Grana, 2008; 47: 185–210 Pollen morphology within the Monodora clade, a diverse group of five African Annonaceae genera THOMAS L. P. COUVREUR1, MARLEEN BOTERMANS1, BERTIE JOAN VAN HEUVEN2 & RAYMOND W. J. M. VAN DER HAM2 1 Nationaal Herbarium Nederland, WU Branch/Biosystematics Group, Wageningen University, Wageningen, The Netherlands, and 2Nationaal Herbarium Nederland, Leiden University, The Netherlands Abstract Pollen morphology has played a major role in elucidating infrafamiliar-level systematics and evolution within Annonaceae, especially within the African genera. The Monodora clade is composed of five genera, Asteranthe, Hexalobus, Isolona, Monodora and Uvariastrum, which are restricted to Africa and contain together c. 50 species. A molecular phylogeny of the family showed that the monophyly of the Monodora clade is strongly supported and that it is part of a larger clade of 11 African genera. In order to support classification a detailed survey was made of the pollen morphological variation within the Monodora clade, using scanning and transmission electron microsopy. For the two most species-rich genera, Isolona and Monodora, a molecular species-level phylogeny was used to assess the taxonomic usefulness of the pollen characters. The survey showed a wide range of pollen morphological diversity. The most conspicuous variation concerned the occurrence of monads without a thicker outer foliation in the basal exine layer in Isolona in contrast to tetrads with a thicker outer foliation in Asteranthe, Hexalobus, Monodora and Uvariastrum. At the infrageneric level, Hexalobus, Isolona and Monodora showed the largest diversity, with various pollen types based on tectum morphology. Hexalobus is exceptional with three types within only five species. The pollen types defined in this study are hardly useful in characterizing major groups identified within both Isolona and Monodora, but they do illustrate relationships within smaller groups. Keywords: Annonaceae, pollen, tetrads, monads, pollen ornamentation, exine structure Annonaceae is a pantropical family of trees, shrubs and lianas belonging to the order Magnoliales (APGII, 2003). With c. 130 genera and c. 2 500 species (Chatrou et al., 2004) it is the most diverse family of the order, not only at the macromorphological level but also at the pollen morphological level (Sampson, 2000). African Annonaceae have been relatively understudied and many genera require updated revisions. However, the pollen morphology of the African genera (Le Thomas & Lugardon, 1976; Le Thomas, 1980, 1981), as well as that of the rest of the family (Walker, 1971a, b, 1972) received significant attention, which has played a considerable role in understanding the evolution of the family (Doyle & Le Thomas, 1994, 1997; Doyle et al., 2000). The genus Anaxagorea is sister to the rest of the family on the basis of morphological (Doyle & Le Thomas, 1994, 1996), molecular (Richardson et al., 2004) and combined data (Doyle et al., 2000, 2004). Anaxagorea is characterized by monosulcate pollen with a granular infratectum. This ancestral pollen type gave rise to columellate monosulcate pollen (malmeoids, Malmea), disulcate pollen (miliusoids, Miliusa), and inaperturate pollen in tetrads (e.g. Annona, Monodora) and polyads (e.g. Xylopia), with a reversal to monads with a granular infratectum in the uvarioid clade (Doyle & Le Thomas, 1996; Doyle et al., 2000; Doyle, 2005). The genera with inaperturate pollen (Doyle & Le Thomas, 1996) represent a strongly supported monophyletic clade referred to as the ‘‘long branch clade’’ (LBC) Correspondence: Raymond W. J. M. van der Ham, Nationaal Herbarium Nederland, Leiden University, P.O. Box 9514, 2300 RA Leiden, The Netherlands. E-mail: ham@nhn.leidenuniv.nl (Received 26 March 2008; accepted 30 May 2008) ISSN 0017-3134 print/ISSN 1651-2049 online # 2008 Collegium Palynologicum Scandinavicum DOI: 10.1080/00173130802256913 186 T. L. P. Couvreur et al. by Richardson et al. (2004). The LBC showed more molecular divergence than the other well supported ‘‘short branch clade’’ (SBC), which is equivalent to the Malmea-Piptostigma-Miliusa (MPM) clade of Doyle & Le Thomas (1996). Asteranthe Engl. & Diels, Hexalobus A.DC., Isolona Engl., Monodora Dunal and Uvariastrum Engl. are five tropical African genera of trees and shrubs that form a well supported clade within the LBC (Table I, Figure 1). Isolona is also found in Madagascar, while Asteranthe is restricted to East Africa (Kenya and Tanzania). Most of the species grow in lowland and montane rainforests although a few species are adapted to slightly more xeric conditions, especially those found in East Africa (e.g. Asteranthe asterias, Hexalobus mossambicensis, Uvariastrum hexaloboides). Isolona and Monodora are unique in Annonaceae in having a syncarpous gynoecium (Couvreur et al., 2008; Deroin, 1997), which is also rare within the Magnoliales (Endress, 1982). In the past, numerous morphological studies have indicated that Asteranthe, Hexalobus, Isolona, Monodora and Uvariastrum are closely related (floral morphology: Van Heusden, 1992; fruit and seed morphology: Van Setten & Koek-Noorman, 1992). Walker (1971b, 1972) was the first to propose an informal classification of the Annonaceae based on a large generic pollen survey using especially light microscopical (LM) characters. Together with seven other African genera and the South American genus Diclinanona, the above five genera were united in the Hexalobus tribe. Most genera in this tribe have tetragonal tetrads, while Cleistochlamys and Isolona have monads. In a cladistic analysis based on pollen and macromorphological characters, Doyle & Le Thomas (1994) recovered Hexalobus and Uvariastrum as sister to Isolona and Monodora (Asteranthe was not included in the analysis). A recent molecular phylogeny based on six plastid Table I. Species richness, geographical distribution and morphological diversity of the Monodora clade genera. Genus Number of species Geographic distribution Gynoecium type Petals Asteranthe 2 East Africa apocarpic fused Hexalobus 5 West-Central/ East Africa apocarpic fused Isolona 20 West-Central/ East Africa and Madagascar syncarpic fused Monodora 14 West-Central/ East Africa syncarpic fused 8 West-Central/ East Africa apocarpic free Uvariastrum markers including many previously unavailable