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ANGIOSPERM Dowd^ OF and M. STUDIES Martin^ p. G. J. PHYLOGENY PROTEIN USING SEQUENCES^ Abstract samples In previous papers we have reported tlie N-terminal 40 amino acids of the small subunit of rubisco for We from four families of gymnosperms, nine families of monocotyledons, and 26 families of dicotyledons. expanded program 335 The main computing 122 families of dicots and derived a phylogenetic tree for all species. this to list major usead was HENNIG86, with which a reliable result can be assured with only 17 taxa or less, so a part of this i paper concerned with the strategy adopted to divide the 335 species and then to build the parts into an overall is and Comparison with other taxonomy suggests that, at the level of tree that as accurate objective as possible. is 90% genera our methods give resuhs that are at least accurate. At higher taxonomic levels placing into families, working hypothesis accuracy may decrease, and the result should be regarded not as a firm conclusion but as a for heterogeneity within subsequent using the longer sequences from nucleic acids. Topics discussed include testing N-terminus rubisco-SSU, and evidence that natural selection powerful in determining species, the nature of the of is amino acid sequence. The rate of evolution has been shown to vary between major taxa, and data suggest that angiosperms originated in the Jurassic. angiosperm phylogeny Palmer 1988; The problems of angiosperm phylogeny are well (e.g., et al., Zimmer an appropriate 1989). therefore by a consideration of the differences et al., It is illustrated when sequencing supplanting between four classifications, less than a decade time, nucleic acid is all and by respected and experienced protein sequencing, to set out the results of a de- highly old all 335 authors. The dicotyledons are divided into six sub- cade of work that has produced partial protein These and seven by Takh- sequences from a wide range of angiosperms. by Cronquist (1981) classes sequences tajan (1983), while, for the other two authors, the sequences are shorter than nucleic acid major groupings are superorders, Thorne (1983) already published and therefore contain less infor- having 19 and Dahlgren (1983) 25. The number mation and are less able to resolve the sequential we Nevertheless, dicotyledonous orders recognized respective- divergences of early radiations. of is, 58, 72, 41, and 83; these figures alone indicate believe that our phylogenetic trees will indicate ly, the resulting diversities of names and content, all likely relationships and profitable working hypoth- which our comparative ignorance of the eses for future investigations, of reflect course that evolution has taken in the angiosperms. down next the In contrast to this, at the level A Summary of Published Investigations hierarchy, there basic agreement about the *'core" is Protein Sequences USING be recognized (Heywood, 1978). families to Macromolecular sequences provide taxonomic The pioneer of the use in botany of protein whose homology over widely diverse sequences for investigating plant phylogeny was D. characters Durham, England, can be assumed with some confidence. Se- Boulter of the University of species quence data can be analyzed objectively with com- During the 1970s, Boulter, along with his col- We probably see the next decade leagues and students, published 25 sequences of puters. in will sequences long and cytochrome 12 complete and 58 partial se- the publication of nucleic acid c, enough some problems of quences of plastocyanin and seven sequences of variable to solve of the We acknowledge with thanks grants from the Australian Research Grants Scheme, the Australian Research ' Council, the Missouri Botanical Garden, the Potter Foundation, the Utah Foundation, and the National Botanic We Gardens (Canberra). are indebted to Richard Norrish who, for many years, has kept our machinery in working order. As indicated in Table 2, we liave obtained leaves from many sources, to all of whom we are grateful; we are Gardens especially grateful to the Adelaide Botanic Gardens, the Missouri Botanical Garden, the National Botanic Kew. and Royal Botanic Gardens, (Canberra), the 5001. Department of Botany, University of Adelaide, Box 498, G.P.O., Adelaide, South Australia 2 199L 296-337. Ann. Missouri Bot. Gard. 78: 1 Volume Number 2 & Dowd 297 78, Martin 1991 Angiosperm Phylogeny Using Protein Sequences RNA These have been ferrodoxin. collated, with ref- ribosomal (available for of the families) s Ramshaw erences, by (1982), and Scogin (1981) was added. has reviewed the results from the taxonomic point This result indicated the need for longer se- Although work much of view. this generated in- quences and better sampling of families. Although terest, also gave rise to