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Proc. Nati. Acad. Sci. USA Vol. 87, pp. 4640-4644, June 1990 rbcL sequence divergence and phylogenetic relationships in lato (DNA sequencing/evolution/) DOUGLAS E. SOLTISt, PAMELA S. SOLTISt, MICHAEL T. CLEGGt, AND MARY DURBINt tDepartment of , Washington State University, Pullman, WA 99164; and tDepartment of Botany and Sciences, University of California, Riverside, CA 92521 Communicated by R. W. Allard, March 19, 1990 (received for review January 29, 1990)

ABSTRACT Phylogenetic relationships are often poorly quenced and analyses to date indicate that it is reliable for understood at higher taxonomic levels ( and above) phylogenetic analysis at higher taxonomic levels, (ii) rbcL is despite intensive morphological analysis. An excellent example a large gene [>1400 base pairs (bp)] that provides numerous is Saxifragaceae sensu lato, which represents one of the major characters (bp) for phylogenetic studies, and (iii) the rate of phylogenetic problems in angiosperms at higher taxonomic evolution of rbcL is appropriate for addressing questions of levels. As originally defined, the family is a heterogeneous angiosperm phylogeny at the familial level or higher. assemblage of herbaceous and woody taxa comprising 15 We used rbcL sequence data to analyze phylogenetic subfamilies. Although more recent classifications fundamen- relationships in a particularly problematic group-Engler's tally modified this scheme, little agreement exists regarding the (8) broadly defined family Saxifragaceae (Saxifragaceae circumscription, , or relationships of these sensu lato). Based on morphological analyses, the group is subfamilies. The recurrent discrepancies in taxonomic treat- almost impossible to distinguish or characterize clearly and ments of the Saxifragaceae prompted an investigation of the taxonomic problems at higher power of chloroplast gene sequences to resolve phylogenetic represents one of the greatest relationships within this family and between the Saxifragaceae levels in the angiosperms (9, 10). Following the traditional and other major plant lineages. Sequence data from the gene interpretation (8), the family is a large, morphologically rbcL (ribulose-1,5-bisphosphate carboxylase, large subunit) diverse assemblage of annual, biennial, and perennial herbs, reveal that (i) Saxifragaceae sensu lato is at least paraphyletic, , , and comprising 15 subfamilies; this was and probably polyphyletic, (ii) the generaParnassia andBrexia later increased to 17 subfamilies (11). The morphological are only distantly related to other members of Saxifragaceae, diversity encompassed by this group is so great that subse- and (iii) representatives of the Solanaceae (subclass Asteridae) quent workers have provided substantially modified con- appear more closely related to Saxifragaceae (subclass Rosidae) cepts of relationships (9, 12-16). However, these recent than traditionally maintained. These data illustrate the value of schemes often differ dramatically; little agreement exists chloroplast gene sequence data in resolving genetic, and hence regarding the circumscription, taxonomic rank, or relation- phylogenetic, relationships among members of the most taxo- ships of Engler's subfamilies. The differences among taxo- nomically complex groups. nomic treatments are too substantial to review here (see reviews in refs. 10 and 12). We illustrate the magnitude of In many groups of angiosperms phylogenetic relationships at these discrepancies, however, in a simplified comparison of higher taxonomic levels (family and above) have remained relationships (Fig. 1). enigmatic despite detailed morphological analyses. Presum- Most recent workers have considered the Englerian con- ably, these