1827.fullmolecular phylogenetics of suborder cactineae (caryophyllales), including insights into...

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

American Journal of Botany 97(11): 18271847, 2010; http://www.amjbot.org/ 2010 Botanical Society of America

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 Ba-sellaceae, Cactaceae, Didiereaceae, Halophytaceae, and Portu-

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 Portu-lacaceae ( 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 ; M ller and Borsch, 2005 ; Applequist et al., 2006 ; Nyffeler, 2007 ; Brockington et al., 2009 ; Nyffeler and Eggli, 2010 ) and have led to a new classifi cation of families, particularly involving members of Portulacaceae s.l. Hershkovitz and Zimmer (1997) , using ribo-somal 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 relation-ships were confi rmed later by Applequist and Wallace (2001) using sequences from the chloroplast gene ndhF , which led to the transfer of Ceraria and Portulacaria , along with Calyp-trotheca , to Didiereaceae ( Applequist and Wallace, 2003 ). In addition, these authors uncovered a clade that grouped Cacta-ceae with Talinum + Talinella , Portulaca , and tribe Anacamp-seroteae of Portulacaceae, although without internal resolution. Subsequent studies using other chloroplast and mitochondrial regions ( Applequist et al., 2006 ; Nyffeler, 2007 ) have recov-ered this monophyletic group, known as the ACPT clade ( Nyffeler, 2007 ), with strong support, but internal relationships

1 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 fi nanced by Rancho Santa Ana Botanic Garden and the Botanical Society of America. Financial support to G. O. was provided by The Fletcher Jones Foundation, Comisi n Nacional de Ciencia y Tecnolog a (Mexico), Fundaci n Prywer (Mexico), and the Instituto de Ecolog a, A. C. (Mexico).

2 Author for correspondence (e-mail: [email protected]);

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

MOLECULAR PHYLOGENETICS OF SUBORDER CACTINEAE (CARYOPHYLLALES), INCLUDING INSIGHTS INTO

PHOTOSYNTHETIC DIVERSIFICATION AND HISTORICAL BIOGEOGRAPHY 1

Gilberto Ocampo 2 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 (= Portu-lacineae) of the Caryophyllales. In addition, photosynthesis diversifi cation and historical biogeography were addressed.

Methods: Chloroplast DNA sequences, mostly noncoding, were used to estimate the phylogeny. Divergence times were cali-brated using two Hawaiian Portulaca species, due to the lack of an unequivocal fossil record for Cactineae. Photosynthetic pathways were determined from carbon isotope ratios ( 13 C) 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 mono-phyletic; however, relationships among families remain uncertain. The age of Cactineae was estimated to be 18.8 Myr. Leaf anatomy and 13 C and were congruent in most cases, and inconsistencies between these pointed to photosynthetic intermedi-ates. Reconstruction of photosynthesis diversifi cation showed C 3 to be the ancestral pathway, a shift to C 4 in Portulacaceae, and fi ve independent origins of Crassulacean acid metabolism (CAM). Cactineae were inferred to have originated in the New World.

Conclusions: Although the C 3 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 in-creasing aridity in the Miocene.

Key words: C 4 photosynthesis; Cactineae; Cactaceae; Caryophyllales; Crassulacean acid metabolism divergence times; his-torical biogeography; photosynthesis diversifi cation; Portulacaceae; Portulacineae.

1828 American Journal of Botany [Vol. 97

criminates less against the 13 C isotope than Rubisco ( Lajtha and Marshall, 1994 ; Winter and Holtum, 2002 ); thus, carbon iso-tope ratios ( 13 C) can be used to distinguish plants that have C 4 and CAM photosynthesis from those that use the C 3 pathway. Although the use of 13 C can serve as the fi rst step in determin-ing the photosynthetic pathway in a group, additional evidence may be needed to discriminate C 4 from CAM photosynthesis, because their 13 C values overlap ( O Leary, 1988 ; Winter and Holtum, 2002 ; Sage et al., 2007 ), or to detect photosynthetic intermediates (e.g., C 3 -C 4 ; 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 ro-bust phylogenetic estimate of relationships within Cactineae by using different, noncoding chloroplast markers from those used in previous studies. Specifi cally, the rpl14 rps8 infA rpl36 re-gion, atpI atpH intergenic spacer, and ndhA intron ( Shaw et al., 2007 ) were used to explore their utility for resolving relation-ships among the families of Cactineae. Using the phylogenetic estimate, we studied the diversifi cation of the photosynthetic pathway (inferred from 13 C values and leaf anatomy) and his-torical 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 refer-ence the age of specifi c 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 sampling Fifty-one species were sampled from all families recog-nized 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 Phy-tolaccaceae 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 ; Cu noud 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 sequencing Sources 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 ge-nomic DNA was extracted from 10 mg of dried material or 20 mg of fresh tissue using the modifi ed 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 suffi cient for performing the polymerase chain reaction (PCR) with the selected markers, although the DNeasy kits out-performed the CTAB method when samples were highly mucilaginous. DNA extracted using the CTAB method was quantifi ed 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 superma-trix approach does not improve internal nodal support within the suborder. Therefore, we conducted a study using the chloroplast rpl14 rps8 infA rpl36 region (comprising coding and spacer sequences), atpI atpH intergenic spacer,

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 phyloge-netic studies to be part of Cactineae ( Cu noud et al., 2002 ; M ller 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 hemi-sphere, although Portulaca has a worldwide distribution, mainly in tropical and subtropical regions of both hemispheres. Cacta-ceae 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 unam-biguous 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 esti-mated to be 8 16 Myr (as western American Portulacaceae; Hershkovitz and Zimmer, 2000 ), but to date there are no hy-potheses of the age of the suborder, which impedes understand-ing of its evolution and historical biogeography.

