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 inflexed 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,
perennial herbs with simple leaves and actinomorphic flowers
having five or more petaloid sepals, numerous stamens with
usually tricolpate pollen, and several distinct carpels. The most
unique morphological feature of the genus is diplophylly—the
presence of distinctly inflexed 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 confined to the adaxial surface
of 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 and
has been utilized in both species delimitation and intrageneric
classification.
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. Caltha
has a strong preference for cooler climates (or an avoidance
of warmer climates) and is distributed in the moist temperate
and 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 first comprehensive taxonomic treatment of the genus
was that of de Candolle (1818), in which he recognized two
sections. Section Psychrophila in the Southern Hemisphere
was characterized as having a persistent calyx, leafless 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 for
their comments on earlier versions of this manuscript, C. K. Hoot for help
with graphics and leaf illustrations, and two anonymous reviewers for their
advice. Support for this project was provided in part by a University of Wis-
consin–Milwaukee 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, North
Carolina 27708 USA (e-mail: ejs7@duke.edu).
247
inflorescences, 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 cordate
or reniform leaves lacking upturned auricles. Subsequent au-
thors (Huth, 1892; Smit, 1973) maintained these two sections,
but with considerable variation in composition. In the most
recent revision of the genus, 10 species are recognized (Smit,
1973): four in the Northern Hemisphere (section Caltha) and
six in the Southern Hemisphere (section Psychrophila).
Caltha palustris, the most widespread species, has a circum-
boreal distribution across much of Europe, Asia, and North
America. This species displays a considerable amount of mor-
phological variation, prompting the recognition of many seg-
regate taxa. However, most of this morphological diversity has
been shown to be the result of environmental conditions, and
there is little support for many of the previously recognized
segregates (Smit, 1967, 1968, 1973; Woodell and Kootin-San-
wu, 1971). Caltha natans, unique because of its floating or
creeping aquatic habit, also has a distribution on multiple con-
tinents (northwestern North America and northeastern Asia)
but is relatively invariable morphologically and has not been
divided into segregate taxa.
The two remaining northern species have relatively broad
distributions, 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. The
latter presents a unique problem. In the southern portion of
the species range, two distinct taxa are clearly present: plants
in California have leaves that are wider than long, two flowers
per inflorescence, and pantoporate pollen grains whereas
plants in Colorado have leaves that are longer than wide, sol-
itary flowers, and tricolpate pollen grains. However, in the
northern portion of the species range, the two forms are in-
distinguishable. In the past, these taxa have been recognized
as 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
248 AMERICAN JOURNAL
these, C. sagittata has the broadest distribution (primarily in
the southern Andes, but with several disjunct northern popu-
lations) and the most morphological variation. The two other
South American endemics (C. appendiculata and C. dionaei-
folia) have more restricted distributions in the southernmost
regions 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 the
South Island. A single species, C. introloba, is endemic to the
alpine regions of Australia and Tasmania.
Aside from the division of the genus into two sections, the
evolutionary relationships within Caltha have not been for-
mally addressed. For this reason, past notions regarding the
evolution 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 the
evolutionary history of diplophylly and other morphological
characters within the genus, and (5) explore explanations for
the current geographical distribution of the genus, in hopes
that 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 internal
transcribed 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 noncoding
component and is thus more variable and more useful at lower
taxonomic levels.
MATERIALS AND METHODS
Sampling—This study included all species of Caltha recognized in the
most recent revision of the genus (Smit, 1973). Three samples of C. palustris
covering the geographical range of the species, two samples of C. sagittata,
and both subspecies of C. leptosepala were included. Because of their putative
affinities with Caltha (Hoot, 1995; Johansson, 1995), Trollius, Helleborus,
and two species of Callianthemum were included as outgroups to root the
tree. Species sampled, their geographical sources, voucher information, and
GenBank accession numbers are listed in the Appendix (see Supplemental
