Phylogenetic relationships of - Springer Link

Pl. Syst. Evol. 214:65-89 (1999)

--Plant Systematics and Evolution © Springer-Verlag 1999 Printed in Austria

Phylogenetic relationships of Polemoniaceae inferred from 18S ribosomal DNA sequences LHöH A. JOHNSON, DOUGLASE. SOLT~S,and PAMELAS. SOLTIS Received May 18, 1997; in revised version October 26, 1997

Key words: Asteridae, Polemoniaceae.- 18S rDNA sequences, systematics, phylogenetics. Abstraet: Cladistic analyses of chloroplast DNA disagree with current classifications by placing Polemoniaceae near sympetalous families with two staminal whorls, including Fouquieriaceae and Diapensiaceae, rather than near sympetalous families with a single staminal whorl, such as Hydrophyllaceae and Convolvulaceae. To explore further the affinities of Polemoniaceae, we sequenced 18S ribosomal DNA for eight genera of Polemoniaceae and 31 families representing a broadly defined Asteridae. The distribution of variation in these sequences suggest some sites are hypervariable and multiple hits at these sites have obscured rauch of the hierarchical structure present in the data. Nevertheless, parsimony, least-squares minimum evolution, and maximum likelihood methods all support a monophyletic Polemoniaceae that is placed near Fouquieriaceae, Diapensiaceae and related "ericalean" families.

Polemoniaceae have been largely accepted as a natural group since the early nineteenth century. With the exception of a few extraneous taxa (e.g. Diapensia L., Fouquieria KUNTH,Amphilophium KUYTH, and Cyanathus WALLICnex BENTn.) that were placed briefly in Polemoniaceae by various authors (see NASH 1903, GRANT 1959), the bounds of Polemoniaceae have been relatively stable. Of the currently recognized genera of Polemoniaceae, only Cobaea is still occasionally separated as a monogeneric family (DAHLGREN 1980, WATSON & DALLWITZ 1992). The monophyly of the Polemoniaceae-Cobaeaceae lineage remains essentially undisputed, however, given that Cobaeaceae is placed next to Polemoniaceae even in these classifications. Although the monophyly of Polemoniaceae is largely accepted, the phylogenetic position of the family among other angiosperms is not well understood. Past hypotheses of relationships primarily involve families considered part of CRONQUIST'S (1981) Asteridae, including Bignoniaceae, Boraginaceae, Convolvulaceae, Dipsacaceae, Hydrophyllaceae, Lennoaceae, and Solanaceae. However, affinities of Polemoniaceae with Caryophyllaceae, Cucurbitaceae, Diapensiaceae, Fouquieriaceae, and Geraniaceae have also been postulated (reviewed by GRANT 1959). Contemporary angiosperm classifications by DAHLGREN (1980), CRONQUIST(1981), TAKHTAJAN(1997), and THORNE(1992), while differing in

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L.A. JOHNSON8Z;al.:

Table 1. Comparison of recent classifications of Polemoniaceae in relation to other angiosperm families, qn other treatments, THORNE(1977, 1983) included Fouquieriaceae in Polemoniineae DAttLGREN ( 1 9 8 0 )

CRONQUIST (1981)

Solanales Solaniflorae Duckeodendraceae Solanales Nolanaceae Solanaceae Sclerophylacaceae SoIanaceae Convolvulaceae Goetzeaceae Cuscutaceae Convolvulaceae Cuscutaceae Retziaceae Cobaeaceae Menyanthaceae Polemoniaceae Polemoniaceae Hydrophyllaceae Boraginales Hydrophyllaceae Ehretiaceae Boraginaceae Wellstediaceae Lennoaceae Hoplesti gmataceae

TAKHTAJAN (1997)

THORNE (1992)

