Plant Syst. Evol. 231: 143±151 -2002)

Multiple origins of Southern Hemisphere ) 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 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 ˆ 8). Possible the nuclear ribosomal internal transcribed spacer phytogeographical links of the Southern Hemi- regions -ITS), the taxonomic anities of two sphere 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 Anemone s.str. consists of approx- manian A. crassifolia, whereas the anity 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 largely congruent with past analyses based sphere. Three of these species, A. on morphology and plastid restriction site data. -=), A. ca€ra -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 ˆ 8). From this analysis, it was hypothesized monidium s.lat., chromosome base number x ˆ 7), that all species found in the Southern Hemi- including A. narcissi¯ora, A. obtusiloba, A. keiske- ana and A. -=) 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. ca€ra -both South African), material from two additional anemo- form a separate clade, sister to A. -=) 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 anity 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 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 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 anities 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 ˆ 28, making the base chromosome studies -Golenberg et al. 1993, Hoot and number most likely x ˆ 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 ˆ 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 anities 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 anities, Clematis was included as an outgroup were aligned -providing complete or near complete to root the -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- from either fresh, silica-dried, or herbarium leaf ed motifs -Type Ib indels) and runs of the same material for each sample. When sucient amounts nucleotide -Type Ia indels). Using MacClade of material were present, DNA was extracted using -Maddison and Maddison 1992), insertions and the procedure of Doyle and Doyle -1987). When deletions -indels) were scored as single events, then only small amounts of leaf material were available, deleted if otherwise uninformative. Regions of DNA easy columns -Qiagen) were utilized accord- ambiguous alignment were removed from the data ing to the manufacturer's protocol. If necessary, set without scoring. DNA was further puri®ed using DNA easy col- Parsimony analyses of the atpB-rbcL spacer umns -Qiagen). and ITS data were conducted for each gene The ITS regions, including the 5.8S gene, were independently -results not shown) and in combina- ampli®ed by the polymerase chain reaction -PCR) tion using PAUP* version 4.0b2 -Swo€ord 1999) using primers 1830F, located in the 18S gene, and and the branch and bound search option. In the 25R, located in the 26S gene -for primer sequences, case of multiple shortest trees, strict consensus trees see Nickrent et al. 1994). Double-stranded ampli- were constructed. To estimate the con®dence to be ®cations in 100 lL reactions were conducted with placed in the topology, bootstrap values were the following reagents: 20 mM Tris -pH8.3), 50 calculated using the branch and bound search mM KCl, 1.5 mM MgCl2, 10 M DMSO, 50 lM option and 1000 replications -Felsenstein 1985). each dNTP, 0.25 lM of each ampli®cation primer, Before combining the data sets, several meth- and 2.5 U Taq polymerase. After overlaying each ods of assessing congruence among the two data reaction with mineral oil, genomic DNA -210±500 sets were implemented: visual comparison of the ng) was added. Reaction conditions consisted of 40 various clades found in the minimal trees, their cycles of 94 °C for 30 sec., 45 °C for 30 sec., and bootstrap support, and implementation of the 72 °C for 2 min. for the denaturation, annealing, incongruence length di€erence -ILD) test -Farris and extension steps, respectively. The ®rst cycle was et al. 1995), which tests whether the prede®ned preceeded by a 4 min denaturation step and the last partitions in the data di€er signi®cantly from cycle by a 5 min extension step. The atpB-rbcL random partitions of the combined data set. The 146 E. Schuettpelz et al.: Multiple origins of Southern Hemisphere Anemone

