Variation in anther and pollen morphology inLeucaenaBenth. (Leguminosae-Mimosoideae)

Botanical Journal of the Linnean Society (1997), 123: 177–196. With 37 figures

Variation in anther and pollen morphology in Leucaena Benth. (Leguminosae-Mimosoideae) COLIN E. HUGHES Department of Plant Sciences, University of Oxford, South Parks Rd., Oxford, OX1 3RB Received September 1996, accepted for publication November 1996

Variation in anther and pollen morphology is described and illustrated for species of Leucaena and a selection of species from closely related genera. This is discussed in relation to the variation found in the closely related genera of the informal Leucaena, Dichrostachys and Xylia groups of the tribe Mimoseae. A number of characters for cladistic analysis are identified and putative homologies assessed. Preliminary taxonomic conclusions about the status and relationships of L. multicapitula are discussed. Mode of anther dehiscence is also described and illustrated. The observation of persistent intact tapetal membranes after anther dehiscence in some species of Leucaena is discussed in relation to pollen unit.  1997 The Linnean Society of London

ADDITIONAL KEY WORDS:—apiculum – polyad – tapetal membrane. CONTENTS Introduction . . . . . Material and methods . . Anthers . . . . . Pollen . . . . . Results . . . . . . . Anthers . . . . . Anther hairs . . . Apicula . . . . . Anther dehiscence . Pollen . . . . . Apertures . . . . Pollen unit . . . . Pollen wall architecture Discussion . . . . . . Acknowledgements . . . References . . . . . . Appendix . . . . . .

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INTRODUCTION

As part of a wider systematic study of Leucaena Benth., the comparative morphology of anthers and pollen has been investigated using scanning electron microscopy (SEM). The study will evaluate species delimitation and relationships as well as generic boundaries. However, given the considerable diversity of pollen and anther 0024–4074/97/030177+20 $25.00/0/bt960083

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morphology encountered, and the observation of unusual persistent intact tapetal membranes after anther dehiscence, results are presented here in advance of the complete morphological analysis, in the expectation that they may be of general interest. In addition, variation in pollen and anther morphology provides new data of immediate taxonomic importance for the resolution of long-standing confusion about the status and relationships of L. multicapitula Schery, and the alliance of closely related species that comprises L. trichodes ( Jacq.) Benth. L. macrophylla Benth. and L. nelsonii Britton & Rose.

MATERIAL AND METHODS

Anthers Anthers were prepared following the procedures of Endress & Stumpf (1991). Anthers of 17 species of Leucaena along with a selection of species from the closely related genera Alantsilodendron Villiers, Calliandropsis H.M. Hern. & Guinet, Schleinitzia Warb. and Xylia Benth. were dissected either from flowers fixed in the field in FAA (1:1:18 mixture of 40% formaldehyde, glacial acetic acid and 70% alcohol), or from dried herbarium specimens which were gently rehydrated by boiling in water for 3 minutes and lightly fixed in FPA (1:1:18 mixture of 40% formaldehyde, glacial propionic acid and 70% ethanol). Anthers were dehydrated using a 20% step ethanol series (20–100%), critical point dried, mounted on aluminium stubs with a thin layer of Araldite, sputter-coated with gold palladium and viewed in a Hitachi S800 SEM. Vouchers for anther samples are listed in the Appendix. The remaining species of Leucaena which were not included in the SEM survey were examined using light microscopy. Additional SEM pictures of anthers and/or anther glands of Desmanthus balsensis J.L. Contreras and Dichrostachys cinerea (L.) Wight & Arn. from Luckow (1993: 7), Neptunia plena (L.) Benth. and Alantsilodendron glomeratum Villiers from Luckow (1995: 66) and a wider range of mimosoid genera illustrated and described by Luckow & Grimes (in press) were also examined and are discussed below.