Solananae Solanales Solanales Solanineae SoIanaceae Solanaceae Sclerophylacaceae Duckeodendraceae Duckeodendraceae Goetzeaceae Nolanaceae Goetzeaceae Convolvulales Convolvulaceae Convolvulaceae Boraginae Hydrophyllaceae Cuscutaceae Polemoniales Boraginaceae Hoplestigmataceae Polemoniaceae Boraginales Lennoaceae Tetrachondraceae HydrophyIIaceae PoIemoniineae ~, Boraginaceae Tetrachondraceae Polemoniaceae Hoplestigmataceae Lennoaceae Limnanthales Limnanthaceae

the circumscription of higher taxonomic categories, all place Polemoniaceae with or near Hydrophyllaceae, Convolvulaceae, and other families of C~oyQuiST'S (1981) Solanales (Table 1). Hydrophyllaceae and Convolvulaceae were also endorsed by GRA~T (1959) as the most plausible close relatives of Polemoniaceae. However, GRANTalso acknowledged the ideas of BROWN(1938) and STEBBINS(in GRANT 1959) that the entire Tubiflorae (i.e. roughly equivalent to the Solanales, Scrophulariales, and Lamiales sensu CRONQUIST, 1981) might have arisen from primitive members of the Ericales or Ebenales, respectively. Earlier, WERNHAM (1911) suggested that the Tubiflorae arose from an "apocynal plexus" (i.e. the Contortae sensu EN~I~ER, including Apocynaceae, Asclepiadaceae, Gentianaceae, and related families). Polemoniaceae were not included in WERNHAM'STubiflorae, however, but rather represented an independent phyletic line that diverged much earlier from "geranial stock" as did both the "apocynal plexus" and the Ericales. In WERYHAM'S hypothesis, Polemoniaceae share similar morphological features with Hydrophyllaceae and Convolvulaceae only through convergence. Several comparative and cladistic studies largely agree with WERYHAM(1911) by suggesting that Polemoniaceae are allied with families outside of Asteridae sensu CRONQUIST (1981). KOI~BE & JOHN (1980) for example, observed close serological relationships among Polemoniaceae and Actinidiaceae, Ericaceae, Primulaceae, and Theaceae - families placed by CRONQUIST(1981) in Dilleniidae. A cladistic analysis of morphological and chemical data (HuvvoRD 1992) places Polemoniaceae as sister to Pittosporaceae; these two families are, in turn, sister to remaining Asteridae, with a clade including Ericaceae and Fouquieriaceae

Affinities of Polemoniaceae- 18S rDNA

67

forming the sister of the clade comprising Polemoniaceae, Pittosporaceae, and Asteridae. ANDEaBEa~(1992) also used morphological and chemical characters in a cladistic analysis that places Polemoniaceae as part of a large polytomy that includes Asteridae and some Rosidae (i.e. Apiaceae, Pittosporaceae, Cornaceae, and Hydrangeaceae). However, a search for multiple islands of most parsimonious trees using ANDERBERG'S (1992) data recovers 1161 trees on five islands and the integrity of this Asteridae clade collapses in the strict consensus of these trees. Analyses of chloroplast DNA restriction sites place Polemoniaceae with Fouquieriaceae outside of a large clade that includes all other Asteridae plus Apiaceae, Hydrangeaceae, and Cornaceae, among other families (DowYiE & PALMER 1992). Analyses of rbcL sequences (C~ASE & al. 1993, OLMSTEAD & al. 1993) show Diapensiaceae to be sister to Polemoniaceae in an "ericalean group" including, among other families, Fouquieriaceae, Ericaceae, Ebenaceae, Styracaceae, and Actinidiaceae. Although the "ericalean group" appears as part of a broadly defined Asteridae (Asteridae sensu lato), Polemoniaceae are still well removed from Solanales and other higher Asteridae with which they have been traditionally allied. We sequenced the nuclear 18S ribosomal RNA gene (rDNA) to provide an independent DNA-based phylogenetic hypothesis for the placement of Polemoniaceae among angiosperms. NICKRENZ& SOLTIS (1995) reviewed the utility of complete and partial 18S rDNA sequences for inferring phylogenetic relationships among angiosperms. A few studies (e.g. KaoN 1996, SOLT~S& SOLrTS 1997) have subsequently used 18S rDNA sequences to resolve relationships within specific groups of angiosperms. KRON (1996), for example, found sufficient variation to explore the relationships among Empetraceae, Epacridaceae, and Ericaceae. These families, though offen separated as independent lineages, are shown by both 18S rDNA and rbcL sequences to form a monophyletic group. Based on the results of an analysis of 223 angiosperm species (SOLT~S & al. 1997), the scope of taxonomic sampling used hefe was confidently limited to representatives of Asteridae s. 1. The objectives of this study were: (1) to characterize the nature of 18S rDNA sequence variation within Polemoniaceae and allies and thereby assess the confidence with which the relationships of Polemoniaceae can be determined by 18S rDNA sequences; and (2) to elucidate the placement of Polemoniaceae among Asteridae s. 1. by analyzing 18S rDNA sequence variation using different tree-building algorithms.