African genera (Couvreur et al., 2008) confirmed that Asteranthe, Hexalobus, Isolona, Monodora and Uvariastrum form a strongly supported monophyletic group, called the Monodora clade, nested within the LBC (Figure 1). The Monodora clade grouped within a clade composed of 11 African genera, which is referred to as the African long branch clade (ALBC, Couvreur et al., 2008). This clade comprises most genera of Walker’s Hexalobus tribe (Cleistochlamys and Diclinanona not included) and Sanrafaelia (described in 1996). Dennettia tripetala appeared to be nested in Uvariopsis and was sunken into that genus by Kenfack et al. (2003). The close match between the taxa included in Walker’s Hexalobus tribe (1971b) and the molecular phylogeny (Couvreur et al., 2008) indicate the value of pollen characters at infrafamilar-level classification (Doyle & Le Thomas, 1997). Here, we take a more in depth look at the Monodora clade to determine the value of these characters within the ALBC (Figure 1). Additionally, using a species-level phylogeny of the two most species-rich genera Isolona and Monodora (Table I), we also assess the usefulness of these characters for infrageneric classification. Material and methods Pollen sampling and preparation In total 78 samples were analyzed representing 46 of the 49 species found within the Monodora clade (see Species investigated: Pollen samples). All species within Asteranthe (2), Hexalobus (5) and Monodora (14) were sampled, whereas 19 of the 20 Isolona species and six of the eight Uvariastrum species were studied. Pollen samples were taken from herbarium or alcohol collections preserved at the following herbaria: BR, C, COI, EA, FHO, G, MO, P and WAG. Annonaceae pollen is very fragile and the acetolysis method for pollen preparation (Erdtman, 1960) is often too drastic and damages the pollen grains making observations difficult. Following Couvreur et al. (2006), we used an alternative method based on three consecutive baths of crushed mature stamens in n-hexane, an organic solvent. Material preserved in 70% alcohol was given an extra bath in 100% alcohol prior to the n-hexane baths. The samples were then gold-coated and examined using scanning electron microscopy (SEM). When possible, the size of five pollen grains per sample was measured. In addition, transmission electron microscopy (TEM) was carried out for a limited number of species within Hexalobus, Isolona Pollen of five African Annonaceae 187 Figure 1. Strict consensus tree of the seven most parsimonious trees based on six plastid markers indicating the major clades recognized within Annonaceae (Couvreur et al., 2008). Monodora clade indicated in bold. Bootstrap support values under 100% are indicated above the branches as well as the major groups recognized within Annonaceae. 188 T. L. P. Couvreur et al. and Monodora. For a few specimens, marked with an asterisk (*) in the section Specimens investigated: Pollen samples, results were derived from SEM and TEM images (mostly unpublished) provided by A. Le Thomas (Muséum National d’Histoire Naturelle, Paris). The delimitation of pollen types is based on ornamentation only, as this is the only informative character available for all specimens examined. Pollen terminology In Annonaceae, the delimitation of the various exine layers is still unclear (Doyle, 2005; Gabarayeva, 1995). The main problem comes from the presence of conspicuous foliations under the infratectum. Different interpretations have been proposed based on different criteria (Figure 2). A first view defines the exine of Annonaceae pollen as lacking an endexinous part, thus consisting only of an ectexinous part composed of three layers: the tectum, the infratectum and a layer composed of conspicuous foliations termed the basal layer (Le Thomas, 1980; Le Thomas & Lugardon, 1976). These conclusions were based on the observation that the tectum, infratectum and basal layer have the same electron density (Le Thomas, 1980). Gabarayeva (1995) had a different view. She took an ontogenetic approach and defined the thick outer foliation of the basal layer as the foot layer (ectexinous) and the thinner inner foliations together as the endexine, the foot layer developing earlier than the endexine. For the sake of consistency we adopt the definition of Le Thomas (1980) in the present paper. Le Thomas (1980, 1981, 1983) used scanning electron microscopy (SEM) as well as transmission electronic microscopy (TEM) to study the variation of tectal and infratectal characters in African Annonaceae genera. She described three major types of infratectum: 1. granular, 2. columellate and 3. intermediate. The intermediate state includes a range of cases, all considered to be intergrading (Doyle, 2005): columellae composed of fused granules, columellae mixed with granules, and infratectum consisting of radially elongated, ellipsoidal elements. Further terminology follows Punt et al. (2007). Molecular phylogeny and character optimization An almost complete species level molecular phylogeny of Isolona and Monodora based on five plastid markers (trnL-trnF, psbA-trnH, trnS-trnG, ndhF and trnD-trnT; Couvreur, unpublished data) was available for assessing the taxonomic utility of the different pollen types defined. Of Isolona, 14 of the 20 recognized species were included, with I. zenkeri and I. congolana represented by two specimens each. Of the 14 species recognized in Monodora, 13 were sampled, with M. myristica represented by two specimens (see Species investigated: Molecular samples). The phylogenetic analysis was run under a Bayesian framework using the Metropolis-coupled Monte Carlo Markov chain (MCMCMC) algorithm implemented in MrBayes, Vers. 3.1.2 (Ronquist & Huelsenbeck, 2003) with the program’s default parameters for the priors. Three separate runs of five million generations each were undertaken and stationarity as well as convergence between the MCMC runs was checked using both Tracer v. 1.3 (Rambaut & Drummond, 2003). The resulting Bayesian majority rule consensus tree is presented in Figure 15. Maximum parsimony optimization of the pollen types was undertaken on the majority rule consensus tree from the Bayesian analysis (see above) using Mesquite, Vers. 1.11 (Maddison W. D. & Maddison D. R., 2006). Pollen types were treated as unordered. The optimized pollen types are represented in