Proc. Natl. Acad. Sci. USA Vol. 83, pp. 5554-5557, August 1986 Evolution Chloroplast DNA evidence for the origin of the Heterogaura from a species of () (chloroplast DNA phylogeny/restriction-site variation/ ) KENNETH J. SYTSMA*t AND LESLIE D. GOTTLIEB*t *Department of Genetics, University of California, Davis, CA 95616 Communicated by Peter H. Raven, April 7, 1986

ABSTRACT Restriction-site variation in chloroplast DNA section of another genus, is particularly noteworthy because was examined in the morphologically distinct and monotypic the Onagraceae is one of the best-known plant families (4). genus Heterogaura and the related speciose genus Clarkia The circular DNA molecule of chloroplasts has been (Onagraceae), both native to California. Of the 605 restriction particularly useful in phylogenetic studies of . cpDNA sites surveyed, a total of 119 mutations were identified. Of is relatively large (140-175 kilobase pairs) and is present in these, 55 were shared by at least two species and were used to high copy number within leaf cells, permitting its ready construct a most parsimonious phylogenetic tree. This analysis, isolation (5). Its high degree of sequence conservation facil- as well as one based on a distance metric, provided evidence itates use of heterologous probes to visualize restriction that Heterogaura and , a member of a fragments on Southern blots. Comparative mapping of phylogenetically advanced section, share a more recent com- cpDNA among congeneric species has provided detailed mon ancestor than either does with any other species. The two phylogenetic relationships in a number ofAngiosperm genera species are more closely related than nearly all pairs of Clarkia (6-11). Mapping of restriction sites in cpDNA of Heterogau- tested. The origin of Heterogaura from within another genus ra and in eight sections of Clarkia demonstrated that raises important questions about the adequacy of morpholog- Heterogaura cpDNA resembled the cpDNA of species in ical data and suggests that the relationships of other well- sect. peripetasma and not that of the other sections (unpub- known monotypic plant genera should be reinvestigated. lished data).

Many plant taxa have been described that possess unusual and distinctive morphologies, which so sharply delimit them MATERIALS AND METHODS that they are given generic rank. Implicit in such a classifi- Leaves from 4- to 6-week-old plants of the eight diploid cation is that the common ancestor of two related genera is species of sect. peripetasma, H. heterandra, Clarkia more ancient than the common ancestor ofany pair ofspecies xantiana (sect. phaeostoma), and (sect. within either genus. However, dramatic morphological di- rhodanthos) were frozen in liquid nitrogen and powdered vergence need not imply much genetic divergence (1) and can with mortar and pestle. Total DNA was extracted by a obscure a close phylogenetic relationship. In this paper, we described protocol (12) modified to include a 5% polyvinyl- use restriction enzyme analysis ofchloroplast DNA (cpDNA) polypyrrolidone grinding buffer and a buffer/powder weight to examine the relationship of the monotypic genus ratio (ml/g) of 40:1. The DNAs -were digested with 29 Heterogaura (Onagraceae) to the related and speciose genus restriction endonucleases that recognize 6-base nucleotide Clarkia (which includes =33 diploid and 10 polyploid spe- sequences and were subjected to electrophoresis in 0.6%, cies). 0.8%, and 1.0% agarose/Tris/EDTA/acetate gels. After The monotypic Heterogaura heterandra (Torr.) Cov. is a denaturation and neutralization, the fragments were blotted self-pollinating annual plant limited to California and Oregon. (13) onto BioDyne membranes in a bidirectional fashion. Its floral and fruit characters are so unusual that it has been Membrane filters were individually and sequentially probed maintained as a genus since 1866. Its flowers possess four with each of 12 Pst I and two Sal I clones, representing the fertile and four sterile anthers and a smooth unlobed capitate entire cpDNA genome of Petunia (kindly provided by J. stigma. Its ovaries contain up to four ovules and mature into Palmer and E. Clark). Also used were clones from Lactuca round nut-like indehiscent fruits with one or two seeds. In and Phaseolus (kindly provided by J. Palmer and R. Jansen), contrast, Clarkia flowers have four-lobed stigmas (the self- which represent distinct portions of