Carex Pseudobrizoides (Cyperaceae) in Poland: Patterns of Isozymatic Phenotypes

Fragm. Flor. Geobot. 45(1–2): 265–271, 2000

Carex pseudobrizoides (Cyperaceae) in Poland: patterns of isozymatic phenotypes

WALDEMAR Z˙ UKOWSKI AND MARLENA LEMBICZ

Z˙ UKOWSKI, W. AND LEMBICZ, M. 2000. Carex pseudobrizoides (Cyperaceae) in Poland: patterns of isozymatic phenotypes. Fragmenta Floristica et Geobotanica 45(1–2): 265–271. Kra- ków. ISSN 0015–931x.

ABSTRACT. The genetic differences among three species – Carex pseudobrizoides Clavaud, C. brizoides L. and C. arenaria L. – were assessed on the basis of isozymatic phenotypes determined by electrophoresis of enzymatic protein. Four enzymes putatively encoded by the eight loci PX-1, PX-2, DIF-1, DIF-2, DIF-3, GOT-1, GOT-2 and IDH were studied. Five of them were polymorphic while the others were monomorphic for all the species studied. The highest numbers of isozymatic phenotypes were noted for PX-1 and IDH. Different phenotypic patterns were found for PX-1, DIF-1 and DIF-3 in populations of C. pseudobrizoides The same phenotypic patterns of C. pseudobrizoides and C. brizoides ramets were found for DIF-2, GOT-1, GOT-2 and DIF-3 in one of the populations. The phenotypes of C. arenaria differed from those of C. pseudobrizoides in only two enzymes: PX-1 and IDH.

KEY WORDS: isozymatic phenotypes, electrophoresis, sedges, Carex pseudobrizoides, C. brizoides, C. arenaria, Poland W. Z˙ukowski and M. Lembicz, Department of Plant Taxonomy, A. Mickiewicz University, Al. Niepodległos´ci 14, PL–61–713 Poznan´, Poland; e-mail: [email protected]; lembicz@ main.amu.edu.pl

INTRODUCTION

Carex pseudobrizoides Clavaud (Carex reichenbachii Bonnet) is one of seven species in the sedge group Carex sect. Ammoglochin Dummert; the others are C. arenaria L., C. brizoides L., C. disticha Hudson, C. ligerica Gay, C. praecox Schreber and C. repens Bell. (Chater 1980). C. pseudobrizoides, originally described from southwestern France, is rather difficult to discern from other species of the section Ammoglochin. It occurs in France, Belgium, Holland, Germany, the Czech Republic and Poland (cf. Chater 1980), and recently it has been reported from the Kaliningrad region (Egorova 1999). The species is also known from the Basque country in Spain (Aseginolaza et al. 1984). It grows at scattered localities within or at the limits of the range of C. arenaria and C. brizoides (Fig. 1). In Poland C. pseudobrizoides is met at a single locality in the Bory Dolnos´la˛skie wood on sandy sites mainly in pine forests and on riverbanks. 266 Fragm. Flor. Geobot. Ann. 45, Pars 1–2, 2000

Carex pseudobrizodes Carex brizoides

Carex arenaria

Fig. 1. Distribution of Carex pseudobrizoides Clavaud, C. brizoides L. and C. arenaria L. (according to Meusel et al. 1965; Haeupler & Schönfelder 1989; Benkert et al. 1996, modified).

According to Egorova (1999), C. pseudobrizoides is a species of hybrid origin which probably appeared as a result of introgressive hybridization between C. brizoides and C. arenaria. Presently the species is in the process of stabilization and the plants are partly fertile. In the areas of C. pseudobrizoides occurrence either one or both of its putative parents do not occur. Egorova supports this hypothesis with interesting morphological data concerning in particular the morphology of flowers and inflorescences. In many specimens she found three zones of flowers instead of the two observed in the other species of the subgenus Vignea, confirming the earlier observations of Kükenthal (1909). The spikes are all gynaecandrous, or male at the apex and base and female in the middle, or completely male. Egorova (1999) claims that this differentiation confirms the involvement of C. brizoides and C. arenaria in the origin of C. pseudobrizoides. C. brizoides has gynaecandrous and male spikes, while C. arenaria has androgynous spikes. However, the hypothesis of the hybrid origin of C. pseudobrizoides requires confirma- tion on the basis of genetic data. The two putative ancestral species of C. pseudobrizoides and many intermediate forms grow in Poland. The present study is part of a project aimed at determining the taxonomic status and origin of C. pseudobrizoides and the other species of the section Ammoglochin in Poland. W. Z˙ ukowski & M. Lembicz: Carex pseudobrizoides: patterns of isozymatic phenotypes 267

The particular aim of this work is to describe the enzymatic phenotypes and identify the markers differentiating C. pseudobrizoides from its putative ancestral species C. arenaria and C. brizoides.