A fascinating aspect of evolution within Cactineae is adapta-tion to xeric habitats, especially in plant morphology but also extending to the physiological level in terms of photosynthetic pathways. All three primary photosynthetic variants C 3 , C 4 , 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 C 3 plants, 1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the reaction where 1,5-bisphosphate (RuBP) reacts with atmospheric CO 2 as the fi rst step of the photosyn-thetic reaction ( Ehleringer and Monson, 1993 ). Atmospheric O 2 and CO 2 are competitive substrates for Rubisco, but the enzyme has more specifi city for the latter. However, the concentration of CO 2 is signifi cantly reduced as it diffuses from the atmo-sphere into the photosynthetic tissue, favoring the oxygenation of RuBP. This condition, in addition to an eventual liberation of the CO 2 , is termed photorespiration, which reduces the overall photosynthetic effi ciency ca. 33% in C 3 plants ( Ehleringer and Monson, 1993 ). In C 4 and CAM plants, phosphoenolpyruvate carboxylase (PEP) catalyzes the fi rst step of the photosynthetic reaction instead of Rubisco. These photosynthetic pathways de-pend upon structural (C 4 ) or temporal (CAM) separation of PEP and Rubisco activity to concentrate CO 2 for the Calvin cycle while reducing photorespiration (C 4 ) and water loss (CAM). In C 4 photosynthesis, fi xed carbon is transported in the form of malate or aspartate to special cells that form the vascular bundle sheath, where it is released as CO 2 and enters the Calvin cycle ( Kanai and Edwards, 1999 ). In the CAM pathway, CO 2 is fi xed 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, CO 2 concentration remains high because it cannot escape through the closed sto-mata ( Ehleringer and Monson, 1993 ). PEP carboxylase dis-

1829November 2010] Ocampo and Columbus Phylogenetics of Cactineae

Estimation of divergence times Estimation of divergence times was con-ducted 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 fi le for BEAST was prepared for the combined data matrix in the Bayesian Evolutionary Analysis Utility (BEAUti), and by manu-ally assigning the best-fi t 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 identifi cation), 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., Chac n et al., 2006 ; VanderWerf et al., 2010 ). The age of particular is-lands 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 dat-ing nodes with these volcanic hotspots overlooks the possibility that the se-lected 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 K Ar method ( Naughton et al., 1980 ; Sherrod et al., 2003 ). The closest relative of P. molokinensi s is P. howellii , en-demic to the Gal pagos Islands ( Wiggins et al., 1971 ), thus the divergence be-tween 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. moloki-niensis is found. Calibration using the Hawaiian Islands ( P. molokiniensis ) was preferred over the Gal pagos ( P. howellii ) because the latter species is distrib-uted throughout the Gal pagos Archipelago ( Wiggins et al., 1971 ), and the ages of these islands differ greatly ( Bailey, 1976 ), making it diffi cult 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 Po opo o 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 lognor-mal; 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 genera-tions at Cornell University s Computational Biology Service Unit (http://cbsua-pps.tc.cornell.edu/beast.aspx), saving every 1000th tree. Trace fi les 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 fi le 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 fi rst 14 000 trees. The program FigTree version 1.2.3 ( Rambaut, 2009 ) was used for visualizing results on divergence dates.

Historical biogeography Analysis of potential ancestral distribution areas for taxa of Cactineae used a Bayesian approach to dispersal vicariance 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 ac-counts for uncertainty in the phylogenetic estimate. The Bayes DIVA 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 re-construction 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 biogeo-graphical reconstruction, simulations were run to evaluate the impact of out-group distribution on the results. The distributions of the outgroup species were modifi ed to restrict them to the southern hemisphere, where apparently the Caryophyllales have their origin ( Raven and Axelrod, 1974 ), specifi cally: (1) South America, (2) Africa, and (3) both continents.