difficulties reflect the more frequent occurrence cept ofSaxifragaceae too broad. Takhtajan (15), for example, of parallel and convergent character state change (ho- distributed the same taxa among more than 10 families and moplasy) at higher taxonomic ranks. Below the familial level, suggested that some members of Saxifragaceae sensu lato restriction site analysis of chloroplast DNA has been excep- were only very distantly related. He placed most families in tionally valuable in resolving phylogenetic affinities (1, 2), two orders (Cunoniales and , superorder Rosa- even when extreme morphological divergence has obscured nae; subclass Rosidae) and considered these to be distantly a close relationship (3). At higher taxonomic levels, however, related to Brexiaceae (, superorder Celastranae, restriction site analysis typically is inadequate for phyloge- subclass Rosidae) and (Hydrangeales, super- netic inference, owing to excessive homoplasy and length Cornanae; subclass Asteridae). His Saxifragaceae are mutation; DNA sequencing appears to be the tool of choice tribe In contrast, in such instances (2, 4, 5). Sequencing of the slowly evolving essentially limited to Engler's Saxifrageae. ribosomal RNA genes, for example, has unequivocally dem- Cronquist (12) placed several of Engler's woody subfamilies onstrated that chloroplasts are of endosymbiotic origin (33). in Grossulariaceae (Fig. 1); he maintained that woody taxa In , DNA sequence comparisons of chloroplast- (his Grossulariaceae and Hydrangeaceae) were more usefully encoded genes will be particularly useful in phylogenetic associated with other woody families of Rosidae. Cronquist's analyses at higher taxonomic levels (4, 5). Although few Saxifragaceae encompasses several of Engler's original her- major studies ofchloroplast gene sequences and phylogenetic baceous subfamilies. relationships have been published (6, 7), the chloroplast gene To understand completely the phylogenetic affinities of all rbcL, encoding the large subunit ofribulose-1,5-bisphosphate members of Saxifragaceae sensu lato would require analysis carboxylase, has emerged as the preferred gene for examin- of a large portion of Rosidae, as well as taxa in other ing higher-level phylogenetic relationships (2, 4). The reasons subclasses. Here we concentrate on a suite of taxa that for this preference include (i) rbcL has been widely se- represent well the morphological diversity of the group and ask: can rbcL sequence data elucidate phylogenetic relation- be used to The publication costs of this article were defrayed in part by page charge ships in this enigmatic group? If so, this tool could payment. This article must therefore be hereby marked "advertisement" unravel the evolutionary history ofother equally problematic in accordance with 18 U.S.C. §1734 solely to indicate this fact. groups of plants. 4640 Downloaded by guest on September 25, 2021 Evolution: Soltis et al. Proc. Nati. Acad. Sci. USA 87 (1990) 4641

Takhtajan (1987) Engler (1964) Cronquist (1981) (28), a , distantly related to the other taxa, which are all ; Nicotiana otophora (Solanaceae, ROSANAE SAXIFRAGACEAE subclass Asteridae) (29); and Pisum sativum (, CUNONIALES subclass Rosidae, but placed in a different order than Saxi- BAUERACEAE Penthoroideae --- , SAXIFRAGACEAE fragaceae) (30). These served as outgroups in the SAXIFRAGALES // Saxifragoideae parsimony analysis. PENTHORACEAE Ae Vahlioideae , UPGMA dendrograms were calculated from distance ma-

