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(American Journal of Botany. 2008;95:608-625.) doi: 10.3732/ajb.2007346 © 2008 Botanical Society of America, Inc. |
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Systematics and Phytogeography |
2 Department of Biology, University of Washington, Box 355325, Seattle, Washington 98195-5325 USA 3 Herbarium, Burke Museum of Natural History, University of Washington, Box 355325, Seattle, Washington 98195-5325
Received for publication 24 October 2007. Accepted for publication 28 February 2008.
ABSTRACT
Variation in life history strategies is a fundamental question in evolutionary biology, and the cooccurrence of annual and perennial habits in Castilleja and Castillejinae provides the opportunity to study the evolution of plant life history in a phylogenetic context. Molecular phylogenetic analysis of two chloroplast (rps16 and trnL/F) and two nuclear ribosomal (internal and external transcribed spacers) DNA regions support the monophyly of subtribe Castillejinae (Orobanchaceae). A well-supported phylogeny of the six genera (Castilleja [
180 spp.], Clevelandia [1 sp.], Cordylanthus [18 spp.], Ophiocephalus [1 sp.], Orthocarpus [9 spp.], and Triphysaria [5 spp.]) comprising the subtribe is presented, and morphological synapomorphies are identified for the major lineages recovered. Orthocarpus and Triphysaria are both monophyletic; Cordylanthus is biphyletic. Clevelandia and Ophiocephalus are derived from within Castilleja. The perennial Castilleja clade (160 spp.) is derived from a grade of annual taxa including Castilleja sect. Oncorhynchus (16 spp.), Cordylanthus, Orthocarpus, and Triphysaria. This suggests that the perennial habit evolved a single time from an annual ancestral lineage that persisted throughout the diversification of Castillejinae, contrary to classical interpretations of life history evolution in plants. Given the prevalence of polyploidy among perennial Castilleja species, perenniality may have played an important role in the origin and establishment of polyploidy in Castilleja.
Key Words: annual • Castilleja • Castillejinae • Cordylanthus • Orobanchaceae • Orthocarpus • perennial • polyploidy
Species belonging to Castilleja (Orobanchaceae), commonly referred to as the paintbrushes, are some of the most emblematic wildflowers of western North America. Castilleja consists of 180 species distributed from coastal dunes to alpine meadows. The large majority of paintbrushes are herbaceous perennials (160 species), but Castilleja also includes 20 annual species. In addition to Castilleja, subtribe Castillejinae includes five small genera of annuals: Clevelandia (1 species), Cordylanthus (18 species), Ophiocephalus (1 species), Orthocarpus (9 species), and Triphysaria (5 species). There is an abundance of biosystematic data available for many species (e.g., Heckard, 1968
; Holmgren, 1970, 1971, 1976; Heckard and Chuang, 1977; Heckard et al., 1980; Chuang and Heckard, 1982, 1983, 1992a, 1993), and Castillejinae have received considerable attention taxonomically (e.g., Chuang and Heckard, 1991). Each of the genera (excluding many species of Castilleja) have been treated in detail from a morphological, anatomical, and cytogenetic perspective (Heckard, 1968; Chuang and Heckard, 1972, 1973, 1975a,1975b, 1976; Heckard and Chuang, 1977; Heckard et al., 1980; Chuang and Heckard, 1982, 1983, 1986, 1992a,1992b, 1993). Despite this, the lack of a robust phylogenetic hypothesis for the paintbrushes and their relatives precludes the evolutionary study of the fascinating morphological variation and patterns of speciation both within Castilleja and among the genera of Castillejinae.
The cooccurrence of annual and perennial habits in Castilleja and Castillejinae provides the opportunity to study the evolution of plant life history in a phylogenetic context. Variation in life history strategies is a fundamental question in evolutionary biology, and the dichotomy between semelparity—characterized by a single reproductive episode—and iteroparity—characterized by repeated reproductive output—has received considerable attention from a theoretical standpoint (Young and Augspurger, 1991; Stearns, 1992). Perhaps the most common form of semelparity in plants is the annual habit, which is in contrast to the iteroparous life history exhibited by most perennial plants. Most investigations of life history variation in plants have focused on the mathematical modeling of ecological and evolutionary scenarios (e.g., bet-hedging, reproductive effort, demography) for the evolution of semelparity in an iteroparous background (see Young and Augspurger [1991] for a review). However, there have been very few studies that have investigated the evolution of the annual and perennial habits in a phylogenetic context (Bena et al., 1998, Conti et al., 1999, Andreasen and Baldwin, 2001; Verboom et al., 2004). Despite the lack of phylogenetic studies investigating this fundamental trait, there is a widely held opinion among plant evolutionary biologists that annuals are derived from perennial ancestors and that the shift between these two strategies is unidirectional (e.g., Stebbins, 1957).
A robust phylogenetic hypothesis provides a framework in which to explore patterns of phenotypic evolution. In this paper, we focus on the evolution of life history variation in Castillejinae, but also consider morphological characteristics that have been important historically for circumscribing taxonomic groups. In addition, we identify diagnostic traits for the major lineages of Castillejinae and discuss systematic implications. We report results based on data from both the chloroplast and nuclear genome. The chloroplast data are from the trnL/F region (Gielly and Taberlet, 1994) and the intron of the ribosomal protein rps16 (Oxelman et al., 1997). The nuclear genome is represented by nrDNA sequences of the internal transcribed spacer (ITS) and external transcribed spacer (ETS) regions (Baldwin et al., 1995; Baldwin and Markos, 1998). Because hybridization has been suggested to be important in the evolution of Castilleja and relatives, obtaining data from both the chloroplast and nuclear genomes may permit the identification of potential problems in the resulting phylogenetic reconstruction from one or the other source of data.
MATERIALS AND METHODS
Taxon sampling
In total, 76 species of subtribe Castillejinae and three outgroup taxa were used in this study (Table 1). We follow the subtribe classification of Chuang and Heckard (1991) because it is the most recent and complete reorganization of Castillejinae. The majority of species in the subtribe are represented by the genus Castilleja, in which the taxonomic complexity is extremely challenging. The treatment of Castilleja in preparation for the Flora of North America project (J. M. Egger [WTU], M. Wetherwax [JEPS], and D. C. Tank, unpublished manuscript) is based on Chuang and Heckard’s broad view of the genus. However, J. M. Egger (unpublished data) has tentatively reinstated an infrageneric classification based on detailed field and herbarium observations. Within Castilleja, Egger’s classification represents the only recent and comprehensive infrageneric work and, therefore, was used here as a guide for sampling. The taxa on which our analyses are based represent all six genera comprising subtribe Castillejinae, as well as three outgroups. Within Castilleja, 46 species representing all five subgenera and 11 of 19 sections were sampled. Efforts were made to include multiple species for most infrageneric groups within Castilleja, except for small or monotypic groups (Table 1). Given the goals of this study, special attention was given to Castilleja subg. Colacus sect. Oncorhynchus (11 of 16 species sampled), which includes species previously recognized as Orthocarpus (Chuang and Heckard, 1991). All species of Orthocarpus and Triphysaria were sampled, as well as the two monotypic genera Clevelandia and Ophiocephalus. Cordylanthus is represented by 14 of 18 species, including representatives from all infrageneric groups (sensu Chuang and Heckard, 1973, 1975a, 1986). On the basis of previously published molecular systematic studies of Orobanchaceae and Scrophulariaceae s.l. (e.g., Young et al., 1999; Olmstead et al., 2001; Wolfe et al., 2005; Bennett and Mathews, 2006; reviewed in Tank et al., 2006), Lamourouxia, Pedicularis, and Seymeria were chosen as outgroups.
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) and purified using QIAquick spin-columns following the protocols of the manufacturer (QIAGEN, Valencia, California, USA).
DNA for sequencing the regions of interest was generated via polymerase chain reaction (PCR) using the trn-c and trn-f primers (Taberlet et al. 1991) for the trnL/F region, rps16_ F and rps16_2R primers (Oxelman et al., 1997) for the rps16 intron, and its4 and its5 primers (Baldwin, 1992) for the entire ITS region. To amplify a portion of the 3' end of the ETS region, the 5' primer ETS-B (Beardsley and Olmstead, 2002) and the 18S-IGS 3' primer of Baldwin and Markos (1998) were used. For some taxa, we had difficulty amplifying the trnL/F, rps16, and ITS regions as one fragment. For these taxa, internal primers were used to amplify the fragments in two parts. For the trnL/F region, the trn-c/trn-d and trn-e/trn-f primer pairs were used to amplify the trnL intron and trnL-trnF spacer, respectively (Castilleja tenuis, Cordylanthus eremicus subsp. eremicus, C. ramosus, Ophiocephalus angustifolius, Orthocarpus purpureo-albus, and O. imbricatus). For the rps16 intron, the primer pairs rps16_F/rps16_iR and rps16_iF/rps16_2R were used to amplify the region in two pieces for one species (Castilleja racemosa). The ITS1 and ITS 2 regions were amplified separately for eight taxa using the its5/its2 and its3/its4 primer pairs, respectively (Castilleja racemosa, C. cusickii, C. peckiana, Cordylanthus capitatus, Triphysaria floribunda, Orthocarpus pachystachyus, Clevelandia beldingii, and Ophiocephalus angustifolius). Amplified PCR products were purified by precipitation from a 20% polyethylene glycol solution and washed in 70% ethanol prior to sequencing. After repeated attempts, we were unable to obtain any PCR products for some taxa for the rps16 intron, ITS, and ETS DNA regions (Table 1); however, all taxa included in this study are represented by at least three of the four DNA regions sequenced.