Carbon isotope ratio tests and leaf anatomy Determination of photosyn-thetic pathways in Cactineae was accomplished by 13 C data complemented by leaf anatomy, an approach that has been used by Sage et al. (2007) to discriminate

and ndhA intron to explore their utility for resolving relationships among fami-lies of Cactineae. These loci are among the 12 most variable chloroplast mark-ers recommended by Shaw et al. (2007) . Primers for amplifi cation via PCR were as in Shaw et al. (2007) . Amplifi cations were performed in 25 L reac-tions with 0.62 units of Taq DNA polymerase (Promega, Madison, Wisconsin, USA), 2.5 L of (NH 4 ) 2 SO 4 buffer, 0.5 pM of forward and reverse primers, 0.25 mM MgCl 2 , 0.25 mM dNTPs, and 0.25 L of BSA 100 for a fi nal con-centration of 1%, plus 1 L of genomic DNA in a Robocycler 96 or RoboCy-cler Gradient 96 thermal cycler (Stratagene, La Jolla, California, USA). PCR cycles were as follows: (1) initial denaturation at 94 C for 4 min; (2) 35 cycles of denaturation at 94 C for 1 min, primer annealing at 50 54 C for 1 min, and primer extension for 1 min 30 s at 72 C; and (3) fi nal elongation for 7 min at 72 C. PCR products were purifi ed by the PEG precipitation protocol ( Johnson and Soltis, 1995 ). Alternatively, amplifi cation products were cleaned by adding 3 L of a solution containing 0.2 L each of Antarctic phosphatase and exonu-clease I (New England Biolabs, Ipswich, Massachusetts, USA) and incubating for 30 min at 37 C then for 20 min at 80 C. 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 rpl14 rps8 infA rpl36 region (rps8F: 5 GYR AGA AAA CAT CAA GAA AGA AA 3 ; rps8R: 5 TCC CGA TCH GTC ATT ATA CC 3 ) and atpI atpH intergenic spacer (atpIF1: 5 ATG GRC RGT TTA CGT TAT GGA 3 ). Products were cleaned using Sephadex G-50 columns (GE Healthcare, Anaheim, California, USA) and read on an ABI Prism automated sequencer 3130xl (Applied Biosys-tems, 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 analyses DNA 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 ) fol-lowing 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 rpl14 rps8 infA rpl36 was incongruent with atpI atpH and the ndhA intron ( P = 0.014 and P = 0.001, respectively, thus rejecting the null hypothesis of congruent data at the 0.05 confi dence level). Therefore, each marker was analyzed separately to assess incongruence in tree topology. Phylogenetic analyses of individual markers yielded incongruent re-lationships 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). Indi-vidual 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 esti-mated 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-fi t model for rpl14 rps8 infA rpl36 was a transversional model (TVM) plus a gamma-distributed rate variation (G; Yang, 1993 ); for atpI atpH 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-fi t model of evolution provided by MrModeltest version 2.3 ( Nylander, 2004 ), under the AIC. The model selected for all data sets was GTR + I. Bayes-ian 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 fi les were visually examined to check convergence between runs, and the burnin value for obtaining the majority rule consensus tree was set to ignore the fi rst 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 probabili-ties ( Rannala and Yang, 1996 ; Li et al., 2000 ).

The Shimodaira Hasegawa (SH; Shimodaira and Hasegawa, 1999 ) test was performed to test selected alternative topologies from different analyses. Con-straint topologies were prepared in the program MacClade version 4 ( Maddison and Maddison, 2000 ) and loaded into Garli. Estimated constrained and uncon-strained topologies were then loaded into PAUP*, and their likelihood scores were compared using the SH test with the RELL option.


thetic pathway in the samples because 13 C 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 avail-able; 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 fi xed and stored in a solution of formalin propionic acid alcohol (FPA; Ruzin, 1999 ). When fresh material was not avail-able, 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 de-hydrated and embedded in paraffi n 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 : paraffi n oil, 2 h; 1 : 2 xylene : paraffi n oil, 2 h; in 58 C oven: paraffi n step 1, 6 h, and paraffi n step 2, 6 h, followed by fi nal paraffi n 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; H 2 O, 2 min; 2% aqueous ZnCl 2 , 1 min; H 2 O, 5 s; 1 : 25 000 aqueous safranin O, 5 min; H 2 O, 5 s; 10 g orange G + 25 g tannic acid + 20 drops HCl + 0.2 g thymol + 500 mL H 2 O, 1 min; H 2 O, 5 s; 25 g tannic

C 4 from C 3 and CAM. Leaf material was used for photosynthetic pathway de-termination by 13 C analysis except for Opuntia vestita , for which leaf material was not available, and Rhipsalis baccifera , which does not have leaves; there-fore, 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 13 C analysis were prepared for all species except Portulaca elatior by drying plant material in an oven at ca. 50 C 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). Sam-ples 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 80 C for 24 h and analyzed in a EuroVector elemental analyzer (EuroVector S.p.A., Milan, Italy). The three photosynthetic pathways discrimi-nate in different proportions against the isotope 13 C (isotope fractionation; O Leary, 1988 ). The following scale was used to identify the photosynthetic pathway. C 3 : typically 25 per mil ( ; O Leary, 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 ); C 4 : 10 to 16 ( O Leary, 1988 ; Sage et al., 2007 ); CAM: 9 to 20 ( O Leary, 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 confi dently determine the photosyn-

Fig. 1. Bayesian allcompat tree of Cactineae, using a combined data matrix of the rpl14 rps8 infA rpl36 region, atpI atpH intergenic spacer, and ndhA intron. The ML topology is identical to the Bayesian estimate. p.p. = posterior probability.