Materials and methods Plant samples. Forty-seven species representing 32 families (following CRONQU~ST,1981)

of Asteridae s. 1. were included in this study (Table 2). This sampling includes eight genera of Polemoniaceae representing all of the major lineages of this family (cf. JOHNSON& al. 1996). Camptotheca, Hydrangea, and Philadelphus were chosen as outgroups based on their phylogenetic position relative to the ingroup taxa both in the larger 18S rDNA analysis (SoLTIS& al. 1997) and in the rbcL phylogeny of CnASE& al. (1993). Sequences of Ipomoea (ConvoIvulaceae) and Ardisia (Myrsinaceae) were kindly donated by D. NICg_R~YT (Southern Illinois UniversitI~) and K. KRON(Wake Forest University), respectively. The 18S rDNA sequence of Gilia (NICKRENT& SOLT~S1995; GenBank #L28238) was corrected for

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Fig. 1. Sequence variation and alternative alignments in the V7 loop region. A rRNA loop structure showing five loop nucleotides in Polemoniaceae and Fouquieriaceae versus six loop nucleotides in Diapensiaceae (and all other taxa examined except Tabernaemontana). Nucleotide numbering corresponds to Glycine (see secondary structure in NICKRENT& SOLT~S 1995). B Three alternative alignments for the nucleotides in the V7 loop region of rDNA. Alignment I and II maintain homology of stem characters 1361 and 1368 across all taxa. Alignment I considers the deletion in Fouquieria and Polemoniaceae to be homologous at the expense of maximum nucleotide matching (maximum parsimony = 627 steps). Alignment II maximizes nucleotide matching at the expense of matching the gap (maximum parsimony = 626 steps). Alignment III, used in the tree-building analyses in this paper, maximizes nucleotide matching at the expense of matching the gap, and further

(contd.)