MATERIALS AND METHODS

The genetic differences among the three species Carex pseudobrizoides, C. brizoides and C. arenaria were assessed on the basis of isozymatic phenotypes determined by electrophoresis of enzymatic protein on starch gel (Soltis & Soltis 1989). The analyses were performed for seven populations (Table 1).

Table 1. List of localities and number of ramets studied from populations of Carex pseudobrizoides Clavaud, C. brizoides L. and C. arenaria L.

Popu- Ramets Species Locality Tufts Total lation old new C. pseudobrizoides ADolnos´la˛skie District, S´wie˛toszów 6 472572 BDolnos´la˛skie District, Piaseczna 4 32 20 52

C. brizoides C Wielkopolskie District, Teresiny 3 24 15 39 DDolnos´la˛skie District, Osiecznica 3 20 15 35 EDolnos´la˛skie District, S´wie˛toszów 2 251035

C. arenaria F Kujawsko-Pomorskie District, Torun´-Bielawy 2 4 2 6 G Pomorskie District, Władysławowo 6 21 16 37

All plants collected from original sites were grown in an experimental garden in the same conditions. Fragments of lower leaves from old ramets were taken for analysis immediately after collection and transportation. Some of the plants were moved to a glasshouse after all of their aboveground parts were removed. In the spring of the following year, fragments of lower leaves appearing on newly formed ramets were analyzed in order to check the stability of the enzymatic pattern. Leaves from each old ramet from the tufts collected (69 old ramets of C. brizoides, 79 of C. pseudobrizoides and 25 of C. arenaria) and selected newly formed ramets (40 ramets of C. brizoides, 45 of C. pseudobrizoides and 18 of C. arenaria) were analyzed. The enzymatic phenotypes of selected enzymes were determined for 109 ramets of C. brizoides, 124 of C. pseudobrizoides and 43 of C. arenaria (Table 1). Four enzymes putatively encoded by eight loci were the subjects of this study. The loci scored were PX-1, PX-2, DIF-1, DIF-2, DIF-3, GOT-1, GOT-2, IDH.

Analytical procedure

Material preparation Each 3 cm long leaf fragment was homogenized with 50 μl extraction buffer (1.21 g tris, 37 mg EDTA, 75 mg KCL, 203 mg MgCl2 × 6 H2O, 1 ml 10% triton X-100 in 80 ml distilled water). Before extraction, 5 μl 2-mercaptoethanol was added. The extracts were placed on Whatmann tissues 3MM (3 × 6 or 3 × 19 mm, depending on gel thickness), and then in 10–12% gel (depending on starch lot). The extract from one leaf was placed onto two tissues. 268 Fragm. Flor. Geobot. Ann. 45, Pars 1–2, 2000

Separation Only one type of separating buffer gel was used. It contained 11.6 g tris, 2.88 g citric acid, 2.28 g anhydrous boric acid and 0.32 g LiOH × H2O in 1000 ml distilled water (pH 8.2). The electrode buffer was made of 45.6 g anhydrous boric acid and 6.4 g LiOH × H2O in 4000 ml distilled water (pH 8.3). The electrophoresis was conducted for 3–4 h using current of 160 V and 32.1 mA or 260 V and 41.4 mA at ca 4oC.

