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.
References
Akeroyd, J. R. 1993. Rumex L. – In: Tutin, T.G. & al. (eds.),
Flora Europaea. Ed. 2, vol. 1, pp. 99-107. Cambridge Univ. Press,
Cambridge.
Andreev, N. 1992. Polygonaceae. In: Kozhuharov, S. (ed.), Field
Guide to the Vascular Plants in Bulgaria. Pp. 629-638. Naouka &
Izkoustvo, Sofia (in Bulgarian).
Baev, S. 1947. Botanical Excursions. Bulgarian Acad. Sci. Press,
Sofia (in Bulgarian).
Baltisberger, M. 1991. Cytological investigation of some Greek
plants. – Fl. Medit., 1: 157-173.
Baranyi, M. & Greilhuber, J. 1996. Flow cytometric and Feulgen
densitometric analysis of genome size variation in Pisum. – Theor.
Appl. Genet., 92: 297-307.
Bennett, M.D., Bhandol, P. & Leitch, I.J. 2000. Nuclear DNA
amounts in angiosperms and their modern uses: 807 new esti- mates.
– Ann. Bot. (Oxford), 86: 859–909.
Bennett, M.D. & Leitch, I.J. 2004. Angiosperm DNA C-values
database (release 5.0, Dec. 2004) http://www.rbgkew.org.uk/
cval/homepage.html.
Cullen, J. 1967. Rumex L. – In: Davis, P.H. (ed.), Flora of Turkey
and the East Aegean Islands. Vol. 2, pp. 281-293. Edinburgh Univ.
Press, Edinburgh.
Dahlgren, R., Karlsson, Th. & Lassen, P. 1971. Studies on the
flora of the Balearic Islands I. Chromosome numbers in Balearic
Angiosperms. – Bot. Not., 124(2): 249-269.
Davidov, B. 1905. Contribution to the investigation of the flora of
Varna district. – Sborn. Nar. Umotv. Naouka Knizhn., 21: 1-73 (in
Bulgarian).
Degraeve, N. 1975. Contribution a l’etude cytotaxonomique des Rumex
– I. Le genere Rumex L. sensu stricto. – Caryologia, 28(1):
187-201.
Delipavlov, D. 2003. Polygonaceae. – In: Delipavlov, D. &
Cheshmedzhiev, I. (eds), A Key to the Plants in Bulgaria. Pp.
97-103. Agrarian Univ. Acad. Press, Plovdiv (in Bulgarian).
Doleel, J., Greilhuber, J., Lucretti, S., Meister, A., Lysák, M.
A., Nardi, L. & Obermayer, R. 1998. Plant genome size
estimation by flow cytometry: inter-laboratory comparison. – Ann.
Bot. (Supplement A), 82: 17-26.
Feulgen, R. & Rossenbeck, H. 1924. Mikroskopisch-chemischer
Nachweis einer Nucleinsäure vom Typus der Thymonucleinsäure und die
darauf beruhende elektive Färbung von Zellkernen in mikroskopischen
Präparaten. – Hoppe Seyler´s Z. Physiol. Chem., 135: 203-248.
Galbraith, D.W., Harkins, K.R., Maddox, J.M., Ayres, N.M., Sharma,
D.P. & Firoozabady, E. 1983. Rapid flow cytometric analysis of
the cell cycle in intact plant tissues. – Science, 220:
1049-1051.
Garcia, C., Pastor, J. & Lique, T. 1989. Contribucion to the
kary- ologycal study of Rumex (Polygonaceae). – Acta Bot. Malac.,
14: 129-140 (in Spanish).
Gregory, T.R. 2005. The C-value enigma in plants and animals: a
review of parallels and an appeal for partnership. – Ann. Bot.
(Oxford), 95: 133-146.
Greilhuber, J. 1986. Severely distorted Feulgen DNA amounts in
Pinus (Coniferophytina) after nonadditive fixations as a result of
meristematic self-tanning with vacuole contents. – Can. J. Genet.
Cytol., 28: 409-415.