Affinities of PoIemoniaceae - 18S rDNA

71

four nucleotide positions. The remaining sequences were generated from total DNAs isolated from fresh, frozen, or silica-dried plant material following the CTAB buffer method (DOYLE& DOYLE 1987) as modified by SoI~T~s & al. (1991) or CULLINGS(1992). Amplifieation and sequeneing. Amplification of 18S rDNA was accomplished using primers and reaction conditions described by BuLT & al. (1992) with a modified thermal cycle profile cousisting of 30 cycles of denaturation for 1 min at 94 °C, annealing for 2 min at 48 °C, and extension for 2 min at 72 °C. Double stranded PCR products were precipitated in 20% PEG/2.5M NaC1, washed in 80% and 90% ethanol, and resuspended in 25 gl distilled H20. These purified double standard DNAs were subsequently used as templates in cycle sequencing reactions employing the PRISM Ready Reaction Dye-Deoxy Terminator Cycle Sequencing Kit (Applied Biosystems, Inc.) and primers described by BuLT & al. (1992) and NICKRENT & STARR (1994). All cycle sequencing reagents were proportionally scaled to 3/4 or 1/2 of the manufacturer's specified volumes. Additionally, 5% DMSO was used in each reaction. Automated sequencing of the reaction products after purification (following SoLTIS & SOLTIs 1997) was accomplished using the 373A DNA Sequencing System (Applied Biosystems, Inc.). Sequence fragments were assembled and proofread using Sequencher 2.1 (Gene Codes Corporation, Inc.). This procedure was followed for all taxa (Table 2) except Gilia, Ardisia, and Ipomoea; sequences from these three taxa were generated via radiolabeled dideoxy sequencing (e.g. NICKRENT& SOLTIS 1995; D. N~C~:t~NT, pers. comm., K. KRoN, pers. comm). With the exception of the unpublished sequence of Ardisia, all sequences have been deposited in GenBank (Table 2). Sequenee alignment and the data matrix. The 18S rDNA sequences used in this study range from 1807 to 1811 base-pair (bp) in length as determined by aligning the sequences against Glycine max MERR. (GenBank #X02623). Forty-nine bp from the 5 ~ end and 66 bp from the 31 end were excluded because these bases could not be confidently determined for all taxa. Length variation, primarily attributable to single nucleotide insertions or deletions, was infrequently encountered; 16 characters required the inclusion of a gap state ("-") for one or more taxa. With the exception of two hypervariable regions of four-bp each, and a six bp region (beginning at nucleotides 496, 667, and 1363, respectively, in the unaligned sequence of Glycine max), the gaps were easily and unambiguously positioned by eye. The secondary structure of the rRNA molecule was considered in aligning these three loop regions (cf. NICKm~NT& SOLT~S1995), as well as an appeal to parsimony (BAUM& al. 1994). For example, three plausible alignments of a single nucleotide deletion in PoIemoniaceae and Fouquieriaceae exist in the aforementioned six bp region (Fig. lA, B). Homology assessments based on secondary structure (i.e. forcing the equivalent of Glycine max stem nucleotide 1368 in all taxa to be the same aligned character in the sequence data matrix) favor two of the alignments (Fig. 1BI, BII). However, the third alignment (Fig. 1BIII), which does not maintain the homology of this stem nucleotide in the data matrix, requires fewer steps in maximum parsimony analyses. Because the lack of stem-nucleotide homology in the third alignment (Fig. 1BIII) also has a plausible molecular evolutionary explanation (Fig. 1C), this most-parsimonious alignment was used for the analyses in this paper. Importantly, analyses of several different suggests nucleotide 1368 in Polemoniaceae is not homologous to nucleotide 1368 in all of the other taxa (maximum parsimony = 625 steps):. C Sequence of events justifying the plausibility of alignment III. Step I. "C" nucleotide in ancestral sequence is lost and adjacent "U" nucleotide assumes position in stem. Step II. The "U" nucleotide that moved from the loop to the stem retains base-pairing with "G" nucleotide (G-U pairs are stable in rRNA). Step III. Compensatory point mutation from "G" to "A" maintains stable stem nucleotide pair and gives rise to the sequence observed in Acanthogilia

72

L . A . JoHYSON& al.:

alignments in all three of the difficult loop regions revealed similar relationships. Of the 1702 aligned characters, 229 (13.5%) are variable, and 133 (7.8%) are potentially informative for parsimony analyses. Of the 79994 character-state cells in this matrix, only 116 (0.15%) are scored ambiguously ("?"); this number increases to 518 (0.65%) when cells scored as gaps ("-") are treated as ambiguous rather than as a fifth-character state (see below). Charaeterization of variation. Several values describing the nature and distribution of variation in this 18S rDNA data set were calculated either directly from the data matrix or by reconstructing character stares over an arbitrarily selected most parsimonious tree. PAUP* 4.0 (development version d54-59; D. SWOFFORD, Smithsonian Institution), MacClade 3.05 (MADDISON& MADDISON1992), and Claris Works (Claris Corp.) facilitated computations. Although some of these values are merely descriptive, most were calculated to aid in choosing appropriate models of sequence evolution for the least-squares distance and maximum likelihood tree search methods described below. The homogeneity of base frequencies across taxa for the entire data set, as well as for only the variable characters, was assessed using the BASE FREQUENCIES option in PAUE The transition: transversion (ti:tv) ratio was calculated based on unambiguous changes reconstructed over a most parsimonious tree, as well as estimated via maximum likelihood. The distribution of numbers of nucleotide substitutions per site, as reconstructed over a most parsimonious tree, was tested for goodness-of-fit to uniform rates (following WA~LEY 1993) and negative-binomial distributions (BLIsS & FISHER 1953, UZZELL & CORBIN 1971). The location of all variable characters with respect to the secondary structure of the 18S rRNA and the designation of these characters as either "stem" or "loop" characters (e.g. DIXON & HILLIS 1993) were determined using the secondary structure model published by NICKRENT& SOLTIS(1995) as a guide (mispaired characters in the stem region were included in the stem category because a compensatory nucleotide change in one or both characters will restore Watson-Crick base-pairing). Substitutions reconstructed on the branch that unites Polemoniaceae and substitutions that are reconstructed within this family on one of the most parsimonious trees were also characterized with respect to the secondary structure of 18S rRNA. Lastly, the homoplasy excess ratio (HER; ARCmE 1989) was calculated to assess the degree of randomness in the distribution of character states both in the entire data matrix and in subsets of taxa representing the "ericalean group" (see below) and Polemoniaceae. Phylogenetie analyses. All phylogenetic analyses were conducted using PAUP* 4.0. In addition to the parsimony method, least-squares minimum evolution and maximum likelihood methods were also used to investigate alternative phylogenetic hypotheses recovered when models of sequence evolution other than methodological parsimony were employed. Although convergence on similar topologies by different tree-building techniques does not guarantee that the "true" tree has been identified, such convergence provides additional support for the relationships that are mutually revealed (KIM 1993, BAUM & al. 1994). Least-squares minimum evolution was selected over other distance measures because this method is better at identifying the minimum-evolution tree when the number of taxa is large (RznETSKY& NEI 1993). Simulation studies using both least-squares minimum evolution and maximum likelihood analyses show that both methods provide reliable estimates of the true phylogeny when the models of sequence evolution employed match the pattern of evolution that gave rise to the data (HuELSENBECK& HILLIS 1993, HUELSE~ECK 1995). P a r s i m o n y . Each of the following parsimony analyses was conducted using HEURISTIC searches employing 100 replications of RANDOM taxon addition with MULPARS and TBR branch swapping. Unweighted (Fitch) parsimony was used initially; however, four general weighting schemes were also employed to explore the effects of

