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Systematics |
2Department of Botany, University of Oklahoma, Norman, Oklahoma 73019-0245 USA; 3Department of Biological Sciences, Moorpark College, Moorpark, California 93021-1695 USA
Received for publication February 21, 2003. Accepted for publication August 29, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Key Words: ETS • Gaura • ITS • molecular systematics • Onagraceae • Stenosiphon • trnL-F
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). This analysis of Gaura is part of an ongoing and detailed study of the Onagraceae, which has generated a comprehensive base of information from morphology-anatomy (Rothrock, 1864
; Munz, 1938
, 1965
; Raven, 1964
; Raven and Gregory, 1972a
; Keating, 1982
; Carr et al., 1990
; Heslop-Harrison, 1990
; Hoch et al., 1993
), embryology (Tobe and Raven, 1983
, 1985
, 1986
), reproductive biology (Raven, 1979
), palynology (Patel et al., 1984
), chromosomes (Kurabayashi et al., 1962
; Raven and Gregory, 1972b
), molecular markers (Sytsma and Gottlieb, 1986
; Hoggard et al., 2000
; Levin et al., 2003
) and pollination ecology (Clinebell and Hoch, 2001
; R. R. Clinebell, A. Crowe, D. P. Gregory, and P. C. Hoch, unpublished manuscript). Phylogenetic relationships between Gaura and the other genera within Onagreae are still in the process of being clarified and elements of its infrageneric classification have been disputed in previous accounts (Carr et al., 1990
; Levin et al., 2003
). Hybrid speciation has been suggested as an important evolutionary mechanism within the genus (Raven and Gregory, 1972a
, b
; Carr et al., 1986a
, b
, 1988a
, b
), but the status of the putative hybrids has never been verified. Cytological evidence of polyploidy as well as varying degrees of translocation heterozygosity have been detected within Gaura, including permanent translocation heterozygosity. However, what role these phenomena play in stabilizing hybrid complexes in Gaura remains unclear. The present paper uses molecular sequence data to (1) elucidate phylogenetic relationships within the genus, (2) reassess the sectional delimitations proposed by Raven and Gregory (1972a)
and Carr et al. (1990)
, (3) reexamine the segregate status of Stenosiphon Spach, another genus in tribe Onagreae with indehiscent fruit similar to Gaura, and (4) investigate hybrid speciation within Gaura.
Gaura is a North American genus that extends across the southwestern and central United States to the east coast, north to southern Canada, and south to include Mexico and Guatemala with its center of diversity in Texas (Raven, 1979
). Representatives of the genus are annual, biennial, or perennial herbs with one to several branched or unbranched stems extending from a taproot or a woody caudex (Rothrock, 1864
; Munz, 1938
, 1965
; Raven and Gregory, 1972a
). The alternate leaves decrease in size upward from a basal rosette that is usually deciduous by the time of flowering. The flowers of most species of Gaura are white to pink, four-merous, zygomorphic, night-blooming, moth-pollinated, and have an appendage at the base of the staminal filaments. The ovary is inferior with a narrow floral tube and the style is deflexed and filiform. The non-commissural stigma is most often four-lobed and has an indusium (a cuplike ring of tissue) surrounding its base. The fruit is a nutlike, indehiscent capsule with absent or incomplete septa that in some species narrows to a sterile base called the stipe. Seeds are brown, ovoid, and often flattened. The basic chromosome number is n = 7 (Raven and Gregory, 1972b
). Eighteen species are diploid and three are polyploid.
Linnaeus (1753)
first described Gaura based on plants grown from seeds of G. biennis collected in eastern Pennsylvania. Over the next 70 years, six additional species were identified, including G. linifolia Nutt. (Nuttall, 1823
). Spach (1835)
determined the filiform hypanthium and one-seeded capsule of G. linifolia justified its placement outside of Gaura, thus he created a segregate genus, Stenosiphon Spach, represented solely by Stenosiphon virgatus Spach (= G. linifolia). However, Spach failed to adopt the earliest available epithet for his species, and this illegitimate name persisted until Heynhold (1840)
published the combination Stenosiphon linifolius (Nutt. ex E. James) Heynh.