Pollen Pollen of 23 taxa of Leucaena, as well as two species of Xylia and one of Schleinitzia, was surveyed in the present study (herbarium vouchers listed in Appendix 1). Pollen was extracted from mature unopened buds taken from dried herbarium specimens by soaking in water and macerating to release the pollen. Pollen was sieved through a steel 125 lm mesh into labelled centrifuge tubes with approx. 2 ml distilled water. Pollen was acetolysed using the hot acetolysis method of Ertdman (1960), mounted on aluminium stubs with exposed film mount attached with Araldite glue, sputter coated with gold palladium and viewed using a Hitachi S800 SEM. For species with very loosely acalymmate polyads which disintegrate during acetolysis, more or less intact polyads were extracted with care from the anthers and observed without acetolysis. Guinet’s (1969, 1981b) surveys of Mimosoideae pollen along with pollen analysis and pollen photographs of Desmanthus Willd. and Neptunia Lour. (Luckow, 1993:

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9–16), analysis of other genera in the Dichrostachys group (Luckow, 1995: 68), pictures of pollen of three species of Schleinitzia (Nevling & Niezgoda, 1978: 349, 351), descriptions of Calliandropsis pollen (Herna´ndez & Guinet, 1990: 614, 616), of Gagnebina Neck. ex DC. pollen (Lewis & Guinet, 1985: 467) and pollen of Kanaloa (Lorence & Wood, 1994: 142), have been examined in addition to analysis of pollen of Xylia and Schleinitzia. These sources allow intepretation of the morphological variation found within Leucaena, in the broader systematic context of the informal Leucaena and Dichrostachys groups sensu Lewis & Elias (1981), and permit delimitation of character states for phylogenetic analysis.

RESULTS

Anthers The anthers of Leucaena are uniformly ovate and dorsifixed, although in some species they are dorsifixed so near the base as to appear basifixed. Anthers vary in size from 0.3 to 1.2 mm long and have two C-shaped thecae bound together by a variable connective, with two microsporangia per theca (Figs 1–7) (see also Jain & Vijayaraghavan, 1992 for description and illustration of anthers of L. leucocephala (Lam.) de Wit). The position of the thecae is introrse in the majority of species of Leucaena but strongly latrorse in the three species L. macrophylla, L. trichodes and L. nelsonii, such that the connective is clearly visible between the thecae in both ventral and dorsal views. Variation in anther morphology occurs within Leucaena in the variable occurrence of an extension of the connective which affords the anther with an apiculum, the presence or absence of hairs and the variable stage at which the tapetal membrane is broken during anther dehiscence.

Anther hairs Anthers bearing hairs are relatively infrequent among legumes (Tucker, 1987), occurring only in the genera Bauhinia L., Moldenhauera Schrad. (Caesalpinioideae), Crotalaria L., Indigofera L., Harleyodendron Cowan, Mucuna Adans. (Papilionoideae) and Leucaena (Mimosoideae). Thus, given that within the subfamily Mimosoideae, Leucaena is the only genus with hairy anthers, this is a notable character, particularly as the hairs are visible to the naked eye or at least with a hand lens. They are therefore useful for field identification, although not all species of Leucaena have hairy anthers. Hairy anthers were noted before the genus was established, as section Trichodae of Willdenow’s Acacia, and in the descriptions of Acacia trichandra Zucc. and Acacia trichodes ( Jacq.) Willd., species later transferred to Leucaena, which were named with reference to their hairy anthers. The hairs on the anthers of Leucaena trichodes are particularly conspicuous, accounting for its earlier description as the “hairy-anthered Acacia” (Miller, 1834). Within Leucaena anthers may be hairy, sometimes being described as pilose (most species) (Figs 1–4), or glabrous (L. pulverulenta (Schltdl.) Benth., L. greggii S. Watson and L. retusa Benth. only) (Figs 5–7). For the species with hairy anthers, hairiness varies from sparsely hairy in L. cuspidata Standl., L. esculenta (Moc¸. & Sesse´ ex DC.) Benth., L. pallida Britton & Rose and L. diversifolia (Schltdl.)