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 S1 S3, wherein the to-pological 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 sup-ported, as are all the families except for Didiereaceae, which has low bootstrap support. Montiaceae are the fi rst 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 Tali-naceae 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 acu-leata , P. lychnidifl ora , 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 pre-sented in Table 2 . According to the analysis, the age of the sub-order is 18.8 (6.7 33.7) Myr, which corresponds to early Miocene ( IUGS, 2009 ).

Historical biogeography The optimal reconstruction from the Bayes DIVA 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 phy-logenetic relationships among the families of Cactineae are not highly supported, the ancestral distribution for each family ex-cept Portulacaceae had high probability values ( Fig. 2 ). Monti-aceae are North American in origin, but dispersed to South America and Australia. Didiereaceae are inferred to have Afri-can Madagascan origin. Anacampserotaceae were found to have originated in the Americas and Cactaceae in South Amer-ica and the Caribbean region. The rest of the taxa in the subor-der have a South American origin (although equivocal in Portulacaceae), with multiple dispersals to other continents.

Two simulation analyses of species with hypothetical distri-butions partially supported the origin of Cactineae in the Amer-icas (48.19 56.12% 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. 3 5 . Leaf anatomy corresponded to the photosynthetic pathways suggested by 13 C values ( Table 3 ) with some excep-tions. Grahamia bracteata (Anacampserotaceae), Quiabentia verticillata (Cactaceae), and Decarya madagascariensis , Di-dierea madagascariensis , and D. trolli (Didiereaceae), have CAM-like 13 C values, but the leaves were considered to have C 3 anatomy. Therefore, the photosynthetic pathway for these species was scored as facultative CAM for character recon-struction. CAM photosynthesis (including facultative CAM)

acid + 0.2 g thymol + 500 mL H 2 O, 5 min; H 2 O, 1 3 s; 1% aqueous iron alum, 2 min; H 2 O, 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 sali-cylate, 2 min; xylene, 2 min; xylene, until permanently coverslipped using Cytoseal (Richard Allan Scientifi c, 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), specifi cally for background subtraction and image lev-els adjustment; scale bars were added to the fi nal image using ImageJ software ( Rasband, 1997 ). A set of slides is deposited at RSA, and original digital image fi les are available upon request from the fi rst author.

C 3 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 C 4 photosynthesis ( Guti rrez 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 meso-phyll) ( Furbank, 1998 ; Kanai and Edwards, 1999 ). CAM photosynthesis is cor-related 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 evolution Evolution of photosynthetic pathways was traced us-ing the program Mesquite version 2.72 ( Maddison and Maddison, 2009 ), esti-mated by ML (Markov k -state 1 parameter model, which corresponds to Lewis s [2001] Mk model) over the ML tree from the combined data matrix.

RESULTS

Phylogenetic relationships of major clades Aligned lengths for the loci were: rpl14 rps8 infA rpl36 region, 1351 bp; atpI atpH intergenic spacer, 906 bp; and the ndhA intron, 1395 bp. Sequences were unambiguously aligned except for rpl14 rps8 , 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 Ba-sellaceae, Didiereaceae, Hallophytaceae, and Montiaceae, and a strongly supported ACPT clade. Families of Cactineae were resolved as monophyletic (except Didiereaceae in rpl14 rps8 infA rpl36 ; see Appendix S1 at http://www.amjbot.org/cgi/content/full/ajb.1000227/DC1) and have moderate (75 89% bootstrap) to strong ( 90% bootstrap; 0.95 posterior probabil-

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

Cannot reject (Diff ln L = 8.58767, P = 0.189)

Halophytum part of Montiaceae Cannot reject (Diff ln L = 3.77526, P = 0.243)

Halophytum basal within Cactineae Cannot reject (Diff ln L = 0.43672, P = 0.366)

Halophytum sister to Basellaceae Cannot reject (Diff ln L = 0.98831, P = 0.421)

Didiereaceae basal within Cactineae Cannot reject (Diff ln L = 3.21613, P = 0.330)

Cactaceae sister to Portulacaceae + Talinaceae + Anacampseroteae

Cannot reject (Diff ln L = 6.38097, P = 0.120)

Portulacaceae sister to Anacampseroteae Cannot reject (Diff ln L = 0.85346, P = 0.318)


Portulaca is the only member of Cactineae with C 4 photosyn-thesis, with a shift to C 3 in P. cryptopetala.

DISCUSSION

Relationships within Cactineae All analyses of the chloro-plast data show suborder Cactineae and, in nearly every case, each of its eight families (Anacampserotaceae, Basellaceae, Cactaceae, Didiereaceae, Halophytaceae, Montiaceae, Portu-lacaceae, 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 confl ict and low clade support, relationships among the families, except for the grouping of the ACPT families, are incongruent among

was inferred only for species of Anacampserotaceae, Cacta-ceae, and Didiereaceae. Leaf anatomy and 13 C values for Por-tulaca cryptopetala (Portulacaceae) both indicate that it undergoes C 3 photosynthesis ( Fig. 4K ), unlike all other species of Portulaca examined, which were C 4 .

