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Systematics and Phytogeography |
2Department of Botany, University of Toronto, Toronto, Ontario, M5S1A1, Canada; 3Department of Natural History, Royal Ontario Museum, 100 Queen's Park, Toronto, Ontario, M5S 2C6, Canada
Received for publication April 2, 2005. Accepted for publication August 16, 2005.
ABSTRACT
A well-resolved phylogeny of Flaveria is used to infer evolutionary relationships among species, biogeographical distributions, and C4 photosynthetic evolution. Data on morphology, life history, and DNA sequences (chloroplastic trnL-F, nuclear ITS and ETS) for 21 of 23 known species were collected. Each data set was analyzed separately and in combination using maximum parsimony and Bayesian analyses. The phylogeny of Flaveria is based on the combined analysis of all data. Our phylogenetic evidence indicates that C3 Flaveria are all basal to intermediate (C3-C4 and C4-like) and fully expressed C4 Flaveria species. Two strongly supported clades (A and B) are present. Using this phylogeny, we evaluate the current systematics of the genus and suggest the removal and reevaluation of certain taxa. We also infer the center of origin and dispersal of Flaveria species. Multiple origins of photosynthetic pathway intermediacy in Flaveria are recognized. C3-C4 intermediacy has evolved twice in the genus and is found to be evolutionarily intermediate in clade A, but not necessarily in clade B. C4-like photosynthesis is also derived once in each clade. In addition, fully expressed C4 photosynthesis may have evolved up to three times within clade A.
Key Words: Asteraceae • C3-C4 intermediates • C4 photosynthesis • ETS • Flaveria • ITS • phylogeny • trnL-F
C4 photosynthesis is a complex adaptation that has generated interest for decades and produced a wealth of research, yet details of the origins and modes of C4 evolution have remained somewhat enigmatic. Numerous lines of evidence suggest that C4 photosynthesis evolved from C3 photosynthesis in response to low levels of atmospheric CO2 and environmental conditions promoting the energetically wasteful oxygenase reaction (photorespiration) of the photosynthetic enzyme ribulose 1,5-bisphosphate carboxylase oxygenase (Rubisco) (reviewed in Sage, 2001
, 2004
). In C4 plants, CO2 enters the C4 cycle first and is incorporated into a 4-carbon organic acid by phosphoenolpyruvate carboxylase (PEPCase). The C4 acid diffuses to the site of Rubisco and is decarboxylated to release and concentrate CO2 near Rubisco, thereby suppressing the enzyme's oxygenase activity and essentially eliminating photorespiration. A significant advantage for plants with this complex adaptation is surviving and thriving in environmental conditions that favor photorespiration, such as warm, saline, or arid habitats.
The C4 syndrome is extremely successful in the monocotyledonous families Poaceae and Cyperaceae, reflected in the global diversity and widespread distribution of C4 grasses and sedges. C4 photosynthesis is documented in one additional monocotyledonous family and 16 dicotyledonous families and is currently known to have at least 45 origins in the angiosperms as a whole, based on molecular phylogenetic data (Sage, 2004
). Studies show that C4 photosynthesis has arisen more than once in some orders or families (Sinha and Kellogg, 1996
; Soros and Bruhl, 2000
; Giussani et al., 2001
; Kadereit et al., 2003
) and even within a single genus (Kopriva et al., 1996
; Pyankov et al., 2001
; Kadereit et al., 2003
; Sage, 2004
). In addition, 10 genera (four monocot and six dicot) include species that have features that are between C3 and C4 values (e.g., degree of inhibition by photorespiration) and may include a combination of characteristics that reflect a partially to nearly complete C4 photosynthetic cycle (Bruhl et al., 1987
; Edwards and Ku, 1987
; Monson and Moore, 1989
; Bruhl and Perry, 1995
; Sage et al., 1999
; Monson and Rawsthorne, 2000
). These species are classified as photosynthetic "intermediates" and have been used in numerous comparative studies of physiology and molecular biology that have formed the basis of evolutionary models hypothesizing the stepwise acquisition of C4 traits (Edwards and Ku, 1987
; Monson and Moore, 1989
; Monson, 1999
, 2003
; Monson and Rawsthorne, 2000
; Svensson et al., 2003
; Westhoff and Gowik, 2004
; Sage, 2004
).
The genus Flaveria Juss. (Asteraceae) in particular has been used extensively in research of C4 photosynthesis evolution. This small genus of 23 known species includes both strictly C3 and C4 species (NADP-malic enzyme type [Ku et al., 1983
; Bauwe, 1984
]), in addition to a large number of intermediate species (C3-C4 and C4-like) (Table 1), and is believed to have evolved C4 photosynthesis at least twice (Powell, 1978
; Kopriva et al., 1996
; Monson, 1996
; Kellogg, 1999
). Compared to C3 Flaveria species, intermediate Flaveria species have reduced photorespiration, lower CO2 compensation concentrations, partial to complete Kranz anatomy, and certain intermediates also have increased PEPCase and NADP-malic enzyme activities, and a partially functional C4 cycle (Apel and Maass, 1981
; Ku et al., 1983
; Bassüner et al., 1984
; Bauwe, 1984
; Holaday et al., 1984
; Rumpho et al., 1984
; Reed and Chollet, 1985
; Bauwe and Chollet, 1986
; Brown et al., 1986
; Monson et al., 1986
; Edwards and Ku, 1987
; Moore et al., 1987
, 1988
; Cameron et al., 1989
; Cheng et al., 1988
; Hylton et al., 1988
; Brown and Hattersley, 1989
; Ku et al., 1991
, Dai et al., 1996
; Drincovich et al., 1998
). While the physiological properties of the intermediate Flaveria species fall between those of C3 and fully expressed C4 Flaveria species, the evolutionary position of the intermediates still remains unclear (Kopriva et al., 1996
). It is possible the intermediates represent stabilized, surviving taxa at various stages of C4 photosynthetic evolution; however, others have also suggested that these species represent stable endpoints in themselves and are not evolving towards full C4 photosynthesis (Edwards and Ku, 1987
; Monson, 1989
; Monson and Moore, 1989
).