To ensure accuracy, we sequenced both strands of the cleaned PCR products using the DYEnamic ET Terminator Cycle Sequencing Kit (Amersham Biosciences, Piscataway, New Jersey, USA) on an ABI 377 DNA sequencer (Applied Biosystems, Foster City, California, USA). For the trnL/F region, the internal primers trn-d and trn-e of Taberlet et al. (1991) and trnL-2C and trnL-2F (located just internal to the PCR primers) of Beardsley and Olmstead (2002) were used to improve sequence quality. The rps16 intron was sequenced using the external PCR primers, rps16_F and rps16_2R, and two internal primers, rps16_iF (5'-GGTATGTTGCTGCCATTTTG-3') and rps16_iR (5'-CAAAATGGCAGCAACATACC-3'), designed using Castillejinae sequences as a reference. The ITS region was sequenced using the PCR primers and two internal primers, its2 and its3 (Baldwin, 1992). The ETS region was sequenced using the 5' ETS-B PCR primer, and the sequencing primer 18S-E (Baldwin and Markos, 1998), that is slightly internal to the 3' PCR primer. Sequence data were assembled and edited for each region using the program Sequencher (Gene Codes, Ann Arbor, Michigan, USA), and consensus sequences were generated.
Phylogenetic analyses
Sequence alignments for all four gene regions were performed manually using the program Se-Al version 2.0a11 (Rambaut, 1996). For each region, parsimony-informative gaps were coded as presence/absence characters using simple gap coding (Graham et al., 2000; Simmons and Ochoterena, 2000). Complex gaps were not included as additional characters in our analyses.
Both the trnL/F and rps16 intron regions are part of the haploid chloroplast genome, and thus, their histories are linked, and there is no a priori reason to not combine the data in one analysis. Likewise, the nrDNA ITS and ETS regions are tightly linked in the rDNA repeat and can, like the chloroplast, be treated as one locus. However, differences in base composition and rates of evolution among loci could result in incongruencies between gene trees produced from different data sets (Bull et al., 1993). To determine whether the individual data sets are significantly different from random partitions of the combined data, we used the incongruence length difference test (ILD; Farris et al., 1994) in the program PAUP* version 4.0b10 (Swofford, 2002) on the combined cpDNA and nrDNA data set. All parsimony uninformative sites were excluded from the partition homogeneity test (Cunningham, 1997), and 1000 replicates of heuristic searches (each with 10 replicates of stepwise random taxon addition and tree-bisection-reconnection [TBR] branch swapping) were performed.
Parsimony analyses were conducted on each of the four data sets individually, as well as the cpDNA, nrDNA, and combined data sets as implemented in PAUP* (Swofford, 2002). Heuristic searches were performed with 1000 replicates of stepwise random taxon addition and TBR branch swapping. Maximum likelihood (ML) analyses were performed on the cpDNA, nrDNA, and combined data sets using PAUP* (Swofford, 2002). The program MODELTEST version 3.6 (Posada and Crandall, 1998) was used to determine the model of sequence evolution best fit to the data by the hierarchical likelihood ratio test. Sequence parameters were estimated by an iterative approach (e.g., Swofford et al., 1996). First, parameters were estimated from one of the most parsimonious trees. This tree was then used as a starting tree for a heuristic search with nearest neighbor interchange (NNI) branch swapping, and the estimated parameter values fixed for the search. Parameter values were then estimated from the resulting tree, and the process was repeated. Once the parameter values estimated from the resulting tree stabilized, these values were used for the full ML search. The full ML search was conducted using the fixed parameter values with 10 replicates of stepwise random taxon addition and TBR branch swapping. Bayesian phylogenetic analyses were conducted using the program MrBayes version 3.1.2 (Ronquist and Huelsenbeck, 2003) on the cpDNA, nrDNA, and combined data sets. Each analysis was conducted using the same models as used for the ML analyses and consisted of two runs of 10000000 generations from a random starting tree using the default priors and four Markov chains (using the default heating values) sampled every 100 generations. Convergence of the chains was determined by examining the plot of all parameter values and the –ln L against generation using the program Tracer version 1.3 (Rambaut and Drummond, 2004). Stationarity was assumed when all parameter values and the –ln L had stabilized. Burn-in trees were then discarded and the remaining trees, and their associated parameter values, were saved. To increase the chance of exploring more of tree space and decrease the chance of obtaining stationarity on local optima, we ran two independent analyses for each data set. In addition, for the combined data set both single-model (i.e., where the two data sets, cpDNA and nrDNA, are modeled by the same parameter values) and partitioned (i.e., two data partitions corresponding to the cpDNA and nrDNA data sets, where each is modeled under different parameter values) analyses were performed. Following Nylander et al. (2004), Brandley et al. (2005), and Brown and Lemmon (2007) we used the Bayes factor to choose between the single-model and partitioned Bayesian analyses.
Nonparametric bootstrapping (Felsenstein, 1985) was used to evaluate relative support for particular clades recovered in the phylogenetic analyses. Parsimony bootstrapping was performed with 500 replicates, each with 20 replicates of stepwise random taxon addition and TBR branch swapping with MULTREES off (DeBry and Olmstead, 2000). Maximum likelihood bootstrapping was performed with 250 replicates, each with three replicates of stepwise random taxon addition and NNI branch swapping. In addition to parsimony and ML bootstrap values, posterior probabilities (PP) resulting from Bayesian phylogenetic analyses were also used to evaluate relative branch support. A majority rule consensus tree showing all compatible partitions from the resulting posterior distribution of tree topologies was used to recover the Bayesian PP values for each clade. The Shimodaira–Hasegawa test (SH; Shimodaira and Hasegawa, 1999), as implemented in PAUP* (Swofford, 2002), was used to evaluate whether major topological differences between results of the separate cpDNA and nrDNA analyses were significant, as well as to evaluate whether alternative topological hypotheses based on traditional taxonomy were significantly different than the topology resulting from phylogenetic analyses of the combined cpDNA and nrDNA data (100000 bootstrap replicates using RELL optimization).
RESULTS
Phylogenetic analyses
The two noncoding chloroplast regions, trnL/F and rps16, aligned unambiguously, although numerous short gaps were introduced. For the trnL/F region, all taxa were included, but Castilleja septentrionalis was missing for the rps16 region (sequences for the other three regions for C. septentrionalis were downloaded from GenBank; Table 1). The tightly linked nrDNA ITS and ETS regions were more difficult to align, but we were still able to align the majority of both data sets. Regions that could not be unambiguously aligned were excluded from the phylogenetic analyses (positions 258–265 in the ITS alignment and positions 192–195 in the ETS alignment). Characteristics of the cpDNA and nrDNA regions sequenced for this study are summarized in Table 2.
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) and has been criticized as too conservative (e.g., Graham et al., 1998; Barker and Lutzoni, 2002; Darlu and Lecointre, 2002). Furthermore, the trees resulting from separate analyses of the cpDNA and nrDNA data sets are largely consistent (Figs. 1 and 2). When the nrDNA topology was constrained to include the well-supported nodes recovered by phylogenetic analyses of the cpDNA data (i.e., Bayesian PP values 
0.95 and bootstrap values 70%; see Fig. 1), the Shimodaira–Hasegawa (SH) test indicated that the resulting likelihood values were not significantly different (P = 0.12). Likewise, when the cpDNA topology was constrained to include the well-supported nodes recovered by analyses of the nrDNA data, the SH test indicated no significant difference between the two topologies (P = 0.08). Therefore, in the absence of any indication that such an analysis is compromised by conflicting histories in the two genomes, we conducted a combined analysis to take advantage of the greater resolution that the larger data set can provide.
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) resulted in the GTR+I+
(GTR = general time reversible, I = proportion of invariable sites, = gamma distributed variable sites) model of sequence evolution. The iterative approach taken to estimate parameters for the maximum likelihood (ML) analyses (see Materials and Methods, Phylogenetic analyses) took three rounds of parameter estimates for each of the three data sets before the parameter values stabilized; the parameter values fixed for the ML analyses are shown in Table 3. Maximum likelihood analyses of the cpDNA data resulted in two equally optimal trees that only differ in the placement of one taxon in a clade where there is little or no branch support. The resulting ML trees for each of the data sets (not shown) were congruent in overall topology with those resulting from parsimony and Bayesian analyses. Bayesian analysis of the cpDNA and nrDNA data achieved apparent stationarity after 1000000 generations, however, because this analysis contained long chains (10000000 generations) and a high sampling frequency (every 100 generations), a conservative burn-in of 2000000 generations was used. The majority rule consensus trees calculated from the posterior distribution (excluding burn-in trees) with mean branch lengths are shown in Fig. 2. To avoid the pitfall of achieving apparent stationarity on a local optimum, we ran two independent Bayesian analyses for each data set; in both analyses, all parameters reached stationarity at the same level. The results presented here (Fig. 2, Table 4) are those of one of the independent runs.
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), results of the partition homogeneity test and ML parameter estimates (e.g., widely differing base compositions and substitution rates between the two data sets; Table 3) suggest that the two DNA regions may be more appropriately modeled as two data partitions. In addition, the Bayes factor result from the comparison of the single-model and partitioned analyses provides very strong evidence (i.e., >10; Kass and Raftery, 1995) for the partitioned analysis (Bayes factor = 371.42). Therefore, we have chosen to show the resulting trees from the partitioned analysis only; Figs. 3 and 4 show the majority rule consensus tree from the partitioned Bayesian analysis with PP values and mean branch lengths, respectively.
