Gilberto Ocampo and J. Travis Columbus

American Journal of Botany 97(11): 18271847. 2010.
MOLECULAR PHYLOGENETICS OF SUBORDER CACTINEAE

(CARYOPHYLLALES), INCLUDING INSIGHTS INTO
PHOTOSYNTHETIC DIVERSIFICATION AND
HISTORICAL BIOGEOGRAPHY1
Gilberto Ocampo2 and J. Travis Columbus

Rancho Santa Ana Botanic Garden and Claremont Graduate University, 1500 North College Avenue, Claremont,
California 91711-3157 USA
Premise of the study: Phylogenetic relationships were investigated among the eight families (Anacampserotaceae, Basellaceae,
Cactaceae, Didiereaceae, Halophytaceae, Montiaceae, Portulacaceae, Talinaceae) that form suborder Cactineae (= Portulacineae) of the Caryophyllales. In addition, photosynthesis diversification and historical biogeography were addressed.
Methods: Chloroplast DNA sequences, mostly noncoding, were used to estimate the phylogeny. Divergence times were calibrated using two Hawaiian Portulaca species, due to the lack of an unequivocal fossil record for Cactineae. Photosynthetic
pathways were determined from carbon isotope ratios (13C) and leaf anatomy.
Key results: Maximum likelihood and Bayesian analyses were consistent with previous studies in that the suborder, almost all
families, and the ACPT clade (Anacampserotaceae, Cactaceae, Portulacaceae, Talinaceae) were strongly supported as monophyletic; however, relationships among families remain uncertain. The age of Cactineae was estimated to be 18.8 Myr. Leaf
anatomy and 13C and were congruent in most cases, and inconsistencies between these pointed to photosynthetic intermediates. Reconstruction of photosynthesis diversification showed C3 to be the ancestral pathway, a shift to C4 in Portulacaceae, and
five independent origins of Crassulacean acid metabolism (CAM). Cactineae were inferred to have originated in the New
World.
Conclusions: Although the C3 pathway is inferred as the ancestral state in Cactineae, some CAM activity has been reported in
the literature in almost every family of the suborder, leaving open the possibility that CAM may have one origin in the group.
Incongruence among loci could be due to internal short branches, which possibly represent rapid radiations in response to increasing aridity in the Miocene.
Key words: C4 photosynthesis; Cactineae; Cactaceae; Caryophyllales; Crassulacean acid metabolism divergence times; historical biogeography; photosynthesis diversification; Portulacaceae; Portulacineae.
The suborder Cactineae of order Caryophyllales (sensu

Thorne and Reveal, 2007) comprises ca. 2000 species in 130
genera and eight families (estimated from Kubitzki et al., 1993),
distributed mainly in the Americas, Africa, and Australia. This
group (also known as Portulacineae) includes families Basellaceae, Cactaceae, Didiereaceae, Halophytaceae, and Portu1
Manuscript received 22 June 2010; revision accepted 14 September 2010.
The authors thank the following persons and institutions who kindly
provided plant material for this study: Jennifer Cruse-Sanders, Urs Eggli,
Holly Forbes, Naomi Fraga, Patricia Jaramillo, Sean Lahmeyer, James
Matthews, Clifford Morden, Robert Nicholson, Mark Porter, Ernesto
Sandoval, John Trager, National Tropical Botanical Garden, Pretoria
National Herbarium (PRE), and the Waimea Arboretum Foundation.
Lucinda McDade, Wendy Applequist, Mark Simmons, and two anonymous
reviewers provided helpful comments on the manuscript. Elena
Voznesenskaya kindly provided the carbon isotope ratio value for Portulaca
elatior. The study was financed by Rancho Santa Ana Botanic Garden and
the Botanical Society of America. Financial support to G. O. was provided
by The Fletcher Jones Foundation, Comisin Nacional de Ciencia y
Tecnologa (Mexico), Fundacin Prywer (Mexico), and the Instituto de
Ecologa, A. C. (Mexico).
2 Author for correspondence (e-mail: gocampo@calacademy.org);
present address: California Academy of Sciences, Botany Department, 55
Music Concourse Drive, Golden Gate Park, San Francisco, CA 94118
USA
doi:10.3732/ajb.1000227
lacaceae (Thorne and Reveal, 2007). Recently, it has been

proposed that Portulacaceae should be split into four families
(Nyffeler and Eggli, 2010), resulting in a monogeneric Portulacaceae (Portulaca) and Anacampserotaceae, Montiaceae, and
Talinaceae. Molecular data have provided important insights
into the relationships within the strongly supported, monophyletic
Cactineae (Applequist and Wallace, 2001; Mller and Borsch,
2005; Applequist et al., 2006; Nyffeler, 2007; Brockington
et al., 2009; Nyffeler and Eggli, 2010) and have led to a new
classification of families, particularly involving members of
Portulacaceae s.l. Hershkovitz and Zimmer (1997), using ribosomal internal transcribed spacer (ITS) sequences, found that
Portulacaceae were not monophyletic. In particular, Cactaceae
were nested within the family, and Ceraria and Portulacaria,
considered genera of Portulacaceae at the time, were more
closely related to Didiereaceae and Basellaceae. These relationships were confirmed later by Applequist and Wallace (2001)
using sequences from the chloroplast gene ndhF, which led to
the transfer of Ceraria and Portulacaria, along with Calyptrotheca, to Didiereaceae (Applequist and Wallace, 2003). In
addition, these authors uncovered a clade that grouped Cactaceae with Talinum + Talinella, Portulaca, and tribe Anacampseroteae of Portulacaceae, although without internal resolution.
Subsequent studies using other chloroplast and mitochondrial
regions (Applequist et al., 2006; Nyffeler, 2007) have recovered this monophyletic group, known as the ACPT clade
(Nyffeler, 2007), with strong support, but internal relationships
American Journal of Botany 97(11): 18271847, 2010; http://www.amjbot.org/ 2010 Botanical Society of America
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[Vol. 97
American Journal of Botany
have low support values. Other relationships within Cactineae