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), subtribe Flaveriinae (Turner and Powell, 1977
; Baldwin et al., 2002
), with two sister genera Sartwellia and Haploësthes (Turner, 1971
, 1975
; Turner and Powell, 1977
). Flaveria remains the sole C4 genus in the Flaveriinae, although another occurrence of C4 photosynthesis exists in the Tageteae (Pectis; Kellogg, 1999
). Two C4 Flaveria species are cosmopolitan, especially as weeds in tropical and subtropical areas, and one C4 species occurs in Australia; however, the majority of Flaveria species are located in the southern USA, Mexico, and the West Indies (Table 1). Powell (1978)
hypothesized evolutionary relationships within the genus based on morphological and hybridization data. The 21 then-known Flaveria species were segregated into two subgeneric groups based on the number of large floral bracts (involucral phyllaries) surrounding each head-type inflorescence (capitulescence), and species were placed either into the 3–4 phyllary or the 5–6 phyllary lineage. Intermediate and C4 (or C4-like) photosynthetic Flaveria species are found within both lineages; therefore, Powell (1978)
suggested the possibility of two separate origins of C4 photosynthesis within the genus. A morphological phylogeny produced by Monson (1996)
with 15 characters obtained from Powell's monograph (1978)
suggested three origins of C4 photosynthesis in Flaveria; however, the phylogenetic tree was only moderately resolved and homoplasy was high. The first molecular-based phylogeny of Flaveria used the H-protein of the glycine cleavage system (338 bp fragments) from cDNA clones of 12 Flaveria species (Kopriva et al., 1996
). The resulting tree topology supported Powell's phyllary-based groupings of Flaveria, although the 5–6 phyllary lineage was nested within the 3–4 phyllary group. Furthermore, the tree supported the hypothesis of C4 or C4-like photosynthesis evolving twice (once in each Flaveria "clade"), but only one origin of photosynthetic intermediacy was suggested based on H-protein marker evidence. High bootstrap support (>75%) was only achieved at a few nodes of the tree, and the authors stated that the addition of Flaveria species and molecular markers was necessary to fully understand the evolutionary history of Flaveria. Another molecular phylogeny by Westhoff and Gowik (2004)
using PEPCase (500 bp ppcA1 promoter sequences) produced a phylogenetic tree similar to that of Kopriva et al. (1996)
; however, this study also included only 12 Flaveria species, did not achieve high bootstrap support (>75%) at most nodes on the tree, and did not resolve the phylogeny of Flaveria better than the previous molecular study. In both molecular phylogenetic studies, the gene markers chosen would not be considered neutral, but under selection pressure for C4 photosynthesis.
The presence of intermediate species within the genus Flaveria has provided the basis for several models involving C4 genetic, enzyme, and physiological evolution (Rosche and Westhoff, 1995
; Drincovich et al., 1998
; Monson, 1999
, 2003
; Engelmann et al., 2003
; Sage, 2004
; Westhoff and Gowik, 2004
). It is evident, however, that a robust phylogeny is necessary to provide an evolutionary framework for previous and future research on Flaveria thus enabling a further understanding of the evolution of C4 photosynthesis in this genus. We infer phylogenetic relationships among 21 of the 23 known Flaveria species using morphological, life history, chloroplastic (trnL-F), and nuclear (ITS, ETS) DNA sequence data. This study has three objectives: (1) to provide a phylogeny of Flaveria, (2) to reassess Flaveria systematics and biogeography, and (3) to evaluate previous hypotheses concerning the phylogenetic placement of photosynthetically intermediate Flaveria species and diversification of photosynthetic pathways in the genus in an evolutionary framework.
MATERIALS AND METHODS
Sampling strategy and material
A large collection of Flaveriinae specimens (Flaveria, Sartwellia, Haploësthes) was studied from various herbaria and sampled for genetic, morphological, and biogeographical data. Specimens were from the University of Toronto Flaveria research collection (grown by F. Kocacinar/voucher specimens housed at the Royal Ontario Museum [TRT]); Arnold Arboretum, Harvard University (AA); Gray Herbarium, Harvard University (GH); Royal Botanical Gardens, Kew (K); New York Botanical Gardens (NY); Sul Ross State University (SRSC); University of Texas (TEX/LL); Texas A & M University (TAES); and Museum of Natural History, Vienna (W) (Table 2; DNA GenBank codes listed in Appendix). Additional plant materials of Flaveria for DNA extraction were donated by A. M. Powell (Sul Ross State University, Alpine, Texas, USA) and E. Sudderth (Harvard University, Cambridge, Massachusetts, USA). Biogeographical data were recorded for specimens from these institutions and compared to the web-based Missouri Botanical Garden Vascular Tropicos Nomenclatural database (W3TROPICOS) and Mexican Biodiversity Information Network database (Red Mundial de Información Sobre Biodiversidad, REMIB). Sequence and morphological data were obtained for specimens of 21 of the 23 described Flaveria species (Powell, 1978
; Dimitri and Orfila, 1986
; Turner, 1995
). Of the two Flaveria species missing from this study, F. intermedia J. R. Johnst. (type specimen collected in 1896, located at GH) was studied for morphology, but is very similar to F. palmeri and remains questionable as a distinct species (Powell, 1978
; A. D. McKown, personal observation). The other species, F. haumanii Dimitri and Orfila, is located in northern Argentina and considered to be closely related to F. bidentis (Dimitri and Orfila, 1986
), but was unavailable for this study. Haploësthes greggii, Sartwellia flaveriae, and S. mexicana were used as outgroup taxa to root the Flaveria phylogeny, because Haploësthes and Sartwellia are both considered to be closely related to Flaveria (Turner and Powell, 1977
; Baldwin et al., 2002
).
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). Gene sequences were amplified from the chloroplast trnL-F spacer region, and the nuclear ribosomal internal (ITS) and external (ETS) transcribed regions. Morphological data from over 600 Flaveriinae samples (most are independent acquisitions), including type specimens, and life history characters were coded with symbols (0, 1, 2, etc.). Only discrete characters were selected and used for analysis, eliminating bias from characters occurring on a gradient, such as length or height measurements.