the small single-copy have reduced region between the inverted repeats ofthe cpDNA molecule. pollinating species lobing), generally eight Restriction sites for 7 enzymes (Pst I, Kpn I, Sal I, Sma I, Pvu , and an elongated many-seeded capsule, which splits I, Nru I, Xho I) were completely mapped. Restriction site along four sutures to release the seeds. Our analysis shows losses and gains for the additional 22 enzymes were detected that H. heterandra is closely related to the outcrossing annual from fragment patterns. Clarkia dudleyana, which belongs to sect. peripetasma. The For restriction site mutations, the computer program two species occupy similar habitats on the western slope of "Phylogenetic Analysis Using Parsimony" (PAUP, version the Sierra Nevada in California and have the same chromo- 2.3), developed by D. Swofford (Illinois Natural History some number (n = 9). Sect. peripetasma is known with Survey, Urbana) was used to find the shortest phylogenetic certainty to be phylogenetically advanced within Clarkia trees; i.e., those requiring the fewest convergent or back- because it possesses duplicated genes encoding the cytosolic mutations. The trees were rooted by using C. xantiana and C. isozyme of phosphoglucose isomerase (2, 3). This result, amoena as out-groups. C. xantiana possesses the phospho- indicating the origin of a new genus within an advanced Abbreviation: cpDNA, chloroplast DNA. The publication costs of this article were defrayed in part by page charge tPresent address: Department of Botany, University of Wisconsin, payment. This article must therefore be hereby marked "advertisement" Madison, WI 53706. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 5554 Downloaded by guest on October 1, 2021 Evolution: Sytsma and Gottlieb Proc. Natl. Acad. Sci. USA 83 (1986) 5555 Table 1. Sequence differences among cpDNAs from Clarkia sect. peripetasma and H. heterandra EPI ROS LEW CYL MOD DUD LIN BIL HET C. epilobioides 23 45 40 42 41 49 47 46 C. rostrata 0.65 50 45 47 46 54 52 51 C. lewisii 1.29 1.44 15 41 42 48 48 45 C. cylindrica 1.14 1.29 0.42 36 35 43 43 40 C. modesta 1.20 1.35 1.17 1.02 21 25 25 24 C. dudleyana 1.17 1.32 1.20 0.99 0.59 28 28 17 C. lingulata 1.41 1.56 1.38 1.23 0.70 0.79 6 33 C. biloba 1.35 1.50 1.38 1.23 0.70 0.79 0.17 33 H. heterandra 1.32 1.47 1.29 1.14 0.67 0.48 0.93 0.93 The number ofrestriction site mutations between any two cpDNAs appears in the upper right portion of the matrix. Total number of restriction sites surveyed in each species is -605. Values of divergence (19) appear as 100 x p in the lower left portion of the matrix. glucose isomerase gene duplication and is assigned to a required only six additional mutations to account for all the related section. C. amoena lacks the duplication and belongs observed data (Fig. 1). These included two parallel site gains, to a section thought to represent the ancestral stock of the three convergent or parallel site losses, and one gain/loss (24, genus (14). This PAUP program is based on Wagner parsi- 25), for a rate of parallelisms or convergences of 4.8%. This mony (15) and therefore will treat convergent site gains value is nearly 2 times the average rate obtained in Brassica equally with convergent site losses. Dollo parsimony (16), (8, 9), Lycopersicon (6), and Pisum (10). If cpDNA evolution which discriminates against convergent site gains and favors is at all uniform in rate, this suggests a greater age for the convergent site losses, was also used to construct trees. The Clarkia/Heterogaura lineage. computer program "Phylogeny Inference Package" This tree of 125 steps had a consistency index value (26) of (PHYLIP, version 2.7) with the Dollo parsimony option, 0.95 on a scale from 0 to 1.0, where 1.0 means perfect developed by J. Felsenstein, was used. The distance algo- consistency. It placed H. heterandra firmly alongside C. rithm of Fitch and Margoliash (17) was used to construct dudleyana and not at the base of the phylogenetic tree of unrooted networks based on overall measures of cpDNA Clarkia where a distinct genus would be expected. The two sequence divergence. species shared nine derived mutations, of which four were unique to them. In all, 16% of the phylogenetically useful mutations were shared by the two species. Thus, they have RESULTS AND DISCUSSION a more recent common ancestor than either has with any other species tested. Although the species differed by 17 Restriction Site Variation. Approximately 605 restriction mutations, this difference between them was small relative to sites (6-base sequences) were surveyed in each of the the total differences among all species within Clarkia sect. cpDNAs, for a total sample of 2.1% of the chloroplast peripetasma plus H. heterandra.