The earliest taxonomic treatment of Gaura (Rothrock, 1864
) examined 14 Gaura species and Stenosiphon. In this treatment, Rothrock used two character states to delimit Gaura, an appendage at the base of the staminal filaments and an indusium surrounding the base of the stigma. He determined that the absence of these characters in G. heterandra was cause to establish a new, monotypic genus, Heterogaura Rothr., represented by H. californica Rothr., a mistake in the specific epithet later corrected by Coville (1893)
, who published the combination H. heterandra (Torr.) Cov. Rothrock also observed that the habit of Heterogaura was more like Clarkia than Gaura, an observation later supported by molecular sequence data (Sytsma and Gottlieb, 1986
; Ford and Gottlieb, 2003
), which placed Heterogaura within Clarkia. Regarding Stenosiphon, Rothrock noted the lack of appendages at the base of the filaments but the presence of an indusium and made no changes in the classification. Interestingly, because specimens of it were not available for him to examine, Rothrock did not include a previously described Mexican species, G. mutabilis (Cavanilles, 1795
), which, like S. linifolius, lacks an appendage at the base of the filaments but has an indusium. Had Rothrock been able to include G. mutabilis in his study, this similarity would have complicated his support for maintaining Stenosiphon as a genus.
The second revision of Gaura (Munz, 1938
) recognized 18 species grouped into two sections; sect. Gauridium consisting of the yellow-flowered G. mutabilis and sect. Gaura including the remaining 17 species with white to pink flowers. Munz did not include or mention Stenosiphon in this revision other than to say his opinions regarding this genus would be addressed in a forthcoming, comprehensive treatment of the Onagraceae. In his monograph of the North American species of the Onagraceae, Munz (1965)
made only minimal changes to his original treatment of Gaura. He discussed Stenosiphon, noting the lack of staminal appendages and the presence of an indusium but did not suggest a change in classification.
The most recent revision of Gaura (Raven and Gregory, 1972a
) recognized 21 species (Table 1) and delimited eight sections based on fruit morphology, floral symmetry, and plant growth form. Four of these sections are monotypic and 17 species are distributed unevenly over the remaining four sections. A cladistic analysis (Carr et al., 1990
) using morphological information derived from Raven and Gregory's treatment of Gaura (1972a) tested the sectional divisions and evaluated some of the hypotheses concerning phylogenetic relationships within the genus. Calylophus Spach (tribe Onagreae) was chosen as the outgroup based on two embryological characters it shares with Gaura. Stenosiphon was not included in this study. Twenty characters were selected from plant growth characteristics and fruit and flower morphology. This analysis supported the majority of Raven and Gregory's phylogenetic hypotheses regarding the infrageneric classification of Gaura. Six of the eight sections that they recognized were monophyletic in the cladistic analysis. However, sections Xerogaura and Gaura did not form monophyletic groups.
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; Baldwin et al., 1995
; Vargas et al., 1998
, 1999
; Molvray et al., 1999
). The ETS region was selected to augment our ITS data because it often has a higher rate of substitution than ITS (Linder et al., 2000
), and the combination of data from both regions can improve resolution and support (Baldwin and Markos, 1998
; Bena et al., 1998
; Clevinger and Panero, 2000
; Markos and Baldwin, 2001
). The chloroplast trnL-F region was included because its utility at the generic and infrageneric level has been demonstrated (Gielly and Taberlet, 1994
; Kores et al., 2001
; Levin et al., in press
) and because its uniparental, rather than biparental, mode of inheritance can facilitate the detection of hybrids. |
MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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, Carr et al. (1990)
, and Levin et al. (2003)
. For taxon names and voucher documentation, see the Appendix (accompanying the online version of this article). Voucher specimens are deposited at OKL.
Total genomic DNA was extracted from fresh or silica-dried plant material using a standard 2% m/v hexadecyltrimethylammonium bromide (CTAB) protocol (Doyle and Doyle, 1987
). All DNA samples were purified by ultracentrifugation using a CsCl2-ethidium-bromide density gradient (1.55 g/mL). Amplification was carried out in a Perkin-Elmer (Norwalk, Connecticut, USA) model 9700 thermal cycler using 100 µL polymerase chain reactions (PCR). The protocol used 2.5 units of Taq polymerase (Promega, Inc., Madison, Wisconsin, USA), 2 µL 4% bovine serum albumin, 2.5 µmol/L MgCl2, and 100 ng of the two PCR primers. The ITS primers used for the initial PCR reaction were AB101 and AB102 (Douzery et al., 1999
). In a few cases when the genomic DNA appeared partially degraded, the internal primers ITS2 and ITS3 (White et al., 1991
) were used in conjunction with AB101 and AB102 to amplify the ITS region as two fragments. The PCR profile included an initial premelt of 1 min 30 s at 94°C, followed by 26 cycles of 1 min denaturation (95°C), 1 min annealing (48°C), and 2 min 30 s elongation (72°C), followed by a 7 min final extension at 72°C. Amplified products were purified using Promega Wizard PCR Minicolumns in accordance with the manufacturer's protocols.