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0.95 and bootstrap values 70% are indicated with an asterisk. Figure 2 shows a comparison of the Bayesian majority rule consensus trees with mean branch lengths. In all analyses, the monophyly of subtribe Castillejinae as well as the two genera Orthocarpus and Triphysaria was well supported. However, the genus Cordylanthus was biphyletic, forming two well-supported clades, with one clade corresponding to subg. Cordylanthus and the other comprised of the monotypic subg. Dicranostegia sister to subg. Hemistegia. Results from analyses of the cpDNA data provided moderate support for a sister-group relationship between Cordylanthus subg. Cordylanthus and the genus Orthocarpus (parsimony bootstrap = 85%; ML bootstrap = 63%; Bayesian PP = 0.95). However, this relationship was not recovered in the nrDNA analyses, in which Orthocarpus forms a clade with the rest of the subtribe (parsimony bootstrap = 73%; ML bootstrap = 58%; Bayesian PP = 0.86). When the nrDNA topology was constrained to match the cpDNA topology with respect to these lineages (i.e., Cordylanthus subg. Cordylanthus sister to Orthocarpus), the Shimodaira–Hasegawa test indicated that the resulting likelihood values were not significantly different (P = 0.41). Likewise, when the cpDNA topology was constrained to that of the nrDNA analyses, the SH test indicated no significant difference between the two topologies (P = 0.26). Therefore, the results of the cpDNA and nrDNA analyses are not in strong conflict with each other. In all analyses, the large genus Castilleja also was paraphyletic with the two monotypic genera Clevelandia and Ophiocephalus nested within Castilleja. Monophyly of Castilleja including Clevelandia and Ophiocephalus (Castilleja s.l.) was well supported in the analyses of the nrDNA data set (parsimony bootstrap = 98%; ML bootstrap = 86%; Bayesian PP = 1.0), but only weakly supported by the cpDNA data (parsimony bootstrap = 59%; ML bootstrap = 60%; Bayesian PP = 0.60). In contrast, results from both DNA regions provided strong support for the clade comprised of Castilleja s.l. and Triphysaria. Figures 3 and 4 show the majority rule consensus tree resulting from the partitioned Bayesian analysis of the combined cpDNA and nrDNA data with PP values and mean branch lengths, respectively. This tree was largely congruent with those resulting from the separate analyses of the cpDNA and nrDNA regions (Figs. 1 and 2) with all analytical methods, as well as the parsimony, ML, and single-model Bayesian analyses of the combined data (trees not shown), and represents our best hypothesis for the phylogeny of subtribe Castillejinae. In agreement with the results from the cpDNA data set (Figs. 1 and 2), Cordylanthus subg. Cordylanthus, and Orthocarpus were resolved as monophyletic and sister to the remainder of the subtribe. However, as a result of the disagreement between the nrDNA and cpDNA data, support for this relationship decreased in the combined analyses relative to the cpDNA results. Within Castilleja s.l., some relationships that were unresolved or poorly supported by the individual analyses were recovered with increased support in the combined analysis. This was most evident with respect to the annual lineages of Castilleja s.l., including Castilleja subg. Colacus sect. Oncorhynchus (Table 1, Fig. 5) and the monotypic annual genera Clevelandia and Ophiocephalus. In the combined analyses, these taxa were resolved as a basal grade of annual lineages leading to a large clade of perennial Castilleja species (Figs. 3 and 4). Of these annual lineages, Clevelandia and Ophiocephalus were resolved as the sister group to the perennial Castilleja clade, albeit with low support. Nevertheless, a strongly supported clade including Clevelandia, Ophiocephalus, and the perennial Castilleja species was recovered by all analytical methods (parsimony bootstrap = 81%; ML bootstrap = 99%; Bayesian PP = 1.0). When the topology recovered in the combined analyses (Figs. 3 and 4) was constrained to correspond to traditional taxonomic groups (i.e., monophyly of Castilleja, monophyly of Castilleja sect. Oncorhynchus, and monophyly of Cordylanthus), the Shimodaira–Hasegawa test indicated that these alternative topologies were significantly less likely (Table 4).
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Throughout the history of the subtribe, there has been difficulty defining generic boundaries in Castillejinae. This difficulty has been especially evident between Castilleja and Orthocarpus, where numerous species have been shifted between the two genera (e.g., Gray, 1862; Watson, 1871; Eastwood, 1909; Jepson, 1925; Keck, 1927; Chuang and Heckard, 1991, 1992b). The most recent treatment of Castillejinae, based on extensive morphological study including floral morphology, seed and seed-coat morphology, and cytological analyses, led to a major realignment of generic boundaries in the subtribe (Chuang and Heckard, 1991). In this treatment, Orthocarpus was redefined to include only nine annual species, elevating Orthocarpus subg. Triphysaria to genus, and assigning 12 other annual Orthocarpus species to Castilleja (Chuang and Heckard, 1991, 1992b). They also concluded that the monotypic genus Gentrya did not differ enough from Castilleja to warrant generic status and reassigned it to Castilleja (C. racemosa) as the monotypic subg. Gentrya (Chuang and Heckard, 1991).
In addition to their realignment of generic boundaries within Castillejinae, Chuang and Heckard (1991) proposed an "intuitive phylogeny" depicting relationships among the major lineages of the subtribe (Fig. 6). A morphological cladistic analysis (Chuang, 1993) with 11 terminal taxa (genera and major infrageneric groups) resulted in a nearly identical topology. They relied on these estimates of phylogenetic relationship to interpret the evolution of some major morphological characters, including the evolution of the annual and perennial life history strategies.
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, 1975a, 1986, 1991, 1992b). Finally, we discuss systematic implications that these phylogenetic results suggest and identify diagnostic morphological traits (Fig. 5) that will serve as a guide for a taxonomic revision of Castillejinae.
Evolution of perenniality
Among the genera of Castillejinae, perenniality is limited to Castilleja, in which perennial species are the large majority of the approximately 180 species. Although Chuang and Heckard (1991) recognized the unique Castilleja floral structure (i.e., elongate upper corolla lip, lower corolla lip reduced to teeth) as a derived morphology in the subtribe, rather than interpreting the evolution of perennial habit as derived, they hypothesized at least five independent origins of annual lineages from a perennial ancestor (Fig. 6). In their generic realignment, Chuang and Heckard (1991) moved 12 annual species from Orthocarpus (sensu Keck, 1927) and the monotypic annual genus Gentrya into their expanded Castilleja, breaking from the tradition of Castilleja as a strictly perennial genus. However, Castilleja sect. Oncorhynchus, Castilleja racemosa, Clevelandia, Ophiocephalus, and Triphysaria were all viewed as annual lineages independently derived from an ancestral perennial lineage (Cordylanthus and Orthocarpus were left unresolved with respect to the other lineages; Fig. 6). This interpretation likely stems from the conventional wisdom that annual taxa are derived from perennial ancestors. Historically, this belief is seated in two lines of evidence. First, morphologists and plant evolutionary biologists have long held that the woody habit, and thus the perennial life history strategy, is the ancestral condition in angiosperms (e.g., Jeffrey, 1916; Ames, 1939; Stebbins, 1957), and therefore an herbaceous annual would necessarily be derived. Second, the majority of studies investigating the annual habit in plants are devoted to the evolution of the annual habit as a bet-hedging strategy with respect to their perennial relatives in extreme and unpredictable environments. Multiple authors have discussed the origin of desert annuals from perennial ancestors (e.g., Gleason and Cronquist, 1964; Johnson, 1968; Axelrod, 1979), and this common view may be an extension of some of these ideas. Surprisingly, there have been few studies on the evolution of the annual and perennial habits in a phylogenetic context (e.g., Bena et al., 1998; Conti et al., 1999; Andreasen and Baldwin, 2001; Verboom et al., 2004).
These results demonstrate that, contrary to the classical interpretation, the perennial Castilleja clade is derived from a grade of annual taxa including the annual Castilleja species comprising sect. Oncorhynchus (Fig. 5). A straightforward parsimony reconstruction of life history strategy on our best estimate of Castillejinae phylogeny suggests that the perennial habit evolved a single time from an annual ancestral lineage that persisted throughout the diversification of Castillejinae. Since the evolution of perenniality in Castilleja, there have been some reversions to the annual habit. Castilleja arvensis, C. exilis, and C. racemosa (formerly Gentrya racemosa) are all annual species sampled in this study that are derived from within the perennial Castilleja clade (Fig. 5), and the annual sect. Epichroma (five spp., not sampled here) is likely another. However, with the exception of the annual habit, these species are more similar morphologically to other perennial Castilleja species, including their floral morphology, which conforms to the derived flower structure seen in the perennial Castilleja clade. In addition, the two Baja California endemics, Clevelandia and Ophiocephalus, may be derived from within the perennial clade, although the optimal trees place them as sister to the perennial clade with weak support (Fig. 3). Therefore, it is likely that Clevelandia and Ophiocephalus retain the ancestral annual habit and are either the sister lineage or successive sister lineages to the perennial Castilleja clade.
Polyploidy is found only in Castilleja, and the large majority of polyploid species are found in the perennial Castilleja clade (Fig. 4), where the number of polyploid species and intraspecific variation in ploidy level is great. Some species are reported to have a single ploidy level (e.g., C. arvensis [N = 12], C. pruinosa [n = 24]), while others have both diploid (n = 12) and tetraploid (n = 24) counts reported (e.g., C. chromosa,C. linariifolia,C. tenuiflora), and some include multiple ploidy levels (e.g., C. lutescens [n = 24, 48, 60], C. miniata [n = 12, 24, 36, 48, 60], C. peckiana [n = 36, 48, 60], C. septentrionalis [n = 12, 24, 48]) (Heckard, 1968; Heckard and Chuang, 1977; Spellenberg, 1986; Chuang and Heckard, 1993; Chambers et al., 1998). Based on chromosome counts from approximately 120 species of Castilleja, it was estimated that, in western North America, more than 50% of the species have polyploid representatives, while in Mexico 25% of the species were estimated to be polyploids (Chuang and Heckard, 1993).