Data accompanying the online version of this article).
DNA extraction and amplification—Total DNA was extracted from either
fresh, silica-dried, or herbarium leaf material for each sample. When sufficient
amounts of material were present, the DNA was extracted using the procedure
of Doyle and Doyle (1987). When limited amounts of leaf material were
available, DNeasy columns (Qiagen, Valencia, California, USA) were utilized
according to the manufacturer’s protocol. If necessary, DNA was further pu-
rified using DNeasy columns. For each of the samples, the atpB-rbcL spacer,
trnL-F region, and ITS regions were separately amplified using the polymer-
ase chain reaction (PCR). Amplifications of the nuclear ITS regions were
carried out using primers 1830F (located in the 18S gene) and 25R (located
in the 26S gene), originally designed by Nickrent et al. (1994). The PCR
protocol was as described in Schuettpelz et al. (2002). When amplifications
using this program were not successful, the annealing temperature was low-
ered to 348C.
Amplification protocols for the two chloroplast regions differed from one
another only in the primers used. Amplifications for the trnL-F region were
carried out using primers A50272 (located in the trnF gene) and B49317
(located in the trnL gene: 59 exon), originally designed by Taberlet et al.
OF BOTANY [Vol. 91
(1991). Amplifications 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 as
in Hoot et al. (1995), differing only in MgCl2 concentration (3.0 mmol/L),
number of cycles (30), and annealing temperature (458C). When amplifica-
tions using this program were not successful, the annealing temperature was
lowered to 408C.
All PCR products were purified using one of two methods: (1) the PCR
products were separated from impurities on a low-melt agarose gel, excised
from the gel as a plug, and separated from the agarose and concentrated using
Wizard Columns (Promega, Madison, Wisconsin, USA) according to the man-
ufacturer’s protocol; or (2) the PCR products were separated from impurities
and concentrated using QIAquick Spin Columns (Qiagen) according to the
manufacturer’s protocol.
DNA sequencing—Sequence reactions were carried out in both directions
for each purified double-stranded PCR product using Dye Terminator Cycle
Sequencing or Big Dye Terminator Cycle Sequencing reagent (Applied Bio-
systems, Foster City, California, USA) and primers identical to those utilized
in PCR, according to the manufacturer’s 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 amplification
primer (S385R).
Alignment—The sequences obtained as chromatograms for each sample
were 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). All
consensus sequences for a given region were aligned manually using Mac-
Clade 4.01 (Maddison and Maddison, 2001). Alignment procedures were as
described in Hoot and Douglas (1998), paying careful attention to repeated
motifs (Type Ib indels) and runs of the same nucleotide (Type Ia indels).
Because of the presence of many indels, homology was at times difficult to
assess. In such instances, the regions of ambiguous alignment were excluded
from the analyses, as were portions of the alignment containing large amounts
of missing data. Indels were not scored for inclusion in the analyses, but were
mapped 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 was
analyzed 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-down
approach (i.e., starting with the most complex model and moving towards
models of decreasing complexity) and a topology generated through equally
weighted parsimony analysis (1000 random addition sequence replicates, TBR
branch swapping, and MULTREES in effect). For each nested comparison,
parameters were estimated using PAUP 4.0b10 (Swofford, 2002), and the
resulting likelihoods were compared using the likelihood ratio test. The B/
MCMC analyses were conducted using the selected model of evolution (with
flat priors) and four chains (one cool and three heated). Chains were allowed
to run for 1 3 106 generations, and trees were sampled from the cool chain
every 100 generations. Following completion, sampled trees were plotted
against their likelihood in order to recognize the point where the likelihoods
converged on a maximum value, and all trees prior to this convergence were
discarded as the ‘‘burn-in’’ phase. The remaining trees were combined in a
majority rule consensus. The consensus trees resulting from each of the three
analyses (with posterior probabilities) were then compared visually for conflict
supported by a posterior probability $0.95.
The three data sets were then combined and analyzed in unison. Searches
for optimal trees used three methods: equally weighted parsimony, unequally
weighted parsimony, and maximum likelihood. For the unequally weighted
parsimony analysis, a symmetrical step matrix was obtained using STMatrix
(F. Lutzoni and S. Zoller, Duke University), which determines the frequency
of possible character state changes a priori from the data and converts these
frequencies to costs with the negative natural logarithm (Felsenstein, 1981;
February 2004] SCHUETTPELZ AND HOOT—PHYLOGENY OF CALTHA 249

