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Significance of palynology for phylogeny ofAnnonaceae: Experiments with removal of pollen characters

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Significance of palynology for phylogeny of : experiments with removal of pollen characters*

JAMES A. DOYLE and ANNICKLE THOMAS

Received July 12, 1996; in revised version December 11, 1996

Key words: , Annonaceae. - Palynology, phylogeny, cladistics. Abstract: Phylogenetic analyses based on morphology place Anaxagorea and other taxa with granular monosulcate pollen, as in other Magnoliales, at the base of Annonaceae. Taxa with columellar tetrads, granular tetrads, and inaperturate monads form a derived clade. To test the systematic importance of palynology, we analyzed the data set with pollen characters removed. The result was lowei" resolution and a different rooting of the family, between Uvariopsis and other groups with columellar tetrads. Anaxagorea and other monosulcates are higher in the tree, implying that granular monosulcate pollen, laminar stamens, and irregular endosperm ruminations are reversals. This rooting is highly unparsimonious when pollen characters are included, and only weakly supported over the Anaxagorea rooting when pollen is excluded. Together with preliminary molecular analyses, these experiments confirm the special value of palynology in systematics of Annonaceae.

Palynology has played a major role in attempts to clarify higher-level relation- ships in Annonaceae, the largest family of primitive angiosperms ("Magnolii- dae"). Classification of Annonaceae has long been unstable, with little agree- ment on basal relationships and few generally accepted major subgroups. For example, FRIES (1959) proposed many groups of genera and sorted most of them (except the aberrant neotropical genus Tetrameranthus and the parasyn- carpous genera Monodora and Isolona) into the tribes Uvarieae and Unoneae, based on imbricate vs. valvate petals, but he acknowledged exceptions to this distinction in both tribes. In the first comprehensive palynological study, WALKER (1971), using light and scanning electron microscopy, divided the family into three informal subfamilies and seven tribes. He argued that the most primitive group is the Malmea tribe, with single monosulcate pollen grains and an exine with columellar infratectal structure, and he recognized independent trends to tetrad

* This paper is dedicated to emer. Univ.-Prof. Dr FR1EDRICH EHRENDORFERon the occasion of his 70th birthday 134 J.A. DOYLE 8,: A. LE THOMAS: pollen in his Fusaea and Annona subfamilies. LE THOMAS & LUGARDON (1974, 1976; see also LE THOMAS 1980/1981), using transmission electron microscopy (TEM), found that monosulcate and other taxa that WAL~R (1971) called "microtectate" have granular infratectal structure, which they considered primitive based on its occurrence in other and gymnosperms. Important TEM data have also been provided by HESSE & al. (1985), WAnA (1985, 1988), and WAHA & MO~WETZ (1988). In DOYLE & LE THOMAS (1994), we undertook to test these ideas by means of a cladistic analysis of Annonaceae, based on 11 pollen characters and 68 other micro- and macromorphological characters, and using other Magnoliales as out- groups. In selecting taxa of Annonaceae, we attempted to obtain a global sampling of major groups, while limiting our attention to taxa in which exine structure is known. This led to a certain bias in favor of African taxa, which are best studied with TEM. In general, this analysis confirmed the views of WALKER (1971) and LE THOMAS (1980/1981) concerning pollen evolution. The Asian-American genus Anaxagorea, with granular monosulcate pollen, was basal (i.e. the sister group of all other Annonaceae), followed by four small genera with similar pollen, the ambavioids. Tetrads originated in two main lines: the xylopioids (part of WALKZR'S Fusaea subfamily), in which exine structure remains granular, and the a n n o n o i d s (WALKER'SAnnona subfamily), where it becomes columellar. The xylopioids were linked with the u v a r i o i d s, which have granular inaperturate pollen; the annonoids, with Artabotrys, which has columellar single grains with a reduced aperture. The palynologically diverse genus Polyalthia was polyphyletic, with members scattered among other taxa with similar pollen. Other important trends were a shift to columellar monosulcate pollen in the m almeoids (WALKER'S Malmea tribe) and multiple origins of sulculate pollen. Many of the groups recognized by VAN SETTEN & KOEK-NOORMAN(1992) based on fruit and seed characters are monophyletic or paraphyletic on these trees. Subsequent studies extended or modified aspects of this analysis. LE THOMAS & al. (1994) described the ultrastructure of Fusaea and Duguetia and discussed the relationships of these and other pseudosyncarpous genera, which formed a clade within the uvarioids. LE THOMAS & DOYLE (1996) compared the cladistic results with geographic distributions in order to reconstruct the geographic history of the family. In a discussion of the phylogenetic position of African Annonaceae, DOYLE & LE THOMAS (1995) added two more African genera, Afroguatteria and Mkilua, and rescored some characters based on more recent information. The most important change (a result of a shift of the echinate Monanthotaxis group from the xylopioid line into the uvarioids, linked with Uvaria via Afroguatteria) was that the annonoid and xylopioid tetrad groups and the inaperturate uvarioids all formed a single clade. Since the Annona group and the other "annonoids" with columellar tetrads formed a basal paraphylefic group relative to the xylopioids and uvarioids, we extended the term annonoids to this whole clade. Most recently, molecular analyses by VAN ZUILEN(1996) and P. BYGRAVEand M. W. CHASE (pers. comm.) have provided an independent source of evidence on phylogeny of Annonaceae. These studies differ in the genes studied, the taxa included, and the precise arrangement of groups, but they both confirm the basal Palynology and phylogeny of Annonaceae 135 position of Anaxagorea and the conclusion that Artabotrys and the groups with tetrads and inaperturate single grains form a clade. The fact that pollen characters often define major clades and are generally consistent with other characters (as discussed in detail by DOYLE & LE THOMAS 1994, 1995, and LE THOMAS & al. 1994) indirectly supports the view that palynology is of major importance in systematics of Annonaceae, as palynologists have argued for many other groups (e.g., WALI~R & DOYLE 1975, NowIc~ & SKVARLA 1979). The present study is intended to provide a more direct empirical test of the relative systematic value of pollen and other characters, by comparing trees derived from the whole data set with trees based on a data set from which pollen characters have been removed. We undertook this exercise in response to a question by A. A. ANDE~ERG (pers. comm.), who asked if we would have obtained the same results if we had not included pollen characters. For these experiments, we have used a new version of our data set, presented in DOYLE & LE THOMAS (1996), which incorporates a considerable number of changes in delimitation of taxa and character analysis, based especially on VAN HEUSDEN'S (1992) monograph on floral morphology and recent work on relationships within groups. Detailed justification and documentation of character definitions and scoring and implications of the results for evolution of the entire spectrum of characters may be found in DOYLE & LE THOMAS (1996).

Data and analyses Our "complete" data set (Appendix; DOYLE & LE THOMAS 1996) includes 42 taxa of Annonaceae and 79 characters, 51 of which are binary and 28 multistate, requiring a minimum of 113 character state changes for the whole data set. Most terminal taxa are genera, others groups of clearly related genera, or, in the variable genus Polyalthia, four palynologically homogeneous infrageneric groups. In some cases, we dealt with variation within groups by scoring them as uncertain (e.g., 0/1); in other cases, where there is evidence on internal relationships, we assumed that one or another state is basic. Multistate characters are unordered, except in 10 cases where there is a clear basis for ordering (e.g., pollen size, chromosome number). In continuous characters, we defined states in terms of average rather than extreme numbers and attempted to set limits between states at natural gaps in the distribution of measures. When two or more potential states often co-occur within taxa (e.g., leaf epidermal crystals), we tried to combine related states in a way that reduces the number of uncertain (polymorphic) scorings. Among the most important modifications, we replaced the character of (maximum) number of monocarps in the fruit stage (documented by VAN SETTEN ~ KOEK-NOORMAN 1992) with (average) carpel number in the flower; these two characters are correlated, but the latter seems less likely to be affected by phenotypic plasticity. We redefined the "floral type" character (spreading vs. connivent petals, a simplification of MORAWETZ 1988) to reflect what appear to be more natural categories in petal form and orientation. Since studies by CHATROU(1997) indicate that Malmea as previously delimited is heterogeneous, we rescored this genus to correspond to CHATROU'Sconcept of Malmea s. str. Because the unity of the three genera included in our Ancana group (Ancana, Fitzalania, Haplostichanthus) was questioned by VAN HEUSDEN (1992), we replaced this group with Ancana alone. A cladistic analysis of the Cymbopetalum group (Bocageeae) by JOHNSON& MURRAY (1995) allowed us to decide which states are basic in three characters that we previously scored as uncertain. 136 J.A. DOYLE 8¢ A. LE THOMAS:

The "non-palynological" data set consists of the "complete" data set with the 11 palynological characters (42-52) removed. Data sets were analyzed with PAUP (SwoFFORD 1990, version 3.1.1). In searching for most parsimonious trees, we performed varying numbers of replicate analyses (usually 20-30) with stepwise random addition of taxa and TBR branch swapping. This increases the likelihood of finding different "islands" of most parsimonious trees (MADDISON1991), within which other equally parsimonious trees can be found by swapping one taxon at a time, but between which more complex (and therefore inaccessible) rearrangements are required. We used the constraints option in PAUP (with constraint trees in which groups under consideration are forced together as polychotomies) to investigate the relative parsimony of alternative hypotheses. We used MacClade (MADDISON8,: MADDISON 1992) to analyze character evolution and the implications of alternative arrangements. When we state that particular clades are united by particular characters, these are characters that unambiguously change at that point on the tree, as indicated by MacClade.