SAXIFRAGACEAE Al Francooideae trices based on the 3ST model (31). Maximum-likelihood and VAHUACEAE , / Eremosynoideae parsimony analyses used the DNAML and DNAPARS programs, 0 Lepuropetaloideae respectively, provided in PHYLIP 3.2 (J. Felsenstein); the EREMOSYNACEAE Pamassioideae DNABOOT program (PHYLIP 3.1) was used to obtain confi- LEPUROPETALACEAE Baueroideae * CUNONIACEAE dence levels for the parsimony analysis. For the DNAML Ribesoideae GROSSULARIACEAE analysis, the transition/transversion ratio was set to 2.0 and GROSSULARIACEAE Pterostemonoideae the empirical frequencies of the bases were used (F option); * Hydrangeoideae HYDRANGEACEAE runs were conducted with and without Z. mays and the Tetracarpaeoideae CELASTRANAE categories option (C). Because of the length of time needed Iteoideae CELASTRALES to run the program with Z. mays and the C option, these were Escallonioideae not included when the jumble option (J) was used. BREXIACEAEBREXIACEAE ~~Brexioideae CORNANAE Montinioideaegegoideae HYDRANGEALES Phyllonomoideae RESULTS AND DISCUSSION PFEROSTEMONACEAE rbcL in Saxifragaceae sensu lato is 1407 bp long, comparable HYDRANGEACEAE W to the size reported for other taxa (5, 7). Sequences for the TETRACARPACEAE eight taxa analyzed are given in Fig. 2. Due to EcoRI* activity, we did not obtain the full length of the rbcL gene MONTINIACEAE from (our sequence for this begins 471 bp from DULONGIACEAE the 5' end of the gene). Thus, we have sequenced approxi- FIG. 1. Schematic representation of relationships among taxa of mately two-thirds of rbcL from Francoa. Detailed sequence Saxifragaceae sensu lato depicting some of the discrepancies that comparisons (4) indicate, however, that use of a partial gene exist among the traditional (11) and more recent (12, 14) taxonomic sequence (particularly one this large) should not affect phy- treatments. logenetic reconstruction. In fact, we obtained the same results when we used only the last two-thirds ofthe rbcL gene MATERIALS AND METHODS for all taxa in a parsimony analysis. We obtained rbcL sequence data for the following members of A matrix of 3ST distances with standard errors for each Saxifragaceae sensu lato (see Fig. 1): taquetii (Saxi- codon position is provided in Table 1. As expected (4, 5), fragoideae/Saxifrageae), madagascariensis (Brexio- most ofthe base substitutions are in the third codon position. ideae), Carpenteria californica (Hydrangeoideae), Francoa Relative rate tests provided no evidence for differential rates sonchifolia (Saxifragoideae/Francoeae), micrantha of evolution of rbcL in any of the lineages analyzed. These (Saxifragoideae/Saxifrageae), virginica (Iteoideae), results are similar to those of earlier studies (6, 7). sedoides (Penthoroideae), and fimbri- In 100% of the UPGMA analyses, (i) Nicotiana (Solan- ata (Parnassioideae). Astilbe, Heuchera, Penthorum, Fran- aceae) clustered more closely with Saxifragaceae than did coa, and Parnassia are herbaceous; Brexia, Itea, and Car- Pisum (Fabaceae); (ii) Heuchera and Astilbe appeared as penteria are woody. closest relatives; (iii) Penthorum, Francoa, Carpenteria, and Total were isolated (17, 18) and digested with Itea clustered with Astilbe and Heuchera; and (iv) Brexia and EcoRI. Genomic libraries were constructed by using the Parnassia formed a distinct cluster well separated from the cluster containing Penthorum, Francoa, Carpenteria, Itea, Lambda ZAP II/EcoRI cloning kit (Stratagene) and Gigapack Astilbe, and Heuchera. In 45% of the analyses, Nicotiana packaging extracts (Vector Cloning Systems). The genomic (Solanaceae) actually appeared between the Brexia-Parnas- libraries (unamplified) were screened (19) with an rbcL probe sia cluster and the Penthorum-Francoa-Carpenteria-Itea- from . Recombinant phage containing DNA inserts Heuchera-Astilbe cluster. In 94% of the analyses, Itea ap- that hybridized to the rbcL probe were subsequently cloned peared as the closest relative of Astilbe and Heuchera. The into the plasmid pBlueScript following the in vivo