are not known with certainty, because the branching order is
ambiguous or poorly supported (Applequist and Wallace, 2001;
Applequist et al., 2006; Nyffeler, 2007; Nyffeler and Eggli,
2010). Halophytaceae, a monotypic family from Patagonia and
traditionally considered a member of Chenopodiaceae Vent.
(e.g., Cronquist, 1981), have been shown in molecular phylogenetic studies to be part of Cactineae (Cunoud et al., 2002; Mller
and Borsch, 2005; Applequist et al., 2006; Brockington et al.,
2009; Nyffeler and Eggli, 2010), but its placement within the
suborder is unclear.
Cactineae tend to be better represented in the southern hemisphere, although Portulaca has a worldwide distribution, mainly
in tropical and subtropical regions of both hemispheres. Cactaceae and Montiaceae have important centers of diversity in
North America (Barthlott and Hunt, 1993; Hershkovitz, 1993).
This distribution pattern suggests that the origin of the suborder
may have been in South America (Applequist and Wallace,
2001), but the temporal scale is unknown. The lack of an unambiguous fossil record for Cactineae has been a limiting factor in
studying its origins (see Hershkovitz and Zimmer, 2000). Age
estimates for cacti range from ca. 100 million years (Myr; Croizat,
1952; Mauseth, 1990; Wallace and Gibson, 2002) to ca. 30 Myr
(Hershkovitz and Zimmer, 1997), while Montiaceae are estimated to be 816 Myr (as western American Portulacaceae;
Hershkovitz and Zimmer, 2000), but to date there are no hypotheses of the age of the suborder, which impedes understanding of its evolution and historical biogeography.
A fascinating aspect of evolution within Cactineae is adaptation to xeric habitats, especially in plant morphology but also
extending to the physiological level in terms of photosynthetic
pathways. All three primary photosynthetic variantsC3, C4,
and Crassulacean acid metabolism (CAM)are present in the
suborder (e.g., Winter, 1979; Nobel and Hartsock, 1986; Sage
et al., 1999; Guralnick and Jackson, 2001; Sayed, 2001; Guralnick
et al., 2008). In C3 plants, 1,5-bisphosphate carboxylase/oxygenase
(Rubisco) catalyzes the reaction where 1,5-bisphosphate (RuBP)
reacts with atmospheric CO2 as the first step of the photosynthetic reaction (Ehleringer and Monson, 1993). Atmospheric O2
and CO2 are competitive substrates for Rubisco, but the enzyme
has more specificity for the latter. However, the concentration
of CO2 is significantly reduced as it diffuses from the atmosphere into the photosynthetic tissue, favoring the oxygenation
of RuBP. This condition, in addition to an eventual liberation of
the CO2, is termed photorespiration, which reduces the overall
photosynthetic efficiency ca. 33% in C3 plants (Ehleringer and
Monson, 1993). In C4 and CAM plants, phosphoenolpyruvate
carboxylase (PEP) catalyzes the first step of the photosynthetic
reaction instead of Rubisco. These photosynthetic pathways depend upon structural (C4) or temporal (CAM) separation of PEP
and Rubisco activity to concentrate CO2 for the Calvin cycle
while reducing photorespiration (C4) and water loss (CAM). In
C4 photosynthesis, fixed carbon is transported in the form of
malate or aspartate to special cells that form the vascular bundle
sheath, where it is released as CO2 and enters the Calvin cycle
(Kanai and Edwards, 1999). In the CAM pathway, CO2 is fixed
as malate in the mesophyll at night, when the stomata are open,
and stored inside the cell vacuoles. The malate is subsequently
decarboxylated during light hours, when the stomata are closed,
and enters the Calvin cycle in the mesophyll (Winter and Smith,
1996; Nelson and Sage, 2008). In this case, CO2 concentration
remains high because it cannot escape through the closed stomata (Ehleringer and Monson, 1993). PEP carboxylase dis-
criminates less against the 13C isotope than Rubisco (Lajtha and
Marshall, 1994; Winter and Holtum, 2002); thus, carbon isotope ratios (13C) can be used to distinguish plants that have C4
and CAM photosynthesis from those that use the C3 pathway.
Although the use of 13C can serve as the first step in determining the photosynthetic pathway in a group, additional evidence
may be needed to discriminate C4 from CAM photosynthesis,
because their 13C values overlap (OLeary, 1988; Winter and
Holtum, 2002; Sage et al., 2007), or to detect photosynthetic
intermediates (e.g., C3-C4; Monson et al., 1984; Rawsthorne
and Bauwe, 1998). Other sources of evidence include stem and
leaf anatomy and biochemical assays (e.g., Ku et al., 1983;
Rajendrudu et al., 1986; Brown and Hattersley, 1989; Sage et al.,
2007).
The overarching aim of this study was to obtain a more robust phylogenetic estimate of relationships within Cactineae by
using different, noncoding chloroplast markers from those used
in previous studies. Specifically, the rpl14rps8infArpl36 region, atpIatpH intergenic spacer, and ndhA intron (Shaw et al.,
2007) were used to explore their utility for resolving relationships among the families of Cactineae. Using the phylogenetic
estimate, we studied the diversification of the photosynthetic
pathway (inferred from 13C values and leaf anatomy) and historical biogeography of the group. In addition, age estimates of
the major groups were calculated based on a relaxed molecular
clock model and indirect calibration methods using as a reference the age of specific Hawaiian Islands inhabited by endemic
Portulaca species. These age estimates were used to address
questions of place and time of origin of the families of
Cactineae.
MATERIALS AND METHODS
Taxon samplingFifty-one species were sampled from all families recognized in Cactineae by Nyffeler and Eggli (2010) and corresponding to the major
clades recovered in recent studies (Applequist and Wallace, 2001; Applequist
et al., 2006; Nyffeler, 2007; Brockington et al., 2009; Nyffeler and Eggli, 2010)
(Appendix 1). Early-diverging species in all families (see Edwards et al., 2005;
Applequist et al., 2006; Nyffeler, 2007) were included in the study, although
relationships within Basellaceae are not known, and only one genus (of four)
was sampled. Species of Aizoaceae, Molluginaceae, Nyctaginaceae, and Phytolaccaceae were selected for rooting the phylogenies because they are known
to be close relatives of the suborder (Rettig et al., 1992; Downie and Palmer,
1994; Applequist and Wallace, 2001; Cunoud et al., 2002; Applequist et al.,
2006; Brockington et al., 2009). Unfortunately, attempts to obtain sequences
from Mollugo (Molluginaceae) failed, so the outgroup taxa employed were
from the remaining three families (Appendix 1).
DNA extraction and sequencingSources of DNA included leaves taken
directly from live plants, leaves dried in silica gel, herbarium specimens, and in
one instance (Portulaca sclerocarpa), a DNA aliquot (Appendix 1). Total genomic DNA was extracted from 10 mg of dried material or 20 mg of fresh tissue
using the modified CTAB method of Doyle and Doyle (1987) or DNeasy kits
(Qiagen, Valencia, California, USA). In general, quality of the genomic DNA
obtained by these two methods was sufficient for performing the polymerase
chain reaction (PCR) with the selected markers, although the DNeasy kits outperformed the CTAB method when samples were highly mucilaginous. DNA
extracted using the CTAB method was quantified and diluted to a concentration
of 10 ng/L, whereas the concentration of DNA extracted using the DNeasy
kits was not measured because typically a concentration of 10 ng/L is obtained
by this method.
Preliminary analyses of a data matrix comprising sequences from GenBank
representing 10 loci and more than 100 Cactineae taxa showed that a supermatrix approach does not improve internal nodal support within the suborder.
Therefore, we conducted a study using the chloroplast rpl14rps8infArpl36
region (comprising coding and spacer sequences), atpIatpH intergenic spacer,
November 2010]
Ocampo and Columbus Phylogenetics of Cactineae
and ndhA intron to explore their utility for resolving relationships among families of Cactineae. These loci are among the 12 most variable chloroplast markers recommended by Shaw et al. (2007). Primers for amplification via PCR
were as in Shaw et al. (2007). Amplifications were performed in 25 L reactions with 0.62 units of Taq DNA polymerase (Promega, Madison, Wisconsin,
USA), 2.5 L of (NH4)2SO4 buffer, 0.5 pM of forward and reverse primers,
0.25 mM MgCl2, 0.25 mM dNTPs, and 0.25 L of BSA 100 for a final concentration of 1%, plus 1 L of genomic DNA in a Robocycler 96 or RoboCycler Gradient 96 thermal cycler (Stratagene, La Jolla, California, USA). PCR
cycles were as follows: (1) initial denaturation at 94C for 4 min; (2) 35 cycles
of denaturation at 94C for 1 min, primer annealing at 5054C for 1 min, and
primer extension for 1 min 30 s at 72C; and (3) final elongation for 7 min at
72C. PCR products were purified by the PEG precipitation protocol (Johnson
and Soltis, 1995). Alternatively, amplification products were cleaned by adding
3 L of a solution containing 0.2 L each of Antarctic phosphatase and exonuclease I (New England Biolabs, Ipswich, Massachusetts, USA) and incubating
for 30 min at 37C then for 20 min at 80C. Cycle sequencing was carried out
with ABI Prism Big Dye Terminator solution (Applied Biosystems, Foster
City, California, USA) using reactions half the volume recommended by the
manufacturer. Internal sequencing primers were designed for the rpl14rps8
infArpl36 region (rps8F: 5GYR AGA AAA CAT CAA GAA AGA AA3;
rps8R: 5TCC CGA TCH GTC ATT ATA CC3) and atpIatpH intergenic
spacer (atpIF1: 5ATG GRC RGT TTA CGT TAT GGA3). Products were
cleaned using Sephadex G-50 columns (GE Healthcare, Anaheim, California,
USA) and read on an ABI Prism automated sequencer 3130xl (Applied Biosystems, Foster City, California, USA). Sequences were contiged and edited using
the program Sequencher 4.2.2 (Genes Codes Corp., Ann Arbor, Michigan,
USA) and deposited in GenBank.
Sequence alignment and phylogenetic analysesDNA sequences were
aligned using the program MUSCLE version 3.7 (Edgar, 2004), followed by
manual alignment with the program Se-Al version 2.0a11 (Rambaut, 2002) following methods discussed by Morrison (2006). To assess congruence among
genetic markers, we performed the incongruence length difference test (ILD;
Farris et al., 1994) as implemented in the program PAUP* version 4.0b10
(Swofford, 2002), with 1000 replicates and 10 random addition sequences. The
ILD test results indicated that rpl14rps8infArpl36 was incongruent with
atpIatpH and the ndhA intron (P = 0.014 and P = 0.001, respectively, thus
rejecting the null hypothesis of congruent data at the 0.05 confidence level).
Therefore, each marker was analyzed separately to assess incongruence in tree
topology. Phylogenetic analyses of individual markers yielded incongruent relationships among families of Cactineae, although with low nodal support
(<75% bootstrap; <0.95 posterior probability); thus, a data matrix including
combined sequences of the three regions was prepared (archived in TreeBASE,
study accession S10818 and matrix accession M6498, http://treebase.org). Individual markers and the combined data matrix were analyzed using maximum
likelihood (ML; Felsenstein, 1973) in the program Garli version 0.951 (Zwickl,
2006) and Bayesian inference under Markov chain Monte Carlo (MCMC; Yang
and Rannala, 1997) in the program MrBayes version 3.1.2 (Ronquist and
Huelsenbeck, 2003). ML analyses used the model of molecular evolution estimated by the program MODELTEST version 3.7 (Posada and Crandall, 1998),
following the recommendation provided by the Akaike information criterion
(AIC; Akaike, 1974). The best-fit model for rpl14rps8infArpl36 was a
transversional model (TVM) plus a gamma-distributed rate variation (G; Yang,
1993); for atpIatpH and ndhA, it was general time reversible (GTR; Tavar,
1986) plus G; and for the combined data set it was GTR plus a proportion of
invariant sites (I; Reeves, 1992). Bayesian analyses were conducted using the
best-fit model of evolution provided by MrModeltest version 2.3 (Nylander,
2004), under the AIC. The model selected for all data sets was GTR + I. Bayesian analyses were run with two replicates of 10 000 000 generations; trees were
saved every 100th generation, unlinking data partitions in the combined data
matrix; log files were visually examined to check convergence between runs,
and the burnin value for obtaining the majority rule consensus tree was set to
ignore the first 25% of the trees to only include trees after stationarity was
reached. Clade support was determined using nonparametric bootstrapping
(Felsenstein, 1985) from 100 ML replicates and Bayesian posterior probabilities (Rannala and Yang, 1996; Li et al., 2000).
The ShimodairaHasegawa (SH; Shimodaira and Hasegawa, 1999) test was
performed to test selected alternative topologies from different analyses. Constraint topologies were prepared in the program MacClade version 4 (Maddison
and Maddison, 2000) and loaded into Garli. Estimated constrained and unconstrained topologies were then loaded into PAUP*, and their likelihood scores
were compared using the SH test with the RELL option.
1829
Estimation of divergence timesEstimation of divergence times was conducted using a series of programs that are part of the Bayesian Evolutionary
Analysis Sampling Trees package (BEAST) version 1.5.1 (Drummond and
Rambaut, 2007). The XML file for BEAST was prepared for the combined data
matrix in the Bayesian Evolutionary Analysis Utility (BEAUti), and by manually assigning the best-fit model of evolution suggested by MODELTEST for
each locus. As the fossil record for the suborder is uncertain (see Chaney [1944]
for a putative cactus fossil from the Eocene that was refuted by Brown [1959];
Muller [1981] and Ravn [1987] cite fossil pollen records for Portulacaceae and
Montiaceae from the upper Miocene to the Pliocene, although Hershkovitz and
Zimmer [2000] question their correct identification), an indirect approach using
estimations of geological events was undertaken. The approach relies on the
dates of geological events in the Hawaiian Islands, as used by other researchers
(e.g., Chacn et al., 2006; VanderWerf et al., 2010). The age of particular islands or groups of islands was taken as the age of Portulaca species endemic to
those islands. However, the results should be viewed with caution, because dating nodes with these volcanic hotspots overlooks the possibility that the selected species existed before the islands on which they presently occur arose
(see Heads, 2005). Portulaca molokiniensis is a narrow endemic found in the
Maui volcanic islands complex (Naughton et al., 1980) of Molokini, Puukoae
Islet, and Kahoolawe (Wagner et al., 1999). The ages of these islands range
from 0.148 to 1.03 Myr according to the KAr method (Naughton et al., 1980;
Sherrod et al., 2003). The closest relative of P. molokinensis is P. howellii, endemic to the Galpagos Islands (Wiggins et al., 1971), thus the divergence between these two species was set at 1.03 0.18 million years ago (Ma) (Naughton
et al., 1980), which is the age of Kahoolawe, the oldest island where P. molokiniensis is found. Calibration using the Hawaiian Islands (P. molokiniensis) was
preferred over the Galpagos (P. howellii) because the latter species is distributed throughout the Galpagos Archipelago (Wiggins et al., 1971), and the ages
of these islands differ greatly (Bailey, 1976), making it difficult to choose one
calibration date. Another Hawaiian endemic is P. sclerocarpa. The node for P.
sclerocarpa and P. villosa was calibrated to 0.43 0.02 Myr (McDougall and
Swanson, 1972), the oldest hypothesized age for the island of Hawaii (Kohala
volcano) where P. sclerocarpa is endemic (Wagner et al., 1999). Although one
Portulaca specimen in Poopoo matches the description of P. sclerocarpa,
Wagner et al. (1999) suggest that it may be a recent dispersal from Hawaii or
that the capsule characteristics in this specimen have converged with P.
sclerocarpa.
To estimate divergence times, we used a relaxed clock (uncorrelated lognormal; Drummond et al., 2006) and a Yule prior on birth rate of new lineages
(Drummond and Rambaut, 2007), enforcing Mirabilis, Rivina, and Sesuvium as
the outgroup. Fourteen independent analyses were run for 10 000 000 generations at Cornell Universitys Computational Biology Service Unit (http://cbsuapps.tc.cornell.edu/beast.aspx), saving every 1000th tree. Trace files were loaded
into the program Tracer version 1.4.1 (Rambaut and Drummond, 2007) looking
for an effective sample size (ESS) >200 for all parameters sampled from the
MCMC. Trees from the 14 independent analyses were combined in the program
LogCombiner, and the resulting tree file was run in the program TreeAnnotator
to summarize tree information in a maximum clade credibility tree (the tree
with the highest product of all the posterior clade probabilities), discarding the
first 14 000 trees. The program FigTree version 1.2.3 (Rambaut, 2009) was used
for visualizing results on divergence dates.
Historical biogeographyAnalysis of potential ancestral distribution areas
for taxa of Cactineae used a Bayesian approach to dispersalvicariance analysis
(DIVA; Ronquist, 1997), following the method of Nylander et al. (2008) as
implemented in the program S-DIVA version 1.5c (Yu et al., 2010), which accounts for uncertainty in the phylogenetic estimate. The BayesDIVA analysis
was done using 1000 random trees after the burn-in period from the BEAST run
and using the topology of the maximum clade credibility tree, allowing the reconstruction of four maximum ancestral areas at each node. Distribution areas
were considered at the continental level. Widespread species were coded as
present in multiple regions, and only the natural distributions were taken into
account (Appendix 1). Because the outgroup is small in this study for biogeographical reconstruction, simulations were run to evaluate the impact of outgroup distribution on the results. The distributions of the outgroup species were
modified to restrict them to the southern hemisphere, where apparently the
Caryophyllales have their origin (Raven and Axelrod, 1974), specifically: (1)
South America, (2) Africa, and (3) both continents.
Carbon isotope ratio tests and leaf anatomyDetermination of photosynthetic pathways in Cactineae was accomplished by 13C data complemented by
leaf anatomy, an approach that has been used by Sage et al. (2007) to discriminate
1830
[Vol. 97
Fig. 1. Bayesian allcompat tree of Cactineae, using a combined data matrix of the rpl14rps8infArpl36 region, atpIatpH intergenic spacer, and
ndhA intron. The ML topology is identical to the Bayesian estimate. p.p. = posterior probability.
C4 from C3 and CAM. Leaf material was used for photosynthetic pathway determination by 13C analysis except for Opuntia vestita, for which leaf material
was not available, and Rhipsalis baccifera, which does not have leaves; therefore, stem material was used for these two species. In addition, leaves were not
available for Portulaca sclerocarpa; however, as explained already, the species
was important for calibrating the phylogeny; thus, it was included only in the
estimation of divergence times. Samples for 13C analysis were prepared for all
species except Portulaca elatior by drying plant material in an oven at ca. 50C
for 24 h, grinding ca. 1 mg of dried sample, and placing it in a 5 9 mm tin
capsule (Costech Analytical Technologies, Valencia, California, USA). Samples were sent to the University of California at Davis Stable Isotope Facility,
which uses a PDZ Europa ANCA-GSL elemental analyzer interfaced to a PDZ
Europa 20-20 isotope ratio mass spectrometer (Sercon, Cheshire, UK). Leaf
material of P. elatior was processed at Washington State University, where it
was dried at 80C for 24 h and analyzed in a EuroVector elemental analyzer
(EuroVector S.p.A., Milan, Italy). The three photosynthetic pathways discriminate in different proportions against the isotope 13C (isotope fractionation;
OLeary, 1988). The following scale was used to identify the photosynthetic
pathway. C3: typically 25 per mil (; OLeary, 1988; Raven et al., 2008;
Guralnick et al., 2008), although it can be as high as 20 due to some CAM
activity (Winter and Holtum, 2002); C4: 10 to 16 (OLeary, 1988; Sage
et al., 2007); CAM: 9 to 20 (OLeary, 1988; Winter and Holtum 2002).
The carbon isotope discrimination ratios were compared to leaf anatomy
(when samples were available), which likewise is predictive of photosynthetic
pathway. This approach was used to more confidently determine the photosyn-
thetic pathway in the samples because 13C values of different photosynthesis

