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American Journal of Botany 91(2): 247253. 2004.

P HYLOGENY AND BIOGEOGRAPHY OF C ALTHA(R ANUNCULACEAE ) BASED ON CHLOROPLAST AND

NUCLEAR DNA SEQUENCES 1

ERIC SCHUETTPELZ 2 AND SARA B. H OOT

Department of Biological Sciences, University of WisconsinMilwaukee, Milwaukee, Wisconsin 53201 USA

The genus Caltha (Ranunculaceae) consists of 10 species of low-growing, perennial herbs distributed throughout the moist temperateand 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 leaveslacking inexed appendages. This study uses chloroplast and nuclear DNA sequences to determine the relationships among the 10species, 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 dispersalto 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,perennial herbs with simple leaves and actinomorphic owershaving ve or more petaloid sepals, numerous stamens withusually tricolpate pollen, and several distinct carpels. The mostunique morphological feature of the genus is diplophyllythepresence of distinctly inexed appendages formed by the au-ricles of the leaf laminae (Fig. 3). The function of these ap-pendages is not completely understood, but in most diplo-phyllous species, stomata are conned to the adaxial surfaceof the leaves. The auricles may thus serve to prevent the wet-ting of the stomata (Goebel, 1891; Hill, 1918). In any case,

diplophylly is a highly variable character within the genus andhas been utilized in both species delimitation and intragenericclassication.

All species of Caltha prefer wet habitats. At lower altitudes,the genus is found in marshes and other wetlands, and at high-er altitudes, it is commonly associated with melt water. Calthahas a strong preference for cooler climates (or an avoidanceof warmer climates) and is distributed in the moist temperateand cold regions of both the Northern and Southern Hemi-spheres. The distribution of the genus in the Southern Hemi-sphere is rather restricted, presumably by the scarcity of suit-able habitat (Hoffmann, 1999).

The rst comprehensive taxonomic treatment of the genuswas that of de Candolle (1818), in which he recognized twosections. Section Psychrophila in the Southern Hemispherewas characterized as having a persistent calyx, leaess solitary

1 Manuscript received 1 April 2003; revision accepted 19 August 2003.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.Zoller for assistance with the data analyses, W. C. Taylor and J. Karron fortheir comments on earlier versions of this manuscript, C. K. Hoot for helpwith graphics and leaf illustrations, and two anonymous reviewers for theiradvice. Support for this project was provided in part by a University of Wis-consinMilwaukee Graduate School Fellowship and a Joseph G. Baier Me-morial Scholarship from the Department of Biological Sciences at UWM.

2 Present address: Department of Biology, Duke University, Durham, NorthCarolina 27708 USA (e-mail: [email protected]).

inorescences, and sagittate basal leaves with upturned auri-cles. Section Populago in the Northern Hemisphere was char-acterized as having a deciduous calyx, leafy stems, and cordateor reniform leaves lacking upturned auricles. Subsequent au-thors (Huth, 1892; Smit, 1973) maintained these two sections,but with considerable variation in composition. In the mostrecent revision of the genus, 10 species are recognized (Smit,1973): four in the Northern Hemisphere (section Caltha ) andsix in the Southern Hemisphere (section Psychrophila ).

Caltha palustris , the most widespread species, has a circum-boreal distribution across much of Europe, Asia, and NorthAmerica. This species displays a considerable amount of mor-phological variation, prompting the recognition of many seg-regate taxa. However, most of this morphological diversity hasbeen shown to be the result of environmental conditions, andthere is little support for many of the previously recognizedsegregates (Smit, 1967, 1968, 1973; Woodell and Kootin-San-wu, 1971). Caltha natans , unique because of its oating orcreeping aquatic habit, also has a distribution on multiple con-tinents (northwestern North America and northeastern Asia)but is relatively invariable morphologically and has not beendivided into segregate taxa.

The two remaining northern species have relatively broaddistributions, but on single continents: C. scaposa is distrib-uted throughout the Himalayas and C. leptosepala is distrib-

uted in mountainous regions of western North America. Thelatter presents a unique problem. In the southern portion of the species range, two distinct taxa are clearly present: plantsin California have leaves that are wider than long, two owersper inorescence, and pantoporate pollen grains whereasplants in Colorado have leaves that are longer than wide, sol-itary owers, and tricolpate pollen grains. However, in thenorthern portion of the species range, the two forms are in-distinguishable. In the past, these taxa have been recognizedas distinct species, but are currently recognized as merely sub-species (Smit, 1973) or not at all (Ford, 1997).

