Plant Syst. Evol. 231: 143±151 -2002) Multiple origins of Southern Hemisphere Anemone Ranunculaceae) based on plastid and nuclear sequence data E. Schuettpelz1, S. B. Hoot1, R. Samuel2, and F. Ehrendorfer2 1Department of Biological Sciences, University of Wisconsin-Milwaukee, USA 2Department of Higher Plant Systematics and Evolution, Institute of Botany, University of Vienna, Austria Received April 23, 2001 Accepted October 4, 2001 Abstract. Using two molecular data sets, the occidentalis and other Northern Hemisphere anem- plastid atpB-rbcL intergenic spacer region and ones -subgenus Anemone s.lat., x 8). Possible the nuclear ribosomal internal transcribed spacer phytogeographical links of the Southern Hemi- regions -ITS), the taxonomic anities of two sphere species are discussed. newly available Anemone species from the South- ern Hemisphere were tested. From previous work Key words: Anemone, Ranunculaceae, atpB-rbcL based on morphology and geographic distribution, intergenic spacer, ITS, phylogeny, biogeography. it was assumed that A. tenuicaulis from New Zealand was most closely related to the Tas- The genus Anemone s.str. consists of approx- manian A. crassifolia, whereas the anity of imately 150 species -Tamura 1995) with the A. antucensis from Chile and Argentina was vast majority of species found in the Northern regarded as uncertain. Analyses of molecular sequence data from these and 18 other species of Hemisphere. However, a few species also occur Anemone s.lat. -with Clematis as outgroup) result in the cooler regions of the Southern Hemi- in trees largely congruent with past analyses based sphere. Three of these species, A. knowltonia on morphology and plastid restriction site data. -=Knowltonia capensis), A. cara -both South They strongly support A. richardsonii and Africa), and A. crassifolia -Tasmania) were A. canadensis -with boreal distributions in the included in a recent phylogenetic study of the Northern Hemisphere) as paraphyletic to a well genus based on both morphological and mo- supported Southern Hemisphere clade consisting lecular data -Hoot et al. 1994). It was found of A. antucensis and A. tenuicaulis. This group of that these species formed a well-supported, four species is part of an otherwise predominantly monophyletic group within subgenus Anemone Northern Hemisphere assemblage -subgenus Ane- -x 8). From this analysis, it was hypothesized monidium s.lat., chromosome base number x 7), that all species found in the Southern Hemi- including A. narcissi¯ora, A. obtusiloba, A. keiske- ana and A. -=Hepatica) americana. All other sphere were somewhat closely related, possibly austral species included in the present sampling, re¯ecting a former Gondwanan distribution A. crassifolia -Tasmania), A. knowltonia -=Knowl- -Hoot et al. 1994, Hoot 1995). tonia capensis), and A. cara -both South African), Leaf material from two additional anemo- form a separate clade, sister to A. -=Pulsatilla) nes from the Southern Hemisphere, A. tenui- 144 E. Schuettpelz et al.: Multiple origins of Southern Hemisphere Anemone caulis -New Zealand) and A. antucensis -South and A. crassifolia into sect. Crassifolia, subgen. America), recently became available, allowing Rivularidium, though far away from his two independent research teams to test this subgen. Pulsatilloides. Ehrendorfer -1995) hypothesis. Based on atpB-rbcL spacer and expressed doubts about this anity of A. ten- ITS sequence data and a sampling of 17 species uicaulis because of its deviating chromosome of Anemone, Schuettpelz and Hoot -2000) base number. reported the inclusion of A. tenuicaulis within A. antucensis occurs in mountain forests of a N. Hemisphere clade, most closely related to central Chile and Neuque n, Argentina. It is a A. canadensis. Ehrendorfer and Samuel -2000, low-growing, somewhat rhizomatous, herba- 2001), using atpB-rbcL spacer sequence data ceous perennial with basal, tripartite leaves and sampling seven species of Anemone -in- with long -5±14 cm) petioles. In¯orescences cluding the South American A. antucensis) are solitary to two-¯owered, with involucral obtained similar results and were able to leaves three-lobed and similar to basal leaves demonstrate a sister group relationship be- -Lourteig 1951). Flowers are whitish and have tween A. antucensis and A. tenuicaulis. Both of ®ve elliptical to suborbicular sepals, 20±33 these preliminary works highlighted the need stamens with threadlike ®laments and slightly to pool data and publish a joint paper with a extended connectives, 25±35 glabrous carpels broader sampling than either could obtain with a relatively long hooked style. Pollen is alone. either tricolpate or 6- to 9-pantocolpate -Huynh A. tenuicaulis is found on both the South 1970a). No chromosome count is