skepticism, some of which rubisco-SSU was always multiply represented, it in can, with hindsight, be attributed to the inadequa- 17 of the 33 samples of other macromolecules cies of computing methods that were being devel- there was only a single sequence. This situation is The oped concurrently. mostly unfavorable reac- precarious because, the average distance from if tion of systematists, epitomized by the review of a familial node to a species N, then on the average is Cronquist (1976), influenced the cessation of re- a single sequence will misrepresent the familial node search in Boulter's laboratory about 1980. by N. This source of error might be responsible Before this, however, partial sequences (up to for part of the poor agreement observed. Sampling 25 N-terminal amino acids) of the small subunit of a family at least twice, preferably from widely ribulose-l,5-bisphosphate carboxylase/oxygenase divergent representatives should give a better es- (rubisco-SSU) were obtained from six species (Has- timate of the familial node (see phase 5). lett et al., 1976; Strobaek et al., 1976). This work In phase 2 we sequenced rubisco-SSU from 1 & SSU led to a complete sequence from spinach (Mar- members of Onagraceae (Martin Dowd, 1986a), & 1979), a forerunner of the work presented 5 monocotyledons (Martin Dowd, 1986b), and tin, 1 We here which concerns the N-terminal 40 amino acids 14 species of Solarium (Martin 1986). et al., of this protein. (The complete sequencing of a reasoned that the reliability of our methods might protein requires prior purification of several frag- be estimated by comparison with taxonomically well ments and at least an order of magnitude more understood groups. The results were similar to olh- is time-consuming than the direct sequencing of the er taxonomic treatments. Additional species of As- N-termlnus of the whole protein using an automatic teraceae were also studied and those results be will sequencer.) Nucleotide sequences of rubisco-SSU presented paper. in this from a few species have been published, and of To estimate the rate of evolution, Proteaceae, all them have been studied using our method. The Solanaceae, Fagaceae, and Winteraceae were sam- only new data comparable to our 334 species are pled in phase 3 using species whose ancestors are from two closely related orchids and their hybrid thought to have been separated by continental drift We Martin unaware known (G. C. et al., 1987). are of at times. This led to a preliminary publi- & phylogenetically useful sequences of other proteins cation (Martin Dowd, 1984b), and the derivation & Grund Nakano since those of et (1981) and et of a molecular evolutionary clock (Martin Dowd, al. (1981) 1988), which indicated that on average one nu- al. Work in our laboratory has proceeded in five cleotide difference arose between two diverging phases. In phase species were chosen because lines once in seven million years. 1 Boulter had already published their complete se- In phase 4 we tested the hypothesis that leghe- quences of cytochrome c and partial sequences of moglobin had evolved in plants by lateral transfer When plastocyanin. a pattern failed to emerge from from animals. This led to an investigation of all we analyses of these data, decided to sample each species for which leghenioglobin sequences had been family with sequences from at least two more rep- published, and was shown that the pathway of it resentative genera. Thus, the families Apiaceae, evolution in those species was closely parallel in & Asteraceae, Brassicaceae, Caprifoliaceae, Cheno- hemoglobin and rubisco-SSU (Martin Dowd, podiaceae, Fabaceae, Malvaceae, Poaceae, Polyg- 1986c), suggesting that there was no need to in- A onaceae, Ranunculaceae, and Solanaceae have each voke novel evolutionary processes. consequence been sampled These was we number at least three times. early of study that increased the of this results were published in a series of papers (Martin species of Fabaceae sequenced to eight (see Group & 1983; Martin Dowd, 1984a, 14 below) and obtained sequences from et al., b, c). several The sequences rubisco-SSU, cytochrome Many were for c, additional families. of these too small and plastocyanin were analyzed for these families to be studied in the normal course of this investi- by Martin, Boulter, and Penny (1985) using de- gation but were obtained either because they are node known rived estimates of familial sequences. Anal- to include nitrogen-fixers or thought to be yses of data from single macromolecules were not relatives of the legumes; these include Betulaceae, consistent with one another but, for nine of the Casuarinaceae, Chrysobalanaceae, Coriariaceae, families, a phylogenetic tree derived from combined Crossosomataceae, Datiscaceae, Elaeagnaceae, when data remained consistent ferrodoxin or 5S- Moringaceae, and Myricaceae. 