Greilhuber, J. 1988. „Self-tanning“ – a new and important source of
stochiometric error in cytophotometric determination of nuclear DNA
content in plants. – Plant Syst. Evol., 158: 87-96.
Greilhuber, J. & Baranyi, M. 1999. Feulgen densitometry: impor-
tance of a stringent hydrolysis regime. – Plant Biol., 1:
538-540.
Greilhuber, J. & Ebert, I. 1994. Genome size variation in Pisum
sativum. – Genome, 37: 646-655.
Greilhuber, J. & Temsch, E.M. 2001. Feulgen densitometry: some
observations relevant to best practice in quantitative nuclear DNA
content determination. – Acta Bot. Croat., 60: 285-298.
Hayek, A. 1924. Rumex L. – In: Prodromus Florae Peninsulae
Balcanicae. Band 1. Repert. Spec. Nov. Regni Veg. Beih., 30(1): 102
– 109.
Himi, H., Iwatsubo, Y. & Naruhashi, N. 1999. Chromosome numbers
of 11 species in Japanese Rumex subg. Rumex (Polygonaceae). – J.
Phytogeogr. Taxon., 47: 121-130.
Ichikawa, S.A., Sparrow, H., Frankton, C., Nauman, Anne, F., Smith,
E.B. & Pond, V. 1971. Chromosome number, volume and nuclear
volume, relationships in a polyploid series (2x-20x) of the genus
Rumex. – Can. J. Genet. Cytol., 13: 842-863.
Kovachev, V. 1903. Additon to the flora of Rousse district. –
Period. Spis. Bulg. Knizh. Druzh., 63: 717-725 (in
Bulgarian).
Kozhuharov, S., Peev, D. & Nikolov, N. 1983. Conservation, rep-
resentation and use of the current chorological information. –
Fitologiya, 22: 61-80 (in Bulgarian).
Löve, Á. 1967. Reports. – In: Löve, Á. (ed.), IOPB Chromosome
number reports XIII. – Taxon, 16(5): 445-461.
Löve, Á. 1986. Reports. – In: Löve, Á. (ed.), IOPB Chromosome
number reports XCII. – Taxon, 35(3): 611-613.
330 Raycheva, Tz. & al. • Rumex pulcher in the Bulgarian
flora
Löve, Á. & Löve, D. 1961. Chromosome numbers of Central and
Northwest European plant species. – Opera Bot., 5: 1-581.
Melander, Y. & Wingstrand, K. G. 1953. Gomori’s haematoxylin as
a chromosome stain. – Stain Technol., 28: 217-223.
Neichev, I. 1908. Materials on the flora of Gabrovo and the Balkan
(from Kademlia to Bedek). – Sborn. Nar. Umotv. Naouka Knizhn., 24:
1-83 (in Bulgarian).
Panov, P. 1987. New plants of Bulgarian flora. – In: Kuzmanov, B.
(ed.), Proc. Fourth Natl. Conf. Bot. Vol. 1, pp. 103-107 (in
Bulgarian).
Raycheva, Tz. 2005. Chromosome counts in some Bulgarian taxa of
Rumex L., subgenus Rumex (Polygonaceae). – In: Gruev, B., Nikolova,
M. & Donev, A. (eds.) Proc. Balkan Sci. Conf. Biol.. May 19-21,
2005, Plovdiv, Bulgaria. P. 397-408.
Rechinger, K.H. 1932. Vorarbeiten zu einer Monographie der Gattung
Rumex. I. – Beih. Bot. Centralbl., Abt. 2, 49(1): 1-132.
Rechinger, K.H. 1933. Ergebnisse einer botanischen Reise nach
Bulgarien. – Magyar Bot. Lapok, 32: 5-58.
Stoeva, M. 1987: Chromosome numbers of Bulgarian an- giosperms. –
Fitologiya, 33: 65-66.
Stojanov, N. & Stefanov, B. 1924. Flora of Bulgaria. Vol. 1.
State Printing House, Sofia (in Bulgarian).
