ACTA BIOLOGICA CRACOVIENSIA Series Botanica 49/2: 15–26, 2007 RETICULATE EVOLUTION OF HIGH-ALPINE ACONITUM (RANUNCULACEAE) IN THE EASTERN SUDETES AND WESTERN CARPATHIANS (CENTRAL EUROPE) JÓZEF MITKA1*, AGNIESZKA SUTKOWSKA2, TOMASZ ILNICKI3, AND ANDRZEJ J. JOACHIMIAK3 1Botanical Garden, Jagiellonian University, ul. Kopernika 27, 31–501 Cracow, Poland 2Department of Plant Breeding and Seed Science, Agricultural University, ul. Łobzowska 24, 31–140 Cracow, Poland 3Department of Plant Cytology and Embryology, Jagiellonian University, ul. Grodzka 52, 31–044 Cracow, Poland Received April 12, 2007; revision accepted July 30, 2007 The chromosomal and molecular PCR-ISSR+RAPD pattern of high-mountain Aconitum sect. Aconitum in the Sudetes and Carpathians were analyzed to test whether the taxon has common markers in these two mountain systems. In the Sudetes the taxon forms an autopolyploid chromosome complex (2n = 32), and allopolyploid in the neighboring Western Carpathians. The chromosome Giemsa C banding pattern of the allotetraploid Carpathian A. firmum was found to be common to both Sudetic autopolyploid A. plicatum and diploid A. var- iegatum. The Sudetes are geologically older than the Carpathians, and it is argued that an ancient Sudetic taxon may have contributed to the genome of the Carpathian taxon. The Quaternary glaciations and corresponding range extensions of alpine floras may have facilitated their secondary contact(s). This is supported by a molec- ular ISSR+RAPD pattern that points to introgression between the Sudetic A. plicatum subsp. sudeticum and Carpathian A. firmum subsp. maninense. Key words: C-banding, heterochromatin, historical biogeography, hybridization, ISSR, phenetics, polyploidy, RAPD. INTRODUCTION 1995; Yang, 1999; Mitka and Starmühler, 2000; Mitka, 2003; Luo and Yang, 2005). Have historical Several cytological studies have been revealed that events contributed to such a morphological pattern? high-mountain Aconitum L. sect. Aconitum in Recently, several attempts have been made to Europe forms a tetraploid complex (e.g., Schafer understand the molecular phylogeny and phylogeog- and La Cour 1934; Leszczak 1950; Zieliński 1982; raphy of Aconitum by sequencing the internal tran- Okada 1991). In the Carpathians and Sudetes it is scribed spacer regions of nuclear ribosomal DNA represented by two distinct endemics, A. firmum (ITS) or noncoding regions of chloroplast DNA Rchb. (Starmühler and Mitka, 2001) and A. plica- (cpDNA) (Kita et al., 1995; Kita and Ito, 2000; Utelli tum Koehl. (Mitka 2003). Each of them has geo- et al., 2000; Luo et al., 2005). It has been shown that graphical races at the rank of subspecies (Mitka, many taxa that are morphologically well-defined or 2003). Two of them – the narrow endemics A. plica- geographically disjunct possess identical or nearly tum subsp. sudeticum Mitka and A. firmum subsp. identical ITS and cpDNA sequences. Neither ITS nor maninense (Skalický) Starmühl. – show intriguing cpDNA sequencing have revealed much information Sudetic-Carpathian affinities. They share a charac- on hybridization and introgression within the ana- ter unusual in A. sect. Aconitum, glandular-hairy lyzed groups of taxa. Hence, hypervariable, arbitrar- indumentum and carpels (Starmühler and Mitka, ily amplified dominant markers (AFLP, ISSR, 2001). Type of indumentum hairiness has proven to RAPD) seem to provide more useful data to generate be a key character in recent Linnaean taxonomic phylogenies (Archibald et al., 2006), including treatments of Aconitum (Kadota, 1987, Kita et al., potential hybridization and introgression events *e-mail: [email protected] PL ISSN 0001-5296 © Polish Academy of Sciences, Cracow 2007 16 Mitka et al. (e.g., Bartish et al., 2000; Cole and Kuchenreuther, MATERIALS AND METHODS 2001, Rieseberg et al., 2000). For example, RAPD analysis has proven to be a useful tool for clear sys- Tetraploid Aconitum sect. Aconitum (former Napellus tematic separation of taxa belonging to subgen. group, Seitz, 1969) in Europe encompasses mostly Aconitum sect. Aconitum, sect. Cammarum, and high-mountain taxa, which are all endemic and often A. subgen. Lycoctonum from the Italian Alps (Fico restricted to narrow geographical ranges. In the et al., 2003), and for analyzing the genetic differen- Western Carpathians and Sudetes, the Carpathian A. tiation and taxonomy of the A. delavayi Franch. firmum and the Sudetic-Hercynian A. plicatum belong complex in the Hengduan Mts. of China (Zhang et to the section (Starmühler, 2001; Starmühler and al., 2005). Mitka, 2001; Mitka, 2003; Tab. 1, Fig. 1a). The historical-biogeographical affinities In the Sudetes and Carpathians, two endemic between the two bordering mountain systems forms are worthy of mention: A. p. subsp. could reach far back in time. It is known from the sudeticum and A. f. subsp. maninense. The former palaeogeological record that parts of the Eastern is endemic to the Eastern Sudetes (Králický Carpathians and Western Carpathians were Snežnik, Czech Republic; Śnieżnik Mt., Poland; formed at the Oligocene/Miocene boundary (26–22 Hrubý Jeseník Mts., Czech Republic), and