Results and discussion Complete data set. Analysis of the complete data set yielded 180 most parsi- monious trees of 425 steps, which belong to two islands (MADDISON 1991), island A consisting of 40 trees, island B of 140. The strict consensus of all trees is shown in Fig. la. The two islands differ only in the arrangement of taxa in the annonoid clade, which includes the columellar tetrads and Artabotrys. In island A (e.g., Fig. lb), Artabotrys is linked with Uvariastrum, Hexalobus, and the parasyncarps Monodora and Isolona, while the Annona and Asimina groups are associated with Uvariodendron and Uvariopsis. In island B (e.g., Fig. lc), Artabotrys is linked with the Annona group, the Asimina group with Mkilua and the Cymbopetalum group. In both islands, the sister group of Annonaceae may be Magnoliaceae, Myristicaceae, Magnoliaceae plus Myristicaceae, Magnoliaceae plus Degeneria, or the three taxa together. Sketches of the principal pollen types are superimposed on lines shaded with MacClade (MADDISON & MA~)DISON 1992) to show the evolution of infratectal structure. These results reaffirm the conclusion of DOYLE & LE THOMAS (1995) that groups with tetrads and inaperturate single grains form a clade. However, relationships within this clade contradict our previous conclusion that all its members were derived from an ancestor with annonoid columellar tetrads. The Annona group is once again nested among the other taxa with columellar tetrads, reconstituting the annonoid clade in nearly the original sense of DOYLE & LE THOMAS (1994). In contrast, our former "uvarioid" clade is split into two well-separated groups. The first is the p s e u d o s y n c a r p s, linked with the African liana Toussaintia by large sepals and a rudimentary aril. The Toussaintia-pseudosyncarp clade is nested among groups with tetrads, linked with the xylopioids, indicating that tetrads (retained in Toussaintia and Fusaea) reverted to single grains within the clade. This implies that the tetrads of Fusaea and the xylopioids, which WALKER (1971) grouped in his Fusaea subfamily, are homologous after all, contrary to LE THOMAS & al. (1994). The other clade, the uvarioids in a restricted sense (Uvaria, Afroguatteria, Monanthotaxis group), is the sister group of all the other inaper- turates, implying that its single grains are primitive. We will refer to the entire tetrad-inaperturate group as the inaperturates or the inaperturate clade. Palynology and phylogeny of Annonaceae 137

Inaperturates Outgps Arab Plalmeoid Pip Hilu Uva Psgn XU! Annonoids I I I I ( ~1 If II il i l If a

~-~ ~ ~ 'd,e.~ Z~ ). 0 ~ ~.~ ~ 0 ~,~ 0 ~ C: 0-~ 0 ~'~ - ~1~ ~ ~ ~" ~" ~" ¢1;

0 :IISUS o set

Inaperturate$ i i Outgps Amb Malmeoid Pip Hilu Uva Psgn Xgi Annonoids i i i i i i ii II li II I I i i li I

-

== ~;..'~:]¢- I \'~, ~) ?/ ~, Y =:o= , .,.i,,

Fig. 1. a Strict consensus of 180 most parsimonious trees found in the "complete" analysis (including pollen characters), b Representative most parsimonious tree from island A (40 trees), showing distribution and evolution of the infratectal structure character, with sketches of major pollen types superimposed at the point where they originate, c Portion of a representative tree from island B (140 trees), showing different relationships in the annonoid clade. Outgps magnolialian outgroups, Arab ambavioids, Pip piptostigmoids, Milu miliusoids, Uva uvarioids, Psyn pseudosyncarps, Xyl xylopioids 138 J.A. DOYLE & A. LE THOMAS:

In DOYLE & LE THOMAS (1995), Artabotrys was the sister group of the inaperturate clade and Annickia its second outgroup. However, in the present analysis Artabotrys is nested within the annonoids, and Annickia is nested within the columellar monosulcate malmeoids. This implies that the single grains of Artabotrys are secondarily derived from tetrads, and the fact that they have an aperture is a reversal rather than a primitive state (suggesting the possibility that the aperture is homologous with the thin proximal exine of the tetrads: DOYLE& LE THOMAS 1996). The sister group of the inaperturates is the m i 1 i u s o i d s, with monosulcate [Polyalthia longifolia (SONNERAT) THWAITES] or disulculate pollen, which are linked with the inaperturates by a shift from elongate to globose pollen and from spiniform to lamelliform endosperm ruminations. The sister group of the malmeoids (including the sulculate Guatteria group) is the granular monosulcate p i p t o s t i g m o i d s (which include another sulculate derivative, Sapranthus). This arrangement implies that the granular exine structure of the piptostigmoids is primitive, whereas on some of our previous trees it was a reversal. The four genera just above Anaxagorea, called "ambavioids" by DOYLE & LE THOMAS (1994, 1995), formed a clade in some of our previous trees, but a paraphyletic group in others. In all the present trees, Ambavia, Tetrameranthus, and Cleistopholis form a clade, but Greenwayodendron is located at the next node; hence we have restricted the name "ambavioids" to the first three genera. These changes from one analysis to the next may seem cause for despair that relationships in Annonaceae will ever be resolved. However, closer examination shows that the changes are not as radical as they first appear. In all analyses, Anaxagorea is the sister group of other Annonaceae, followed by the ambavioids. Most major clades and other key relationships also remain intact: the piptostigmoid, miliusoid, xylopioid, and pseudosyncarp clades, the association of Artabotrys in one way or another with the annonoids, and the group consisting of Hexalobus and the parasyncarpous genera Monodora and Isolona (cf. WALKER 1971). Furthermore, many aspects of the trees, notably the basal position of Anaxagorea and the unity of the inaperturate clade, are confirmed by the molecular analyses of VAN ZUILEN (1996) and BYCRAVE & CHASE (pers. comm.). As noted by DOYLE & LE THOMAS (1994), these instabilities reflect the high level of homoplasy in Annonaceae. Another indication is the consistency index (CI) (an inverse measure of homoplasy), 0.27, which is below average for this number of taxa (0.36: SANDERSON& DONOGHUE 1989). AS a result, different trees and scenarios for character evolution can be expected with addition or reinterpretation of characters and taxa, and new character sets (especially molecular sequence data) may be required before higher-level relationships are resolved. However, the general results seem sufficiently robust for purposes of the present exercise. Consistency indices for different subsets of characters give some indication of their probable systematic reliability. Values of three different consistency measures for the tree in Fig. lc (from island B) are summarized in Table 1, with characters (omitting cytology) divided into four categories. Based on all three measures, vegetative characters have the lowest consistency (highest homoplasy), despite the fact that seven of these do not vary within Annonaceae (e.g., nodal anatomy, vessel perforations, sieve tube plastids), tied with or followed by inflorescence and floral Palynology and phylogeny of Annonaceae 139

Table 1. Three measures of consistency (inversely related to homoplasy) for subsets of characters, based on the tree in Fig. 1c. CI Consistency index, RI retention index, RC rescaled consistency index