Stebbins (1938, 1950) noted an association between herbaceous perennials and the frequency of polyploidy, and these results suggest a link between perenniality and polyploidy in Castilleja. Although these two traits seem to be correlated, the order in which they occur has been debated. Among herbaceous perennials, polyploidy has been associated with taxa that have effective means of vegetative reproduction and, furthermore, that perenniality and vegetative reproduction are the result of polyploidization (Gustafsson, 1948). Alternatively, it has been suggested that polyploidization is easier in a perennial background, owing to the possibility that perenniality and effective vegetative reproduction may allow the negative effects of polyploidization (e.g., reproductive isolation, meiotic irregularity) to be buffered for several generations (Stebbins, 1950). Neopolyploids commonly go through a period of reduced fertility, but fertility has been shown to increase rapidly in early generation polyploids (Ramsey and Schemske, 2002), and the formation of tetraploids through triploid intermediates (triploid bridge) may be facilitated by perenniality (Ramsey and Schemske, 1998).
In Castilleja (which is primarily outcrossing), it is likely that perenniality has played an important role in the origin and establishment of polyploidy. Because polyploid complexes are common throughout perennial Castilleja (Heckard, 1968; Heckard and Chuang, 1977; Chuang and Heckard, 1993), perenniality could be important for the maintenance of spontaneous polyploids in populations from which they otherwise would be reproductively isolated. Reproductive isolation of these individuals could then be alleviated by the spontaneous formation of other individuals of the same ploidy level. Contrary to classical view, the recurrent formation of polyploids is now considered the rule, rather than the exception (Soltis and Soltis, 1993; Soltis et al., 2003). Despite the prevalence of polyploidy among perennial Castilleja species, the lack of resolution of interspecific relationships and limited sampling (interspecific and intraspecific) within this clade prevents any inference as to the importance of polyploidy to speciation, but will be the focus of future research.
In addition, these analyses revealed a marked difference between the amount of sequence divergence (as evidenced by the mean branch length estimates) within each of the annual lineages of Castillejinae and the clade comprising the perennial Castilleja species (Figs. 2 and 4). Although the amount of inferred sequence divergence from the most recent common ancestor of Castillejinae is roughly equivalent between the perennials and any of the annual lineages, the inferred sequence divergence among species in each terminal clade varies dramatically between annuals and perennials. Given the representative, but incomplete, sampling of perennial Castilleja species, it is clear that the perennial clade has dramatically less divergence among species than the annual clades. This conspicuously lower sequence divergence at both chloroplast and nuclear loci suggests that the diversification of perennial Castilleja species in western North America represents a recent event in the evolution of Castillejinae.
Morphological evolution and systematic implications
The results from molecular phylogenetic analysis of the chloroplast and nuclear ribosomal DNA regions support the monophyly of subtribe Castillejinae. Figure 5 shows a summary of relationships among the major lineages of Castillejinae, their current taxonomic circumscription, and a list of diagnostic morphological traits for each clade. Table 4 provides a summary of morphological characteristics traditionally used to recognize genera in Castillejinae and whether the group was recovered as monophyletic in our analyses. In this section, each genus is discussed with reference to the most recent reorganization of generic boundaries in Castillejinae (Fig. 6; Chuang and Heckard, 1991) and the evolution of morphological characteristics important for the recognition of the major lineages recovered (sensu Scotland et al., 2003).
Orthocarpus
Orthocarpus sensu Chuang and Heckard (1992b) represents a well-supported monophyletic group (Fig. 3). Chuang and Heckard (1991) recognized that Orthocarpus s.l. (Keck, 1927) comprised three distinct groups of species that did not necessarily belong together. Molecular data support this assertion, resolving Orthocarpus s.s. as one of the basal lineages of the subtribe, which is not particularly close to either Triphysaria or sect. Oncorhynchus of Castilleja (Figs. 1 and 3). The nine species of Orthocarpus can be recognized by the morphology of the lower corolla lip, which is only minutely three-toothed, and their unequally cleft calyx in which the dorsal cleft is cut more deeply than that of the ventral calyx cleft (Fig. 5; see Figs. 2 and 8 in Chuang and Heckard, 1991).
Cordylanthus
Cordylanthus (sensu Chuang and Heckard, 1986) was not monophyletic in any phylogenetic analysis (Table 4), but rather was recovered in two distinct, well-supported clades corresponding to Cordylanthus subg. Cordylanthus and a clade containing the monotypic Cordylanthus subg. Dicranostegia plus Cordylanthus subg. Hemistegia (Figs. 1, 3, and 5). This result was surprising given that Cordylanthus has been considered one of the most distinctive genera of Castillejinae, due primarily to the presence of a unique calyx which is cleft completely (or nearly so) to the base abaxially and fused adaxially, forming a spathe-like structure that surrounds the corolla (Chuang and Heckard, 1973, 1986; see Fig. 1 in Chuang and Heckard, 1991 and Fig. 5 in Chuang and Heckard, 1986). However, based on the results presented here, a parsimony reconstruction of this distinctive feature on our best estimate of Castillejinae phylogeny suggests that the spathe-like calyx has arisen independently in the two lineages. Chuang and Heckard (1986), following Pennell (1947), hypothesized that the origin of the spathe-like calyx of Cordylanthus is an evolutionary modification of the four-lobed calyx found throughout tribe Pedicularideae, where fusion of the two lateral lobes and the deepening of the abaxial cleft is followed by further fusion of the adaxial cleft to produce the one-parted, spathe-like calyx typical of most Cordylanthus species. Two species, Cordylanthus orcuttianus of subg. Dicranostegia and C. capitatus of subg. Cordylanthus, have a deeply cleft calyx, which can be interpreted as the incomplete fusion of the adaxial cleft following the fusion of the lateral calyx lobes and deepening of the abaxial cleft (Chuang and Heckard, 1975a, 1986). Cordylanthus capitatus is resolved as the sister species to the remainder of Cordylanthus subg. Cordylanthus (Fig. 3), and in addition to its bifid calyx, it also has a definite tubular base, indicating that the deepening of the abaxial calyx cleft is not complete. Within subg. Cordylanthus, nearly all of the species have a calyx with a ± tubular base and a bifid tip (sometimes only minutely so). Likewise, within the second Cordylanthus clade, C. orcuttianus (subg. Dicranostegia) is resolved as the sistergroup to the Cordylanthus subg. Hemistegia clade (Figs. 1, 3, and 5). Along with its deeply bifid calyx, C. orcuttianus also has a nearly tubular base (Chuang and Heckard, 1975a). The four species of subg. Hemistegia all have the derived, spathe-like calyx that lacks a tubular base, but is usually minutely cleft at the tip (Chuang and Heckard, 1973). Given the phylogenetic relationships within the two Cordylanthus clades presented here (Fig. 3), the evolutionary modification of the calyx hypothesized by Chuang and Heckard (1986) is consistent with the distribution of calyx features within Cordylanthus, but would have occurred two times independently.
The two Cordylanthus clades comprise two additional basal lineages of Castillejinae. Cordylanthus subg. Cordylanthus is resolved as the sister group to Orthocarpus in all analyses of the cpDNA data (Figs. 1 and 2) and the combined Bayesian and parsimony analyses (Figs. 3 and 4), or as the sister group to the remainder of Castillejinae in analyses of the nrDNA data (Figs. 1 and 2) and the combined ML analysis (tree not shown); however, neither of these relationships were well supported, and there are no apparent morphological synapomorphies for the monophyly of Orthocarpus and Cordylanthus subg. Cordylanthus. Members of subg. Cordylanthus can be recognized by two morphological synapomorphies. First, the architecture of the inflorescence has undergone an evolutionary reduction from the basic spike common throughout the subtribe (and the majority of Orobanchaceae) to single-flowered florescences and the subsequent clustering of these into capitate or spike-like synflorescences. Members of this clade possess inflorescences that are a reduced spike (<2 cm), single-flowered florescences, or capitate or spike-like synflorescences (Fig. 5; see Fig. 2 in Chuang and Heckard, 1986). The second morphological synapomorphy is a tightly revolute tip of the middle lobe of the lower corolla lip (Fig. 5).
The second Cordylanthus clade, comprised of the monotypic subg. Dicranostegia and Cordylanthus subg. Hemistegia, was resolved unambiguously as the sister lineage to the remainder of Castillejinae (exclusive of subg. Cordylanthus and Orthocarpus) in all analyses (Figs. 1, 3, and 5), although there is no apparent morphological synapomorphy for this more inclusive clade. Throughout Cordylanthus, there has been a tendency toward a reduction of the androecium, putatively correlated with increased specialization in pollination (Chuang and Heckard, 1986). This characteristic is most pronounced in the clade containing Dicranostegia and Hemistegia (Fig. 5), where the androecium is reduced to two fertile stamens. The majority of the 13 species of subg. Cordylanthus possess four didynamous stamens, each with two anther sacs (the upper, larger anther sac attached medially and the lower, smaller anther sac attached apically, as in most members of Castillejinae; Fig. 5). In subg. Cordylanthus, the four fertile stamens of C. nevinii A. Gray and C. laxiflorus A. Gray (neither sampled in this study) have been reduced to only the upper anther sac, and C. capitatus, which is sister to the rest of the clade, has been reduced to only two fertile stamens, each possessing only the upper anther sac (Chuang and Heckard, 1986). The remaining 10 species of subg. Cordylanthus have undergone no reduction of the androecium. Given the sister-group relationship of C. capitatus and the remainder of subg. Cordylanthus (Fig. 3), the reduction of the androecium in those three species may represent a synapomorphy for a subgroup or autapomorphies for individual taxa (if C. nevinii, C. laxiflorus, and C. capitatus are not monophyletic). In contrast, a reduced androecium in the clade containing subg. Dicranostegia and subg. Hemistegia can be interpreted as a morphological synapomorphy (Fig. 5). In the monotypic subg. Dicranostegia, the androecium of C. orcuttianus consists of two fertile stamens (the longer, anterior stamens), each with two anther sacs, while the posterior pair is reduced to filaments bearing sterile appendages (Chuang and Heckard, 1975a, 1986). In the sister group of C. orcuttianus, Cordylanthus subg. Hemistegia, three of the four species possess two fertile anterior stamens (with two anther-sacs each) with the posterior pair of stamens reduced to two rudimentary filaments (Chuang and Heckard, 1986). The posterior stamens of the fourth species, C. maritimus, each bear one fertile anther sac (rather than just a rudimentary filament), and this species is the only exception to the pattern of reduction that unites subg. Dicranostegia and subg. Hemistegia.
