Ann. Bot. Fennici 37: 105–123 ISSN 0003-3847 Helsinki 15 June 2000 © Finnish Zoological and Botanical Publishing Board 2000 Genetic polymorphism detected with RAPD analysis and morphological variability in some microspecies of apomictic Alchemilla Silvia Sepp, Vera K. Bobrova, Alex K. Troitsky & Klavdiya P. Glazunova Sepp, S., Institute of Botany and Ecology, University of Tartu, Lai 40, 51005 Tartu, Estonia Bobrova, V. K. & Troitsky, A. K., A. N. Belozersky Institute of Physicochemical Biology, Moscow State University, Moscow 119899, Russia Glazunova, K. P., Biological Faculty of Moscow State University, Moscow 119899, Russia Received 1 September 1999, accepted 11 February 2000 Sepp, S., Bobrova, V. K., Troitsky, A. K. & Glazunova, K. P. 2000: Genetic polymor- phism detected with RAPD analysis and morphological variability in some microspecies of apomictic Alchemilla. — Ann. Bot. Fennici 37: 105–123. Alchemilla L. (Rosaceae) contains numerous agamospermous microspecies, which are often treated as species. However, many of them are not clearly morphologically dis- tinct, and their genetic variability is practically not investigated. In the present study, we used RAPD analysis to assess the genetic relatedness between Alchemilla micro- species. In all, 51 plants from 12 Alchemilla microspecies were analysed, and 116 char- acters were considered (68 RAPD bands over three primers and 48 morphological char- acters). Phylogenetic trees were constructed by the unweighted pair-group method, neighbour-joining and maximum parsimony methods. The genetic data supported most Fröhner’s system of sections. Despite the use of a limited set of data in the investigation and weak support values, some tentative conclusions could be based on congruence of the RAPD analysis and morphological data. Alchemilla acutiloba Opiz and A. micans Buser should be united as a single microspecies, A. micans; section Plicatae should be divided into two series Pubescentes and Barbulatae; and A. heptagona Juz. may be separated in Exuentes series of Ultravulgares. Keywords: genetic variation, molecular taxonomy, morphological variation, random amplified polymorphic DNA 106 Sepp et al. • ANN. BOT. FENNICI 37 INTRODUCTION continuous variation also in nature. Considering the fact that several microspecies normally occur Genus Alchemilla L. (Rosaceae) contains numer- in the same habitat, and the conclusions of Tures- ous microspecies, which are often treated as spe- son (1943), one may assume that they could have cies. Most of the microspecies are high polyploids parallel variation, but this is not proved. Genetic (Wegener 1967) reproducing apomictically (Stras- variation within and among microspecies should bourger 1905), though in the recent works of Gla- be investigated to decide if it could be so, and if zunova (1987) and Izmailow (1994) it was stated the morphologically indistinct pairs or even com- that the apomixis is facultative and probably hy- plexes of microspecies should be taxonomically bridisation takes place from time to time. This united. makes the taxonomy very complicated. In fact, in The development of “molecular markers”, practical geobotanical fieldwork all microspecies which reveal extensive polymorphism at the DNA under investigation here are identified as a col- or protein level, has greatly facilitated research in lective species A. vulgaris L. coll. taxonomy, phylogeny and genetics. In recent The microspecies have very different distri- years, a molecular technique called the random butions. Endemic ones probably represent single amplified polymorphic DNA (RAPD) assay (Wil- or few clones, while widespread species are cer- liams et al. 1990), is increasingly used for detect- tainly not genetically homogenous. Experimen- ing and estimating genetic diversity, in agamo- tal works of Turesson (1943, 1956, 1957) and sperms and other species (e.g. Van Coppenolle et Lundh-Almestrand (1958), where genetic variants al. 1993, Wachira et al. 1995, Marillia & Scoles were detected within microspecies prove the lat- 1996, Brunell & Whitkus 1997, Crawford 1997). ter fact. According to Turesson (1943), the geneti- Intraspecific genetic variability and species bor- cally distinct types within microspecies, called ders are successfully investigated using RAPD agamotypes, are specialised to different habitats. (Weising et al. 1995, Bachmann 1997, Kokaeva The genus has not been very intensely inves- et al. 1998). RAPD markers are generated by the tigated in the last few decades; the only notewor- amplification of anonymous genomic DNA seg- thy modern considerations of Alchemilla origi- ments with single, 10 base pair, arbitrary primers. nate from Fröhner (1995 and earlier works). The Amplified DNA fragments are size-fractionated latter are based on morphology and cytology by agarose gel electrophoresis, and polymorphism (chromosome numbers and shapes), but they are is detected as the presence or absence of a par- purely classical, empirical works. We analysed ticular band. The method is based on the statisti- the variation of the morphological features, and cal probability that complementary