excision differences among the dendrograms mainly involved the method provided by Stratagene. We used a double-stranded alteration of relationships ofPenthorum, Carpenteria, Fran- dideoxynucleotide sequencing protocol (20, 21) and used a coa, and Itea to Heuchera and Astilbe. series of synthetic oligonucleotide primers based on the rbcL Parsimony analysis yielded two equally most-parsimo- sequence of mays (obtained from G. Zurawski, DNAX). nious trees that differ only in the placement ofFrancoa. One Sequence data were verified by sequencing both strands of places Francoa in a with Itea, Penthorum, Astilbe, the rbcL clones. and Heuchera; the other places Francoa as the sister species rbcL sequences were compared by three methods: (i) ofa large clade having two groups: Itea, Penthorum, Astilbe, unweighted pair group method ofanalysis (UPGMA; ref. 22), and Heuchera in one and Carpenteria and Nicotiana in the (ii) a maximum-likelihood procedure developed for nucleo- other. We also analyzed the data by using 100 bootstrap tide sequence data (23), (iii) Wagner parsimony. For UPGMA replicates; the tree obtained (Fig. 3) provides confidence we used a bootstrap procedure with 500 replicates. Several intervals for the nodes. investigators (24-26) have reviewed the methods ofanalyzing The maximum-likelihood solution yielded a topology (Fig. sequence data. We also performed relative rate tests (27) to 4) that differed from the parsimony tree only in the positions determine whether rbcL evolves in a clock-like fashion ofPenthorum and Itea relative to Heuchera and Astilbe. The among the taxa sequenced. maximum-likelihood solution is also very similar to the We included in these analyses previously published rbcL UPGMA dendrogram that appeared most frequently. Use of sequences from taxa that show varying degrees ofrelatedness thejumble option (but with Zea omitted to shorten run times) to Saxifragaceae (based on refs. 12-16): Z. mays () yielded several different solutions with similar likelihood Downloaded by guest on September 25, 2021 4642 Evolution: Soltis et al. Proc. Natl. Acad. Sci. USA 87 (1990)

NUCL HE AS PE PA BR CA IT FR NUCL HE AS PE PA BR CA IT FR scores. These solutions always support the following: (i) 0015 C 0757 C Heuchera and Astilbe are closely allied, (it) Brexia and Par- 8027 G C 0759 G G G G G G 8042 G G 0762 A A A nassia always appear together and are distantly related to 0045 C 0763 C A A 0048 C C 07764 other Saxifragaceae sensu lato, (iii) Nicotiana is more similar 8057 T T T T T T T 0771 C C C C C C 0060 T T T 0778 C to Saxifragaceae than is Pisum. These solutions differ in their 0068 A A 0084 T T C C C C C 0783 placement of Carpenteria, Itea, Francoa, and Penthorum 0087 T T T T T T T 0785 C relative to Heuchera and Astilbe. 0088 G G G G G 0786 C C 8095 C 0789 T T A T T T C T The three methods ofanalysis are concordant in suggesting 0096 A A A A 0801 C C that (i) Saxifragaceae sensu lato is at least paraphyletic and 0108 C C C C C C C 0804 T T 0111 A - 0813 A A C A A A 0120 T 0816 T A probably polyphyletic, (ii) Brexia and Parnassia are well 0124 A 0825 T T T T T T TT separated from the remaining taxa ofSaxifragaceae analyzed, 0126 G A genera and closely 0138 A A 0834 T () the herbaceous Astilbe Heuchera are 0141 G 0147 G G 0840 A A A allied-both herbaceous (Penthorum) and woody (Itea) taxa 0153 G G G G 0843 C T show a close 0159 G G C 084 T T Tclslgerarelationship to these two genera, and (iv) 0162 A 0845 T TT Solanaceae (Asteridae) are more closely related to Saxi- 0165 A A T G fragaceae (Rosidae) than are of 0168 T 0878 T T T TT T T T members Fabaceae (Ros- 0177 T T T TT T 0882 T A A A A T T idae). The phylogenetic of taxa 0186 C 8097 AAAAAA A A placementpaeet 3A~G0~~Usaxifragaceous 0195 C 0915 A A closer to Solanaceae than to Fabaceae is not due to any 0207 C G G G G G G 097 A A A A G A A A 0213 T 0933 T TT TT T peculiarities in the sequences of either Pisum or Nicotiana. 