Affinities of

Polemoniaceae

-

18S rDNA

73

various weights on the recovery of minimal-length trees: 1) a posteriori weighting of characters by their maximum rescaled consistency index (RC) on the trees recovered by Fitch parsimony; 2) weighting transversions 1.8, 2, and 2.2 times as heavily as transitions (NIc~CRENr & SOLTIS 1995 report an unambiguous ti:tv ratio of 1.91:1 in their analysis of 62 seed-plant 18S rDNA and rRNA sequences); 3) scoring gap states ("-") as a fifth character state and weighting changes to or from this state 0.5, 1, and 2 times as heavily as nucleotide character-state changes; and 4) with respect to the secondary structure of the 18S rRNA molecule (see below), weighting stem characters 0.8 times as heavily as loop characters (following DIxoN & HILLIS 1993), and weighting loop characters 0.8 times as heavily as stem characters. Both ti:tv weighting and gap-state weighting were accomplished using step matrices. Support for clades revealed on the minimal-length Fitch trees was assessed using both bootstrap (FELsENSTEIN 1985, see also FELSENSTHY& KISHINO 1993, HILLIS& BtmL 1993) and decay (BI~MEa 1988, DOYOGH~ & al. 1992) analyses. Bootstrap analyses used only the potentially informative characters (HARsI-IMAN1994) and consisted of 500 replications using HEURISTIC searches with TBR branch swapping, MULPARS, and four replications with RANDOM addition in which no more than 100 trees were saved per addition replication. Decay analyses were performed individually for each branch by saving only trees from heuristic searches (employing 100 RANDOM addition replications) that failed to satisfy a constraint topology in which only the branch of interest was resolved (e.g. BAUM & al. 1994). D i s t a n c e . Minimum evolution trees were found using HEURISTIC searches employing 100 replications of RANDOM addition with MULPARS and TBR branch swapping. Missing data were ignored in pairwise comparisons between taxa, and negative lengths (if any) were set to zero. A relatively simple model of sequence evolution, the Kimura 2-parameter model (I~MURA 1980; K-2), was initially employed using a uniform substitution rate at each nucleotide site to provide a basis for comparison with other, more complex evolutionary models. Two additional models were employed, the HASEGAWA• al. (1985) HKY85 model and the TN model of TAMURA& NEI (1993). In addition to providing different rate coefficients to transitions and transversions (i.e. K-2) HKY85 applies different coefficients to each nucleotide according to its relative abundance. The TN model further specifies rates for directionality of change (e.g. the frequency of a change from "A" to " C " is not necessarily equivalent to the frequency of change from "C" to "N'). Searches using each of these three models were also conducted after specifying a gamma shape parameter of a = 0.0886 for site-to-site rate variation. This shape parameter was estimated empirically for this data set via maximum likelihood using per site nucleotide substitutions reconstructed over one of the most parsimonious trees (UzzELL & CORBIN 1971). The negative-binomial (gamma) distribution for site-to-site rate heterogeneity was used becanse substitutions in DNA sequences have been repeatedly suggested to be more accurately modeled by gamma-distributed rates than the uniform rate specified by the Poisson distribution (e.g. UZZELI~& CORB~N1971, WAI~LEY 1993, YANG 1994). This same conclusion was reached by goodness-of-fit tests for the 18S rDNA data reported here (see Results). M a x i m u m 1i k e 1i h o o d. Despite improvements in computational speed implemented in PAUP* 4.0, deriving shortest trees via maximum likelihood is still time-intensive for a data set of this size. The nature of variation in these 18S rDNA sequences was therefore considered before specifying the parameters employed by the maximum likelihood algorithm, and a single model of sequence evolution was used. This model specified a ti:tv ratio of 2.0 because this value matches closely the range of values estimated empirically via maximum likelihood over the set of most parsimonious trees. The HKY85 model with unequal nucleotide frequencies was also specified using the nucleotide