DNA isolation
Total genomic DNA for all taxa was obtained from dried herbarium leaf material, dried fruit material, or alcohol-preserved leaf tissue (Table 2). Herbarium specimens were sampled with as little destruction as possible using 0.5 cm2 of tissue or less. Recent collections were chosen (the oldest sample was 50 yr old), and green portions of the leaf were sampled. Samples were ground in 1 mL of 2.5% SDS with 1.0 mm zirconia/silica beads and a single ceramic bead (Bio Spec Products, Bartlesville, Oklahoma, USA) in a Fast Prep FP120 mixer mill (Thermo Electron Corp., Milford, Massachusetts, USA). Following complete pulverization of tissues, samples were centrifuged for 4–5 min at 13 000 rpm. Supernatants were collected and washed twice with an equivalent volume of 24 : 1 chloroform : isoamyl alcohol for removal of proteins, lipids, pigments, and debris. Approximately 800 µL of supernatant was recovered for each sample, and DNA was precipitated from this solution by adding 500 µL of isopropanol. DNA precipitates were rinsed with 1 mL 80% ethanol at –20°C, resuspended in 100 µL distilled, deionized water and stored at –20°C. Extractions with poor amplifications were further cleaned by "re-extracting" the DNA using a DNeasy Plant mini kit (QIAGEN, Mississauga, Ontario, Canada) following the manufacturer's instruction.
Gene markers and amplification
Gene markers selected for this study included chloroplastic trnL-F (primers "c" and "f" from Taberlet et al., 1991
donated by S. Malcomber, University of Missouri, St. Louis, Missouri, USA) (Bruneau et al., 2001
), and nuclear ITS (primers ITS1 and ITS4 from White et al., 1990
) and ETS (primers Hel-1 and 18S from Baldwin and Markos, 1998
). All gene markers were amplified with a polymerase chain reaction (PCR) using the same PCR "cocktail": 4 µL 10 mM dNTPs, 3.5 µL ddH2O, 2.5 µL 10x PCR buffer (QIAGEN), 2.5 µL bovine albumin serum, 0.5 µL Taq DNA polymerase (QIAGEN), and 1 µL each of forward and reverse 10 µM primers. Some samples with low PCR product yield required lowering the water content by 1 µL and adding 1 µL 25 mM MgCl2 solution (QIAGEN). All PCR amplifications were performed with the same thermal cycler settings (10 s at 95°C, 36 cycles of 1 min at 95°C, 30 s at 50°C, 4 min at 72°C, followed by a 10-min final extension at 72°C). PCR products were purified with QIAquick PCR purification kit (QIAGEN) and concentrated to approximately 10 ng DNA/µL for direct sequencing. Samples from Flaveria brownii showed allelic variation of ITS, and samples from F. pringlei and F. anomala had multiple alleles of both ITS and ETS, all of which required cloning with a QIAGEN PCR cloning kit using QIAGEN competent cells prior to sequencing.
DNA sequencing
Cleaned PCR products were sequenced in both directions using Big Dye Terminator ver. 3.5 (Applied Biosystems, Foster City, California, USA). Sequencing products were cleaned with Sephadex G-50 Fine DNA grade (Amersham Biosciences, Uppsala, Sweden), heat and vacuum-dried, and resuspended in Hi-Di formamide (Applied Biosystems). Sequences were read with an ABI PRISM 3100 genetic analyzer (Applied Biosystems, Foster City, California, USA), and the resulting gene sequences were edited with Sequencher ver. 2.2 (Gene Codes, Ann Arbor, Michigan, USA). Sequences containing single nucleotide polymorphisms were marked for the polymorphism. All sequences were submitted to GenBank (samples listed in Appendix).
Phylogenetic analysis
Nucleotide sequences were aligned using ClustalW (Chenna et al., 2003
). Sequence alignments were optimized by eye in Se-Al manual sequence alignment editor (Rambaut, 1996
), and ambiguously aligned regions were removed from the phylogenetic analyses. Aligned files were analyzed with maximum parsimony using PAUP* version 4.0 (Swofford, 2003
) and Bayesian inference using MrBayes version 3.0 (Huelsenbeck and Ronquist, 2001
; Ronquist and Huelsenbeck, 2003
). Parsimony searches were conducted for each data set separately and in combination assuming gaps as missing data, multiple states as uncertainty, and with a minimum F value for character state optimization. Full heuristic searches of alignment files were run with 1000 repetitions of random addition sequences and using tree-bisection-reconnection (TBR) branch swapping. To estimate support and robustness of the parsimony tree, 1000 bootstrap replicates were run with 100 full heuristic searches per replicate. All data sets were compared in PAUP* 4.0 for congruency with the partition homogeneity test (PHT) using 1000 replicates with 10 full heuristic searches per replicate. Bayesian analyses were run for separate and combined data sets with one million generations using four Markov chains and assuming a general model of DNA substitution/character change with gamma-distributed (DNA) or beta-distributed (morphological) rate variation across sites. Trees were sampled and saved at every 100th generation, and the initial 500 trees were discarded (as burn-ins). Bayesian consensus trees were constructed from 9500 trees and compared to parsimony consensus trees for congruence by eye. A single tree from parsimony analysis is presented for each data set indicating branch lengths (where applicable), and bootstrap/posterior probability values.
RESULTS
Alignment and tree statistics (alignment lengths, number of variables and phylogenetically informative sites, number of most parsimonious trees found, tree length, consistency, and retention indices) are given in Table 3.
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), have identical trnL-F sequences, such as F. australasica and F. trinervia, or F. campestris and F. palmeri. Multiple samples from different species of Flaveria cluster together, except samples of F. chloraefolia and F. linearis, which do not demonstrate strict monophyletic species groupings. All C3 Flaveria (F. cronquistii, F. mcdougallii, F. pringlei, and F. robusta) are located at the base of the tree and C4 Flaveria (F. australasica, F. bidentis, and F. trinervia) are found at the tips of the tree. Flaveria campestris (C4) and F. palmeri (C4-like) are also at the tips of the tree, but are not clustered with the other true C4 species where they have traditionally been grouped (Powell, 1978
).