types may overlap (see Winter and Holtum, 2002; Guralnick et al., 2008).
Leaves of 42 of the 54 species in the study were sectioned transversely and
examined. Material of Opuntia vestita and Portulaca sclerocarpa was not available; Rhipsalis baccifera lacks leaves; and leaf sections from nine species proved
to be inadequate for study. The central portion of mature leaves was cut into
small segments ca. 5 mm long and fixed and stored in a solution of formalin
propionic acidalcohol (FPA; Ruzin, 1999). When fresh material was not available, leaf samples from herbarium specimens or dried in silica gel for molecular
study were treated in a solution of 10% Aerosol OT or boiled in water for 10 min
for rehydration; however, these samples displayed tissue expansion and were
inadequate for anatomical characterization. The remaining leaf samples were dehydrated and embedded in paraffin via the following steps: 70% ethanol (EtOH),
2 h; 90% EtOH, 2 h; 95% EtOH, 2 h; 100% EtOH with 1% safranin, overnight;
100% EtOH, 2 h; 2 : 1 100% EtOH : xylene, 2 h; 1 : 2 100% EtOH : xylene, 2 h;
xylene, 2 h; xylene, 2 h; 2 : 1 xylene : paraffin oil, 2 h; 1 : 2 xylene : paraffin oil,
2 h; in 58C oven: paraffin step 1, 6 h, and paraffin step 2, 6 h, followed by final
paraffin embedding. Thick sections (10 m) were cut using an American Optical
820 rotary microtome (American Optical, now part of Carl Zeiss Vision, San
Diego, California, USA). The staining schedule, based on Sharman (1943), was as
follows: xylene, 10 min; xylene, 10 min; 1 : 1 100% EtOH : xylene, 5 min; 100%
EtOH, 5 min; 95% EtOH, 2 min; 90% EtOH, 2 min; 70% EtOH, 2 min; 50%
EtOH, 2 min; 30% EtOH, 2 min; H2O, 2 min; 2% aqueous ZnCl2, 1 min; H2O,
5 s; 1 : 25 000 aqueous safranin O, 5 min; H2O, 5 s; 10 g orange G + 25 g tannic
acid + 20 drops HCl + 0.2 g thymol + 500 mL H2O, 1 min; H2O, 5 s; 25 g tannic
November 2010]
Table 1.
Evaluation of alternative hypotheses regarding the placement

of select taxa of the suborder Cactineae. The difference between
the ln likelihood score of the most likely tree and the constrained
topology is reported along with results of the RELL test (Shimodaira
and Hasegawa, 1999).
Hypothesis
Outcome
Halophytum sister to Ceraria +

Portulacaria
Halophytum part of Montiaceae
Halophytum basal within Cactineae
Halophytum sister to Basellaceae
Didiereaceae basal within Cactineae
Cactaceae sister to Portulacaceae +
Talinaceae + Anacampseroteae
Portulacaceae sister to Anacampseroteae
Cannot reject
(Diff ln L = 8.58767, P = 0.189)
Cannot reject
(Diff ln L = 3.77526, P = 0.243)
Cannot reject
(Diff ln L = 0.43672, P = 0.366)
Cannot reject
(Diff ln L = 0.98831, P = 0.421)
Cannot reject
(Diff ln L = 3.21613, P = 0.330)
Cannot reject
(Diff ln L = 6.38097, P = 0.120)
Cannot reject
(Diff ln L = 0.85346, P = 0.318)
acid + 0.2 g thymol + 500 mL H2O, 5 min; H2O, 13 s; 1% aqueous iron alum,
2 min; H2O, 15 s; 30% EtOH, 5 s; 50% EtOH, 5 s; 70% EtOH, 5 s; 90% EtOH,
5 s; 95% EtOH, 5 s; 100% EtOH, 5 s; 100% EtOH, 10 s; 3 : 1 xylene : methyl salicylate, 2 min; xylene, 2 min; xylene, until permanently coverslipped using
Cytoseal (Richard Allan Scientific, Kalamazoo, Minnesota, USA).
Slides were examined with a light microscope and images recorded with a
SPOT digital camera (Diagnostic Instruments, Sterling Heights, Minnesota,
USA). Resulting images were edited in Photoshop CS3 (Adobe Systems, San
Jose, California, USA), specifically for background subtraction and image levels adjustment; scale bars were added to the final image using ImageJ software
(Rasband, 1997). A set of slides is deposited at RSA, and original digital image
files are available upon request from the first author.
C3 leaf anatomy is distinguished by the presence of palisade mesophyll and
usually intercellular spaces between spongy mesophyll cells (Cutler et al.,
2008). Kranz anatomy, which is correlated with C4 photosynthesis (Gutirrez
et al., 1974; Furbank, 1998; Dengler and Nelson, 1999), is characterized by a
sheath of large cells surrounding each vascular bundle (= bundle sheath), each
cell containing large and abundant chloroplasts; outside the bundle sheath is a
layer of mesophyll cells, each cell usually radially elongated (= radiate mesophyll) (Furbank, 1998; Kanai and Edwards, 1999). CAM photosynthesis is correlated with leaves having a thick cuticle, large cell vacuoles, and minimal
intercellular space between mesophyll cells (Cushman, 2001; Nelson et al.,
2005; Nelson and Sage, 2008).
Character evolutionEvolution of photosynthetic pathways was traced using the program Mesquite version 2.72 (Maddison and Maddison, 2009), estimated by ML (Markov k-state 1 parameter model, which corresponds to Lewiss
[2001] Mk model) over the ML tree from the combined data matrix.
RESULTS
Phylogenetic relationships of major clades Aligned
lengths for the loci were: rpl14rps8infArpl36 region, 1351
bp; atpIatpH intergenic spacer, 906 bp; and the ndhA intron,
1395 bp. Sequences were unambiguously aligned except for
rpl14rps8, although exploratory ML analyses using different
alignments of this region yielded the same topology. Analyses
of individual loci showed the suborder to be highly supported
as monophyletic, with a non-ACPT group comprising Basellaceae, Didiereaceae, Hallophytaceae, and Montiaceae, and
a strongly supported ACPT clade. Families of Cactineae were
resolved as monophyletic (except Didiereaceae in rpl14rps8
infArpl36; see Appendix S1 at http://www.amjbot.org/cgi/
content/full/ajb.1000227/DC1) and have moderate (7589%
bootstrap) to strong (90% bootstrap; 0.95 posterior probabil-
1831
ity) support in each phylogeny. However, besides the ACPT