Dying After electrophoresis the gel samples were cut into a few layers 2 mm thick, and each of them was dyed for a different enzymatic system. Depending on the enzyme, either the overlay method was used (all components dissolved in buffer and added to 7.5 ml agar at ca 60oC), or dying was performed or the cells were dyed directly. The patterns developed on the gel samples were described and interpreted on the same day, and some of them were fixed by standard methods. The following enzymes were analyzed: GOT-1 and GOT-2 (transaminases of glutamic acid), PX-1, PX-2 (peroxidases), IDH (isocitrate dehydrogenase), DIA-1, DIA-2 and DIA-3 (diaphorases). Dying followed the method of Soltis and Soltis (1989) with modifications (Ford et al. 1998). The results were interpreted in terms of the patterns of isozymatic phenotypes and not individual isozyme loci and alleles. The constant and characteristic pattern of bands developed as a result of electrophoresis was assumed to be the isozymatic phenotype.

RESULTS

Five of the eight loci examined were polymorphous; the others were monomorphous for Carex pseudobrizoides, C. brizoides and C. arenaria (Table 2). The greatest number of isozymatic phenotypes (four) was determined for PX-1 and IDH. Three phenotypes were identified for DIF-1, two for DIF-3 and PX-2, and one for DIF-2 and GOT-2 (Fig. 2). No differences in phenotype were found between the old ramets and the young ones produced in a given tuft the next spring. All populations of C. brizoides and C. arenaria were characterized by the same isozymatic patterns. However, two populations of C. pseudobrizoides differed in respect to isozymatic phenotypes for PX-1, DIF-1 and DIF-3. The

Table 2. List of determined isozymatic phenotypes in populations of Carex pseudobrizoides Clavaud, C. brizoides L. and C. arenaria L. Numbers refer to phenotypes shown schematically in Fig. 2.

Enzymes and their phenotypes Species Population PX-1 PX-2 DIF-1 DIF-2 DIF-3 GOT-1 GOT-2 IDH A 31312114 C. pseudobrizoides B 41111114 C 22211111 C. brizoides D 22211111 E 22211111, 2 F 11111113 C. arenaria G 11111113 W. Z˙ ukowski & M. Lembicz: Carex pseudobrizoides: patterns of isozymatic phenotypes 269

123 412

123 4

123 112

Fig. 2. Patterns of isozymatic phenotypes for three species of Carex sect. Ammoglochin: Carex arenaria L., C. brizoides L. and C. pseudobrizoides Clavaud. Banding patterns of each enzyme are marked with numbers corresponding to the ones in Table 2. All enzymes migrated towards the anode. same phenotypes of C. pseudobrizoides, C. brizoides and C. arenaria ramets were determined in population A for DIF-2, GOT-1, GOT-2. In population B of C. pseudobrizoides the number of phenotypes identical with those of C. arenaria was greater than the number of those identical with C. brizoides. Different phenotypes were determined only for PX-1 and IDH. Good enzymatic markers (PX, DIF and IDH) differentiating C. pseudobrizoides from its putative ancestral species C. brizoides and C. arenaria were found. For these enzymes the number of isozymatic phenotypes was the greatest.

DISCUSSION

The evolutional relations within the numerous and widespread genus Carex have been relatively poorly recognized, mainly because of the lack of well-documented phylogenetic reconstructions among particular species within and between individual sections. There are still problems in establishing the hybrid origin of many taxa and their differentiation within a section. The use of molecular characters (DNA fragments and allozymes) to reconstruct phylogenetic relations, combined with analysis of morphological and anatomical characters, can help identify within-genus taxonomical relations (Szweykowski & Krzakowa 1977; 270 Fragm. Flor. Geobot. Ann. 45, Pars 1–2, 2000

Starr & Ford 1995; Ford et al. 1998; Starr et al. 1999; Reinhammar 1999; Vellend & Waterway 1999). In Poland, species from the section Ammologchin occur in marginal populations in which ecological and genetic processes differ from those in populations at the center of the species’ distribution, and this is the principal cause of phytogeographi- cal, phylogenetic and taxonomic problems. In particular the taxonomic status of such species as Carex repens (C. posnaniensis Sprib.) and C. pseudobrizoides has not been resolved. The study of this group of sedges began with the search for markers differentiating particular taxa (Szweykowski & Krzakowa 1977). Our study provided the patterns of enzymatic phenotypes for C. pseudobrizoides and its potential ancestor species C. arenaria and C. brizoides. At the present stage of investigations we have identified three enzymatic markers distinguishing C. pseudobrizoides from its potential ancestors, but this finding does not allow the proposed hybrid origin of C. pseudobrizoides (Egoro- va 1999) to be verified. According to Egorova the parent species of C. pseudobrizoides are not noted in the area of occurrence of this taxon. In Russia they do not occur, sugges- ting that the formation of the species may have begun in Poland. The many intermediate forms make it difficult to interpret the patterns obtained. Future studies must be based on material collected from a much larger area of the species’ occurrence.