Three species of Caltha are endemic to South America. Of


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these, C. sagittata has the broadest distribution (primarily inthe southern Andes, but with several disjunct northern popu-lations) and the most morphological variation. The two otherSouth American endemics ( C. appendiculata and C. dionaeifolia ) have more restricted distributions in the southernmostregions of the continent. An additional two species are endem-ic to New Zealand: C. novae - zelandiae found on both the

North and South Islands and C. obtusa found only on theSouth Island. A single species, C. introloba , is endemic to thealpine regions of Australia and Tasmania.

Aside from the division of the genus into two sections, theevolutionary relationships within Caltha have not been for-mally addressed. For this reason, past notions regarding theevolution of diplophylly or the events responsible for the cur-rent distribution of the genus have been somewhat speculative.This phylogenetic study has the following objectives: (1) de-termine the phylogenetic relationships among the 10 species,(2) test the monophyly of sections Psychrophila and Caltha ,(3) provide limited insight into species delimitation within C. palustris , C. leptosepala , and C. sagittata , (4) determine theevolutionary history of diplophylly and other morphologicalcharacters within the genus, and (5) explore explanations forthe current geographical distribution of the genus, in hopesthat this information will provide insight into the biogeograph-ical histories of other related and unrelated taxa.

DNA sequence data from the chloroplast atpB-rbcL inter-genic spacer ( atpB-rbcL spacer), the chloroplast trnL intron/ trnL-trnF intergenic spacer ( trnL-F region), and the internaltranscribed spacers of nuclear ribosomal DNA (ITS regions)were used to reconstruct the phylogeny of the genus. Each of these regions is either noncoding or includes a noncodingcomponent and is thus more variable and more useful at lowertaxonomic levels.

MATERIALS AND METHODS

Sampling This study included all species of Caltha recognized in themost recent revision of the genus (Smit, 1973). Three samples of C. palustriscovering the geographical range of the species, two samples of C. sagittata ,and both subspecies of C. leptosepala were included. Because of their putativeafnities with Caltha (Hoot, 1995; Johansson, 1995), Trollius , Helleborus ,and two species of Callianthemum were included as outgroups to root thetree. Species sampled, their geographical sources, voucher information, andGenBank accession numbers are listed in the Appendix (see SupplementalData accompanying the online version of this article).

DNA extraction and amplication Total DNA was extracted from eitherfresh, silica-dried, or herbarium leaf material for each sample. When sufcientamounts of material were present, the DNA was extracted using the procedureof Doyle and Doyle (1987). When limited amounts of leaf material wereavailable, DNeasy columns (Qiagen, Valencia, California, USA) were utilized

according to the manufacturers protocol. If necessary, DNA was further pu-ried using DNeasy columns. For each of the samples, the atpB-rbcL spacer,trnL-F region, and ITS regions were separately amplied using the polymer-ase chain reaction (PCR). Amplications of the nuclear ITS regions werecarried out using primers 1830F (located in the 18S gene) and 25R (locatedin the 26S gene), originally designed by Nickrent et al. (1994). The PCRprotocol was as described in Schuettpelz et al. (2002). When amplicationsusing this program were not successful, the annealing temperature was low-ered to 34 C.

Amplication protocols for the two chloroplast regions differed from oneanother only in the primers used. Amplications for the trnL-F region werecarried out using primers A50272 (located in the trnF gene) and B49317(located in the trnL gene: 5 exon), originally designed by Taberlet et al.

(1991). Amplications for the atpB-rbcL spacer were carried out using prim-ers S385R (located in the atpB gene) and RBCL1R (located in the rbcL gene),as in Hoot et al. (1995). Reaction mixtures and cycling parameters were asin Hoot et al. (1995), differing only in MgCl 2 concentration (3.0 mmol/L),number of cycles (30), and annealing temperature (45 C). When amplica-tions using this program were not successful, the annealing temperature waslowered to 40 C.

All PCR products were puried using one of two methods: (1) the PCRproducts were separated from impurities on a low-melt agarose gel, excisedfrom the gel as a plug, and separated from the agarose and concentrated usingWizard Columns (Promega, Madison, Wisconsin, USA) according to the man-ufacturers protocol; or (2) the PCR products were separated from impuritiesand concentrated using QIAquick Spin Columns (Qiagen) according to themanufacturers protocol.

DNA sequencing Sequence reactions were carried out in both directionsfor each puried double-stranded PCR product using Dye Terminator CycleSequencing or Big Dye Terminator Cycle Sequencing reagent (Applied Bio-systems, Foster City, California, USA) and primers identical to those utilizedin PCR, according to the manufacturers protocol. In the case of the atpB-rbcL spacer, one of two sequencing primers, S2R (Hoot et al., 1995) or S85R(sequence available from S. Hoot), was often substituted for the amplicationprimer (S385R).