Primer development for ETS required amplification of the entire intergenic spacer region using long-distance PCR. Universal PCR primers located at the 3' end of the 26S and the 5' end of the 18S were used for the initial amplification (Bena et al., 1998
). The PCR was performed with 50-µL reactions using 2.25 units of Taq polymerase and 0.25 units of Pfu polymerase (Promega), 5 µL of Pfu reaction buffer (containing 2 mmol MgSO4), 3 µL 4% bovine serum albumin, 5 µL of 5 mol betaine, and 50 ng of the two PCR primers. A modified two-step PCR protocol was used (Perkins, 2001
), which included an initial premelt of 1 min at 94°C, followed by 35 cycles of 15 s denaturation (94°C), 15 min annealing, and extension at 68°C. The PCR products were column purified, sequenced from the 3' end, and a new forward primer (Gaura-ETS-F) was developed approximately 600 base pairs (bp) upstream from the beginning of the 18S region. Gaura-ETS-F (5'-CCG GAC GAC GCA CGT GGA-3') and 18SNS1R (5'-TTG AGA CAA GCA TAT GAC TAC-3') were used in subsequent PCR amplifications to generate the ETS sequences used in this study. Reaction conditions were similar to ITS, however, the cycling protocol was modified as follows: an initial premelt of 2 min at 94°C, followed by 28–30 cycles of 30 s denaturation (94°C), 30 s annealing (44–52°C), and 1 min extension at 72°C, followed by a final extension of 7 min at 72°C.
The two PCR primers for trnL-F were c and f (Taberlet et al., 1991
). The PCR protocol differed from that of ITS and ETS in that it included an initial premelt of 2 min at 94°C, followed by 30 cycles of 1 min denaturation (94°C), 30 s annealing (50°C), and 1 min elongation (72°C), and ending with a final extension at 72°C.
Cycle sequencing was carried out directly on the purified PCR product using the ABI Prism Big Dye Terminator Cycle Sequencing Ready Reaction kit (Applied Biosystems, Foster City, California, USA), with 10 ng of primer, 3 µL of sequence dilution buffer, and 2 µL of cycle sequence mix in a 20-µL reaction volume. Cycle sequencing conditions were as follows: 30 cycles of 15 s denaturation (96°C), 1 s annealing (50°C), and 4 min elongation (60°C) using a Perkin-Elmer 9700 thermal cycler. Sequencing reactions were purified by ethanol precipitation and run on an ABI Prism 377 automated sequencer (PE-Applied Biosystems). Electropherograms were assembled and edited with Sequencher 3.1 software (GeneCodes, Ann Arbor, Michigan, USA). Sequences were aligned manually.
Two of the sequenced regions, ITS and ETS, used in this study are part of a tandem repeat within the diploid nuclear genome, while the third region, trnL-trnF, is part of the haploid chloroplast genome. Because the modes of inheritance are different for these two genomes, biparental vs. uniparental, their evolutionary histories are not linked (Doyle, 1992
; Moore, 1995
). Thus, there is no a priori reason to assume that the three resulting gene trees will be identical. Possible conflict between the three data sets was evaluated with an incongruence length difference test (ILD) (Farris et al., 1994
, 1995
) prior to combining the data. This test, implemented as the partition homogeneity test in PAUP* (Swofford, 2001
), determines whether the original data partitions differ significantly from randomly shuffled partitions of the combined data set. One hundred replicates were performed on parsimony-informative characters using TBR (tree bisection and reconnection) branch swapping, simple sequence addition, MULTREES on, Steepest Descent in effect, and MaxTrees set at 100. The ILD test was chosen because recent studies have shown that it performs better than other tests designed for the same purpose under a wide range of conditions (Cunningham, 1997a
, b
). However, Liu and Miyamoto (1999)
caution that the ILD test is prone to Type 1 errors (findings of significant incongruence when none actually exists) and recommend that data partitions should be provisionally combined and the results evaluated. In this study, trees obtained for each gene region were also examined for "hard" or "soft" incongruencies (Seelanan et al., 1997
), and the data were combined following the suggestions of Liu and Miyamoto (1999)
. This approach has been used to increase phylogenetic resolution in studies of other plant groups (Whitten et al., 2000
; Cameron et al., 2002
; Hall et al., 2002
) and in family level studies of Onagraceae (Levin et al., 2003
).