Acknowledgements. The authors thank Professor J. Szweykowski (A. Mickiewicz University, Poznan´) for providing access to his laboratory for our isozymatic studies. Financial support from the State Committee for Scientific Research (KBN grant no. 6 P04G 015 14) is gratefully acknowledged.

REFERENCES

ASEGINOLAZA C., GOMEZ D., LIZAUR X., MONTSERRAT G., MORANTE G., SALAVERRIA M. R., URIBE- ECHEBARRIA P. & A LEJANDRE J. A. 1984. Catalogo floristico de Alaya. 1149 pp. + map. Graficás Santamaria S.A., Vitoria – Gasteiz.

BENKERT D., FUKAREK F., KORSCH H. 1996. Verbreitungstatlas der Farn-und Blütenpflanzen Ost- deutschlands. 615 pp. Gustav Fischer, Jena, Stuttgart, Lübeck, Ulm.

CHATER A. O. 1980. Carex. – In: T. G. TUTIN, V. H. HEYWOOD, N. A. BURGES, D. M. MOORE, D. H. VALEN- TINE, S. M. WALTERS & D. A. WEBB (eds), Flora europaea. 5. Alismataceae to Orchidaceae (Mono- cotyledones), pp. 290–323. Cambridge University Press, Cambridge.

EGOROVA T. V. 1999. The sedges (Carex L.) of Russia and adjacent States (within the limits of the former USSR). 772 pp. St.-Petersburg State Chemical-Pharmaceutical Academy, St.-Petersburg & Missouri Botanical Garden Press, St. Louis.

FORD B. A., STARR J. R., MCQUEEN D. A. R. & NACZI F. C. R. 1998. Relationships among species in Carex sect. Phyllostachys (Cyperaceae) based on divergence. – Pl. Syst. Evol. 212: 31–51.

HAEUPLER H. & SCHÖNFELDER P. 1989. Atlas der Farn-und Blütenpflanzen der Bundesrepublik Deutschland. 778 pp. Eugen Ulmer, Stuttgart.

KÜKENTHAL G. 1909. Cyperaceae-Caricoideae. – In: A. ENGLER (ed.), Das Pflanzenreich, IV. 20(38), 824 pp. Wilhelm Engelmann, Leipzig.

MEUSEL H. , JÄGER E. & WEINERT F. 1965. Vergleichende Chorologie der zentraleuropaischen Flora. 1. 258 pp. G. Fischer, Jena. W. Z˙ ukowski & M. Lembicz: Carex pseudobrizoides: patterns of isozymatic phenotypes 271

REINHAMMAR L. G. 1999. Allozyme differentiation between the lowland Carex capitata and the alpine Carex arctogena (Cyperaceae) in Scandinavia. – Biol. J. Linn. Soc. 67(3): 377–389. SOLTIS D. E. & SOLTIS P. S. 1989. Isozymes in Plant Biology. 4. 220 pp. Discorides Press, Portland, Oregon. STARR J. R. & FORD B. A. 1995. Phylogeny and character evolution in Carex section Phyllostachys (Cyperaceae). – Amer. J. Bot. 82: 164–165. STARR J. R., BAYER R. J. & FORD B. A. 1999. The phylogenetic position of Carex section Phyllostachys and its implications for phylogeny and subgeneric circumscription in Carex (Cyperaceae). – Amer. J. Bot. 86(4): 563–577. SZWEYKOWSKI J. & KRZAKOWA M. 1977. Peroxidases as taxonomic characters in Carex sectio Vignea (Cyperaceae, Angiospermae). – Bull. Soc. Amis. Sci. Poznan´ Ser. D 17: 29–32. VELLEND M., WATERWAY M. J. 1999. Geographic patterns in the genetic diversity of a northern sedge, Carex rariflora. – Can. J. Bot. 77(2): 269–278.