TABLE 1. Summary of DNA sequences and alignments for each molecular region used in this study of Caltha (Ranunculaceae).

Parsimony
Mean sequence Alignment Included Variable informative Missing
Marker length length characters characters charactersa data (%)b

ITS regions 621 675 573 189 120 7.87

atpB-rbcL spacer 676 867 581 102 46 2.34
trnL-F region 869 1072 769 108 55 2.56
Total 2166 2614 1923 399 221 4.08
a Number of characters informative for equally weighted parsimony analyses.
b From included regions.

Wheeler, 1990; Miadlikowska et al., 2002). The resulting step matrix was
implemented across all sites. For the maximum likelihood analysis, the ap-
propriate model of sequence evolution was selected using the top-down ap-
proach as described. All searches were implemented in PAUP* 4.0b10 with
1000 random addition sequence replicates, TBR branch swapping, MUL-
TREES in effect, and gaps treated as missing data. Various outgroup com-
binations were tested for their effect on the ingroup topology.
Branch support was assessed using maximum likelihood bootstrapping (Fel-
senstein, 1985) and B/MCMC analysis. The maximum likelihood bootstrap
analysis, implemented in PAUP* 4.0b10 using the model selected for the
original data set, consisted of 1000 replicates, each with 10 random addition
sequence replicates, TBR branch swapping, and MULTREES in effect. The
B/MCMC analysis of the combined data set was conducted using three models
(one for each molecular marker, as determined above), flat priors, and four
chains. Chains were allowed to run for 1 3 106 generations, and trees were
sampled every 100 generations. Following completion, the burn-in trees were
discarded and the remaining trees were combined in a majority rule consensus,
to determine the posterior probability of each node.
RESULTS
Alignments—The mean sequence length, alignment length,
number of characters included after pruning regions of ambig-
uous alignment, numbers of variable and parsimony informa-
tive characters, and percentage missing data are given for each
sequenced region in Table 1.
Individual analyses—Through comparisons using the like-
lihood ratio test, a submodel of the general time reversible
model was selected for each of the molecular regions (Table
2). A gamma distribution for among-site rate variation was
also included for the ITS regions and trnL-F region. In
MrBayes, the number of substitution types was set to six for
each of the three regions, and rates 5 gamma was specified
for the ITS regions and trnL-F region.
The Bayesian analyses for each of the three data sets con-
verged quickly on a maximum value and excluding the first
500 trees (50 000 generations) was sufficient to remove the
burn-in trees. Analysis of the ITS regions yielded a well-re-
solved and well-supported topology (Fig. 1A). Ten nodes re-
ceived significant support (posterior probability $0.95). None
of the remaining nodes received posterior probability support
greater than 0.81. The analysis of the atpB-rbcL spacer pro-
vided significant support for only six nodes, with the remain-
ing nodes receiving #0.76 posterior probability (Fig. 1B). The
analysis of the trnL-F region recovered nine nodes with sig-
nificant support, and in this case, the remaining nodes all re-
ceived posterior probability support #0.65 (Fig. 1C). No sig-
nificant conflict was detected among the three resulting topol-
ogies, and the data sets were combined.
Combined analyses—Through comparisons using the like-
lihood ratio test, a submodel of the general time reversible
model with a gamma distribution for among-site rate variation
was selected for the combined data set (Table 2). Analysis
using this model resulted in a single most likely topology
(2lnL 5 5765.25821; Fig. 2). The equally weighted and un-
equally weighted parsimony analyses both resulted in three
most parsimonious trees (550 and 898.19 steps, respectively).
For each of these two analyses, the strict consensus of the three
trees was identical in topology to the most likely tree. Various
combinations of the outgroup taxa did not have an effect on
the ingroup topology.
This resulting topology was well resolved and well sup-
ported by both Bayesian posterior probabilities (PP) and max-
imum likelihood bootstrap values (BS). The genus Caltha is
strongly supported as monophyletic (PP 5 1.00; BS 5 100),
and C. natans is resolved and well supported as sister to the
remaining species. Within the large Caltha clade, two well-
supported groups are present: a clade consisting of C. palustris
and C. scaposa (PP 5 1.00; BS 5 100) and a clade consisting
of C. leptosepala and all of the Southern Hemisphere species
(PP 5 1.00; BS 5 99). Within the second clade, the two sub-
species of C. leptosepala are resolved as basal and paraphy-
letic but with poor support (PP 5 0.65; BS 5 52). Among
the Southern Hemisphere species (PP 5 1.00; BS 5 92), C.
sagittata branches first and the five remaining species form
two well-supported clades: one clade consisting of C. appen-
diculata plus C. dionaeifolia (PP 5 1.00; BS 5 95) and the
other clade consisting of C. novae-zelandiae, C. obtusa, and
C. introloba (PP 5 1.00; BS 5 97).

