Eastern): near Ivailovgrad – Doupkata Protected Area, 104 m, 14.07.2005, MF-29 (Raycheva) SEM, Map, M
6 Rhodopi Mts (Eastern): village Odrintsi, 194 m, 15.07.2005, MF-28 (Raycheva) Fe, Map, M
7 Thracian Lowland: near Pyasuchnik dam, 300 m, 22.06.2003, KG-99 (Raycheva) SOA 56407 Fl, Fe, K, Map, M
R. pulcher subsp. woodsii 8. Black Sea Coast (Southern): light oak forests near Sinemorets village, 40 m, 03.07.2004, NG-85 (Raycheva) SOA 57068 Fl, Fe, K, Map, M
9* Rhodopi Mts (Eastern): betwen Siv Kladenets and Odrintsi villages, 194 m, 15.07.2005, MF-28 (Raycheva) SOA 56931 Fl, Fe, Map, M
10* Rhodopi Mts (Eastern): near Belopolyane village, in sunflower fields, 170 m, 15.07.2005; MF-38 (Raycheva) SOA 56929 SEM, Fe, Map, M
11* Rhodopi Mts (Eastern): abandoned lands and pastures near Ivailovgrad, 160 m, 15.07.2005, MF-29 (Raycheva) SOA 56930 Fe, Map, M
R. pulcher subsp. raulinii 12* Black Sea Coast (Southern): rare oak forests near Sinemorets village, 40 m, 03.07.2004, NG-85 (Raycheva) SOA 57067 Fl, Fe, K, Map, M
13* The Valley of Strouma River: Kresna town, 180 m, 18.06.2005, FM-72 (Raycheva) SOA 56926 K, Map, M
14* Belasitsa Mts: between Skrut and Klyuch villages, 300 m, 18.06.2005, F-L68 (Raycheva) SOA 56927 SEM, Fl, Fe, Map, M
15* Rila Mts: Dupnitsa town, above the road tunnels, 510 m, 19.06.2005, FM-78 (Raycheva) SOA 56928 K, Map, M
Fl – flow cytometry; Fe – Feulgen densitometry; SEM – scanning electron microscopy; K – karyological studies with classical haema- toxylin method; Map – mapped locality; M – gross-morphological studies; * – plants from a new floristic region.
For quantitative Feulgen staining (Feulgen & Rossenbeck 1924), seedlings of R. pulcher were co- fixed in 4 % neutral formaldehyde solution, togeth- er with primary root tips of the standard organism Pisum sativum 'Kleine Rheinländerin' (1C = 4.42 pg DNA, Greil hu ber & Ebert 1994). These samples were post-fixed with methanol acetic acid (3:1) and after- wards stored in 96 % ethanol in the freezer until use. Following the preparation method of Greilhuber & Ebert (1994), rehydrated material was hydrolyzed in 5N HCl for 75 min at 20.0 °C (Greilhuber & Baranyi 1999; Greilhuber & Temsch 2001) and stained in Schiff´s reagent for 1.5 hours at room temperature. After washing out in SO2-water, the plant tissue was squashed in 45 % acetic acid, briefly rinsed in 96 % ethanol, and finally air-dried. Three slide pairs of R. pulcher and the standard tissue were produced in par- allel per accession. For each slide, 20 G1/0 or 10 te- lophase plus 10 prophase nuclei were measured. We have used the Cell Image Retrieval and Evaluation System (CIRES, Kontron, Munich, Germany) for measurement.
Results and discussion
Chorology and ecology
Rumex pulcher subsp. pulcher occurs in the Medi te- r ra nean region, Balkan Peninsula, Asia Minor, Hun- garian lowlands, Atlantic West Europe. It has spread into Asia and got naturalized in America (Akeroyd 1993). In Bulgaria, the subspecies grows in open grassy habitats, along roads, and as a weed in agri- cultural phytocoenoses across the country, from 0 up to 1500 m a.s.l. (Fig. 1, Table 3).