Table 4 shows the 13 C values obtained in this study. C 3 val-ues ranged between 20.44 and 33.52 ; for C 4 , 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 diversifi cation of photosynthetic pathways in Cactineae is shown in Fig. 6 . ML reconstruction recovered the C 3 pathway as ancestral to the suborder. CAM photosynthesis is inferred to have evolved independently fi ve times, including facultative CAM in Anacampserotaceae, Cactaceae, and Didiereaceae.

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 Huelsen-beck, 2003 ), except for the relationships among Pereskia aculeata , P. lychnidifl ora , and P. sacharosa (Cactaceae). Dates in millions of years. Arrows indi-cate 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 fi ve or more reconstructed ancestral ranges with similar probability values. Letters after each taxon name represent the distribution of the species.


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 confi rmed 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; M ller 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 affi nities 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 atpI atpH , where Talinaceae have a bootstrap value of 32% and a 0.73 posterior probability and Cactaceae have 71% bootstrap support; see on-line 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 in-tron recovers Cactaceae as basal (online Appendix S3) in con-cordance 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 consider-able uncertainty remains about its evolutionary history. Famil-ial relationships (besides the ACPT grouping) remain uncertain despite the use of a variety of molecular markers for phyloge-netic reconstruction. This uncertainty is associated with short branch lengths. Internal short branches have been related to rapid radiations ( Whitfi eld and Lockhart, 2007 ), where ances-tral polymorphisms can persist and lead to incongruence among loci ( Maddison, 1997 ; Degnan and Rosenberg, 2006 , 2009 ). Phylogenetic reconstruction involving short branches can also be infl uenced by a few homoplastic characters, which are suf-fi cient to mask the signal because relationships are supported by a limited number of characters ( Rokas and Carroll, 2006 ; Whitfi eld 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 S1 S3), it could be hypothesized that these are due to rapid radiations, as

analyses of individual markers (online Appendices S1 S3) 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 , psbA trnH , trnK matK , 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. Cu noud 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 fami-lies (except Calyptrotheca Gilg [Didiereaceae], which has up to six ovules; Nyffeler and Eggli, 2010 ), a unique trait in Cactineae and putative synapomorphy. However, Nyffeler s (2007) re-sults 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 .

Node MRCA of

95% Highest posterior density

Mean Lower Upper

1 Cactineae 18.8 6.7 33.72 Montiaceae 13 3.4 25.43 Basellaceae 3.8 0.4 94 Didiereaceae 12.1 2.4 24.45 Basellaceae + Didiereaceae 14.9 3.9 28.56 Cactineae except Montiaceae 17.6 6.5 31.97 Halophytaceae + ACPT clade 17.1 6.1 31.48 ACPT clade 15.2 5.4 27.89 Talinaceae 9.1 2 18.310 Anacampserotaceae 11.4 3.2 22.611 Anacampserotaceae + Cactaceae +

Portulacaceae14.3 5.1 26.6

12 Cactaceae 10 3.1 19.113 Portulacaceae 9.6 3.0 18.514 Cactaceae + Portulacaceae 13.9 4.9 26.5


characterizing CAM activity using leaf anatomical traits, but their criteria could not predict the correct photosynthetic path-way in all cases. These studies show that predicting photosyn-thetic pathways based solely on leaf anatomy cannot be achieved with confi dence in some cases. Our primary basis for differen-tiating C 3 leaf anatomy from CAM in this study was the pres-ence 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 13 C 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 rep-resent photosynthetic variants that are diffi cult to identify with 13 C and leaf anatomical data in tandem, such as facultative CAM and CAM-cycling. In our study, fi ve facultative CAM taxa were detected, which are distributed in Anacampserota-ceae, 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 C 3 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 ). C 4 13 C values for Portulaca species with Kranz anat-omy are consistent with reported values for C 4 photosynthesis ( 10 to 16 ; O Leary, 1988 ; Sage et al., 2007 ). Only P. cryp-topetala has leaf anatomy and a 13 C value corresponding to C 3 photosynthesis; however, the species has been shown to be a C 3 C 4 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 oc-currence of various degrees of CAM activity within Cactineae, limit our ability to reconstruct photosynthetic pathway evolu-tion. Despite that, our results show that the C 3 pathway is well distributed in the outgroup and Cactineae ( Fig. 6 ), and other possible topologies do not seem to affect the inference of mul-tiple origins of CAM photosynthesis. Character reconstruction analysis recovers the C 3 pathway as ancestral for the suborder. C 4 , CAM, and facultative CAM are all derived from the C 3 pathway, in agreement with the hypothesis that C 3 is the ances-tral 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,