Phylogenetic analyses were performed using PAUP* (Swofford, 2001
). Gaps were inserted at positions where insertions-deletions (indels) occurred, and these areas were treated as missing data in the nucleotide matrix (Swofford, 2001
). Indels were scored as binary characters using PaupGap (Cox, 1997
) and appended to the sequence matrix for inclusion in the maximum parsimony (MP) analyses. Starting trees were obtained using random sequence addition, searched using equally weighted MP (Fitch, 1971
) with TBR branch swapping, MULPARS on, Steepest Descent not in effect, and MaxTrees set at 5000. Maximum likelihood (ML) scores for all the equally parsimonious trees obtained were calculated using a GTR + 
+ I substitution model (Rodriguez et al., 1990
) with indel characters excluded. This model used the following parameters: rate matrix AC = 1.5430, AG = 1.1717, AT = 0.6396, CG = 0.51329, CT = 2.67615, GT = 1.0; a discrete gamma rate distribution with four categories with the shape parameter 
= 0.8488; the proportion of invariable sites I = 0.55411 (all estimated from the MP trees).
Internal support was assessed by nonparametric bootstrapping (Felsenstein, 1985
) using equally weighted characters. Bootstrap percentages (BP) for each node were computed after resamplings followed by a maximum parsimony (MP) reconstruction (bootstrap option in PAUP* with 1000 replicates of heuristic search, one random sequence addition per replicate, and TBR branch swapping and MaxTrees set to 1000).
Separate analyses were performed for each gene region, with and without the binary-coded characters representing indels, and bootstrap support was calculated. A total evidence analysis incorporating data from all three regions was performed with and without the binary-coded indel characters. Because of conflicts between the three gene trees in the placement of G. drummondii, this species was excluded from the combined analysis.
Alternative topologies designed to investigate traditionally defined relationships within Onagreae and to assess the monophyly of the sections within Gaura (Raven and Gregory, 1972a
) were constructed using MacClade (Maddison and Maddison, 2000
). The cost of these alternate topologies in parsimony was assessed by implementing the TOPOLOGICAL CONSTRAINTS function in PAUP*. Support for the alternative branching hypotheses was evaluated with a one-tailed Shimodaira-Hasegawa nonparametric test (SH) (Shimodaira and Hasegawa, 1999
; Goldman et al., 2000
) of the MP trees against the alternative trees using the same substitution model and likelihood settings described earlier. To reduce the computational time, we used the resampling estimate log-likelihood method (RELL option in PAUP*) (Kishino and Hasegawa, 1989
) with 1000 bootstrap replicates. When more than one MP tree was obtained, the trees were sorted according to their likelihood scores (Sort Trees option in PAUP*), and a separate SH test was performed on the tree with the highest and lowest ML scores (Stefanovic et al., 2002
).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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, b
; Raven, 1979
), their inclusion in a parsimony analysis is potentially disruptive. The degree of disruption to phylogenetic restructuring depends on the degree of relationship between the parent species (McDade, 1992
; Judd et al., 1999
). Disruption is minimal when parental species are closely related as is the case for G. sinuata. Our results show G. sinuata occupies the same position in the trees from all three gene regions and its exclusion from any of the analyses has no effect on other portions of the topology. Conversely, the degree of disruption to phylogenetic restructuring can be problematic when parental species are from widely separate lineages. Raven and Gregory (1972a)
postulated that G. drummondii arose from an interspecific hybridization between G. coccinea (sect. Campogaura) and a representative from sect. Stipogaura, possibly G. mckelveyae. Our molecular data support the hypothesis that G. drummondii arose via interspecific hybridization between parental species from widely separate lineages within Gaura. As a result, its position varies in each of the three separate trees (Fig. 2) and its inclusion in a combined analysis is disruptive. For this reason, the taxon has been excluded from the combined analysis.
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The ETS analysis
The analysis of the ETS data set produced 1716 equally parsimonious trees with a length of 162 steps, CI = 0.8580 and RI = 0.9102. In this tree, there is strong support for the monophyly of Gaura plus Stenosiphon (BP = 99%) and the G. coccinea-G. boquillensis clade (BP = 97%). Oenothera speciosa (sect. Xylopleurum) is sister to the Gaura-Stenosiphon clade but this relationship is only weakly supported (BP = 66%). The six species of Gaura included in sect. Gaura (G. lindheimeri, G. angustifolia, G. neomexicana, G. longiflora, G. biennis, and G. demareei) form a weakly supported group (BP = 67%), but the other sections of the genus are not supported.