298 Annals of the Garden Missouri Botanical In phase 5 we surveyed the dicotyledons which are available are arranged by families and Groups, number from 24 and sources and sequences are given, increased the of famihes studied their 124. to Methods Biochemical A Survey of the Dicotyledons The methods published by Martin and Jennings There are about 250 families of dicots. Because (1983) have stood the test of time, so, rather than them be was impractical sample of them, a decision repeat here, a general description will to all it was made to sample about half, to increase given and the few modifications mentioned, i.e., ^1 number from 24 mentioned above 124. Two methods were described, one for "pungent the the to Three (Acanthaceae, Loranthaceae, San- leaves with high concentrations of phenolics or families make talaceae) failed for reasons that will be discussed other substances that protein purification whose later. The additional 97 families were chosen pri- difficuh, the other for "bland" species leaves The much more amenable. The bland method gives marily on the basis of size. majority of families are sampled have more than 20 genera. To cover as better quality protein and therefore to be pre- is wide a range of variation as possible, some small ferred. However, because the pungent method works was were sampled. For example, the order well with bland leaves, but not vice versa, families also it has only three genera, so the family Schi- preferred when there was doubt or too few leaves Illiciales sandraceae (two genera) was chosen to represent for extractions. trial Only three orders are unrepresented out of Both procedures started with maceration of about it. Thome's 41 (two of which are parasitic and devoid 100 g of leaves from which the midribs were re- of rubisco), 10 out of Cronquist's 58, 19 out of moved practicable. For bland leaves the extract- if 85 was Takhtajan's 72, and 21 out of Dahlgren's ing buffer essentially a reducing, saline tris- pH impractical, mainly because computers are HCl buffer at 7.4, while for pungent leaves a It is pH limited in their capacities to analyze large numbers reducing, saline borate buffer at 8.6 and con- X-100 of taxa simuhaneously, to contemplate building a taining the detergent Triton was used. After remove phylogenetic tree for 122 families (comprising 310 crude straining and centrifugation to solids, We species) without some subdivision into groups. the extract was passed through a succession of two A Sephadex G-25 column was have done this by referring to all four current liquid gel columns. phylogenies. Thorne (1983) and Dahlgren (1983) used to remove low molecular weight sub- first A 6B have superorders as their major groups, the former stances. Sepharose column was used to remove nominating 19 and the latter 25. If these two remaining low molecular weight substances mem- authors agree that families are in the same super- and high molecular weight nucleic acids and were order then they have been grouped together our brane fragments. Eluting buffers different for in scheme, with one proviso. Takhtajan (1983) and the two extraction procedures and for the different Cronquist (1981) have respectively seven and columns used. The protein was precipitated with six ammonium subclasses as their major groups, and these two sulfate for the bland method and with authors have been allowed a veto; either them acetone for pungent. Procedures after the second if if does not also agree that families are in the same column were the same for both types of leaves, pH subfamily, then they are ungrouped. In The protein was S-carboxymethylated at 8.6 this left way we have divided 102 of the studied families to break disulphide bridges between cysteine res- 25 Groups, leaving 20 ungrouped because idues and then passed through a long column of into We there disagreement. are reluctant to use a Sephadex G-lOO in an eluting buffer containing is make formal term like superorder but need to sodium dodecyl sulfate. This separated the large it clear that our use of Group does have a defined subunit from the small subunit, which was precip- meaning, so we have used a capital G. The Groups itated in acetone and dried before sequencing. (A are shown in Table 1. variation of this procedure was to use a column of G75 was sample each new family followed by G-lOO,) practicable to It and we have done by choosing two The methods are rather crude but are successful only twice, this species not only from different genera but, pos- because rubisco a very large protein and, by a if is sible, from different subfamilies or tribes. Some- considerable margin, the most abundant protein in down times criterion has broken because fresh leaves. this mg have been About 5 of small subunit 0.5 ml of water leaves not available. (in Beckman