clade, relationships among the families are equivocal and have
low support (Bayesian allcompat trees of each individual
marker are shown in online Appendices S1S3, wherein the topological differences with the ML estimates are indicated). The
ML and Bayesian analyses of the combined data set (3652 bp)
yielded identical topologies (Fig. 1), although relationships
among families have low support. Cactineae are strongly supported, as are all the families except for Didiereaceae, which
has low bootstrap support. Montiaceae are the first diverging
member of the order, and Basellaceae are sister to Didiereaceae.
The combined analysis shows Halophytaceae as sister to the
ACPT clade. Relationships inside the ACPT clade, with Talinaceae basal and Cactaceae sister to Portulacaceae, are weakly
supported. The SH test could not reject alternative hypotheses
of relationships involving placement of Halophytaceae, a basal
position of Didiereaceae, and the relationships within the ACPT
clade (Table 1).
Divergence times A chronogram obtained using BEAST is
shown in Fig. 2 and has an identical topology to the ML tree
and Bayesian allcompat consensus tree of the combined data
matrix, except for the branching pattern among Pereskia aculeata, P. lychnidiflora, and P. sacharosa (Cactaceae). The mean
values for the age of the most recent common ancestor (MRCA)
and the maximum and minimum values for the 95% highest
posterior density interval (HPD) for selected nodes are presented in Table 2. According to the analysis, the age of the suborder is 18.8 (6.733.7) Myr, which corresponds to early
Miocene (IUGS, 2009).
Historical biogeography The optimal reconstruction from
the BayesDIVA analysis showed 47 dispersals. The MRCA of
Cactineae was recovered as widely distributed from South to
North America (69.3% probability; Fig. 2). Although the phylogenetic relationships among the families of Cactineae are not
highly supported, the ancestral distribution for each family except Portulacaceae had high probability values (Fig. 2). Montiaceae are North American in origin, but dispersed to South
America and Australia. Didiereaceae are inferred to have AfricanMadagascan origin. Anacampserotaceae were found to
have originated in the Americas and Cactaceae in South America and the Caribbean region. The rest of the taxa in the suborder have a South American origin (although equivocal in
Portulacaceae), with multiple dispersals to other continents.
Two simulation analyses of species with hypothetical distributions partially supported the origin of Cactineae in the Americas (48.1956.12% probability). The exception was when the
three outgroup species were restricted to South America, which
resulted in an ancestor distributed in the New World, Africa,
and Madagascar (52.09%).
Photosynthetic pathway determination Representative
leaf anatomy micrographs of the examined species are shown in
Figs. 35. Leaf anatomy corresponded to the photosynthetic
pathways suggested by 13C values (Table 3) with some exceptions. Grahamia bracteata (Anacampserotaceae), Quiabentia
verticillata (Cactaceae), and Decarya madagascariensis, Didierea madagascariensis, and D. trolli (Didiereaceae), have
CAM-like 13C values, but the leaves were considered to have
C3 anatomy. Therefore, the photosynthetic pathway for these
species was scored as facultative CAM for character reconstruction. CAM photosynthesis (including facultative CAM)
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[Vol. 97
Fig. 2. Chronogram and biogeographical analysis of Cactineae. Maximum clade credibility tree from a BEAST (Drummond and Rambaut, 2007)
analysis of the combined data matrix. Topology is identical to the trees obtained from ML and Bayesian analyses using MrBayes (Ronquist and Huelsenbeck, 2003), except for the relationships among Pereskia aculeata, P. lychnidiflora, and P. sacharosa (Cactaceae). Dates in millions of years. Arrows indicate calibration points. Information for selected nodes (black boxes) is provided in Table 2. Biogeographical reconstructions are displayed in the form of a
pie chart at each node, representing the probability for each alternative ancestral area derived from the dispersal-vicariance analysis (DIVA; Ronquist,
1997) optimizations over 1000 trees randomly sampled from the BEAST run, as implemented in the program S-DIVA (Yu et al., 2010). Black portions of
the pie charts represent five or more reconstructed ancestral ranges with similar probability values. Letters after each taxon name represent the distribution
of the species.
was inferred only for species of Anacampserotaceae, Cactaceae, and Didiereaceae. Leaf anatomy and 13C values for Portulaca cryptopetala (Portulacaceae) both indicate that it
undergoes C3 photosynthesis (Fig. 4K), unlike all other species
of Portulaca examined, which were C4.
Table 4 shows the 13C values obtained in this study. C3 values ranged between 20.44 and 33.52; for C4, between
10.41 and 15.56; and for CAM, from 15.05 to 19.48
(including facultative CAM taxa).
Evolution of photosynthetic pathways Reconstruction of
the diversification of photosynthetic pathways in Cactineae is
shown in Fig. 6. ML reconstruction recovered the C3 pathway
as ancestral to the suborder. CAM photosynthesis is inferred to
have evolved independently five times, including facultative
CAM in Anacampserotaceae, Cactaceae, and Didiereaceae.
Portulaca is the only member of Cactineae with C4 photosynthesis, with a shift to C3 in P. cryptopetala.
DISCUSSION
Relationships within Cactineae All analyses of the chloroplast data show suborder Cactineae and, in nearly every case,
each of its eight families (Anacampserotaceae, Basellaceae,
Cactaceae, Didiereaceae, Halophytaceae, Montiaceae, Portulacaceae, and Talinaceae) to be monophyletic, most with strong
support. In addition, a clade comprising Anacampserotaceae,
Cactaceae, Portulacaceae, and Talinaceae (ACPT clade) is
strongly supported. However, owing to topological conflict and
low clade support, relationships among the families, except for
the grouping of the ACPT families, are incongruent among
November 2010]
analyses of individual markers (online Appendices S1S3) and

weakly supported in the analysis of the combined data matrix
using both ML and Bayesian reconstruction methods (Fig. 1).
These results are consistent with Hershkovitz and Zimmer
(1997; ITS), Applequist and Wallace (2001; ndhF), Edwards
et al. (2005; combined analysis of phyC, psbAtrnH, trnKmatK,
rbcL, and cox3), Applequist et al. (2006; ndhF), Nyffeler (2007;
combined analysis of matK, ndhF, and nad1), and Nyffeler and
Eggli (2010; combined analysis of matK and ndhF) in which
monophyly of Cactineae, the ACPT group, and all families are
statistically supported, but the relationships among families are
unresolved or have low support.
Different hypotheses of relationships were evaluated with
the SH test, in particular relationships of Halophytaceae and
Didiereaceae, as well as the relationships inside the ACPT
clade, but none could be rejected (Table 1). Montiaceae are the
earliest-diverging lineage within the suborder in analyses of the
combined data matrix, which agrees with Brockington et al.
(2009) based on a combined analysis of the chloroplast inverted
repeat, nine chloroplast and two nuclear regions, and the results
of Nyffeler and Eggli (2010); however, neither of these studies
show statistical support for this relationship. Cunoud et al.
(2002), in their combined rbcL and matK analysis, and Nyffeler
(2007) recovered Basellaceae as basal within Cactineae. The
combined analysis reveals Basellaceae as sister to Didiereaceae,
in agreement with Brockington et al. (2009). Although a sister
relationship of Basellaceae and Didiereaceae has low statistical
support, the relationship between these families is supported by
the presence of a solitary basal ovule in members of both families (except Calyptrotheca Gilg [Didiereaceae], which has up to
six ovules; Nyffeler and Eggli, 2010), a unique trait in Cactineae
and putative synapomorphy. However, Nyffelers (2007) results showed Didiereaceae as sister to the ACPT clade (although
with low support), in agreement with Ogburn and Edwards
(2009), who suggested that Didiereaceae share parallelocytic
leaf stomata, tannin cells, and pericyclic sclereids with the
ACPT clade.
The position of Halophytaceae has been controversial for
a long time. Its single species was described in Aizoaceae
(Spegazzini, 1899), but based on different characters it has been
Table 2.
Estimated ages for the most recent common ancestor (MRCA)

of select taxa of Cactineae, expressed in millions of years. Nodes
labeled in Fig. 2.
95% Highest
posterior density
Node
1
2
3
4
5
6
7
8
9
10
11
12
13
14
MRCA of
Cactineae
Montiaceae
Basellaceae
Didiereaceae
Basellaceae + Didiereaceae
Cactineae except Montiaceae
Halophytaceae + ACPT clade
ACPT clade
Talinaceae
Anacampserotaceae
Anacampserotaceae + Cactaceae +
Portulacaceae
Cactaceae
Portulacaceae
Cactaceae + Portulacaceae
Mean
Lower
Upper
18.8
13
3.8
12.1
14.9
17.6
17.1
15.2
9.1
11.4
14.3
6.7
3.4
0.4
2.4
3.9
6.5
6.1
5.4
2
3.2
5.1
33.7
25.4
9
24.4
28.5
31.9
31.4
27.8
18.3
22.6
26.6
10
9.6
13.9
3.1
3.0
4.9
19.1
18.5
26.5
1833
proposed to be closely related to Chenopodiaceae (Spegazzini,