The trnL-F analysis
After excluding G. calcicola, G. mckelveyae, and G. neomexicana because of missing sequences, the analysis of the trnL-F data produced 12 equally parsimonious trees with a length of 113 steps, CI = 0.8230 and RI = 0.8261. In this analysis, the monophyly of Gaura plus Stenosiphon has moderate support (BP = 78%). The Gaura-Stenosiphon clade forms a polytomy with O. speciosa (sect. Xylopleurum) and O. macrocarpa (sect. Megapterium) with strong bootstrap support (BP = 92%). Within Gaura, only the sects. Stipogaura (BP = 56%) and Gaura (BP = 61%) are monophyletic. There is also moderate support for the G. coccinea-G. boquillensis clade (BP = 77%) and weak support for the G. parviflora-Stenosiphon clade (BP = 50%). However, the remaining portions of the tree are largely unresolved.
Combined analysis
The ILD test indicated that there is no significant incongruence between the ITS and ETS data sets (P = 0.44–0.57 with and without the binary characters representing the indels) or the ITS and trnL-F data sets (P = 0.82–0.86), however, there is significant incongruence between the ETS and trnL-F data sets (P = 0.01). Because of the ILD test results, the topologies obtained in the separate analyses for each region were compared for hard incongruencies. Because no hard incongruencies were found between the three trees and the ILD test is known to be prone to Type I errors (Liu and Miyamoto, 1999
), the three data sets were combined.
The combined analysis using all three regions across 28 taxa (G. drummondii excluded) resulted in 504 most parsimonious trees. After three rounds of successive weighting, the number of most parsimonious trees was reduced to 10 (weighted length = 317 082, base weight = 1000, Fitch length = 445; CI = 0.8546; RI = 0.9480). These 10 trees differ in the branching order of three clades within Gaura represented by G. macrocarpa, G. boquillensis-G. coccinea, and G. parviflora-Stenosiphon. Six of the 10 successively weighted trees have a slightly higher maximum likelihood score, and a representative of one of these trees is shown (Fig. 2). In the combined analysis, there is strong support for the monophyly of Gaura plus Stenosiphon (BP = 100%), but there is only weak bootstrap support for O. macrocarpa as the sister group to the Gaura-Stenosiphon clade (BP = 60%). However, there is strong bootstrap support for O. speciosa as the sister group to the O. macrocarpa-Gaura-Stenosiphon clade (BP = 100%). Although sampling within Oenothera is limited in the present study, our results strongly indicate that the closest relative to Gaura is a representative from within Oenothera sects. Megapterium or Xylopleurum.
Within the Gaura-Stenosiphon clade, the monotypic sect. Gauridium represented by G. mutabilis is sister to all other species of Gaura and Stenosiphon with strong bootstrap support (BP = 100%). In the tree depicted (Fig. 2), G. macrocarpa (sect. Xerogaura) is sister to a clade comprising the remaining species within Gaura and Stenosiphon although this relationship is only weakly supported (BP = 58%). Gaura boquillensis, the other representative from sect. Xerogaura, is sister to G. coccinea (sect. Campogaura) (BP = 100%), while G. parviflora (sect. Schizocarya) is sister to S. linifolius. However, three of the nodes supporting these relationships lack bootstrap support and collapse in a strict consensus resulting in a five-way polytomy comprised of G. macrocarpa, G. parviflora, S. linifolius, the G. coccinea-G. boquillensis clade, and a clade made up of sects. Stipogaura, Pterogaura, and Gaura. Despite the lack of resolution in this portion of the tree, our results indicate that section Xerogaura is either paraphyletic or polyphyletic.
Within the more derived members of Gaura, there is moderate support (BP = 68%) for a monophyletic clade made up of representatives from sects. Stipogaura, Pterogaura, and Gaura, but only two of these sections are monophyletic. Gaura sect. Stipogaura (G. villosa, G. calcicola, G. filipes, G. mckelveyae, and G. sinuata) is monophyletic with strong support (BP = 98%) and is sister to a clade made up of representatives from the other two sections (BP = 68%). The monophyly of the clade comprising sects. Pterogaura and Gaura is strongly supported (BP = 96%), but only sect. Gaura (G. lindheimeri, G. angustifolia, G. neomexicana, G. longiflora, G. biennis, and G. demareei) is monophyletic (BP = 98%). Section Pterogaura is rendered paraphyletic by the position of G. hexandra outside of a strongly supported clade (BP = 89%) comprised of the remaining three representatives of this section, G. triangulata, G. brachycarpa, and G. suffulta.