Character sets CI RI RC Whole data set 0.27 0.55 0.15 Vegetative (characters 1-21) 0.24 0.43 0.10 Floral, inflorescence (22-41, 53-57) 0.24 0.44 0.11 Pollen (42-52) 0.26 0.73 0.19 Fruit, seed (58-75) 0.33 0.62 0.21

morphology. Fruit and seed characters have the highest consistency as measured by the simple and rescaled consistency indices, but pollen characters score higher in terms of the retention index. This confirms previous subjective impressions concerning the relatively high systematic value of pollen, fruit, and seed characters in Annonaceae, and the low value of traditionally emphasized floral characters (WALKER 1971; LE THOMAS 1980--1981; VAN SErrEN & KOEK-NOORMAN1992; DOYLE & LE THOMAS 1994, 1995). Removal of pollen eharaeters. Analysis of the data set with pollen characters removed yielded more than 12000 equally parsimonious trees (the maximum number that could be stored with available memory) of 341 steps. All trees found in several replicate analyses appear to belong to a single island. A strict consensus is shown in Fig. 2a. As is often the case, the huge near-basal polychotomy largely reflects rearrangements among adjacent clades and "jumping" of individual taxa to alternative positions in a small number of most parsimonious trees. The obvious decrease in resolution in itself attests to the value of pollen characters in reducing the number of alternative trees. A representative tree (Fig. 2b), which corresponds to the majority rule consensus (with a trichotomy of the three lines above the miliusoids resolved arbitrarily) will be used as a basis for discussion. However, it should be noted that statements about the character support for particular groups may not be valid for other equally parsimonious trees. In all most parsimonious trees, the sister group of all the remaining Annonaceae is Uvariopsis, an African genus with columellar tetrad pollen, which belonged to the annonoid clade in the complete analysis. In most trees (c. 95%), Magnoliaceae are the sister group of Annonaceae. Uvariopsis is followed by several palynologically similar annonoid groups. These groups apparently share enough non-palynological similarities with Uvariopsis and each other that they are "pulled" toward the base of the tree when Uvariopsis is basal. The other annonoids (labeled "A" in Fig. 2) are scattered higher in the tree, showing the importance of columellar tetrad pollen in uniting the annonoid clade as a whole. Anaxagorea, which is basal in the complete analysis, is now nested well within the family, associated with the ambavioids, Greenwayodendron, and usually Artabotrys, based on flowers in cymes and (originally) two basal ovules. The continued association of the three ambavioids and Greenwayodendron reflects the fact that these taxa were already united primarily by non-palynological characters, such as two lateral ovules, tuberculate seeds, and (except for Greenwayodendron, which has not been 140 J.A. DOYLE & A. LE THOMAS:

Outgps Ann0noid Pip Psun Melm Milu A Xu! Uvar A Arab , ,, ni"~ u , , ='Iit==='~i'~in + i'' , '"n ¢, n Co

.~_,.?.,t",,,~.~J~,,,5,?..~,g-o-,~o"C ~.~,=.,~ "g" .'* "go~.,,~ "r. x .~'~g~,.,,~ :~.s.,, mc.~ +_+_,="~ .~2"+~~'~-~-:~o ~'',~~° ~2

a

~nsus • set

Outgps Annonoid Pip Psyn Haim Hilu A XgI Uver A Arab I II |1 I I I I • mm~t. II II I ~mm m II I C: I I

~® _..o c o. ~ 'r-- o.~ 4' ~ ,.n :,o,= ,,, .~.~o.. ~F. ...,m

~.,~ S.. ~o~;7 ,V"~ C X tO.~ 0. ,,~ ',':'. 0 0 o~.(~, tn 4, ~,-~.~',-%" o ,";.~,~',~" E "-,~ ~: 0 "" ~ :; ~: S.. "'+" :..~"~ ."~.'" :;.~ :r,a, ,v • >. ~ o ~ >.~ ,v o.~C ,v :~ 4, = :; ,~_m o o c ~.'---- 0 o m..~ :~2 ,v ,s,~ >.OO~..~ DDnDnnunnnnnnnnnmmmmnnnmnuunnnBnmmnnBoB~Bun.x:]D~

[ Stigma ITreelenc~ I 16 steps L~.~.de red ir-~ I ~ oapitate II elongate ~on- [~ uncertain

Fig. 2. a Strict consensus of more than 12 000 most parsimonious trees found in the "non- palynological" analysis (after removal of pollen characters), b Representative most parsimonious tree, showing distribution and evolution of the stigma character. Outgps magnolialian outgroups, Pip piptostigmoids, Psyn pseudosyncarps, Malta malmeoids, Milu miliusoids, A groups belonging to the annonoid clade in the complete analysis, XyI xylopioids, Uvar uvarioids, Amb ambavioids Palynology and phylogeny of Annonaceae 141 counted) the unusual chromosome number of n = 7, although they also have more or less similar granular monosulcate pollen. Many other groups are similar to those found in the complete analysis, but they are often associated with genera previously located elsewhere. In most trees Piptostigma, Polyceratocarpus, and Sapranthus still form the piptostigmoid clade, united by percurrent tertiary veins, but they are often associated with Ancana, which belongs to the miliusoid clade in the complete analysis, based on spiniform ruminations. The other miliusoids remain together but also include the malmeoid genus Ephedranthus. The other malmeoids often form a clade, but not always. The pseudosyncarps are still strongly supported, but they are associated with the Guat- teria group rather than Toussaintia or the uvarioid lianas, based on complex midrib histology and imbricate petals. Mkilua and the Cymbopetalum group are still associated with each other, based on reticulate tertiary venation (a reversal), locellate anthers, numerous ovules, and dorsally dehiscent fruit, but they are separated from other annonoids. In most trees, the Annona group is linked with the uvarioids, based on globose-cylindrical receptacle and idioblasts in the nucellus. The three xylopioid genera form a clade in nearly 100% of the trees obtained (the exceptions presumably reflect the fact that they have only two non-palynological synapomorphies, elongate petals and elongate stigmas). However, their sister group is usually the malmeoid genus Annickia, based on strongly curved secondary veins and apical articulation of the monocarp stipe. Irnpaet of pollen characters on tree topology. The changes between trees found before and after removal of pollen characters imply that palynology is of major value not only in improving resolution of relationships in the Annonaceae, but also in affecting schemes for their higher-level systematics and evolution. Its most striking contribution is in "rooting" the family, which would in turn define the first subdivision in a phylogenetic classification. The reasons why pollen characters have such a radical effect on tree topology can be understood by considering what the non-palynological trees would imply about pollen evolution (Fig. 3). The resulting scenario is highly implausible from a palynological point of view. Thus the basal position of Uvariopsis and other annonoids would imply that monosulcate pollen with a continuous tectum, granular infratectal structure, and one or two nexine foliations, as seen in other Magnoliales, was transformed into highly advanced inaperturate tetrads with well-developed columellae, tectal perforations, and numerous foliations at the base of the Annonaceae. The only potentially intermediate condition is the columellar structure of (some) Magnoliaceae. These changes would then be reversed within Annonaceae to produce granular monosulcate pollen essentially identical to that of other Magnoliales in Anaxagorea, Greenwayodendron, and the ambavioids, and independently in the piptostigmoids. Many specific associations of taxa are also implausible from a palynological point of view. In the complete analysis, Annickia is nested among the malmeoids, which have similar reticulate, columellar, monosulcate pollen, but in the non- palynological analysis, it is linked with the xylopioids, which have smooth, granular, inaperturate tetrads. In the complete analysis, the Annona group is nested among other annonoids, which also have reticulate-columellar, inaperturate tetrads with multiple nexine foliations; when pollen characters are removed, it is 142 J.A. DOYLE ~; A. LE THOMAS:

Ontgp$ Annonoid Pip Psyn Ilelm Itil. Am XU! Uvor A' Arab . . . ~ m ~ , ""'If • .--. , i"== •'~''if='='=i . ,

oor.o.r.m&mmumooom o,~, mmmommmmum. ]rim O.¢r~oo~rnr-moODO

granular i~ intermed nen-palynological dora sot m colume,lar m,L,_,_,_,iuncertain [---I equivocal

Fig. 3. Implications of a tree found after removal of pollen characters (Fig. 2b) for pollen evolution, with exine structure plotted a posteriori on the tree and transitions between single grains and tetrads indicated with symbols. Abbreviations of taxa as in Fig. 2

associated with the uvarioids, which have single grains with granular exine structure. It may be noted, however, that uvarioids are like the Annona group in having inaperturate pollen and multiple foliations, characters that place both groups in the same large inaperturate clade in the complete analysis. In the complete analysis, the Guatteria group is nested among the malmeoids, but when pollen is removed it is linked with the pseudosyncarps. Its position in the malmeoids is not obviously due to pollen characters, since it has highly autapomorphic sulculate pollen with a reduced exine, which seems no more (or less) at home in the malmeoids than in the pseudosyncarps. Its new position is presumably a subtle effect of changes in the overall tree topology. A quantitative measure of the inconsistency of the non-palynological trees with pollen data can be obtained by comparing the number of steps (state changes) in pollen characters on the tree based on the whole data set, 62; and the number of steps on the tree found after removal of pollen, 105 (see Appendix for steps in individual characters). These numbers can be compared with the minimum possible number of steps in pollen characters if there were no homoplasy at all, 16. On the complete trees, there are therefore 46 excess (homoplastic) steps in pollen characters; on the non-palynological trees, 89. Thus the trees found after pollen is removed would imply almost twice as much homoplasy in pollen characters as the trees found in the complete analysis. Palynology and phylogeny of Annonaceae 143