earlier suggested by Hershkovitz and Zimmer (2000) for Mon-tiaceae (as western American Portulacaceae). The study by Arakaki et al. (2010a) using low copy nuclear markers (phyto-chromes B and C) and a number of chloroplast loci is of great interest because some relationships are apparently being re-solved within the suborder, although that work is at a prelimi-nary 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 Previ-ous studies have shown that all three major photosynthetic pathways C 3 , C 4 , and CAM are 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 , re-spectively), or switch pathways depending on the environmen-tal 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 fi rst time) (e.g., Guralnick and Jackson, 2001 ; Guralnick et al., 2002 , 2008 ), including facultative CAM (the plants can switch to CAM or C 3 depending on water availability; they use CAM when water-stressed and C 3 photo-synthesis when water is abundant; Cushman, 2001 ) and CAM-cycling (during the day, the plants do not completely close their stomata, and they fi x atmospheric CO 2 ; at night, the stomata are closed and the plants fi x respiratory CO 2 ; Cushman, 2001 ). In this study, leaf anatomy was important in helping to predict the photosynthetic pathway with more accuracy than with 13 C 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 distri-bution of the photosynthetic pathways in the suborder, and fur-ther biochemical characterization could yield additional insights.

Determination of photosynthetic pathways from leaf anat-omy alone was challenging in some cases, in particular discrim-inating C 3 from CAM, which was also borne out in previous studies. Nyananyo (1988) concluded that Portulacaria afra , Talinopsis frutescens , and Talinum paniculatum undergo C 3 photosynthesis based on anatomy, whereas Landrum (2002) considered them to undergo CAM; here we coded the fi rst spe-cies as CAM and the other two as C 3 using leaf anatomy. Nyananyo (1988) also described the leaf anatomy of Portulaca cryptopetala as C 4 , while Voznesenskaya et al. (2010) and we in the present study showed that the species has C 3 leaf anat-omy. Nelson et al. (2005) proposed a quantitative method for

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 (C 3 ; Basellaceae). (D) A. ramosa (C 3 ). (E) Calandrinia caespitosa (C 3 ; Montiaceae). (F) Calyptridium parryi (C 3 ; Montiaceae). (G) Ceraria namaquensis (C 3 ; Didiereaceae). (H) Claytonia parvifl ora (C 3 ; Mon-tiaceae). (I) Decarya madagascariensis (C 3 ; Didiereaceae). (J) Didierea madagascariensis (C 3 ; Didiereaceae). (K) D. trolli (C 3 ). (L) Grahamia bracteata (C 3 ; Anacampserotaceae). (M) Lewisia rediviva (C 3 ; Montiaceae). (N) Maihuenia patagonica (C 3 ; Cactaceae). (O) Mirabilis sanguinea (C 3 ; Nyctag-inaceae). PM = palisade mesophyll. Black scale bar = 0.75 mm; gray scale bar = 0.25 mm.


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 as-sociated 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 ). Muller s (1981) list of fossil pollen records includes Monti-aceae ( Hedlund and Engelhardt, 1970 ; Martin, 1973 ) and Por-tulacaceae ( Van Campo, 1976 ) from the Miocene-Pliocene period (ca. 6 4 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, Po-lygonaceae Juss.; Erdtman, 1952 ). If these fossil pollen records can be confi dently 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 Bayes DIVA ap-proach and a limited outgroup sample places the origin of Cactineae in the Americas. The results of the simulation analy-ses showed that alternate distribution areas assigned to the out-group do impact the reconstruction of the ancestral distribution of the suborder, in particular increasing its range. DIVA optimi-zations become less reliable as the root node is approached ( Ronquist, 1996 ) and have the tendency to yield wide distribu-tions that include all analyzed areas, as is the case in one simu-lation 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 hemi-sphere. 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 cool-temperate-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

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 ; G erere 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 C 3 and CAM pho-tosynthesis, while the ACPT clade has in addition the C 4 path-way. It is interesting to note that all leafy Cactaceae sampled here (except Opuntia vestita , for which only stem material was available) have C 3 leaf anatomy. This may represent a symple-siomorphy, although the photosynthetic functions of the leaf may vary (see Nobel and Hartsock, 1986 ; Martin and Wallace, 2000 ). The C 4 pathway has evolved only once in Cactineae, specifi cally in Portulacaceae ( Portulaca ); the MRCA of the family is reconstructed as having C 4 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 CO 2 concentrations from the Oligocene into the Pliocene were a critical factor for the evolution of C 4 photosynthesis ( Ehleringer and Monson, 1993 ; Sage, 2005 ). A transition to an intermediate C 3 C 4 pathway from C 4 is found in P. cryptopetala ( Voznesenskaya et al., 2010 ), which can be classifi ed as type I intermediacy, characterized by an absence of C 4 cycle activity and enhanced reassimilation of photorespired CO 2 ( Edwards and Ku, 1987 ). Fixation of CO 2 is accomplished exclusively by Rubisco, which explains a 13 C value within the C 3 range ( Sage et al., 2007 ). Portulaca cryptopetala is distrib-uted in central South America (Bolivia to Argentina and Brazil) and is generally associated with rivers and streams. What drives the shift from C 4 to C 3 C 4 intermediacy is not understood, al-though it has been hypothesized that it may represent a physi-ological response to environmental selection pressure ( Duvall et al., 2003 ; McKown et al., 2005 ).