Cordylanthus subg. Hemistegia is comprised of a specialized group of species adapted to the coastal and inland saline habitats of western North America (Chuang and Heckard, 1973, 1986). The distinctive nature of this group of species has been recognized by numerous authors (e.g., Gray, 1867; Ferris, 1918; Chuang and Heckard, 1973) and has been described as the distinct genus Chloropyron Behr (Behr, 1855). Subgenus Hemistegia is further distinguished from Cordylanthus subg. Cordylanthus by having the typical inflorescence type found throughout subtribe Castillejinae, an elongate spike with only one floral bract associated with each flower (in contrast to the synflorescence described earlier; Chuang and Heckard, 1973). In addition to the halophytic nature of this group, Cordylanthus subg. Hemistegia is marked by the following morphological synapomorphies: (1) the terminal stigmatic surface is bent backward at maturity, and (2) the middle lobe of the lower corolla lip is erect (Fig. 5; Chuang and Heckard, 1973, 1986).
Triphysaria
The elevation of Orthocarpus subg. Triphysaria (sensu Keck, 1927) to generic status (as Triphysaria) was based on chromosome number, seed and seed-coat morphology, floral morphology, and experimental hybridization studies (Chuang and Heckard, 1991). In the phylogenetic analyses presented here, Triphysaria was recovered as a well-supported lineage (Figs. 1 and 3) and the sister group of Castilleja s.l. The monophyly of Triphysaria and Castilleja s.l. is supported by a number of morphological synapomorphies (Fig. 5). In both Orthocarpus and Cordylanthus the galea (formed by the fusion of the two petals comprising the upper corolla lip of the zygomorphic corolla) is closed at the tip, being the product of the two fused corolla lobes that are folded downward forming a true galea, or hood (Chuang and Heckard, 1991). In both Triphysaria and Castilleja s.l. the upper corolla lip is open at the tip, and there is no true galea (Fig. 5; see Figs. 7–12 in Chuang and Heckard, 1991). Chuang and Heckard (1991) noted that the use of the term galea throughout the Castilleja literature is in error and instead promoted the use of the term beaked to describe this condition. Triphysaria and Castilleja s.l. also share the derived characteristics of an expanded stigma that is either capitate or bilobed (Fig. 5; the stigma is unexpanded in both Cordylanthus and Orthocarpus) and a terminal attachment of the ovule to the placenta (Fig. 5; Cordylanthus and Orthocarpus both have a lateral hilum).
Morphologically, the species of Triphysaria are very similar to the annual species of Castilleja subg. Colacus sect. Oncorhynchus (also removed from Orthocarpus s.l. by Chuang and Heckard, 1991). However, a number of the characteristics used by Chuang and Heckard (1991) as justification for the elevation of Triphysaria to generic status provide morphological synapomorphies for the genus, given the relationships presented here (Fig. 5). In all five species of Triphysaria, the stamens are reduced to a single anther sac, whereas the sister group (Castilleja s.l.) have no such reduction in the androecium. With the exception of the tiny (4–6 mm) corolla of T. pusilla, the throat of the corolla is abruptly indented, forming a distinct fold below the lower corolla lip, distinguishing Triphysaria morphologically from the annual species of Castilleja sect. Oncorhynchus (see Figs. 9–11 in Chuang and Heckard, 1991). Lastly, Triphysaria are cytologically unique among members of Castillejinae with a chromosome number of n = 11 (Fig. 5).
Castilleja
Despite extensive taxonomic study of this iconic group of western North American wildflowers, infrageneric classification in Castilleja has been difficult. Most efforts to classify this group (Bentham, 1846; Gray, 1862; Wettstein, 1891; Eastwood, 1909; Rydberg, 1917; Pennell, 1935, 1951; Ownbey, 1959; Holmgren, 1970, 1971, 1976, 1984; Nesom, 1992a–1992c, 1994) have been ignored largely due to their failure to delimit cohesive units within Castilleja or to their narrow use of small or monotypic infrageneric groups. Chuang and Heckard (1991) proposed a new infrageneric classification for Castilleja including the three subgenera, Castilleja, Colacus, and Gentrya. The majority of Castilleja species were included in subg. Castilleja (150 mostly perennial species), which includes species with flowers primarily modified for hummingbird pollination. Subgenus Colacus includes the majority of Castilleja species that have flowers modified for insect pollination. Colacus includes two sections of perennials that have been assigned historically to Orthocarpus and Castilleja (sects. Pilosae and Pallescentes) and all of the annual Castilleja species (sect. Oncorhynchus) previously assigned to Orthocarpus by Keck (1927). Subgenus Gentrya comprises the single species Castilleja racemosa (= G. racemosa Breedlove & Heckard).
Castilleja was not monophyletic in any of the separate or combined analyses of the cpDNA and nrDNA regions because the two monotypic annual genera Clevelandia and Ophiocephalus are derived from within the mostly perennial genus Castilleja (Table 4). The clade containing Castilleja, Clevelandia, and Ophiocephalus was recovered by separate and combined analyses of the cpDNA and nrDNA data (Figs. 1 and 3). The only potential morphological synapomorphy for this clade is the presence of a loose-fitting seed coat (i.e., the reticulate outer seed coat forms a network that encloses a freely suspended body made up of the inner seed coat, endosperm, and embryo; see Figs. 25–49 in Chuang and Heckard, 1983). Morphological characteristics of the seed coat have been used extensively for infrageneric classification throughout Castillejinae (e.g., Chuang and Heckard, 1972, 1983, 1992b) and as one of the major characteristics separating Cordylanthus,Orthocarpus, and Triphysaria from the annual species of Castilleja (Chuang and Heckard, 1991). However, of the five species of Triphysaria, two species, Triphysaria micrantha and T. eriantha (Fig. 3), have a loose-fitting seed coat like Castilleja (Chuang and Heckard, 1991). Therefore, without knowing the underlying developmental mechanism for this morphological change, whether a tight-fitting seed coat was a reversal in one lineage of Triphysaria or the loose-fitting seed coat arose independently in Castilleja and the two species of Triphysaria is equivocal (Figs. 3 and 5). Although there is no unequivocal morphological synapomorphy for the Castilleja s.l. clade, this group is cytologically distinct from the remainder of the subtribe with a basic haploid chromosome number of x = 12 (Fig. 5).
Clevelandia & Ophiocephalus
Clevelandia and Ophiocephalus have been maintained as distinct genera in Castillejinae despite numerous similarities to Castilleja, including chromosome number (n = 12), vegetative morphology, seed coat morphology, and the ability to make fertile hybrids in experimental crosses (only Ophiocephalus was tested for cross compatibility; Chuang and Heckard, 1991). Both of these genera possess unique corolla morphologies that formed the primary justification of their status as separate genera (Chuang and Heckard, 1991). Clevelandia has a small corolla (1–2 cm) in which the lobes of the upper corolla lip are not fused, and the lower corolla lip has three triangular, spreading lobes that are wider than the upper corolla lobes, forming a slightly zygomorphic corolla (Greene, 1885, 1886). Likewise, the inflated corolla of Ophiocephalus, with its long, exserted stamens, is only slightly bilabiate; however, the upper corolla lip is fused into a beak, as in Castilleja (Wiggins, 1933). Field observations of a population of Ophiocephalus angustifolius suggest that the distinct floral morphology of this species is likely the result of increased specialization to pollinators (J. M. Egger [WTU], personal communication), and we postulate the same is true for the unusually small, open corolla of Clevelandia beldingiii.
The sister-group relationship of Clevelandia and Ophiocephalus received a significant Bayesian PP value (0.99) but low ML and parsimony bootstrap support (50% and 41%, respectively) and, therefore, may not represent the true relationship of these two species (Fig. 3). Based on the combined analyses of the cpDNA and nrDNA data (Fig. 3), these two annual genera are more closely related to perennial Castilleja than they are to any of the annual species transferred to Castilleja from Orthocarpus (sensu Keck, 1927) and may represent the sister lineage (or successive sister lineages) to the perennial Castilleja clade.
Castilleja subgenus Colacus section Oncorhynchus
Chuang and Heckard (1991) were correct in their interpretation that the two former sections of the annual genus Orthocarpus (sects. Castillejoides and Cordylanthoides, sensu Keck, 1927) belonged with Castilleja, where they were placed in subg. Colacus sect. Oncorhynchus. However, these annuals form a basal grade within the Castilleja s.l. clade (Fig. 5), rather than the monophyletic group hypothesized by Chuang and Heckard (Fig. 6; Table 4). The majority of the annual species were recovered in two well-supported clades (Fig. 3), which correspond to the two sections of Keck (1927); the clade containing Castilleja lineariloba and C. attenuata is equivalent to sect. Castillejoides (with the exception of Castilleja rubicundula), while the clade containing C. campestris and C. tenuis is equivalent to sect. Cordylanthoides. Castilleja exserta subsp. exserta (formerly of sect. Castillejoides) represents an additional independent lineage comprising the grade of annual Castilleja species approaching the remainder of the Castilleja s.l. clade (Fig. 3).