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Figures 1–7. Anther morphology and occurrence of anther hairs in Leucaena. Fig. 1. Leucaena involucrata, anther from ventral side. Fig. 2. L. nelsonii, anther from above (dorsal side) showing short dorsi-ventrally flattened apiculum. Fig. 3. L. esculenta anther from dorsal side. Fig. 4. L. macrophylla anther from above left side showing flattened, ‘hooded’ apiculum. Fig. 5. L. pulverulenta, anther from ventral side. Fig. 6. L. greggii, anther from ventral side. Fig. 7. L. pulverulenta, anther from dorsal side showing small rounded apiculum. Scale bars=0.25 mm.

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Benth. to moderately or densely so in the remaining species. The hairs arise mainly on the ventral face of the anthers from the epidermis of the unopened thecae, are sometimes concentrated along the stomial furrow, and are uniformly distributed from base to tip or, in some species, concentrated on the distal half or around the apex of the anther. The hairs range from 0.3 to 0.7 mm in length and in some species are longer than the anthers. Apicula In six species of Leucaena, L. pulverulenta, L. cuspidata, L. retusa, L. macrophylla, L. trichodes and L. nelsonii, the anthers have a short distal protrusion of the connective between the thecae, termed an apiculum by Lewis & Guinet (1985), the anthers being described as apiculate (Figs 2, 4, 7 and 9–11), while in the remaining species the apiculum is lacking (Figs 3, 5, 6 and 8). The apiculum in the Mimoseae has variously been described as “a short point” or “glandular prolongation” (Villiers, 1994), or a “sub-cylindrical apical gland” (Herna´ndez & Guinet (1990), and the anthers as “appendiculate” (thus referring to an “appendage”) (Luckow, 1995); more generally apicula have been simply described as “short connective protrusions” (Parkin, 1951; Hufford & Endress, 1989). The apiculum in Leucaena varies from a small pointed or rounded extension of the connective (as in L. pulverulenta, L. cuspidata and L. retusa, Figs 7, 9 and 10) to a relatively larger, broader, dorsi-ventrally flattened lip that extends forwards over the ventral face of the anther in the form of a small ‘hood’ as in L. macrophylla, L. trichodes and L. nelsonii (Figs 2, 4 and 11). The apiculum arises directly from the connective with no disjunction, and consists of closely-packed fusiform cells with marked reticulate thickenings. Luckow & Grimes (in press) surveyed variation in anther glands across virtually all the genera of the tribes Parkieae and Mimoseae and divided them into three types: the piptadenioid type which are spherical, ellipsoid or clavate and usually stipitate, the gagneboid type which are apiculate, and the pentaclethroid type which are unique to Pentaclethra and are large, dorsally furrowed, with a specialized conical structure on the ventral surface. The apicula observed on the anthers of some species of Leucaena fall into the gagneboid type sensu Luckow & Grimes (1997). Gagneboid apicula have been recorded on the anthers of some species of Alantsilodendron (Figs 13 and 20 and Villiers, 1994: 68–69; Luckow, 1995: 66; Luckow & Grimes, 1997), all species of Gagnebina (Fig. 12 and Lewis & Elias, 1981; Lewis & Guinet, 1985: 468; Luckow, 1995; Luckow & Grimes, in press), some species of Mimosa (Barneby, 1991) and sporadically in Calliandropsis (Herna´ndez & Guinet, 1990: 614). Luckow & Grimes note the unusual occurrence of cells at different angles in the apicula of Gagnebina; this was not observed in Leucaena. The function of the apiculum in Leucaena is unknown, as indeed it appears to be in other mimosoid legume genera. The only investigation of similar swollen anther tips found in some species of Caesalpinia sect. Poincianella (Caesalpinioideae) by Rudall, Myers & Lewis (1994), showed that the tip has mucilage-filled secretory ducts forming discrete regions of secretory tissue, justifying the description of the apiculum as a type of gland, but it is doubtful if this can be extended to other genera without detailed examination. Luckow (1995) treated the appendiculate tips (gagneboid gland type) of the anthers of Alantsilodendron and Gagnebina as homologous with the stipitate anther glands (piptadenioid type) that are commonly found in a wide range of genera in the

