American Journal of Botany 91(2): 247±253. 2004. PHYLOGENY AND BIOGEOGRAPHY OF CALTHA (RANUNCULACEAE) BASED ON CHLOROPLAST AND NUCLEAR DNA SEQUENCES1 ERIC SCHUETTPELZ2 AND SARA B. HOOT Department of Biological Sciences, University of Wisconsin±Milwaukee, Milwaukee, Wisconsin 53201 USA The genus Caltha (Ranunculaceae) consists of 10 species of low-growing, perennial herbs distributed throughout the moist temperate and cold regions of both the Northern and Southern Hemispheres. Traditionally, the species have been divided into two sections: section Psychrophila in the Southern Hemisphere with diplophyllous leaves and section Caltha in the Northern Hemisphere with leaves lacking in¯exed appendages. This study uses chloroplast and nuclear DNA sequences to determine the relationships among the 10 species, test the monophyly of sections Psychrophila and Caltha, trace the evolutionary history of diplophylly, and explore biogeo- graphical hypotheses for the genus. Analysis of these data resulted in a well-resolved and well-supported phylogeny. Section Psy- chrophila (C. sagittata, C. appendiculata, C. dionaeifolia, C. obtusa, C. introloba, and C. novae-zelandiae) was resolved as mono- phyletic, indicating a single origin of diplophylly. The species of section Caltha (C. natans, C. scaposa, C. palustris, and C. leptosepala) formed a paraphyletic grade. The resulting phylogeny strongly supports a Northern Hemisphere origin for Caltha, followed by dispersal to the Southern Hemisphere (Gondwanaland). A vicariance model is invoked to explain present-day distributions in South America, Australia, and New Zealand. Key words: biogeography; Caltha; diplophylly; Gondwanaland; molecular systematics; phylogeny; Ranunculaceae. The genus Caltha (Ranunculaceae) consists of low-growing, in¯orescences, and sagittate basal leaves with upturned auri- perennial herbs with simple leaves and actinomorphic ¯owers cles. Section Populago in the Northern Hemisphere was char- having ®ve or more petaloid sepals, numerous stamens with acterized as having a deciduous calyx, leafy stems, and cordate usually tricolpate pollen, and several distinct carpels. The most or reniform leaves lacking upturned auricles. Subsequent au- unique morphological feature of the genus is diplophyllyÐthe thors (Huth, 1892; Smit, 1973) maintained these two sections, presence of distinctly in¯exed appendages formed by the au- but with considerable variation in composition. In the most ricles of the leaf laminae (Fig. 3). The function of these ap- recent revision of the genus, 10 species are recognized (Smit, pendages is not completely understood, but in most diplo- 1973): four in the Northern Hemisphere (section Caltha) and phyllous species, stomata are con®ned to the adaxial surface six in the Southern Hemisphere (section Psychrophila). of the leaves. The auricles may thus serve to prevent the wet- Caltha palustris, the most widespread species, has a circum- ting of the stomata (Goebel, 1891; Hill, 1918). In any case, boreal distribution across much of Europe, Asia, and North diplophylly is a highly variable character within the genus and America. This species displays a considerable amount of mor- has been utilized in both species delimitation and intrageneric phological variation, prompting the recognition of many seg- classi®cation. regate taxa. However, most of this morphological diversity has All species of Caltha prefer wet habitats. At lower altitudes, been shown to be the result of environmental conditions, and the genus is found in marshes and other wetlands, and at high- there is little support for many of the previously recognized er altitudes, it is commonly associated with melt water. Caltha segregates (Smit, 1967, 1968, 1973; Woodell and Kootin-San- has a strong preference for cooler climates (or an avoidance wu, 1971). Caltha natans, unique because of its ¯oating or of warmer climates) and is distributed in the moist temperate creeping aquatic habit, also has a distribution on multiple con- and cold regions of both the Northern and Southern Hemi- tinents (northwestern North America and northeastern Asia) spheres. The distribution of the genus in the Southern Hemi- but is relatively invariable morphologically and has not been sphere is rather restricted, presumably by the scarcity of suit- divided into segregate taxa. able habitat (Hoffmann, 1999). The two remaining northern species have relatively broad The ®rst comprehensive taxonomic treatment of the genus distributions, but on single continents: C. scaposa is distrib- was that of de Candolle (1818), in which he recognized two uted throughout the Himalayas and C. leptosepala is distrib- sections. Section Psychrophila in the Southern Hemisphere was characterized as having a persistent calyx, lea¯ess solitary