0219 G 0936 T T T T T T T 0227 A 094 C C C C C C C C The seunesequences of these twow taxa arer extremely similariia to 0228 T T 094 T T T G G T T T other representatives of the same two families (e.g., Medi- 0231 G 094 G C 0237 G 0951 A C C C C cago sativa and Petunia hybrida, respectively). In fact, the 0243 G 80952 C C 0246 A A A A A A 09572 C C C C same results were obtained when rbcL sequences for M. 0235 T T T T 0257 A A A A A A A - 809810976 T TG Tanls.sativa and P. hybrida were included in a UPGMA analysis. 0264 A 0982 G The relationships suggested by rbcL sequence data have 0266 C C C C C C C 0267 C C C C C C 1008 G T important evolutionary and systematic implications. For 0270 G G G G G 0272 C C C C C C C 10201020 C GGC ATCA T TGT C G C example, morphological parallelism and/or convergence 0280 G G G GG C G 1023 C C C C C C T C 0283 A A A A A A A 1029 A A may have prompted erroneous assessments of evolutionary 0284 G G 1047 A A A A A A A relationships. Furthermore, the woody vs. herbaceous habit 02S0 T T C TT 0293 C °058 A C is not always a reliable indicator ofrelationship in this group. 0309 T T 1059 C 0312 CC C A C 106 A A A A A A A AabltImportantly, our data also demonstrate the ability of rbcL 0324 C 1866 A A A A A A A A sequence data to clarify relationships in a group notorious for 0342 T T TT G T T 1071 C C C C C 0345 T 1077 C C its taxonomic difficulty. The implications of rbcL sequence 0357 T 080 T T data regarding the validity of recent classification schemes 0369 T T T T T T T 1095 C 0372 C C 1098 G are discussed below. 0378 A - 1101 ~~~~~~~C 0390 A 1111 C C C C C C C Cronquist's (12) system in particular is called into question 0393 T T A T 0405 A A 1123 T T by rbcL sequence data, as are some aspects ofthe systems of 0408 G G C C G C G 1125 G G G G G G GG 0412 T T 1137 C C G Thorne (16) and Dahlgren (13). The system of Takhtajan (15) 0414 T 1140 G G G C G G is largely supported. Not surprisingly, Heuchera and Astilbe 0423 C C 0424 G G G G A 1167 C C are closely allied; they form the core of the Saxifragaceae 0425 T T T 1168 T 0433 T T T 1170 A according to most investigators. However, Cronquist in- 0434 C C C 1185 A A A T T A A cludes in Saxifragaceae not only Astilbe and Heuchera, but 0444 T 1180 C 0443 G 1194 T also Penthorum, Francoa, and Parnassia; he considered the 0450 G C G C C CC C 0456 G G 203 T A A A woody Itea to be distantly related to these herbaceous 0439 T T T TT T T T 1209 taxa and and together his 0462 C C T T CC G G placed Brexia Itea in Grossulari- 0468 G 1227 C C C C C C aceae. These concepts ofrelationship seem untenable in light 0474 G G G GG G G 1236 A A A A A AA A 0483 G 1242 A AA A A A AA ofrbcL sequence data because (i) Parnassia is well separated 0489 T 1243 C G C C T G G G 0498 A A G C 12515 G G G T T G G G from the other members of his Saxifragaceae, (ii) Itea is just 0501 C C 1264 G G G G C G G G 0504 TT 1266 T T T as closely related to Astilbe and Heuchera as are Penthorum 0307 A A A A A A A 1269 C and Francoa, and (iii) Brexia and Itea appear to be distantly 0310 A 1209 G G G GG G 0513 C 1302 G related. 0332 C 0537 C A A 1317 T T T T T T TT Both Thorne (16) and Dahlgren (13) considered Parnassia 0543 T T T T 1320 G G G G G G to represent a distinct family; Dahlgren even placed Parnas- 0549 G G G G G G G 1335 C CC A 0552 T T 1338 T T T T T T T siaceae in a separate order. rbcL data support the view that 0555 TT 0564 A A A C A G G A 34C5 G A A A Parnassia is well differentiated from those taxa with which it 0579 C A A C C C C A 1346 C 0582 T T T T T 1356 T T T T T T T T traditionally has beenbe allied; rbcLu~. data asalso suggest a rela- 0394 C C C 1359 T T T T T T T T tionship, heretofore unrecognized, of this genus to Brexia. 