primer sites the distinctness of some microspecies and sections occur repeatedly in the genome. There may be according to them, with multivariate statistical problems with repeatability of the experiments, methods (Sepp & Paal 1998). However, we did and with compatibility between laboratories, but not thoroughly investigate genetic variability these can be overcome by ensuring that the tem- within the genus. The rather old experimental perature profiles inside the tubes are identical works mentioned above and the very small-scale (Penner et al. 1993). The main problem is that the research of Baeva et al. (1998) are so far the only markers are anonymous and one cannot be sure attempts. Walters (1987) stressed the need to use whether the annealing sites are really homologous so-called biological systematics for solving the (Quiros et al. 1995). Nevertheless, in comparison problems of this genus, but his advice is not fol- with some other analogous methods (restriction lowed very enthusiastically. fragment polymorphism, minisatellite DNA fin- According to the statistical analysis of mor- gerprinting) RAPD is much faster and simpler. In phological features (Sepp & Paal 1998), lots of comparison with isozyme electrophoresis, the the pairs of microspecies are mutually indistinct. RAPD markers are always dominant and they give Experienced botanists know that several pairs of more information involving the whole genome microspecies (e.g. Alchemilla acutiloba and A. mi- (Penner et al. 1993, Hillis 1996). cans, A. glaucescens and A. hirsuticaulis) have In the current study, we used RAPD analysis ANN. BOT. FENNICI 37 • Genetic polymorphism of Alchemilla 107 to assess the genetic relatedness of Alchemilla mi- 2 min, 72 °C for 2 min; 40 cycles of 94 °C for 1 crospecies. Preliminary data showing the variabil- min, 36 °C for 1 min, 72 °C for 1 min; and 1 cycle ity of RAPD patterns for 10 accessions of A. vul- of 94 °C for 1 min, 36 °C for 1 min, 72 °C for 4 garis s. lato (Baeva et al. 1998) revealed dissimi- min. All the PCR reactions were repeated at least larity of populations within microspecies. twice to confirm repeatability and if discrepan- cies occurred, PCR was repeated until two identi- cal results were achieved. Amplified fragments MATERIAL AND METHODS were run on 2% NuSieve 3:1 agarose gels (FMC), stained with ethidium bromide and photographed In total, 51 plants from 12 Alchemilla microspecies on an UV transilluminator. (A. acutiloba, A. baltica, A. cymatophylla, A. gla- Altogether, 116 characters were considered: bricaulis, A. glaucescens, A. micans, A. hepta- 68 RAPD bands over three primers and 48 mor- gona, A. hirsuticaulis, A. monticola, A. sarmatica, phological characters (characters in Table 2, data A. semilunaris, and A. subcrenata) were analysed. matrix in Appendix). Forty-two samples were collected in Estonia (Ta- Phylogenetic trees were constructed with the ble 1). Voucher specimens are deposited in the unweighted pair-group method with the arithme- Herbarium of the Institute of Botany and Ecol- tic average (UPGMA) and neighbour-joining (NJ) ogy of Tartu University (TU). On these plants, methods with the TREECON package (Van de the same morphological characters as in phenetic Peer & De Wachter 1994). The genetic distances analysis (Sepp & Paal 1998) were measured and GD were calculated as follows (Link et al. 1995): coded for cladistic analysis (Table 2). Nine sam- GD = (N + N )(N + N + N )–1 (1), ples were collected at the Biological station of xy x y x y xy Moscow University in Zvenigorod, Moscow dis- where Nx is the number of bands in lane x and not trict, Russia (Table 1), but without voucher speci- in lane y, Ny is the number of bands in lane y and mens, hence no morphological data were recorded not in lane x, and Nxy is the number of bands in for those. All plants were identified or their iden- lanes x and y. For the NJ tree, bootstrap values tification verified by K. P. Glazunova. Classifica- were calculated. tions of the studied microspecies according to the Maximum parsimony (MP) analysis was car- different systems of genus Alchemilla are pre- ried out with the PAUP 3.1.1 programme (Swof- sented in Table 3. ford 1993). Heuristic search settings: random addi- DNA was extracted from quickly dried (40 °C) tion sequence (10 replicates), tree bisection-recon- or frozen leaves according to a slightly modified nection branch swapping, MULPARS option, and protocol of Doyle and Doyle (1987). The concen- accelerated transformation were used for character tration of the template was estimated on agarose state optimisation. Bootstrap values and Bremer’s minigel in comparison with a previously known decay indices (Bremer 1988) were calculated. MP DNA sample. DNA was amplified in 20 µl reac- analyses were performed on three different data sets: tion mixtures containing 67 mm Tris-HCl (pH RAPD data separately, morphological data sepa- 8,4), 16,6 mm (NH4)2SO4, 2.5 mm MgCl2, 0,01% rately and the combined data. gelatin, 100 mm each of dATP, dCTP, dGTP and We used the functional outgroup method in dTTP, 10 pmol primer, 2 units Taq polymerase NJ and MP analysis.