the latter myr BP), that the whole of the Carpathians were is a Western Carpathian endemic restricted to two united some 14 myr BP (in the Middle Miocene), regions: the Stražovské vrchy Mts. in Slovakia, and and that this event was accompanied by regression the High Tatras and adjacent Rów Podtarzański of the sea from the Alpine-Carpathian Foredeep trench in Poland (Mitka, 2003; Tab. 1, Fig. 1a). (Rögl, 1998; Golonka et al., 2006). Following uplift The basal chromosome set of Aconitum is of the mountain range it could be colonized from bimodal (2 long and 6 short chromosomes), and the adjacent Sudetic, Paleogene flora (Syabryay evolutionarily stable with respect to general chro- and Stuchlik, 1994). mosome morphology but not to heterochromatin The floristic relationships between the banding patterns (Okada, 1991, Joachimiak et al., Carpathians and Sudetes could also be of more 1999). Different C-banding chromosome variants recent age. Palaeobotanical, palaeoenvironmental appeared to be of diagnostic value in analysis of and molecular phylogeographical studies support Aconitum species and hybrids (Joachimiak et al., the idea that even during the most severe glacial 1999, Mitka and Starmühler, 2000; Ilnicki, 2005). maxima the alpine flora of Central Europe persist- ed in peripheral periglacial refugia in intermoun- PLANT MATERIAL tain lowlands or restricted to a few ice-free moun- tain tops – nunataks (Starkel, 1988; Abbott et al., Thirty randomly chosen specimens of Aconitum 1995; Obidowicz, 1996; Stehlik, 2000; Stehlik et sect. Aconitum were collected in the Sudetes and al., 2002; Holderegger et al., 2002; Kropf et al., Western Carpathians from late July to early 2003; Valero-Garces et al., 2004, Schönswetter et September 2002–2003, and kept in the Botanical al., 2004, 2005). Moreover, cooling episodes, push- Garden of the Jagiellonian University in Cracow. ing out part of the high-mountain flora to periph- The sample size was limited by the scarcity of most eral refugia, could facilitate secondary contacts taxa. DNA from fresh leaves of plants under cultiva- between otherwise isolated genetic stocks and thus tion was extracted in 2004 just before flowering. The originate hybridogenous taxa. geographic localities and information on the popula- The aim of our study was to investigate cytoge- tions sampled are given in Figure 1b and Table 1. netic (Giemsa C-banding) and molecular ISSR and RAPD markers in Sudetic and Western Carpathian KARYOTYPE ANALYSIS high-mountain Aconitum sect. Aconitum, and to compare those data with predictions of species In the summer of 2005, all planted specimens were relationships based on biogeographical evidence. dug up, transferred to the laboratory and kept in In the European Alps, molecular studies have shed pots, and root tips were cut for cytological analysis. some light on the origin of high-mountain flora Chromosomes were studied in squash preparations (Stehlik et al., 2002, Comes and Kadereit, 2003, made according to Grabowska-Joachimiak and Schönswetter et al., 2005), but hardly any robust Joachimiak (2002) and stained according to the C- phylogenetic analyses have been performed for banding method of Schwarzacher et al. (1980). other European mountain systems such as the From each plant, three complete metaphase plates Sudetes and Carpathians. Here, we sampled bio- showing maximum banding response were selected geographically representative areas of species for detailed analysis. An additional sample of A. var- occurrence. Their taxonomic treatment is dis- iegatum (A. sect. Cammarum DC.) from the cussed in terms the phylogeographic scenario Slovakian and Polish Carpathians was also cytoge- adopted. netically studied (Ilnicki, 2005). Reticulate evolution of Aconitum 17 Fig 1. (a) Geographic distribution of A. firmum and A. plicatum in Central Europe, modified from Mitka (2003), (b) Geographic locations of studied populations (see Tab. 1). DNA ISOLATION AND PCR AMPLIFICATION at 72°C; and for primers ISSR1, ISSR3 and ISSR6 DNA was isolated from fresh leaves using Plant in an initial cycle of 5 min at 94°, followed by 40 DNAzol Reagent (Invitrogen) according to the manu- cycles of 30 sec at 94°C, 30 sec at 47°C, and 30 sec facturer's instructions. DNA quality and quantity at 72°C. For RAPD primers the thermal cycler was were determined in 1% agarose LMP gel (Invitrogen). programmed for an initial cycle of 5 min at 94°C, fol- PCR reactions for ISSR and RAPD were per- lowed by 40 cycles of 40 sec at 94°C, 40 sec at 35°C formed in 25 μl buffer supplied by the Taq poly- and 40 sec at 72°C. The programs were terminated merase manufacturer (Invitrogen), 1.5 mM MgCl2, with an additional step of 7 min at 72°C before cool- 0.19 mM of each dNTP, 27 pmol primer, 10 ng tem- ing down to 4°C. plate DNA, and 1.4 units of Taq polymerase. Six The amplification products were separated on oligonucleotides (according to Stepansky 1999) 2.5% agarose gel by electrophoresis, stained in were used for ISSR DNA amplification; the primers ethidium bromide and stored with Imagemaster used in RAPD analysis were according to Oxelman VDS (Pharmacia, Amersham).