To assess the seriousness of the conflict in rooting of the tree, we investigated the relative strength of alternative trees in terms of each data set. With the complete data set, the shortest trees found when Uvariopsis is forced to the base of the Annonaceae are four steps longer (429 steps) than the shortest trees. An example is shown Fig. 4a, with the inferred sequence of aperture evolution - from mono- sulcate in the outgroups, to inaperturate, and back to monosulcate. In contrast, with the non-palynological data set, the shortest trees found when Anaxagorea is forced to the base of the family are only two steps longer (343 steps) than the shortest trees. One such tree is illustrated in Fig. 4b, with one of the non-palynological characters that supports a basal position of Anaxagorea, stamen morphology. Thus pollen data contradict the result obtained when pollen is excluded more strongly than non-palynological data contradict the result obtained when pollen is included. To maintain that the pollen results should be rejected would require assuming that pollen characters are consistently much less reliable than other characters. Further evidence for the reliability of the pollen characters is the fact that Anaxagorea is basal and annonoids (including Uvariopsis) are nested within the family in the molecular analyses of VAN ZUILEN (1996) and BYGRAVE • CHASE (pers. comm.). Role of specific characters in rooting. The reasons for these results may be evaluated in more detail by observing the behavior of characters on the two sorts of trees, using MacClade (MADDISON & MADDISON 1992). We will emphasize the problem of basal relationships, examining which characters support the two contradictory roofings. In trees based on the complete data set, which characters are primitive (retain the outgroup state) in Anaxagorea but derived in Uvariopsis and other annonoids? Conversely, when pollen characters are removed, which characters are primitive in Uvariopsis but derived in Anaxagorea and its relatives? We will evaluate the impact of particular characters by counting numbers of steps (state changes) on trees derived from the two data sets. We have concentrated on the trees in Fig. lc (island B) and Fig. 2b; numbers of steps may differ on other trees from the same analyses. Numbers of steps for each character are presented after character definitions in the Appendix. The relevant characters can be grouped into three main categories. (1) Some non-palynological characters agree with pollen in favoring a basal position of Anaxagorea (and often the ambavioids) and a derived position of Uvariopsis (i.e. nested well within the family). One such character is stamen morphology. According to the complete analysis (Fig. 5a), Anaxagorea retains the laminar stamens of other Magnoliales (cf. WAL~R 1971). These are modified to narrower stamens that still have a tongue-like extension of the connective in the ambavioids and Greenwayodendron, and to stamens with the typical peltate-truncate connective of most Annonaceae (includ- ing Uvariopsis) above this. Counting a separate origin of the peltate connective in Cleistopholis, a reversal in Miliusa, and origin of a peltate-apiculate connective in the xylopioids, the total number of state changes is five. In the non-palynological analysis (Fig. 5b), where Uvariopsis is basal, laminar stamens are replaced by stamens with a peltate connective at the base of the family (a two-step change, since this is an ordered multistate character), which reverse to stamens with a tongue-like connective in the ambavioids, and to laminar stamens in Anaxagorea. This results in two more steps (seven) than there are in the complete analysis. 144 J.A. DOYLE & A. LE THOMAS:

Inaperturotos i i Oatgps Annonotds Ps!ln X!ll UYa HiIu Pip Halmeaid Amb a li il i (~immll II II II I I I ~i. ~ Ul ~ :~ O~

== ~ a= =~ ® o _ .m~ v, o~ c ~ ...c .~.~ :~ :a~.~ ,,,~ ,,,o*~,~, ,,,=-~ ~_ ~ ~ ~ ~ ,-. 7o= o,,, ~ ~ ~ ~J~ t&'~' ~ 0.~ "~C ~.~ "~ ~--~ -~ '.~. ~. X 0 ~.;Z ~ "~ ~ ~.~ o ,,...-. =_ ,, ~-v, ~~ o~ ~ ~ .:,,,e ~ ~-g ._, o: i ~,I ~ ~ ~ ~-~ =~ o .== ~ ~ ,', ~..,=..,.. uDDDDnnnuunun[::)nuuuNnununnnnnmnmDnDDDr~[:)Dr~DmD[:)DD[:)D

Outgps Arab Xgl A Uva Annonoids Pip Psgn Holm Hilu . u u t cu i i"='. . f %n . . i ~='~i 0 qh 4a O.,v tg ® '~ .,= = ,=, =-." o .~,= ~,~ ~ .=.o~ ~o~

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Stamen morph

J Treelenc~th:343 J (HP+2) prolonged i=~m peR-truno peR-apJcul nan-pal!lnological data set uncertain ~=====J equivocal

Fig. 4. a Tree based on the complete data set with Uvariopsis forced to the base of the Annonaceae. b Tree based on the non-palynological data set with Anaxagorea forced to the base of the Annonaceae. MP most parsimonious. Abbreviations of taxa as in Figs. 1 and 2 Palynology and phylogeny of Annonaceae 145

, Inaper,,turates Ir J Outgps Arab MalmeoN Pip Htlu Uva Psgn Xgi Annonoids I '' I ~ I I! ' II " -I[ II ' =1 I "if 'l

on or~ [] Br~ [] ommn n nr~ ~~I. [] nun mE

Outgps Annono|d Pip , Psgn Maim Milu A Xgl Uvar A Arab i ' Hi il I ie, ' i i 2 .mill. II 7| t ~e li~ii~ I .... I

L .~ '~' a. ~P¢:: ,.~

o ~.~ ~,,"~.._.q o,~. "~'~o~L:~om~ "~w~-*."~ ~.,.,~ w,~m°'~",~

.~ ~.nnnuunuuuunnuunnnnunnnur~nu BG ,

" 7 ~tdp~ ordered L'--] laminar prolonged b iJ pelt-trunc IIOfl- pel~-apioul :N uncertain equivocal

Fig. 5. Evolution of stamen morphology (a) before and (b) after removal of pollen characters. Abbreviations of taxa as in Figs. 1 and 2 146 J.A. DOYLE& A. LE THOMAS:

Anaxagorea is also the only member of the Annonaceae that retains the adaxial plate of vascular tissue in the midrib found in other Magnoliales. This is a reversal in the non-palynological trees, resulting in a one-step difference (six vs. seven) between the two footings. According to the complete analysis, Anaxagorea and the ambavioids retain the irregular endosperm ruminations of other Magnoliales, which are replaced by spiniform ruminations in Greenwayodendron and other intermediate groups, then lamelliform ruminations in the miliusoids and inaperturates (Fig. 6a). Independent advances in Sapranthus and Ephedranthus, reversals in Ancana and Cananga, and absence of ruminations in two outgroups (Himantandraceae, Magnoliaceae) give a total of eight steps. However, when Uvariopsis is basal (Fig. 6b), lamelliform ruminations are derived from irregular ruminations or none at the base of the family, the irregular ruminations of Anaxagorea and the ambavioids are a reversal, and spiniform ruminations originate four times, resulting in ten state changes. There are other characters that do not directly favor a basal position of Anaxagorea over Uvariopsis but do show fewer state changes on trees derived from the complete data set. For example, in the complete analysis, glass-like endosperm originates twice, in Greenwayodendron and the malmeoid-piptostigmoid clade, with reversals in Ephedranthus and Sapranthus (four steps). In the non- palynological trees, Annickia and the piptostigmoids are separated from the malmeoids, so this character shows six steps. The position of Annickia among the malmeoids in the complete analysis is also supported by its columellar monosulcate pollen. Silica cells also unite the malmeoids and thus show one less step in the complete analysis. Large sepals originate one less time in the complete analysis, where they link Toussaintia and the pseudosyncarps; this relationship is reinforced by the tetrads of Toussaintia, xylopioids, and the Fusaea group. Other characters that show one less state change on the tree in Fig. lc include petal estivation, presence and orientation of fibers in the testa, arils, and distribution of idioblasts in the seed. Anaxagorea has other features that appear to be primitive but do not positively favor the basal position of Anaxagorea, because of mixed states and/or ambiguous arrangements in the outgroups. For example, Anaxagorea and Xylopia are the only Annonaceae with inner staminodes, and the complete analysis indicates that these are homologous in Anaxagorea and other Magnoliales. This character shows three state changes on trees based on both data sets, because inner staminodes do not occur in Magnoliaceae. In the non-palynological analysis, when Magnoliaceae are the sister group of Annonaceae, staminodes are lost in the common ancestor of the two families, followed by reappearances in Anaxagorea and Xylopia. Another potentially primitive feature is the dehiscent fruit of Anaxagorea, also seen in Magnoliaceae and Myristicaceae. In trees where Magnoliaceae and Myristicaceae are the first two outgroups, this condition is unambiguously primitive in Annonaceae, but in Fig. lc, because Degeneria is indehiscent, the dehiscent state is equivocally homologous, and the character undergoes three state changes with both rootings. (2) Other characters favor a basal position of Uvariopsis and undergo more state changes when Anaxagorea is basal. It is these characters that overrule the primitive features of Anaxagorea when pollen is ignored. Palynology and phylogeny of Annonaceae 147