Perennial Castilleja and subgenus Gentrya
The majority of the Castilleja species sampled here are included in the large perennial Castilleja clade (Figs. 3 and 5). Because of the lack of resolution with the cpDNA and nrDNA regions used in this study, few conclusions regarding the relationships of perennial Castilleja species can be drawn. Perennial Castilleja are a morphologically complex group with numerous infrageneric assemblages and intraspecific taxa in which the taxonomic complexity is extremely challenging. This complexity stems from (1) complex morphological variation often attributed to the formation of polyploid complexes (Heckard, 1968; Heckard and Chuang, 1977) and from (2) the difficulty in circumscribing taxa due to overlapping variation in nearly every character (Holmgren, 1984). These two sources of confusion in Castilleja taxonomy are not mutually exclusive. More than half of the perennial species are known to have polyploid populations, with ploidy levels ranging from 4x to 12x (Heckard, 1968; Heckard and Chuang, 1977), and polyploidy in Castilleja is often attributed to hybridization (Heckard and Chuang, 1977). Apparent hybrid swarms and morphological intergradation are common between populations of some species, especially those belonging to polyploid complexes (Heckard, 1968; Heckard and Chuang, 1977; Chuang and Heckard, 1993; Hersch and Roy, 2007). In experimental hybridization studies, hybrids of varying degrees of fertility were obtained from a wide variety of interspecific crosses, including those between parents with different ploidy levels (Heckard, 1968; Chuang and Heckard, 1991). Thus, interspecific hybridization may be responsible for much of the conspicuous overlapping variation observed between many Castilleja species (Holmgren, 1984; Chuang and Heckard, 1991). It is clear that to investigate interspecific relationships within the perennial Castilleja clade, it will be necessary to generate a much larger cpDNA data set or sequence DNA regions that are more variable than cpDNA or nrDNA (i.e., single or low-copy nuclear genes).
In their realignment of generic boundaries, Chuang and Heckard (1991) included the monotypic Mexican genus Gentrya in Castilleja (as C. racemosa), creating a new monotypic subgenus (Fig. 6). In its original description, the corolla of Gentrya racemosa was described as "more truly galeate" than Castilleja because of its curved, helmet-shaped upper corolla lip, rather than the straight, beak-like upper corolla lip of Castilleja (Breedlove and Heckard, 1970, p. 23). This trait formed the basis for its status as a distinct genus in Castillejinae, despite the many other noted similarities to Castilleja. However, Chuang and Heckard (1991) felt that this distinction alone did not warrant generic status, given their expanded view of Castilleja. In their intuitive view of the phylogenetic relationships of the major lineages of Castillejinae, Chuang and Heckard (1991) placed Castilleja subg. Gentrya as the sister lineage to the remainder of Castilleja and viewed the species as an isolated lineage in Castilleja (Fig. 6). Our phylogenetic analyses place C. racemosa sister to a group of other Mexican and Central American Castilleja species (Fig. 3) within the perennial Castilleja clade (Fig. 5). This relationship received significant Bayesian PP values (0.98 and 1.0), but relatively low ML (66% and 86%) and parsimony (48% and 84%) bootstrap support (Fig. 3); therefore, the precise relationship of this species to the remainder of Castilleja s.l. is still tentative. Nevertheless, C. racemosa is a member of the clade containing perennial Castilleja, Clevelandia, and Ophiocephalus (Fig. 5), and not an isolated lineage of Castillejinae (e.g., Breedlove and Heckard, 1970) or Castilleja (e.g., Chuang and Heckard, 1991) as previously hypothesized.
Systematic conclusions
(1) Subtribe Castillejinae is monophyletic and includes the six genera traditionally recognized as members of this group (sensu Chuang and Heckard, 1991). With the exception of Ophiocephalus and those species that have undergone a reduction to only one anther sac per stamen (e.g., Triphysaria and some species of Cordylanthus), members of Castillejinae are characterized by having anther sacs that are unequal in size and unequally attached (Fig. 5).
(2) Orthocarpus and Triphysaria are monophyletic and represent two separate evolutionary lineages within Castillejinae. Castilleja subg. Colacus sect. Oncorhynchus, also previously recognized as Orthocarpus, is not monophyletic, but rather, forms a basal grade of annual species within Castilleja s.l. Although all three of these groups were previously included in Orthocarpus (sensu Keck, 1927) as suggested by Chuang and Heckard (1991), they are more closely related to other members of the subtribe than they are to each other.
(3) Cordylanthus is biphyletic; subg. Cordylanthus and subg. Hemistegia are each monophyletic, and the monotypic subg. Dicranostegia is sister to subg. Hemistegia. All these clades can be recognized by morphological synapomorphies (Fig. 5).
(4) Castilleja is not monophyletic because the two monotypic genera Clevelandia and Ophiocephalus are derived from within this clade (Castilleja s.l.). The majority of Castilleja species are perennial, and these taxa, along with Clevelandia and Ophiocephalus, form a well-supported clade derived from the grade of annual taxa in subg. Colacus sect. Oncorhynchus. This clade includes Castilleja racemosa, which was formerly isolated as the monotypic genus Gentrya.
(5) Based on the well-supported phylogenetic hypothesis for Castillejinae presented here, it will be necessary to revise the current circumscription of generic boundaries. In the formal treatment that will be presented elsewhere, Orthocarpus and Triphysaria will retain their current circumscriptions, Castilleja will be expanded to include the two monotypic genera Clevelandia and Ophiocephalus, and Cordylanthus will be split into three genera corresponding to the three previously recognized subgenera.
Conclusions
The results of this study place the generic realignment of Castillejinae presented by Chuang and Heckard (1991) in the context of a robust phylogenetic hypothesis for all of the major lineages comprising the subtribe. In addition to providing a basis for the systematics of this group, we were able to interpret morphological characters that are important for recognizing major lineages (sensu Scotland et al., 2003) and to draw inferences regarding the evolution of the perennial habit. The observation of a seemingly tight association between perenniality and polyploidy in Castilleja sets the stage for future research focused on the detection of correlates between organismal traits (e.g., chromosomal change and shifts in life history) and shifts in the rate of diversification. The restriction of hummingbird pollination to the perennial clade of Castilleja suggests another possible association that may reflect underlying constraints of perenniality. Thus, the findings presented here serve as valuable background information for future research not only in Castillejinae and the large, complex genus Castilleja, but also for investigating commonalities that may have been important for plant diversification in western North America.
FOOTNOTES
1 The authors thank J. Ammirati, T. Bradshaw, M. Egger, and three anonymous reviewers for critical comments on earlier drafts of the manuscript; M. Donoghue, K. Karol, B. Moore, T. Near, and S. Smith for helpful discussions; and S. Collier, P. Lu-Irving, and P. Reeves for laboratory assistance. This research was supported by a Graduate Fellowship in Molecular Systematics from the University of Washington Department of Botany, the Karling Graduate Student Research Award from the Botanical Society of America, the Research Award for Graduate Students from the American Society of Plant Taxonomists, the Award for Graduate Student Research from the Society of Systematic Biologists, a Sigma-Xi Grants in Aid of Research from the University of Washington Chapter, and the Giles Award for Graduate Student Field Research from the University of Washington Department of Botany to D.C.T., and the NSF Doctoral Dissertation Improvement Grant DEB-0412653 to R.G.O. for D.C.T. 
4 Author for correspondence (e-mail: david.tank{at}yale.edu); present address: Division of Botany, Peabody Museum of Natural History, Yale University, P.O. Box 208118, New Haven, CT 06520-8118 USA
LITERATURE CITED
Ames, O. 1939. Economic annuals and human cultures. Botanical Museum of Harvard University, Cambridge, Massachusetts, USA.
Andreasen, K., AND B. G. Baldwin. 2001. Unequal evolutionary rates between annual and perennial lineages of checker mallows (Sidalcea, Malvaceae): Evidence from 18S-26S rDNA internal and external transcribed spacers. Molecular Biology and Evolution 18: 936–944.
Axelrod, D. I. 1979. Age and origin of the Sonoran desert vegetation. Occasional Papers of the California Academy of Sciences 132: 1–74.
Baldwin, B. G. 1992. Phylogenetic utility of the internal transcribed spacers of nuclear ribosomal DNA in plants: An example from the Compositae. Molecular Phylogenetics and Evolution 1: 3–16.[CrossRef][Medline]
Baldwin, B. G., AND S. Markos. 1998. Phylogenetic utility of the external transcribed spacer (ETS) of 18S-26S rDNA: Congruence of ETS and ITS trees of Calycadenia (Compositae). Molecular Phylogenetics and Evolution 10: 449–463.[CrossRef][Web of Science][Medline]
Baldwin, B. G., M. J. Sanderson, J. M. Porter, M. F. Wojciechowski, C. S. Campbell, AND M. J. Donoghue. 1995. The ITS region of nuclear ribosomal DNA: A valuable source of evidence on angiosperm phylogeny. Annals of the Missouri Botanical Garden 82: 247–277.[CrossRef][Web of Science]
Barker, F. K., AND F. M. Lutzoni. 2002. The utility of the incongruence length difference test. Systematic Biology 51: 625–637.
Beardsley, P. M., AND R. G. Olmstead. 2002. Redefining Phrymaceae: The placement of Mimulus, tribe Mimuleae and Phryma. American Journal of Botany 89: 1093–1102.