Alignment The sequences obtained as chromatograms for each samplewere aligned, providing complete or nearly complete sequence overlap. Am-biguous bases were corrected and consensus sequences created using the com-puter program Sequencher 4.1 (Gene Codes, Ann Arbor, Michigan, USA). Allconsensus sequences for a given region were aligned manually using Mac-Clade 4.01 (Maddison and Maddison, 2001). Alignment procedures were asdescribed in Hoot and Douglas (1998), paying careful attention to repeatedmotifs (Type Ib indels) and runs of the same nucleotide (Type Ia indels).Because of the presence of many indels, homology was at times difcult toassess. In such instances, the regions of ambiguous alignment were excludedfrom the analyses, as were portions of the alignment containing large amountsof missing data. Indels were not scored for inclusion in the analyses, but weremapped on the resulting topologies (Table 3).

Data analysis To assess phylogenetic relationships based on the individ-ual markers and to assess the combinability of the data sets, each region wasanalyzed independently using a Bayesian Markov chain Monte Carlo (B/ MCMC) approach, as implemented in MrBayes 3.0 (Huelsenbeck and Ron-quist, 2001). Models of sequence evolution were selected using a top-downapproach (i.e., starting with the most complex model and moving towardsmodels of decreasing complexity) and a topology generated through equallyweighted parsimony analysis (1000 random addition sequence replicates, TBRbranch swapping, and MULTREES in effect). For each nested comparison,parameters were estimated using PAUP 4.0b10 (Swofford, 2002), and theresulting likelihoods were compared using the likelihood ratio test. The B/ MCMC analyses were conducted using the selected model of evolution (withat priors) and four chains (one cool and three heated). Chains were allowedto run for 1 10 6 generations, and trees were sampled from the cool chainevery 100 generations. Following completion, sampled trees were plotted

against their likelihood in order to recognize the point where the likelihoodsconverged on a maximum value, and all trees prior to this convergence werediscarded as the burn-in phase. The remaining trees were combined in amajority rule consensus. The consensus trees resulting from each of the threeanalyses (with posterior probabilities) were then compared visually for conictsupported by a posterior probability 0.95.

The three data sets were then combined and analyzed in unison. Searchesfor optimal trees used three methods: equally weighted parsimony, unequallyweighted parsimony, and maximum likelihood. For the unequally weightedparsimony analysis, a symmetrical step matrix was obtained using STMatrix(F. Lutzoni and S. Zoller, Duke University), which determines the frequencyof possible character state changes a priori from the data and converts thesefrequencies to costs with the negative natural logarithm (Felsenstein, 1981;


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Fig. 1. Results of Bayesian analyses of three individual molecular datasets used in this study of Caltha (Ranunculaceae). All topologies shown areconsensus trees of 9500 sampled trees. Branch lengths are averages. Posteriorprobabilities 0.50 are given at the nodes. Internal branches with posteriorprobability support 0.95 are thickened. (A) ITS regions. (B) atpB-rbcL spac-er. (C) trnL-F region.

DISCUSSION

Phylogeny Analyses of the individual molecular data setsprovided signicant support (posterior probability 0.95) forat most 10 nodes (ITS data). When these data were combined,11 nodes received posterior probability support of 1.00 (Fig.2). This is not surprising, as nonconicting data sets often

provide more support and resolution when combined (Bremeret al., 1999; Hahn, 2002; Matheny et al., 2002). In the topol-ogy resulting from the combined analysis, only one trichotomyis present (species from Australia and New Zealand), and onlytwo resolved nodes receive poor support (the node that re-solves C. leptosepala as paraphyletic and the node at the baseof two populations of C. palustris ). These superior levels of resolution and support allow one to draw conclusions from thephylogeny with reasonable condence.

Caltha natans was resolved as the earliest branching spe-cies. This is surprising based on its presumably derived aquatichabit but is supported by its relatively low chromosome num-ber (n 16; Hoffmann, 1999). Among the remaining species,C. scaposa is resolved as sister to the widespread C. palustris .Although the scapose habit of C. scaposa suggests an afnityto the Southern Hemisphere species, the presence of broadlyobovate sepals suggests afnities with either C. natans or C. palustris (Fig. 3). Indel data (Table 3) further support the sis-ter-group relationship of C. scaposa and C. palustris . Calthaleptosepala (although potentially paraphyletic) is resolved assister to the Southern Hemisphere species. This relationship issupported by a common chromosome number ( n 24; Hoff-mann, 1999), the presence of oblong sepals, and indel data(Fig. 3; Table 3).