Stojanov, N. & Stefanov, B. 1933. Flora of Bulgaria. Ed. 2.
Guttenberg Press, Sofia (in Bulgarian).
Stojanov, N. & Stefanov, B. 1948. Flora of Bulgaria. Ed. 3. Pp.
344- 350. Univ. Press, Sofia (in Bulgarian).
Stojanov, N., Stefanov, B. & Kitanov, B. 1966. Flora of
Bulgaria. Ed. 4, vol. 1. Naouka & Izkoustvo, Sofia (in
Bulgarian).
Stoyanov, K. 2003. Documentation system in the herbarium of the
Agricultural University of Plovdiv. – Bulg. J. Balkan Ecol., 6:
28-34.
Strid, A. 1971. Chromosome numbers in some Albanian Angiosperms. –
Bot. Not., 24: 490-496.
Terziisky, D. 1981. Scanning electron microscopy: problems, appli-
cation and perspectives for developing in the biological sciences
in Bulgaria. – Nauchni Trudove Selskost. Inst. “Vasil Kolarov”,
26(4): 115-121 (in Bulgarian).
Terziisky, D. & Atanasov, A. 1977. Scanning
electron-microscopic study of pollen morphology in Bulgarian Cicer
arietinum L. populations. – Fitologiya, 7: 51-58 (in
Bulgarian).
Urumov, I. 1897. Materials on the flora of Lovech district. –
Sborn. Nar. Umotv. Naouka Knizhn., 14: 3-85 (in Bulgarian).
Urumov, I. 1898. Materials on the flora of Turnovo district. –
Sborn. Nar. Umotv. Naouka Knizhn., 15: 3-90 (in Bulgarian).
Urumov, I. 1901. A contribution to the Bulgarian flora. – Sborn.
Nar. Umotv. Naouka Knizhn., 18: 1-124 (in Bulgarian).
Urumov, I. 1902. Second contribution to the Bulgarian flora. –
Period. Spis. Bulg. Knizh. Druzh., 62: 293-409 (in
Bulgarian).
Urumov, I. 1904. Third contribution to the Bulgarian flora. –
Sborn. Nar. Umotv. Naouka Knizhn., 20: 1-103 (in Bulgarian).
Urumov, I. 1905a. Fourth contribution to the Bulgarian flora. –
Period. Spis. Bulg. Knizh. Druzh., 65: 661-712 (in
Bulgarian).
Urumov, I. 1905b. Fifth contribution to the Bulgarian flora. –
Sborn. Nar. Umotv. Naouka Knizhn., 21: 1-125 (in Bulgarian).
Urumov, I. 1906. Sixth contribution to the Bulgarian flora. –
Sborn. Nar. Umotv. Naouka Knizhn., 22: 1-126 (in Bulgarian).
Urumov, I. 1908a. Seventh contribution to the Bulgarian flora. –
Sborn. Nar. Umotv. Naouka Knizhn., 24: 1-113 (in Bulgarian).
Urumov, I. 1908b. Ninth contribution to the Bulgarian flora. –
Sborn. Nar. Umotv. Naouka Knizhn., 24: 1-112 (in Bulgarian).
Urumov, I. 1909. Tenth contribution to the Bulgarian flora. –
Sborn. Nar. Umotv. Naouka Knizhn., 25: 1-159 (in Bulgarian).
Urumov, I. 1910. Eleventh contribution to the Bulgarian flora. –
Sborn. Blg. Acad. Nauk., 26: 1-224 (in Bulgarian).
Urumov, I. 1913. Twelfth contribution to the Bulgarian flora. –
Sborn. Blg. Akad. Nauk., 3: 1-243 (in Bulgarian).
Urumov, I. 1917. Thirteenth contribution to the Bulgarian flora. –
Sborn. Blg. Akad. Nauk., 7: 1-225 (in Bulgarian).
Urumov, I. 1923. Materials on the flora of the Pirin Mts. – Spis.
Bulg. Akad. Nauk., 28: 109-178 (in Bulgarian).
