Review of Palaeobotany and Palynology, 50 (1987): 63 95 63 Elsevier Science Publishers B.V., Amsterdam Printed in The Netherlands

THE IMPORTANCE OF FOSSILS IN ELUCIDATING SEED PLANT PHYLOGENY AND MACROEVOLUTION

JAMES A. DOYLE and MICHAEL J. DONOGHUE

Department of Botany, University of California, Davis, CA 95616 (U.S.A.) Department of Ecology and Evolutionary Biology, University of Arizona, Tucson, AZ 85721 (U.S.A.)

(Revised and accepted August 19, 1986)

Abstract

Doyle, J.A. and Donoghue, M.J., 1987. The importance of fossils in elucidating seed plant phylogeny and macroevolution. Rev. Palaeobot. Palynol., 50: 63--95.

In order to gain insights on the controversial question of the value of fossils in understanding phylogeny and macroevolution, we used numerical parsimony methods to analyze a data set amassed for a cladistic study of living and fossil seed plants, variously modified by subtraction and re-addition of fossil groups. Some cladograms based on extant groups alone are consistent with relationships derived from the whole data set, but the direction of leaf and sporophyll evolution in seed plants and floral evolution in angiosperms and Gnetales is equivocal. However, use of Carboniferous seed ferns as outgroups favors the concept that all seed plants were derived from ancestors with fern- like leaves, that there was a double trend to linear-dichotomous leaves in coniferopsids and Gnetales, and that gnetalian flowers are reduced relative to those of angiosperms and Bennettitales, as inferred from the whole data set. With the whole data set, it is slightly more parsimonious to assume that coniferopsids were derived from Callistophyton-like platyspermic seed ferns than from Archaeopteris-like progymnosperms, but if Archaeopteris were unknown the seed fern hypothesis would be strongly favored, while if Callistophyton were unknown both hypotheses would be equally parsimonious. Bennettitales strengthen relationships between Gnetales and angiosperms, which are only slightly stronger than links between Gnetales and coniferopsids when extant data alone are considered, while Caytonia clarifies reproductive homologies of angiosperms, Bennettitales, and Gnetales and their origin from platyspermic ancestors. Although fossil information does not radically alter inferred relationships among extant taxa in seed plants, it may in other groups, and it provides unique evidence on the sequence of events and possible adaptive factors involved in the origin of groups. Both contributions are especially important when there has been extensive homoptasy and/or when living groups are isolated from each other by large gaps, as is often true of higher taxa.

Introduction relationships (Simpson, 1961; Hughes, 1976; Gingerich, 1979), cladists have assigned them a The role of fossil evidence in elucidating the more limited role. Cladistic analysis provides a relationships of major groups and evolutionary set of logical principles for formulating and processes involved in their origin has recently testing phylogenetic hypotheses whether or become the subject of much debate, closely tied not fossils are available: using shared derived with the general revival of interest in phylo- traits (synapomorphies) as evidence of common geny stimulated by cladistics, or phylogenetic ancestry, and most commonly determining po- systematics (Hennig, 1966). Whereas many larities (ancestral and derived character states) paleontologists have considered fossils the best by outgroup comparison and deciding among or even the only valid source of evidence on alternative hypotheses on the basis of parsi-

0034-6667/87/$03.50 © 1987 Elsevier Science Publishers B.V. 64 mony (preferring the scheme that requires the others (Fisher, 1980; Doyle et al., 1982). Paleon- fewest character state changes). Hennig (1966) tologists have also come under much criticism argued that fossils provide phylogenetic infor- for thinking in terms of ancestor-descendant mation only in conjunction with comparative relationships: "ancestral groups" are paraphy- analysis of all characters, and as such they have letic (incomplete) and therefore rejected by many disadvantages relative to living organ- cladists as artificial, and even if rephrased in isms because of their incompleteness, making terms of species, ancestor descendant relation- their detailed relationships difficult to deter- ships are thought to be less readily testable mine (Hennig, 1966, pp.140-142). At the same than sister-group relationships (Engelmann and time, he granted that fossils can be of great Wiley, 1977; Platnick, 1977; Eldredge, 1979). importance in the area of character analysis, Patterson (1981) argues that because of their which forms the basis of cladistic analysis of emphasis on ancestors paleontologists have both living and fossil groups, and for this reason more often hindered understanding of phylo- he urged closer study of those characters that genetic relationships than advanced them. are preserved in fossils (Hennig, 1966, pp.142, However, even if paleontologists have used 165). He also accepted the stratigraphic order of debatable concepts, this says nothing about the appearance of character states as one source of potential of fossils themselves if analyzed prop- evidence on polarity (Hennig, 1966, p.95). He erly. In a discussion covering several of these noted that fossils are often valuable in filling points, Schaeffer et al. (1972) claim that horse the gaps in transformation series and revealing phylogeny could be reconstructed by treating the sequence of acquisition of derived features, all known living and fossil taxa as if they came which may be decisive in determining the from one time plane, so that the information relationships of highly derived living groups that Hyracotherium is Eocene and Equus is and in showing that features that might be extant is therefore superfluous. However, since assumed to be homologous actually evolved all horses except Equus are extinct, this is quite independently within separate clades (Hennig, different from saying that fossils are of no value 1966, pp.142- 145). in reconstructing phylogeny. More recent discussions have explored the An analysis that seems to us more directly issues raised by Hennig to varying degrees, damaging to the view that fossils are valuable giving some points ample attention but largely in reconstructing phylogeny was presented by neglecting others. For example, some authors Patterson (1981). Like Hennig (1966), Patterson (Nelson, 1978; Stevens, 1980) have criticized argues that fossils might sometimes be ex- the use of stratigraphic evidence in polarity pected to change ideas on homology or polarity assessment, based in part on the fact that more within living groups, and to reveal details of advanced members of a group may appear the sequence of origin of features that charac- before more primitive ones because of the terize them. However, after consideration of a incompleteness of the fossil record. However, series of examples he concludes that in prac- more recently Eldredge and Novacek (1985) tice "instances of fossils overturning theories have argued that stratigraphy as well as of relationship based on Recent organisms are outgroup comparison and ontogeny can pro- very rare, and may be nonexistent" (p.218). It vide valid evidence on polarity when the should be noted that almost all of his discus- record of a group meets independently testable sion deals with the effect of fossils on topolo- criteria of completeness. Others have suggested gies of cladograms and therefore classification; that the degree of agreement between the order he says less about their effects on ideas of of appearance of character combinations character evolution, which might have as predicted by phylogenetic hypotheses and the much or more interest for an understanding of stratigraphic record can be used as an indepen- evolutionary processes and adaptive factors dent basis for favoring some hypotheses over involved in the origin of major groups (i.e., 65 scenarios: Eldredge, 1979). Patterson's (pers. stitution of scale leaves (cataphylls) for fronds. comm., 1980) discussion is of particular in- It is less clear that fossils have had impor- terest to us because among other examples he tant effects on ideas of angiosperm phylogeny. cites C.R. Hill as confirming that these con- Traditionally, the view that fossils provide the clusions hold for plants. only definitive evidence on phylogeny has been Although we agree with much of Patterson's widespread among angiosperm systematists, discussion, his claim that fossils have had little but they have used fossils very little in practice impact on ideas of plant relationships seems because of the supposedly uninformative na- counterintuitive from a historical point of ture of the early angiosperm record, which was view, especially when non-angiospermous believed to show the simultaneous appearance groups are considered. It could be argued that of diverse taxa in the Early Cretaceous. one of the first major paleobotanical discov- Together, these perceptions have often led to a eries of this century, the recognition of seed dismissal of phylogeny in general as idle ferns (Oliver and Scott, 1903), did not have speculation. The discovery of bisexual flowers much effect on phylogenetic concepts. These in Mesozoic Bennettitales led Arber and Par- fossils seemed to fit the idea that seed plants kin (1907) to propose a relationship between are related to ferns, which was strongly Bennettitales and angiosperms and to support promoted at about the same time on primarily the "euanthial" concept of angiosperm evolu- neontological grounds by Jeffrey (1902, 1910), tion: that showy, insect-pollinated, bisexual who combined the two groups in his class flowers with numerous free parts, as in Magno- Pteropsida. However, the work of Florin lia, are primitive in angiosperms, and that the (1938 1945, 1951) on Paleozoic cordaites and apetalous, wind-pollinated, unisexual flowers conifers was required to eliminate once and for of the Amentiferae, considered primitive by all ideas that conifers were related to lycop- Von Wettstein (1907) and many members of the sids, although the same homologies of conifer "Englerian" school, were derived by reduction cones had been inferred by some morpholo- and aggregation. However, Arber and Parkin's gists. The concept of a close relationship views on Bennettitales have subsequently been between ferns and seed plants began to lose largely abandoned. The euanthial concept was favor with recognition of the Devonian already widespread and it has persisted, but it "progymnosperms" (Beck, 1960, 1970, 1981), has been defended on other grounds, for example which show that gymnospermous anatomy was the association of euanthial floral structure acquired before seeds or fern-like fronds. with features such as vesselless wood and Studies of progymnosperms were taken to monosulcate pollen, whose primitive status support the already existing idea that the two was inferred from their almost universal occur- widely recognized subgroups of seed plants, rence in living as well as fossil gymnosperms. cycadopsids and coniferopsids, were separately Cretaceous paleobotanical studies have caused derived from ancestors without seeds, since the excitement in showing trends in pollen and leaf progymnosperm Archaeopteris shows conifer- morphology consistent with the primitive status opsid-like advances that cycadopsids and aneu- of magnoliids and in elucidating the timing of rophytalian progymnosperms lack. More re- early angiosperm evolution (Doyle, 1969, 1978; cently, however, recognition of conifer-like Muller, 1970, 1981; Hickey and Doyle, 1977; features such as platyspermic seeds and sac- Upchurch, 1984). However, it can be argued that cate pollen in the Late Carboniferous seed fern these studies have largely confirmed phyloge- Callistophyton led Rothwell (1982) to propose netic ideas already assumed by most students of instead that conifers (and possibly other coni- modern angiosperms, and that they have not feropsids) were derived from platyspermic seed changed the views of supporters of unorthodox ferns by a radical shift in leaf morphology and theories (e.g., Meeuse, 1972). A possible excep- habit, perhaps mediated by heterochronic sub- tion concerns the subclass Hamamelididae of 66

Cronquist (1968) and Takhtajan (1969), which tives the crocodilians. Using numerical cladis- includes both "core" members of the former tic methods, Gauthier et al. obtained results Amentiferae with basically triporate pollen similar to Gardiner's when they analyzed (Juglandaceae, Betulaceae, Myricaceae, Urti- modern groups only, but when they added cales, etc.) and groups with more primitive fossil groups and osteological characters that tricolpate pollen (Trochodendron, Hamameli- they provide, cladograms corresponding to the dales, etc.). The Late Cretaceous Normapolles classical view became more parsimonious. This pollen complex appears to provide a link appears to be a direct contradiction of Patter- between triporate amentiferous pollen and tri- son's (1981) contention. angular tricolporate pollen characteristic of the Our approach differs somewhat from that of subclass Rosidae, a concept confirmed by dis- Gauthier et al. in that we began with a data set covery of Normapolles pollen in situ in juglan- amassed for a numerical cladistic analysis of dalian flowers (Friis, 1983). This suggests that both living and fossil seed plants and progym- the Hamamelididae are polyphyletic: the tripo- nosperms (Doyle and Donoghue, 1986). We rate Amentiferae are actually rosids, only very then modified this matrix by subtracting and distantly related to the tricolpate groups (Wolfe re-adding fossil groups, including both extinct et al., 1975; Hickey and Doyle, 1977). However, relatives (outgroups) of the clade made up of even this example is unclear: Wolfe (1973) had all living seed plants and extinct seed plant inferred that Juglandaceae are rosids based on taxa (belonging to the ingroup), with charac- the leaf architecture of extant members (al- ters subtracted or re-added where appropriate. though he did not extend this to other Amenti- Essentially, these experiments ask what differ- ferae because of conflicting similarities between ence it would make to a hypothetical cladist if leaves of Betulaceae and Hamamelidaceae), and particular fossil groups were or were not it has not been widely accepted by students of known. This approach is not precisely analo- living plants (e.g., Cronquist, 1981). gous to Patterson's (1981), which emphasized In the present paper, we will not attempt to comparisons of pre-Darwinian and molecular- resolve the philosophical issues involved in the or cytological-based classifications with clas- controversy over the role of fossils, nor to sifications influenced by knowledge of fossils. analyze in detail the historical role of paleobo- However, we believe that our approach ad- tany in plant phylogeny. Instead, we have dresses the central issue more directly. With adopted an experimental approach to the Patterson's approach, it is easy to dismiss problem, using numerical cladistic methods to contradictions between the two sorts of clas- probe the effects of fossil taxa on both clado- sifications on the grounds that the neontologi- gram topology and hypotheses of character cal classifications were poorly constructed in evolution. The only comparable study that we the first place. In order to avoid confusion of know was recently conducted by Gauthier, issues, we will not address the role of stratigra- Kluge, and Roe (pers. commun., 1985), stimu- phy in polarity assessment; all of our decisions lated by a cladistic study of amniotes by on polarity are based on outgroup comparison. Gardiner (1982), which emphasized extant Throughout our discussion, we will attempt to groups. Gardiner's study led to the conclusion relate our results to macroevolutionary prob- that birds and mammals are more closely lems by considering their implications for related to each other than either is to any adaptive scenarios and evolutionary processes living group of "reptiles". This contrasts involved in the origin of groups, with particu- sharply with the view of most paleontologists lar emphasis on coniferopsids, Gnetales, and that mammals are derived from (nested within) angiosperms. Finally, we will attempt to make Permo-Triassic therapsids, whereas birds are some generalizations on recognition of cases derived from Mesozoic archosaurs (specifically where fossil evidence is more or less critical for dinosaurs), making their closest living rela- inference of relationships. 67