The Southern Hemisphere species have always been rec-ognized as a natural group based on the presence of diplo-phyllous leaves, and the results of this study strongly support(PP 1.00; BS 92) this relationship (Fig. 2). Caltha sag-ittata , with the broadest distribution in the Southern Hemi-sphere and relatively simple diplophylly (Fig. 3), is resolvedas basal. Caltha dionaeifolia and C. appendiculata , both fromSouth America, are resolved as sister species. They both havehighly modied and divergent leaf morphologies (Fig. 3). Thespecies from Australia and New Zealand also form a well-supported, and geographically consistent, clade (PP 1.00;BS 97).

Sectional classication Of the two sections of Caltha pre-viously recognized, only section Psychrophila is monophylet-ic. The species of section Caltha form a paraphyletic grade(Fig. 2). For this reason, it is necessary to revise the previousclassications of the genus. Three phylogenetically sound, in-formal groupings are suggested (Fig. 2). The Natans group ( C.natans ) has an aquatic creeping habit and small ( 1 cm di-ameter) owers with obovate white sepals; the Caltha group(C. palustris and C. scaposa ) has a generally upright habit andlarger ( 2 cm diameter) owers with obovate sepals; and thePsychrophila group ( C. leptosepala , C. sagittata , C. dionaeifolia , C. appendiculata , C. introloba , C. obtusa , and C. novae - zelandiae ) has a generally scapose habit, oblong sepals (morethan twice as long as wide), and mostly diplophyllous leaves(Fig. 3).

Subspecic taxa The three geographically disparate sam-ples of C. palustris included in this study are, on a molecularlevel, quite distinct from other species and are united by sev-


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Fig. 2. Single most likely tree (lnL 5765.258 21) resulting from combined analysis of three molecular data sets used in this study of Caltha (Ranun-culaceae). Numbers at nodes before slashes are Bayesian posterior probabilities, and numbers at nodes after slashes are maximum likelihood bootstrap values(%). For both estimates of support, only values above 0.50 (50%) are shown. Internal branches with posterior probabilities 1.00 are thickened. Speciesdistributions are indicated, as are the sections recognized by Smit (1973) and the informal groupings recognized in the current study.

eral substitutions and a three base-pair indel (Fig. 2; Table 3).However, within this extremely polymorphic and widespread

species, there is relatively little variation, supporting Smits(1973) recognition of one species (rather than the numeroussegregates that had been proposed previously).

On the other hand, the two populations of C. sagittata in-cluded in this study are relatively divergent from each otheron a molecular level, despite their geographical proximity.Such differentiation could indicate the presence of additionalspecies as Hill (1918) suggested, but further study at a moredetailed level is required.

The results based on the inclusion of both subspecies of C.leptosepala are inconclusive but intriguing. The two popula-tions sampled here (from Colorado and Oregon) are quite dis-tinct on a molecular level and are actually resolved as para-phyletic. This result, although poorly supported (PP 0.65;BS 52), is contradictory to the three indels that support themonophyly of the species (Table 3). These results illustrate theproblematic nature of these taxa, which are morphologicallydistinct in the southern portion of their geographical range, butindistinguishable in the northern portion. A more detailed mo-lecular study examining many individuals across westernNorth America is needed.

Evolution of diplophylly and other morphological features Because the diplophyllous species are resolved asmonophyletic, it is quite reasonable to believe that this traitevolved only once within the genus (Fig. 3). However, it isinteresting and important to note that the basal lobes of the

other species of Caltha often show tendencies toward an up-turned morphology, especially in their younger leaves (Troll,

1932; E. Schuettpelz and S. B. Hoot, personal observations).In any case, it is clear that the earliest form of this characteris represented by inexed lobes and that appendages that coverthe entire lamina or that rise from the lamina itself are morederived (Fig. 3).

Most other morphological features of the genus appear tobe homoplastic, including some that were previously used forintrageneric and intraspecic classication. A scapose habithas evolved three times (Fig. 3), pantoporate pollen twice (Fig.3), and sepal color has toggled between white and yellow nu-merous times. Aside from strongly diplophyllous leaves, theonly character that appears to be uniquely derived and notsubsequently lost is the presence of oblong (greater than twiceas long as wide) sepals (Fig. 3).