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L.A. JOHNSON&al.:

frequencies determined from only the variable characters (see below). Although site-to-site rate variation in these 18S rDNA sequences conforms to the gamma distribution (see below), employing the gamma distribution in conjunction with the other parameters proved to be too time and memory-intensive for this data set (on a Power Macintosh 7100 with 8 megabytes of RAM). Characters were therefore divided into three rate classes: (1) characters that are not reconstructed over a most parsimonious tree (1473 characters); (2) characters that require from one to five substitutions for reconstruction over a most parsimonious tree (200 characters); and (3) characters that require six or more substitutions (29 characters). Relative rates (r) for each class (class one, r = 0.0000; class two, r = 4.4162; class three, r = 28.2330) were then estimated using PAUP via maximum likelihood over a set of most parsimonious trees. Because the rate for class one is zero, these characters were excluded by PAUP before the tree searches began. Starting trees for a HEURISTIC search using TBR branch swapping were found by calculating likelihoods for each of the 2832 topologies found by the various parsimony and distance analyses described above. Three of the shortest trees, as well as a neighbor-joining tree, were then swapped to completion. The trees with the best likelihood resulting from this search were used as estimates of the trees that fit the data with the maximum likelihood.

Results Characterization of variation. The frequency of each nucleotide in these 18S rDNA sequences (24.6% A, 22.4% C, 27.4% G, and 25.6% T) is homogeneous across taxa (X: = 9.63, d f = 138, P = 1.00). Homogeneity across taxa in nucleotide frequencies is also maintained when only the variable positions are considered ( x a = 66.42, d f = 138, P = 1.00); however, the proportion of adenosine and cytosine is markedly different (14.6% A, 31.6% C, 26.6% G, and 27.2% T). Although adenosine is relatively low in frequency among variable sites, there are more changes to adenosine than from adenosine (Fig. 2). In contrast, there are more changes from guanine than to guanine. Additionally, while similar numbers of transition and transversion changes occur either to or from adenosine and

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Fig. 2. Proportions of unambiguous nucleotide changes in the 18S rDNA sequences reconstructed over the most parsimonious tree shown in Fig. 3. The area of each circle is scaled relative to the number of observed unambiguous changes over the most parsimonious tree

Affinities of Polemoniaceae- 18S rDNA

75

guanine, transitions far outweigh transversions for cytosine and thymine (Fig. 2). Overall, unambiguous transitions (352) and transversions (181) provide a ti:tv ratio of 1.94:1, similar to the 1.91:1 reported by NIC~ENT & SOLTIS (1995) for 62 seed plant 18S rDNA sequences and to the 2.0:1 ratio estimated via m a x i m u m likelihood over a set of most parsimonious trees. The most parsimonious reconstruction of base substitutions over an arbitrarily chosen most parsimonious trees shows a highly significant non-uniform distribution of substitution rates (P