1902; Eckardt, 1976; Blackwell, 1977; Cronquist, 1981; Rodman
et al., 1984; Rodman, 1990), to Aizoaceae or Phytolaccaceae
(Gibson, 1978), to Amaranthaceae Juss. or Chenopodiaceae
(Skvarla and Nowicke, 1976), and to families of suborder
Cactineae (Behnke, 1994). Molecular studies have confirmed
the family to be a member of Cactineae and have recovered
Halophytaceae as sister to Basellaceae (Savolainen et al.,
2000; Hilu et al., 2003), sister to Basellaceae + Didiereaceae
(Brockington et al., 2009), or sister to the ACPT clade (although
no non-ACPT families were sampled; Mller and Borsch, 2005).
Results here are incongruent but weakly supported among the
different markers and methods, yet the placement of the family
is always among the non-ACPT families. Morphological traits
that may indicate affinities of this family with other taxa of the
suborder include its cube-shaped pollen (Skvarla and Nowicke,
1976), which is shared with Basellaceae. More studies are
needed to further clarify its evolutionary relationships within
Cactineae.
Other molecular studies have recovered the ACPT clade,
usually with moderate to strong statistical support (Hershkovitz
and Zimmer, 1997; Applequist and Wallace, 2001; Edwards et
al., 2005; Applequist et al., 2006; Nyffeler, 2007; Nyffeler and
Eggli, 2010). Interestingly, there are no known morphological
or anatomical synapomorphies for the ACPT clade (Ogburn
and Edwards, 2009). The monophyly of each family is strongly
supported in almost every analysis (except atpIatpH, where
Talinaceae have a bootstrap value of 32% and a 0.73 posterior
probability and Cactaceae have 71% bootstrap support; see online Appendix S2 for the Bayesian tree), but the relationships
lack strong support. Most analyses show Talinaceae as basal
within the ACPT clade, although ML analysis of the ndhA intron recovers Cactaceae as basal (online Appendix S3) in concordance with Hershkovitz and Zimmer (1997). The clade
comprising Anacampserotaceae, Cactaceae, and Portulacaceae
has a potential synapomorphy of nodal trichomes and bristles
(Ogburn and Edwards, 2009). The ML and Bayesian analyses
of the combined data matrix resolved Portulacaceae as sister to
Cactaceae, as in the phyC phylogeny of Edwards et al. (2005)
and in a combined chloroplast, mitochondrial, and nuclear loci
tree of Butterworth and Edwards (2008). Morphological and
anatomical traits that may serve as indicators of relationships
among these three families are homoplastic (Ogburn and
Edwards, 2009); thus further study of the group is clearly needed.
As we have detailed, there have been a number of efforts to
clarify relationships within suborder Cactineae, but considerable uncertainty remains about its evolutionary history. Familial relationships (besides the ACPT grouping) remain uncertain
despite the use of a variety of molecular markers for phylogenetic reconstruction. This uncertainty is associated with short
branch lengths. Internal short branches have been related to
rapid radiations (Whitfield and Lockhart, 2007), where ancestral polymorphisms can persist and lead to incongruence among
loci (Maddison, 1997; Degnan and Rosenberg, 2006, 2009).
Phylogenetic reconstruction involving short branches can also
be influenced by a few homoplastic characters, which are sufficient to mask the signal because relationships are supported
by a limited number of characters (Rokas and Carroll, 2006;
Whitfield and Lockhart, 2007). Because short branch lengths
are manifest in Cactineae phylogenies using different loci
(Hershkovitz and Zimmer, 1997; Applequist et al., 2006; Nyffeler,
2007; Brockington et al., 2009; Fig. 1; Appendices S1S3), it
could be hypothesized that these are due to rapid radiations, as
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[Vol. 97
November 2010]
earlier suggested by Hershkovitz and Zimmer (2000) for Montiaceae (as western American Portulacaceae). The study by
Arakaki et al. (2010a) using low copy nuclear markers (phytochromes B and C) and a number of chloroplast loci is of great
interest because some relationships are apparently being resolved within the suborder, although that work is at a preliminary stage. These results will allow analysis of a supermatrix
comprising additional chloroplast, nuclear, and mitochondrial
data to better understand the relationships among Cactineae,
although preliminary analyses show that missing data may be a
problem in resolving relationships within Cactaceae (Arakaki
et al., 2010a), and this may apply to the suborder as well.
Evolution of photosynthetic pathways in Cactineae Previous studies have shown that all three major photosynthetic
pathwaysC3, C4, and CAMare represented in the suborder
(e.g., Winter, 1979; Sage et al., 1999; Guralnick and Jackson,
2001; Sayed, 2001; Guralnick et al., 2008). In addition, there
are taxa that use more than one photosynthetic pathway in the
same or different organs (e.g., Portulaca and Quiabentia, respectively), or switch pathways depending on the environmental conditions (Koch and Kennedy, 1980, 1982; Ku et al., 1981;
Nobel and Hartsock, 1986; Kraybill and Martin, 1996; Martin
and Wallace, 2000; Guralnick and Jackson, 2001; Guralnick
et al., 2002, 2008). There is evidence of some degree of CAM
activity for all families of Cactineae except Basellaceae and
Halophytaceae (photosynthetic pathway data for the latter family
are here reported for the first time) (e.g., Guralnick and Jackson,
2001; Guralnick et al., 2002, 2008), including facultative
CAM (the plants can switch to CAM or C3 depending on water
availability; they use CAM when water-stressed and C3 photosynthesis when water is abundant; Cushman, 2001) and CAMcycling (during the day, the plants do not completely close their
stomata, and they fix atmospheric CO2; at night, the stomata are
closed and the plants fix respiratory CO2; Cushman, 2001). In
this study, leaf anatomy was important in helping to predict the
photosynthetic pathway with more accuracy than with 13C
data alone, although it is evident that biochemical data are
needed to detect photosynthetic variants. Therefore, the results
presented herein may provide an incomplete view of the distribution of the photosynthetic pathways in the suborder, and further biochemical characterization could yield additional
insights.
Determination of photosynthetic pathways from leaf anatomy alone was challenging in some cases, in particular discriminating C3 from CAM, which was also borne out in previous
studies. Nyananyo (1988) concluded that Portulacaria afra,
Talinopsis frutescens, and Talinum paniculatum undergo C3
photosynthesis based on anatomy, whereas Landrum (2002)
considered them to undergo CAM; here we coded the first species as CAM and the other two as C3 using leaf anatomy.
Nyananyo (1988) also described the leaf anatomy of Portulaca
cryptopetala as C4, while Voznesenskaya et al. (2010) and we
in the present study showed that the species has C3 leaf anatomy. Nelson et al. (2005) proposed a quantitative method for
1835
characterizing CAM activity using leaf anatomical traits, but

their criteria could not predict the correct photosynthetic pathway in all cases. These studies show that predicting photosynthetic pathways based solely on leaf anatomy cannot be achieved
with confidence in some cases. Our primary basis for differentiating C3 leaf anatomy from CAM in this study was the presence of palisade parenchyma, which means a certain degree of
shape differentiation among cells inside the leaf.
For those Cactineae examined in this study, leaf anatomy and
13C values are concordant in most samples, and the predicted
photosynthetic pathways partially agree with previous studies
that employed data besides leaf anatomy alone (Nobel and
Hartsock, 1986; Ziegler, 1996; Martin and Wallace, 2000;
Guralnick et al., 2008). The inconsistencies with other studies represent photosynthetic variants that are difficult to identify with
13C and leaf anatomical data in tandem, such as facultative
CAM and CAM-cycling. In our study, five facultative CAM
taxa were detected, which are distributed in Anacampserotaceae, Cactaceae, and Didiereaceae (Fig. 6). The results for these
species are consistent with other studies (Ziegler, 1996; Guralnick
et al., 2008), except Quiabentia verticillata has been reported
as CAM-cycling (Martin and Wallace, 2000). On the other
hand, Alluaudia ascendens, A. humbertii, and Portulacaria afra
(Didiereaceae) are species considered to be CAM in this study,
but are known to behave as facultative CAM (Kluge and Ting,
1978; Guralnick and Ting, 1987). Other species coded here as
C3 have been found to have some CAM activity as well (Fig. 6;
Rayder and Ting, 1981; Winter and Smith, 1996; Martin and
Wallace, 2000; Guralnick and Jackson, 2001; Guralnick et al.,
2008). C4 13C values for Portulaca species with Kranz anatomy are consistent with reported values for C4 photosynthesis
(10 to 16; OLeary, 1988; Sage et al., 2007). Only P. cryptopetala has leaf anatomy and a 13C value corresponding to C3
photosynthesis; however, the species has been shown to be a
C3C4 intermediate based on biochemical and gas exchange
data (Voznesenskaya et al., 2010).
Uncertainty about relationships within the ACPT clade and
among the non-ACPT families, as well as the widespread occurrence of various degrees of CAM activity within Cactineae,
limit our ability to reconstruct photosynthetic pathway evolution. Despite that, our results show that the C3 pathway is well
distributed in the outgroup and Cactineae (Fig. 6), and other
possible topologies do not seem to affect the inference of multiple origins of CAM photosynthesis. Character reconstruction
analysis recovers the C3 pathway as ancestral for the suborder.
C4, CAM, and facultative CAM are all derived from the C3
pathway, in agreement with the hypothesis that C3 is the ancestral type from which the other pathways evolved (Monson,
1989; Ehleringer and Monson, 1993). However, some CAM
activity has been reported for most of the families in Cactineae
(Anacampserotaceae [Guralnick and Jackson, 2001; Guralnick
et al., 2008], Cactaceae [Rayder and Ting, 1981; Nobel and
Hartsock, 1986; Guralnick and Ting, 1987; Gibson, 1996; Martin
and Wallace, 2000], Didiereaceae [Kluge and Ting, 1978;
Winter and Smith, 1996; Ziegler, 1996; Guralnick and Jackson,
Fig. 3. Light micrographs of transectional leaf anatomy in Cactineae. Photosynthesis type inferred from anatomy is indicated within parentheses. (A)
Alluaudia ascendens (CAM; Didiereaceae). (B) A. humbertii (CAM). (C) Anredera cordifolia (C3; Basellaceae). (D) A. ramosa (C3). (E) Calandrinia
caespitosa (C3; Montiaceae). (F) Calyptridium parryi (C3; Montiaceae). (G) Ceraria namaquensis (C3; Didiereaceae). (H) Claytonia parviflora (C3; Montiaceae). (I) Decarya madagascariensis (C3; Didiereaceae). (J) Didierea madagascariensis (C3; Didiereaceae). (K) D. trolli (C3). (L) Grahamia bracteata
(C3; Anacampserotaceae). (M) Lewisia rediviva (C3; Montiaceae). (N) Maihuenia patagonica (C3; Cactaceae). (O) Mirabilis sanguinea (C3; Nyctaginaceae). PM = palisade mesophyll. Black scale bar = 0.75 mm; gray scale bar = 0.25 mm.
1836
2001; Veste et al., 2001], Montiaceae [Martin et al., 1988; Harris