Historical biogeography of Cactineae Our study is the fi rst to employ calibration points inside Cactineae, using two endemic Hawaiian Portulaca species, which yields an estimate of 18.8 (6.7 33.7) 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 ( Wikstr m et al., 2001 , 2004 ). This calibration approach has been criticized be-cause 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 Gal pagos Is-lands. Even more, some researchers contend that calibrating phylogenies using this method may result in signifi cant (tens of millions of years) age underestimations ( Heads, 2009 ). Prelimi-nary 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

Fig. 4. Light micrographs of transectional leaf anatomy in Cactineae. Photosynthesis type inferred from anatomy is indicated within parentheses. (A) Montiopsis andicola (C 3 ; Montiaceae). (B) Parakeelya pleiopetala (C 3 ; Montiaceae). (C) Pereskia aculeata (C 3 ; Cactaceae). (D) P. grandifolia (C 3 ). (E) P. lychnidifl ora (C 3 ). (F) P. quisqueyana (C 3 ). (G) P. sacharosa (C 3 ). (H) Phemeranthus multifl orus (C 3 ; Montiaceae). (I) Portulaca amilis (C 4 ; Portulacaceae). (J) P. bicolor (C 4 ). (K) P. cryptopetala (C 3 ). (L) P. echinosperma (C 4 ). (M) P. elatior (C 4 ). (N) P. guanajuatensis (C 4 ). (O) P. molokiniensis (C 4 ). PM = pali-sade 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.


by the high number of dispersal events inferred from the Bayes DIVA analysis. It is not clear how ancestral taxa may have dis-persed successfully around the world. Dispersal mechanisms known in the suborder include zoochory ( Ridley, 1930 ; Barth-lott and Hunt, 1993 ; Hershkovitz and Zimmer, 2000 ), anemo-chory ( Barthlott and Hunt, 1993 ; Carolin, 1993 ; Kubitzki, 1993 ;

Fig. 5. Light micrographs of transectional leaf anatomy in Cactineae. Photosynthesis type inferred from anatomy is indicated within parentheses. (A) P. pilosa (C 4 ; Portulacaceae). (B) P. umbraticola subsp. lanceolata (C 4 ). (C) Portulacaria afra (CAM; Didiereaceae). (D) Quiabentia verticillata (C 3 ; Cactaceae). (E) Rivina humilis (C 3 ; Phytolaccaceae). (F) Sesuvium portulacastrum (C 3 ; Aizoaceae). (G) Talinopsis frutescens (C 3 ; Anacampserotaceae). (H) Talinum caffrum (C 3 ; Talinaceae). (I) T. fruticosum (C 3 ). (J) T. lineare (C 3 ). (K) T. paniculatum (C 3 ). (L) T. polygaloides (C 3 ). 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.

Sperling and Bittrich, 1993 ), hydrochory ( Ridley, 1930 ; Danin et al., 1978 ; Barthlott and Hunt, 1993 ), and voluntary or invol-untary 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.


Applequist and Wallace (2001) showed that the place of ori-gin of Montiaceae is uncertain, but it is here recovered as North America, with a MRCA age of 13 Myr, consistent with Hersh-kovitz and Zimmer s (2000) estimate of 8 16 Myr. Phemeran-thus , 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 ac-cording to the phylogeny in Applequist et al. (2006) it may rep-resent another independent long-distance dispersal event to the islands of the Pacifi c.

The biogeographical analysis postulates Didiereaceae as Af-rican Malagasy in origin (fi ve 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 fami-ly s Old World distribution is likely the result of an early long-distance dispersal from South America to the Old World. These authors ( Applequist and Wallace, 2000 , 2001 ) provided evi-dence that Calyptrotheca , a genus of Didiereaceae (Calyptroth-ecoideae 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

Table 3. Photosynthetic pathways inferred from 13 C values and leaf anatomy data derived from this study.

Species 13 C ( )Pathway

based on 13 C

Leaf anatomy

Pathway for sample

Anacampserotaceae Anacampseros

vulcanensis 24.53 C 3 C 3

Grahamia bracteata 19.48 CAM C 3 Fac. CAM Talinopsis frutescens 27.10 C 3 C 3 C 3 Basellaceae Anredera cordifolia 30.16 C 3 C 3 C 3 A. ramosa 26.31 C 3 C 3 C 3 Cactaceae Maihuenia patagonica 25.10 C 3 C 3 C 3 Opuntia vestita 16.87 CAM CAM Pereskia aculeata 27.56 C 3 C 3 C 3 P. grandifolia 30.18 C 3 C 3 C 3 P. lychnidifl ora 24.52 C 3 C 3 C 3 P. quisqueyana 29.97 C 3 C 3 C 3 P. sacharosa 24.16 C 3 C 3 C 3 Quiabentia verticillata 16.25 CAM C 3 Fac. CAM Rhipsalis baccifera 17.08 CAM NA CAMDidiereaceae Alluaudia ascendens 17.16 CAM CAM CAM A. humbertii 15.05 CAM CAM CAM Ceraria namaquensis 20.44 C 3 C 3 C 3 Decarya

madagascariensis 15.52 CAM C 3 Fac. CAM

Didierea madagascariensis

19.37 CAM C 3 Fac. CAM

D. trollii 18.38 CAM C 3 Fac. CAM Portulacaria afra 18.35 CAM CAM CAMHalophytaceae Halophytum ameghinoi 24.75 C 3 C 3 Montiaceae Calandrinia caespitosa 25.67 C 3 C 3 C 3 Calyptridium parryi 25.08 C 3 C 3 C 3 Claytonia parvifl ora 33.52 C 3 C 3 C 3 Lewisia rediviva 31.27 C 3 C 3 C 3