Figure 16. Results Measurements and descriptions for each species are summarized in Table II and Table III. 1. Asteranthe (Figure 3) Previous observations. — LM: Walker (1972), Le Thomas (1974); SEM and TEM: Le Thomas (1974, 1980). Present observations. — 2/2 species studied. Figure 2. Alternative terminologies used for pollen wall structures in Annonaceae by Gabarayeva (1995) and Le Thomas (1980). In the present study the terms used by Le Thomas (1980) are applied. C5columella, G5granule, F5foliations, OF5outer foliation. SEM: Pollen in acalymmate tetragonal tetrads, 105 – 140 mm in diameter. Constituent monads inaperturate, P550 – 63 mm, E566 – 84 mm, P/E50.75 – 0.76. Ornamentation foveolate; foveolae 0.9 – 1.8 mm. TEM (A. asterias): Exine 3.4 mm thick. Tectum 0.9 mm. Infratectum 1 mm, columellate. Basal layer consisting of 2 – 4 loose, undulate foliations; outer Table II. SEM pollen data for species of Asteranthe, Hexalobus, Isolona, Monodora and Uvariastrum (Monodora clade). All measurements in mm; P5length polar axis, E5equatorial diameter. An asterisk (*) indicates data obtained from the archive of A. Le Thomas. Taxa Pollen type Asteranthe A. asterias A. lutea Tetrad size P monad E monad P/E monad 105 140 50 63 66 84 0.76 0.75 foveolate foveolate granular to gemmate granular to gemmate areolate-verrucate to/or rugulate areolate-verrucate to/or rugulate psilate, with perforations 79 56 64 59 61 42 28 31 29 28 61 52 55 53 38 0.69 0.54 0.56 0.55 0.74 Isolona I. capuronii A — — — — I. deightonii A — 32 25 1.28 I. dewevrei A — 40 31 1.29 I. heinsenii A — 41 38 1.08 I. humbertiana A — 46 41 1.12 I. madagascariensis* A — 33 26 1.27 I. perrieri A — 45 35 1.29 I. pilosa A — 37 33 1.12 I. thonneri A — 35 29 1.21 I. I. I. I. I. I. I. I. I. I. B B B B B C C C C C — — — — — — — — — — 41 44 45 36 45 36 36 40 46 37 33 33 39 27 42 30 32 34 35 31 1.24 1.33 1.15 1.33 1.07 1.20 1.13 1.18 1.31 1.19 campanulata cooperi hexaloba pleurocarpa zenkeri cauliflora congolana ghesquierei lebrunii linearis scabrate, scabrae not fused, at same level scabrate, scabrae not fused, superimposed scabrate, scabrae rarely fused, at same level scabrate, scabrae not fused, at same level verrucate, verrucae often fused, at same level verrucate, verrucae often fused, at same level scabrate, scabrae somet. fused, at same level scabrate, scabrae rarely fused, superimposed scabrate, scabrae often fused, somet. elongate finely rugulate, perforations small finely rugulate, perforations small finely rugulate, perforations small finely rugulate, perforations small finely rugulate, perforations small rugulate, rarely with perforations rugulate, with perforations rugulate, somet. with perforations rugulate, with perforations rugulate, with perforations Size of gemmae, scabrae, verrucae Size of perforations, foveolae — — — — 1–1.8 0.9–1.7 — — 0.6–1.9 1.0–1.9 — 0.4–1.5 0.4–1.5 — — — — — — — 0.1–0.4 — 0.7–1.2 — — 0.1–0.6 — — 0.2–0.8 — — 0.8–1.1 — — 1–3 — — 0.6–2.1 — — 0.5–1 — — 0.1–0.5 — — 0.5–1 — 0.5–0.8 0.3–0.5 0.3–0.6 0.3–0.5 0.2–0.5 0.6–1.0 0.6–1.2 1.0–1.5 0.8–1.5 0.8–1.2 — — — — — — — — — — 0.2.–0.3 0.2 0.1–0.3 0.2–0.3 v0.1 v0.2 0.4–0.7 0.2–0.5 0.2–0.4 v0.1 189 A A B B C Width of muri Pollen of five African Annonaceae Hexalobus H. bussei H. mossambicensis H. crispiflorus H. salicifolius H. monopetalus Ornamentation 190 Taxa Pollen type Tetrad size P monad E monad P/E monad A1 A1 A1 A1 A1 A1 A2 A2 A2 A2 B 92 72 82 54 110 104 71 66 70 85 63 48 41 42 27 60 52 45 39 33 40 31 52 45 48 31 52 44 41 34 38 47 33 0.93 0.92 0.87 0.87 1.15 1.18 1.10 1.15 0.87 0.85 0.94 M. globiflora B 80 44 48 0.92 M. hastipetala B 54 24 30 0.80 M. junodii B 86 40 44 0.91 52 60 81 63 107 82 22 30 35 30 47 41 33 40 47 44 64 52 0.67 0.75 0.74 0.68 0.73 0.79 Monodora M. laurentii M. minor M. myristica M. stenopetala M. tenuifolia M. undulata M. carolinae M. crispata M. grandidieri M. zenkeri M. angolensis Uvariastrum U. germainii U. hexaloboides U. insculptum U. pierreanum U. pynaertii U. zenkeri Ornamentation Width of muri Size of gemmae, scabrae, verrucae Size of perforations, foveolae psilate, perforations regular in size psilate, perforations regular in size psilate, perforations regular in size psilate, perforations regular in size psilate, perforations regular in size psilate, perforations regular in size rugulate, locally psilate rugulate, locally psilate rugulate, locally psilate rugulate, locally psilate rugulate, perfor. bigger and denser than in type A rugulate, perfor. bigger and denser than in type A rugulate, perfor. bigger and denser than in type A rugulate, perfor. bigger and denser than in type A — — — — — — 0.5–3.0 0.3–2.5 1.0–3.0 1.0–2.0 0.7–1.0 — — — — — — — — — — — 0.2–0.4 0.2–0.5 0.1–0.2 0.1–0.3 0.1–0.3 v0.1 0.2–0.4 0.1–0.4 0.1–0.2 0.1–0.5 0.3–0.7 1.0–1.5 — 0.3–1 0.5–0.7 — 0.3–0.5 0.8–1.1 — 0.3–0.9 rugulate, rarely with perforations rugulate, rarely with perforations rugulate, with perforations rugulate, with perforations rugulate, without perforations rugulate, locally psilate, with perforations 0.8–1.0 2.0–2.5 0.7–1.0 2.1–3.0 1.5–2.1 1.5–2.0 — — — — — — 0.1–0.2 0.2–0.3 0.1–0.2 0.2–0.5 — 0.1 T. L. P. Couvreur et al. Table II. Continued. 191 Pollen of five African Annonaceae Table III. TEM pollen data for species of Asteranthe, Hexalobus, Isolona, Monodora and Uvariastrum (Monodora clade). All measurements in mm. An asterisk (*) indicates data obtained from the archive of A. Le Thomas. Genus Species Tectum thickness Columella length Columella width Granule size Outer foliation Asteranthe asterias* 0.9 1.0 0.7 — 0.35 Hexalobus bussei crispiflorus monopetalus 0.7 1.2 1.4 — 1.5 1.9 — 1.1 1.2 0.5–1.3 0.1–0.3 0.1–0.3 0.15 0.1–0.3 0.1–0.4 Isolona campanulata congolana ghesquierei hexaloba humbertiana thonneri* 0.4 0.6 0.4–0.8 0.4 0.5–1.5 0.4 0.5 0.9 — 0.7 — — 0.4 0.6 — 0.5 — — 0.2–0.3 0.4–0.8 1.3–1.5 0.1–0.4 0.4–0.9 0.1–0.4 v0.1 — — v0.1 v0.1 v0.1 Monodora angolensis crispata myristica* 0.6 0.8 1.7 1.1 0.7 0.6 0.8 0.7 0.7 0.5 0.3–0.8 0.15–0.35 0.4 0.4 0.3 Uvariastrum pierreanum* pynaertii* 1.1 1.8 2.0 3.2 1.3 1.8 0.2–0.7 0.3–0.9 0.7 0.6 foliation thicker, 0.35 mm. Tectum, infratectum and thick outer foliation of basal layer absent between monads, the contact zones consisting of thin foliations only. Species included: A. asterias, A. lutea. 