and Martin, 1991; Guralnick and Jackson, 2001; Guralnick et al.,
2001], Portulacaceae [Koch and Kennedy, 1980, 1982; Kraybill
and Martin, 1996; Guralnick and Jackson, 2001; Guralnick et al.,
2002], and Talinaceae [Herrera et al., 1991; Gerere et al., 1996;
Guralnick and Jackson, 2001]), which may suggest that the
CAM pathway has only one origin within the suborder. More
biochemical studies are needed to determine the extent of the
CAM pathway, which might enhance our understanding of the
origin and age of this photosynthetic type in Cactineae.
The non-ACPT families of Cactineae have C3 and CAM photosynthesis, while the ACPT clade has in addition the C4 pathway. It is interesting to note that all leafy Cactaceae sampled
here (except Opuntia vestita, for which only stem material was
available) have C3 leaf anatomy. This may represent a symplesiomorphy, although the photosynthetic functions of the leaf
may vary (see Nobel and Hartsock, 1986; Martin and Wallace,
2000). The C4 pathway has evolved only once in Cactineae,
specifically in Portulacaceae (Portulaca); the MRCA of the
family is reconstructed as having C4 photosynthesis (Fig. 6),
and the divergence times analysis estimates that it evolved ca.
9.5 Ma (Fig. 2). This is concordant with the hypothesis that
decreasing atmospheric CO2 concentrations from the Oligocene
into the Pliocene were a critical factor for the evolution of C4
photosynthesis (Ehleringer and Monson, 1993; Sage, 2005). A
transition to an intermediate C3C4 pathway from C4 is found in
P. cryptopetala (Voznesenskaya et al., 2010), which can be
classified as type I intermediacy, characterized by an absence of
C4 cycle activity and enhanced reassimilation of photorespired
CO2 (Edwards and Ku, 1987). Fixation of CO2 is accomplished
exclusively by Rubisco, which explains a 13C value within the
C3 range (Sage et al., 2007). Portulaca cryptopetala is distributed in central South America (Bolivia to Argentina and Brazil)
and is generally associated with rivers and streams. What drives
the shift from C4 to C3C4 intermediacy is not understood, although it has been hypothesized that it may represent a physiological response to environmental selection pressure (Duvall
et al., 2003; McKown et al., 2005).
Historical biogeography of Cactineae Our study is the
first to employ calibration points inside Cactineae, using two
endemic Hawaiian Portulaca species, which yields an estimate
of 18.8 (6.733.7) Myr (early Miocene) for the MRCA of the
suborder. This implies a recent radiation in the Caryophyllales,
whose MRCA is estimated at ca. 100 Myr (Wikstrm et al.,
2001, 2004). This calibration approach has been criticized because it overlooks the possibility that the taxon may be older
than the strata to which it is endemic (Heads, 2005), as shown
by Rassmann (1997) in a study of iguanas in the Galpagos Islands. Even more, some researchers contend that calibrating
phylogenies using this method may result in significant (tens of
millions of years) age underestimations (Heads, 2009). Preliminary results by Arakaki et al. (2010b) yield an age of the MRCA
of Cactaceae of ca. 30 Myr when calibrating their phylogeny
with fossils distributed across the angiosperms. On the other
[Vol. 97
hand, Klak et al. (2004) showed that ca. 85% (more than 1500
species) of the diversity of the Aizoaceae originated ca. 5 Ma,
which was hypothesized to be the result of key innovations associated with adaptations to arid environments (e.g., wide-band
tracheids, which are also found in some species of Cactineae;
Mauseth, 2004; Landrum, 2002, 2006). This suggests that late
radiations may not be unusual within Caryophyllales, although
this case involves the intrafamiliar level.
The estimated age of the MRCA of Cactineae from this study
is younger than the age proposed for a putative Cactaceae fossil
record from the Eocene (Chaney, 1944) and Montia-like fossil
pollen from the Late Cretaceous (Montiaceae; Ravn, 1987).
Mullers (1981) list of fossil pollen records includes Montiaceae (Hedlund and Engelhardt, 1970; Martin, 1973) and Portulacaceae (Van Campo, 1976) from the Miocene-Pliocene
period (ca. 64 Myr; IUGS, 2009), which is younger than the
estimated ages of the MRCA of those two families in our study
(13 and 9.6 Myr, respectively). Further studies are required to
clarify the interpretation of the putative fossil pollen record of
taxa of Cactineae, whose pollen may be confused with that of
other families in the Caryophyllales (e.g., Nyctaginaceae, Polygonaceae Juss.; Erdtman, 1952). If these fossil pollen records
can be confidently attributed to the suborder and unequivocally
assigned to families, they can be used to calibrate deeper nodes
of the phylogeny, which may provide more reliable dating and
reduce the 95% HPD intervals recovered in the divergence
times estimate (see Table 2).
Biogeographical reconstruction using the BayesDIVA approach and a limited outgroup sample places the origin of
Cactineae in the Americas. The results of the simulation analyses showed that alternate distribution areas assigned to the outgroup do impact the reconstruction of the ancestral distribution
of the suborder, in particular increasing its range. DIVA optimizations become less reliable as the root node is approached
(Ronquist, 1996) and have the tendency to yield wide distributions that include all analyzed areas, as is the case in one simulation restricting the outgroup to South America. With a larger
outgroup sample, Applequist and Wallace (2001) recovered
Cactineae as South American in origin, but they mentioned a
potential bias due to the wide distribution of outgroup species,
though the suborder is well represented in the southern hemisphere. A more accurate biogeographical reconstruction of the
suborder may be obtained in a Caryophyllales-wide study,
which would place Cactineae farther from the root node.
The recovered dates indicate that taxa of Cactineae were not
present in the Americas until well after the separation of South
America and Africa between 84 Ma and 106 Ma (Pitman et al.,
1993) and after the separation of South America and Antarctica
ca. 45 Ma (Raven and Axelrod, 1974). The proximity of the
latter two continents may have permitted dispersal of some
plants to Australia, but this may have been restricted to cooltemperate-adapted taxa (Raven and Axelrod, 1974). Therefore,
intercontinental disjunctions in Cactineae are better explained
by long-distance dispersal, in agreement with Raven and Axelrod
(1974) and Hershkovitz and Zimmer (1997), and also supported
Montiopsis andicola (C3; Montiaceae). (B) Parakeelya pleiopetala (C3; Montiaceae). (C) Pereskia aculeata (C3; Cactaceae). (D) P. grandifolia (C3). (E) P.
lychnidiflora (C3). (F) P. quisqueyana (C3). (G) P. sacharosa (C3). (H) Phemeranthus multiflorus (C3; Montiaceae). (I) Portulaca amilis (C4; Portulacaceae).
(J) P. bicolor (C4). (K) P. cryptopetala (C3). (L) P. echinosperma (C4). (M) P. elatior (C4). (N) P. guanajuatensis (C4). (O) P. molokiniensis (C4). PM = palisade mesophyll. Black arrows indicate bundle sheaths (with abundant chloroplasts) surrounded by radiate mesophyll, characteristic of Kranz anatomy.
Black scale bar = 0.75 mm; gray scale bar = 0.25 mm.
November 2010]
1837
1838
[Vol. 97
P. pilosa (C4; Portulacaceae). (B) P. umbraticola subsp. lanceolata (C4). (C) Portulacaria afra (CAM; Didiereaceae). (D) Quiabentia verticillata (C3;
Cactaceae). (E) Rivina humilis (C3; Phytolaccaceae). (F) Sesuvium portulacastrum (C3; Aizoaceae). (G) Talinopsis frutescens (C3; Anacampserotaceae).
(H) Talinum caffrum (C3; Talinaceae). (I) T. fruticosum (C3). (J) T. lineare (C3). (K) T. paniculatum (C3). (L) T. polygaloides (C3). PM = palisade mesophyll.
Black arrows indicate bundle sheaths (with abundant chloroplasts) surrounded by radiate mesophyll, characteristic of Kranz anatomy. Black scale bar =
0.75 mm; gray scale bar = 0.25 mm; white scale bar with black background = 0.1 mm.
by the high number of dispersal events inferred from the Bayes

DIVA analysis. It is not clear how ancestral taxa may have dispersed successfully around the world. Dispersal mechanisms
known in the suborder include zoochory (Ridley, 1930; Barthlott and Hunt, 1993; Hershkovitz and Zimmer, 2000), anemochory (Barthlott and Hunt, 1993; Carolin, 1993; Kubitzki, 1993;
Sperling and Bittrich, 1993), hydrochory (Ridley, 1930; Danin

et al., 1978; Barthlott and Hunt, 1993), and voluntary or involuntary dispersal by humans (e.g., weedy species of Portulaca
and Talinum and species of Pereskia escaped from cultivation),
but the exact mechanisms for successful dispersal of these
plants throughout the world are not known.
November 2010]
Table 3.
1839
Photosynthetic pathways inferred from 13C values and leaf anatomy data derived from this study.
Species
Anacampserotaceae
Anacampseros
vulcanensis
Grahamia bracteata
Talinopsis frutescens
Basellaceae
Anredera cordifolia
A. ramosa
Cactaceae
Maihuenia patagonica
Opuntia vestita
Pereskia aculeata
P. grandifolia
P. lychnidiflora
P. quisqueyana
P. sacharosa
Quiabentia verticillata
Rhipsalis baccifera
Didiereaceae
Alluaudia ascendens
A. humbertii
Ceraria namaquensis
Decarya
madagascariensis
Didierea
madagascariensis
D. trollii
Portulacaria afra
Halophytaceae
Halophytum ameghinoi
Montiaceae
Calandrinia caespitosa
Calyptridium parryi
Claytonia parviflora
Lewisia rediviva
13C ()
Pathway
based
on 13C
Leaf
anatomy
Pathway
for sample
24.53
C3
C3
19.48
27.10
CAM
C3
C3
C3
Fac. CAM
C3
30.16
26.31
C3
C3
C3
C3
C3
C3
25.10
16.87
27.56
30.18
24.52
29.97
24.16
16.25
17.08
C3
CAM
C3
C3
C3
C3
C3
CAM
CAM
C3
C3
C3
C3
C3
C3
C3
NA
C3
CAM
C3
C3
C3
C3
C3
Fac. CAM
CAM
17.16
15.05
20.44
15.52
CAM
CAM
C3
CAM
CAM
CAM
C3
C3
CAM
CAM
C3
Fac. CAM
19.37
CAM
C3
Fac. CAM
18.38
18.35
CAM
CAM
C3
CAM
Fac. CAM
CAM
24.75
C3
C3
25.67
25.08
33.52
31.27
C3
C3
C3
C3
C3
C3
C3
C3
C3
C3
C3
C3
Species
Montiopsis andicola
Parakeelya pleiopetala
Phemeranthus multiflorus
Portulacaceae
P. amilis
P. bicolor
P. californica
P. cryptopetala
P. echinosperma
P. elatior
P. guanajuatensis
P. howellii
P. massaica
P. molokiniensis
P. pilosa
P. quadrifida
P. umbraticola subsp.
lanceolata
P. villosa
Talinaceae
Talinum arnottii
T. caffrum
T. fruticosum
T. lineare
T. paniculatum
T. polygaloides
T. tenuissimum
Outgroups
Mirabilis sanguinea
(Nyctaginaceae)
Rivina humilis
(Phytolaccaceae)
Sesuvium portulacastrum
(Aizoaceae)
13C ()
Pathway
based
on 13C
Leaf
anatomy
Pathway
for sample
27.52
27.42
27.39
C3
C3
C3
C3
C3
C3
C3
C3
C3
11.96
13.75
13.15
26.55
10.41
12.74
14.25
12.27
15.21
15.56
14.05
13.03
14.09
C4
C4
C4
C3
C4
C4
C4
C4
C4
C4
C4
C4
C4
C4
C4
C4
C3
C4
C4
C4
C4
C4
C4
C4
C4
C3
C4
C4
C4
C4
C4
C4
C4
C4
C4
15.05
C4
C4
24.96
23.29
29.34
26.28
28.22
26.70
23.69
C3
C3
C3
C3
C3
C3
C3
C3
C3
C3
C3
C3
C3
C3
C3
C3
C3
C3
C3
27.02
C3
C3
C3
29.33
C3
C3
C3
25.62
C3
C3
C3
Notes: Fac. = facultative; NA = Not applicable; = leaf anatomy not available for the sample.
Applequist and Wallace (2001) showed that the place of origin of Montiaceae is uncertain, but it is here recovered as North
America, with a MRCA age of 13 Myr, consistent with Hershkovitz and Zimmers (2000) estimate of 816 Myr. Phemeranthus, a genus with most species in North America and one
disjunct species in Argentina [P. punae (R. E. Fr.) Eggli &
Nyffeler], is basal. Although only seven of the 15 recognized
genera in the family were sampled (Nyffeler and Eggli, 2010),
at least two early, independent long-distance dispersal events to
South America and one to Australia are inferred. Hectorella,
which is not sampled here, is endemic to New Zealand, and according to the phylogeny in Applequist et al. (2006) it may represent another independent long-distance dispersal event to the
islands of the Pacific.
The biogeographical analysis postulates Didiereaceae as AfricanMalagasy in origin (five of seven genera included in this
study; Applequist and Wallace, 2003), with a MRCA age of ca.
12 Myr. Applequist and Wallace (2001) showed that the familys Old World distribution is likely the result of an early longdistance dispersal from South America to the Old World. These
authors (Applequist and Wallace, 2000, 2001) provided evidence that Calyptrotheca, a genus of Didiereaceae (Calyptrothecoideae Pax & Gilg) endemic to east tropical Africa (not
sampled here), is the closest relative to Didiereoideae Appleq.
& R. S. Wallace (including Alluaudia, Decarya, and Didierea
in our study). On the basis of this and the low sequence divergence between the two subfamilies, they concluded that it is
more plausible that the clade was introduced via dispersal from
Africa to Madagascar.
Although only one genus was sampled for Basellaceae, the
analysis shows it originated in South America, in agreement
with Raven and Axelrod (1974), with a MRCA age of ca. 4 Myr
for Anredera. The family has four genera (Nyffeler and Eggli,
2010), but only one with representatives in the Old World,
which suggests a New World origin. Halophytaceae include a
single species from the southern part of Argentina; the age of
the MRCA of the Halophytaceae + ACPT clade is ca. 17 Myr.
Table 4.
13C statistics for Cactineae and outgroup taxa.
Statistic
C3
C4
CAM
30
26.78
2.69
33.52
20.44
13
13.5015
1.44912
15.56
10.41
10
17.35
1.52
19.48
15.05
No. species
Average ()
Standard deviation ()
Minimum ()
Maximum ()
Notes: Photosynthetic pathways were assigned using 13C values and