Data and methods of analysis every attempt to include as many potentially useful characters from all organs as possible In the analysis of living and fossil groups on and to code certain controversial characters in which the present study is based (Table I), we ways consistent with alternative hypotheses used two similar programs: the Wagner parsi- (Tables II and III). For instance, we coded mony algorithm in PHYSYS (Mickevich and platyspermic seeds X1, so that they can be Farris, 1982), and the Mixed Method parsi- derived by one step from no seeds (00), as in mony algorithm with the Wagner option in Beck's (1970, 1981) hypothesis that cycadopsids PHYLIP (Felsenstein, 1985), with global and coniferopsids were derived from different branch-swapping. Both algorithms begin with groups of progymnosperms, and from radio- a matrix of binary characters, scored 0, 1, and spermic seeds (10), as under Rothwell's (1982) X (missing information, coded 9 in PHYSYS hypothesis that conifers were derived from and ? in PHYLIP), and attempt to find the Callistophyton-like seed ferns. Likewise, we tree(s) requiring the smallest number of char- coded the linear-dichotomous leaves of conifer- acter state changes (steps), treating forward opsids as X01, derivable by one step from either changes and reversals equally. Since one of our the progymnosperm condition (000) or the seed main purposes was to evaluate the relative fern one (100). We attempted to evaluate merits of current hypotheses, and since we alternative hypotheses by adding "dummy" wished to overcome problems of previous synapomorphies to the matrix to force particu- cladistic studies of seed plants (Hill and Crane, lar groups together and then subtracting the 1982; Crane, 1985), which omitted many charac- corresponding numbers of steps after analysis, ters that might support alternative results and or by employing the user tree option in coded others in ways that seemed to make PHYLIP, which allows one to specify whole restrictive assumptions on homology, we made trees and determine their length.

TABLE I

Terminal taxa used (Doyle and Donoghue, 1986), with abbreviations used in figures

Taxa Abbreviation

Aneurophyton s. lat., including Triloboxylon and Eospermatopteris An Archaeopteris s. lat., including Svalbardia Ar Early Carboniferous protostelic lyginopterids with multiovulate cupules ML "Higher" lyginopterids, including Heterangium and Lyginopteris HL Medullosa, not including Quaestora and Sutcliffia Md Callistophyton Ca Glossopteridales GI Peltaspermum (Lepidopteris, Antevsia) PI Corystospermaceae (Dicroidium, Rhexoxylon, Umkomasia, Pteruchus) Cs Caytonia (Sagenopteris, Caytonanthus) Ct Cycadales, including Nilssoniales Cy Bennettitales ( = Cycadeoidales) Bn Pentoxylon Pn Euramerican cordaites, including Cordaites, Cordaianthus, and Mesoxylon Cd Ginkgoales, including Baiera, Karkenia, and Ginkgo Go Coniferales, including Lebachiaceae, Podocarpaceae, and Taxaceae Cn Ephedra Ep Welwitschia We Gnetum Gn Angiosperms Ag 68

TABLE II

Characters used (Doyle and Donoghue, 1986). Throughout, 0 is used for the presumed ancestral state, 1 for the derived state, and X for missing information (also used in multistate characters for states where the precursor state is unknown). When only one state is listed in the definition of a character, it is the derived state

1. 0 = branching apical; 1 = axillary 2. 0 = axillary buds single; 1 = multiple 3. Leaves on (homologs of) progymnosperm penultimate order branches 4. 0 = phyllotaxy spiral; 1 = opposite-decussate or whorled 5 7. 000=simple, dichotomous leaves only; 100=pinnately compound leaves and cataphylls; ll0=simple, pinnately veined leaves and cataphylls; X01, XX1 = pointed cataphyll-like leaves only, or simple, linear or dichotomous leaves and cataphylls 8. 0 = rachis regularly bifurcate; 1 = usually or always simple 9, 10. 00 = one order of laminar venation, open; 10 = one order of laminar venation, reticulate; 11 = two or more orders of laminar venation, at least finest order reticulate 11. 0 = poles of guard cells raised; 1 = level with aperture 12. 0 = stomata entirely haplocheilic; 1 = some or all syndetocheilic 13. Apical meristem with differentiation of tunica and corpus 14, 15. 00 = protostele (including vitalized types); 10 = eustele usually with external secondary xylem only; Xl = eustele with regular internal secondary xylem 16. 0 = some or all stem bundles mesarch or exarch; 1 = all endarch 17, 18. 00 = leaf traces from one stem bundle or protoxylem strand (one-trace unilacunar node); 10 = from more than two bundles (multilacunar node); X1 = from two adjacent bundles (two-trace unilacunar node) 19. 0 = some scalariform pits in metaxylem; 1 = no scalariform metaxylem, circular bordered pits in protoxylem 20. 0 = only circular bordered pitting or perforations in secondary xylem; 1 = at least some scalariform 21. Vessels in the secondary xylem 22. 0 = rays uniseriate or rarely biseriate; 1 = at least some multiseriate 23. Secretory canals 24. M/iule reaction 25 27. 000=dichotomous megasporangiate fertile appendages (cupules) on radial axis; 100=pinnately compound megasporophyll; 110 = once-pinnate megasporophyll, with two rows of simple leaflets or cupules bearing ovules; X01, XX1 = ovule on one-veined megasporophyll or sessile 28 30. 000 = dichotomous microsporangiate fertile appendages on radial axis; 100 = pinnately compound microsporophyll; 110= once-pinnate ~nicrosporophyll, with two rows of simple leaflets or stalks bearing pollen sacs; X01, XX1 = one- veined microsporophyll 31. 0 = ovule on lateral appendage; 1 = terminal 32. 0 = homologs of progymnosperm fertile branchlets on homologs of lower order axes; 1 = on homologs of last order axes 33-35. 000, 010 = ovule(s) in radial cupule; 100 = ovules directly on more or less laminar sporophyll (terminal, abaxial, or adaxial); ll0=ovules in anatropous cupule, or anatropous and bitegmic; X01=ovule with second integument derived from two appendages lower on axis 36. 0 = several ovules per anatropous cupule or potential homolog; 1 = one 37. 0 = microsporangia terminal, marginal, or adaxial; 1 = abaxial 38. 0=microsporangia free; 1 = fused at least basally into microsynangia 39. 0 = microsporophylls spirally arranged; 1 = whorled 40. 0 = strobili on undifferentiated axes, or only female aggregated into compound strobili; 1 = both male and female strobili aggregated 41, 42. 00 = no seeds; 10 = radiospermic seeds; X1 = platyspermic seeds 43, 44. 00 = megasporangium with unmodified apex; 10 = lagenostome with central column; 11, X1 = pollen chamber without central column 45. Micropylar tube 46. Nucellar vasculature 47. 0 = nucellar cuticle thin; 1 = thick, maceration-resistant 48. Heterospory 49, 50. 00= tetrad scar, no sulcus/pollen tube; 10= sulcus/pollen tube; 11 = pollen tube but no sulcus 51. 0 = pollen radially symmetrical or mixed; 1 = strictly bilateral 69

TABLE II (continued)

52. 0 = pollen nonsaccate or subsaccate; 1 = saccate 53. 0 = infratectal structure alveolar; 1 = granular or columellar 54. Pollen striate 55. 0 = megaspore tetrad tetrahedral; 1 = linear 56. 0 = megaspore wall thick; 1 = thin or lacking sporopollenin 57. 0 = microgametophyte with prothallial(s) and sterile cell; 1 = with prothallial but no sterile cell 58. 0-motile sperm; 1 = siphonogamy, nonmotile sperm 59. 0 = megagametophyte monosporic; 1 = tetrasporic 60. Apex of megagametophyte free-nuclear or with multinucleate cells; wall formation irregular, resulting in polyploid cells at maturity; egg a free nucleus 61. 0-early embryogenesis free nuclear; 1 = entirely cellular 62. Embryo with feeder

TABLE III

Data matrix for extant and fossil taxa (Doyle and Donoghue, 1986)

1 2 3 4 5 6 Aneurophyton 0X00000X00XXX0000000000X000000XO000XO00000000x×00000OO00X00×XX Archaeopteris 0X10000X00XXXIO0000000XX000000X1000X000000000XXI000000OOX00XXX Multiov. lyg. XXO010000000X0000000010X00000000000X010X101000X1000OX000X000XX Higher lygin. 100010000000X0000000010XIO010000000X010XI01000XI0000XO00X000XX Medullosa 100010000000XX101000011X10010000100X010X101101Xl001000XOXOXOXX Callistophyton 1000100100XOXl000000010X100100001000110XXlllOOXll01100X00XX0×X Glossopterids 10X0110XIOXOXIOXXX0000XXXXOXX000X000000XXIXIO011101101XOXXXOXX Peltaspermum XXOXIO010OXOXXXXXXXXXXXXI10100001000100XXIIIXX011010XOXOXXXXXX Corystosperms XX0010000000XX101000000XI00110001101100XXlllXX011011XOXIXXXXXX Caytonia 1000100X1010XXXXXXXXXXXXII0100001100X10XXlll0011101100XIXXXXXX Cycads XXX011OX000OO101100001101X01X00X100X100X10Xl010110100010000000 Bennettitales 10X0110X0001Xl010001011XXXlllOOX010XOll0XXXll0Xll0101011XX00XX Pentoxylon 10X011OX0000XXIXX101010XXXIOIOOX010XOX10XIX100111010XOXIXXXXXX Cordaites 1010X01X000OX1000000000XX01X0101X00X0001XIX10XXI000100X000XOXX Ginkgos 1010XOiXO0000101XllO0000X01X0101X00XX000XiX1000110100010000000 Conifers 1010X01X00OO010100100010X01X0101×00XlO00XiX1000100110010000000 Ephedra llXiXXiX000Oll01Xll01101XXlXXll×X01X0111XlXll00111101111010000 Welwitschia liXiXXIXll010101Xll01111XXiXXllXX01X01llXiXll001101011Xlllllll Cnetum llXlll0Xll011101101Xll01XXiXXllXX01XOXlll0Xll001110010Xlllllll Angiosperms 10X0110Xllllll011001Xl011101100Xll01Xl00XXXX001110101011X10010