Urumov, I. 1925. Forteenth contribution to the Bulgarian flora. –
Spis. Bulg. Akad. Nauk., 21: 97-208 (in Bulgarian).
Urumov, I. 1926. Fifteenth contribution to the Bulgarian flora. –
Sborn. Blg. Akad. Nauk., 22: 3-128 (in Bulgarian).
Urumov, I. 1928. Sixteenth contribution to the Bulgarian flora. –
Sborn. Blg. Acad. Nauk., 23: 1-128 (in Bulgarian).
Urumov, I. 1929a. The flora of Mt Lyulin. – Spis. Bulg. Akad.
Nauk., 15: 1-117 (in Bulgarian).
Urumov, I. 1929b. Flora von Karlova Kreis. – Sborn. Blg. Akad.
Nauk., 25: 3-132 (in Bulgarian).
Urumov, I. 1930. The flora of Mt Vitosha. – Sborn. Blg. Akad.
Nauk., 26: 1-143 (in Bulgarian).
Urumov, I. 1935a. The flora of Vratza district. – Sborn. Blg. Akad.
Nauk., 29: 1-205 (in Bulgarian).
Urumov, I. 1935b. The flora of Kjustendil district. – Sborn. Blg.
Akad. Nauk., 30: 1-235 (in Bulgarian).
Vulev, S. 1966. Genus Rumex L. – In: Jordanov, D. (ed.), Fl.
Reipubl. Popularis Bulgaricae. Vol. 3, pp. 188-126. In Aedibus
Acad. Sci. Bulgaricae, Serdicae (in Bulgarian).
Velenovský, J. 1891. Flora Bulgarica. Descriptio et enumeratio sys-
tematica plantarum vascularium in principatu Bulgariae sponte
nascentium. Prague.
Velenovský, J. 1898. Flora Bulgarica. Supplementum I. Prague.
<< /ASCII85EncodePages false /AllowTransparency false
/AutoPositionEPSFiles true /AutoRotatePages /All /Binding /Left
/CalGrayProfile (Dot Gain 20%) /CalRGBProfile (sRGB IEC61966-2.1)
/CalCMYKProfile (U.S. Web Coated \050SWOP\051 v2) /sRGBProfile
(sRGB IEC61966-2.1) /CannotEmbedFontPolicy /Warning
/CompatibilityLevel 1.4 /CompressObjects /Tags /CompressPages true
/ConvertImagesToIndexed true /PassThroughJPEGImages true
/CreateJDFFile false /CreateJobTicket false /DefaultRenderingIntent
/Default /DetectBlends true /DetectCurves 0.0000
/ColorConversionStrategy /LeaveColorUnchanged /DoThumbnails false
/EmbedAllFonts true /EmbedOpenType false
/ParseICCProfilesInComments true /EmbedJobOptions true
/DSCReportingLevel 0 /EmitDSCWarnings false /EndPage -1
/ImageMemory 1048576 /LockDistillerParams false /MaxSubsetPct 100
/Optimize true /OPM 1 /ParseDSCComments true
/ParseDSCCommentsForDocInfo true /PreserveCopyPage true
/PreserveDICMYKValues true /PreserveEPSInfo true /PreserveFlatness
true /PreserveHalftoneInfo false /PreserveOPIComments false
/PreserveOverprintSettings true /StartPage 1 /SubsetFonts true
/TransferFunctionInfo /Apply /UCRandBGInfo /Preserve /UsePrologue
false /ColorSettingsFile () /AlwaysEmbed [ true ] /NeverEmbed [
true ] /AntiAliasColorImages false /CropColorImages true
/ColorImageMinResolution 300 /ColorImageMinResolutionPolicy /OK
/DownsampleColorImages true /ColorImageDownsampleType /Bicubic
/ColorImageResolution 300 /ColorImageDepth -1
/ColorImageMinDownsampleDepth 1 /ColorImageDownsampleThreshold
1.50000 /EncodeColorImages true /ColorImageFilter /DCTEncode
/AutoFilterColorImages true /ColorImageAutoFilterStrategy /JPEG
/ColorACSImageDict << /QFactor 0.15 /HSamples [1 1 1 1]
/VSamples [1 1 1 1] >> /ColorImageDict << /QFactor 0.15
/HSamples [1 1 1 1] /VSamples [1 1 1 1] >>
/JPEG2000ColorACSImageDict << /TileWidth 256 /TileHeight 256
/Quality 30 >> /JPEG2000ColorImageDict << /TileWidth
256 /TileHeight 256 /Quality 30 >> /AntiAliasGrayImages false
/CropGrayImages true /GrayImageMinResolution 300
/GrayImageMinResolutionPolicy /OK /DownsampleGrayImages true