Rumex pulcher subsp. woodsii is abundant and often occurs as a ruderal plant in the Aegean region, SW Asia and the central part of East Mediterranean. The subspe- cies has been introduced in Central and West Europe and different parts of America and South Africa. In Bulgaria, the taxon has been reported in literature from the Black Sea Coast (Southern), Balkan Range (Eastern) and Toundzha Hilly Country. In the course of the cur- rent study, the subspecies was confirmed for the Black Sea Coast (Southern), and registered for the first time
324 Raycheva, Tz. & al. • Rumex pulcher in the Bulgarian flora
Table 3. Chorological data on Rumex pulcher s.l. in Bulgaria. Literature sources Herbarium specimens Floristic regions Altitude Floristic regions Altitude R. pulcher L. subsp. pulcher 1.1 (Urumov 1908b); 1.2 (Urumov 1901; Davidov 1905; Rechinger 1932, 1933); 2 (Urumov 1901; 1905a; Kovachev 1903; Rechinger 1932; Stoeva 1987); 3 (Urumov 1898, 1901, 1902, 1910, 1917, 1925, 1926, 1928, 1935a); 4.1 (Velenovský 1898; Urumov 1898, 1901, 1905a, 1917, 1925, 1926, 1928, 1935a; Rechinger 1932, 1933); 4.2 (Urumov 1897, 1926, 1928); 5.1 (Urumov 1902, 1905b, 1935a); 5.2 (Urumov 1897, 1898, 1917, 1929b; Neichev 1908; Baev 1947); 5.3 (Velenovský 1891; Urumov 1909); 6 (Velenovský 1891; Urumov 1905b, 1906, 1910, 1929a, 1930, 1935b); 7 (Urumov 1905b, 1913, 1935b); 8 (Urumov 1929a, 1930); 9 (Urumov 1904, 1913, 1935b); 11 (Urumov 1905a); 14.1 (Urumov 1923); 15 (Urumov 1904, 1906); 16.1 (Urumov 1902, 1905a, 1908a, 1935b); 16.2 (Urumov 1904; 1910, 1929b); 17.1 (Urumov 1906, Rechinger 1932); 18 (Velenovský 1891; Urumov 1906, 1908b, 1917, 1929b; Rechinger 1932); 19 (Urumov 1909, 1910). Vulev (1966), Andreev (1992), and Delipavlov (2003) report the taxon for whole territory of the country
up to 2000 m 1.1; 1.2; 2; 3; 4; 4.2; 5.2; 5.3; 6; 7; 8; 9; 10*; 11; 12*;
13*; 14.1; 15; 16.1; 16.2; 17.1; 17.2*; 17.3*; 18; 19; 20*
up to 1100 m
R. pulcher subsp. woodsii 1.1 (Panov 1987); 5.3 (Panov 1987); 19 (Panov 1987). Andreev (1992) and Delipavlov (2003) report the taxon for the Black Sea Coast, Balkan Range (Eastern) and Toundzha Hilly Country.
– 1.1; 17.3* up to 200 m
R. pulcher subsp. raulinii Vulev (1966), Andreev (1992), and Delipavlov (2003) report the taxon for Black Sea Coast, Forebalkan (Eastern) and Thracian Llowland.
– 1.1; 10*; 11*; 15* up to 500 m
1 – Black Sea Coast (1.1 – Southern, 1.2 – Northern); 2 – NE Bulgaria; 3 – Danubian Plain; 4 – Forebalkan (4.1 – Western, 4.2 – East- ern); 5 – Balkan Range (5.1 – Western, 5.2 – Central, 5.3 – Eastern); 6 – Sofia region; 7 – Znepole region; 8 – Vitosha region; 9 – West Frontier Mts; 10 – The Valley of Strouma River (10.1 – Southern, 10.2 – Northern); 11 – Mt Belasitsa; 12 – Mt Slavyanka; 13 – The Val- ley of Mesta River; 14 – Pirin Mts (14.1 – Southern, 14.2 – Northern); 15 – Rila Mts; 16 – Mt Sredna Gora (16.1 – Western, 16.2 – East- ern); 17 – Rhodopi Mts (17.1 – Western, 17.2 – Central, 17.3 – Eastern); 18 – Thracian Lowland; 19 – Toundzha Hilly Country; 20 – Mt Strandzha; * – new data.
Fig. 1. Distribution of R. pulcher subsp. pulcher in Bulgaria. Note: Th is fi gure includes also data on R. pulcher s.l. from the Bulgarian fl oristic literature prior to Vulev (1966), where the subspecifi c variation has not been considered.
– new and unpublished data
325Phytol. Balcan. 13(3) • Sofia • 2007
in the Rhodopi Mts (Eastern) (Fig. 2, Table 3). The sub- species occurs in secondary herbaceous plant commu- nities, with Lolium perenne L., Dactylis glomerata L., Aegilops triuncialis L., Alopecurus myosuroides Huds., Cirsium sp., Filago vulgaris Lam., Plantago lanceolata L., Eryngium campestre L., Hypericum perforatum L., Daucus guttatus Sm., and Potentilla pedata Willd.