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Figures 8–13. Variation in the occurrence and morphology of apicula on the anthers of Leucaena, Gagnebina and Alantsilodendron. Fig. 8. L. esculenta, distal portion of anther from dorsal side showing lack of apiculum. Fig. 9. L. pulverulenta, distal portion of anther from dorsal side showing small rounded apiculum. Fig. 10. L. cuspidata, distal portion of anther from dorsal side showing small pointed apiculum. Fig. 11. L. macrophylla, distal portion of anther from above left side showing short dorsi-ventrally flattened apiculum. Fig. 12. Gagnebina commersoniana, distal portion of linear-elongate anther from dorsal side, showing small pointed apiculum. Fig. 13. Alantsilodendron alluaudiana, distal portion of anther from dorsal side showing small pointed apiculum. Scale bars=0.1 mm.

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Figures 14–19. Stipitate anther glands in Xylia and Schleinitzia and anther dehiscence in Leucaena. Fig. 14. Xylia torreana, distal portion of anther from dorsal side showing round stipitate anther gland. Fig. 15. Schleinitzia novoguineensis, anther (lateral view) showing stipitate rounded anther gland. Fig 16. L. macrophylla, lateral view of dehisced anther. Fig. 17. Schleinitzia insularum, distal portion of anther from dorsal side showing clavate stipitate anther gland. Fig. 18. L. pallida, dehisced anther from ventral side showing free monad pollen grains in a single cavity. Fig. 19. L. macrophylla, dehisced anther (lateral view). Scale bars in Figs 14, 15, 17 and 19=0.1 mm; Figs 16 and 18=0.25 mm.

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Mimoseae. This appears to be justified based on the similarity ‘test’ of topographic correspondence (Patterson, 1982) in that both are appendages that arise at the apex as direct extensions of the connective. The glandular nature of these stipitate appendages was demonstrated for Prosopis juliflora (Sw.) DC. (Chaudhry & Vijayaraghavan, 1992) and more recently for more genera (Luckow & Grimes, 1997). Bentham (1875) relied on the presence or absence of stipitate anther glands to characterize tribes and more recently Lewis & Elias (1981) used this as one character to separate Schleinitzia and Leucaena. Within the Dichrostachys, Xylia and Leucaena groups sensu Lewis & Elias (1981), stipitate, terminal, often caducous, anther glands are found in species of Schleinitzia (Figs 15 and 17 and Nevling & Niezgoda, 1978: 347), some species of Xylia (Fig. 14), Calpocalyx, one species of Desmanthus (Luckow, 1993: 7), some species of Neptunia and Dichrostachys sensu stricto (Lewis & Elias, 1981; Luckow, 1995: 66). Luckow (1995) recognized two types of stipitate anther glands based on shape (round or claviform), packing of the cells (dense or loose) and prominence of the reticulate thickenings on the cells (prominent or not). Considerable variation in shape and sculpture pattern of the cell surfaces was observed in the wider survey of piptadenioid anther glands of Luckow & Grimes (1997). Anther dehiscence The anthers of Leucaena and other Mimoseae, dehisce along a simple, linear, longitudinal, stomial furrow, dividing the thecae into two almost equal halves. In most plant species and most species of Leucaena, the inter-locular zone, consisting of the septum and the tapetum, is disrupted along the ‘break-through zone’ (sensu Hufford & Endress, 1989), prior to dehiscence along the stomium (Keijzer, 1987), thereby creating a single pollen-containing chamber prior to dehiscence (Figs 18 and 22) with the pollen grains free within that chamber at the time of dehiscence. Breakdown of the septum and tapetum prior to dehiscence was ascertained and illustrated for L. leucocephala by Dnyansagar (1949) and Jain & Vijayaraghavan (1992). In L. macrophylla, L. nelsonii and L. trichodes, the locules remain separate, as discrete elliptical ‘units’ surrounded by what appears to be an intact tapetal membrane that surrounds the contents of the locule during spore development forming a ‘culture sac’. The tapetal membrane is usually broken before anther dehiscence, either due to expansion of the pollen mass beyond the capacity of the locule or simultaneously with dehiscence of the stomium due to the inward-bending locule walls (Keijzer, 1987). Although it is generally accepted that breakdown of the tapetal membrane is mechanical rather than metabolic, Keijzer (1987) suggests that breakage occurs as a result of only slight mechanical force, while Heslop-Harrison (1969) emphasizes the strength of the membrane as it resists the pressure of the developing spores. In the case of L. macrophylla, the tapetum apparently remains intact until after dehiscence of the anther (Figs 16 and 19) and forms a ‘sac’ which holds the whole locular contents together as a unit. The intact tapetal membrane of L. macrophylla was observed on fixed flower material prior to critical-point drying, and is not therefore an artefact of the drying procedure. As far as can be ascertained, this has not been observed in other plants before. Pettit (1966), Banerjee (1967) and Heslop-Harrison (1969) showed that the tapetal membranes of some species are resistant to acetolysis, suggesting that, like the pollen