Inaperturates

Outgps Arab Ilelmeoid Pip Hilu ' Ova Psgn Xgl &nnonoid$ l i u u l ii ii in u i n 'Ul a

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unordered absent irregular a BI~ spiniform lamelliform complete data set uncertain equivocal

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no~ommmmmmmmnnummmm~nmmunnmr, mmmmmmnmmmmBn~mnm~n

--.,-o,-,-,

Fig. 6. Evolution of endosperm ruminations (a) before and (b) after removal of pollen characters. Abbreviations of taxa as in Figs. 1 and 2 148 J.A. DOYLE & A. LE THOMAS:

One potentially primitive feature of Uvariopsis is the Tetrameranthus nucleus type (Mor~w~zz 1986), in which chromosomes condense homogeneously rather than proximally to distally, as in the prochromosomal type of most Annonaceae. This condition also occurs in Himantandraceae and Degeneria, and MORAWETZ (1986) therefore suggested that it is primitive in Annonaceae. However, another outgroup, Eupomatia, is prochromosomal, and conditions in Magnoliaceae and Myristicaceae are not readily assignable to either state. According to the complete analysis (Fig. 7a), the basal state in the family is ambiguous, since ambavioids have the Tetrameranthus nucleus type but Anaxagorea is prochromosomal. However, all groups studied between ambavioids and Uvariopsis are prochromosomal, so the Tetrameranthus nucleus type of Uvariopsis is unambiguously a reversal. In contrast, in the non-palynological analysis (Fig. 7b), the Tetrameranthus nucleus type of Uvariopsis is primitive and homologous with the same condition in other Magnoliales, while the Tetrameranthus type of the ambavioids is a reversal. In the complete analysis, this character undergoes four state changes; when pollen is subtracted, the number falls to three. A problem is that this character is documented in less than half of the genera - it may therefore be highly sensitive to addition of new data. A similar case is the elongate receptacle of Uvariopsis, which is reminiscent of Magnoliaceae. We treated receptacle form as two characters, for the stamen- bearing and carpel-bearing portions, since these often vary independently. Both characters favor a basal position of Uvariopsis, but this effect is stronger for the stamen-bearing portion. The picture is complicated by the fact that the outgroups are variable; only Magnoliaceae have an elongate receptacle. In the complete analysis (Fig. 8a), the basal state in Annonaceae is flat-conical rather than elongate. This is homologous with the same state in Degeneria and Eupomatia, and the elongate receptacle of Uvariopsis and Magnoliaceae is a convergence. When pollen characters are removed (Fig. 8b), the elongate receptacle is a synapomorphy of Magnoliaceae and Annonaceae, which is modified to globose-cylindrical above Uvariopsis. The flat-conical receptacle of Anaxagorea, ambavioids, and related groups is a reversal to the ancestral condition of Magnoliales other than Magnoliaceae. Counting all state changes, the character contributes 14 steps in the complete analysis, 11 when pollen is removed. Ovule number also supports the basal positon of Uvariopsis, despite variation in the outgroup states. Anaxagorea has two basal ovules, whereas Uvariopsis and related annonoids have numerous ovules. According to the complete analysis (Fig. 9a), the basal state in Annonaceae is ambiguous: because several states occur in the outgroups, it can be either numerous, two lateral, or two basal. The numerous ovules of Uvariopsis and intervening groups can be either homologous with the same state in the outgroups or derived from one basal ovule, which arises within Annonaceae. The total number of state changes is 13. However, when pollen characters are removed (Fig. 9b), the numerous ovules of Uvariopsis are unambiguously ancestral, the two basal ovules of Anaxagorea and Artabotrys and the two lateral ovules of Greenwayodendron and the ambavioids are derived, and the number of steps decreases to nine. The inflorescence character shows a similar pattern. In the complete analysis, the cymes of Anaxagorea and the ambavioids are derived relative to the solitary Palynology and phylogeny of Annonaceae 149

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NO onooo mn niiii • • inn • []

e 4 steps

complete data set ~ Te~r~merar Ji i equiv'ocal

Outgp$ Annonoid Pip Psun Maim I'filu A XUI Uvar A Amb , . . , m + : "'"if" .~ : I""""~l"--I : ..... ,

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ooo -,..,

Nuoleo'tgpe 3 s'ceps

.on-palgnological data set ~k~'~N'~ " IL..J "r+'trpmeranl - - "I~.\\\\"~'+7 I i proonromo I II~ equ+~ooal i

Fig. 7. Evolution of nucleus type (a) before and (b) after removal of pollen characters. Abbreviations of taxa as in Figs. 1 and 2 150 J.A. DOYLE & A. LE THOMAS:

Iliaperturates I ! Outgps Arab I'lalmeoid Pip rlilu Uva Psgn Xyl Anlionotds I l I I l II I| II II I I II I

0 ~ ~h 0 0 • '~ -~ O. @ I= iS. 0 m a, ~ ~ ," "~,"~'o =~ x. ~ o ,..q .~ ~. X ~ ~ ~ m "z: o ,t,. E

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Receptacle 14 steps unordered flat-conical It glob-cglind I elongate complete data set ~,~ uncertain r--i equivocal Z Outgpa Annonotd Pip Psyn Ilolm Ililli A Xg] Uvor k Arab , . .. j . . ~ ='"it = nr----. ' , r-==--='qP=--I , ,

,* '*'~ ~,-e~ '~ o":' ~ '*~ ~-~ ,* °'~ ~ - ~- x . ° v~ ~ ~ -'°'~ ~. ',' ~ ~'~ -'~ ~ ,*~ '~ ~'- = o*" ~"~ ~- o I'.~ ~-~,~.~.~ ~ ~ ~ ¢ ~"~ ~-5 ,;~ 3"-5 t~ o ,*'~" ;--,~-~.~ tn G ~c.~ X ~= -> ~

on onmno~oonnnnomnoonnunnnnmnnDooooo.m.uuoooono

Receptacle 1 1 steps unordered

liOn- ~ glob-cylind plil~to set ~~~ I i gell~bn;;)e ~)_,L,L,L( uncertain

Fig. 8. Evolution of the stamen-bearing portion of the receptacle (a) before and (b) after removal of pollen characters. Abbreviations of taxa as in Figs. 1 and 2 Palynology and phylogeny of Annonaceae 151

Inaperturates i i Outgps Arab I'lalmeoid Pip Illlu gva Psgn Xgl Anaonoids i i i i i ii ii Ii ii i i ii i

E ~ ~ ~ m'~,"~Z"~ e,-~ e,'~_.¢ ¢:_ ~,~ o ~,~ =. o_ ,.., ;.~ ,., .~.i.. ~_ o ~.~ ',~ -~ ? ~,-~-- E-~-.,': ~- .'f" ~. ,'v ~ X ~_. o

n mmmnno nrmral~rm mnmmmnm mmnmmmo000~mmn O~ omm mmnm on OOO0 O mm U ono O0 0

13 steps unordered numerous t::::::::l t~'o lateral a t~vo basal complete data set one basal uncertain equivocal /, Outgps Annonoid Pip Psgn Maim Plilu A Xgl Uvar A Arab I I, II I I I i . ma~f. I~mm~I I ~ i =~ I I

_~o_®~c ,^,~L_~o ~- ~ xo+"c ~: += ~G~ ~ .~o.u, ~ Q ~-, ¢~h 3. ++ +.C;

~ n;.~.- ¢L. ~ ~ ~,..~ __. ~ ~. E ~. :~.~ ~.¢:: :~ eL. ,~.,.. ::;~ ~ ~ C e = ~ ~ ~ 0 + ~.~ ~ ~ ~ 0 0 ~ ~, ,V :,. ~

ormmmoB O O0 O 000 DO oom mmn mmnrn mmn nm mm m~ mmooo on mm O OOl~UUnrmr~r~l~ N"

~ numerous b t::::::::l two lateral two basal non- one basal uncertain r===j equivocal