Table 4. 13 C statistics for Cactineae and outgroup taxa. Statistic C 3 C 4 CAM

No. species 30 13 10Average ( ) 26.78 13.5015 17.35Standard deviation ( ) 2.69 1.44912 1.52Minimum ( ) 33.52 15.56 19.48Maximum ( ) 20.44 10.41 15.05

Notes: Photosynthetic pathways were assigned using 13 C values and leaf anatomical data, when available. CAM includes facultative CAM taxa as scored in this study.

in our study). On the basis of this and the low sequence diver-gence 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.

Notes: Fac. = facultative; NA = Not applicable; = leaf anatomy not available for the sample.

Species 13 C ( )Pathway

based on 13 C

Leaf anatomy

Pathway for sample

Montiopsis andicola 27.52 C 3 C 3 C 3 Parakeelya pleiopetala 27.42 C 3 C 3 C 3 Phemeranthus multifl orus 27.39 C 3 C 3 C 3 Portulacaceae P. amilis 11.96 C 4 C 4 C 4 P. bicolor 13.75 C 4 C 4 C 4 P. californica 13.15 C 4 C 4 C 4 P. cryptopetala 26.55 C 3 C 3 C 3 P. echinosperma 10.41 C 4 C 4 C 4 P. elatior 12.74 C 4 C 4 C 4 P. guanajuatensis 14.25 C 4 C 4 C 4 P. howellii 12.27 C 4 C 4 P. massaica 15.21 C 4 C 4 P. molokiniensis 15.56 C 4 C 4 C 4 P. pilosa 14.05 C 4 C 4 P. quadrifi da 13.03 C 4 C 4 P. umbraticola subsp.

lanceolata 14.09 C 4 C 4 C 4

P. villosa 15.05 C 4 C 4 Talinaceae Talinum arnottii 24.96 C 3 C 3 T. caffrum 23.29 C 3 C 3 C 3 T. fruticosum 29.34 C 3 C 3 C 3 T. lineare 26.28 C 3 C 3 C 3 T. paniculatum 28.22 C 3 C 3 C 3 T. polygaloides 26.70 C 3 C 3 C 3 T. tenuissimum 23.69 C 3 C 3 Outgroups Mirabilis sanguinea

(Nyctaginaceae) 27.02 C 3 C 3 C 3

Rivina humilis (Phytolaccaceae)

29.33 C 3 C 3 C 3

Sesuvium portulacastrum (Aizoaceae)

25.62 C 3 C 3 C 3


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 = G erere 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. C 3 -C 4 intermediate: M = Voznesenskaya et al., 2010. *As Talinum triangulare (Jacq.) Willd.; **as Quiabentia chacoensis Backeb.; ***as Portulaca mundula I. M. Johnst.


conjectured that this location would explain the near absence of Cactaceae in the Old World and would facilitate the early dis-persal 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 Bayes DIVA analysis, though re-stricted 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., Rodr guez-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 World Australian clade ( P. bicolor and P. quadrifi da ) with opposite leaves and a primarily New World clade with alternate to subopposite leaves. The biogeo-graphical analysis is not clear about the origin of Portulaca , but Applequist and Wallace (2001) indicate that it is of South Ameri-can 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 Pacifi c Ocean (e.g., Gal pagos, 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 fl oating 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 evo-lutionary relationships, including when loci are analyzed in combi-nation ( 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 organ-isms 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 al-ready present across the central Andes ( Hartley, 2003 ). Population-level studies and complete chloroplast genome sequence analysis may contribute to a better understanding of the evolutionary his-tory of the suborder by providing greater insights into adaptations to aridity, as well as the mechanisms that triggered those adapta-tions in this charismatic lineage of fl owering plants.

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The ACPT clade, which has high statistical support in almost all analyses ( Fig. 1 ; Appendices S1 S3), 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 Wallace s 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 distrib-uted 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.

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Our results show that the MRCA of Cactaceae was distrib-uted in South America and the Caribbean region, but other stud-ies 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) com-mented 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 (fi ve of 126 recognized genera sampled; Anderson, 2001 ) are in concordance with oth-ers (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 Af-rica 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 111 85 Myr ( Wikstr m et al., 2001 , 2004 )]. Hersh-kovitz and Zimmer (1997) suggested a mid-Tertiary (Oligo-cene) 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 (3.1 19.1) Myr for the MRCA of Cactaceae supports Hershkovitz and Zimmer s (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


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