TABLE 2. Model parameters estimated using PAUP* for phylogenetic study of Caltha (Ranunculaceae). Models of evolution determined using the
likelihood ratio test coupled with a top-down approach. All values rounded to three decimal places. Rates were equal among sites for atpB-
rbcL spacer.

Marker AC AG AT CG CT GT A C G T alpha

ITS regions 1.000 2.260 2.260 0.000 5.586 1.000 0.250 0.250 0.250 0.250 0.381
atpB-rbcL spacer 1.000 1.000 0.217 1.000 1.000 1.000 0.381 0.141 0.147 0.332 —
trnL-F region 1.000 1.000 0.160 1.000 1.000 1.000 0.307 0.163 0.163 0.367 0.287
Combined 1.000 1.429 0.448 0.448 2.779 1.000 0.319 0.175 0.186 0.320 0.291
250 AMERICAN JOURNAL
Fig. 1. Results of Bayesian analyses of three individual molecular data
sets used in this study of Caltha (Ranunculaceae). All topologies shown are
consensus trees of 9500 sampled trees. Branch lengths are averages. Posterior
probabilities $0.50 are given at the nodes. Internal branches with posterior
probability support $0.95 are thickened. (A) ITS regions. (B) atpB-rbcL spac-
er. (C) trnL-F region.
OF BOTANY [Vol. 91
DISCUSSION
Phylogeny—Analyses of the individual molecular data sets
provided significant support (posterior probability $0.95) for
at 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 nonconflicting data sets often
provide more support and resolution when combined (Bremer
et al., 1999; Hahn, 2002; Matheny et al., 2002). In the topol-
ogy resulting from the combined analysis, only one trichotomy
is present (species from Australia and New Zealand), and only
two resolved nodes receive poor support (the node that re-
solves C. leptosepala as paraphyletic and the node at the base
of two populations of C. palustris). These superior levels of
resolution and support allow one to draw conclusions from the
phylogeny with reasonable confidence.
Caltha natans was resolved as the earliest branching spe-
cies. This is surprising based on its presumably derived aquatic
habit but is supported by its relatively low chromosome num-
ber (n 5 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 affinity
to the Southern Hemisphere species, the presence of broadly
obovate sepals suggests affinities 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. Caltha
leptosepala (although potentially paraphyletic) is resolved as
sister to the Southern Hemisphere species. This relationship is
supported by a common chromosome number (n 5 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 5 1.00; BS 5 92) this relationship (Fig. 2). Caltha sag-
ittata, with the broadest distribution in the Southern Hemi-
sphere and relatively simple diplophylly (Fig. 3), is resolved
as basal. Caltha dionaeifolia and C. appendiculata, both from
South America, are resolved as sister species. They both have
highly modified and divergent leaf morphologies (Fig. 3). The
species from Australia and New Zealand also form a well-
supported, and geographically consistent, clade (PP 5 1.00;
BS 5 97).
Sectional classification—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 previous
classifications 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) flowers with obovate white sepals; the Caltha group
(C. palustris and C. scaposa) has a generally upright habit and
larger (.2 cm diameter) flowers with obovate sepals; and the
Psychrophila group (C. leptosepala, C. sagittata, C. dionaei-
folia, C. appendiculata, C. introloba, C. obtusa, and C. novae-
zelandiae) has a generally scapose habit, oblong sepals (more
than twice as long as wide), and mostly diplophyllous leaves
(Fig. 3).
Subspecific taxa—The three geographically disparate sam-
ples of C. palustris included in this study are, on a molecular
level, quite distinct from other species and are united by sev-
February 2004] SCHUETTPELZ AND HOOT—PHYLOGENY OF CALTHA 251