0600 CC CC C C C 1378 G C G C C C C 0612 A A A A A 1389 G G GG G GGG Although both Thorne (16) and Dahlgren (13) included Fran- 0628 A A 13962 A AAA cai '''~~~~ 0624 C CC C C C C T 139 A A A A A A AA coa and Penthorum in Saxifragaceae (Thorne's Saxifrago- 0648 C C C C C C C 1397 A A A A A A A A ideae) and excluded Itea, rbcL data fail to support this 0663 C 1402 C CC GG C C G 0666 A T A 1404 A A A A A A A A concept of relationships. 0642 T T G T G 1407 CC C CC CC 0673 A A A 0677 T 0684 T rbcL differences between N. and 0687 AA A A FIG. 2. sequence otophora (29) 0688 G G G C G G G Heuchera (HE), Astilbe (AS), Penthorum (PE), Parnassia (PA), 0696 G 070S T Brexia (BR), Carpenteria (CA), Itea (IT), and Francoa (FR). The 0711 A A nucleotide positions (NUCL) at which differences exist between N. 0717 T 0720 G G G G G G G otophora and the taxa analyzed here are given followed by the 0732 G C nucleotides found in the members of the sensu lato at 0738 G Saxifragaceae 0751 C C those positions. Minus symbols indicate portion of the gene for 0753 G G G G Francoa that was not available for sequence analysis. Downloaded by guest on September 25, 2021 Evolution: Soltis et al. Proc. Natl. Acad. Sci. USA 87 (1990) 4643

Table 1. Matrix of Kimura 3ST distance partitioned by codon position Codon Taxon position ZE PI NI HE AS PE PA BR CA IT FR ZE 1 0.079 0.084 0.077 0.074 0.079 0.081 0.081 0.072 0.074 0.084 2 0.039 0.046 0.044 0.042 0.051 0.031 0.033 0.037 0.037 0.037 3 0.479 0.448 0.426 0.428 0.404 0.482 0.448 0.457 0.459 0.460 PI 1 0.0135 0.055 0.042 0.048 0.044 0.055 0.048 0.042 0.046 0.041 2 0.0094 0.028 0.019 0.017 0.024 0.017 0.019 0.013 0.019 0.012 3 0.0455 0.292 0.246 0.240 0.237 0.268 0.246 0.240 0.261 0.228 NI 1 0.0140 0.0112 0.033 0.035 0.042 0.046 0.042 0.033 0.026 0.041 2 0.0102 0.0079 0.024 0.022 0.031 0.015 0.017 0.017 0.022 0.009 3 0.0435 0.0306 0.199 0.198 0.183 0.266 0.231 0.170 0.200 0.193 HE 1 0.0133 0.0096 0.0085 0.006 0.017 0.028 0.026 0.009 0.015 0.012 2 0.0099 0.0065 0.0073 0.006 0.017 0.013 0.015 0.006 0.011 0.006 3 0.0420 0.0275 0.0238 0.044 0.106 0.216 0.209 0.123 0.077 0.106 AS 1 0.0131 0.0104 0.0088 0.0037 0.019 0.030 0.028 0.011 0.017 0.022 2 0.0096 0.0061 0.0069 0.0037 0.011 0.011 0.013 0.004 0.013 0.009 3 0.0418 0.0270 0.0236 0.0099 0.096 0.200 0.184 0.110 0.074 0.091 PE 1 0.0135 0.0099 0.0097 0.0061 0.0065 0.037 0.037 0.019 0.022 0.025 2 0.0107 0.0072 0.0082 0.0061 0.0048 0.019 0.022 0.015 0.024 0.022 3 0.0397 0.0268 0.0225 0.0162 0.0153 0.219 0.211 0.139 0.129 0.109 PA 1 0.0138 0.0112 0.0102 0.0079 0.0082 0.0091 0.024 0.028 0.024 0.031 2 0.0082 0.0061 0.0057 0.0053 0.0048 0.0065 0.002 0.006 0.006 0.003 3 0.0463 0.0286 0.0291 0.0251 0.0236 0.0253 0.098 0.214 0.199 0.173 BR 1 0.0138 0.0104 0.0097 0.0076 0.0079 0.0091 0.0072 0.022 0.019 0.034 2 0.0085 0.0065 0.0061 0.0057 0.0053 0.0069 0.0021 0.009 0.009 0.003 3 0.0435 0.0273 0.0264 0.0249 0.0227 0.0249 0.0154 0.194 0.201 0.165 CA 1 0.0128 0.0096 0.0085 0.0043 0.0048 0.0065 0.0079 0.0069 0.011 0.018 2 0.0091 0.0053 0.0061 0.0037 0.0030 0.0057 0.0037 0.0043 0.009 0.003 3 0.0455 0.0270 0.0214 0.0179 0.0166 0.0193 0.0251 0.0237 0.127 0.133 IT 1 0.0131 0.0102 0.0076 0.0057 0.0061 0.0069 0.0072 0.0065 0.0048 0.022 2 0.0091 0.0065 0.0069 0.0048 0.0053 0.0072 0.0037 0.0043 0.0043 0.003 3 0.0448 0.0284 0.0238 0.0135 0.0132 0.0181 0.0199 0.0240 0.0182 0.121 FR 1 0.0141 0.0095 0.0095 0.0052 0.0069 0.0074 0.0083 0.0087 0.0063 0.0069 2 0.0091 0.0051 0.0044 0.0036 0.0044 0.0069 0.0026 0.0026 0.0026 0.0026 3 0.0451 0.0264 0.0233 0.0165 0.0148 0.0165 0.0217 0.0212 0.0190 0.0178 Standard errors appear