Rumex pulcher subsp. raulinii is distributed in the Aegean region. The subspecies has been reported in the Bulgarian literature from the Black Sea Coast, Forebalkan, Balkan Range (Central), and Thracian Lowland. Our revision of the Bulgarian herbaria has revealed one specimen belonging to R. pulcher sub- sp. raulinii (SV: coll. B. Davidov, SOM 17832, in hu- midis Deli Orman, July 1903) that had been first de- termined as R. palustris, and subsequently revised by B. Achta rov as R. pulcher. We have confirmed the taxon for the Black Sea Coast (Southern), and disco- vered it for the first time in the Valley of River Strou- ma, Mt Belasi tsa, and Rila Mts (Fig. 3, Table 3). It occurs seldom in thin oak forests (Quercus cerris L., Q. frainetto Ten.), accom panied by Briza maxima L., Poa bulbosa L., P. sylvicola Guss., Bromus tectorum L., Lolium perenne L., Festuca pratensis Huds., Cistus in- canus L., and Cynosurus cristatus L.
Morphological characteristics
Rumex pulcher s.l. shows a typical tendency to xero- phytization, e.g, formation of fleshy tubers on the main root, reduction in the leaf size, distinctly fiddle- shaped leaves, and indumentum on stems and leaves. These features appear in all three subspecies but are most clearly expressed in the typical one.
Rumex pulcher is a perennial plant. In R. pulcher subsp. woodsii, tubers can be frequently found on the main root. Typical for this taxon is the fiddle- like shape of the basal leaves, but our observations have shown that this cannot be used as a diagnostic feature on suspecific level, since it is regularly found in the three subspecies distributed in Bulgaria. This is contrary to Rechinger (1932), according to whom the fiddle-shaped leaves distinguish R. pulcher subsp. woodsii from R. pulcher subspp. pulcher and raulinii. The leaf and stem indumentum varies considerably in the three taxa. It is mostly papillose, and seldom (in subsp. woodsii) consists of unicellular trichomes. The stem height also varies between the subspecies but in ge neral the typical subspecies has shorter, flexuose, often intricate stems, while R. pulcher subsp. woodsii has high stems with long lateral divaricate branches.
– new and unpublished data
– herbarium specimens
– literature data Fig. 2. Distribution of R. pulcher subsp. woodsii in Bulgaria.
326 Raycheva, Tz. & al. • Rumex pulcher in the Bulgarian flora
Our observations have shown that most of the taxo nomically reliable morphological features for sub ge nus Rumex can be found in the valves of the mature plants (length/width of valves, tuberculae and ochreae, size and number of spines) (Table 4).
Table 4. Morphological parameters of the valves of R. pulcher s.l.
Character
±S (mm)
±S (mm)
±S (mm)
Valve length 4.73±0.02 5.46±0.03 5.34±0.03 Valve width 3.00±0.03 4.65±0.03 2.96±0.02 Tubercule length 2.38±0.03 2.81±0.02 2.48±0.03 Tubercule width 1.18±0.01 1.55±0.01 1.13±0.02 Ochrea length 2.33±0.03 2.84±0.02 2.00±0.02 Ochrea width 1.46±0.02 1.84±0.02 1.50±0.02
The identification key given below is based on morphological features that have proven to be most discrete and reliable for identification of the three sub- species. The data related to plant habitus, morphology of basal leaves and indumentum type are considered reliable for the purpose and are not used, in contrast to Akeroyd (1993).
Identification key
1 Valves clearly dentate, oblong to narrowly ovate, 4.5–5.5 mm long, 3(–3.4) mm wide, with 4–6 irregular spines longer than 1 mm; branches short, flexuose, often intricate…………………………………………2
1* Valves slightly dentate, rounded in shape, 5.2–6 mm long, 4.2–5.2 mm wide, with 6–8 almost regular 0.5–0.8 mm long spines; branches long, divari- cate……………………………………subsp. woodsii
2 Some spines 3–3.5 mm long ………subsp. raulinii
2* Some spines 1.5–2 mm long ………subsp. pulcher
Scanning microscopy supports the significance of the valves morphology for identification of the three subspecies. Furthermore, the surface of the tubercu- lae also shows some differences that correspond to the subspecific taxonomic concept. In R. pulcher subsp. raulinii their structure is more robust and papillose as compared to the other two taxa.
The surface of the valves is similar in all three sub- species, but when compared to other species in subge- nus Rumex, their thick and robust venation outlines
– new and unpublished data
Fig. 3. Distribution of R. pulcher subsp. raulinii in Bulgaria.