Table 1. Species included in the study, sectional anities -informal, from Hoot et al. 1994), geographic distribution, vouchers, and GenBank numbers -atpB-rbcL spacer ®rst line, ITS second line). RBGE = Royal Botanical Garden, Edinburgh Species Sectional anities Vouchers GenBank numbers Anemone americana Hepatica S. Hoot 883, MICH AY055407 DC. [=Hepatica americana AY055386 -DC.) H. Hara] Anemone canadensis L. Anemonidium S. Hoot 867, MICH AY055408 AY055387 A. richardsonii Hook. f. Anemonidium C.L. Parker 9801, ALA AY055409 AY055388 A. antucensis Poeppig Anemonidium L. & F. Ehrendorfer: AF311735 Chile, Concepcion, AY056049 Nahuelbuta, 24.01.98, WU A. tenuicaulis -Cheeseman) Anemonidium Garnock-Jones 2147, CHR AY055410 Parkin & Sledge AY055389 A. ¯accida F. Schmidt Keiskea S. Hoot 8952, MICH AY055412 AY055391 A. keiskeana Ito Keiskea S. Hoot 8951, MICH AY055411 AY055390 A. narcissi¯ora L. Homalocarpus R. Meyers 88±8 AY055414 AY055393 A. demissa Homalocarpus RBGE 841910 AY055413 Hook. f. & Thomson AY055392 A. obtusiloba D. Don Homalocarpus RBGE 851867 AY055415 AY055394 A. trullifolia Homalocarpus RBGE 812614 AY055416 Hook. f. & Thomson AY055395 A. rivularis Buch.-Ham.ex DC. Anemonospermos S. Hoot 8853, MICH AY055417 AY055396 A. hupehensis Lemoine Anemonospermos S. Hoot 911, MICH AY055418 AY055397 A. crassifolia Hook.f. Pulsatilloides S. Hoot 8855, MICH AY055419 AY055398 A. ca€ra -Eckl. & Zeyh.) Pulsatilloides RBGE 770617 AY055420 Harvey AY055399 A. knowltonia Pulsatilloides Univ. Of Copenhagen, AY055421 Burtt-Davy [=Knowltonia Bot. Garden, Denmark AY055401 capensis -L.) Huth Anemone occidentalis Pulsatilloides S. Hoot 8817, MICH AY055426 S. Waston [=Pulsatilla AY055400 occidentalis -S. Watson) Freyn] A. blanda Schott & Kotschy Anemone Matthaei Bot. Garden, AY055422 Ann Arbor, MICH AY055402 A. caroliniana Walter Anemone L. Raymond s.n. AY055423 AY055403 A. drummondii S. Watson Anemone R. Meyers 88±7 AY055424 AY055404 A. multi®da Poir Anemone B. Polastri s.n. AY055425 AY055405 Clematis hexapetala Pall. S. Hoot 9150, MICH AY055406 AY055385 E. Schuettpelz et al.: Multiple origins of Southern Hemisphere Anemone 147

ILD analysis was conducted using PAUP* with the Combined analysis. The ILD test revealed following settings: 1000 replications, heuristic no signi®cant di€erence -P ˆ 0.78) between the search with simple addition, TBR -tree bisection- partition de®ned by the two genes and random reconnection) branch swapping, and saving up to partitions, indicating a high degree of data 2000 trees for each replicate. congruence. Because of these results and the largely congruent topologies of the ITS and Results atpB-rbcL spacer trees, the data sets were combined. Analysis of the combined data -196 Nuclear internal transcribed spacers parsimony informative characters) resulted in ITS). The average length of the sequences a single, most parsimonious tree -Fig. 1) with obtained for this region was 570 bases, with the CI ˆ 0.72 and RI ˆ 0.81 -Fig. 1). Two large longest at 599 bases -Anemone ca€ra) and the clades are evident, corresponding to the two shortest at 494 bases -A. hupehensis). Thirteen subgenera Anemone and Anemonidium previ- insertions/deletions -indels) were scored. After ously found by Hoot et al. -1994): 1) Subgenus the removal of unalignable regions and unin- Anemone -base chromosome number x ˆ 8) formative characters, the data set consisted of consisting of A. -=Pulsatilla) occidentalis, 193 variable characters, 134 of these were A. knowltonia -=), and parsimony informative. The parsimony analy- assorted other Anemone species from South sis of the ITS data resulted in a single most Africa, Tasmania, and the Northern Hemi- parsimonious tree -tree not presented) with a sphere and 2) Subgenus Anemonidium -x ˆ 7), consistency index excluding parsimony-un- including three well-supported clades -boot- informative characters -CI) of 0.65, and a strap ˆ 100%): sect. Keiskea -A. ¯accida and retention index -RI) of 0.74. The clades found A. keiskeana), sect. Anemonidium consisting of with the ITS data are largely identical to those the Northern Hemisphere species A. canaden- found in the tree resulting from the combined sis/A. richardsonii and the Southern Hemi- data sets -Fig. 1). They di€er only in the sphere species A. antucensis/A. tenuicaulis, and placement of A. -=Hepatica) americana;itis section Homalocarpus consisting of various sister to all other with the ITS data. species from the Northern Hemisphere Chloroplast atpB-rbcL intergenic spac- -Fig. 1). er. The average length of the sequences was 793 bases, with the longest at 884 bases -A. ¯accida) and the shortest at 632 bases Discussion -A. hupehensis). 