2. Hexalobus (Figures 4–6) Previous observations. — LM: Walker (1971b, 1972), Le Thomas (1974); SEM and TEM: Le Thomas & Lugardon (1976), Le Thomas (1980). Present observations. — 5/5 species studied. SEM: Pollen in acalymmate, tetragonal tetrads, 56 (63.8) 79 mm in diameter. Constituent monads inaperturate, P528 (33.6) 42 mm, E538 (51.8) 61 mm, P/E50.54 (0.65) 0.74. Ornamentation granular to gemmate (type A), areolate-verrucate to/or rugulate (type B), or psilate with perforations (type C). TEM: Exine 2.4 – 3.9 mm thick. Tectum 0.7 – 1.4 mm. Infratectum 1.5 – 1.9 mm, granular or columellate/granular. Basal layer consisting of 2 – 4 undulate foliations; outer foliation not too clearly thicker, 0.1 – 0.4 mm. Tectum, infratectum and thick outer foliation of basal layer absent between monads, the contact zones consisting of tightly packed thin foliations only. Hexalobus – type A (Figure 4) SEM: Ornamentation granular to gemmate; granules/gemmae 0.4 – 1.5 mm in diameter. TEM (A. bussei): Exine 2.1 – 4.0 mm thick. Tectum 0.7 mm, consisting of granules/gemmae, hardly or not distinguishable from the granular infratectum. Basal layer consisting of 2 – 4 undulate foliations; outer foliation not much thicker, 0.15 mm. Species included: H. bussei, H. mossambicensis. Hexalobus – type B (Figure 5) SEM: Ornamentation areolate-verrucate to/or rugulate; muri 0.6 – 1.9 mm wide. TEM (H. crispiflorus): Exine 2.4 – 3.9 mm thick. Tectum 1.2 mm. Infratectum 1.5 mm, columellate/ granular. Basal layer consisting of 2 – 3 undulate foliations; outer foliation not or slightly thicker, 0.1 – 0.3 mm. Species included: H. crispiflorus, H. salicifolius. Note: Both species included into the Hexalobus – type B exhibit a continuous ornamentation range from areolate-verrucate (Figure 5D, E, H) to rugulate (Figure 5B, F, I). Hexalobus – type C (Figure 6) SEM: Ornamentation psilate; perforations 0.1 – 0.4 mm in diameter. TEM: Exine 3.6 mm thick. Tectum 1.4 mm. Infratectum 1.9 mm, columellate/granular. Basal layer consisting of 2 – 3 undulate foliations; outer foliation hardly to clearly thicker, 0.1 – 0.4 mm. Species included: H. monopetalus. R Figure 3. Pollen of Asteranthe. A, B. A. asterias (Sacleux 712): cross-sections of pollen wall showing columellate infratectum (SEM and TEM). C, D. A. asterias (Robertson 3878): tetrad and detail of foveolate tectum. E–G. A. lutea (Couvreur 46): tetrad, detail of foveolate tectum, and four tetrads in anther locule. Scale bars – 10 mm (C, E, G); 5 mm (F), 1 mm (A, B, D). C5columella, G5granule, F5foliation, OF5outer foliation, T5tectum. 192 T. L. P. Couvreur et al. Pollen of five African Annonaceae 193 Figure 4. Pollen type A of Hexalobus. A–C. H. bussei (Bos 5370): cross-section of pollen wall (TEM), tetrad and detail of granular to gemmate tectum. D, E. H. mossambicensis (Gomes e Sousa 4897): tetrad and detail of granular to gemmate tectum. Scale bars – 10 mm (B, D), 1 mm (A, C, E). C5columella, G5granule, F5foliation, OF5outer foliation, T5tectum. 194 T. L. P. Couvreur et al. Figure 5. Pollen type B of Hexalobus. A & F. H. crispiflorus (Liben 2390): cross-section of pollen wall (TEM) and detail of rugulate tectum. B. H. crispiflorus (Hoyle 789): detail of areolate-verrucate to rugulate tectum. C, D. H. crispiflorus (J. J. de Wilde 7909): tetrad and detail of Pollen of five African Annonaceae 195 Figure 6. Pollen type C of Hexalobus. A–C. H. monopetalus (Breteler 7288): cross-section of pollen wall (TEM), tetrad and detail of psilateperforate tectum. Scale bars – 10 mm (B); 1 mm (A, C). C5columella, G5granule, F5foliation, OF5outer foliation, T5tectum. 3. Isolona (Figures 7–9, 14) Previous observations. — LM: Walker (1971b); SEM and TEM: Le Thomas & Lugardon (1976), Le Thomas (1980). Present observations. — 19/20 species studied. SEM: Pollen grains solitary, inaperturate, apolar, subspheroidal, L (length)532 (39.7) 46 mm, B (width) 525 (33.0) 42 mm, L/B51.07 (1.20) 1.33 mm. Ornamentation scabrate to verrucate (type A), finely rugulate (type B) or more coarsely rugulate (type C). TEM: Exine 1.0 – 2.3 mm thick proximally reduced. Tectum 0.4 – 1.5 mm, sometimes hardly or not distinguishable as a separate layer. Infratectum granular to columellate/granular, 0.5 – 1.5 mm. Basal layer consisting of 2 – 3 undulate foliations; outer foliation not thicker, v0.1 mm. r areolate-verrucate tectum. E. H. crispiflorus (Chevalier 13385): detail of areolate-verrucate tectum. G, H. H. salicifolius (Zenker 3330): tetrad and detail of areolate-verrucate tectum. I. H. salicifolius (Letouzey 8122): detail of areolate-verrucate to rugulate tectum. Scale bars – 10 mm (C, G); 1 mm (A, B, D–F, H, I). C5columella, G5granule, F5foliation, OF5outer foliation, T5tectum. 196 T. L. P. Couvreur et al. Figure 7. Pollen type A of Isolona. A. I. thonneri (Letouzey 10205): cross-section of pollen wall (TEM). B, C. I. thonneri (Letouzey 12111): pollen grain (monad) and detail of scabrate tectum. D–F. I. humbertiana (Perrier 1511): cross-section of pollen wall (TEM), two pollen grains (monads), detail of verrucate tectum. G. I. capuronii (Service Forestier de Madagascar 8941): detail of scabrate tectum. H. I. pilosa (Le Testu 8602): detail of scabrate tectum. Scale bars – 10 mm (B, E); 1 mm (A, C, D & F–H). C5columella, G5granule, F5foliation, OF5outer foliation, T5tectum. Pollen of five African Annonaceae 197 198 T. L. P. Couvreur et al. Figure 9. Pollen type C of Isolona. A, B & H. I. ghesquierei (Service Forestier de Madagascar 8587): cross-section of pollen wall (TEM), pollen grain (monad) and detail of rugulate tectum. C, D. I. congolana (Leeuwenberg 9550): cross-section of pollen wall (TEM) and detail of rugulate tectum. E. I. cauliflora (Polhill 4782): detail of rugulate tectum. F. I. linearis (Frimodt-Möller TZ59): detail of rugulate tectum. G. I. lebrunii (Deville 234): detail of rugulate tectum. Scale bars – 10 mm (B); 1 mm (A, C–H). C5columella, G5granule, F5foliation, OF5outer foliation, T5tectum. r Figure 8. Pollen type B of Isolona. A, D & F. I. campanulata (De Koning 6748): cross-section of pollen wall (TEM), two pollen grains (monads) and detail of finely rugulate tectum. B, E. I. hexaloba (J. J. de Wilde 839 WALK-B): pollen grain (monad) and detail of finely rugulate tectum. C. I. hexaloba (Letouzey 10419): cross-section of pollen wall (TEM). G. I. cooperi (Bos 1609): detail of finely rugulate tectum. Scale bars – 10 mm (B, D); 1 mm (A, C, E–G). C5columella, G5granule, F5foliation, OF5outer foliation, T5tectum. Pollen of five African Annonaceae Isolona – type A (Figures 7, 14A) SEM: Ornamentation scabrate to verrucate; scabrae/ verrucae 0.1 – 3.0 mm in diameter. TEM (I. humbertiana, I. thonneri): Exine 1.0 – 1.4 mm thick. Tectum 0.4 – 1.5 mm, consisting of scabrae/verrucae, hardly or not distinguishable from the granular infratectum. Basal layer consisting of 2 – 3 foliations; outer foliation not thicker, v0.1 mm. Foliations tightly packed at the distal side, but loose and undulate proximally. Species included: I. capuronii, I. deightonii, I. dewevrei, I. heinsenii, I. humbertiana, I. madagascariensis, I. perrieri, I. pilosa, I. thonneri. Isolona – type B (Figures 8, 14C) 199 granular. Basal layer consisting of 2 – 5 loose, undulate foliations; outer foliation thicker, 0.3 – 0.4 mm. Tectum, infratectum and thick outer foliation of basal layer absent between monads, the contact zones consisting of tightly packed thin foliations only. Monodora – type A1 (Figure 10) SEM: Ornamentation psilate; perforations up to 0.4 mm in diameter. TEM (M. myristica): Exine 3 mm thick. Tectum 1.6 mm. Infratectum 0.6 mm, columellate/granular. Basal layer consisting of 2 – 4 loose, undulate foliations; outer foliation thicker, 0.3 mm. Species included: M. laurentii, M. minor, M. myristica, M. stenopetala, M. tenuifolia, M. undulata. Monodora – type A2 (Figure 11) SEM: Ornamentation finely rugulate; muri 0.2 – 0.8 mm wide. TEM (I. campanulata, I. hexaloba): Exine 1.0 – 1.9 mm thick. Tectum 0.4 mm. Infratectum columellate/granular, 0.5 – 0.7 mm. Basal layer consisting of 2 – 3 moderately undulate foliations; outer foliation not thicker, v0.1 mm; proximal foliations loose. Species included: I. campanulata, I. cooperi, I. hexaloba, I. pleurocarpa, I. zenkeri. SEM: Ornamentation rugulate/locally psilate; perforations 0.1 – 0.5 mm in diameter. TEM (M. crispata): Exine 3.2 mm thick. Tectum 0.8 mm. Infratectum 0.7 mm, columellate/granular. Basal layer consisting of 3 – 4 loose, undulate foliations; outer foliation thicker, 0.4 mm. Species included: M. carolinae, M. crispata, M. grandideri, M. zenkeri. Isolona – type C (Figures 9, 14B, D) Monodora – type B (Figure 12) SEM: Ornamentation rugulate; muri 0.6 – 1.5 mm wide. TEM (I. congolana, I. ghesquierei): Exine 2.0 – 2.3 mm thick. Tectum 0.4 – 0.8 mm. Infratectum granular to columellate/granular, 0.9 – 1.5 mm. Basal layer consisting of 2 – 3 moderately undulate foliations; outer foliation not thicker, v0.1 mm. In I. ghesquierei the proximal exine consists of 2 – 3 loose foliations, often with the inclusion of granules (data proximal exine I. congolana not available). Species included: I. cauliflora, I. congolana, I. ghesquierei, I. lebrunii, I. linearis. 4. Monodora (Figures 10–12) Previous observations. — LM: Walker (1971b), SEM and TEM: Le Thomas (1980, 1983). Present observations. — 14/14 species studied. SEM: Pollen in acalymmate, tetragonal tetrads, 54 (77.8) 110 mm in diameter. Constituent monads inapertuarate, P524 (40.4) 60 mm, E530 (41.9) 52 mm, P/E50.80 (0.96) 1.18. Ornamentation psilate with small perforations (type A1), rugulate/locally psilate with small perforations (type A2) or rugulate with relatively large perforations (type B). TEM: Exine 3.0 – 3.6 mm thick. Tectum 0.6 – 1.6 mm. Infratectum 0.6 – 1.1 mm, columellate/ SEM: Ornamentation rugulate; perforations 0.3 – 1.0 mm in diameter. TEM (M. angolensis): Exine 3.6 mm thick. Tectum 0.6 mm. Infratectum 1.1 mm, columellate/ granular. Basal layer consisting of 4 – 5 loose, undulate foliations; outer foliation thicker, 0.4 mm. Species included: M. angolensis, M. globiflora, M. hastipetala, M. junodii. 5. Uvariastrum (Figure 13) Previous observations. — LM: Walker (1971b), Le Thomas (1974); TEM and SEM: Le Thomas (1980, 1983). Present observations. — 6/8 species studied. SEM: Pollen in acalymmate, tetragonal tetrads, 52 (74.2) 107 mm in diameter. Constituent monads inaperturate, P522 (34.2) 47 mm; E533 (46.7) 64 mm, P/E50.67 (0.73) 0.79. Ornamentation rugulate, sometimes locally psilate; perforations up to 0.5 mm. TEM (U. pierreanum, U. pynaertii): Exine 3.5 – 5.6 mm thick. Tectum 1.1 – 1.8 mm. Infratectum 2.0 – 3.2 mm, columellate/granular. Basal layer consisting of 4 – 6 loose, undulate foliations; outer foliation thicker, 0.6 – 0.7 mm. Tectum, infratectum and thick outer foliation of basal layer absent between 200 T. L. P. Couvreur et al. Figure 10. Pollen type A1 of Monodora. A. M. myristica (Letouzey 11474): cross-section of pollen wall (TEM). B, F. M. undulata (Bos 2306): tetrad and detail of psilate-perforate tectum. C, E. M. myristica (De Koning 1146): tetrad and detail of psilate-perforate tectum. D, G. M. minor (Mgaza 783): tetrad anddetailofpsilate-perforatetectum.Scalebars–10 mm(B–D);1 mm(A,E,F).C5columella,G5granule,F5foliation,OF5outerfoliation,T5tectum. Pollen of five African Annonaceae 201 Figure 11. Pollen type A2 of Monodora. A–D. M. crispata (W. J. de Wilde 867): cross-section of pollen wall (TEM), contact zone between two monads showing presence of thin foliations and absence of thicker outer foliation (TEM), tetrad and detail of rugulate (locally psilate) 202 T. L. P. Couvreur et al. Figure 12. Pollen type B of Monodora. A–D. M. angolensis (Van Valkenburg 2688): (A) cross-section of pollen wall (TEM); (B) contact zone between two monads showing presence of thin foliations and absence of thicker outer foliation (TEM); (C) tetrad; (D) detail of rugulate Pollen of five African Annonaceae 203 Figure 13. Pollen of Uvariastrum. A. U. pyneartii (Le Testu 8473): cross-section of pollen wall (TEM). B, F. U. insculptum (Breteler 5811): detail of rugulate tectum and tetrad. C. U. pierreanum (Letouzey 10225): cross-section of pollen wall (TEM). D, H. U. zenkeri (Bos 6266): 204 T. L. P. Couvreur et al. monads, the contact zones consisting of thin foliations only. Species included: U. germainii, U. hexaloboides, U. insculptum, U. pierreanum, U. pynaertii, U. zenkeri. Discussion Intergeneric variation The almost complete species-level sampling of the five genera within the Monodora clade showed a wide pollen morphological diversity. The most conspicuous variation concerns the occurrence of monads without a thicker outer foliation in Isolona in contrast to tetrads with a thicker outer foliation in the basal exine