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Figures 20–25. Anther dehiscence in Alantsilodendron and Leucaena and occurrence of pollen in polyads in Leucaena. Fig. 20, Alantsilodendron sp. dehisced anther from ventral side. Fig. 21. L. nelsonii, nearly intact loosely associated acalymmate polyad prepared without acetolysis. Fig. 22. L. multicapitula, dehisced anther from ventral side showing a single polyad in open anther. Fig. 23. L. nelsonii, pantoporate, asymmetric monad unit derived from acalymmate polyad. Fig. 24. L. multicapitula, 16-grained acalymmate tetrad polyad, the individual pollen grains tricolporate. Fig. 25. L. nelsonii, loosely associated acalymmate polyad fragment after acetolysis. Scale bars in Figs 20 and 22= 0.25 mm; Figs 21 and 24=24 lm; Figs 23 and 25=15 lm.

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grain exine, they also contain material with the chemical properties of sporopollenin. When whole anthers of L. macrophylla were destroyed using the hot acetolysis method of Erdtman (1960) for a longer than normal period of 15 minutes as well as pollen grains, sheets or webs of reticulate material remained. If resistance to acetolysis is accepted as diagnostic of materials of the general class of sporopollenin, then this indicates that the membrane in Leucaena is of this character. The function of the intact tapetal membranes surrounding the locules remains unknown and it is unclear at what stage the tapetal membrane breaks up after dehiscence of the anthers. Sorensson (1988) reports that L. macrophylla pollen is “shed in clumps” and that “these aggregations of pollen are not spread easily”, possibly lending weight to the idea that the tapetal membrane persists after anther dehiscence and potentially after the pollen is shed. Persistence of an intact tapetal membrane after anther dehiscence was also observed in species of Schleinitzia and Alantsilodendron (Figs 15 and 20), but in this case anthers were derived from dried herbarium material and the drying procedure may have caused premature dehiscence of the anthers (see also Nevling & Niezgoda, 1978: 347, Plate 1.1 for photograph showing similar intact tapetal membranes in Schleinitzia novoguineensis (Warb.) Verdc.). Similar membranes have also been observed in Acacia subulata Bonpl. by Kenrick & Knox (1989) and Parkia and Dichrostachys (Luckow, pers. comm.). What the taxonomic and functional significance of the tapetal membrane in the Mimoseae may be, if any, remains to be investigated. The present study provided insufficient data to delimit any characters for analysis. Pollen The tremendous morphological diversity in mimosoid legume pollen and its practical value for systematic studies were reviewed by Guinet (1981a,b) and Guinet & Ferguson (1989). Variation in apertural type, the structure of the exine and particularly the tectum and its ornamentation, and the high frequency of compound grains (polyads and tetrads) have provided useful characters in systematic studies of the Mimosoideae. The pollen of Leucaena was first studied in depth by Guinet (1966) who examined pollen of 17 species (although several of these are here treated as conspecific) using light microscopy and was the first to show the occurrence of both polyads and eumonads within the genus. Guinet’s (1966) study was limited by reliance on light microscopy, by taxonomic confusion surrounding the material used and incomplete sampling of species within the genus; voucher specimens were cited later in Guinet (1969); the majority of these have been seen and identified during the present work. Apertures Pollen in the majority of Leucaena species occurs in symmetric tricolporate eumonads (Figs 26–29). Pollen occurs in polyads in four Leucaena species, one of which, L. multicapitula, has individual pollen grains which are tricolporate (Fig. 24), while the other three (L. trichodes, L. macrophylla and L. nelsonii) have pantoporate monad units (Figs 23 and 25). Thus in Leucaena there are two types of aperture, colporate and porate. Pollen of Leucaena material from Hispaniola, originally described as L.