23 indels were scored. After removal of unalignable regions, the data set The combined phylogeny resulting from the consisted of 125 variable characters, 62 of present study is nearly identical to that derived which were parsimony informative. The parsi- by Hoot et al. -1994) using morphological and mony analysis of the atpB-rbcL intergenic plastid restriction site data. Only two clades spacer data resulted in 42 equally parsimoni- have somewhat di€erent placements. In the pre- ous trees with CI ˆ 0.91 and RI ˆ 0.97. vious study, subg. Anemone Sect. Anemonosp- The strict consensus tree of the 42 most ermos -including A. rivularis and A. hupehensis) parsimonious trees is identical in topology to was resolved as sister to sect. Pulsatilloides the tree derived from the combined data including A. pulsatilla -=Pulsatilla vulgaris), -Fig. 1) but exhibits less resolution. The clade A. crassifolia, A. knowltonia -=Knowltonia consisting of A. -=Pulsatilla) occidentalis capensis), and A. ca€ra, but with weak through to A. caroliniana is a polytomy with bootstrap support -64%). In the present the following clades recognized: --A. rivularis, study, sect. Anemonospermos is moderately A. hupehensis), -A. drummondii -A. blanda, supported -73%) as sister to sect. Anemone A. multi®da, A. caroliniana))). -Fig. 1). 148 E. Schuettpelz et al.: Multiple origins of Southern Hemisphere Anemone

Fig. 1. Single most parsimonious tree for Anemone s.lat. resulting from analysis of the combined atpB-rbcL spacer and ITS data, using Clematis hexapetala as the outgroup. Abbreviated geographical distributions are indicated in shaded column; Southern Hemisphere species in bold. Informal sectional names -Hoot et al. 1994) are listed after geographical distributions

The second di€erence relates to the posi- sister to sect. Homalocarpus -including tion within subgen. Anemonidium of sect. A. narcissi¯ora, A. demissa, A. obtusiloba, Keiskea -including A. keiskeana and A. ¯acci- and A. trullifolia). In the present study, sect. da) which in Hoot et al. -1994) is placed as Keiskea is sister to both sect. Homalocarpus E. Schuettpelz et al.: Multiple origins of Southern Hemisphere Anemone 149 and sect. Anemonidium -including A. canaden- pollen). According to Parkin and Sledge -1935), sis, A. richardsonia, A. antucensis, and A. tenuicaulis di€ers from A. antucensis by single A. tenuicaulis). In both studies, these relation- ¯owered scapes and thread-like rather than ships received moderate -93%) to high -99%) ¯attened ®laments. However, examinations of bootstrap support. Both of these contradic- herbarium specimens -Hoot, preliminary data) tions may be due to sampling di€erences. do not support this: both A. antucensis and A. Available data on Anemone support a tenuicaulis have fairly similar ®lament widths Northern Hemisphere origin of the genus and and single to two-¯owered scapes. two major clades: subgenus Anemone -x ˆ 8) Considering the numerous examples of and subgenus Anemonidium -x ˆ 7). The com- links between Tasmania -and Southeast Aus- bined sequence data -Fig. 1) clearly support tralia) and New Zealand -Wardle 1978), the the close anities of the South African lack of closer phylogenetic relationships be- A. ca€ra and A. knowltonia with the Tasma- tween A. crassifolia and A. tenuicaulis from nian A. crassifolia -bootstrap ˆ 94%) as these two areas is remarkable. For an expla- already suggested by morphological similari- nation of the present day distribution of the ties and plastid restriction data -Hoot et al. relevant Anemone clades, one has to account 1994, bootstrap ˆ 92%). This veri®es the tax- for links between South Africa and Tasmania onomic placement of these taxa into the -for sect. Pulsatilloides) and between North informal sect. Pulsatilloides -sensu Hoot et al. America, South America, and New Zealand 1994). -for sect. Anemonidium). If one includes the The combined sequence data -Fig. 1) also three other Ranunculaceae genera with taxa in demonstrate that the New Zealand A. tenui- both hemispheres -Caltha, Clematis, and Ra- caulis is sister to the South American nunculus; Hoot 1995; Schuettpelz, preliminary A. antucensis -bootstrap ˆ 100%) and that data), at least three additional Southern