Alternative topologies
Five alternative hypotheses were tested concerning the relationships within tribe Onagreae including Gaura (Table 3). Three of these alternative topologies were designed to test different taxa as possible sister groups to Gaura. Constrained topologies that force representatives from Oenothera sect. Megapterium and sect. Xylopleurum to occupy positions more distant to Gaura resulted in trees that are 20 steps longer. Similarly, alternate topologies that place Calylophus as sister to Gaura imposed a length penalty of 25 additional steps. Both of these topologies were rejected as significantly worse solutions by the SH test (Table 3). However, the alternate topology, in which Stenosiphon is sister to Gaura (rather than embedded within it), had a length penalty of only two steps and could not be rejected by the SH test (Table 3). Two alternative topologies were designed to test the possible monophyly of traditionally described sects. Pterogaura and Xerogaura, which are reported as being polyphyletic or paraphyletic in this study. In the case of sect. Pterogaura, an alternate topology that forces the section to be monophyletic accrued a length penalty of only one step. This topology could not be rejected by the SH test as a significantly worse solution (Table 3). Conversely, topological constraints that render sect. Xerogaura monophyletic increased the tree length by 12 steps and can be rejected as a significantly worse solution by the SH test (Table 3).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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; Carr et al., 1990
), our molecular phylogeny and that of Levin et al. (2003)
strongly support Oenothera as its sister genus. Tobe et al. (1987)
used fruit and seed characters to describe two lineages of Oenothera, one characterized by sharply angled or winged fruits and the other by subterete, unwinged fruits. The molecular phylogeny of Levin et al. (2003)
confirmed these separate lineages and determined the lineage characterized by taxa with sharply angled or winged fruits is sister to Gaura, while Oenothera taxa with subterete, unwinged fruits are more closely related to Calylophus. Our analysis supports these findings showing two taxa with winged fruits, O. macrocarpa and O. speciosa, closely related to Gaura and four taxa with subterete, unwinged fruits, O. albicaulis, O. laciniata, O. drummondii, and O. heterophylla, more closely related to Calylophus.
Raven and Gregory (1972a)
recognized eight sections within Gaura. Of these, the monophyly of sections Gauridium, Stipogaura, and Gaura is strongly supported in our combined molecular phylogeny. There is also strong support for the position of G. mutabilis (sect. Gauridium) as the sister group to the other species of Gaura. This placement is congruent with Raven and Gregory's hypothesis that G. mutabilis is one of the less-derived species of Gaura (Raven and Gregory, 1972a
) and is supported by the morphological phylogeny of Carr et al. (1990)
. Our phylogeny also indicates there is strong support for the inclusion of Stenosiphon linifolius within Gaura; however, its sister taxon position to G. parviflora (sect. Schizocarya) lacks bootstrap support. Historically, Spach's treatment of G. linifolia Nutt. as a segregate genus, Stenosiphon, remained unchallenged in subsequent revisions (Rothrock, 1864
; Munz, 1938
, 1965
; Raven and Gregory, 1972a
); however, its placement within Gaura in our molecular phylogeny and that of Levin et al. (2003)
requires a close examination of Spach's criteria for the generic recognition of Stenosiphon and morphological character states that support the placement of Stenosiphon within Gaura. The morphological and cytological similarities that Stenosiphon and Gaura share are illustrated in Table 4. Of the 14 character states listed in this table, only one, a unilocular ovary, is clearly unique to Stenosiphon. Although Spach (1835)
segregated Stenosiphon from Gaura on the basis of two morphological characters (a filiform hypanthium and a one-seeded capsule), the difference between the filiform hypanthium of Stenosiphon and the narrow tubular hypanthium of Gaura seems rather subjective. As for the one-seeded capsule, all Gaura species and Stenosiphon produce ovaries with four ovules, three of which abort in Stenosiphon and often, one or two abort in Gaura. Levin et al. (2003)
view the reduced ovule number as a synapomorphy uniting the two genera. Stenosiphon lacks staminal appendages and Rothrock (1864)
interpreted this as support for Spach's generic recognition of Stenosiphon; however, his study did not include G. mutabilis, which also lacks these appendages. Further, he noted in G. parviflora, the sister taxon to Stenosiphon in our molecular phylogeny, that these appendages are reduced in size from scales to papillae. Other synapomorphies that Stenosiphon and Gaura share include the presence of an indusium, indehiscent fruits, and absent or incomplete fruit septa. Because there is little morphological support for the continued recognition of Stenosiphon as a segregate genus and the molecular phylogenies of this study and Levin et al. (2003)
place Stenosiphon within Gaura, we conclude that Stenosiphon linifolius should once again be recognized as Gaura linifolia Nutt. and either be placed in a new monotypic section within Gaura or included in section Schizocarya with G. parviflora.