below the diagonal. ZE, Z. mays; PI, P. sativum; NI, N. otophora; other abbreviations are the same as in Fig. 2. rbcL data support Takhtajan's (15) narrowly defined Sax- ASTILBE ifragaceae, which he limits to only 30 genera. Astilbe and Heuchera represent well the morphological extremes of this group. This narrow circumscription of Saxifragaceae is also HEUCHERA supported by chloroplast DNA restriction site data (D.E.S., A. Grable, P.S.S., and M. Edgerton, unpublished data). ITEA Furthermore, all taxa examined corresponding to Takhta- jan's Saxifragaceae have lost the intron for the chloroplast gene rpl2 (S. Downie, R. G. Olmstead, G. Zurawski, D.E.S., P.S.S., J. C. Watson, and J. D. Palmer, unpublished data). This intron is present, however, in Parnassia, Francoa, Penthorum, Brexia, Itea, close relatives of Carpenteria, and other taxa representing many of the original subfamilies of Saxifragaceae sensu lato. Takhtajan considered Itea (but not

FRANCOA Brexia) and Penthorum (his Iteaceae and Penthoraceae, respectively) to be close relatives of his narrowly defined PARNASSIA Saxifragaceae; rbcL data are consistent with these hypoth- eses. Takhtajan (15) also placed Brexiaceae in a separate superorder; rbcL data support the distinctiveness of Brexi- BREXIA aceae. However, Parnassia (Parnassiaceae) and Francoa PISUM (Francoaceae) were considered close allies of Saxifragaceae by Takhtajan, and these views are not supported by sequence data. ZEA One point of disagreement among the three methods of data analysis involves the woody genus Carpenteria. UP- FIG. 3. Parsimony tree obtained using DNABOOT for taxa of GMA indicates an association of Carpenteria with five other Saxifragaceae sensu lato and outgroup taxa. The branch lengths have been drawn to reflect the number of mutations that support each members of Saxifragaceae sensu lato. However, parsimony branch. The branch length for Francoa is an estimate for the entire analysis and some maximum-likelihood solutions suggest a gene based on the number of mutations detected in that portion ofthe particularly close relationship of Carpenteria with Solan- gene actually sequenced. The percentages provide confidence levels aceae (Figs. 3 and 4). As noted earlier, the relationship of based on 100 bootstrap replicates. woody and herbaceous members of Engler's Saxifragaceae Downloaded by guest on September 25, 2021 4644 Evolution: Soltis et al. Proc. Natl. Acad. Sci. USA 87 (1990)

ASTILBE We thank Ed Golenberg, Jerry Learn, Amy MacRae, Joel Davis, and David Henderson for their assistance in the laboratory. This research was supported in part by National Science Foundation Grants BSR-8717471 and BSR-8500206. .PENTHORUM 1. Palmer, J. D. (1987) Am. Nat. 130, S6-S29. 2. Palmer, J. D., Jansen, R. K., Michaels, H. J., Chase, M. W. & Manhart, J. R. (1988) Ann. Mo. Bot. Gard. 75, 1180-1206. 3. Sytsma, K. J. & Gottlieb, L. D. (1986) Proc. Natl. Acad. Sci. USA 83, 5554-5557. 4. Ritland, K. & Clegg, M. T. (1987) Am. Nat. 130, S74-5100. 5. Zurawski, G. & Clegg, M. T. (1987) Annu. Rev. Plant Physiol. NICOTIANA 38, 391-418. 6. Doebley, J., Durbin, M., Golenberg, E. M., Clegg, M. T. & FRANCOA Ma, D. P. (1990) Evolution, in press. 7. Giannasi, D. E., Zurawski, G. & Clegg, M. T. (1990) Syst. Bot., PARNASSIA in press. 8. Engler, A. (1930) in Die Naturlichen Pflanzenfamilien, eds. Engler, A. & Prantl, K. (Engelmann, Leipzig), Vol. 18a, pp. BREXIA 74-226. PISUM 9. Dahlgren, R. M. T. (1980) J. Linn. Soc. Bot. 80, 19-124. 