Finding most parsimonious trees with both the order of entry based on an advancement programs requires considerable experimenta- index, but with PHYLIP the order of entry is tion. Because the number of possible trees specified by the user. The shortest trees that we increases rapidly with increasing numbers of obtained with PHYSYS (124 steps) were found taxa (Felsenstein, 1978), present methods can- only by forcing together taxa with dummy not guarantee finding the most parsimonious characters. Several of our shortest trees (123 tree(s) with large data sets. In Wagner algo- steps) were obtained with PHYLIP, by judi- rithms, taxa are added sequentially to the cious shuffling of the order of entry of taxa (as analysis in the most parsimonious position, recommended by Felsenstein in the on-line and what trees are found depends in part on PHYLIP documentation), and by use of the the order of entry of taxa. PHYSYS determines user tree option. In general, we obtained the 70 best results by entering taxa roughly in order iferous seed fern Medullosa, and a major clade of increasing advancement, but placing pos- that includes all extant groups, initially united sible alternative "linking" taxa in various by platyspermic seeds and saccate pollen, arrangements before specialized and problem- within which Callistophyton has the largest atical ones. We emphasize that this required number of primitive traits. Cordaites, conifers, much familiarity with the data and potential and ginkgos form a group at (or near) the base alternative arrangements of groups, many of of this clade; this result supports Rothwell's which were first seen during experimentation (1982) hypothesis that conifers were derived with PHYSYS. Other 123-step trees were from platyspermic, saccate seed ferns rather kindly brought to our attention by W.E. Stein than Archaeopteris-like progymnosperms (Beck, (pers. commun., 1986), using the PAUP pro- 1970, 1981), but it implies that this concept gram of D.L. Swofford. should be extended to coniferopsids as a whole One of our most parsimonious trees is shown (cf. Crane, 1985). In Fig.1 cycads are associated in Fig.l; others differ in reversing the order of with the Permo-Triassic seed fern Peltasper- Bennettitales and Pentoxylon and/or rearrang- mum, not with medullosans as often suggested ing Callistophyton, coniferopsids, corysto- (e.g., Crane, 1985); with this arrangement it is sperms, and cycads in various ways. In general, most parsimonious to interpret cycads as our trees are similar to the preferred tree of secondarily radiospermic, which is consistent Crane (1985). Arranged sequentially from the with the fact that seeds of Permian taeniopter- base are the progymnosperms Aneurophyton ids considered primitive cycads by Mamay and Archaeopteris, two groups of Carbonifer- (1976) appear to be flattened and Cycas seeds ous lyginopterid seed ferns, the Late Carbon- have bilateral symmetry (interpreted as sec-

An Ar ML HL Md Go Cn Cd Ca Cs Cy P1 G1 Ct Ag Bn Pn Ep We Gn \

Fig.1. One of several most parsimonious 123-step trees derived from analysis of the whole data set (Doyle and Donoghue, 1986). Taxa and their abbreviations are defined in Table I; characters in Table II. Minus signs before characters indicate reversals. 71 ondary by Meyen, 1984). However, in other 123- Gnetales and Amentiferae were secondarily step trees cycads are linked with Medullosa or reduced and aggregated. Our trees imply that form the sister group of the platyspermic clade. the first anthophytes had pinnate mega- and The angiosperms also belong in the platysper- microsporophylls, but the megasporophylls mic group, forming a clade with Bennettitales, were reduced to single ovules in the common Pentoxylon, and Gnetales; since all four groups ancestor of Bennettitales and Gnetales, have flower-like reproductive structures, we whereas the microsporophylls were indepen- refer to them as the anthophytes. Crane (1985) dently simplified in angiosperms and Gnetales. recognizes the same clade and connects it with Experiments show that some connections in the Mesozoic seed ferns (corystosperms, Cayto- this scheme are very strong, but others are nia, glossopterids), based largely on the hy- weaker. For example, only one step is added if pothesis that the reflexed cupule of Mesozoic coniferopsids are associated with Archaeop- seed ferns is homologous not only with the teris (Beck, 1970, 1981), which would imply that anatropous bitegmic ovule of angiosperms the seed originated twice. The position of the (Gaussen, 1946; Stebbins, 1974; Doyle, 1978) but anthophytes and relationships among members also with the orthotropous cupulate ovule of of the platyspermic clade are also relatively Bennettitales and Pentoxylon. All of our most unstable: we found many trees only one step parsimonious trees differ from Crane's in longer (124 steps) with cycads the sister group relating anthophytes more closely to Caytonia of the platyspermic clade, coniferopsids linked and glossopterids than to corystosperms, and with Peltaspermum and/or glossopterids, and in treating angiosperms as the sister group of anthophytes linked with Caytonia and corys- other anthophytes, rather than linked directly tosperms, Caytonia and glossopterids, corysto- with Gnetales. sperms alone, or all three groups in various Implications of our results for evolution of orders (cf. Crane, 1985). Three extra steps are key characters in seed plants are summarized needed to link anthophytes with Medullosa in Fig.2, with less favored relationships indi- and cycads. Most disconcertingly, we found cated by dotted lines. On a broad scale, our trees of only 125 steps with anthophytes nested most parsimonious trees imply that there were in the coniferopsids, with Gnetales the sister two trends in seed plants from a "cycadopsid" group of angiosperms, Pentoxylon, and Bennet- to a "coniferopsid" habit, one in the Carbon- titales, suggesting that the latter groups origi- iferous leading to the coniferopsids and the nated from coniferopsids via Gnetales-like other in the Mesozoic leading to Gnetales. intermediates (a ~'neo-englerian" arrange- Elsewhere we argue that both trends may be ment). On the other hand, the anthophytes tied to a shift to drier habitats (Doyle and appear to be a more robust group. Moving Donoghue, 1986). Although our results indi- Gnetales (the least securely associated group) cate that Gnetales are the closest living into the coniferopsids, their next-best position, relatives of angiosperms, they in no way adds four steps, and other arrangements that support suggestions that angiosperms were break up the anthophytes are still less parsimo- derived from a gnetalian prototype, as in Von nious. Angiosperms can be linked directly with Wettstein's (1907) theory that the catkins of Caytonia with addition of only one step, but in Amentiferae are homologous with gnetalian this case the two groups are still most closely compound strobili and that bisexual flowers associated with other anthophytes. originated by aggregation of unisexual units. In the present study, all analyses were Rather, they are more consistent with the performed with PHYLIP (version 2.8), using an views of Arber and Parkin (1907, 1908), who Eagle PC microcomputer. For data sets with proposed that angiosperms, Bennettitales, and larger numbers of taxa, we used the Wagner Gnetales were derived from a common ancestor option in the Mixed Method Parsimony algo- with bisexual flowers and that the flowers of rithm, as discussed above. When the number of 72 taxa was small (up to nine), we used the or on addition of fossils to modern groups, we Wagner option in the Penny algorithm in left in all varying characters even when the PHYLIP, which uses a "branch and bound" original derived state occurred in only one search strategy (Hendy and Penny, 1982) that taxon, since in these experiments polarities of guarantees finding all most parsimonious trees many characters become equivocal or are even (with larger numbers of taxa, the search time reversed, depending on the rooting. The most becomes prohibitively long). We tested alterna- complex decisions concerned recoding of mul- tive hypotheses by means of the user tree tistate characters, where some but not all option and/or the dummy character method states disappear or become autapomorphic discussed above. with subtraction of groups; as discussed in With PHYLIP, trees can be rooted either by individual experiments, we attempted to re- specifying one of the included taxa as outgroup code such characters in the light of informa- to the rest or by specification of a set of tion that could be obtained from the groups ancestral states (0, 1, or X). In experiments included in the analysis. with the whole data set and subtraction of fossil taxa, we rooted trees by specifying Experiments Aneurophyton (which by comparison with out- groups appears to be primitive in all characters Analyses of extant groups only considered) as the outgroup, but in experi- ments with the modern data set and addition of Our first experiments were designed to fossils to it we used the ancestral states option, assess whether and to what extent fossils specifying primitive states on the basis of clarify seed plant phylogeny by providing various concepts of outgroup relationships (as closer extinct relatives (outgroups) of extant discussed further below). When the ancestral seed plants as a clade (the ingroup). As argued states option is invoked, the program provides by Maddison et al. (1984), addition of closer the most parsimonious "best guesses of ances- outgroups may require changes in ideas on tral states" for all characters (which may of polarity and therefore relationships in the course still be X), given the rooting defined by ingroup, and this should hold whether the those characters that were polarized. We used outgroups are living or fossil. Outgroups could this feature extensively in evaluating the have been entered into the analysis as addi: implications of knowledge of fossil groups for tional taxa and ingroup and outgroup relation- character evolution. ships resolved simultaneously. However, be- In compiling characters for our previous cause we wished to consider particular sets of study, we consistently eliminated autapomor- outgroups separately and to keep the total phies (where the derived state is limited to one number of taxa small enough to use the Penny taxon), since they provide no information on algorithm, we instead employed the two-step cladistic relationships of groups and give a outgroup procedure described by Maddison et misleading impression of the amount of data al. (1984), using different outgroup arrange- supporting a scheme. In the present study we ments to assess character states of a hypothet- followed a somewhat different procedure. In all ical ancestor, and then using the resulting list experiments we eliminated characters that of states to root the ingroup tree with the were invariant in the subset of taxa repre- ancestral states option. If one of the two sented, and in experiments involving subtrac- character states (0, 1) found in the ingroup is tion of fossil groups from the whole data set we found in both of the two nearest outgroups, it also eliminated autapomorphies, since polari- is most parsimonious to assume that that state ties were the same as with the whole data set, occurred in the hypothetical ancestor, but if being determined by Aneurophyton. However, the first two outgroups differ in state and in experiments based on modern groups only, additional outgroup information is unavail- 73

N

b l °,

Fig.2. Major transformations in leaf morphology and reproductive structures of seed plants inferred from the cladogram shown in Fig.1. Groups indicated: (a) progymnosperms; (b) seed ferns; (c) primitive coniferopsids; (d) hypothetical common ancestor of anthophytes (angiosperms, Bennettitales, Pentoxylon, Gnetales); (e) angiosperms; (f) Bennettitales; (g) Gnetales. Less parsimonious alternative transformations are indicated with dotted lines ("Beck": independent derivation of cycadopsids and coniferopsids from progymnosperms; "NE": a "neo-englerian" arrangement, with anthophytes nested in coniferopsids).

able, the ancestral state for the character is Many characters become invariant in the taxa equivocal and is thus coded X. remaining and were therefore eliminated (e.g., Removal of the 13 fossil taxa from the 20 axillary branching, endarch primary xylem, groups in our original analysis resulted in heterospory). Some multistate characters col- reduction of 62 characters to 40 (Table IV). lapse to binary characters (e.g., lagenostome 74

TABLE IV

Experiments with extant taxa. "Conservative" through "seed fern" designate sets of user-specified ancestral states based on different assumptions concerning outgroup relationships. "BGAS" represents "best guesses" of ancestral states provided by PHYLIP for the most parsimonious trees obtained with the conservative rooting

1 2 3 4 5 6 Conservative -0-O--X-0-100---000XOXX0--X--XX--XX-XXX0-X--XXX--XXXXX-O000010 Moderate fern -O-O--O-O-100---O00XOXXO--0--00--XX-XXOO-X--XXX--XXXXX-0000010 Extreme fern -0-O--O-O-IO0---000101X0--O--O0--XX-XXOO-X--XXX--XXXXX-O000010 Progymnosperm -0-O--I-0-100---00000000--I--IO--X0-00OO-X--XXX--XO000-0000010 Seed fern -0-O--O-O-O00---X00001X0--O--00--XO-0100-0--OXX--XXOOO-OOOOOXO

Cycads -X-O--O-0-OOO---10OOOII0--O--OO--OO-IOOX-0--OIO--OIO00-O000000 Ginkgos -O-O--I-O-O00---XIIOOO00--I--10--O0-XO00-1--O00--OIO00-O000000 Conifers -0-0--i-0-000---00100010--i--I0--00-I000-i--000--01100-0010000 Ephedra -I-I--I-O-O01---XII01101--I--II--OI-OIII-I--100--II011-1010000 Welwitschia -I-I--I-I-OIO---XIIOIIII--I--II--OI-OIII-I--100--OIOII-IIIIIII Gnetum -I-I--O-I-011---101XII01--1--II--OI-0XII-O--100--10010-1111111 Angiosperms -O-O--0-1-111---IO01XI01--O--OO--10-XIO0-X--OOI--OI010-IXIO010