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but are congruent with the results of the morphological analysis by Carr et al. (1990)
, who also found sect. Xerogaura to be paraphyletic. Changes in the current circumscription of sect. Xerogaura should be considered. Our suggestion is to transfer G. boquillensis from sect. Xerogaura, where it is currently assigned, to sect. Campogaura maintaining sect. Xerogaura as a monotypic section represented by G. macrocarpa. The placement of G. boquillensis as sister to G. coccinea is strongly supported by the molecular data, and the two taxa are also morphologically similar. When G. boquillensis was first described, the authors noted the new species is morphologically similar to G. coccinea but "differs markedly [from G. macrocarpa] in its smaller, more irregular flowers and shorter, somewhat stipitate fruits" (Raven and Gregory, 1972a
).
There is strong support for the monophyly of sect. Stipogaura, a group of five species (G. calcicola, G. filipes, G. mckelveyae, G. sinuata, and G. villosa) (Fig. 2). All the species in this section have fruits with a long, slender stipe, a synapomorphy that characterizes this section (Raven and Gregory, 1972a
; Carr et al., 1990
). Our results validate the sectional classification of Raven and Gregory (1972a)
and the morphological phylogeny of Carr et al. (1990)
. Section Pterogaura is paraphyletic in our phylogeny (Fig. 2) as G. suffulta, G. brachycarpa, and G. triangulata form a strongly supported monophyletic clade that does not include G. hexandra. Our study does not support the sectional classification of Raven and Gregory (1972a)
or the morphological phylogeny of Carr et al. (1990)
and suggests changes in the current circumscription of sect. Pterogaura are required. A possible solution to this paraphyly is to recognize a new monotypic section that includes G. hexandra.
There are differences in distribution and flowering time between the three members of the strongly supported clade and G. hexandra (Raven and Gregory, 1972a
; Carr et al., 1990
). Gaura suffulta, G. brachycarpa, and G. triangulata are spring ephemerals, flowering from March to June with a distribution that is centered in Texas and Oklahoma with some extension into central New Mexico. The flowering time of G. hexandra is from July to October, and it is found at middle elevations in the mountainous areas from central Arizona and southwestern New Mexico to southern Mexico and Guatemala.
In our molecular phylogeny (Fig. 2), sect. Gaura is monophyletic and consists of two clades, one with strong support including G. neomexicana, G. lindheimeri, and G. demareei and one lacking support represented by G. longiflora, G. angustifolia, and G. biennis. There is a sister taxa relationship with limited support between G. demareei and G. lindheimeri, which is of interest as they are the only two morning-flowering taxa within the genus. The monophyly of sect. Gaura is congruent with the sectional delimitation of Raven and Gregory (1972a)
but not with the morphological study of Carr et al. (1990)
, who found sect. Gaura to be paraphyletic because G. lindheimeri did not cluster with the other five species of sect. Gaura.
When Raven and Gregory (1972a)
monographed Gaura, they stressed speciation via hybridization as an important evolutionary mechanism within the genus. These authors postulated that four species of Gaura, 19% of the total number of species, arose via ancient diploid hybridization events. Species hypothesized to be of hybrid origin included G. demareei (G. lindheimeri x G. longiflora) (Carr et al., 1986), G. drummondii (G. mckelveyae x G. coccinea), G. sinuata (G. calcicola x G. mckelveyae), and G. triangulata (G. brachycarpa x G. suffulta). These taxa have all been studied cytologically and have some interesting adaptations (Raven and Gregory, 1972b
; Raven, 1979
). Gaura drummondii and G. sinuata are both autotetraploid, while G. triangulata and G. demareei are both diploid. However, the former is a permanent translocation heterozygote and the latter has varying degrees of translocation heterozygosity (Raven and Gregory, 1972b
). Either of these mechanisms, if they arose after interspecific hybridization, could act to stabilize the newly formed hybrid complex.