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Figures 26–31. Tricolporate monad pollen of Leucaena and pollen of Schleinitzia. Fig. 26. L. salvadorensis, equatorial view. Fig. 27. L. salvadorensis, polar view. Fig. 28. L. trichandra, equatorial view. Fig. 29. L. trichandra, polar view. Fig. 30. Schleinitzia novoguineensis, rugulate tectal ornamentation on pollen grain. Fig. 31. Schleinitzia novoguineensis, polyad of acalymmate tetrads. Scale bars in Figs 26–29 and 31=15 lm; Fig 30=3 lm.

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pseudotrichodes (DC.) Britton & Rose, shows unusual variation in aperture type; some grains are clearly colporate; others show only weak development of the colpi and would be described as colpoidorate. Tricolporate eumonads or monad units were considered by Guinet (1981a) to be the basic (my emphasis) pollen aperture type in the Leguminosae. In species of the Dichrostachys, Leucaena and Xylia groups sensu Lewis & Elias (1981), both porate (Alantsilodendron, Dichrostachys, Gagnebina and some species of Xylia) and colporate (Calliandropsis, Desmanthus, Kanaloa, Neptunia, Schleinitzia (Fig. 31) and some species of Xylia) apertures occur. In her phylogenetic analyses of both Desmanthus and the Dichrostachys group, Luckow (1993 and 1995) postulated porate apertures to be plesiomorphic and tricolporate apertures to be independently derived at least twice in both groups. Pollen unit The pollen unit is the grouping in which pollen is found at maturity within anther locule of the stamen (Walker & Doyle, 1975). The occurrence of compound grains (polyads and tetrads) is a most conspicuous feature of mimosoid pollen. As pointed out by Guinet (1981a,b), the compound grain is a more or less compact unit made by the permanent assemblage of a variable number of individual cells remaining more or less independent of each other. The degree of asymmetry in shape and exine development is a function of the closeness of the grouping. Within the Mimosoideae, there is a complete spectrum from calymmate (the external layer of the exine is common to all the cells instead of being restricted to each individual cell) to acalymmate (polyads in which the ectexine is not completely continuous around members of the dispersal unit) compound grains. This means that simply distinguishing between monads and polyads in assessment of primary homology does not properly account for the morphological variation encountered, given the variety of types of compound grains that can be recognized. Luckow (1995), recognized this problem in relation to variation in types of compound grain within the Dichrostachys group, and divided polyads into three types, calymmate, acalymmate tetrads (where individual cells are tightly bound within tetrads and the tetrads are loosely associated into larger compound grains) and acalymmate monads where monad units are themselves loosely associated into compound grains. The number of cells per compound grain is sometimes extremely variable and also of limited value due to intraspecific variation (Guinet, 1981a,b); the tetrad represents in most genera, only a sporadic variant occurring along with 1-, 8- and 16-celled grains, often in the same anther. The polyads found by Guinet (1966) in Leucaena were all acalymmate, corresponding to the category of acalymmate monads of Luckow (1995), but Guinet further differentiated Leucaena polyads as more or less loosely associated. For the species with very loosely associated grains (which disintegrate following normal acetolysis or even modified procedures of Wodehouse (1935) and taking great care in dissecting the anthers), Guinet (1966) deduced their occurrence in polyads from the asymmetry of the loosened monad units. The progression (my emphasis) from eumonads through very loosely associated polyads to acalymmate polyads was interpreted by Guinet (1966) as a transitional series (acalymmate polyads – very loosely acalymmate polyads shed as single grains but with clear asymmetry of monad