leaf anatomical data, when available. CAM includes facultative CAM taxa
as scored in this study.
1840
[Vol. 97
Fig. 6. Maximum likelihood (ML) ancestral character reconstruction for photosynthetic pathways in Cactineae. Analysis based on the ML tree from
the combined data matrix. Proportional likelihoods in the form of a pie chart are shown at each node of the ML reconstruction. Superscript capital letters
by some taxa names are references to other studies reporting different photosynthesis pathways than found here. Facultative CAM or CAM-cycling activity:
A = Kluge and Ting, 1978; B = Rayder and Ting, 1981; C = Nobel and Hartsock, 1986; D = Guralnick and Ting, 1987; E = Herrera et al., 1991; F = Gerere
et al., 1996; G = Kraybill and Martin, 1996; H = Winter and Smith, 1996; I = Ziegler, 1996; J = Martin and Wallace, 2000; K = Guralnick and Jackson,
2001; L = Guralnick et al., 2008. C3-C4 intermediate: M = Voznesenskaya et al., 2010. *As Talinum triangulare (Jacq.) Willd.; **as Quiabentia chacoensis
Backeb.; ***as Portulaca mundula I. M. Johnst.
November 2010]
The ACPT clade, which has high statistical support in almost

all analyses (Fig. 1; Appendices S1S3), has its origin in South
America, in agreement with Applequist and Wallace (2001),
with a MRCA date of ca. 15 Myr. The clade has Talinaceae as
basal with a MRCA age of ca. 9 Myr. Applequist and Wallaces
analysis (2001) showed the place of origin of the family as
equivocal, while here it is recovered as South America. The
family comprises three genera (one sampled here; Nyffeler and
Eggli, 2010) and has been very successful in terms of dispersal,
colonizing almost all continents. The early-divergent clade
comprising T. fruticosum and T. paniculatum is widely distributed in the Americas and is naturalized in the Old World (e.g.,
Phillips, 2002; Dequan and Gilbert, 2003). The analysis shows
that T. lineare, a species endemic to the North American
deserts, is sister to the South American T. polygaloides and is
derived from a vicariance event involving a widely distributed
ancestor. It is also interesting that the African taxa form a clade,
suggesting a single dispersal event to the continent.
The origin of Anacampserotaceae is shown to be in the
Americas, with the MRCA age of ca. 11 Myr. The analysis,
which includes all three genera recognized in the family
(Nyffeler and Eggli, 2010), shows an early vicariance event,
resulting in the North American endemic Talinopsis frutescens
and the South American Anacampseros vulcanensis and Grahamia bracteata. Other members of the family, such as the Old
World Anacampseros, as well as the Australian endemic A.
australiana J. M. Black, are derived taxa of Anacampserotaceae (Nyffeler, 2007); thus, it can be hypothesized that they are
result of long-distance dispersals from South America.
Our results show that the MRCA of Cactaceae was distributed in South America and the Caribbean region, but other studies have suggested an exclusively South American origin (e.g.,
Hershkovitz and Zimmer, 1997; Applequist and Wallace, 2001;
Edwards et al., 2005; Nyffeler, 2007). Croizat (1952) commented on a possible Old World origin of Cactaceae because of
the presence of Rhipsalis in tropical Africa, Madagascar, and
Sri Lanka. Results from our analysis (five of 126 recognized
genera sampled; Anderson, 2001) are in concordance with others (e.g., Nyffeler, 2002; Wallace and Gibson, 2002; Crozier,
2005), in which the genus is derived and likely its distribution
is the result of long-distance dispersal, probably via birds, as
suggested by Barthlott and Hunt (1993). On the other hand,
there is a controversy about the age of the family. Croizat (1952)
proposed the origin of Cactaceae before the separation of Africa and South America in the early Cretaceous, while others
suggest it was soon after the separation of the continents ca. 100
Ma (Mauseth, 1990; Leuenberger, 1986; Wallace and Gibson,
2002) to explain the absence of cacti in the Old World (with the
exception of Rhipsalis). These dates imply that the age of the
family is similar to, or even older, than the estimated age of
the order that it belongs to [Caryophyllales, with an estimated
MRCA of 11185 Myr (Wikstrm et al., 2001, 2004)]. Hershkovitz and Zimmer (1997) suggested a mid-Tertiary (Oligocene) origin ca. 30 Ma for Cactaceae, which has been adopted
by Nyffeler (2002) and Edwards et al. (2005), and supported by
the preliminary results of Arakaki et al. (2010b). In our study,
an age of ca. 10 (3.119.1) Myr for the MRCA of Cactaceae
supports Hershkovitz and Zimmers (1997) hypothesis that the
family is relatively young. Leuenberger (1986) speculated a
probable origin of the family in northwestern South America in
the late Cretaceous, distant from the closest point where the
continent was in contact with Africa (presently northeast Brazil
and Gabon, respectively; Raven and Axelrod, 1974); the author
1841
conjectured that this location would explain the near absence of

Cactaceae in the Old World and would facilitate the early dispersal of the leafy Pereskia and other subfamilies to North
America and the Caribbean. In contrast, Wallace and Gibson
(2002) hypothesized that the family originated in the central
Andes based on the presence there of early-diverging lineages.
Portulacaceae, reduced to the single genus Portulaca, have an
uncertain place of origin in the BayesDIVA analysis, though restricted to southern hemisphere continents, with a MRCA age of
ca. 9.5 Myr. Preliminary divergence dates analysis using a wider
sampling of the family and a different set of molecular markers
(except for the ndhA intron) estimates an older age for the MRCA
of Portulacaceae (ca. 15 Myr; G. Ocampo and J. T. Columbus,
unpublished data). Differences in divergence estimates have been
observed using different loci for the same set of samples (e.g.,
Rodrguez-Trelles et al., 2004), attributable to differences in rates
of evolution of the loci; in addition, it has been shown that increased
taxon sampling may produce older estimates (Pirie et al., 2005).
Therefore, results obtained in each study should be considered
with caution, although in general terms it can be concluded that the
MRCA of the family coincided with the Miocene. Results indicate
an early split of the genus into an Old WorldAustralian clade (P.
bicolor and P. quadrifida) with opposite leaves and a primarily
New World clade with alternate to subopposite leaves. The biogeographical analysis is not clear about the origin of Portulaca, but
Applequist and Wallace (2001) indicate that it is of South American origin and has successfully colonized all continents except
Antarctica. In a separate study with a wider sampling of the genus
(G. Ocampo and J. T. Columbus, unpublished data), it is evident
that taxa of different subclades have independently dispersed to
other continents (e.g., Africa, Australia) and to the islands of the
Pacific Ocean (e.g., Galpagos, Hawaiian Islands) from South and
North America. Although there is no clear dispersal mechanism,
there is some evidence of long-distance dispersal via birds (Ridley,
1930), by floating across bodies of water (Danin et al., 1978), and
by tropical storms (Matthews et al., 1991). Like the other families
of Cactineae, more studies are desirable to better understand the
distribution of Portulaca.
This study adds to the knowledge of the evolution of Cactineae,
but a strongly supported hypothesis of relationships within the
group remains elusive. Phylogenetic studies of the suborder have
yielded incongruent gene trees, in particular within the ACPT and
non-ACPT clades, likely owing to short branches that obscure evolutionary relationships, including when loci are analyzed in combination (Degnan and Rosenberg, 2006). Although the branching
pattern is not clearly known, it seems plausible that the short
branches may represent rapid radiations as a response of the organisms to increased aridity. These processes may have occurred above
all in the species of the ACPT clade, which is South American in
origin and whose MRCA coincides with an arid environment already present across the central Andes (Hartley, 2003). Populationlevel studies and complete chloroplast genome sequence analysis
may contribute to a better understanding of the evolutionary history of the suborder by providing greater insights into adaptations
to aridity, as well as the mechanisms that triggered those adaptations in this charismatic lineage of flowering plants.
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1846
[Vol. 97
Appendix 1. Taxon name, voucher information, source of sample material.
Taxon
Anacampserotaceae Eggli & Nyffeler
Anacampseros vulcanensis An
Grahamia bracteata Gill. ex Hook. & Arn.
Talinopsis frutescens A. Gray
Basellaceae Raf.
Anredera cordifolia (Ten.) Steenis
A. ramosa (Moq.) Eliasson
Cactaceae Juss.
Maihuenia patagonica (Phil.) Britton & Rose
Opuntia vestita Salm-Dyck
Pereskia aculeata Mill.
P. grandifolia Haw.
Collection number (herbarium)

Leuenberger 3534 (ZSS)
Ocampo et al. 1665 (RSA, SI)
Ocampo 1480 (RSA)
Argentina; cultivated at ZSS

Argentina
Mexico
Ocampo 1781cv (RSA)
South America; cultivated at University of

California, Davis
Mexico
Ocampo & Morales 1459 (RSA)

Leuenberger et al. 4091 (ZSS)
Swoda 169 (ZSS)
NA (ZSS)
UCBerk54006 (living collection)
P. lychnidiflora DC.
P. quisqueyana Alain
P. sacharosa Griseb.
Ocampo 1775cv (RSA)
Quiabentia verticillata Borg
Accession HBG65934 (HNT)
Rhipsalis baccifera (J. S. Muell.) Stearn

Didiereaceae Radlk.
Alluaudia ascendens (Drake) Drake
A. humbertii Choux
SC-380-03 (RSA)
Ocampo 1777cv (RSA)
Ceraria namaquensis Pears. & Steph.
Ocampo 1774cv (RSA)
Decarya madagascariensis Choux
Ocampo 1778cv (RSA)
Didierea madagascariensis Baill.
Ocampo 1780cv (RSA)
D. trollii Capuron & Rauh
Ocampo 1779cv (RSA)
Portulacaria afra Jacq.