Fig. 9. Evolution of ovule number and position (a) before and (b) after removal of pollen characters. Abbreviations of taxa as in Figs. 1 and 2 152 J.A. DOYLE & A. LE THOMAS: flowers in most other Magnoliales. Cymes reverse to solitary flowers above Greenwayodendron, but they reappear six times in higher groups, not counting the many cases of variation within taxa, for a total of ten steps. When pollen characters are removed, the solitary flowers of Uvariopsis and most other Annonaceae are ancestral, the cymes of Anaxagorea and the ambavioids are derived, and the number of steps decreases to eight. Again, several characters are not especially primitive in Uvariopsis but do undergo fewer changes in the non-palynological analysis. Examples include ray width (a highly homoplastic character), petal orientation, carpel number, base and articulation of monocarps, thickness of the fruit wall, seed surface, and chromosome number (see Appendix). (3) Finally, there are several characters in which both Anaxagorea and Uvariopsis have the ancestral state for Annonaceae. When Anaxagorea is basal, the state in Uvariopsis is a reversal, but when Uvariopsis is basal, the state in Anaxagorea is a reversal. These characters recall the problem of rooting the angiosperms as a whole. Based on morphological data (DoNoc~ & DOYLE 1989, DOYLE & al. 1994), trees with Magnoliales basal and Nymphaeales basal are equally or almost equally parsimonious, because both groups have features such as boat-shaped monosulcate pollen with granular exine structure that are primitive based on outgroup comparison, but lacking in intervening groups. However, there are fewer such cases in Annonaceae - the potentially primitive features of Anaxagorea and Uvariopsis are usually different. In some of these cases, the character still favors trees in which Uvariopsis is basal. An example is secondary venation. Other Magnoliales have straight secondary veins, which are modified to moderately and then strongly curved within Annonaceae. In the complete analysis (Fig. 10a), the point at which straight changes to moderately curved is ambiguous because the state Anaxagorea is un- certain (0/1) and ambavioids are variable. However, the tree is consistent with the view that straight secondaries in Anaxagorea, Ambavia, and Polyalthia stuhlmannii (EN~L) VE~C. are primitive, while the straight secondaries of Uvariopsis are clearly a reversal. Advances to strongly curved occur in several groups, plus various reversals, for a total of 14 steps. In the non-palynological analysis (Fig. 10b), it is Uvariopsis that retains the straight secondary veins of other Magnoliales. The shift to moderately curved occurs just above Uvariopsis, followed by three reversals, for a total of ten steps. The fact that Annickia and the xylopioids (both strongly curved) are associated saves a step; in the complete analysis, pollen characters help place them in the malmeoids and inaperturates, respectively. In other cases, both rootings involve the same number of steps. For example, in the complete analysis, the sessile stigma of the outgroups is retained in Anaxagorea, ambavioids, and Greenwayodendron, but the stigma becomes capitate above Greenwayodendron and reverses to sessile in Uvariopsis. When pollen charac- ters are removed (Fig. 2b), the sessile stigma of Uvariopsis is primitive; it changes to capitate immediately above this genus and reverses to sessile in Anaxagorea and the ambavioids. This character shows six state changes on both trees. Several considerations suggest that the characters in category 2 may be intrinsically less reliable than those in category 1, thus strengthening the con- clusions reached when pollen characters are included. Many characters supporting Palynology and phylogeny of Annonaceae 153

Inaperturate$ Outgp$ Arab Halmeoid P~p Hilu Uva Psgn Xljl Annonoids I n I I I II II II II I I [ II I

~,*_® ~,.~ ~ ~ ~ ~ = ~ .~.~'r- ~. ~* ~ S~- ~oc

n[~r~l~DBnnnnB[~nlunr~numl~unnnnlBununmnuuununnununn

Secondaries 14 steps complete data set N~.~.~N~x~ l curved ~%%~%%%%"N~ I'i'i'i'L#uncertain I I equivocal

0utgps Annono|d Pip Psgn Italm Hilu A Xgl Uvar A Arab

Lo ~ ~=n_~o~- ~ Xo~ E ~,.c ~ ~o~ ~ .~ o~L "~

~-. ~: .~ U'. ~" Ip ~ 0 ,.: ,~ :~ ..,-: Q.,.-~ Q. ~ 0 0~,~.. Vj go q; ,-2_ ,..4 ,-i 0 ,- ;.~ ,.*1 ,.< ~: .~ ,-~ ~ 0 ¢: ~ = ¢: L ¢: -*." ~ .~ .*., "~

Secondaries 1 0 steps ordered , [--7 straight medium D011- B curved [01014 uncertain r-~ equivocal

Fig. 10. Evolution of secondary venation (a) before and (b) after removal of pollen characters. Abbreviations of taxa as in Figs. 1 and 2 154 J.A. DOYLE & A. LE THOMAS:

the Uvariopsis rooting show very high levels of homoplasy on all trees: e.g., secondary venation (number of steps --- 14, 10 in the two analyses), inflorescence type (10, 8), receptacle (14, 11), and ovule number (13, 9). Reliability of the nucleus type character can also be questioned because of the large proportion of missing data (24 out of 42 taxa have not been studied). The inflorescence character shows especially high variation within taxa (nine taxa have both states), plus problems in definition; as emphasized by L. CHATROU (pers. comm.), all Annonaceae except the Cymbopetalum group are potentially cymose, since they have bracteoles on the pedicel. The character as defined here expresses the number of flowers seen at any one time and thus suffers from general problems in the use of quantitative characters in cladistic analysis. Furthermore, implications of many of these characters depend on outgroup relationships, which are poorly resolved. However, these "a posteriori weighting" arguments are secondary to the fact that the complete data set strongly favors the basal position of Anaxagorea when characters are treated equally, as do the molecular data of VAN ZUtLEN (1996) and BYGRAVE & CHASE (pers. comm.).

Conclusions These results confirm the value of palynology in recognizing major groups and their relationships in Annonaceae. There is still much uncertainty about the exact relationships among pollen types - within the inaperturates (number of origins of tetrads, whether single grains are derived from tetrads or vice versa, and how many times), among granular and columellar monosulcates (piptostigmoids, malmeoids, miliusoids), and between these two general pollen classes (via Annickia, Artabotrys, or miliusoids). However, the rooting of the family between Anaxagorea and other groups with granular monosulcate pollen and the advanced status of the annonoids, xylopioids, and uvarioids with inaperturate tetrads and monads seem relatively robust. The only other large complex of characters that appears to have a comparable level of systematic informativeness and reliability is fruit and seed morphology, plus perhaps stamen morphology and chromosome characters. Pollen features overcome several highly homoplastic characters that might lead to an incorrect concept of rooting of the family and directions of character evolution. Finally, the conclusion that it is the pollen characters that are reliable is independently confirmed by preliminary molecular analyses. We thank MICHAELHESSE for inviting us to present this paper in honor of Prof. FRITZ EHRENDORFER, who has been a major stimulus to our interest in primitive angiosperms and a supporter of the value of palynological studies. We are also grateful to the Ecole Pratique des Hautes Etudes for financial support, to ARNE ANDERBERG(Stockholm) for urging us to conduct the experiment of removing pollen characters, and to PAUL BYGRAVEfor sharing molecular results.

References CI~ATROU, L., 1997: Preliminary remarks on the subdivision of Malmea. - Annonaceae Newslett. 11 (in press). Palynology and phylogeny of Annonaceae 155