layer in Asteranthe, Hexalobus, Monodora and Uvariastrum. Canright (1963) described the monads of Isolona as monosulcate, while Walker (1971b) characterized them as inaperturate. Le Thomas (1980) confirmed the latter view, stating that the pollen grains do not possess any distinct (distal) apertural structure, but instead show a clearly reduced proximal exine in crosssection (I. hexaloba, I. thonneri; TEM). We also observed such a (probably) proximal thinning in I. campanulata, I. ghesquierei and I. humbertiana (Figure 14). We did not find it in I. congolana, however, possibly because the plane of sectioning was not through the proximal pole (Figure 14B). The phylogenetic analysis by Couvreur et al. (2008) clearly showed Isolona to be nested in the ‘African long branch clade’ (ALBC; Figure 1), which, except for Isolona, is characterized by tetrad pollen. This topology demonstrates that the Isolona monads are not transitional between aperturate monads and inaperturate tetrads (Le Thomas, 1980, 1981), but that they represent a derived state relative to the tetrads, which confirms conclusions reached by Doyle & Le Thomas (1996). The reduced proximal exine of Isolona pollen would then be a relic of the thin proximal exine of an ancestral tetrad condition. The observation of Le Thomas (1980: p. 322, 340) that I. thonneri pollen has a prolonged developmental tetrad stage fits very well in this view: the longer the tetrad stage lasts, the less space/time there is for proximal exine growth. An explanation for the occurrence of tetrads and monads within the Monodora clade could come from the involvement of different pollen vectors. Unfortunately, very little is known about the pollination biology within the Monodora clade; more data is needed in order to adequately tackle these questions. A similar case of evolution from tetrads to monads, also unexplained, occurred in the Winteraceae, in the genus Zygogynum s. s. (Van der Ham & Van Heuven, 2002). The locally reduced exine of Isolona pollen seems to be fundamentally different from that found in the miliusoid clade of the ‘short branch clade’ (SCB, Mols et al., 2004). Most genera in the miliusoid clade have monad pollen, in which an exine thinning, if present, probably has a distal position, and therefore would represent an apertural structure. The scarce tetrads in the miliusoid clade, present in Mitrephora, Petalolophus and Pseuduvaria, appeared to be derived (twice), being nested in monad subclades (Mols et al., 2004). Further intergeneric variation pertains to the structure of the infratectum. Both basic angiosperm types of infratectal structure, columellate and granular (Le Thomas, 1980, 1981), are represented within the Monodora clade. Asteranthe is the only genus characterized by a strictly columellate infratectum, while Isolona and Hexalobus contain species with a strictly granular infratectum. All other representatives of the clade possess an intermediate infratectum type, showing columellae mixed with granules. So, the latter type is the commonest within the Monodora clade. H. monopetalus was previously thought to have an exclusively columellate infratectum (Le Thomas, 1980, 1981; Le Thomas & Lugardon, 1976), but granules are clearly present in the material studied by us (different from that used by Le Thomas). Thus, H. monopetalus is better defined as having an intermediate infratectum type, though with a dominance of columellae. All genera of the Monodora clade share a basal layer consisting of foliations which is, however, also common in numerous other African genera with tetrad pollen (Le Thomas, 1980). Except for Isolona, all genera in the Figure 11. Cont. tectum. E, F. M. zenkeri (Breteler 2747): tetrad and detail of folded rugulate (locally psilate) tectum. G. M. carolinae (Philipson 4940): detail of rugulate (locally psilate) tectum. Scale bars – 10 mm (C, E); 5 mm (F); 1 mm (A, B, D, G). C5columella, G5granule, F5foliation, OF5outer foliation, T5tectum. Figure 12. Cont. tectum. E, F. M. junodii (Torre and Paiva 9035): tetrad and detail of rugulate tectum. G. M. globiflora (Luke 3136): detail of rugulate tectum. Scale bars – 10 mm (C, E); 2 mm (A, B); 1 mm (D, F, G). C5columella, G5granule, F5foliation, OF5outer foliation, T5tectum. r detail of rugulate tectum and tetrad. E, I. U. germainii (Lebrun 5977): tetrad and detail of rugulate tectum. G. U. hexaloboides (Breteler 11894): detail of rugulate tectum. Scale bars – 10 mm (E, F, H); 1 mm (A–D, G, I). C5columella, G5granule, F5foliation, OF5outer foliation, T5tectum. Pollen of five African Annonaceae 205 Figure 14. Pollen grains (monads) of Isolona (TEM). A. I. humbertiana (Perrier 1511): pollen wall showing (probably) proximal thinning (arrow). B. I. congolana (Leeuwenburg 9550): note absence of proximal thinning, which might be due to orientation of section. C. I. campanulata (De Koning 6748): pollen wall showing (probably) proximal thinning (arrow). D. I. ghesquieri (Service Forestier de Madagascar 8587): pollen wall showing (probably) proximal thinning (arrow). Scale bar – 5 mm (A–D). Monodora clade show a relatively thick outer foliation. Contrary to Doyle and Le Thomas (1994), Hexalobus also shows a thickened outer foliation, though less obviously so than the other genera. The deviating pollen of Isolona within the Monodora clade implies that the monophyly of this clade as indicated by molecular evidence, cannot be demonstrated using a pollen morphological criterium. 