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The consensus tree topology of the morphological characters from both parsimony and Bayesian analyses does not retrieve the exact tree topology of any of the gene analyses (Fig. 4). The consistency index of this data set is very low (Table 3), indicating extensive homoplasy in morphological features; however, the consensus tree topology does retrieve Powell's subgeneric phyllary groups (excepting the position of Flaveria cronquistii). Portions of the morphological tree correspond to the gene tree topologies, such as the placement of F. mcdougallii at the base of the morphological tree, along with the glabrous 3–4 phyllary F. cronquistii. A clade comprising species from F. ramossisima upwards is similar to clade A from the gene analyses (excepting the placement of F. angustifolia). Species designated as 5–6 phyllary by Powell (1978)
are found in more basal positions along the tree, but the group is paraphyletic. In contrast with the ITS analysis, the morphological tree suggests that species in clade A (ITS) are more derived compared to species placed in clade B (ITS). In addition, while the gene markers differentiate among F. chloraefolia samples collected in northern areas of Mexico/southern USA and the F. chloraefolia specimen from Nuevo León, Mexico (labeled C in the gene analyses) (Figs. 1–3), no single character used in the morphological analysis distinguishes between these specimens. In contrast, morphological differences among collections of F. linearis were observed, suggesting agreement between the morphological data and the non-monophyletic grouping of samples labeled F. linearis in the gene analyses (Figs. 1–3). The F. linearis sample from Belize (labeled "Yucatan" in Fig. 4) is more robust in appearance with broader leaves and loosely aggregated inflorescences, compared to samples from Florida and the Bahamas (Appendix). Among the West Indies samples, the most striking difference is the presence or absence of ray florets in the inflorescence. While this character varies among species of Flaveria, it does not vary within any other Flaveria species, suggesting at least two entities in the Caribbean are included under the taxonomic label "F. linearis."
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) were given twice the weight assigned to other morphological characters. The resulting parsimony tree topology is identical to the tree from analysis of equally weighted characters except that it resolves the basal polytomy shown in Fig. 4, and bootstrap supports are lower (tree not shown). Excluding the outgroup species and re-rooting the data set with either F. cronquistii or F. pringlei does not yield any tree topology similar to those observed in the gene analyses and lowers the consistency index further (trees not shown).
Combined data
The gene marker data sets were not found to be statistically different from each other using PHT (trnL-F vs. ITS: P = 0.05, trnL-F vs. ETS: P = 0.06, ITS vs. ETS: P = 0.06). The different data partitions were therefore combined and analyzed under the same conditions used for the separate gene marker analyses. The combined data set was constructed with each species represented by one specimen with all three markers sequenced (chosen randomly if multiple specimens were available). Where cloned sequences exist for a specimen (e.g., Flaveria pringlei), the representative sequence was chosen randomly (the resulting tree topology was not affected by changing clone sequences). The consensus tree topologies obtained from either parsimony or Bayesian analyses of the combined gene data are congruent, and most nodes are supported with high posterior probabilities and bootstrap values (Fig. 5, Table 3). In the consensus tree, F. mcdougallii is separated from the other species of Flaveria with 100% bootstrap and posterior probability supports. Flaveria cronquistii is located at the base of the tree along with F. pringlei, and a node with good posterior probability support (85%) separating F. robusta and F. sonorensis is present that was not supported in the separate gene analyses. Following this node are two well-supported clades (previously ascribed as clades A and B; Figs. 2, 3). Although F. angustifolia is placed at the base of clade B, this placement is not strongly supported statistically (65%). A polytomy exists in clade B, indicating insufficient genetic signal to resolve species' relationships.
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DISCUSSION
This study uses nucleotide sequence data from one chloroplastic and two nuclear genes in combination with morphological and life history data (Fig. 6). It is the first to provide a nearly fully resolved phylogeny for virtually all known Flaveria species, and the resulting phylogeny enables us to infer the evolution of C4 photosynthesis in Flaveria in an evolutionary context.
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). Based on the hybridization and morphological studies, Powell suggested that this species might represent a distinct, monotypic genus. The results of our phylogenetic analyses and the previous study by Powell (1978)
concur in the exclusion of F. mcdougallii from the genus Flaveria (Fig. 6).
The removal of Flaveria mcdougallii from Flaveria indicates that the true basal species are the large shrub/short tree species, F. cronquistii and F. pringlei (Fig. 6). This suggests that the ancestral condition of Flaveria was a large, shrubby, self-incompatible perennial with glabrous stems and leaves, loosely aggregated, paniculate corymbs, and inflorescences of solely disc-type flowers with 3–4 phyllaries. Flaveria cronquistii is found in the Tehuacán valley of southern Mexico (southeast Puebla and northwest Oaxaca) (Powell, 1978
; A. D. McKown, collection notes). Flaveria pringlei occurs sympatrically with F. cronquistii in the Tehuacán valley region, but is also located across the southern half of Puebla, the northern part of Oaxaca and west into central Guerrero (Powell, 1978
; A. D. McKown, collection notes). The proximity of these two basal Flaveria species suggests that the origin and distributional center for the genus is the south-central region of Mexico (Puebla-Oaxaca) (Fig. 7). There is no evidence of hybridization between F. cronquistii and F. pringlei; however, F. pringlei forms hybrids with F. angustifolia, as previously suggested by Kopriva et al. (1996)
. Four of our seven F. pringlei samples (labeled A, B, D, and G in Figs. 1–3) show direct genetic evidence of hybridization by possessing chloroplast or nuclear gene sequences that are identical to those from F. angustifolia samples. Further morphological evidence of hybridization is observed in the F. pringlei sample labeled B, because ray florets are present in the inflorescences, stem and leaf surfaces are moderately pubescent, and leaf margins are toothed—characters that are observed in F. angustifolia but are absent in typical F. pringlei. In addition to evidence of hybridization, most of our F. pringlei samples have copies of ITS and/or ETS that are more similar to sequences from F. angustifolia than to the remaining F. pringlei sequences. For instance, one allele of ITS from sample C and one allele of ETS from sample F cluster with F. angustifolia sequences (Figs. 2, 3); however, the clustering of these alleles from F. pringlei samples C and F with F. angustifolia sequences occurs in a basal position, suggesting retention of a more ancestral copy of the gene in F. pringlei. Powell (1978)
considered F. pringlei to have evolved from F. angustifolia; however, according to our phylogeny, F. angustifolia is more derived. Overall, we speculate that hybridization between these two species still occurs, because this genus is relatively young and the geographical ranges of the two species are extremely similar (Powell, 1978
; A. D. McKown, collection notes). Hybridization likely accounts for reports of "F. angustifolia-like" morphological characters and gene copies in some F. pringlei specimens.