BGAS, tree 4a: -0-O--X-0-1OO---OOOOOXXO--X--X0--O0-1000-1--000--OI000-0000010 BGAS, tree 4f: -O-O--X-O-100---0OOO01XO--X--XO--O0-??O0-1--O00--010?0-O000010 vs. normal pollen chamber), or it becomes between the dissected leaves of cycads and the unlikely that distinctions made with the whole entire leaves of angiosperms and Gnetum, but data set would be recognized. For example, ordering the resulting states would be problem- with the loss of groups with simply reticulate atical. Cycad leaves might be considered more venation (Caytonia, glossopterids), two charac- fern-like in being dissected, but they are ters that appear distinct with the whole data basically once-pinnate, with leaflets that have set, reticulate venation and presence of several parallel-dichotomous venation, whereas most vein orders, are invariably associated and fern leaves are several-times compound, with would probably be interpreted as redundant, so pinnules that themselves have pinnate vena- they were combined as a single character. We tion. In such cases, we have assumed that a eliminated two characters that express ad- hypothetical investigator who knew only ex- vances of Archaeopteris over Aneurophyton in tant groups would prefer to err on the side of the arrangement of the leaves (3) and fertile caution and keep the number of distinctions to appendages (32); if coniferopsids are inter- a minimum. This entails little loss of informa- preted in a progymnosperm context, it is clear tion, since the special similarity between the that they have the Archaeopteris state in these leaves of angiosperms and Gnetum is expressed characters, but without reference to progym- by the venation character. We assumed (per- nosperms these characters are less obvious as a haps optimistically) that the angiosperm basis for linking coniferopsids with each other. stamen would be recognized as a basically With the loss of groups with dichotomous pinnate structure because the pollen sacs are leaves and no cataphylls (progymnosperms) arranged as lateral pairs, in contrast to the and pinnately compound leaves (seed ferns), situation in coniferopsids and Gnetales. In only two main leaf conditions remain: simply order to keep this an exercise based on Recent pinnate (cycads, angiosperms, and Gnetum) information only, we rescored conifers as and linear-dichotomous (conifers, Ginkgo, having a sulcus and pollen tube and siphono- Ephedra, Welwitschia). Distinctions might be gamy, although the earliest Paleozoic conifers made within these categories, for instance had a tetrad scar and no sulcus and presum- 75 ably had motile sperm (Mapes and Rothwell, plants were derived from (nested within) ferns, 1984). expressed in the widely adopted grouping of An unrooted analysis of the modern data set the two taxa into Pteropsida (Jeffrey, 1902, gave three unrooted trees ("networks") of 57 1910): a relatively cautious '~moderate fern" steps (Fig.3). One (Fig.3.a) is consistent with rooting and an "extreme fern" rooting based all the shortest trees obtained from the whole on more questionable homologies between data set (e.g., Fig.l), as well as with the rooted ferns and seed l~lants. The last two exercises trees obtained most frequently in analyses of are the first tests of the effect of fossil data on the modern data set with the various rootings seed plant phylogeny, using a '~progymno- described below (Fig.4.a, f). The other two sperm" rooting and a "seed fern" rooting. They (Fig.3.b, c) are consistent with "neo-englerian" model hypothetical situations in which we rooted trees (among others), with both angio- knew (1) about progymnosperms (Aneurophy- sperms and Gnetales nested in coniferopsids ton, Archaeopteris) but no other fossils, and (2) (e.g., Fig.4.d), which are less parsimonious about the most primitive Carboniferous seed than the shortest rooted trees obtained with ferns ("lyginopterids" and Medullosa) but not both modern and complete data sets. Meacham progymnosperms, as was the case early in this (1984) argues that it is best to construct an century. Where information on characters in unrooted tree first and then attempt to root it the fossil groups is lacking (e.g., M/iule re- rather than to begin with assumptions on action, embryology), we relied on living lower polarity. However, this example shows that vascular plants as the next closest outgroups. unrooted analysis may produce some networks For each rooting, besides using the Penny that are globally less parsimonious under any algorithm to find all most parsimonious trees, reasonable outgroup assumptions, so that even we also determined the lengths of several user- minimal a priori polarity assessment is of value specified trees that are of particular theoreti- in reducing the number of alternatives to be cal interest (Fig.4): (a) with coniferopsids the considered and should therefore be attempted sister group of all other taxa and angiosperms (cf. Donoghue and Maddison, 1986). related to Gnetales (consistent with both the Altogether, we did five sets of exercises with most parsimonious tree of all groups shown in the extant data set, with rootings based on Fig.1 and the concept that coniferopsids and different assumptions on outgroup relation- cycadopsids were separately derived from pro- ships (Table IV). The first or "conservative" gymnosperms); (b) with coniferopsids and an- rooting represents what we consider the stron- giosperms plus Gnetales as sister groups, and gest conclusions on polarity that could be with cycads the sister group of both (as in drawn from extant outgroups only, assuming other most parsimonious trees derived from the that ferns and sphenopsids are closer to seed whole data set and the trees of Crane, 1985); (c) plants than lycopsids (for reasons discussed with coniferopsids and cycadopsids as sister below) but not specifying more detailed rela- groups, but with Gnetales nested in coniferop- tionships. The second and third rootings ex- sids; (d) a "neo-englerian" arrangement, with plore the historically important view that seed both angiosperms and Gnetales nested within

Ag Ag Ag

Cn ~ We Cn ~ We We

GO / \ Gn Cy i "~ Gn Gn a b c Fig.3. Most parsimonious unrooted trees (networks) obtained from analysis of extant seed plant taxa (57 steps). 76

Go Cn Cy Ag Ep We Gn C¥ Go Cn Ag Ep We Gn Cy Ag Cn Go Ep We Gn

a C

conserv. 59 60 62 mod. fern 59 60 62 ext. fern 60 61 63 progym. 61 63 64 seed fern 60 59 61

Cy Cn Go Ag Ep We On Ag Cy Cn Go Ep We Gn Go Cn Cy Ag Ep We Gn

conserv. 60 60 59 rood. fern 60 60 59 ext. fern 61 60 60 progym. 63 66 61 seed fern 59 60 58

Fig.4. (a-e) User-specified trees of extant seed plant taxa, and (f) a topology seen among the most parsimonious trees obtained in all experiments, with the number of steps obtained using ancestral states based on different assumptions concerning outgroup relationships (see text). the coniferopsids; and (e) with Gnetales nested (Doyle and Donoghue, 1986), but since this is in coniferopsids but with cycads the sister not evident from any modern data, we have group of coniferopsids, making angiosperms treated ferns, sphenopsids, and seed plants as basal. Figure 4 also includes a tree (Fig.4.f) an unresolved trichotomy. that we found with all rootings, in which Under these assumptions, we reached the cycads are linked with coniferopsids and same decisions on the polarity of 16 characters angiosperms with Gnetales. as with the whole data set, because one of the states does not occur (or if present is clearly Conservative rooting independently derived) in extant non-seed Neontological evidence supporting the as- plants. Most of these are features now re- sumption made here that ferns and/or sphenop- stricted to angiosperms, Gnetales, or both (e.g., sids are closer to seed plants than lycopsids opposite leaves, reticulate venation, syndeto- includes the fact that seed plants, ferns, and cheilic stomata, tunica layer in the apical sphenopsids differ from lycopsids in having meristem, vessels, M~iule reaction, compound multiflagellate sperm, a derived feature on strobili, reduced megaspore wall, siphono- comparison with bryophytes and green algae gamy, and other features associated with (plus mesarch or endarch primary xylem and gametophyte reduction). In a great many other longitudinal sporangial dehiscence, characters characters, we could find no basis for deciding of less certain polarity when only living groups which state was ancestral, either because the are considered). Ferns and sphenopsids may appropriate characters are lacking in the form a clade, based on presence of peripheral outgroups (e.g., features of secondary xylem, loops in early fossil members of both groups seeds), or because both states are present. For 77

example, both pinnately organized and linear- more, the alternative topology of extant groups dichotomous leaves occur in the outgroups, derived from the whole data set, with cycads secretory canals occur in some fern groups but basal (Fig.4.b), is not seen (it is one step less not others, and both trilete and monolete parsimonious). spores (corresponding to radial and bilateral Consideration of the user-specified trees pollen symmetry) exist in ferns and lycopsids. suggests that one effect of elimination of fossil In some cases, the comparisons that led us to groups is poorer resolution of alternatives that leave the ancestral state undefined may seem differ considerably in relative parsimony when farfetched (e.g., fused pollen sacs with synan- fossils are included. Most significantly, one gia of Marattiales, whorled microsporophylls tree with Gnetales separated from angiosperms of Gnetales with sporangiophores of sphenop- and nested in the coniferopsids (Fig.4.e) is only sids, inaperturate pollen with alete spores of one step longer than the shortest trees, while Equisetum, or saccate pollen with the perinate with the whole data set moving Gnetales into condition in various lower groups), but in the coniferopsids adds at least four steps. formulating this rooting we wished to give Nevertheless, the modern data set does allow wide leeway to potential homology. In two rather strong rejection of some alternatives: cases, we reversed polarities assumed with the trees with angiosperms forced into (derived whole data set. There we assumed that flat from) Gnetales, as the sister group of Welwit- guard cells (angiosperms, Caytonia) and cellu- schia and Gnetum, are five steps longer than lar embryogeny (angiosperms, Welwitschia, the shortest trees, and trees with taxa forced Gnetum) were secondarily derived within seed into three major clades based on the scheme of plants, because guard cells with raised poles Meyen (1984) -- Ginkgoopsida (Ginkgo, Ephe- and large egg cells (correlated with free- dra), Pinopsida (conifers), and Cycadopsida nuclear embryogeny in modern plants, presum- (cycads, angiosperms, Welwitschia, Gnetum), ably for functional reasons) are characteristic with the last two related to each other -- are of Carboniferous seed ferns and cordaites, even worse (71 steps). which appear to be basal within seed plants on At first sight, the two 59-step trees might outgroup comparison with progymnosperms. suggest the two major hypotheses on relation- However, fiat guard cells and cellular embryo- ships of seed plant groups based on fossil geny are ubiquitous in living lower vascular evidence: Fig.4.a, the Beck hypothesis that plants, so the elimination of evidence from coniferopsids and cycadopsids were separately fossil groups requires designating these states derived from progymnosperms; Fig.4.f, the as ancestral. Rothwell hypothesis that conifers (extended to Analysis of this data set yields two most coniferopsids as a whole) were derived from a parsimonious 59-step cladograms (Fig.4.a, f). seed fern prototype. However, in fact they have Both agree with trees derived from the whole no clear relation to these two hypotheses in data set in showing coniferopsids (conifers plus terms of either topology (Fig.4.a is equally Ginkgo) and Gnetales as natural groups, Ephe- consistent with our analysis of the whole data dra as the sister group of Welwitschia and set) or character evolution. This can be seen by Gnetum, and angiosperms as the sister group of examination of the "best guesses" of ancestral Gnetales. One (Fig.4.a) is consistent with the character states provided by PHYLIP (BGAS, cladogram of extant and fossil groups shown in Table IV) and the equally parsimonious place- Fig.l, with coniferopsids the sister group of the ments of character state changes on each tree remaining taxa and cycads the sister group of shown in Fig.5. For example, under both angiosperms plus Gnetales (anthophytes). topologies it is equally parsimonious to assume However, in the other (Fig.4.f), cycads are the that either pinnate or linear-dichotomous sister group of coniferopsids, an arrangement leaves are basic in seed plants (Fig.5.a, d vs. never seen with the whole data set. Further- Fig.5.b, c, e). Since the concept that pinnate 78

Go Cn Cy Ag Ep We On Go Cn Cy Ag Ep We On Go Cn Cy Ag Ep We Gn

L,S L,S

Go Cn Cy Ag Ep We On Go Cn Cy Ag Ep We Gn

L leaves L, , S sporophylls r-1 pinnate-~linear-dichot. • linear-dichot.--~pinnate