Biogeography The results of this study reveal that theNorthern Hemisphere species of Caltha are paraphyletic to astrongly supported Southern Hemisphere clade, indicating thatthe origin and early differentiation of the genus most likelyoccurred in the Northern Hemisphere. Because the NorthAmerican species, C. leptosepala , is sister to the SouthernHemisphere clade and the rst-branching member of theSouthern Hemisphere clade is a South American species ( Csagittata ; Fig. 2), it is most probable that dispersal from theNorthern to Southern Hemisphere occurred between North andSouth America. Based on our results, this was followed bylater movement from South America to Australia and New


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Fig. 3. Summary tree based on single most likely tree (Fig. 2) indicatingevolution of morphological characters within the genus Caltha (Ranuncula-ceae). Leaf illustrations are to the right of scientic names. Numbers undernames are haploid chromosome counts (Hoffmann, 1999). D diplophylly,S scapose habit, P pantoporate pollen, O oblong sepals. Note: scaposehabit is not present in all individuals of C. scaposa or C. leptosepala ; pan-toporate pollen is not present in all individuals of C. leptosepala or C. pal-ustris .

T ABLE 3. Unambiguous indel support for various groups resolved in this study of Caltha (Ranunculaceae), including number of indels, sequenceregions containing indels, and range of indel lengths.

Taxonomic groupNo. of indels Regions Length(s)

Genus CallianthemumGenus CalthaC. scaposa C. palustrisC. palustris

7611

ITS regions, atpB-rbcL spacer, trnL-F regionatpB-rbcL spacer, trnL-F regionatpB-rbcL spacertrnL-F region

11711453

C. leptosepalaPsychrophila groupSouthern Hemisphere speciesC. sagittata

3121

ITS regions, atpB-rbcL spacerITS regionsITS regionstrnL-F region

1511, 33

Zealand. The primary biogeographical questions within the ge-nus center on the timing of and the mechanisms involved inthese two events: the movement of Caltha from the NorthernHemisphere to the Southern Hemisphere and the spread of Caltha throughout the Southern Hemisphere. It is interesting

to note that another genus in the Ranunculaceae ( Anemone )poses similar biogeographical questions (Schuettpelz et al.,2002).

In addressing these questions, it is necessary to consider thedispersal strategies of the genus as well as its probable age.The fruits of Caltha are small, unspecialized follicles, makinglong-distance wind or animal dispersal unlikely. Therefore,

scenarios involving repeated short-distance dispersals shouldbe favored. There is no reliable fossil record for the Ranun-culaceae. However, Ranunculales is one of the earliest branch-ing eudicot clades (Hoot et al., 1999; Soltis et al., 2000). Giventhat eudicot pollen has been documented from around the Bar-remian-Aptian boundary of the Lower Cretaceous (approxi-mately 125 mya; Crane et al., 1995) and that angiospermsunderwent a rapid diversication by the mid-Cretaceous(Crane et al., 1995; Magallon et al., 1999), an origin of Calthaby the mid-Cretaceous is possible.

The relative positions of North and South America have notchanged substantially since the Cretaceous (Scotese, 2001).However, the geology of the Caribbean region and CentralAmerica during this time is complicated and not well under-stood. It seems likely that a separate small tectonic plate, theCaribbean plate, formed between North and South America,producing chains of volcanic islands at its eastern and westernmargins (Cox and Moore, 2000). This, together with a cli-mactic cooling trend and lower sea levels during the late Cre-taceous/early Paleocene, may have produced a land link sim-ilar to (if not identical with) the present day Panama Isthmus(Hallam, 1992; Briggs, 1994; Cox and Moore, 2000). Thisscenario is consistent with the distributions of many terrestrialfossil vertebrates, the separation of marine bivalves, and therelatively old age of the Central American biota (Briggs, 1994)and is also congruent with the movement of Caltha from Northto South America indicated by our phylogeny (Fig. 2).

The Southern Hemisphere distribution of Caltha can be bestexplained by invoking a vicariance model. Under such a mod-el, the ancestor of the Southern Hemisphere species (excludingC. sagittata ) would have moved across Gondwanaland whenthe various austral landmasses were relatively contiguous (aslate as the Middle Eocene; Scotese, 2001) and diverged fol-lowing the breakup. This model is supported by similar linksfound within other ranuculacean genera (e.g., Anemone ;Schuettpelz et al., 2002) and within other lower eudicot fam-ilies (e.g., Proteaceae; Hoot and Douglas, 1998).

In summary, based on our phylogeny, it seems most prob-able that Caltha originated in the Northern Hemisphere,moved from North to South America along a land bridge dur-ing the late Cretaceous or early Paleocene (ca. 65 mya), andmoved from South America to New Zealand and Australia via


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Antarctica by the middle Eocene (ca. 49 mya). These move-ments, and their timing, will be further evaluated in futurebiogeographical studies of ranunculacean genera.

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247===caltha (ranunculaceae)+

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