/GrayImageDownsampleType /Bicubic /GrayImageResolution 300
/GrayImageDepth -1 /GrayImageMinDownsampleDepth 2
/GrayImageDownsampleThreshold 1.50000 /EncodeGrayImages true
/GrayImageFilter /DCTEncode /AutoFilterGrayImages true
/GrayImageAutoFilterStrategy /JPEG /GrayACSImageDict <<
/QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >>
/GrayImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples
[1 1 1 1] >> /JPEG2000GrayACSImageDict << /TileWidth
256 /TileHeight 256 /Quality 30 >> /JPEG2000GrayImageDict
<< /TileWidth 256 /TileHeight 256 /Quality 30 >>
/AntiAliasMonoImages false /CropMonoImages true
/MonoImageMinResolution 1200 /MonoImageMinResolutionPolicy /OK
/DownsampleMonoImages true /MonoImageDownsampleType /Bicubic
/MonoImageResolution 1200 /MonoImageDepth -1
/MonoImageDownsampleThreshold 1.50000 /EncodeMonoImages true
/MonoImageFilter /CCITTFaxEncode /MonoImageDict << /K -1
>> /AllowPSXObjects false /CheckCompliance [ /None ]
/PDFX1aCheck false /PDFX3Check false /PDFXCompliantPDFOnly false
/PDFXNoTrimBoxError true /PDFXTrimBoxToMediaBoxOffset [ 0.00000
0.00000 0.00000 0.00000 ] /PDFXSetBleedBoxToMediaBox true
/PDFXBleedBoxToTrimBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ]
/PDFXOutputIntentProfile () /PDFXOutputConditionIdentifier ()
/PDFXOutputCondition () /PDFXRegistryName () /PDFXTrapped /False
/Description << /CHS
<FEFF4f7f75288fd94e9b8bbe5b9a521b5efa7684002000500044004600206587686353ef901a8fc7684c976262535370673a548c002000700072006f006f00660065007200208fdb884c9ad88d2891cf62535370300260a853ef4ee54f7f75280020004100630072006f0062006100740020548c002000410064006f00620065002000520065006100640065007200200035002e003000204ee553ca66f49ad87248672c676562535f00521b5efa768400200050004400460020658768633002>
/CHT
<FEFF4f7f752890194e9b8a2d7f6e5efa7acb7684002000410064006f006200650020005000440046002065874ef653ef5728684c9762537088686a5f548c002000700072006f006f00660065007200204e0a73725f979ad854c18cea7684521753706548679c300260a853ef4ee54f7f75280020004100630072006f0062006100740020548c002000410064006f00620065002000520065006100640065007200200035002e003000204ee553ca66f49ad87248672c4f86958b555f5df25efa7acb76840020005000440046002065874ef63002>
/DAN
<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>
/DEU
<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>
/ESP
<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>
/FRA
<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>
/ITA
<FEFF005500740069006c0069007a007a006100720065002000710075006500730074006500200069006d0070006f007300740061007a0069006f006e00690020007000650072002000630072006500610072006500200064006f00630075006d0065006e00740069002000410064006f006200650020005000440046002000700065007200200075006e00610020007300740061006d007000610020006400690020007100750061006c0069007400e00020007300750020007300740061006d00700061006e0074006900200065002000700072006f006f0066006500720020006400650073006b0074006f0070002e0020004900200064006f00630075006d0065006e007400690020005000440046002000630072006500610074006900200070006f00730073006f006e006f0020006500730073006500720065002000610070006500720074006900200063006f006e0020004100630072006f00620061007400200065002000410064006f00620065002000520065006100640065007200200035002e003000200065002000760065007200730069006f006e006900200073007500630063006500730073006900760065002e>