Fig.5. Selected equally parsimonious placements of the leaf and mega- and microsporophyllcharacters (7, 27, 30) on the two shortest trees obtained with the conservative rooting (Fig.4a, f; 59 steps). leaves are ancestral (favored by analysis of the Gnetales might have arisen from ancestors whole data set) implies that drastic leaf with more complex floral structures by reduc- reduction occurred in both Gnetales and coni- tion, as proposed by Arber and Parkin (1908) feropsids, possibly linked to a shift to arid and supported by analysis of the whole data habitats (Doyle and Donoghue, 1986), whereas set, or angiosperms might have arisen from the alternative concept does not, both trees Gnetales-like ancestors with linear leaves and permit a great range of adaptive scenarios. simple sporophylls (though not such gnetalian Furthermore, without indications (like those advances as opposite leaves and isolated circu- provided by Archaeopteris) that coniferopsids lar-bordered pits in the protoxylem) by elabora- are related to one group of non-seed plants and tion or aggregation of simple floral units, as cycadopsids to another, it is most parsimoni- proposed by Von Wettstein (1907). Without ous to assume that seeds are homologous in knowledge of Mesozoic seed ferns, the idea coniferopsids and cycadopsids even when they that the anatropous bitegmic ovule of angio- are sister groups, rather than originating sperms is a cupule derived from a reflexed leaflet twice, as under the Beck hypothesis (this is might never be considered. Other concepts also true for axillary branching, eustele, pollen might appear equally plausible; we assumed chamber, linear megaspore tetrad, and sulcus that the development of the gnetalian outer and pollen tube, which were eliminated from integument from two perianth-like primordia this data set). Interestingly, given both trees, (Martens, 1971) would be recognized as evi- Fig.4.a and f, it is most parsimonious t O assume dence that it is not homologous with the that the common ancestor of living seed plants angiosperm outer integument, but this may had platyspermic seeds, as in Fig.1 and most have been overly optimistic. other trees based on the whole data set; fossils Our experiments also suggest that ignorance are needed to show that their earlier ancestors of fossils may lead one to underestimate the were radiospermic. amount of homoplasy in some characters. Thus Evolution of characters and corresponding analysis of the whole data set implies that scenarios within higher seed plant groups are saccate pollen existed in the common ancestor also poorly defined. Since it is equally parsimo- of all extant groups and was lost in the lines nious to consider the pinnately organized leading to cycads, Ginkgo, and anthophytes, leaves and sporophylls of angiosperms either but on extant data alone it would appear to be primitive or derived relative to Gnetales, a conifer autapomorphy. In other cases, con- 79 vergences may be misinterpreted as synapo- rather than leaving polarity unspecified. The morphies. Even if vessels are assumed to be extreme fern rooting extrapolates from the fact basic in angiosperms (Young, 1981), the trees that ferns have scalariform primary xylem derived from the whole data set imply that they pitting to the conclusion that the scalariform originated independently in angiosperms and secondary xylem pitting of angiosperms is Gnetales, since they are absent in Bennetti- primitive, and from the observation that the tales and Pentoxylon. However, given the trees stems of ferns look more like the manoxylic based on extant taxa, if vessels are taken as stems of cycads than the pycnoxylic stems of basic in angiosperms, it is most parsimonious conifers and Ginkgo to the conclusion that to assume that they are homologous in the two manoxylic characters such as multiseriate rays modern groups. are ancestral. These extrapolations now seem highly questionable, not only in light of the Fern rootings anatomy of progymnosperms and seed ferns Both '~moderate" and ~'extreme" fern root- (Beck, 1970), but also because they compare the ings assume that seed plants are nested within primary xylem of ferns with the secondary ferns, so that the first two outgroups are both xylem of seed plants. However, there is again fern taxa, without specifying exact relation- historical precedent for considering these ships. In historical terms, this may be a more views; for example, the scalariform pitting of accurate representation of ideas not influenced angiosperms has often been cited as an archaic by fossils than the previous rooting, since it feature suggesting that they were derived from was first articulated by Jeffrey (1902, 1910) on some unknown primitive group of seed ferns primarily neontological grounds and has fallen (e.g., Takhtajan, 1969). out of favor largely as a result of studies on With both fern rootings, the most parsimoni- progymnosperms and seed ferns (Beck, 1970), ous trees included the same two derived from although Beck et al. (1982) argue that it should the conservative rooting (Fig.4.a, f). Here the have been and was questioned on the basis of course of leaf and sporophyll evolution neces- stelar anatomy of extant plants. In considering sarily follows the patterns shown in Fig.5.a, d. this alternative, we are not erecting a straw However, the extreme fern rooting yielded three man: despite paleobotanical critiques, the idea additional trees in which angiosperms are basal that ferns are the closest relatives of seed in seed plants and Gnetales are associated with plants is far from dead. In a synthesis of recent coniferopsids, varying in the arrangement of cladistic studies emphasizing extant groups, conifers and Ginkgo (Fig.6.a). With the conser- Bremer (1985) treats Ophioglossales, Maratti- vative and moderate fern rootings, such trees ales and ~'true ferns", and seed plants as a are one step less parsimonious than the shortest trichotomy, united on megaphyllous leaves trees. In terms of character evolution, these with a midrib (i.e., pinnate leaves) and tri- trees imply that the scalariform pitting, flat chomes. From a paleobotanical point of view, guard cells, and cellular embryogeny of angio- these characters are highly questionable as sperms are all primitive features directly re- synapomorphies, since apparent fossil rela- tained from a fern ancestry, as assumed by tives of both seed plants (progymnosperms) and Takhtajan (1969) for pitting, rather than sec- ferns (Cladoxylales, some coenopterids) have ondary reversals. The last two features were three-dimensional branch systems rather than also coded as ancestral in the previous experi- pinnate fronds, and trichomes are rare in ments, but by themselves they were not enough gymnosperms. to shift angiosperms to a basal position. Both fern rootings differ from the conserva- tive rooting in assuming that pinnate leaves Progymnosperm rooting and sporophylls are ancestral, and terminal The ~progymnosperm" rooting differs from ovules and whorled microsporophylls derived, all others in assuming that the simple, non- 80

Ag Cy Cn Go Ep We Gn Cy Cn Go Ag Ep We Gn Go Cn Cy Ag On Ep We

a b e

Fig.6. Additional most parsimonious trees obtained with (a) the extreme fern rooting (60 steps), (b) the progymnosperm rooting (61 steps), and (c) the seed fern rooting (58 steps). The trichotomy in (a) indicates that all three possible arrangements of the taxa concerned were found. pinnate leaves and sporophylls of coniferopsids that angiosperms are derived from Gnetales- are primitive relative to the pinnate leaves of like ancestors (this is equivocal in Fig.4.a: cycadopsids, being less removed from the contrast Fig.5.b, c). For the same reasons, they dichotomous appendages of progymnosperms. contrast with trees derived from the whole Since progymnosperms have secondary wood data set. However, they also differ from "Beck- and more uniform reproductive morphology type" trees obtained by manipulation of the than living outgroups, they allow uniseriate whole data set, in which coniferopsids are rays, circular-bordered secondary xylem pit- associated with Archaeopteris but the topology ting, free microsporangia, and radial, non- of other groups remains unchanged. In such saccate (pseudosaccate) pollen with alveolar trees, pinnate leaves are still basic within exine structure to be designated as ancestral. cycadopsids and Gnetales are reduced relative However, since they lack seeds, the polarity of to angiosperms. most seed characters cannot be specified (ex- Although our analysis of the whole data set cept terminal ovules, which seem derived assumed that seed plants are ultimately de- under any reasonable homology of ovules with rived from Aneurophyton-like progymno- progymnosperm structures). Since the stoma- sperms, it should not be surprising that coding tal structure of progymnosperms is unknown, ancestral states for exant groups in terms of we follow the modern data in designating progymnosperms gives such different (and raised guard cell poles as derived. presumably incorrect) results, since progym- It might be thought that using progymno- nosperms are more distant from modern seed sperms to root extant groups would favor the plants than seed ferns are (cf. Maddison et al., concept that coniferopsids and cycadopsids are 1984). sister groups, but in fact it results in the same two trees found with the conservative and fern Seed fern rooting rootings (Fig.4.a, f) plus a new tree that places The "seed fern" rooting is based on the cycads within coniferopsids as the sister group assumption that the closest outgroups of the of conifers, leading to the bizarre conclusion clade including all modern seed plants are that cycads are derived from coniferopsid-like Medullosa and lyginopterid seed ferns, as in ancestors (Fig.6.b). Although two of the three many of the shortest trees derived from the trees have topologies seen with the fern whole data set, such as Fig.1. Like the fern rootings, they all differ radically from the fern- rootings, this arrangement implies that pin- rooted trees in terms of character evolution. nate leaves and sporophylls are ancestral. Like Two of them (Figs.4.f and 6.b) imply that the progymnosperm rooting, it allows conclu- pinnate leaves and sporophylls are indepen- sions on the polarity of many characters that dently derived from simple structures in all comparison with modern outgroups left undi- cases where they occur (angiosperms, cycads, rected. It agrees with the progymnosperm Gnetum, as in Fig.5.e), favoring the concept rooting on the polarity of the tracheary 81 pitting, saccate pollen, and exine structure pycnoxylic anatomy is derived from manoxylic. characters, but it differs in treating as ances- However, without additional information from tral multiseriate rays and microsynangia (ap- ingroup fossils such as Mesozoic seed ferns and parently derived from the progymnosperm Bennettitales, the inferred homologies of the conditions in early seed ferns), raised guard angiosperm outer integument and most of the cell poles, and radiospermic seeds. However, course of pollen evolution would still be the polarity of several other characters either obscure. remains or becomes equivocal: presence vs. Since all of our experiments produced one or absence of a cupule (potentially, though prob- more trees with coniferopsids nested in seed ably not actually, represented today by the plants, and since it is most parsimonious to angiosperm outer integument), uni- vs. multila- assume that the seed arose only once even cunar nodes, and radial vs. bilateral pollen; in when coniferopsids are basal, one may ask why each case, the former state occurs in lyginop- historically the idea of diphyletic origin of seed terids, the latter in Medullosa. As noted above, plants flourished before as well as after the the large size of the egg cell suggests that seed recognition of progymnosperms (e.g., Chamber- ferns had free-nuclear embryogeny, but to be lain, 1935; Arnold, 1948). One reason may be cautious we left this character unpolarized. that evidence amassed by Florin (1938-1945, If the correct outgroups are sufficient to 1951) suggested that the leaves and sporophylls establish correct ingroup relationships even of Paleozoic conifers and cordaites are basi- when the ingroup is incomplete, this rooting cally dichotomous, and this was assumed to be should give the trees most consistent with a retention of the primitive dichotomous or- those obtained from the whole data set. Quite ganization of Devonian vascular plants, which to the contrary, for the first time both topolo- lacked seeds. A logical alternative, that both gies of extant groups derived from the whole coniferopsids and cycadopsids were derived data set (Fig.4.a, b) are eliminated: they are from a coniferopsid-like common ancestor with now one or two steps longer than the shortest dichotomous leaves and seeds, was not con- trees, which are the tree with cycads linked sidered, perhaps because seed ferns were as- with coniferopsids (Fig.4.f) and a new tree with sumed to be derived from ferns, and/or because Gnetum basal in Gnetales (Fig.6.c). they appear stratigraphically before coniferop- With this rooting, user-specified trees with sids. Some might cite this as a case where over- angiosperms basal (Fig.4.e) become three steps reliance on fossils obscured true relationships less parsimonious than the shortest trees, (cf. Patterson, 1981). However, it might be implying that knowledge of early seed ferns better attributed to a tendency to assume that should have eliminated the idea that the evolution of certain characters is irreversible, scalariform pitting and other fern-like features an error independent of the question of the of angiosperms are primitive retentions. This value of fossil evidence. Furthermore, it is far effect is even more striking with the progym- from clear that the concept of a diphyletic nosperm rooting, where the difference is six origin of the seed is incorrect, since it is almost steps. as parsimonious when the whole data set is Despite the different placement of cycads, analyzed (Doyle and Donoghue, 1986). the trees obtained with the seed fern rooting In summary, the experiments described so are certainly closest to those derived from the far show that parsimony analysis of extant whole data set in terms of inferred pathways of ingroups and outgroups only does produce one character evolution leading to coniferopsids, topology of seed plant taxa (among several angiosperms, and Gnetales (and presumably others) that is identical to one of the two adaptive scenarios for their origin). Thus with derived from analysis of both living and fossil the topology in Fig.4.f, changes in leaves and groups, but even with this "correct" topology sporophylls follow the pattern in Fig.5.d, and implications on character evolution and adap- 82 tive scenarios for the origin of major groups groups with pinnately compound leaves and a are very poorly constrained. Addition of appro- simple rachis; flat guard cells become an priate fossil outgroups (early seed ferns) clar- angiosperm autapomorphy; and the distinc- ifies many problems of character evolution, but tions among the various sorts of bitegmic and it leaves others unresolved (e.g., evolution of cupulate ovules (lyginopterids, angiosperms, the angiosperm outer integument), and ironi- Bennettitales and Pentoxylon) become prob- cally it does not give either of the topologies lematical, so we collapsed multistate character seen in analysis of the whole data set. 33 34 to a binary character, with cupules These observations indicate that fossils are ancestral. Addition of Callistophyton to the important in providing not only closer out- modern data set (Table V) required rein- groups but also new members of the ingroup. statement of the distinctions between mes- The remaining experiments address the role of arch and endarch primary xylem and between fossil ingroup taxa by adding selected fossils to pinnately compound and once-pinnate leaves the modern data set or subtracting them from and sporophylls (simplified to 00 and 10, the whole data set. The addition experiments respectively). In the whole data set we scored were done with both the conservative rooting coniferopsid leaves X01, one step from either and the seed fern rooting, corresponding to progymnosperms or seed ferns but two steps hypothetical situations where (1) only modern from once-pinnate groups (e.g., cycads), since groups and the added fossil ingroup taxa are there are no current hypotheses that propose known, and (2) both the added ingroup taxa that they are derived from once-pinnate ances- and primitive Carboniferous seed ferns are tors; however, with the present limited data known. set, there would be no grounds for biasing against any sort of ancestry, so we rescored Archaeopteris, Callistophyton, and the them X1 (for XX1). position of coniferopsids As expected, when Archaeopteris is removed from the whole data set, trees with coniferop- Based on the notion that it is the coniferop- sids forced into a sister-group relationship sid features of A rchaeopteris that most strongly with cycadopsids, as under the Beck hypothe- suggest a separate origin of coniferopsids and sis, became three steps less parsimonious (121 cycadopsids from progymnosperms (Beck, steps) than trees with coniferopsids nested in 1970), and the fact that it was recognition of seed plants (118 steps); in both cases, the coniferopsid characters in CaUistophyton that topology of other groups was unaffected (cf. led to the alternative hypothesis that conifer- Fig.l). This is actually an underestimate of the opsids were derived from platyspermic seed difference in parsimony between the two cur- ferns (Rothwell, 1982), we performed a set of rent hypotheses on the origin of coniferopsids, experiments subtracting Archaeopteris, Callis- since the artificial topology with coniferopsids tophyton, and Callistophyton plus other platy- directly below lyginopterids is not a perfect spermic seed fern groups from the whole data model of the Beck hypothesis in terms of set and adding Callistophyton to the extant character evolution. With this topology, the data set. No characters are lost with subtrac- Wagner algorithm assumes that the seed and tion of Callistophyton from the whole data set, axillary branching are homologous in conifer- but subtraction of Archaeopteris entails loss of opsids and cycadopsids, whereas under the characters 3 and 32; as explained above, Beck hypothesis they arise twice. This implies without reference to Archaeopteris these are that without Archaeopteris there would be not obvious as synapomorphies of coniferop- little basis for believing that coniferopsids and sids. When all platyspermic seed ferns are cycadopsids originated separately from pro- removed, the bifurcate vs. simple rachis char- gymnosperms. acter is lost, since there are no longer any When Callistophyton was removed from the 83