DONOOm~E, M. J., DOYLE, J. A., 1989: Phylogenetic analysis of angiosperms and the relationships of Hamamelidae. - In CRANE, R R., BLACKMORE, S., (Eds): Evolution, systematics, and fossil history of the Hamamelidae, 1, pp. 17-45. - Oxford: Clarendon Press. DOYLE, J. A., LE THOMAS, A., 1994: Cladistic analysis and pollen evolution in Annonaceae. - Acta Bot. Gallica 141: 159-170. 1995: Evolution of pollen characters and relationships of African Annonaceae: implications of a cladistic analysis. - In LE THOMAS, A., ROCHE, E., (Eds): 2e Symposium de palynologie africaine, Tervuren (Belgique), pp. 241-254. - Orldans: Centre International pour la Formation et les Echanges G6ologigues. 1996: Phylogenetic analysis and character evolution in Annonaceae. - Bull. Mus. Natl. Hist. Nat., B, Adansonia 18: 279-334. -- DONOGHUE,M. J., ZIMMER, E. A., 1994: Integration of morphological and ribosomal RNA data on the origin of angiosperms. - Ann. Missouri Bot. Gard. 81: 419-450. FRmS, R. E., 1959: Annonaceae. - In MELCHIOR,H., (Ed.): Die nattirlichen Pflanzenfamilien 17alI, pp. 1-171. 2nd edn. - Berlin: Duncker & Humblot. HESSE, M., MORAWETZ, W., EHRENDORFER, E, 1985: Pollen ultrastructure and systematic affinities of Anaxagorea (Annonaceae). - P1. Syst. Evol. 148: 253-285. JOHNSON, D. M., MURRAY, N. A., 1995: Synopsis of the tribe Bocageeae (Annonaceae), with revisions of Cardiopetalum, Froesiodendron, Trigynaea, Bocagea, and Hornschuchia.- Brittonia 47:248-319. LE THOMAS, A., 1980-1981: Ultrastructural characters of the pollen grains of African Annonaceae and their significance for the phylogeny of primitive angiosperms. - Pollen & Spores 22: 267-342, 23: 5-36. - DOYLE,J. A., 1996: Implications d'une analyse cladistique dans l'histoire g6ographique des Annonaceae: famille d'Angiospermes primitives. -In GUILLAUMET,J. L., BELIN, M., PuI~, H., (Eds): Phytog~ographie tropicale, r6alites et perspectives, pp. 171-180. - Paris: ORSTOM. - LU~ARDON, B., 1974: Quelques types de structure grenue dans l'ectexine de pollens simples d'Annonac6es. -Compt. Rend. Hebd. S6ances Acad. Sci., S~r. D 278: 1187- 1190. 1976: De la structure grenue ~ la structure columellaire dans le pollen des Annonac~es. - Adansonia 15: 543-572. - LU~At~DON,B., DOYLE, J. A., 1994: Pollen ultrastructure and relationships of Fusaea (BAILLON) SAFFORO and Duguetia A. SAINT-HILAIRE (Annonaceae). - Rev. Palaeobot. Palynol. 83: 55-64. MADDISON, D. R., 1991: The discovery and importance of multiple islands of most- parsimonious trees. - Syst. Zool. 40: 315-328. MADDISON, W. P., MADDISON, D. R., 1992: MacClade: analysis of phylogeny and character evolution, version 3. - Sunderland, Mass.: Sinauer. MORAWETZ, W., 1986: Systematics and karyoevolution in Magnoliidae: Tetrameranthus as compared with other Annonaceae genera of the same chromosome number. - P1. Syst. Evol. 154: 147-173. - 1988: Karyosystematics and evolution of Australian Annonaceae as compared with Eupomatiaceae, Himantandraceae, and Austrobaileyaceae. - P1. Syst. Evol. 159: 49- 79. NOWICKE, J. W., SKVARLA,J. J., 1979: Pollen morphology: the potential influence in higher order systematics. - Ann. Missouri Bot. Gard. 66: 633-700. SANDERSON, M. J., DONOGIaUE, M. J., 1989: Patterns of variation in levels of homoplasy. - Evolution 43: 1781-1795. 156 J.A. DOYLE & A. LE THOMAS:

Swo~o~, D. L., 1990: PAUP: phylogenetic analysis using parsimony, version 3.0. - Champaign, Ill.: Illinois Natural History Survey. VAN HEUSDEN, E. C. H., 1992: Flowers of Annonaceae: morphology, classification, and evolution.- Blumea, Suppl. 7: 1-218. VAN SETTEN, A. K., KOEK-NoORMAN,J., 1992: Fruits and seeds of Annonaceae. Morphology and its significance for classification and identification. Studies in Annonaceae XVII. - Biblioth. Bot. 142: 1-101. VAN ZtnLEN, C. M., 1996: Patterns and affinities in the Duguetia alliance (Annonaceae). Molecular and morphological studies. - Doctoral Thesis, Utrecht University. WA~, M., 1985: Ultrastruktur und systematische Bedeutung des Pollens bei Bocageopsis, Ephedranthus, Malmea und Unonopsis (Annonaceae). - P1. Syst. Evol. 150: 165-177. - 1988: Different origins of fragile exines within the Annonaceae. - P1. Syst. Evol. 158: 23-27. - MO~WEXZ, W., 1988: Pollen evolution and systematics in Annonaceae with special reference to the disulcate Australian endemic genera. - P1. Syst. Evol. 161: 1-12. WAL~R, J. W., 1971: Pollen morphology, phytogeography, and phylogeny of the Annonaceae.- Contr. Gray Herb. 202: 1-131. - DOYLE,J. A., 1975: The bases of angiosperm phylogeny: palynology. - Ann. Missouri Bot. Gard. 62: 664-723.

Appendix Characters: Numbers of steps for each character on the trees in Fig. lc (complete data set) and Fig. 2b (non-palynological data set) are indicated in parentheses after definitions of states. Unless otherwise indicated, multistate characters are unordered. 1. Habit (0) trees or shrubs, (1) lianas or lianescent shrubs or trees (4, 3). 2. Phyllotaxy (0) spiral, (1) distichous (3, 3). 3. Prophylls (0) adaxial, (1) lateral (4, 3). 4. Nodes (0) trilacunar, (1) multilacunar (2, 3). 5. Histology of midrib (0) simple arc, (1) arc plus adaxial plate, (2) complex, with phloem and/or sclerenchyma intruding xylem body (6, 7). 6. Midrib (0) concave or fiat on adaxial side, (1) convex (5, 5). 7. Secondary veins (0) straight or recurved, (1) moderately curved, (2) strongly curved (ordered) (14, 10). 8. Tertiary venation (0) reticulate, frequent intersecondary veins, (1) partially percurrent (toward margin), some intersecondaries, (2) strongly percurrent, no inter- secondaries (ordered) (13, 12). 9. Hairs (0) simple or absent, (1) stellate or peltate (4, 4). 10. Sclereids (0) absent, (1) astrosclereids, (2) osteosclereids (10, 8). 11. Epidermal crystals (0) druses or absent, (1) solitary rhombic, etc. (6, 6). 12. Silica idioblasts (0) absent, (1) present (3, 4). 13. Oil cells (0) in sponge parenchyma only, (1) in palisade or both palisade and sponge parenchyma (5, 5). 14. Pith (0) normal, (1) septate (1, 1). 15. Vessel perforations (0) scalariform, (1) simple (2, 2). 16. Vessel density (0) < 10/mm2, (1) 10-40/mm 2, (2) > 40/mm 2 (ordered) (8, 8). 17. Rays (0) narrow, (1) wide (widest > 100 pm) (10, 9). 18. Parenchyma (0) diffuse or paratracheal, (1) apotracheal (1, 1). 19. Phloem (0) normal, (1) stratified (1, 1). 20. Sieve tube plastids (0) S or Psc type, (1) Pcs type (1, 1). Palynology and phylogeny of Annonaceae 157