Our molecular data are consistent with the hypothesis that G. drummondii and G. sinuata arose via interspecific hybridization followed by genome doubling as predicted by Raven and Gregory (1972b)
. In G. sinuata, the molecular data indicate that the ovule donor came from the lineage that gave rise to G. calcicola (sect. Stipogaura), while the pollen donor originated from the lineage that gave rise to G. filipes and G. villosa (sect. Stipogaura). A similar pattern is also apparent for G. drummondii. The molecular data suggest that the ovule donor came from the lineage that gave rise to G. mckelveyae (sect. Stipogaura) while the pollen donor came from the lineage that gave rise to the G. neomexicana-G. lindheimeri (sect. Gaura) species complex. In both cases, sufficient time has elapsed since the formation of these hybrid species for their ITS and ETS regions to become completely homogenized (no ambiguous nucleotide sites) and to acquire additional substitutions. Seelanan et al. (1997)
demonstrated that concerted evolution via gene conversion or unequal crossing-over can homogenize different parental genomes in a hybrid so that only one parental genome type may be found in the hybrid, a process that has apparently taken place in the two autotetraploid species of Gaura. These hybrids were detected because of conflicts between their plastid and nuclear ribosomal gene trees.
Our data neither confirm nor refute the hypothesis that G. demareei and G. triangulata originated by interspecific hybridization. Their putative parental species are very closely related phylogenetically, and the plastid data are uninformative at these levels. Because the ITS and ETS trees for these taxa are congruent, it is not possible to determine if they arose via interspecific hybridization.
Several authors have reported the difficulty reticulate evolution poses for phylogenetic reconstruction (Rieseberg, 1991
; McDade, 1992
; Rieseberg and Morefield, 1995
; Sang et al., 1995
; Xu, 2000
). After performing cladistic analyses on data sets containing artificially produced hybrids from the Aphelandra pucherrima species complex, McDade (1992)
concluded that (1) a hybrid would be placed as a basal lineage to the clade containing its most derived parent and (2) the amount of disruption a hybrid would cause the tree topology is positively correlated with the phylogenetic distance between its parental species. Our findings with Gaura agree with these predictions. The putative parental species of Gaura sinuata are closely related within sect. Stipogaura, there is little effect on tree topology, and G. sinuata is placed with G. villosa and G. filipes, the lineage containing the more derived of its putative parents. Gaura drummondii, however, probably represents a hybrid between representatives from the distantly related sections Stipogaura and Gaura and its inclusion in a phylogenetic analysis of the genus disrupts the branching order within the sect. Pterogaura-sect. Gaura clade.
Our findings suggest that ancient hybridization events have taken place within Gaura and that these events are often followed by lineage capture within the biparentally inherited nrDNA resulting in the elimination of one parental genotype. If lineage capture results in an nrDNA phylogeny that is congruent with the cpDNA phylogeny, there may be little evidence that ancient hybridization has taken place. Given that allopolyploidy is known to occur within Camissonia, Clarkia, and Gayophytum and that hybrid speciation has been hypothesized in Gaura and Oenothera (Raven, 1979
), reticulate evolution may be an important evolutionary mechanism within the Onagraceae.
Conclusions
In our paper, the combined analysis of the ITS, ETS, and trnL regions is shown to be useful in assessing the infrageneric relationships of Gaura. This study supports much of the sectional delimitations of previous research based on morphological data and indicates Stenosiphon should once again be included within Gaura. Sections Xerogaura and Pterogaura are not monophyletic and should be reevaluated. The molecular data also provide support for the hypothesis that G. sinuata and G. drummondii arose as a result of interspecific hybridization followed by genome doubling; however, the presence of stabilized diploid hybrid species in the genus has not been confirmed. Future research will include the acquisition of additional data, in particular the trnL-F sequences for G. mckelveyae and G. calcicola, to further address this hypothesis. When reticulate evolution has been detected, our study of Gaura indicates that the resulting phylogeny may be improved when this information is taken into consideration.
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FOOTNOTES |
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4 rghoggard{at}earthlink.net
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Names followed by • indicate taxa known to be permanent translocation heterozygotes. Arrows with dashed lines indicate the position of G. drummondii in the separate analysis for each of the sequenced gene regions