uted in mountainous regions of western North America. The latter presents a unique problem. In the southern portion of the species range, two distinct taxa are clearly present: plants 1 Manuscript received 1 April 2003; revision accepted 19 August 2003. in California have leaves that are wider than long, two ¯owers The authors thank S. Wagstaff, R. Halse, T. Forbis, D. Schimpf, and P. Choler for providing leaf material or DNA for this project. Thanks also to S. per in¯orescence, and pantoporate pollen grains whereas Zoller for assistance with the data analyses, W. C. Taylor and J. Karron for plants in Colorado have leaves that are longer than wide, sol- their comments on earlier versions of this manuscript, C. K. Hoot for help itary ¯owers, and tricolpate pollen grains. However, in the with graphics and leaf illustrations, and two anonymous reviewers for their northern portion of the species range, the two forms are in- advice. Support for this project was provided in part by a University of Wis- distinguishable. In the past, these taxa have been recognized consin±Milwaukee Graduate School Fellowship and a Joseph G. Baier Me- morial Scholarship from the Department of Biological Sciences at UWM. as distinct species, but are currently recognized as merely sub- 2 Present address: Department of Biology, Duke University, Durham, North species (Smit, 1973) or not at all (Ford, 1997). Carolina 27708 USA (e-mail: [email protected]). Three species of Caltha are endemic to South America. Of 247 248 AMERICAN JOURNAL OF BOTANY [Vol. 91 these, C. sagittata has the broadest distribution (primarily in (1991). Ampli®cations for the atpB-rbcL spacer were carried out using prim- the southern Andes, but with several disjunct northern popu- ers S385R (located in the atpB gene) and RBCL1R (located in the rbcL gene), lations) and the most morphological variation. The two other as in Hoot et al. (1995). Reaction mixtures and cycling parameters were as South American endemics (C. appendiculata and C. dionaei- in Hoot et al. (1995), differing only in MgCl2 concentration (3.0 mmol/L), folia) have more restricted distributions in the southernmost number of cycles (30), and annealing temperature (458C). When ampli®ca- regions of the continent. An additional two species are endem- tions using this program were not successful, the annealing temperature was ic to New Zealand: C. novae-zelandiae found on both the lowered to 408C. North and South Islands and C. obtusa found only on the All PCR products were puri®ed using one of two methods: (1) the PCR South Island. A single species, C. introloba, is endemic to the products were separated from impurities on a low-melt agarose gel, excised alpine regions of Australia and Tasmania. from the gel as a plug, and separated from the agarose and concentrated using Aside from the division of the genus into two sections, the Wizard Columns (Promega, Madison, Wisconsin, USA) according to the man- ufacturer's protocol; or (2) the PCR products were separated from impurities evolutionary relationships within Caltha have not been for- and concentrated using QIAquick Spin Columns (Qiagen) according to the mally addressed. For this reason, past notions regarding the manufacturer's protocol. evolution of diplophylly or the events responsible for the cur- rent distribution of the genus have been somewhat speculative. DNA sequencingÐSequence reactions were carried out in both directions This phylogenetic study has the following objectives: (1) de- for each puri®ed double-stranded PCR product using Dye Terminator Cycle termine the phylogenetic relationships among the 10 species, Sequencing or Big Dye Terminator Cycle Sequencing reagent (Applied Bio- (2) test the monophyly of sections Psychrophila and Caltha, systems, Foster City, California, USA) and primers identical to those utilized (3) provide limited insight into species delimitation within C. in PCR, according to the manufacturer's protocol. In the case of the atpB- palustris, C. leptosepala, and C. sagittata, (4) determine the rbcL spacer, one of two sequencing primers, S2R (Hoot et al., 1995) or S85R evolutionary history of diplophylly and other morphological (sequence available from S. Hoot), was often substituted for the ampli®cation characters within the genus, and (5) explore explanations for primer (S385R). the current geographical distribution of the genus, in hopes that this information will provide insight into the biogeograph- AlignmentÐThe sequences obtained as chromatograms for each sample ical histories of other related and unrelated taxa. were aligned, providing complete or nearly complete sequence overlap. Am- DNA sequence data from the chloroplast atpB-rbcL inter- biguous bases were corrected and consensus sequences created using the com- genic