1 2 3 4 5 6 7 1234567890123456789012345678901234567890123456789012345678901234567890123456789 Outg A0?000000A???00?00000AA??0AA?0?0000000A00AA00ABOB0??????0B?A?I?A????00A0?0A?0?? Eupo 01011A00000??00210011A0?C01???2??????01000020100000002200??0?100????0000011?100 Hima 000010001207?11101100101101?7?2?????71100000010000001720111??100????0000?00?000 Dege 01?1100000???101111001011001000000000010000010000000020000?0?100????0010201?0?? Myr i 011010000C0??10A0?10011??Z01?02?????????000000AOA011??0030?0?000????0C0000A??00 Magn 0A11100A01?Z?Z0C01101A01100010A000000000000010B0C0112020A000?000????00102007??? Afro 11???0110????????????00?10010010000000020002010000201120D0?1?1011101000???3???? Amba 0177?00007??????????7111100?00110000100100100000001001101070?1011101100???10000 Anax 010010A001AOA11111??1AZ11000001100002010000010000010012020??001110000A000C11100 Anca 0177?00107???????????101100100110001B002000201102011011100?00101100000077722100 Anni 01???021A?l?Z111011?Z001100100110100200E00001020201101213071111121100000CE217?? Anno 0110A011AIA011111111100110010011000020020111212020211?2C310??1AIC1000A0032E0100 Arta 110000110?0?1111111110112001001100012002000011COC0210111207001AZFA00A00AD231??? Asim 0110011100001111011111001001001000000002011121202021121100?00101100002001231??? Cana 01?00022070?111011111111100100110001B003011121000100022200?11111110002011211100 Clei 01700011070711100111711110000010001110020000000001100120107Al101C10010011210000 Cymb 010?01100?00111101?1700100010AIA0000100?11112120202?A11A00?102AIC00002001232101 Dugu 010?001010AIAZ11011110AZ100110100000000200011110????1?22312??10110A00100?131101 Ephe 010?0A2201110????????10121000010000010020000102020111?2130?10111000000001337??? Fusa 017?2A110210011101?110A1101110100000010201112100002007223117?10110000107??31101 Gree 017??01A070??71111??10111100001100010001000000000010012010?1011100101000132???? Guat ~1~?2~1A~1~11~11111~22~~2~21A?~????~22~3~?1A1111~L~A~12E~1~ Hexa ~1???~1~2~?~1~LA1~?1~1~1~111~?~2~11111C~1~2~111~?~1~12A~323???? Isol 01??01110?07711211711101C001001?100?000200111110102001A10277?1012101000033317?? Lete 11???0110????????????001101110100000000200021110002007223117?1Al11000100113???? Malm 010?00000011011101???0Al1000001000000002000010202011122130?10111C0100A00132???? Mili 01??0011011701110111?1N?20010011010000010002011010102021A0?10111100000001332100 Mkil 01???0100????????????00?1001001?00000002111121?0????012100?072112?00020???3???? Mona 11???0220?0?N?1211?11AAZZ00100110000N002000101?10???NCCN00?101111A0100003231??? Mono 01?001110?A?III?IIIIIA0110010011101?I002011111201021BIAI02???I01CI0100003131??? Neos 01???0220?0?0?1101???0011001001100112003111111000100012230?11111110100003231??? Pach 01??20C01?07011011171Al11001101000001002000101?1????1?22312??10110000100123???? Pipt 01???0120????71101??71111001001101012002000010000000121100?071011010000013277?? Post 017??0000????????????10?1001001100000002000000000010112130?101111?000007??2???? Polo 01700011071??1??????71111001001100010002000001101010111130?10111100000000332100 Pocs 01?070000???0?1?71??110110010011000N000200001020201112C130?10111201000001222100 Podi 01??001A071???1??171710710010011000N00020002011020111121G0?10111AOA0000013E2100 Poly 01???02207077??277???1011101001100000002000010000110121100?00101Al100000112???? Pseu 017701000201111111??11NCC0000010000010020100102020111121G0?Z011110100000332???? Sapr 01??00220210011201?1?0011001001000000002000211000000122100?00101C000000013327?? Tetr 007?201111001110111?710220000010000000010000010000201C1010?0?10121001001?210010 Tous 11???0210????????????101100111N000000002011111?0??2?212000?101111100010??73???? Unon 0100211102010111017??11110010011000010020000102020111C21G0?101112N100N0022227?? Uvar 11000011110?0112011110N110N1~1000000N02000111000020AC2N00?101NIC101NI003231100 Uvas 01??00100????11111??71Nl1001111100000002011111201021011000?0010121010000323???? Uvad 01???0110????????????101200A001100000002011111202021122100?001011A010000323???? Uvop 01???0000?1?11????l?11A11N0?00110000000201111120C02A202000?00101100100003231000 Xylo 0100001N0000N11001111111C00100110001211Ellll1100010002C200?A0?N111000C001C31101 Data matrix: A= 0/1, B = 0/2, C = 1/2, D= 1/3, E = 2/3, F = 0/1/2, G= 0/1/3

21. Isoquinoline alkaloids (0) absent, (1) present (3, 2). 22. Inflorescence position (0) terminal (leaf-opposed, supra-axillary, extra-axillary), (1) axillary (9, 7). 23. Flowers (0) solitary (-2), (1) in cymes (10, 8). 24. Articulation of pedicel (0) absent, (1) (sub) basal, (2) distal (3, 3). 25. Bracteoles (0) absent, (1) 1-3 per flower, (2) > 3 (ordered) (7, 6). 26. Flowers (0) bisexual, (1) male and bisexual or unisexual (4, 4). 27. Sepals (0) distinct or basally fused, (1) calyptrate (3, 2). 28. Sepal estivation (in bud) (0) imbricate, (1) valvate (4, 4). 29. Sepal size (0) small, (1) large, enclosing petals (4, 5). 30. Sepal margins (0) normal, (1) winged (2, 2). 31. Corolla (0) > 2 cycles, (1) 1-2 cycles, (2) absent (ordered) (2, 2). 158 J.A. DOYLE & A. LE THOMAS:

32. Petal estivation (in bud; refers to outer whorl if two whorls differ) (0) imbricate, (1) valvate (8, 9). 33. Petal fusion (0) free, (1) fused at least basally (1, 1). 34. Outer petals (0) subequal, (1) reduced or absent (3, 3). 35. Inner petals (0) subequal, (1) reduced or absent (3, 3). 36. Petal shape (0) oval, (1) elongate, pointed (7, 7). 37. Petals (0) spreading, (1) curved over other floral parts for whole length, (2) with concave basal portion, often connivent (13, 12). 38. Outer staminodes (0) absent, (1) present (3, 3). 39. Inner staminodes (0) absent, (1) present (3, 3). 40. Stamen morphology (0) laminar, (1) narrower but with tongue-like extension of connective, (2) peltate-truncate (cap concealing anthers), (3) peltate-apiculate (ordered) (5, 7). 41. Anthers (0) normal, (1) locellate at maturity (3, 3). 42. Pollen unit (0) single (monads), (1) compound (tetrads, polyads) (5, 9). 43. Proximal exine (0) normal, (1) reduced (4, 8). 44. Aperture (0) sulcate, (1) inaperturate, (2) sulculate (8, 14). 45. Average pollen size (0) small (<45 gin), (1) medium, (2) large (> 90 gm) (ordered) (10, 14). 46. Pollen shape (0) elongate (boat-shaped), (1) globose (4, 9). 47. Tectum (0) imperforate, (1) verrucate, (2) reticulate-perforate (5, 12). 48. Spinules (0) absent, (1) present (2, 2). 49. Infratectal structure (0) granular, (1) intermediate, (2) columellar (6, 10). 50. Base of infratectum (0) undifferentiated, (1) fused granules (3, 3). 51. Nexine foliations (0) absent, (1) 1-2, often discontinuous, (2) multiple, continuous, often contorted (ordered) (8, 12). 52. Outer foliation (0) undifferentiated, (1) thickened (7, 12). 53. Stamen-bearing portion of receptacle (0) flat or conical, (1) globose or short cylindrical, (2) elongate (14, 11). 54. Apex of receptacle (carpel-bearing portion) (0) elongate, (1) flat or convex, (2) concave (10, 9). 55. Average number of carpels (0) 1 (-2), (1) 2-10, (2) > 10 (12, 10). 56. Stigma (0) sessile, (1) capitate, (2) elongate (6, 6). 57. Ovules (0) numerous, (1) two lateral, (2) two basal, (3) one basal (13, 9). 58. Fruit (0) apocarpous, (1) pseudosyncarpous, (2) parasyncarpous (4, 4). 59. Basal ring (in pseudosyncarps) (0) absent, (1) formed from receptacle, (2) formed from carpels (3, 3). 60. Base of monocarp (0) sessile or nearly so, (1) stipitate (5, 4). 61. Articulation of stipe (0) basal, (1) apical (4, 3). 62. Fruit (0) ventrally dehiscent, (1) indehiscent, (2) dorsally dehiscent (4, 4). 63. Fruit wall (0) thick (>1 mm, generally woody or leathery), (1) thin (<1 ram, generally juicy) (8, 4). 64. Chalaza (0) normal, (1) perichalazal (1, 1). 65. Raphe (0) more or less pronounced groove, (1) flat, not manifest, or nearly so, (2) more or less pronounced rib (ordered) (7, 8). 66. Micropylar plug (0) absent or small, (1) large (8, 6). 67. Endosperm (0) normal, (1) glass-like or stony (4, 6). 68. Colored oil cells in endosperm (0) absent, (1) present (4, 5). 69. Seed surface (0) smooth or with low sculpture, (1) tuberculate (2, 1). 70. Aril (0) absent, (1) rudimentary, (2) bilobed (5, 6). Palynology and phylogeny of Annonaceae 159

71. Sarcotesta (0) absent, (1) present (1, 2). 72. Integuments (0) two, (1) three (2, 2). 73. Idioblasts (oil cells) (0) in inner integument, (1) absent, (2) in outer integument, (3) in nucellus (8, 9). 74. Mesotesta fibers (0) absent, (t) longitudinal, (2) crossed, (3) transverse (10, 11). 75. Ruminations (0) absent, (1) irregular plates or pegs, (2) spiniform, (3) lamelliform (including dissected plates) (8, 10). 76. Chromosome number (0) n = 7, (1) n -- 8, (2) n = 9 (ordered) (7, 6). 77. Nucleus type (0) Tetrameranthus type and intermediate, (1) prochromosomal nucleus, chromosomes condensing proximally to distally (4, 3). 78. Centromeres (0) mostly (sub) metacentric, (1) acro- or telocentric (2, 2). 79. Chromosome size distribution (0) continuous, (1) bimodal (3, 3).

Addresses of the authors: JAMESA. DOYLE, Section of Evolution and Ecology, University of California, Davis, CA 95616, USA. - AN~tcK LE THOMAS, Ecole Pratique des Hautes Etudes, Museum National d'Histoire Naturelle, 16 rue Buffon, F-75005 Paris, France.

Accepted December 11, 1996 by M. HESSE and I. KRISAI-GREILHUBER

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