327Phytol. Balcan. 13(3) • Sofia • 2007
a clear tendency towards xerophytization. In R. pul- cher subsp. woodsii the surface of the valves is larger and spines are shorter (0.4–0.8 mm). In the remaining two subspecies the valves are narrower and elongated, while the spines are longer and more robust, reaching 2.8–3.2 mm and exceeding the valve width.
The tuberculae are usually three in number, only in R. pulcher subsp. woodsii two of them are reduced to nodes. The surface of the valve tuberculae is irregular- ly concave-undulate, but distinctly differs in the three taxa. Most clearly differentiated are the concave struc- tures in R. pulcher subsp. raulinii, where the pits are strongly undulated (Plate I, 3b), while in the remain- ing two subspecies the pit surface is smooth. The dif- ference in valve architecture correlates with the gross- morphology of the three taxa and their ecological characteristics. Rumex pulcher subsp. pulcher, which shows the strongest tendency to xerophytization, has the smallest concave structures with irregular shape as compared to the remaining two subspecies.
Karyology and genome size variation
Further studies (in addition to Raycheva 2005) on the karyology of R. pulcher in Bulgaria have shown stable chromosome numbers 2n = 2x = 20 for the three sub- species (Fig. 4).
Apart from classical karyology, we have applied flow cytometry and Feulgen densitometry to test for nuclear C-value variation among the Bulgari- an subspecies of R. pulcher. An earlier comparison with the Feulgen stained samples fixed in formalde- hyde or methanol acetic acid (3:1) revealed the pres- ence of secondary metabolites in the present materi- al (Fig. 5). Such substances are known as inhibitors of DNA staining (Greilhuber 1986, 1988), but sus-
metry diverge widely. However, in our investigation we found good data agreement, when formaldehyde fixed material was used for Feulgen densitometry.
The mean 1C-values were 0.76 pg DNA in R. pul- cher subsp. woodsii, 0.83 pg DNA in R. pulcher subsp. raulinii, and 0.82 pg DNA in R. pulcher subsp. pulcher. The C-value of R. pulcher subsp. woodsii was approx. 7 % lower than the values of the other two subspecies (Table 5). This difference underlines the more specific morphological syndrome of R. pulcher subsp. woodsii and its more divergent position within R. pulcher s.l.
Table 5. Comparative DNA data obtained by flow cytometry and Feulgen densitometry. Locality numbers follow the sequence in Table 2.
Locality No Flow cytometry pg/1C±S.D
Feulgen densitometry pg/1C±S.D
R. pulcher L. subsp. pulcher 2 0.80±0.02 0.83±0.05 4 0.79±0.02 0.81±0.04 6 – 0.81±0.03 7 0.79±0.02 0.88±0.08 R. pulcher subsp. wodsii 8 0.76±0.01 0.76±0.06 9 – 0.70±0.06 10 – 0.81±0.04 11 – 0.81±0.09 R. pulcher subsp. raulinii 12 0.80±0.02 0.82±0.03 14 – 0.85±0.004
Fig. 4. Metaphase plate of R. pulcher subsp. woodsii. Scale bar = 5 μm.
ceptibility of the two approaches (flow cy- tometry and Feulgen densitometry) to that problem is different. A strong influence of in- hibitors can often be demonstrated, when re- sults with flow cytomet- ry and Feulgen densito-
Fig. 5. Root-tip cells of R. pulcher; fi xation in 4 % formaldehyde, Feulgen staining. Arrowhead shows polymerized secondary compounds (probably condensed tannins) in many cells and (n) shows regular staining of nuclei.
7• Phytol. Balcan. 13(3) • 2007
328 Raycheva, Tz. & al. • Rumex pulcher in the Bulgarian flora
SEM of valves and ochreae surface of the three subspecies of R. pulcher: 1a, 1b, subsp. pulcher; 2a, 2b, subsp. woodsii; 3a, 3b, subsp. raulinii.
Plate I
1a 1b
2a 2b
3a 3b
Conclusions
The biosystematic study of R. pulcher in Bulgaria has confirmed the existence of three subspecies in the na- tional flora: R. pulcher subspp. pulcher, woodsii and raulinii. Morphological studies and genome size esti- mations have confirmed the more divergent position of R. pulcher subsp. woodsii in the plant group.
Acknowledgements. The study has been supported financially by Bulgarian National Science Fund (Project VUB 10/5) and FWF Austrian Science Fund (P14607 B03). The authors are grateful to Prof. Johann Greilhuber for his support and useful comments on the paper and to the anonymous reviewer for the constructive and valuable suggestions.
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