/JPN
<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>
/KOR
<FEFFc7740020c124c815c7440020c0acc6a9d558c5ec0020b370c2a4d06cd0d10020d504b9b0d1300020bc0f0020ad50c815ae30c5d0c11c0020ace0d488c9c8b85c0020c778c1c4d560002000410064006f0062006500200050004400460020bb38c11cb97c0020c791c131d569b2c8b2e4002e0020c774b807ac8c0020c791c131b41c00200050004400460020bb38c11cb2940020004100630072006f0062006100740020bc0f002000410064006f00620065002000520065006100640065007200200035002e00300020c774c0c1c5d0c11c0020c5f40020c2180020c788c2b5b2c8b2e4002e>
/NLD (Gebruik deze instellingen om Adobe PDF-documenten te maken
voor kwaliteitsafdrukken op desktopprinters en proofers. De
gemaakte PDF-documenten kunnen worden geopend met Acrobat en Adobe
Reader 5.0 en hoger.) /NOR
<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>
/PTB
<FEFF005500740069006c0069007a006500200065007300730061007300200063006f006e00660069006700750072006100e700f50065007300200064006500200066006f0072006d00610020006100200063007200690061007200200064006f00630075006d0065006e0074006f0073002000410064006f0062006500200050004400460020007000610072006100200069006d0070007200650073007300f5006500730020006400650020007100750061006c0069006400610064006500200065006d00200069006d00700072006500730073006f0072006100730020006400650073006b0074006f00700020006500200064006900730070006f00730069007400690076006f0073002000640065002000700072006f00760061002e0020004f007300200064006f00630075006d0065006e0074006f00730020005000440046002000630072006900610064006f007300200070006f00640065006d0020007300650072002000610062006500720074006f007300200063006f006d0020006f0020004100630072006f006200610074002000650020006f002000410064006f00620065002000520065006100640065007200200035002e0030002000650020007600650072007300f50065007300200070006f00730074006500720069006f007200650073002e>
/SUO
<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>
/SVE
<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>
/ENU (Use these settings to create Adobe PDF documents for quality
printing on desktop printers and proofers. Created PDF documents
can be opened with Acrobat and Adobe Reader 5.0 and later.)
>> /Namespace [ (Adobe) (Common) (1.0) ] /OtherNamespaces [
<< /AsReaderSpreads false /CropImagesToFrames true
/ErrorControl /WarnAndContinue /FlattenerIgnoreSpreadOverrides
false /IncludeGuidesGrids false /IncludeNonPrinting false
/IncludeSlug false /Namespace [ (Adobe) (InDesign) (4.0) ]
/OmitPlacedBitmaps false /OmitPlacedEPS false /OmitPlacedPDF false
/SimulateOverprint /Legacy >> << /AddBleedMarks false
/AddColorBars false /AddCropMarks false /AddPageInfo false
/AddRegMarks false /ConvertColors /NoConversion
/DestinationProfileName () /DestinationProfileSelector /NA
/Downsample16BitImages true /FlattenerPreset <<
/PresetSelector /MediumResolution >> /FormElements false
/GenerateStructure true /IncludeBookmarks false /IncludeHyperlinks
false /IncludeInteractive false /IncludeLayers false
/IncludeProfiles true /MultimediaHandling /UseObjectSettings
/Namespace [ (Adobe) (CreativeSuite) (2.0) ]
/PDFXOutputIntentProfileSelector /NA /PreserveEditing true
/UntaggedCMYKHandling /LeaveUntagged /UntaggedRGBHandling
/LeaveUntagged /UseDocumentBleed false >> ] >>
setdistillerparams << /HWResolution [2400 2400] /PageSize
[612.000 792.000] >> setpagedevice