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; A. D. McKown, collection notes). Flaveria robusta is found northwest of the Puebla-Oaxaca region (in Colima, northern Michoacán, and southern Jalisco) whereas F. sonorensis is substantially further northwest in Sonora and Chihuahua, Mexico (Fig. 7; Powell, 1978
; A. D. McKown, collection notes). Based on the results of the combined analysis, it is feasible that F. robusta is the sister species to F. sonorensis and that long reproductive isolation has resulted in significant genetic variation, as indicated by the long branch lengths observed in our gene marker analyses. The placement of these two species in a separate branch of Flaveria indicates that both Powell's subgeneric phyllary lineages are paraphyletic (Fig. 6).
Clade A
Clade A is the most unambiguous and well-supported group in all analyses, and most species within this clade are well-defined genetically (Figs. 1–3, 5). Clade A includes Flaveria ramosissima, F. palmeri, F. campestris, F. australasica, F. trinervia, F. bidentis (F. haumanii), F. kochiana, and F. vaginata (Fig. 6). Flaveria vaginata and F. kochiana have been suggested as representing a single species as these plants are sympatric and morphologically similar (J. L. Villaseñor, Instituto de Biología, Universidad Nacional Autonóma de México, México D. F., México, personal communication); however, hybridization was not detected from our gene data and the phylogenetic analyses strongly support two independent species. Identical trnL-F sequences and low variability in the nuclear sequences confirm a recent divergence between F. australasica and F. trinervia (Powell, 1978
; Kopriva et al., 1996
). The latter is most likely the result of successful establishment of F. trinervia in Australia and, given the identical morphology and high genetic similarity, could be considered a subspecies of F. trinervia, as suggested by Powell (1978)
.
The species of clade A fall within Powell's 3–4 phyllary lineage and all are annuals, with the exception of F. vaginata (Powell, 1978
) and possibly F. kochiana (E. Sudderth, Harvard University, Cambridge, Massachusetts, USA, personal communication). The only self-compatible Flaveria species are also fully C4, and these species are all included within this clade: F. trinervia, F. australasica, F. campestris, and F. bidentis (Fig. 6; Powell, 1978
). Species in clade A are small to moderately sized herbaceous plants with moderate pubescence, strongly serrated leaves (excepting F. vaginata and F. kochiana), and inflorescences with both ray and disc flowers. In general, floral features are reduced within this clade. For instance, capitulescence shape is contracted to a scorpioid corymb (F. ramosissima, F. campestris, F. palmeri, and F. bidentis) or further reduced to a globose or glomerule shape (F. trinervia, F. australasica, F. vaginata, and F. kochiana). In addition, this clade shows a reduction in phyllary number and shape, from 4–5 broadly elliptic phyllaries in F. ramosissima to 3–4 narrow phyllaries in other species of clade A, and further reduced to two oblong and 1–2 linear phyllaries in F. trinervia and F. australasica. The outermost phyllaries in F. trinervia and F. australasica are narrow and not laterally expanded. These were previously described as "chaff-like setae" (Powell, 1978
); however, these structures are vascularized and are not always completely reduced to narrow, linear structures, but can appear similar to the larger phyllaries.
The basal species of clade A, F. ramosissima, occurs in southern Puebla and northern Oaxaca sympatrically with the basal species of Flaveria and more derived members of clade A (F. vaginata, F. kochiana) (Fig. 7; Powell, 1978
; A. D. McKown, collection notes; E. Sudderth, Harvard University, personal communication). One branch of clade A suggests a northward radiation, as F. palmeri is found in northcentral Mexico and F. campestris occurs across southern and central USA (Fig. 7; Powell, 1978
; A. D. McKown, collection notes). Another branch of clade A represents a possible southern dispersal, as F. bidentis occurs in Central America, the West Indies, and across South America, in addition to being established in other continents as a cosmopolitan weed (Fig. 7; Powell, 1978
; A. D. McKown, collection notes). Flaveria haumanii, the relative of F. bidentis, is located in northern Argentina (Dimitri and Orfila, 1986
) and should also be considered as part of this southern lineage. The other derived species, F. trinervia, is widespread throughout Mexico and the southern USA and is a very successful cosmopolitan weed established on nearly every continent (Powell, 1978
; A. D. McKown, collection notes).
Clade B
Clade B in Flaveria is as diverse in species as clade A, and includes F. angustifolia, F. anomala, F. brownii, F. chloraefolia, F. floridana, F. linearis, F. oppositifolia, and F. pubescens (Fig. 6). In the gene analyses, branch lengths of species in this clade, excepting F. angustifolia and F. anomala, are very short and the lack of genetic variation makes the phylogeny of this clade difficult to resolve without the inclusion of morphological data (Figs. 1–3). The radiation of more derived members in this clade appears to have occurred rapidly, as indicated by lower genetic divergence, and this difficulty in resolution is also evident in the analyses of Kopriva et al. (1996)
and Westhoff and Gowik (2004)
. The basal species, F. angustifolia, is included in clade B by both Bayesian and parsimony analyses of the combined data; however, the posterior probability is low (65%) without the inclusion of morphological and life history data (Fig. 5). Morphological characters observed in F. angustifolia, such as stem pubescence and scorpioid corymb capitulescence, are features that are similar to the basal species of clade A (F. ramosissima); however, other characters, such as shallowly toothed leaf margins, corky phyllaries, campanulate corollas, and pubescent corolla tubes, are shared with the other basal species of clade B (F. anomala). In addition, growth habit and life history of F. angustifolia is more similar to other clade B species than it is to clade A species. The phyllary number of F. angustifolia (3–4) does not correspond with F. ramosissima (4–5) or F. anomala (2–4), and is consistent with species in clade A (3– 4) rather than clade B (5–6), indicating homoplasy in the focal character previously used to designate subgeneric groups (Powell, 1978
).