TABLE V

Experiments with addition of fossils to extant taxa. "Conservative" and "seed fern" designate sets of user-specified ancestral states based on different assumptions concerning outgroup relationships. "BGAS" represents "best guesses" of ancestral states provided by PHYLIP for selected most parsimonious trees obtained with the conservative rooting

Adding Calllstophyton:

i 2 3 4 5 6 Conservative -O-O-OX-O-IOO--XOOOXOXXO-OX-OXX--XX-XXXO-X--XXX--XXXXX-OO00010 Seed fern -O-O-O0-O-OO0--OXOOOOIXO-OO-OOO--XO-OIOO-O--OXX--XXOOO-OOOOOXO

Callistophyton -O-O-O0-O-XOX--OOOOOOIOX-OO-OOO--OO-IIOX-I--OOX--OIIOO-OOXXOXX

BGAS, tree 7c: -O-O-O0-O-IO0--XOOOOOIOO-OO-OOO--OO-IIOO-I--OOO--OIXO0-O000010

Adding Bennettitales (ancestral states specified as with extant taxa):

i 2 3 4 5 6 Bennettitales -O-O--O-O-OIX---OOOIOIIX--I--OO--IO-OIIO-X--IOX--OIOIO-IXXOOXX

BGAS, tree 8a: -O-O--O-O-IOO---O00XOIXO--X--OO--XO-XXOO-I--OOO--OIOXO-OOOO010 BGAS, tree 8d: -O-O--X-O-IOO---OOOOOXXO--X--XO--O0-1000-1--O00--OIOO0-OO00010

Adding Caytonia and Bennettitales:

i 2 3 4 5 6 Conservative -O-0-OX-OOIOO---OOOXOXXO--X-OXX--XX-XXXO-X--XXX--XXXXX-O000010 Seed fern -O-O-OO-O0000---XOOOOIXO--O-OOO--XO-OIOO-O--OXX--XXOOO-OOOOOXO

Caytonia -O-O-O0-1010X---XXXXXXXX--O-OOO--IO-XIOX-I--OOI--OIIOO-IXXXXXX Bennettitales -O-O-IO-O001X---OOOIOIIX--I-IOO--IO-OIIO-X--IOX--OIOIO-IXXOOXX

BGAS, tree 9a: -O-O-O0-O0100---O00XOIXO--0-OOO--XO-IXOO-I--OOX--OIO00-O000010 BGAS, tree 9c: -O-O-OX-OOIOO---OOOOOXXO--X-OXO--O0-1000-1--O00--OIO00-O000010 BGAS, tree 9f: -O-O-O0-O0100---O00XOIXO--O-OOO--XO-XXOO-I--OOX--OIO00-O000010 whole data set, we obtained one tree with groups appears to be needed for "correct" coniferopsids linked to Archaeopteris (Fig.7.a) placement of the coniferopsids, since in Fig.7.b that is just as parsimonious (121 steps) as any coniferopsids move below Medullosa. with coniferopsids nested in seed plants (in the In summary, these experiments indicate that latter, other groups may be arranged as in knowledge of Archaeopteris is more important either Fig.7.a or Fig.l). It might be surmised for the Beck hypothesis that coniferopsids and that the shift in favor of the Beck hypothesis is cycadopsids were independently derived from not greater than this because the presence of progymnosperms than knowledge of Callisto- other platyspermic seed ferns compensates in phyton is for the Rothwell hypothesis that part for removal of Callistophyton. To some coniferopsids originated from platyspermic extent, this appears to be true: when the seed ferns. This is partly because of the remaining platyspermic, saccate groups and existence of other platyspermic seed ferns, but Peltaspermum are also removed, it becomes even if such groups were unknown, both more parsimonious to link coniferopsids with hypotheses would still be viable. Presumably Archaeopteris than to nest them in seed plants the reason that these forms were not recog- (Fig.7.b), but only by one step (96 vs. 97). nized as evidence for a seed fern origin of Interestingly, however, knowledge of such coniferopsids is that they are all considerably 84

An Go Cn Cd Ar ML HL Md Cs P1 Cy GI Ct Ag Bn Pn Ep We Gn

a

HL Go Cn Cd Md Cy Bn Ag Pn Ep We On Ca Go Cn Cy AK Ep We On

Fig.7. Experiments with Archaeopteris and Callistophyton. (a) Most parsimonious tree obtained after subtraction of Callistophyton (121 steps). (b) Tree obtained after subtraction of all platyspermic seed ferns (97 steps); this is one step less parsimonious than the shortest tree found, which linked coniferopsids with Archaeopteris (taxa not shown arranged as in Fig.l). (c, d) Most parsimonious trees obtained after addition of Callistophyton to extant taxa (c and d differ in that cycads are placed as shown by the solid and dashed lines, respectively); with the conservative rooting only tree (c) was found (64 steps), but with the seed fern rooting both trees were found (65 steps). younger than the first coniferopsids, so they ferns and Medullosa were used to root extant seemed less relevant to the problem than a groups, even though these are presumably the Carboniferous fossil with similar features. most appropriate outgroups. The one tree Experiments with addition of Callistophyton found with the conservative rooting links to the modern data set show that knowledge of coniferopsids with cycads (Fig.7.c), but with this genus does not markedly affect relation- the seed fern rooting the topology seen in Fig.1 ships of extant groups, but it does improve also appears (Fig.7.d). Turning to character inferences on character evolution. In both evolution (cf. BGAS, Table V), even with the trees obtained (Fig.7.c, d), Callistophyton is conservative rooting it becomes most parsimo- basal, consistent with several most parsimoni- nious to assume that coniferopsids originated ous trees based on the whole data set (though from ancestors with pinnate leaves, as inferred not Fig.l). This arrangement is supported by from the whole data set. With the seed fern the fact that all extant groups are endarch, rooting, even though radiospermic was spec- whereas Callistophyton is mesarch or exarch. ified as ancestral, it is more parsimonious to Addition of Callistophyton corrects an anomaly assume that platyspermic seeds arose before noted above, namely that the arrangement of the immediate common ancestor of extant extant groups seen in Fig.1 and many other groups and Callistophyton and secondarily trees derived from the whole data set (i.e., reverted to radiospermic in cycads and Gnetum Fig.4.a) was not seen when lyginopterid seed (as in Fig.l) than to assume that platyspermic 85 originated several times. However, it is still Bennettitales play a key role in strengthening most parsimonious to assume that saccate the angiosperm-gnetalian connection. Specifi- pollen arose independently in Callistophyton cally, more features of Gnetales are accounted and conifers. for if they are considered highly modified For symmetry with our experiments with the relatives of Bennettitales rather than conifer- seed fern and progymnosperm rootings of the opsids (e.g., syndetocheilic stomata, whorled modern data set, it might be interesting to microsporophylls, micropylar tube); and Ben- observe effects of subtracting Carboniferous nettitales help to link Gnetales with angio- seed ferns from the whole data set but leaving sperms, since they have several angiosperm- in progymnosperms. However, we did not carry like traits that are lacking in Gnetales, out such an exercise, since we found that the apparently because of secondary loss (e.g., problems of character analysis soon become scalariform pitting, cupulate ovules). Like- intractable, especially in the multistate char- wise, as pointed out by Crane (1985), Mesozoic acters (e.g., cupules, radio- vs. platyspermic seed ferns (Caytonia, corystosperms, and/or seeds, stelar types), for which ideas on the glossopterids) appear to link the resulting ancestral condition and the interrelationships "anthophyte" clade with other seed plants, of derived states depend heavily on knowledge thus reconciling ideas of a relationship be- of early seed ferns. This is itself a measure of tween angiosperms and Mesozoic seed ferns their importance in understanding the evolu- (Gaussen, 1946; Stebbins, 1974; Doyle, 1978) tion of extant groups. and among angiosperms, Bennettitales, and Gnetales. For example, Caytonia, corysto- Bennettitales, Caytonia, and relationships of sperms, and anthophytes have a reduced mega- angiosperms and Gnetales spore wall; Caytonia, glossopterids, angio- sperms, most Bennettitales, and Pentoxylon Another case where fossils appear to provide have a thick nucellar cuticle; and Bennetti- important links among extant taxa concerns tales and Pentoxylon have an orthotropous the relationship of angiosperms and Gnetales cupule that might correspond to the anatro- to each other and to other groups. Although pous cupule of Caytonia and/or corystosperms our analysis of extant taxa links Gnetales with and the outer integument of angiosperms. angiosperms, trees in which Gnetales are To explore the effects of Bennettitales on nested within coniferopsids are only one step relationships of angiosperms and Gnetales, we longer (Fig.4.e). Without parsimony analysis, performed two sets of experiments: adding angiosperm-gnetalian relationships are easy Bennettitales to the modern data set and to dismiss (e.g., Doyle, 1978) because of the subtracting Bennettitales and Pentoxylon many coniferopsid features of Gnetales (wood (which have many of the same features and anatomy, compound strobili), the great mor- hence might be expected to produce similar phological gap between angiosperm and gne- results independently) from the whole data set. talian reproductive structures, and the fact No new characters appear with addition of that many of the most striking similarities Bennettitales. Both anatropous and orthotro- between angiosperms and Gnetales (e.g., the pous cupulate ovules now occur, but we dicot-like leaves of Gnetum, possibly vessels) combined them and left the ancestral state appear to be primitively lacking in some unspecified, since their relations to each other members of one or the other group. Other and to other states would be obscure. Without angiosperm gnetalian similarities remain, but knowledge of the leaflet-like anatropous cu- they tend to be rather cryptic (e.g., the tunica pules of Caytonia and corystosperms or the layer in the apical meristem, M/iule reaction, evidence that these taxa are nested among siphonogamy, granular exine structure). Our groups with non-cupulate, laminar ovules, analysis of the whole data set suggests that there would be less reason to suspect that there 86