In Table 2 the 335 species for which sequences without polybrene) was sequenced on the Volume Number & Dowd 299 2 Martin 78, 1991 Angiosperm Phylogeny Using Protein Sequences L Table Families of dicotyledons grouped because they are placed in the same major taxon by of Cronquist all (1981), Dahlgren (1983), Takhtajan (1983), and Thorne (1983). Group Group 4 Group 9 Group 12 Group 17 Group 21 1 Ulm Magnoli Dipterocarp Eric Connar Lami Mor Winter Elaeocarp Epacrid Sapind Verben Annon Group Urtic 13 Anacardi Group 22 Tili Group Myristic 5 Cunoni Simaroub Sterculi Solan Hamamelid Bombac Schisandr Ros Meli Convolvul Monimi Betul Malv Saxifrag Rut Polemoni Laur Fag Group 10 Group 14 Group 18 Group 23 Aristoloch Casuarin Viol Caesalpini Halorag Scrophulari Group Calycanth 6 Mimos Flacourti Rhizophor Gesneri Group 2 DiUeni Datisc Papilioni Group 19 Bignoni Berberid Thea Cucurbit Group 15 Zygophyll PedaU Ranuncul Ochn Trap Gerani Group 24 Salic Cap Lardizabal Clusi par Lylh Tropaeoli Valerian Menisperm Group Myrt 7 Brassic Malpighi Caprifol Papaver Myric Resed Punic Group 20 Group 25 Group 3 Jugland Moring Onagr Logani Api Cabomb Group 8 Group 11 Melastomat Gentian Arali Nymphae Caryophyll Sapot Combret Apocyn Nyctagin Styrac Group 16 Asclepiad Amaranth Primul Olac Ole Phytolacc Myrsin Rubi Celastr Chenopodi Families that do not into one of the groups fit Aster Coriari Goodeni Nelumbon Polygon Thymelae Bux Crossosomat Hydrophyll Piper Prote Vit Campanul Rhamn Elaeagn Lecythid Plumbagin Chrysobalan Euphorbi Loas Note: -aceae omitted from names all 890C automatic sequencer using Beckman's stan- corrected This problem occurred Onagra- this. in 50% dard quadrol program with quadrol buffer. ceae and a few others with small leaves containing The (PTH) phenylthiohydantoin derivatives of the a high proportion of veins. Insolubility of the pro- HPLC amino were Waters acids identified using a tein, leading to precipitation in columns, could instrument with a C- 18 radially compressed column sometimes be corrected by loading a more dilute M and sodium (pH and C4 eluted with 0. acetate 6.0) extract. Plants with photosynthesis, and rubisco 1 acetonitrile. This did not distinguish two pairs of tightly bound in bundle sheaths, were avoided if amino and was supplemented C3 acids therefore with possible. Plants with photosynthesis often occur TLC. same in the genera or families and were unlikely Using these methods, we could, without assis- to be phylogenetically biased. However, una void- if tance, produce two proteins each week and se- able (e.g., Welwitschla reported to be C4), spe- is quence two others. care was taken during the maceration process. cial common suspected that the most cause of It is was failure the presence of powerful proteases in Failures the leaves and, retrospect, would have been in it 90% Although of attempts led to successful se- profitable to try correcting this with research early 10% known quences, the remaining deserve brief atten- in the project. Species of Ficus^ to have tion. Unless there was an identified reason for fail- leaf proteases, showed symptoms of this failure. are that could be corrected, our policy was to try Large amounts of protein traveled where the small another representative of the family. subunit should have been on the G-lOO column many Faults that could be corrected include the and gave amino acids at each position when amounts of extraction and elution buffers used. sequenced. Another casualty of was Gne- this sort Some plants gave extracts that were mucilaginous turn gnemon, which was particularly desired be- gymnosperm to the point of setting solid. 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Volume Number 2 & Dowd 78, Martin 303 1991 Angiosperm Phylogeny Using Protein Sequences 04 Oi Oi CM Oi CM 0< 01 CM CM CM CM C^ CM CM 04 CM 04 CM Oi 04 04 04 CM CM 01 04 04 M > > > M > > > > > > M M M M M M > > H W > M M M IS 3e X 2 2 2 2 2 2 2 » 2 2 2 2 2 2 » 2 o o K ^ S Z O z ^ t>l4 Hi Hi Hi Hi Hi Hi Hi CO CO Hi Hi CO CO CO s 2 Z Z z Z > Z Z iic: at: te: CO CO CO > z ft< ^ a M M M ;z; ;z; •^ h) h:) .-1 0: 0: cd Oi 0: h^ CO Hi hI i4 CO J H ^ M h^ hI t^ *^ ^^ h^ 1^ »4 *J h1 h1 »4 Hi ^ h) >^ Â k^ >^ »^ h3 t^ •^ 1-1 1^ h^l »4 :) t-1 h3 h1 m ^ h1 >^ >^ M>H M>i >H >H >H >H >* >H >H >H >^ >H >^ >H >H >^ SH >< O O o a Q O Q a Q Q Q Q u U b] C3 CD b) b] C3 C^ > > > > > > > > M > > > > M > > > > M > > w > > > Oi U b] M M M U b] M b] bl bl M b3 U u U M M U U u b] b] < K K M MS be: Hi Hi Hi bd Hi K 000 t<: ad ac ad aii Eld E<: Hi M < < < ^ Hi »< Hi H? < < < < CO .^ CO CO •< ^ ^ 1^ h^ hI aa>A h^ Hi Hi 1^ Hi t^ Hi 1^ Â k1 h1 Hi Hi Hi ^ a a a a a > > u a b] C3 C3 Q Z Q H Q < Q CO CO H > M H H M :& CO CO fr* CO CO CO Eh £h CO CO Fi Eh CO CO CO H H H Eh CO CO q X ^^ h) *-^ 1-1 h) 1^ hI -1 h^ Hi H? 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