The species in clade B are circumscribed in Powell's 5–6 phyllary line, with the exception of F. angustifolia and F. anomala (Fig. 6; Powell, 1978
). Species in clade B species are all self-incompatible and most are perennial, excepting F. anomala, which is annual, although it is suggested that F. brownii and F. floridana might be long-lived annuals (Fig. 6; Powell, 1978
). Unlike clade A, there is not a strong trend in reducing floral features, and extensive homoplasic variation in vegetative and floral characters is present among these species. Plants of clade B species are moderately sized herbaceous or shrubby plants with sessile, linear to narrowly lanceolate leaves (excepting F. chloraefolia). The capitulescence shape tends to be a paniculate corymb, and corolla tubes are sparsely to moderately pubescent in these species. All samples from the different Flaveria species within clade B form respective monophyletic groups except samples labeled F. chloraefolia and F. linearis (Figs. 1–3). The genetically distinct, but morphologically similar F. chloraefolia sample labeled C (Figs. 1–3) is distinguished only by leaf length from other F. chloraefolia specimens and by its disjunct geographical location (Nuevo León). Further study is required to determine the extent of the range of this plant and whether any other morphological differences distinguish it from F. chloraefolia sensu stricto. The other paraphyletic taxon, F. linearis, likely represents more than one distinct entity, evident from both the genetic and morphological data. The more basal specimen according to our phylogeny (sample labeled C; Figs. 1–3, 5) is found in the Yucatán Peninsula area (A. D. McKown, collection notes) and is separated from other F. linearis samples with 100% posterior probability and 93% bootstrap supports (Fig. 5). Powell (1978)
observed that F. linearis plants from this region are similar in morphology to F. brownii, which is consistent with our molecular phylogenetic results and morphological observations (Appendix). Features of these robust plants from the Yucatán Peninsula that are comparable to F. brownii include glabrous stems, glabrous, entire to shallowly toothed, narrowly lanceolate leaves, and loose, paniculate corymbs with ray and disc-type flowers. The other F. linearis specimens (samples labeled A, B, D–F; Figs. 1–3, 5) from the Bahamas and Florida form a clade with F. floridana but do not demonstrate cohesion either genetically or morphologically. Variability in morphological characters of F. linearis has also been noted by Long and Rhamstine (1968)
and Powell (1978)
, in addition to physiological variability (Edwards and Ku, 1987
). In this study, Floridean samples A and F and F. floridana cluster together with 98% posterior probability support and exclude samples labeled B and E (Keys) and D (Bahamas) (Fig. 5). The samples from the Keys and Bahamas have only disc flowers, whereas all other samples demonstrate ray and disc flowers. While samples from the Keys cluster together with 100% posterior probability (Fig. 5), there is little morphological similarity beyond inflorescence characters (Appendix). Therefore, it is clear that further detailed study of these entities, including F. floridana, is required to determine the history of this group and the potential number of taxa that may be included under "F. linearis."
The radiation of clade B has been restricted to North America and the West Indies (Fig. 7; Powell, 1978
; A. D. McKown, collection notes). No species in this clade demonstrates the wide geographic range of the C4 cosmopolitan weeds in clade A. The basal species F. angustifolia is found in the Tehuacán valley, and across the southern half of Puebla, northern part of Oaxaca, and west into central Guerrero (Powell, 1978
; A. D. McKown, collection notes) sympatrically with F. pringlei. The other basal species, F. anomala, is found in the northeastern states of Mexico (in southern Coahuila, Durango, Nuevo León, San Luís Potosí, Queretaro, Tamaulipas, and eastern Zacatecas) (Fig. 7; Powell, 1978
; A. D. McKown, collection notes). Flaveria chloraefolia sensu stricto, F. oppositifolia, and F. pubescens are also found in this area of northeast Mexico, and the range of F. chloraefolia sensu stricto also extends into Chihuahua, Texas, and New Mexico (Fig. 7; Powell, 1978
; A. D. McKown, collection notes). Flaveria brownii occurs northwards from this northeastern area in Mexico in Tamaulipas and along the Texan Gulf of Mexico coast (Fig. 7; Powell, 1978
; A. D. McKown, collection notes). A southeastern branch of clade B Flaveria, including F. floridana and the variable entities of F. linearis, extends from the Yucatán Peninsula to the West Indies and Florida (Fig. 7). The more basal F. linearis (represented by sample C) is found in the Yucatán and Quintana Roo (Mexico), Belize, and Honduras (Powell, 1978
; A. D. McKown, collection notes) and may extend into Cuba based on morphological similarities of the specimens studied. If this entity is indeed the most basal of the F. linearis complex, it supports Powell's (1978)
hypothesis that F. linearis originated in Mexico and spread eastward to the West Indies and into Florida. Consistent with this hypothesis is the recent divergence between F. floridana and F. linearis specimens from Florida.
Evolution of C4 photosynthesis
The results of this study demonstrate two independent origins of C3-C4 intermediacy and C4-like photosynthesis from C3 ancestry. They also provide a phylogenetic context to examine physiological, genetic and ecological factors promoting the evolution of C4 photosynthesis in dicots.
Multiple origins of C3-C4, C4-like, and C4 photosynthesis
All Flaveria species with C3 photosynthesis are restricted to the basal portions of the phylogeny (Figs. 5, 6). This result is also supported by the H-protein phylogeny of Kopriva et al. (1996)
, but is not apparent in the Westhoff and Gowik (2004)
PEPCase phylogeny. The inclusion of all known C3 Flaveria species in our study confirms that the ancestral condition in Flaveria is unambiguously C3 photosynthesis. Photosynthetically intermediate Flaveria species are all placed within clades A and B, excepting C3-C4 F. sonorensis. The phylogenetically disjunct F. sonorensis is also geographically distant from the other intermediate Flaveria species and represents an independent evolution of C3-C4 intermediacy. Physiological characteristics of F. sonorensis indicate that photosynthesis in this species is more C3-like (Moore et al., 1987
; Ku et al., 1991
). The CO2 compensation point of F. sonorensis is lower than that of its sister species F. robusta, but there is relatively little difference in O2 inhibition between the two species. In addition, F. sonorensis does not operate a partial C4 cycle (Ku et al., 1991
) and should be considered a type I intermediate (no C4 cycle [Edwards and Ku, 1987
]). Within clade A and B, the absence of C3 species at the base of both clades suggests a second origin of C3-C4 intermediacy in the shared common ancestor of these clades. This shared origin of intermediacy giving rise to both lineages was also suggested by Kopriva et al. (1996)
. While the basal species of clades A and B (F. angustifolia and F. ramosissima) are C3-C4 photosynthetic intermediates, the physiological characteristics of both species differ. Flaveria angustifolia is similar to F. sonorensis, classifying it as a type I intermediate; however, F. ramosissima has a substantially lower CO2 compensation point, lower O2 inhibition, and a limited C4 cycle compared to C3 species and type I C3-C4 intermediates (Ku et al., 1991
), and is a type II intermediate (with C4 cycle [Edwards and Ku, 1987
]).