Behr, H. H. 1855. A new genus and species, Chloropyron palustre. Proceedings of the California Academy of Sciences 1: 62.
Bena, G., B. Lejeune, J.-M. Prosperi, AND I. Olivieri. 1998. Molecular phylogenetic approach for studying life-history evolution: The ambiguous example of the genus Medicago L. Proceedings of the Royal Society of London, B, Biological Sciences 265: 1141–1151.
Bennett, J. R., AND S. Mathews. 2006. Phylogeny of the parasitic plant family Orobanchaceae inferred from phytochrome A. American Journal of Botany 93: 1039–1051.
Bentham, G. 1846. Scrophulariaceae. In A. de Candolle [ed.], Prodromus systematis naturalis regni vegetabilis, vol. 10, 180–586. Victoris Masson, Paris, France.
Brandley, M. C., A. Schmitz, AND T. W. Reeder. 2005. Partitioned Bayesian analyses, partition choice, and the phylogenetic relationships of scincid lizards. Systematic Biology 54: 373–390.
Breedlove, D. E., AND L. R. Heckard. 1970. Gentrya, a new genus of Scrophulariaceae from Mexico. Brittonia 22: 20–24.[CrossRef][Web of Science]
Brown, J. M., AND A. R. Lemmon. 2007. The importance of data partitioning and the utility of Bayes factors in Bayesian phylogenetics. Systematic Biology 56: 643–655.
Bull, J. J., J. P. Huelsenbeck, C. W. Cunningham, D. L. Swofford, AND P. J. Waddell. 1993. Partitioning and combining data in phylogenetic analysis. Systematic Biology 42: 384–397.
Chambers, K. L., D. Green, S. Potampa, AND L. McMahan. 1998. IOPB chromosome data 13. International Organization of Plant Biosystematists Newsletter 29: 18–22.
Chuang, T. I. 1993. A cladistic analysis of the subtribe Castillejinae (Scrophulariaceae-tribe Pediculareae). Botanical Bulletin of Academia Sinica 34: 155–161.[Web of Science]
Chuang, T. I., AND L. R. Heckard. 1972. Seed coat morphology in Cordylanthus (Scrophulariaceae) and its taxonomic significance. American Journal of Botany 59: 258–265.[CrossRef][Web of Science]
Chuang, T. I., AND L. R. Heckard. 1973. Taxonomy of Cordylanthus subgenus Hemistegia (Scrophulariaceae). Brittonia 25: 135–158.[CrossRef][Web of Science]
Chuang, T. I., AND L. R. Heckard. 1975a. Taxonomic status of Cordylanthus (subg. Dicranostegia) orcuttianus (Scrophulariaceae). Madroño 23: 88–95.
Chuang, T. I., AND L. R. Heckard. 1975b. Re-evaluation of bract morphology in taxonomy of Cordylanthus (Scrophulariaceae). Madroño 23: 169–173.
Chuang, T. I., AND L. R. Heckard. 1976. Morphology, evolution, and taxonomic significance of the inflorescence in Cordylanthus (Scrophulariaceae). American Journal of Botany 63: 272–282.[CrossRef][Web of Science]
Chuang, T. I., AND L. R. Heckard. 1982. Chromosome numbers of Orthocarpus and related monotypic genera (Scrophulariaceae, subtribe Castillejinae). Brittonia 34: 89–101.[CrossRef][Web of Science]
Chuang, T. I., AND L. R. Heckard. 1983. Systematic significance of seed-surface features in Orthocarpus (Scrophulariaceae subtribe Castillejinae). American Journal of Botany 70: 877–890.[CrossRef][Web of Science]
Chuang, T. I., AND L. R. Heckard. 1986. Systematics and evolution of Cordylanthus (Scrophulariaceae-Pedicularieae). Systematic Botany Monographs 10: 1–105.
Chuang, T. I., AND L. R. Heckard. 1991. Generic realignment and synopsis of subtribe Castillejinae (Scrophulariaceae—tribe Pediculareae). Systematic Botany 16: 644–666.[CrossRef][Web of Science]
Chuang, T. I., AND L. R. Heckard. 1992a. New species of bee-pollinated Castilleja from Peru, with a taxonomic revision of South American members of subg. Colacus. Systematic Botany 17: 417–431.[CrossRef][Web of Science]
Chuang, T. I., AND L. R. Heckard. 1992b. A taxonomic revision of Orthocarpus (Scrophulariaceae—tribe Pediculareae). Systematic Botany 17: 560–582.[CrossRef][Web of Science]
Chuang, T. I., AND L. R. Heckard. 1993. Chromosome numbers of neotropical Castilleja (Scrophulariaceae, tribe Pediculareae) and their taxonomic implications. Annals of the Missouri Botanical Garden 80: 974–986.[CrossRef][Web of Science]
Conti, E., D. E. Soltis, T. M. Hardig, AND J. Schneider. 1999. Phylogenetic relationships of the silver saxifrages (Saxifraga, sect. Ligulatae Haworth): Implications for the evolution of substrate specificity, life histories, and biogeography. Molecular Phylogenetics and Evolution 13: 536–555.[CrossRef][Web of Science][Medline]
Cunningham, C. 1997. Can three incongruence tests predict when data should be combined? Molecular Biology and Evolution 14: 733–740.[Abstract]
Darlu, P., AND G. Lecointre. 2002. When does the incongruence length difference test fail? Molecular Biology and Evolution 19: 432–437.
DeBry, R. W., AND R. G. Olmstead. 2000. A simulation study of reduced tree-search effort in bootstrap resampling analysis. Systematic Biology 49: 171–179.[CrossRef][Web of Science][Medline]
Doyle, J. J., AND J. L. Doyle. 1987. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochemical Bulletin 19: 11–15.
Eastwood, A. 1909. Synopsis of the Mexican and Central American species of Castilleja. Proceedings of the American Academy of Arts 44: 563–591.
Farris, J. S., M. Källersjö, A. G. Kluge, AND C. Bult. 1994. Testing significance of incongruence. Cladistics 10: 315–319.[CrossRef][Web of Science]
Felsenstein, J. 1985. Confidence limits on phylogenies—an approach using the bootstrap. Evolution 39: 783–791.[CrossRef][Web of Science]
Ferris, R. S. 1918. Taxonomy and distribution of Adenostegia. Bulletin of the Torrey Botanical Club 45: 399–423.[CrossRef]
Gielly, L., AND P. Taberlet. 1994. The use of chloroplast DNA to resolve plant phylogenies—Noncoding versus rbcL sequences. Molecular Biology and Evolution 11: 769–777.[Abstract]
Gleason, H. A., AND A. Cronquist. 1964. The natural geography of plants. Columbia University Press, New York, New York, USA.
Graham, S. W., J. R. Kohn, B. R. Morton, J. E. Eckenwalder, AND S. C. H. Barrett. 1998. Phylogenetic congruence and discordance among one morphological and three molecular data sets from Pontederiaceae. Systematic Biology 47: 545–567.
Graham, S. W., P. A. Reeves, A. C. E. Burns, AND R. G. Olmstead. 2000. Microstructural changes in noncoding chloroplast DNA: Interpretation, evolution, and utility of indels and inversions in basal angiosperm phylogenetic inference. International Journal of Plant Sciences 161 6 Supplement : S83–S96.[CrossRef][Web of Science]
Gray, A. 1862. Revision of the genus Castilleja. American Journal of Science II 34: 335–339.
Gray, A. 1867. Characters of new plants of California and elsewhere, principally of those collected by H. N. Bolander in the state geological survey. Proceedings of the American Academy of Arts 7: 327–402.
Greene, E. L. 1885. Studies in the botany of California and parts adjacent. Bulletin of the California Academy of Science 1: 66–155.
Greene, E. L. 1886. Studies in the botany of California and parts adjacent II. Bulletin of the California Academy of Science 4: 179–228.
Gustafsson, Å. 1948. Polyploidy, life-form, and vegetative reproduction. Hereditas 34: 1–22.[Web of Science]
Heckard, L. R. 1958. Documented chromosome numbers in plants. Madroño 14: 237.
Heckard, L. R. 1968. Chromosome numbers and polyploidy in Castilleja (Scrophulariaceae). Brittonia 20: 212–226.[CrossRef][Web of Science]
Heckard, L. R., AND T. I. Chuang. 1977. Chromosome numbers, polyploidy, and hybridization in Castilleja (Scrophulariaceae) of the Great Basin and Rocky Mountains. Brittonia 29: 159–172.[CrossRef][Web of Science]
Heckard, L. R., M. I. Morris, AND T. I. Chuang. 1980. Origin and taxonomy of Castilleja montigena (Scrophulariaceae). Systematic Botany 5: 71–85.[CrossRef][Web of Science]
Hersch, E. I., AND B. A. Roy. 2007. Context-dependent pollinator behavior: An explanation for patterns of hybridization among three species of indian paintbrush. Evolution 61: 111–124.[CrossRef][Web of Science][Medline]
Holmgren, N. H. 1970. Castilleja. In D. S. Corell, and M. C. Johnson [eds.], Manual of the vascular plants of Texas, 1439–1442. Texas Research Foundation , Renner, Texas, USA.
Holmgren, N. H. 1971. A taxonomic revision of the Castilleja viscidula group. Memoirs of the New York Botanical Garden 21: 1–63.
Holmgren, N. H. 1976. Four new species of Mexican Castilleja (subgenus Castilleja, Scrophulariaceae) and their relatives. Brittonia 28: 195–208.[CrossRef][Web of Science]
Holmgren, N. H. 1984. Scrophulariaceae. In A. Cronquist, A. H. Holmgren, N. H. Holmgren, J. L. Reveal, and P. K. Holmgren [eds.], Intermountain flora, 344–506. New York Botanical Garden, Bronx, New York, USA.