are two sorts of cupules in seed plants: the Gnetales nested in coniferopsids are only one radial lyginopterid type that appears to be step less parsimonious than trees where they basic in seed plants, and an unrelated type are linked with angiosperms (115 vs. 114 steps), secondarily derived from an enrolled ovule- rather than four steps less parsimonious. bearing leaflet. Similarly, no characters are Arrangements of other groups are not affected lost when Bennettitales and Pentoxylon are (cf. Fig.D; thus when Gnetales are moved into subtracted. coniferopsids, angiosperms remain linked with As expected, these experiments show that Caytonia, as previously assumed by one of us knowledge of Bennettitales does strengthen (Doyle, 1978) after overlooking or rejecting as relationships between angiosperms and Gne- convergences the angiosperm and gnetalian tales. When Bennettitales are added to the features of Bennettitales. modern data set (Fig.8.a-e), the three antho- Knowledge of Bennettitales also has some phyte groups are always associated. With the effect on inferred character evolution in antho- conservative rooting all possible arrangements phytes and seed plants as a whole (BGAS, of the three groups are found (Fig.8.a-e), but Table V). For example, two of the five shortest with the seed fern rooting (i.e., better outgroup trees obtained with the conservative rooting information) Bennettitales are linked with (Fig.8.a, b) imply that pinnate leaves are Gnetales (Fig.8.a), as inferred from the whole ancestral in seed plants and that the linear data set. With Bennettitales present, moving leaves and simple microsporophylls of Gne- Gnetales into the coniferopsids (Fig.8.f) adds tales and coniferopsids are independently re- two or three steps (depending on the rooting), duced, as inferred from the whole data set. In whereas with the modern data set one tree with contrast, the course of evolution in these Gnetales nested in coniferopsids (Fig.4.e) was characters is equivocal in all trees of extant only one step longer than the shortest trees. taxa alone based on the same rooting. If it is When Bennettitales and Pentoxylon are sub- assumed that vessels are basic in angiosperms, tracted from the whole data set, trees with analysis of modern groups alone would imply

Go Cn Cy Ag Bn Ep We Gn Go Cn Cy Bn Ag Ep We Gn

b,c

Go Cn C¥ Ag Bn Ep We Gn Bn Ag Cy Cn Go Ep We On IIII

f

Fig.8. Experiments with addition of Bennettitales to extant taxa (b and d differ from c and e in that the groups indicated are placed as shown by the solid and dashed lines, respectively). With the conservative rooting, five most parsimonious trees were found (a e, 63 steps), while with the seed fern rooting only tree (a) was found (62 steps). (f) Tree with Gnetales forced into coniferopsids (66 steps with the conservative rooting, 64 steps with the seed fern rooting). 87 that vessels are homologous in angiosperms cupule character and recoded Bennettitales, and Gnetales, but in trees with Bennettitales Pentoxylon, and angiosperms as having the added (except Fig.8.b) it becomes equally lyginopterid state, for the same reasons dis- parsimonious to assume that they originated cussed in connection with experiments where independently. However, four of the five trees we added Bennettitales. (Fig.8.b e) imply that the stalked or sessile With both rootings, when Caytonia is added ovules of coniferopsids, Bennettitales, and to the modern groups, it is always linked Gnetales are primitive, whereas our analysis of directly with angiosperms, rather than being the whole data set indicates that they were the sister group of angiosperms plus Gnetales, derived by reduction. and it is equally parsimonious to associate To probe the importance of Caytonia and Gnetales with angiosperms plus Caytonia possibly related forms in elucidating the posi- (Fig.9.b, c) or with coniferopsids (Fig.9.a, d). tion and character evolution of the antho- For comparison, trees derived from the whole phytes, we added Caytonia to the modern data data set with Caytonia linked with angio- set and subtracted Caytonia and Caytonia plus sperms and with Gnetales nested in coniferop- glossopterids and corystosperms from the sids are one and four steps less parsimonious whole data set. In the addition experiments we than the best arrangements, respectively. reinstated the distinction between pinnately These results again confirm the importance of compound and once-pinnate leaves and micro- Bennettitales as a link between angiosperms sporophylls (since leaflets of Caytonia have and Gnetales and the idea that if Bennettitales midribs), and between reticulate venation and were unknown (or overlooked), the clearest presence of several vein orders. In the subtrac- relationships of angiosperms would, be with tion experiments, we eliminated flat guard Mesozoic seed ferns. cells when we subtracted Caytonia, plus one As occurred when Bennettitales were added ovule per cupule when we subtracted all three to modern groups, when Caytonia is added it groups; with these subtractions, these features becomes most parsimonious to assume that become angiosperm autapomorphies. When all pinnately compound leaves and sporophylls three groups were subtracted, we simplified the were basic in seed plants even in some of the

Ct Ag Cy Cn Go Ep We Gn Go Cn Cy Ct Ag Ep We Gn Cy Ct Ag Cn Go Ep We Gn

a

Ct Go Cn Cy Ag Bn Ep We Gn Go Cn Cy Ct Ag Bn Ep We Gn

f,g

Fig.9. Experiments with addition of Caytonia and Bennettitales to extant taxa (b and f differ from c and g in that the groups indicated are placed as shown by the solid and dashed lines, respectively). (a-d) Trees obtained after addition of Caytonia alone; with the conservative rooting trees (a c) were found (66 steps), while with the seed fern rooting trees (b) and (d) were found (65 steps). (e-g) Trees obtained after addition of both Caytonia and Bennettitales; with the conservative rooting trees (e) and (f) were found (71 steps), while with the seed fern rooting all three trees were found (71 steps). 88 trees obtained with the conservative rooting, synapomorphies such as scalariform pitting, namely those where Gnetales are nested in the M/iule reaction, and siphonogamy have no coniferopsids (Fig.9.a; BGAS, Table V). How- effect either way because the condition in ever, both the basic state in seed plants and the Caytonia and/or Bennettitales is unknown. In direction of evolution within the anthophytes the absence of Bennettitales, Gnetales are are still equivocal in trees where Gnetales are unable to dissociate angiosperms and Cayto- linked with angiosperms and Caytonia (Fig.9.b, nia, apparently because several basic antho- c; BGAS, Table V). In the tree with Gnetales phyte features seen in angiosperms and Ben- linked with angiosperms and Caytonia ob- nettitales (syndetocheilic stomata, cupules) tained with the seed fern rooting (Fig.9.b), it is are lacking in some or all Gnetales (probably most parsimonious to assume that gnetalian due to secondary loss). flowers are reduced rather than primitively Even with the conservative rooting, addition simple. However, not even Fig.9.b gives a of both Bennettitales and Caytonia finally wholly satisfactory picture of character evolu- allows many of the same conclusions regarding tion in anthophytes: the angiosperm outer character evolution that were drawn from integument can be homologized with the Cayto- analysis of the whole data set (BGAS, Table V). nia cupule, but it is best interpreted as a Thus it is most parsimonious to assume that synapomorphy of Caytonia and angiosperms the first seed plants had seed fern-like anatomy that never existed in the ancestry of Gnetales, and pinnate leaves and sporophylls, and that rather than a basic feature of the whole Gnetales originated by reduction of Bennetti- Caytonia-anthophyte clade that was lost in tales-like ancestors. Most trees (except Fig.9.g) Gnetales. Similarly, unless it is assumed that imply that flowers are homologous in antho- Caytonia once had flowers and then lost them, phytes and that the Caytonia-anthophyte cu- all these trees imply that flower-like reproduc- pule was lost in Gnetales. However, the origin tive structures originated independently in of the cupule remains obscure; without infor- angiosperms and Gnetales, whereas they are mation on more primitive seed ferns, it is best interpreted as homologous in the shortest equivocal whether it is homologous with the trees derived from the whole data set. original lyginopterid cupule or a secondary This interpretation is confirmed by experi- innovation (as inferred from the whole data ments where both Bennettitales and Caytonia set). are added to the modern taxa (Fig.9.e-g). With The subtraction experiments suggest that if both rootings, trees with Gnetales associated Caytonia and potentially related forms were with coniferopsids are again eliminated, but a unknown there would be little support for curious new topology appears, with Caytonia derivation of anthophytes from seed ferns with basal in seed plants (Fig.9.e). One of the platyspermic seeds and saccate pollen. When remaining trees (Fig.9.g) still links angio- Caytonia alone is removed, two most parsimo- sperms directly with Caytonia, but the other nious positions for the anthophytes are found, (Fig.9.f) interpolates angiosperms between linked with corystosperms or with glossopter- Caytonia and Bennettitales, which is the result ids (119 steps, with various arrangements of obtained from the whole data set (Fig.l). other groups). This is not surprising consider- Caytonia and angiosperms have two potential ing that glossopterids are the next-closest synapomorphies that tend to favor a close relatives of anthophytes in all 123-step trees relationship (flat guard cells, reticulate vena- derived from the whole data set (e.g., Fig.l), tion), but when Bennettitales are present these while corystosperms are their closest relatives are balanced by anthophyte features seen in in some 124-step trees. When Caytonia, corys- Bennettitales but not Caytonia (once-pinnate tosperms, and glossopterids are removed, the leaves, syndetocheilic stomata, granular non- best position for anthophytes is linked with saccate pollen). Other potential anthophyte cycads and Peltaspermum (Fig.10.a, b). This 89 topology is consistent with that derived from tales play in linking angiosperms with Gne- the whole data set, but although it implies that tales. When angiosperms are subtracted from anthophytes are basically platyspermic, it the whole data set, the shortest tree found (113 suggests that they never had ancestors with steps) is of the neo-englerian type, with Ben- saccate pollen. In fact, with removal of so nettitales, Pentoxylon, and Gnetales nested in many saccate groups, it becomes most parsimo- coniferopsids (Fig.10.c). This arrangement sug- nious (by one step) to rearrange coniferopsid gests that the flowers of Bennettitales are taxa so that ginkgos are basal and to assume secondarily elaborated from coniferopsid axil- that saccate pollen originated independently lary fertile shoots via gnetalian intermediates. in Callistophyton and within coniferopsids, With the whole data set, such trees are two rather than being basic for the whole platy- steps longer than the shortest trees (and highly spermic clade. Another conflict with trees based implausible on developmental grounds) be- on the whole data set is that Gnetales are the cause of the numerous secondary elaborations sister group of angiosperms, Bennettitales, and and de novo origins of seed fern-like features Pentoxylon; with removal of outgroups with that must be assumed in angiosperms and/or potentially homologous cupules, it is most Bennettitales (pinnate leaves and microsporo- parsimonious to assume that the cupule (outer phylls, multiseriate rays, scalariform primary integument) of Bennettitales, Pentoxylon, and xylem, cupules, microsynangia). However, angiosperms is an innovation of those groups since Bennettitales and Pentoxylon are more that never existed in the ancestry of Gnetales. coniferopsid-like than angiosperms in having Interestingly, angiosperms seem to play the stalked or sessile ovules, there are fewer steps same role in linking Bennettitales and Gne- to reverse when angiosperms are ignored. tales with Mesozoic seed ferns that Bennetti- Basically, the presence of pinnately organized