Within clade A, type II C3-C4 photosynthesis coincides with the evolution of an annual life cycle in Flaveria ramosissima (Fig. 6). The phylogenetic position of F. ramosissima suggests that its common ancestor with other clade A species (C4-like and C4) was C3-C4, thereby supporting an evolutionary intermediate position for C3-C4 photosynthesis. The common ancestor to the other derived clade A species (C4-like and C4) may have been an advanced intermediate similar to the C4-like species F. palmeri, suggesting that fully expressed C4 photosynthesis may have evolved up to three times in clade A (once each in F. australasica/trinervia, F. bidentis/haumanii, and F. campestris) along with the evolution of self-compatibility (Fig. 6). An alternative hypothesis, although unlikely, is that the common ancestor to C4-like and C4 clade A species was fully expressed C4 photosynthesis and that both C4-like F. palmeri and F. vaginata are reversals from C4 photosynthesis (Monson and Moore, 1989
). Further study to determine the photosynthetic type of F. kochiana (currently known to be either C4-like or C4 [A. D. McKown, unpublished data]) may assist in interpretation of the photosynthetic evolutionary history in this clade.
All species in clade B are C3-C4 intermediate, excepting Flaveria brownii, which is classified as having C4-like photosynthesis, and fully expressed C4 photosynthesis is absent in clade B. The short branch lengths of the more derived portion of clade B in the genetic analyses (Figs. 2, 3) may indicate a rapid evolution and radiation of species and prevent the full reconstruction of the evolutionary history of this clade from our data. As a result, only the basal C3-C4 species F. angustifolia and F. anomala are placed with certainty in a phylogenetically intermediate position between C3 photosynthetic species and C4-like F. brownii. The other C3-C4 species do not demonstrate unequivocal intermediate phylogenetic placements, although the trnL-F and ITS analyses suggest that F. chloraefolia should be considered phylogenetically basal to C4-like F. brownii. The basal species F. angustifolia is designated as a type I intermediate; however, the other basal species, F. anomala, has physiological characteristics supporting its classification as a type II intermediate species (Edwards and Ku, 1987
; Moore et al., 1987
; Ku et al., 1991
). Similar to F. ramosissima of clade A, the appearance of type II intermediacy in F. anomala coincides with annualism (Fig. 6). Of the remaining C4-C4 intermediate species, most demonstrate little C4 cycle activity, except F. floridana (Monson et al., 1986
; Moore et al., 1987
; Chastain and Chollet, 1989
; Ku et al., 1991
). The derived species F. brownii with C4-like photosynthesis is clearly not directly related to the C4-like and C4 species of clade A, supporting Powell's (1978)
hypothesis that C4 (or C4-like) photosynthesis in Flaveria has evolved independently at least twice (Fig. 6). The presence of one "advanced" C3-C4 species (F. floridana) and one C4-like species (F. brownii) at the tips of clade B does not signify that all C3-C4 species in this clade are evolving towards full C4 photosynthesis. It is possible that F. linearis (all entities) and F. floridana are both undergoing photosynthetic-type reversal (Monson and Moore, 1989
), but the recent origin of these species, as indicated by the phylogenetic analyses, does not suggest sufficient evolutionary time for this scenario. The evolutionary intermediate positions of C3-C4 photosynthesis (in F. angustifolia and F. anomala) and C4-like photosynthesis (in F. brownii) is clear; however, caution should be exercised in the interpretation of other clade B Flaveria species.
Photosynthetic intermediate species in comparative studies are generally interpreted to represent evolutionary steps progressing towards the development of fully expressed C4 photosynthesis (Edwards and Ku, 1987
; Monson, 1989
; Monson and Moore, 1989
). Evolution patterns in Flaveria demonstrate this to be the case with intermediacy in clade A, although this is not as clear among intermediates in clade B. Monson and Moore (1989)
discussed three alternatives for the origin of C3-C4 intermediate species. The first considers C3-C4 intermediates as a product of reverse evolution from C4 photosynthesis; however, the ecology of the intermediates and the unapparent adaptive advantage this strategy would confer do not support this hypothesis (Monson and Moore, 1989
). The recent divergence of Flaveria species, especially derived clade B species, suggested by our phylogeny also rejects this hypothesis. A second hypothesis suggests that C3-C4 intermediates are the result of hybridization between C3 and C4 plants. Within Flaveria, reproductive isolation exists among some species, and the only C3 species observed to form hybrids does not do so with C4 plants (Powell, 1978
). In addition to the naturally occurring F. pringlei x F. angustifolia hybrids, only two other hybrids were found. These were between research collection plants but were not hybrids of C3 and C4 species. Therefore, it is highly unlikely that the C3-C4 Flaveria intermediates arose from hybridization between C3 and C4 Flaveria. Despite the recent and rapid radiation inferred by the phylogenetic results of clade B and the high crossability of species in this clade (Powell, 1978
), lack of hybrid evidence suggests that intermediacy was not spread through this clade by means of hybridization. The third hypothesis is that C3-C4 intermediates are evolutionary "dead-ends" and will not evolve fully expressed C4 metabolism (Monson and Moore, 1989
). Phylogenetic evidence suggests that this is not the case in clade A (Figs. 5, 6). The diversity of physiological characteristics in clade B photosynthetic intermediates (type I and type II) demonstrates that C3-C4 intermediacy is not identical in these species (Edwards and Ku, 1987
; Ku et al., 1991
, Dai et al., 1996
), which is also suggestive that these intermediates are not "dead-ends." As mentioned by Monson and Moore (1989)
, niches can exist for intermediate plants, and therefore the potential to evolve fully expressed C4 photosynthesis exists, but is not realized without C4-selecting conditions.