available. and North Islands of New Zealand where it Based on morphology, Hoot et al. -1994) spec- prefers subalpine to lower alpine habitats, ulated that A. antucensis along with three other usually con®ned to damp sites in snow tussock South American species, may have anities grassland and herb®elds -Allan 1961). It is a with both the South African, New Zealand, and low-growing, herbaceous perennial with a Tasmanian species and provisionally placed vertical to rhizomatous rootstock -Parkin and them all into the Knowltonia group of sect. Sledge 1935). Leaves are tripartite with long Pulsatilloides. In contrast, Tamura -1995) listed petioles ->3 cm). In¯orescences are usually A. antucensis in subgen. and sect. Rivularidium. one- or two-¯owered, with an involucre con- To solve these deviating opinions on sisting of three linear, entire or bi- to tri-lobed Southern Hemisphere Anemone species, DNA leaves that are dissimilar to the basal leaves. sequences from the plastid atpB-rbcL intergen- Flowers are dull red in color with 5±6--7) linear ic spacer region and the nuclear ITS regions sepals with acute apices and rounded bases. were chosen as data sources after preliminary Stamens number 6±14, ®laments are threadlike tests indicated the level of variation was with the connectives extending slightly beyond appropriate within the genus. The atpB-rbcL the pollen sacs. Carpel number varies from 14 intergenic spacer of the plastid genome is to 34; achenes are glabrous and have a long approximately 750 bp in length, and has been hooked style. Pollen is spiraperturate -Huyhn used successfully in a variety of phylogenetic 1970b). 2n 28, making the base chromosome studies -Golenberg et al. 1993, Hoot and number most likely x 7 -Hair 1963; Ehren- Douglas 1998). The ITS I and II regions are dorfer 1995). Mainly based on morphology, located in the nuclear genome between the 18S Parkin and Sledge -1935) placed A. tenuicaulis and 26S ribosomal genes. The ITS regions into sect. Rivularidium -comparing it with together with the 5.8S gene are approximately A. antucensis). Hoot et al. -1994) tentatively 600 bp long. Although there are multiple placed A. tenuicaulis together with A. crassifo- copies of this ribosomal array in the genome, lia into the informal Knowltonia group of sect. they often appear to evolve in concert, and are Pullsatilloides, subgenus Anemone -x 8). therefore frequently identical -Baldwin et al. Tamura -1995) also combined A. tenuicaulis 1995). The ITS regions have been used exten- E. Schuettpelz et al.: Multiple origins of Southern Hemisphere Anemone 145 sively to study angiosperms at the species and spacer regions were ampli®ed using the PCR as generic level. described in Hoot and Douglas -1998). The PCR products were puri®ed using one of Materials and methods two methods: 1) the PCR products were separated from impurities on a low-melt agarose gel, excised Sampling. Sampling for the proposed study from the gel as a plug, and separated from the was done using a placeholder approach, selecting agarose and concentrated using Wizard Columns species to represent the major subdivisions of the -Promega) according to the manufacturer's proto- genus based on the molecular and morphological col; or 2) the PCR products were separated from results of Hoot et al. -1994). Once the anities of impurities and concentrated using QIAquick Spin Anemone tenuicaulis and A. antucensis were deter- Columns -Qiagen) according to the manufacturer's mined, additional species were added to further protocol. Sequencing was carried out in both resolve their placement. Included in the sampling directions for each puri®ed double-stranded PCR -Table 1), are one species each of the traditional product using the same ampli®cation primers as genera Hepatica -A. americana), Pulsatilla -A. above and Dye Terminator Cycle Sequencing occidentalis), Knowltonia -A. knowltonia), and 18 -ABI) according to the manufacturer's protocol. species of Anemone s.str. Because of its close Data analysis. The two contigs for each sample anities, Clematis was included as an outgroup were aligned -providing complete or near complete to root the tree -Hoot et al. 1994, Johannson 1995, overlap) and any ambiguous bases corrected using Hoot 1995). The grouping of taxa within Anemone the computer program Sequencher -Gene Codes s.lat. -=Anemoninae) follows the provisional and Corporation). The resulting consensus sequences informal arrangement presented by Hoot et al. for each species were aligned with each other using -1994: Fig. 4 and Appendix 2) of subgenera, Sequencher, then further adjusted manually. Align- sections, and species groups. ment procedures were as described in Hoot and DNAsequencing. Total DNA was extracted Douglas -1998), paying careful attention to repeat-