10. Spongberg, S. A. (1972) J. Arnold Arbor Harv. Univ. 53, FIG. 4. The maximum-likelihood topology for the taxa analyzed 409-498. by thejumble option based on 10 orderings ofthe data (log likelihood 11. Engler, A. (1964) in Syllabus der Pflanzenfamilien, eds. Mel- = -4327.90). Zea was not included in thejumble analysis because of chior, H. & Werdermann, E. (Gebruder Borntraeger, Berlin), the excessive computer run times involved. The relative distances pp. 137-138. between nodes and tips and between internodes are displayed. The 12. Cronquist, A. (1981) An Integrated System ofClassification of placement ofFrancoa is based on the partial sequence data available Flowering Plants (Columbia Univ. Press, New York). for this taxon. All branch lengths are significant at the P = 0.01 level. 13. Dahlgren, R. M. T. (1983) Nord. J. Bot. 3, 119-149. 14. Takhtajan, A. (1980) Bot. Rev. 46, 225-259. has been controversial. The discrepancy involving Carpen- 15. Takhtajan, A. (1987) System of Magnoliophyta (Academy of some that Sciences U.S.S.R., Leningrad). teria is intriguing because (13, 15) have suggested 16. Thorne, R. F. (1983) Nord. J. Bot. 3, 85-117. Hydrangeaceae (which includes Carpenteria) are more 17. Soltis, D. E., Soltis, P. S. & Bothel, K. D. (1990) Syst. Bot. 15, closely related to some members of Asteridae (such as 349-362. Solanaceae) than to Rosidae. 18. Doyle, J. J. & Doyle, J. L. (1987) Phytochem. Bull. 19, 11-15. rbcL sequence data indicate a closer relationship of Sax- 19. Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982) Molecular ifragaceae (Rosidae) to Solanaceae (Asteridae) than to Fa- Cloning:A Laboratory Manual (Cold Spring Harbor Lab., Cold baceae (Rosidae). Although most recent investigators have Spring Harbor, NY). Saxi- 20. Korneluk, R. G., Quan, F. & Gravel, R. A. (1985) Gene 40, considered Asteridae to be derived from Rosidae, 317-323. fragaceae typically have been considered to occupy a 21. Hattori, M. & Sakaki, Y. (1986) Anal. Biochem. 152, 232-238. position in Rosidae, well separated from Solanaceae. A few 22. Sneath, P. H. A. & Sokal, R. R. (1973) Numerical investigators (32) have, however, suggested that Solanaceae (Freeman, San Francisco). may be derived from Saxifragaceae. The suggestion from 23. Felsenstein, J. (1981) J. Mol. Evol. 17, 368-376. rbcL data that some Asteridae and Rosidae are more closely 24. Felsenstein, J. (1983) in Statistical Analysis ofDNA Sequence allied than traditionally maintained merits further investiga- Data, ed. Weir, B. S. (Dekker, New York), pp. 133-150. 25. Felsenstein, J. (1988) Annu. Rev. Genet. 22, 521-565. tion. 26. Nei, M. (1987) Molecular Evolutionary Genetics (Columbia In summary, rbcL sequence data have provided important Univ. Press, New York). phylogenetic information regarding Saxifragaceae sensu lato. 27. Wu, C.-I. & Li, W.-H. (1985) Proc. Natl. Acad. Sci. USA 82, In several instances, these data enabled us to discern among 1741-1745. the competing phylogenetic hypotheses of Cronquist, Dahl- 28. Zurawski, G., Clegg, M. T. & Brown, A. H. D. (1984) Genetics gren, Thorne, and Takhtajan. In other instances,' rbcL se- 106, 735-749. quence data provided insights into evolutionary relation- 29. Lin, C. M., Liu, Z. Q. & Kung, S. D. (1986) Plant Mol. Biol. also illustrates the of using 6, 81-87. ships. This study importance 30. Zurawski, G., Whitfeld, P. R. & Bottomley, W. (1986) Nucleic several different methods of data analysis in the comparison Acids Res. 14, 3973-3974. of rbcL sequences. The ability of rbcL sequence data to 31. Kimura, M. (1981) Proc. Natl. Acad. Sci. USA 78, 454-458. elucidate phylogenetic relationships in a notoriously difficult 32. Hutchinson, J. (1959) The Families ofFlowering Plants (Oxford group such as Saxifragaceae sensu lato indicates the tremen- Univ. Press, New York), 2nd Ed. dous potential of chloroplast gene sequence data for higher- 33. Pace, N. R., Olsen, G. J. & Woese, C. R. (1986) Cell 45, level phylogenetic analyses. 325-326. Downloaded by guest on September 25, 2021