206 T. L. P. Couvreur et al. Figure 15. Bayesian majority rule consensus tree of Isolona and Monodora using five plastid markers. Thick branches indicate posterior probabilities of w0.95. Infrageneric variation Isolona, Hexalobus and Monodora exhibit the largest amount of pollen morphological variation, especially with regard to ornamentation, each of these three genera being subdivided into several pollen types (Figures 4–12). Hexalobus is remarkable in that three types occur in five species only. The two species belonging to Hexalobus type B (H. crispiflorus and H. salicifolius; Figure 5) show large infraspecific variation of the ornamentation, ranging from areolateverrucate to rugulate, which is unique within the Monodora clade. Interestingly, Uvariastrum, the sister genus of Hexalobus, exhibits hardly any variation of the ornamentation (Figure 13). Why there is such a contrast between these two small genera is difficult to explain. They have similar distributions, mainly in the Guineo-Congolian region in West-Central Africa, with one or two species occurring in East Africa. Moreover, they display the same amount of macromorphological variation, e.g. Hexalobus does not present a strikingly larger amount of variation in its flowers than Uvariastrum. As with the presence of tetrads and monads within the Monodora clade, an explanation for the wide ornamentation range within Hexalobus might be the occurrence of different pollination syndromes. For instance, pollen ornamentation has Pollen of five African Annonaceae 207 Figure 16. Maximum parsimony optimization of the various pollen types on the Bayesian majority rule consensus tree. A. Isolona: white: type A, black: type B, gray: type C. B. Monodora: white: type A1, black: type A2, gray: type B. Posterior probabilities w0.90 are indicated below the branches. in some cases been shown to be correlated with the type of pollen vector (e.g. Hesse, 2000; Osborn et al., 1991; Tanaka et al., 2004). However, very little is known about pollinators within the Monodora clade. Taxonomic significance of pollen characters Given that Isolona and Monodora are the two most species-rich genera (together 34 of 49 species) within the Monodora clade, species-level molecular phylogenies should provide a reasonable guideline in assessing the usefulness of pollen characters for infrageneric classification within this clade using the Bayesian majority consensus rule tree (Figure 15). When the different pollen types, which are based on pollen ornamentation, are optimized on the trees using the maximum parsimony method (Figures 16), there appears to be no taxonomic information for the deeper relationships within both genera, i.e. no major clade is characterized by a particular pollen type. The largest clade within Isolona contains all three pollen types. Within Monodora, both the West-Central and the East African clades contain representatives of each pollen (sub)type. However, pollen characters appear more informative within smaller groups of species. Several groups of closely related species have similar pollen morphologies. For example, the West-Central clade within Monodora, excluding M. angolensis, contains species with quite dissimilar macromorphologies, except maybe for M. myristica and M. undulata, which are less disparate. Some species have a unique macromorphology (M. tenuifolia, M. laurentii) or resemble more distantly related species (M. crispata with M. angolensis or M. grandidieri). On the other hand, pollen morphology shows little variation within this group, all species belonging to pollen type A, and most of them to subtype A1, which is in agreement with the molecular data (Figure 16). Species found in the two early diverging clades within Isolona are also united by the same pollen type (B) with the exception of I. congolana (type C). Strongly supported sister species in general possess the same pollen type (Figure 16), except for I. heinsenii and I. linearis, and I. congolana and I. hexaloba. In the latter case, this difference might be explained by a shift in habitat, with I. congolana generally growing in montane forests above 900 m, while I. hexaloba is restricted to lowland rain forests 208 T. L. P. Couvreur et al. below 700 m. Indeed, such a difference in habitat could imply a difference in pollinating vectors which could have led to these differences. In the former, the variation is harder to explain because both species are restricted to the montane forests of the Eastern Arc in Tanzania, although hardly occurring in sympatry (Couvreur et al., 2006). Pollen morphology has also been very useful to distinguish morphologically similar species. Verdcourt (1986) identified a small Eastern Arc Mountain population in Tanzania as being part of the West-Central African species I. hexaloba. Pollen, however, provided support for the description of a new species (Couvreur et al., 2006), I. linearis, which is strongly supported by the molecular phylogeny, i.e. I. linearis does not cluster with I. hexaloba. Thus, pollen characters at the infrageneric level would appear to have a mixed utility. They provide little information for characterizing major clades within genera, but they do seem to contain information regarding closely related species. In addition they can be used to a certain extent to support taxonomic decisions. The taxonomic significance of pollen characters within the other three genera is hard to assess without a molecular phylogeny. A case worth mentioning is that of H. bussei and H. mossambicensis. Both species show many morphological as well as ecological differences. The latter species is a shrub or a small tree distributed in the xeric southern part of East Africa, while the former is a large rain forest tree endemic to Cameroon. Furthermore, H. bussei has the largest flowers within the genus, while H. mossambicensis has the smallest flowers. Despite numerous differences, both species present strong pollen morphological affinities, having a granular to gemmate exine ornamentation (Figure 4). In view of the macromorphological differences as well as the large geographical separation, it is hard to suggest close relationship between both species. Clearly this case deserves further investigation. Acknowledgements Specimens investigated Pollen samples Vouchers and specimens used for TEM (Genus; species; country; herbarium voucher; herbarium acronym; TEM5N). Pollen photos from specimens marked with an asterisk (*) were provided by A. Le Thomas (Muséum National d’Histoire Naturelle, Paris). Asteranthe A. asterias. Kenya; Robertson 3878; WAG A. asterias. Tanzania; Sacleux 712*; P; N A. lutea. Tanzania; Couvreur 46; WAG Hexalobus H. bussei. Cameroon; Zenker 3889; P H. bussei. Cameroon; Bos 5370; WAG; N H. crispiflorus. Congo; Schaijes 3596; BR H. crispiflorus. Sudan; Hoyle 789; FHO H. crispiflorus. Guinea; Chevalier 13385; P H. crispiflorus. Guinea; Pobéguin 844; P H. crispiflorus. Guinea-Bissau; Espirito Santo 3841; WAG H. crispiflorus. Ivory Coast; Jongkind 4386; WAG; N H. crispiflorus. Cameroon; J. J. de Wilde 7909; WAG H. crispiflorus. Congo; Liben 2390; WAG; N H. monopetalus. Tanzania; Gillman 1090; EA H. monopetalus. Zambia; Brenan 7856; FHO H. monopetalus. South Africa; Schlieben 