Fig. 2. Single most likely tree (lnL 5 25765.258 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 5 1.00 are thickened. Species
distributions 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 Smit’s
(1973) recognition of one species (rather than the numerous
segregates 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 other
on a molecular level, despite their geographical proximity.
Such differentiation could indicate the presence of additional
species as Hill (1918) suggested, but further study at a more
detailed 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 5 0.65;
BS 5 52), is contradictory to the three indels that support the
monophyly of the species (Table 3). These results illustrate the
problematic nature of these taxa, which are morphologically
distinct in the southern portion of their geographical range, but
indistinguishable in the northern portion. A more detailed mo-
lecular study examining many individuals across western
North America is needed.
Evolution of diplophylly and other morphological fea-
tures—Because the diplophyllous species are resolved as
monophyletic, it is quite reasonable to believe that this trait
evolved only once within the genus (Fig. 3). However, it is
interesting 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 character
is represented by inflexed lobes and that appendages that cover
the entire lamina or that rise from the lamina itself are more
derived (Fig. 3).
Most other morphological features of the genus appear to
be homoplastic, including some that were previously used for
intrageneric and intraspecific classification. A scapose habit
has 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, the
only character that appears to be uniquely derived and not
subsequently lost is the presence of oblong (greater than twice
as long as wide) sepals (Fig. 3).
Biogeography—The results of this study reveal that the
Northern Hemisphere species of Caltha are paraphyletic to a
strongly supported Southern Hemisphere clade, indicating that
the origin and early differentiation of the genus most likely
occurred in the Northern Hemisphere. Because the North
American species, C. leptosepala, is sister to the Southern
Hemisphere clade and the first-branching member of the
Southern Hemisphere clade is a South American species (C.
sagittata; Fig. 2), it is most probable that dispersal from the
Northern to Southern Hemisphere occurred between North and
South America. Based on our results, this was followed by
later movement from South America to Australia and New
252 AMERICAN JOURNAL OF BOTANY [Vol. 91

to note that another genus in the Ranunculaceae (Anemone)

Fig. 3. Summary tree based on single most likely tree (Fig. 2) indicating
evolution of morphological characters within the genus Caltha (Ranuncula-
ceae). Leaf illustrations are to the right of scientific names. Numbers under
names are haploid chromosome counts (Hoffmann, 1999). D 5 diplophylly,
S 5 scapose habit, P 5 pantoporate pollen, O 5 oblong sepals. Note: scapose
habit 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.
Zealand. The primary biogeographical questions within the ge-
nus center on the timing of and the mechanisms involved in
these two events: the movement of Caltha from the Northern
Hemisphere to the Southern Hemisphere and the spread of
Caltha throughout the Southern Hemisphere. It is interesting
poses similar biogeographical questions (Schuettpelz et al.,
2002).
In addressing these questions, it is necessary to consider the
dispersal strategies of the genus as well as its probable age.
The fruits of Caltha are small, unspecialized follicles, making
long-distance wind or animal dispersal unlikely. Therefore,
scenarios involving repeated short-distance dispersals should
be 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). Given
that 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 angiosperms
underwent a rapid diversification by the mid-Cretaceous
(Crane et al., 1995; Magallon et al., 1999), an origin of Caltha
by the mid-Cretaceous is possible.
The relative positions of North and South America have not
changed substantially since the Cretaceous (Scotese, 2001).
However, the geology of the Caribbean region and Central
America during this time is complicated and not well under-
stood. It seems likely that a separate small tectonic plate, the
Caribbean plate, formed between North and South America,
producing chains of volcanic islands at its eastern and western
margins (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). This
scenario is consistent with the distributions of many terrestrial
fossil vertebrates, the separation of marine bivalves, and the
relatively old age of the Central American biota (Briggs, 1994)
and is also congruent with the movement of Caltha from North
to South America indicated by our phylogeny (Fig. 2).
The Southern Hemisphere distribution of Caltha can be best
explained by invoking a vicariance model. Under such a mod-
el, the ancestor of the Southern Hemisphere species (excluding
C. sagittata) would have moved across Gondwanaland when
the various austral landmasses were relatively contiguous (as
late as the Middle Eocene; Scotese, 2001) and diverged fol-
lowing the breakup. This model is supported by similar links
found 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), and
moved from South America to New Zealand and Australia via

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

No. of
Taxonomic group indels Regions Length(s)

Genus Callianthemum 7 ITS regions, atpB-rbcL spacer, trnL-F region 1–17

Genus Caltha 6 atpB-rbcL spacer, trnL-F region 1–14
C. scaposa 1 C. palustris 1 atpB-rbcL spacer 5
C. palustris 1 trnL-F region 3
C. leptosepala 3 ITS regions, atpB-rbcL spacer 1–5
Psychrophila group 1 ITS regions 1
Southern Hemisphere species 2 ITS regions 1, 3
C. sagittata 1 trnL-F region 3
February 2004] SCHUETTPELZ AND HOOT—PHYLOGENY
Antarctica by the middle Eocene (ca. 49 mya). These move-
ments, and their timing, will be further evaluated in future
biogeographical studies of ranunculacean genera.
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