Jeffrey, E. C. 1916. The anatomy of woody plants. University Press, Chicago, Illinois, USA.
Jepson, J. L. 1925. Manual of flowering plants of California. University of California Press, Berkeley, California, USA.
Johnson, A. W. 1968. The evolution of desert vegetation in western North America. In G. W. J. Brown [ed.], Desert BIOLOGY, 101–140. Academic Press, New York, New York, USA.
Kass, R., AND A. Raftery. 1995. Bayes factors. Journal of the American Statistical Association 90: 430–773.
Keck, D. D. 1927. A revision of the genus Orthocarpus. Proceedings of the California Academy of Science Series 4 16: 517–571.
Lockwood, M., AND M. Forstner. 1991. IOPB chromosome data 3. International Organization of Plant Biosystematists Newsletter 17: 10.
Nesom, G. L. 1992a. Castilleja section Euchroma (Scrophulariaceae) in Mexico: New species and comments on other taxa. Phytologia 73: 384–388.
Nesom, G. L. 1992b. New species and taxonomic evaluations of Mexican Castilleja (Scrophulariaceae). Phytologia 72: 231–252.
Nesom, G. L. 1992c. Taxonomy of the Castilleja tenuiflora group (Scrophulariaceae) in Mexico, with an overview of sect. Castilleja. Phytologia 73: 389–415.
Nesom, G. L. 1994. A new species of Castilleja (Scrophulariaceae) from Chihuahua, Mexico. Phytologia 76: 406–409.
Nylander, J. A. A., F. Ronquist, J. P. Huelsenbeck, AND J. L. Nieves-Aldrey. 2004. Bayesian phylogenetic analysis of combined data. Systematic Biology 53: 47–67.
Olmstead, R. G., C. W. dePamphilis, A. D. Wolfe, N. D. Young, W. J. Elisons, AND P. A. Reeves. 2001. Disintegration of the Scrophulariaceae. American Journal of Botany 88: 348–361.
Ownbey, M. 1959. Castilleja. In C. L. Hitchcock, A. Cronquist, M. Ownbey, and J. W. Thompson [eds.], Vascular plants of the Pacific Northwest, 295–326. University of Washington Press, Seattle, Washington, USA.
Oxelman, B., M. Liden, AND D. Berglund. 1997. Chloroplast rps16 intron phylogeny of the tribe Sileneae (Caryophyllaceae). Plant Systematics and Evolution 206: 393–410.[CrossRef][Web of Science]
Pennell, F. W. 1935. The Scrophulariaceae of eastern temperate North America. Academy of Natural Sciences of Philadelphia Monographs 1: 1–650.
Pennell, F. W. 1947. Some hitherto undescribed Scrophulariaceae of the Pacific states. Proceedings of the Academy of Natural Sciences of Philadelphia 99: 151–171.
Pennell, F. W. 1951. Scrophulariaceae. In L. Abrams [ed.], Illustrated flora of the Pacific states, 686–859. Stanford University Press, Stanford, California, USA.
Pinkava, D. J., T. Reeves, E. Lehto, AND L. A. McGill. 1979. Plants new to Arizona flora. VII. Chromosome counts and new distribution records of noteworthy species. Journal of the Arizona–Nevada Academy of Science 14: 37.
Posada, D., AND K. A. Crandall. 1998. MODELTEST: Testing the model of DNA substitution. Bioinformatics (Oxford, England) 14: 817–818.[CrossRef]
Rambaut, A. 1996. Se-Al: Sequence Alignment Editor. Available at http://tree.bio.ed.ac.uk/software/. Institute of Evolutionary Biology, Edinburgh, UK.
Rambaut, A., AND A. J. Drummond. 2004. Tracer. Available at http://tree.bio.ed.ac.uk/software/. Institute of Evolutionary Biology, Edinburgh, UK.
Ramsey, J., AND D. W. Schemske. 1998. Pathways, mechanisms, and rates of polyploid formation in flowering plants. Annual Review of Ecology and Systematics 29: 467–501.[CrossRef][Web of Science]
Ramsey, J., AND D. W. Schemske. 2002. Neopolyploidy in flowering plants. Annual Review of Ecology and Systematics 33: 589–639.[CrossRef][Web of Science]
Ronquist, F., AND J. P. Huelsenbeck. 2003. MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics (Oxford, England) 19: 1572–1574.[CrossRef]
Reveal, J. L., AND R. Spellenberg. 1976. Miscellaneous chromosome counts of western American plants III. Rhodora 78: 37–52.
Rydberg, P. A. 1917. Flora of the Rocky Mountains and adjacent plains. Published by the author, New York, New York, USA.
Scotland, R. W., R. G. Olmstead, AND J. R. Bennett. 2003. Phylogeny reconstruction: The role of morphology. Systematic Biology 52: 539–548.
Shimodaira, H., AND M. Hasegawa. 1999. Multiple comparisons of log-likelihoods with applications to phylogenetic inference. Molecular Biology and Evolution 16: 1114–1116.[Web of Science]
Simmons, M. P., AND H. Ochoterena. 2000. Gaps as characters in sequence-based phylogenetic analyses. Systematic Biology 49: 369–381.
Soltis, D. E., AND P. S. Soltis. 1993. Molecular data and the dynamic nature of polyploidy. Critical Reviews in Plant Sciences 12: 243–273.[CrossRef][Web of Science]
Soltis, D. E., P. S. Soltis, AND J. A. Tate. 2003. Advances in the study of polyploidy since Plant speciation. New Phytologist 161: 173–191.[CrossRef][Web of Science]
Spellenberg, R. 1986. Chromosome number reports 90. Taxon 35: 197.
Stearns, S. C. 1992. The evolution of life histories. Oxford University Press, Oxford, UK.
Stebbins, G. L. 1938. Cytological characteristics associated with different growth habits in the dicotyledons. American Journal of Botany 25: 189–198.[CrossRef][Web of Science]
Stebbins, G. L. 1950. Variation and evolution in plants. Columbia University Press, New York, New York, USA.
Stebbins, G. L. 1957. Self fertilization and population variability in the higher plants. American Naturalist 91: 337–354.[CrossRef][Web of Science]
Swofford, D. L. 2002. PAUP*: Phylogenetic analysis using parsimony (*and other methods), version 4b10. Sinauer, Sunderland, Massachusetts, USA.
Swofford, D. L., G. J. Olsen, P. J. Waddell, AND D. M. Hillis. 1996. Phylogenetic inference. In D. M. Hillis, C. Moritz, and B. K. Mable [eds.], Molecular systematics, 407–514. Sinauer, Sunderland, Massachusetts, USA.
Taberlet, P., L. Gielly, G. Pautou, AND J. Bouvet. 1991. Universal primers for amplification of three non-coding regions of chloroplast DNA. Plant Molecular Biology 17: 1105–1109.[CrossRef][Web of Science][Medline]
Tank, D. C., P. M. Beardsley, S. A. Kelchner, AND R. G. Olmstead. 2006. L. A. S. Johnson review no. 7. Review of the systematics of Scrophulariaceae s.l. and its current disposition. Australian Systematic Botany 19: 289–307.[CrossRef][Web of Science]
Verboom, G. A., H. P. Linder, AND W. D. Stock. 2004. Testing the adaptive nature of radiation: Growth form and life history divergence in the African genus Ehrharta (Poaceae: Ehrhartoideae). American Journal of Botany 91: 1364–1370.
Watson, S. 1871. United States geological exploration of the fortieth parallel, Clarence King, Geologist-in-Charge. Government Printing Office, Washington, D.C., USA.
Wettstein, R. 1891. Scrophulariaceae. In A. Engler, and K. Prantl [eds.], Die natürlichen Pflanzenfamilien, 39–107. Engelmann, Leipzig, Germany.
Wiggins, I. L. 1933. New plants from Baja California. Contributions from the Dudley Herbarium 1: 161–187.
Wolfe, A. D., C. P. Randle, L. Liu, AND K. E. Steiner. 2005. Phylogeny and biogeography of Orobanchaceae. Folia Geobotanica 40: 115–134.[CrossRef]
Yoder, A. D., J. A. Irwin, AND B. A. Payseur. 2001. Failure of the ILD to determine data combinability for slow loris phylogeny. Systematic Biology 50: 408–424.
Young, N. D., K. E. Steiner, AND C. W. dePamphilis. 1999. The evolution of parasitism in Scrophulariaceae/Orobanchaceae: plastid gene sequences refute an evolutionary transition series. Annals of the Missouri Botanical Garden 86: 876–893.[CrossRef][Web of Science]
Young, T. P., AND C. K. Augspurger. 1991. Ecology and evolution of long-lived semelparous plants. Trends in Ecology &Evolution 6: 285–289.[CrossRef][Web of Science]
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unequal anther-sacs, unequally attached; 
corolla minutely 3-toothed; calyx unequally cleft, with deeper cut adaxially; 
inflorescence a reduced spike (<2 cm), single-flowered florescence, or synflorescence; tip of the middle lobe of lower corolla lip tightly revolute; 
androecium reduced to 2 fertile stamens; 
plants halophytic; terminal stigmatic surface bent backward; middle lobe of lower corolla lip erect; 
upper corolla lip (beak) open at tip; stigma expanded; hilum terminal,; 
stamens reduced to 1 anther sac; corolla throat abruptly indented, forming a fold; n = 11; 
basic chromosome number of x= 12; 
perennial habit. Two asterisks (**) denote the annual members of the perennial Castilleja clade, C. arvensis and C. exilis, as discussed in the text.