§ genome (172 kbp in ). The Clarkia and The derivation of Heterogaura from a common ancestor Heterogaura cpDNAs proved colinear to the Petunia with C. dudleyana was also indicated by the next three most cpDNA. They did not share a large 45-kilobase-pair inversion likely (but longer) trees. These trees required 126, 127, and characteristic of several species of Oenothera (18), also in 128 mutations, respectively. In each of them, the close Onagraceae. A total of 119 mutations were identified among relationship of Heterogaura and C. dudleyana was main- the eight species of sect. peripetasma and H. heterandra. The tained. The trees differed only in the relative placement of highest incidence of change occurred in portions of the large larger branches or in the placement of Clarkia modesta (data single-copy region near the inverted repeat, a region where a not shown). high frequency of mutations has been noted (7, 9). A single These Wagner parsimony trees require six or more addi- mutation was identified within the inverted repeat, confirm- tional mutations (convergences), two of which are unlikely ing its high degree of conservation (6, 9). convergent gains (16, 24, 25). Dollo parsimony, which does Genetic Distance Measures. The proportion of substitutions not allow for convergent gains, gave a most parsimonious per nucleotide position, p (19), estimated from the proportion tree exactly congruent in topology with the Wagner tree. This of site differences between any pair of the eight species in tree, however, required 10 convergent losses or gain/losses Clarkia sect. peripetasma and H. heterandra, ranged from (four additional mutations to account for the two convergent 0.0017 to 0.0156, with an average of 0.0107 (Table 1). This gains in the Wagner parsimony tree). The close relationship value is 3-3.5 times higher than that of other cpDNAs within of C. dudleyana and H. heterandra is maintained by both the a genus in Brassicaceae, Gentianaceae, and Poaceae (9, 11, Wagner and Dollo parsimony methods. 20). The sequence divergence between H. heterandra and Phylogenetic analysis based solely on overall measures of each ofthe eight Clarkia species ranged from 0.0048 (with C. divergence gave exactly concordant results with the analysis dudleyana) to 0.0147 (with C. rostrata), the average being based on parsimony. This was demonstrated by utilizing the 0.0103 (Table 1). The value for the former pair is the third p values in Table 1 in the Fitch and Margoliash algorithm (17). lowest of all 36 pairwise comparisons, demonstrating that H. The result was a network of relationships topologically heterandra and C. dudleyana are more closely related to each consistent with the phylogeny in Fig. 1 (27). The congruence other than are nearly all pairs of Clarkia species tested. of results from Wagner parsimony, Dollo parsimony, and Lower values were found only in the progenitor/derivative distance analyses strengthens our proposed phylogenetic species pair C. bilobaIC. lingulata (21, 22) and the morpho- model. logically similar C. cylindrica and C. lewisii (23). Phylogenetic Inferences from cpDNA Data. Fifty-five of the §In a separate report (27) is a detailed description of all the 119 restriction-site mutations were shared by at least two but restriction-site variations and a detailed comparison of branching not all species examined and, consequently, were phylo- patterns derived from parsimony and distance measures in sect. genetically informative in parsimony analysis. The PAUP peripetasma as these apply to other phylogenetic problems within program found a single most (Wagner) parsimonious tree that the section. Downloaded by guest on October 1, 2021 5556 Evolution: Sytsma and Gottlieb Proc. Natl. Acad Sci. USA 83 (1986)

a