Ecological conditions and life history traits
Sage (2004)
outlines that under current atmospheric conditions and at warm temperatures (>30°C), photosynthesis can be inhibited by 30% due to photorespiration. Some intermediate species of Flaveria (type II with a limited C4 cycle [Edwards and Ku, 1987
]) demonstrate an increase in initial assimilation of CO2 into C4 acids under low CO2 levels, which promotes photorespiration (Chastain and Chollet, 1989
). This supports the hypothesis that CO2 levels play a selective role in C4 photosynthesis in Flaveria. In addition to the lower CO2 levels recorded in recent geological time, other environmental conditions promoting photorespiration could act as selective forces, such as heat, drought, low humidity, or salinity (Sage, 2001
, 2004
). The Puebla-Oaxaca region of Mexico is warm and arid, and two C3-C4 intermediate species in this region (F. angustifolia of clade B and F. ramosissima of clade A) are known to occur in sandy and possibly gypseous soils (Powell, 1978
). The aridity of this environment and the potential nutrient-deficiency of these soils (Jafarzadeh and Zinck, 2000
) could act to select for adaptations compensating for a higher rate of photorespiration and water use efficiency (Sage, 2004
). The two basal C3 species (F. cronquistii and F. pringlei) occur, however, in the same region and in similar habitats as these intermediates (Powell, 1978
). C3-C4 intermediate F. sonorensis and species of clade B are all described as occurring in saline or gyseous soils, but are also generally found in more mesic habitats (Long and Rhamstine, 1968
; Powell, 1978
). In particular, C4-like F. brownii is found along saline, sandy coastal flats and brackish marshes of the Gulf of Mexico. This highly saline environment may have represented a more stringent selective force (e.g., for higher water use efficiency) and propelled F. brownii towards developing C4-like photosynthesis. Supporting this hypothesis are the ecological and physiological data from F. floridana, as this species also occurs in coastal saline, sandy soils near brackish marshy areas in Florida (Long and Rhamstine, 1968
) and is a physiologically advanced C3-C4 intermediate in clade B (Monson et al., 1986
, 1988
; Moore et al., 1987
; Brown and Hattersley, 1989
; Chastain and Chollet, 1989
; Ku et al., 1991
; Dai et al., 1996
). Monson and Jaeger (1991)
demonstrated that C3-C4 intermediacy in F. floridana conveys ecological advantages (e.g., higher rates of photosynthesis) compared to C3 plants in the same natural habitat at high temperatures. Thus, despite variability in habitats, the ecological conditions Flaveria species currently experience (high light intensity, heat, aridity, and saline or gypseous soil) suggest environments that promote photorespiration and might exert adaptive pressure towards evolving the development of full C4 photosynthesis.
Flaveriinae species (Flaveria, Haploësthes, and Sartwellia) are found in gypseous soils, indicating a common ecological niche; however, photosynthetic intermediacy and C4 photosynthesis are only known in Flaveria. Gene duplication and neofunctionalization are outlined in Flaveria as preceding the evolution of many C4 biochemical attributes (reviewed in Monson, 2003
). It is likely that this genetic "preconditioning" exists in Flaveria species of clades A and B, but is lacking in the basal C3 Flaveria, Haploësthes and Sartwellia species. Assuming similar genetic preconditioning for Flaveria species, the rate at which the C4 syndrome can evolve in each species may be determined by the species' life history (Monson, 2003
). This hypothesis is supported by the co-existence of annualism and C4, C4-like or physiologically advanced C3-C4 intermediate photosynthesis in both derived clades of Flaveria species (Fig. 6). In annual plants, generation time is shorter and the rate of gene recruitment and modification for C4 photosynthesis is potentially higher (Monson, 2003
). Gene recruitment processes may be enhanced by inbreeding in Flaveria, as all known C4 species of Flaveria are self-compatible (Powell, 1978
). The recent evolution of C4-like photosynthesis in F. brownii indicates that the intensity of environmental selection pressures, such as heat and salinity, also plays a major selective role in the evolution of C4 photosynthesis in the genus Flaveria. Together, the number of fully developed C4 species in this genus and the multiple evolutions of intermediacy all suggest a latent ability to evolve C4 photosynthesis. Within Flaveria, however, we hypothesize that the evolution of this complex adaptation is realized through the intensity of environmental selection pressure and through modification of a species' life history to exploit the presence of genetic preconditioning outlined in Flaveria.
Concluding remarks
The well-resolved phylogeny of nearly all Flaveria species enables a better understanding of the evolutionary relationships among Flaveria species and the biogeographical patterns of dispersal in this genus. The presence of intermediate (C3-C4 and C4-like) species in separate clades of Flaveria is in agreement with earlier studies that have suggested multiple origins of photosynthetic intermediacy in this genus (Powell, 1978
; Kopriva et al., 1996
; Monson, 1996
; Westhoff and Gowik, 2004
). Our phylogenetic evidence indicates more precisely where, and how many times, multiple and parallel evolutions of C3-C4 or C4-like photosynthesis have occurred. This information will be useful in evaluating the appropriateness of species choice in future comparative studies and supports review of the large body of existing Flaveria research in a phylogenetic context. Flaveria characteristics, especially of the intermediate species, underpin many models of C4 evolution and phylogeny (Rosche and Westhoff, 1995
; Drincovich et al., 1998
; Monson, 1999, 2003;
Engelmann et al., 2003
; Sage, 2004
; Westhoff and Gowik, 2004
). Within the phylogenetic context proposed, characteristics in the progression of C3-C4 to C4-like/C4 photosynthesis in clade A, and traits that have evolved in parallel among species of clade A and B can be identified. This will greatly facilitate testing stepwise evolutionary models for C4 photosynthesis in dicots in general, thereby advancing our understanding of C4 photosynthetic evolution in plants.
|
1 This work was supported by NSERC (NGD) and the Canada Foundation for Innovation/Ontario Innovation Trust (grant no. 7475 to JMM). The authors thank Dr. M. Powell for advice and plant material, Dr. T. Dickinson for curatorial assistance and helpful discussion, Drs. J. Bruhl and R. Sage for manuscript review, S. Margaritescu and O. Haddrath for technical assistance, E. Sudderth for plant material, Dr. S. Malcomber for donation of trnL-F primers, and Drs. J. E. Eckenwalder and J. L. Villaseñor for helpful discussion. 
4 Author for correspondence (e-mail: dengler{at}botany.utoronto.ca
)
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