Md Ca Cn Cd Go PI Cy Bn Ag Pn Ep We Gn

~ a,b

Md Cy PI Ca Cs Ct G1 Cd Cn Go Bn Pn Ep We Gn

Fig.10. Experiments with subtraction of Mesozoic seed ferns and angiosperms (groups not shown arranged as in Fig.l). (a, b) Most parsimonious trees obtained after subtraction of Caytonia, glossopterids, and corystosperms (104 steps; a and b differ in that eyeads are placed as shown by the solid and dashed lines, respectively). (c) Most parsimonious tree obtained after subtraction of angiosperms (113 steps). 90

carpels and anatropous bitegmic ovules in help identify cases where fossil evidence is angiosperms shifts the balance to the scheme more or less likely to be important in the derived from the whole data set. This illus- future. These questions were also addressed by trates graphically the point that although Fortey and Jeffries (1982), who concluded that fossils may be useful or even necessary in fossils are most likely to be significant when understanding the origin and relationships of there has been homoplasy in characters of high living groups, living groups have a similar role "burden" that characterize major groups (i.e., in understanding the relationships of fossils characters on whose presence the existence of (cf. Hennig, 1966). other characters depends). Since complete loss of such characters is unlikely, Fortey and General implications Jeffries suggest that fossils will seldom change topological relationships of extant groups, but In general, our results lead us to a more when they do the effects may be profound positive view of the value of fossils in phylo- because of the rank of the taxa involved. While geny reconstruction than that of Patterson we find this concept intriguing and worth (1981) -- in many respects, a return to the further exploration, we would propose a some- generalizations of Hennig (1966) summarized what different set of generalizations, based on in the introduction. We agree that the primary our present results and the analysis of Maddi- way that fossils provide phylogenetic informa- son et al. (1984), who have considered the tion is just as additional extant groups do, general effects of adding information on new namely by providing new character combina- groups to cladistic analysis. We expect that tions that may necessitate changes in clado- fossils are most likely to lead to changes in gram topology. However, Patterson's empirical topology when (1) the ingroup is separated claim that this rarely occurs in practice seems from the outgroup by large gaps, i.e., numbers overstated. The study of Gauthier, Kluge and of steps (as extant seed plants are separated Roe (pers. commun., 1985) on amniotes shows from extant "pteridophytes"); (2) there are that fossils can dramatically alter conclusions major gaps in the ingroup (as between angio- on relationships. Our results do not contradict sperms, Gnetales, and other seed plants); and Patterson's conclusions so directly, since cla- (3) there is evidence of homoplasy, so that dogram topologies derived from extant taxa alternative placements of groups (e.g., of Gne- alone are either similar or identical to topolo- tales with angiosperms and coniferopsids) are gies derived from our whole data set. However, almost equally parsimonious. Topological our study focuses attention on a more subtle changes are especially likely when more than but equally important point, which we believe one of these conditions hold. Conversely, when has received less consideration than it de- gaps between extant groups are small (as in serves: even when fossils do not change clado- many angiosperm families), or there are few gram topologies, they may greatly clarify and character conflicts, fossils are less likely to in some cases reverse ideas on the nature and have major effects. sequence of character changes involved in the In the present study, despite the large gaps origin of major groups. This may provide between extant seed plants and other groups otherwise quite inaccessible insights into evo- and between angiosperms and Gnetales, fossil lutionary processes involved, especially when data did not overturn relationships inferred combined with other sorts of information from extant groups alone, but they did affect provided by the geologic record. the stability of relationships. Thus knowledge Closer examination of our results from a of Bennettitales greatly strengthens the link theoretical viewpoint may allow more detailed between angiosperms and Gnetales, which is generalizations on ways that fossils can otherwise only slightly more parsimonious change phylogenetic conclusions, and this may than a link between Gnetales and coniferop- 91 sids. With a slightly different choice of charac- are still separated from Mesozoic seed ferns by ters, the relative parsimony of the two arrange- several steps, and both angiosperms and Gne- ments could easily have been reversed. In this tales are isolated from other anthophytes by context, it is worth noting that our approach numerous pomorphies (at least 12 in each case: may have been overly optimistic in modeling Doyle and Donoghue, 1986). Thus it should not how well a hypothetical student of extant be surprising if future discoveries of fossils groups would analyze characters. We derived with unexpected character combinations alter our extant data set from one based on both inferred relationships. For example, discovery living and fossil groups, where character of fossils below the anthophyte node might interpretations were strongly influenced by modify connections between anthophytes and knowledge of fossils, and although we tried to other groups and relationships among antho- reformulate all characters as a hypothetical phytes. Likewise, recognition of closer fossil student of extant groups might do, a real relatives of the angiosperms might force reeva- investigator would probably have interpreted luation of basic angiosperm states and favor some characters in unexpected ways. Perhaps a direct association of angiosperms with Bennet- more realistic model is provided by comparison titales or Gnetales (cf. Crane, 1985; Doyle and of the cladistic analyses of seed plants pre- Donoghue, 1986). sented by Hill and Crane (1982), who considered Our results provide many illustrations of the extant groups only, and by Crane (1985), who point that even when fossils have no effect on included fossils as well. Hill and Crane's cladogram topology, ideas on the evolution of favored cladogram differs from all those dis- characters based on extant groups alone may cussed so far in treating angiosperms as the be nebulous or even incorrect, and addition of sister group of conifers plus Gnetales, and appropriate fossils may eliminate many alter- cycads plus Ginkgo as the sister group of the natives. Thus with extant groups only, the resulting clade. This result was not supported direction of leaf evolution in seed plants is by a numerical parsimony analysis of Hill and entirely ambiguous. However, when informa- Crane's data (Doyle and Donoghue, 1986), but tion from closer fossil outgroups is added the tree that we obtained from their data still (lyginopterid seed ferns and Medullosa, or differed from trees derived from our whole data CaUistophyton), it becomes most parsimonious set (angiosperms were the sister group of either to assume that the first seed plants had Gnetales or Gnetum, and angiosperms and pinnately compound leaves, and that there was Gnetales were nested within coniferopsids, a a double trend from a "cycadopsid" to a neo-englerian arrangement). In contrast, as "coniferopsid" habit and linear-dichotomous discussed above, Crane's (1985) scheme is much leaves in coniferopsids and Gnetales. The same closer to ours in most respects, including not is true when appropriate ingroup taxa are only the relationship of angiosperms, Bennetti- added (Bennettitales, Caytonia, especially tales, Gnetales, and Mesozoic seed ferns, but both). Without fossils, the direction of floral also the arrangement of extant groups. Another evolution in the anthophytes is also ambigu- more realistic example of results obtained from ous, but when seed ferns or Caytonia plus consideration of extant groups alone is pro- Bennettitales are added, the balance shifts to vided by the scheme of Bremer (1985), who the interpretation that gnetalian reproductive linked seed plants with ferns on pinnate leaves structures are reduced and aggregated relative and trichomes, features that fossil evidence to flowers of angiosperms and Bennettitales indicates arose independently. (Arber and Parkin, 1907, 1908), rather than Looking ahead, major gaps remain at several primitively simple (Von Wettstein, 1907). Simi- places in the phylogeny of seed plants even larly, without Mesozoic seed ferns such as when fossils are included, and homoplasy is Caytonia there is little indication of where clearly widespread. In particular, anthophytes structures such as the carpel or the outer 92 integument came from. Without fossil data, pinnately compound leaves, axillary branch- saccate pollen would appear to be a conifer ing, the seed, and the eustele, modernization of autapomorphy, rather than a basic feature of the ovule and pollen-capture system, and origin the ancestors of all modern seed plants that of saccate pollen, basic features of the crown was lost several times. Likewise, without group to which all extant seed plants belong. plesiomorphic vesselless groups of antho- Similarly, Mesozoic seed ferns and Bennetti- phytes (Bennettitales, Pentoxylon) vessels tales fill in steps in the origin of angiosperms, could be considered homologous in angio- such as appearance of scalariform secondary sperms and Gnetales, but with Bennettitales xylem pitting, derivation of the outer integu- and Pentoxylon included in the analysis it is ment from a reflexed leaflet and the carpel from clear that they arose independently. a sporophyll rachis, loss of air sacs in the pollen, All the points made so far apply equally to and a shift to granular exine structure. Bennet- addition of any newly discovered taxa, which titales show steps leading to Gnetales, such as leads us to ask what if anything is "special" reduction of the megasporophylls, whorling of about fossils. Obviously, stratigraphic distribu- the microsporophylls, and origin of a micropy- tion is such a special element, but we would lar tube. Judging from extant data alone, the argue that fossils provide unique insights even gaps between crown groups could conceivably if stratigraphy is ignored, as it was in our be due solely to anagenesis in single stem analysis. This is not because of any intrinsic species, but since stem group taxa often consist properties of fossils but rather because of the of numerous coexisting species (e.g., lyginopter- way evolution seems often to proceed. As was ids, medullosans), only one of which can be recognized by Hennig (1969), Jeffries (1979), directly ancestral to the crown group, it is more and Patterson (1981) in elaborating the con- likely that the origin of gaps involved extensive cept of "stem groups" and "crown groups", cladogenesis followed by extinction, whether most of the extant biota consists of large clades due to displacement of older "experimental (e.g., mammals, birds, angiosperms) that are models" by the crown group as a result of a separated from their closest extant relatives by superior combination of features or to stochas- major gaps (large numbers of apomorphies). tic effects. Plesiomorphic fossil relatives of these crown Given the present state of exploration of the groups (members of their stem groups, which earth's biota, it is highly unlikely that the gaps are by definition paraphyletic) often show that between crown groups will be filled by discov- apomorphies were acquired in a stepwise eries of rare extant taxa. Instead, this will be fashion, culminating in the complex of ad- accomplished primarily by discovery of new vances seen in the crown group. Patterson fossils, or since Meyen (1984) may be correct in (1977, 1981) gives an excellent example of this believing that most major land plant groups phenomenon in the origin of teleost fishes. This are already known at least as isolated organs, pattern might be suspected without fossils, but by reconstruction of new groups from their the actual sequence of origin of apomorphies component parts (as illustrated by Archaeop- would be entirely speculative. Hennig (1966) teris and Callistophyton). Here it is worth argued that such information may alter ideas noting that although plants are notorious for on relationships of extant taxa, and this being preserved as isolated organs, which are expectation is confirmed by the work of difficult to fit into phylogenetic analysis (cf. Gauthier et al. (pers. comm., 1985) on amniotes. Hennig, 1966), plant cells have more or less Relationships among seed plant taxa seem to be resistant walls, so that more organ systems are less affected, but the Devonian progymno- potentially fossilized than in animals (especi- sperms and Carboniferous seed ferns making up ally in the Paleozoic, thanks to the unusual the stem group document in great detail the anatomical preservation seen in coal balls). In sequential acquisition of secondary growth, fact, at least some members of many major 93 fossil groups have been reconstructed almost in Gnetales may also be linked to aridity, since as completely as modern plants. This suggests Gnetales are centered in presumed arid regions that the possibilities of gaining phylogenetic in the Early Cretaceous (Brenner, 1976; Doyle information from fossils are as good or better et al., 1982). The somewhat similar distribu- in plants than in animals, at least at higher tions of Bennettitales and early angiosperms taxonomic levels. suggest that aridity may also have been The greater resolution of character evolu- involved in the strong progenetic tendency tion provided by fossils is relevant not only to seen in anthophytes as a whole, and in the relationships but also to theories of evolution- independent origin of vessels in angiosperms ary mechanisms and adaptive scenarios in- and Gnetales (Doyle and Donoghue, 1986). volved in the origin of major groups, which In other groups, a shift from manoxylic to may be virtually unconstrained when informa- "coniferopsid" pycnoxylic stem anatomy seems tion on the order of acquisition of apomorphies to be correlated with occupation of temperate is lacking. For example, given that angio- areas (glossopterids, corystosperms, Pentoxy- sperms are related to Bennettitales and Gne- lon, possibly ginkgos). Generally similar con- tales, the fact that the latter groups show clusions were reached by Meyen (1984), work- evidence of insect pollination (aggregation of ing in a non-cladistic framework. In contrast, sporophylls into flowers, often bisexual) and modern coniferopsid distributions alone might progenetic acceleration of the reproductive suggest an origin in the mesic temperate zones, cycle (small seed size, reduced megaspore wall: while modern angiosperm distributions have cf. Crane, 1985) indicates that these features often been cited as evidence for an origin in the were established well before carpel closure. wet tropics. This in turn requires revision of suggestions In summary, our analysis indicates that that the origi n of angiosperms was directly tied fossil data can be of great value in altering or to the origin of insect pollination and/or strengthening cladistically inferred relation- pressures for rapid reproduction (e.g., Steb- ships among extant groups, and in providing bins, 1974; Hickey and Doyle, 1977; Doyle, unique evidence on the sequence of events and 1978), although it may of course have been ecological factors involved in their origin. related to changes in these factors. Since these effects are strongest in dealing Fossils provide still more evidence on sce- with the isolated major groups that dominate narios when other information obtainable from the modern biota, our observations amply the fossil record is added: data on minimum justify the high regard in which fossils have ages of groups, their geographic distributions, been traditionally held by students of macro- and associated paleoenvironments, including evolution. both physical and biotic components (e.g., presence or absence of potential herbivores or Acknowledgements pollinators). Thus several lines of evidence support the notion that the origin of coniferop- We wish to thank Jacques Gauthier, Arnold sids was related to aridity. The plesiomorphic Kluge, and Tim Roe for discussion of results of seed fern relatives of coniferopsids were cen- their unpublished study on amniote phylogeny, tered in wet, tropical, lowland areas, but there and Wm. 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