Professional Documents
Culture Documents
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
American Journal of Botany 97(11): 18271847, 2010; http://www.amjbot.org/ 2010 Botanical Society of America
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[Vol. 97
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]
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
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[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-
November 2010]
Table 1.
Hypothesis
Outcome
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-
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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]
Table 2.
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
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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
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.
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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
Fig. 4. Light micrographs of transectional leaf anatomy in Cactineae. Photosynthesis type inferred from anatomy is indicated within parentheses. (A)
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
Fig. 5. Light micrographs of transectional leaf anatomy in Cactineae. Photosynthesis type inferred from anatomy is indicated within parentheses. (A)
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.
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.
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 ()
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]
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[Vol. 97
November 2010]
1843
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1844
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1845
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[Vol. 97
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.
P. lychnidiflora DC.
P. quisqueyana Alain
P. sacharosa Griseb.
SC-380-03 (RSA)
Ocampo 1777cv (RSA)
Argentina
Argentina
USA
USA; cultivated at RSA
USA; cultivated in Portland
Argentina
Australia
Mexico
Argentina
Australia
Mexico
Argentina
Argentina
Caribbean; cultivated at the Huntington
Botanical Gardens
Mexico
Galpagos Islands
Tanzania
Hawaii
USA
Tanzania
Hawaii
Mexico
Hawaii
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)
Mexico
Argentina
Namibia
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;