Halophytaceae A. Soriano
Halophytum ameghinoi (Speg.) Speg.
Montiaceae Raf.
Calandrinia caespitosa Gill. ex Arn.
Calyptridium parryi A. Gray
Claytonia parviflora Douglas ex Hook.
Lewisia rediviva Pursh
Montiopsis andicola (Gill. ex Hook. & Arn.) D. I. Ford
Parakeelya pleiopetala (F. Muell.) Hershkovitz
Phemeranthus multiflorus (Rose & Standl.) Ocampo
Portulacaceae Juss.
Portulaca amilis Speg.
P. bicolor F. Muell.
P. californica D. Legrand
P. cryptopetala Speg.
P. echinosperma Hauman
P. elatior Mart. ex Rohrb.
P. guanajuatensis Ocampo
P. howellii (D. Legrand) Eliasson
P. massaica S. M. Phillips
P. molokiniensis R. W. Hobdy
P. pilosa L.
P. quadrifida L.
P. sclerocarpa A. Gray a
P. umbraticola Kunth subsp. lanceolata J. F. Matthews & Ketron
P. villosa Cham.
Talinaceae Doweld
Talinum arnottii Hook. f.
T. caffrum Eckl. & Zeyh.
T. fruticosum (L.) Juss.
T. lineare Kunth
Provenance; if cultivated, the source is

indicated.
Ocampo 1433 (RSA)
Argentina; cultivated at ZSS

Bolivia; cultivated at ZSS
Americas; cultivated at ZSS
Brazil; cultivated at University of California,
Berkeley
Costa Rica; cultivated at University of
California, Berkeley
Dominican Republic; cultivated at University of
California, Berkeley
South America; cultivated at University of
California, Davis
Brazil; cultivated at the Huntington Botanical
Gardens
Mexico
Ocampo 1714cv (RSA)
Madagascar; cultivated at Smith College

Madagascar; cultivated at University of
California, Davis
Africa; cultivated at University of California,
Davis
California, Davis
California, Davis
California, Davis
Africa; cultivated in Mexico
Porter & Machen 14987 (RSA)
Argentina

Fraga 2010 (RSA)
Ocampo 1716cv (RSA)
Ocampo 1533cv (RSA)
Ocampo et al. 1760 (BRI, RSA)
Argentina
USA
USA; cultivated at RSA
USA; cultivated in Portland
Argentina
Australia
Mexico

Ocampo et al. 1753 (BRI, RSA)
Ocampo & Columbus 1529 (RSA)
Ocampo 1708cv (RSA)
Ocampo 1482 (RSA)
Jaramillo 3332 (CDS)
Cruse-Sanders s.n. (RSA)
Perlman 12643 (RSA)
Nortrup s.n. (UNCC)
Cruse-Sanders s.n. (RSA)
Morden 1828 (HAW)
Perlman 13305 (PTBG)
Argentina
Australia
Mexico
Argentina
Argentina
Caribbean; cultivated at the Huntington
Botanical Gardens
Mexico
Galpagos Islands
Tanzania
Hawaii
USA
Tanzania
Hawaii
Mexico
Hawaii
Bartsch et al. SB70 (PRE)

Zietsman 4050 (PRE)
Ocampo 1463 (RSA)
Namibia
South Africa
Mexico
Mexico
November 2010]
Appendix 1.
1847
Continued.
Taxon
T. paniculatum (Jacq.) Gaertn.
T. polygaloides Gillies ex Arn.
T. tenuissimum Dinter
Outgroups
Mirabilis sanguinea Heimerl (Nyctaginaceae Juss.)
Rivina humilis L. (Phytolaccaceae R. Br.)
Sesuvium portulacastrum (L.) L. (Aizoaceae Martinov)
Collection number (herbarium)
Provenance; if cultivated, the source is

indicated.

Dreyer 476 (PRE)
Mexico
Argentina
Namibia
Ocampo & Prez-Calix 1467 (RSA)

Ocampo 1426 (RSA)
Mexico
Mexico
Mexico
Used only for calibration purposes; 13C and leaf anatomy data not included.
Notes: NA = Not available. Herbaria acronyms: BRI = Queensland Herbarium, Brisbane, Queensland, Australia; CDS = Charles Darwin Research Station,
Puerto Ayora, Ecuador; HAW = University of Hawaii, Honolulu, Hawaii, USA; HNT = Huntington Botanical Gardens, San Marino, California, USA;
PRE = South African National Biodiversity Institute (SANBI), Pretoria, Gauteng Province, South Africa; PTBG = National Tropical Botanical Garden,
Kalaheo, Hawaii, USA; RSA = Rancho Santa Ana Botanic Garden, Claremont, California, USA; SI = Museo Botnico, San Isidro, Buenos Aires,
Argentina; UNCC = Reedy Creek Park and Nature Preserve, Charlotte, North Carolina, USA; ZSS = Sukkulenten-Sammlung Zrich, Switzerland.
Appendix 2. GenBank accessions (atpIatpH, ndhA intron, rpl14rps8infArpl36) for the species used in this study. Taxa are listed in alphabetical order by genus
and species.
Taxon; GenBank accession: atpIatpH, ndhA intron, rpl14rps8infArpl36.
Alluaudia ascendens; HQ241623, HQ241569, HQ241677. Alluaudia
humbertii; HQ241624, HQ241570, HQ241678. Anacampseros
vulcanensis; HQ241676, HQ241622, HQ241730. Anredera cordifolia;
HQ241625, HQ241571, HQ241679. Anredera ramosa; HQ241626,
HQ241572, HQ241680. Calandrinia caespitosa; HQ241627,
HQ241573, HQ241681. Calyptridium parryi; HQ241628, HQ241574,
HQ241682. Ceraria namaquensis; HQ241629, HQ241575, HQ241683.
Claytonia parviflora; HQ241630, HQ241576, HQ241684. Decarya
madagascariensis; HQ241631, HQ241577, HQ241685. Didierea
madagascariensis; HQ241632, HQ241578, HQ241686. Didierea
trollii; HQ241633, HQ241579, HQ241687. Grahamia bracteata;
HQ241634, HQ241580, HQ241688. Halophytum ameghinoi;
HQ241675, HQ241621, HQ241729. Lewisia rediviva; HQ241635,
HQ241581, HQ241689. Maihuenia patagonica; HQ241636, HQ241582,
HQ241690. Mirabilis sanguinea; HQ241637, HQ241583, HQ241691.
Montiopsis andicola; HQ241638, HQ241584, HQ241692. Opuntia
vestita; HQ241639, HQ241585, HQ241693. Parakeelya pleiopetala;
HQ241640, HQ241586, HQ241694. Pereskia aculeata; HQ241641,
HQ241587, HQ241695. Pereskia grandifolia; HQ241642, HQ241588,
HQ241696. Pereskia lychnidiflora; HQ241643, HQ241589, HQ241697.
Pereskia quisqueyana; HQ241644, HQ241590, HQ241698. Pereskia
sacharosa; HQ241645, HQ241591, HQ241699. Phemeranthus
multiflorus; HQ241646, HQ241592, HQ241700. Portulaca amilis;
HQ241647, HQ241593, HQ241701. Portulaca bicolor; HQ241648,

HQ241594, HQ241702. Portulaca californica; HQ241649, HQ241595,
HQ241703. Portulaca cryptopetala; HQ241650, HQ241596, HQ241704.
Portulaca echinosperma; HQ241651, HQ241597, HQ241705. Portulaca
elatior; HQ241652, HQ241598, HQ241706. Portulaca guanajuatensis;
HQ241653, HQ241599, HQ241707. Portulaca howellii; HQ241655,
HQ241601, HQ241709. Portulaca massaica; HQ241654, HQ241600,
HQ241708. Portulaca molokiniensis; HQ241656, HQ241602,
HQ241710. Portulaca pilosa; HQ241657, HQ241603, HQ241711.
Portulaca quadrifida; HQ241658, HQ241604, HQ241712. Portulaca
sclerocarpa; HQ241661, HQ241607, HQ241715. Portulaca umbraticola
subsp. lanceolata; HQ241659, HQ241605, HQ241713. Portulaca villosa;
HQ241660, HQ241606, HQ241714. Portulacaria afra; HQ241662,
HQ241608, HQ241716. Quiabentia verticillata; HQ241663, HQ241609,
HQ241717. Rhipsalis baccifera; HQ241664, HQ241610, HQ241718.
Rivina humilis; HQ241665, HQ241611, HQ241719. Sesuvium
portulacastrum; HQ241666, HQ241612, HQ241720. Talinopsis
frutescens; HQ241667, HQ241613, HQ241721. Talinum arnottii;
HQ241668, HQ241614, HQ241722. Talinum caffrum; HQ241669,
HQ241615, HQ241723. Talinum fruticosum; HQ241670, HQ241616,
HQ241724. Talinum lineare; HQ241671, HQ241617, HQ241725.
Talinum paniculatum; HQ241672, HQ241618, HQ241726. Talinum
polygaloides; HQ241673, HQ241619, HQ241727. Talinum tenuissimum;
HQ241674, HQ241620, HQ241728.

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American Journal of Botany 97(11): 18271847. 2010.

MOLECULAR PHYLOGENETICS OF SUBORDER CACTINEAE

Gilberto Ocampo2 and J. Travis Columbus

The suborder Cactineae of order Caryophyllales (sensu

Manuscript received 22 June 2010; revision accepted 14 September 2010.

lacaceae (Thorne and Reveal, 2007). Recently, it has been

American Journal of Botany

have low support values. Other relationships within Cactineae

Ocampo and Columbus Phylogenetics of Cactineae

American Journal of Botany

thetic pathway in the samples because 13C values of different photosynthesis

Ocampo and Columbus Phylogenetics of Cactineae

Evaluation of alternative hypotheses regarding the placement

Halophytum sister to Ceraria +

ity) support in each phylogeny. However, besides the ACPT

American Journal of Botany

Ocampo and Columbus Phylogenetics of Cactineae

analyses of individual markers (online Appendices S1S3) and

Estimated ages for the most recent common ancestor (MRCA)

proposed to be closely related to Chenopodiaceae (Spegazzini,

American Journal of Botany

Ocampo and Columbus Phylogenetics of Cactineae

characterizing CAM activity using leaf anatomical traits, but

American Journal of Botany

2001; Veste et al., 2001], Montiaceae [Martin et al., 1988; Harris

Ocampo and Columbus Phylogenetics of Cactineae

American Journal of Botany

by the high number of dispersal events inferred from the Bayes

Sperling and Bittrich, 1993), hydrochory (Ridley, 1930; Danin

Ocampo and Columbus Phylogenetics of Cactineae

13C statistics for Cactineae and outgroup taxa.

Notes: Photosynthetic pathways were assigned using 13C values and

American Journal of Botany

Ocampo and Columbus Phylogenetics of Cactineae

The ACPT clade, which has high statistical support in almost

conjectured that this location would explain the near absence of

American Journal of Botany

Applequist, W. L., and R. S. Wallace. 2000. Phylogeny of the

Cutler, D. F., T. Botha, and D. W. Stevenson. 2008. Plant anatomy:

Ocampo and Columbus Phylogenetics of Cactineae

Guralnick, L. J., A. Cline, M. Smith, and R. F. Sage. 2008. Evolutionary

American Journal of Botany

Morrison, D. A. 2006. Multiple sequence alignment for phylogenetic

Rambaut, A. 2009. FigTree version 1.2.3 [computer program]. Website

Ocampo and Columbus Phylogenetics of Cactineae

Savolainen, V., M. F. Fay, D. C. Albach, A. Backlund, M. van der

C4 photosynthesis and a C3C4 intermediate in genus Portulaca L.

American Journal of Botany

Appendix 1. Taxon name, voucher information, source of sample material.

Collection number (herbarium)

Argentina; cultivated at ZSS

Ocampo 1781cv (RSA)

South America; cultivated at University of

Ocampo & Morales 1459 (RSA)

UCBerk902365 (living collection)

UCBerk850030 (living collection)

Ocampo 1775cv (RSA)

Quiabentia verticillata Borg

Accession HBG65934 (HNT)

Rhipsalis baccifera (J. S. Muell.) Stearn

Ceraria namaquensis Pears. & Steph.

Ocampo 1774cv (RSA)

Decarya madagascariensis Choux