Hemi- these two species are included within sect. sphere links must be considered. Anemonidium along with the Northern Hemi- Achene morphology and the relatively re- sphere A. richardsonii and A. canadensis -and stricted ranges of many of the anemones in most certainly, although not sampled, its question make long-distance dispersal events Eurasian sister species A. dichotoma). Species unlikely. Therefore, a more parsimonious ex- in sect. Anemonidium have the following mor- planation invokes a vicariance model: Anemone phological and karyological characters in and other ranunculacean genera must have been common -but not unique to just this section): present in the N. Hemisphere and Gondwana- leaves trilobed, no stomata on adaxial leaf land, probably in the mid to late Cretaceous. surface; in¯orescences 1-, 2-, -3-) ¯owered; From what we know about the fossil record: the sepals in low number -4±7); stamens with oldest de®nite angiosperm pollen dating at narrow ®laments -<0.4 mm); carpels in limit- 130 mya -Brenner 1996), the oldest tricolpate ed number -<40); stigmas slender; and x ˆ 7 pollen at 120 mya -Doyle 1992, Hughes 1994), -not yet documented for A. antucensis). and the rapid diversi®cation of angiosperms by The moderately supported clade of the mid Cretaceous -Drinnan and Crane 1990), A. richardsonii, A. tenuicaulis, and A. antucensis Anemone could have already undergone con- is characterized by low-growing, somewhat siderable radiation by 100 mya. rhizomatous habit and achenes tapering to an Geological reconstructions of the continents extended hooked style. Di€erences between the -Smith et al. 1981, Storey 1995) indicate that as species are found in the nature of the involucral late as the Santonian of the late Cretaceous leaves -similar to basal leaves or not), sepal -80 mya); North America, South America, shape and color, and pollen morphology -A. Antarctica, New Zealand, and Australia were richardsonii and A. antucensis have tricolpate to contiguous to each other or close enough to be eupantocolpate, A. tenuicaulis spiraperturate accessible by short distance dispersal. In addi- 150 E. Schuettpelz et al.: Multiple origins of Southern Hemisphere Anemone tion, North America, Eurasia, and Africa were Botanical Garden, Ann Arbor; and the University more or less contiguous at this time. The only of Copenhagen Botanical Garden. This work was Gondwanan land masses that may not have partially supported by a Ruth Walker Graduating been accessible by short-distance dispersal are Senior Award, University of Wisconsin-Milwaukee, Madagascar and India. Thus, the di€erentia- Department of Biological Sciences to E.S. The team tion of Anemone s.lat. into two major clades, of R. S. & F. E. is grateful for ®nancial support from the Austrian Academy of Sciences, Commission for the subgenera Anemone -x ˆ 8) and Anemoni- Interdisciplinary Ecological Research. Thanks are dium -x ˆ 7) and their subgroups, could have due to U. Jensen for New Zealand plant material and occurred before short distance dispersal was to C. Stanzel for technical assistance. prevented between the austral land masses. In this way ancestors of sect. Pulsatilloides could have linked South Africa to Tasmania, and References ancestors of sect. Anemonidium could have extended from North to South America via Allan H. H. -1961) Flora of New Zealand, VI. Antarctica to New Zealand. Indigenous Tracheophyta. R. E. Owen, Welling- ton, New Zealand. Further support for a vicariance model to Baldwin B. G., Sanderson M. J., Porter J. M., explain geographical distribution patterns in Wojciechowski M. F., Campbell C. S., Don- Anemone comes from the , a family oghue M. J. -1995) The ITS region of nuclear that, like the Ranunculaceae, is among the ribosomal DNA: A valuable source of evidence earliest branching -Hoot et al. 1999). on angiosperm phylogeny. Ann. Missouri. Bot. Proteaceae include several well-supported lin- Gard. 82: 247±277. eages containing ``Gondwanan'' elements, in- Brenner G. J. -1996) Evidence for the earliest stage dicating that the family had substantially of angiosperm pollen evolution: a paleoequato- di€erentiated before gene ¯ow was compro- rial section from Israel. In: Taylor D. W., Hickey mised by the separation of the austral land- L. J. -eds.) origin, evolution and masses -Hoot and Douglas 1998). This is phylogeny. 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