7432; G H. monopetalus. Togo; Breteler 7288; WAG; N H. monopetalus. Mali; Diarra 367*; P; N H. mossambicensis. Mozambique; Gomes e Sousa 4897; COI H. mossambicensis. Mozambique; Pedro 5189; EA H. salicifolius. Cameroon; Letouzey 8122; BR H. salicifolius. Cameroon; Zenker 3330; UPS Isolona I. campanulata. Ivory Coast; De Koning 6748; WAG; N I. capuronii. Madagascar; Service Forestier de Madagascar 8941; P I. cauliflora. Kenya; Polhill 4782; C I. congolana. Cameroon; Leeuwenberg 9550; WAG; N I. congolana. Congo; Lejoly 4961; BR I. cooperi. West Africa; Bos 1609; WAG I. deightonii. Ivory Coast; Bernardi 8691; US I. dewevrei. Ghana; Merello 1346; US I. ghesquierei. Madagascar; Service Forestier de Madagascar 8587; P; N I. heinsenii. Tanzania; Schlieben 1539; G Annick Le Thomas and Thierry Deroin are deeply thanked for allowing access and use of the pollen archives of Annick Le Thomas (Paris). Marc Sosef and James Richardson are thanked for critically reading through earlier versions of the manuscript. Bruce Sampson and one other anonymous reviewer are also thanked for their valuable comments on an earlier version of our paper. We are grateful to Wim Star and Ben Kieft for preparing the TEM views and photo plates, respectively. I. hexaloba. Gabon; J. J. de Wilde 839; WAG I. hexaloba. Gabon; Sosef 2244; WAG I. hexaloba. Cameroon; Letouzey 10419*; P; N I. humbertiana. Madagascar; Perrier 1511; P; N I. madagascariensis. Madagascar; Service Forestier de Madagascar 11409*; P I. lebrunii. Congo; Deville 234; BR; I. linearis. Tanzania; Frimodt-Möller TZ59; C I. perrieri. Madagascar; Du Puy MB512; P I. perrieri. Madagascar; Perrier 18714*; P Pollen of five African Annonaceae I. pilosa. Gabon; Le Testu 8602; WAG I. pilosa. Gabon; Le Testu 8740; WAG I. pleurocarpa. Cameroon; Leeuwenberg 9784; WAG I. thonneri. Cameroon; Letouzey 10205*; P; N I. thonneri. Cameroon; Letouzey 12111; WAG I. zenkeri. Gabon; Sosef 2232; WAG I. zenkeri. Congo; Cabalion 144; WAG Monodora M. angolensis. Congo; Hart 1593; WAG M. angolensis. Gabon; Van Valkenburg 2688; WAG; N M. angolensis. Central African Republic; Tisserant 1858*; P M. carolinae. Tanzania; Philipson 4940; C M. crispata. Ivory Coast; W. J. de Wilde 867; WAG; N M. globiflora. Tanzania; Luke 6724; EA M. globiflora. Tanzania; Luke 3136; MO M. grandidieri. Mozambique; Luke 10104; MO M. grandidieri. Kenya; Lesley 163; WAG M. grandidieri. Kenya; Sacleux 958*; P M. hastipetala. Tanzania; Philipson 4958; MO M. junodii. Mozambique; Torre & Paiva 9035; WAG M. laurentii. Congo; De Giorgi 1617; BR M. minor. Tanzania; Mgaza 783; EA M. myristica. Ivory Coast; De Koning 1146; WAG M. myristica. Cameroon; Letouzey 11474*; P; N I. congolana. DR Congo; Liben 3852; EU216704; EU216613; EU216658; EU216637 209 WAG. I. cooperi. Cultivated; Bot. Tuinen Utrecht, 473; U. AY841704; EU216612; EU216657; EU216636; EU216681 I. dewevrei. Ghana; Merello 1346; MO. EU216715; EU216624; EU216669; EU216645; EU216690 I. ghesquierei. Madagascar; Schatz 3364; MO. EU216709; EU216618; EU216663 I. heinsenii. Tanzania; Couvreur 10; WAG. EU216710; EU216619; EU216664; EU216640; EU216686 I. hexaloba. Cameroon; Burgt 791; WAG. EU169763; EU169808; EU169740; EU169717; EU216683 I. linearis. Tanzania; Couvreur 102; WAG. EU216711; EU216620; EU216665; EU216641; EU216687 I. maitlandii. Cameroon; Leeuwenberg 9550; WAG. EU216714; EU216623; EU216668; EU216644; EU216688 I. perrieri. Madagascar; Carlson 48; K. EU216707; EU216616; EU216661; EU216639; EU216685 I. pleurocarpa. Cameroon; van Andel 4177; WAG. EU216712; EU216621; EU216666; EU216642 I. thonneri. Cameroon; Letouzey 10205; P. EU216713; EU216622; EU216667; EU216643 I. zenkeri(1). Gabon; Sosef 2250; WAG. EU216705; EU216614; EU216659; EU216638; EU216684 I. zenkeri(2). Gabon; Sosef 2322; WAG. EU216706; EU216615; EU216660 M. stenopetala. Mozambique; Simaõ 1196; COI M. tenuifolia. Ivory Coast; De Koning 982; WAG M. tenuifolia. Cameroon; Letouzey 4978*; P M. undulata. Liberia; Bos 2306; WAG M. undulata. Cameroon; Letouzey 10120*; P M. zenkeri. Cameroon; Breteler 2747; WAG Uvariastrum U. hexaloboides. Zambia; Breteler 11894; WAG U. germainii. Congo; Germain 213; P U. germainii. Congo; Lebrun 5977; P U. insculptum. Ivory Coast; Aké Assi 16772; G U. insculptum. Ivory Coast; Breteler 5811; WAG U. pierreanum. Cameroon; Letouzey 10225*; P; N U. pynaertii. Gabon; Le Testu 8473*; P; N U. zenkeri. Cameroon; Thomas 4334; US U. zenkeri. Cameroon; Bos 6266; WAG. Monodora M. angolensis. West Africa; Alpen S4013; WAG. EU216718; EU216627; EU216672; EU216648; EU216696 M. carolinae. Tanzania; Couvreur 54; WAG. EU216723; EU216632; EU216677; EU216653; EU216700 M. crispata. UUBC; Chatrou 476; U. EU169811; EU169743; EU169720; EU216691 AY841715; M. globiflora. Tanzania; Couvreur 99; WAG. EU216724; EU216633; EU216678; EU216654; EU216701 M. grandidieri. Tanzania; Vollesen 3031; WAG. EU216719; EU216628; EU216673; EU216649; EU216697 M. hastipetala. Tanzania; Couvreur 42; WAG. EU216725; EU216634; EU216679; EU216655; EU216702 M. junodii. Tanzania; Couvreur 88; WAG. EU216721; EU216630; EU216675; EU216651; EU216699 M. laurentii. Gabon; Niangadouma 179; WAG. EU216720; EU216629; EU216674; EU216650; EU216698 M. minor. Tanzania; Couvreur 36; WAG. EU216726; EU216635; U216680; U216656; U216703 Molecular samples M. myristica (1). UUBC; Chatrou 477; U. AY743466; EF179347; DQ125129; EF179305; EU216692 Vouchers and GenBank accession numbers (trnL-trnF; trnSG; psbA-trnH; ndhF; trnD-trnT) for each of the five chloroplast markers used. M. myristica (2). Cameroon; Richardson 191; WAG. EU216716; EU216625; EU216670; EU216646; EU216693 UUCB – University Utrecht Botanical Garden; DRC – Democratic Republic of Congo. M. stenopetala. Mozambique; Correia 3840; M. EU216722; EU216631; EU216676; EU216652 Isolona M. tenuifolia. Ghana; Schmidt 2025; MO. EU216717; EU216626; EU216671; EU216647; EU216694 I. campanulata. UUBC; Chatrou 472; U. EF179318, EF179343, DQ125127, EF179301, EU216689 M. undulata. Cultivated; Alpin 4012; WAG. EU169766; EU169813; EU169744; EU169722; EU216695 I. capuronii. Madagascar; Serv. Forest. Madagascar 8941; P. EU216708; EU216617; EU216662 Uvariopsis I. cauliflora. Kenya; Robertson 7555; WAG. EU169762; EU169807; EU169739; EU169716; EU216682 U. vanderystii. Gabon; Sosef 2241; WAG. EU169773; EU169821; EU169752; EU169728 210 T. 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