FIG. 1. Phylogenetic tree for H. heterandra and Clarkia sect. peripetasma based on 119 restriction-site mutations. This tree was generated by PAUP computer Wagner parsimony analysis using the "branch-and-bound" function and thus represents the shortest or most parsimonious tree. The tree requires 125 steps to account for the observed data with two parallel gains, three convergent or parallel losses, and one gain/loss. Numbers refer to mutations assigned to an internode in the phylogenetic tree. For example, C. dudleyana and H. heterandra share 9 mutations of which 4 belong only to the branch leading to them. From their common ancestor, they differ by 7 and 10 mutations, respectively, and they differ from each other by 17 mutations. Branch lengths are not intended to depict relative amounts of divergence. EPI, epilobioides, ROS, rostrata; LEW, lewisii; CYL, cylindrica; DUD, dudleyana; MOD, modesta; LIN, lingulata. The problem remains whether the "sister-species" rela- been uncovered in Brassica (9), Lisianthius (11), and tionship ofC. dudleyana to H. heterandra as seen in the three Tetramolopium (29). Second, mono- and ditypic genera need most parsimonious trees is significantly better in a statistical not stand in a basal position with respect to related and more sense than the placement of H. heterandra elsewhere in the speciose genera, but may be derived from within them. tree. Less parsimonious trees that place the Heterogaura Mono- and ditypic genera are not infrequent within lineage above or below node "a" (see Fig. 1) are each four Angiosperm families; for example, 5 of the 17 genera in the mutations longer than the most parsimonious tree. Felsen- Onagraceae are of this type (4). Third, the relatively simple stein (28) has shown for a clocklike three-species tree that a and conceptually straightforward restriction enzyme analysis tree must be 5-10 steps worse before it is significantly worse. of cpDNA is particularly suitable for testing relationships at Statistical discrimination among these three trees is thus not the level ofthe genus and perhaps also the tribe. We conclude possible yet. However, placement of the Heterogaura lin- that increased utilization of biochemical and molecular evi- eage below all species within sect. peripetasma requires 9 dence will improve the reliability of phylogenetic models in extra steps and can be statistically rejected by Templeton's plants but also will pose new dilemmas for classical taxo- algorithm (24, 25) as modified by Felsenstein (28). It is clear nomic theory. that although the sister-species relationship of C. dudleyana and H. heterandra cannot be statistically supported (despite We are grateful to Rob DeSalle and Bruce Benz for computer their sharing of a number of derived restriction site muta- assistance. We appreciate comments on the manuscript by three tions), Heterogaura can be firmly placed within a specific anonymous reviewers. This research was supported by National subsection (a group of four species) of Clarkia sect. Science Foundation Grant BSR83-15124. peripetasma. Implications for Systematic Biology. The discovery that the 1. Gottlieb, L. D. (1984) Am. Nat. 123, 681-709. morphologically distinct Heterogaura is a Clarkia, albeit an 2. Gottlieb, L. D. (1977) Genetics 86, 289-307. unusual one, is noteworthy. But, even more remarkable is 3. Gottlieb, L. D. & Weeden, N. F. (1979) Evolution 33, that C. dudleyana is more closely related to H. heterandra 1024-1039. than it is to any other Clarkia species. The finding confirms 4. Raven, P. H. (1979) N. Z. J. Bot. 17, 575-593. the recent speculation that Heterogaura might be a derivative 5. Curtis, S. E. & Clegg, M. T. (1984) Mol. Biol. Evol. 1, 291-301. of Clarkia evidence, however, could not 6. Palmer, J. D. & Zamir, D. (1982) Proc. Natl. Acad. Sci. USA (4). Morphological 79, 5006-5010. place Heterogaura near any particular Clarkia species be- 7. Kung, S. D., Zhu, Y. S. & Chen, K. (1982) Theor. Appl. cause their morphological divergence has completely ob- Genet. 61, 73-79. scured their relationship. 8. Erickson, L. R., Straus, N. A. & Beversdorf, W. D. (1983) Several important taxonomic implications follow from Theor. Appl. Genet. 65, 201-206. these results. First, primary reliance on morphological data 9. Palmer, J. D., Shields, C. R., Cohen, D. B. & Orton, T. J. to model phylogenetic relationships may be misleading no (1983) Theor. Appl. Genet. 65, 181-189. matter how many characters are examined. Discrepancies 10. Palmer, J. D., Jorgensen, R. A. & Thompson, W. F. (1985) between morphological and biochemical evidence have also Genetics 109, 195-213. Downloaded by guest on October 1, 2021 Evolution: Sytsma and Gottlieb Proc. Nati. Acad. Sci. USA 83 (1986) 5557

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