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units – eumonads), a transition accompanied by a switch from porate to tricolporate grains. In this survey, polyads were found in four species of Leucaena (Figs 21, 24 and 25): L. multicapitula, L. trichodes, L. macrophylla and L. nelsonii, but these are of two very different types. In L. multicapitula, pollen occurs in calymmate tetrahedral tetrads composed of tricolporate monad units, which are loosely aggregated into 16-celled polyads, equivalent to the acalymmate tetrads sensu Luckow (1995) (Fig. 24). In L. trichodes, L. macrophylla and L. nelsonii pollen occurs in acalymmate polyads with (few–) 26–28 irregularly asymmetric porate monad units per polyad. These compound grains are loosely attached and therefore difficult to observe intact. Evidence for their occurrence comes from the asymmetry of the monad units (Figs 23 and 25), broken polyad fragments, observed after acetolysis, containing several monad units in regular, non-random, arrangements (Fig. 25), and observation of more or less intact polyads when pollen is extracted with great care from the anthers and observed without acetolysis (Fig. 21). It appears that Guinet (1966) did not observe the calymmate polyads of L. multicapitula although his map showing the sources of his material (Guinet, 1966: 45) suggests that one specimen (which he labelled as L. macrophylla) was collected in Panama, where the only known native species of Leucaena is L. multicapitula. Guinet’s locality for L. macrophylla, which closely resembles L. multicapitula in other characters, but is restricted to Mexico, is therefore assumed to be mistaken; none of the vouchers cited in Guinet (1969) are L. multicapitula. The discovery of calymmate polyads in L. multicapitula is significant for several reasons. Firstly, it indicates that the two characters, apertural type and occurrence of polyads, are not directly correlated, as assumed by Guinet (1966), and that his postulated transitional series is an oversimplification. Secondly, it provides evidence to support the recognition of L. multicapitula as a species distinct from L. trichodes and its close allies L. macrophylla and L. nelsonii, a status doubted by several authors ( Janzen & Liesner, 1980; Brewbaker, 1987; Za´rate, 1994). The affinities of Leucaena material from Hispaniola, originally described as L. pseudotrichodes, and subsequently treated, by some authors, as conspecific with L. trichodes, must also be questioned on the basis of pollen evidence. Pollen of L. pseudotrichodes was examined both by Guinet (1966) and during the present survey and occurs in loosely associated polyads composed of asymmetric tricolporate monad units, although some grains are colpoidorate, the colpi being only weakly, but variably, developed. Pollen of L. pseudotrichodes is thus distinct from that of L. trichodes (polyads with pantoporate monad units) and more similar to that of L. multicapitula. The occurrence of species with pollen in monads and others with polyads within the same genus is not uncommon within the Mimosoideae and has been reported for Newtonia Baill., Entada Adans., Dichrostachys, Dinizia Ducke as well as Leucaena (Guinet, 1981b) and recently for Desmanthus (Luckow, 1993). Across the genera of the Dichrostachys, Xylia and Leucaena groups, there is great diversity of pollen unit: calymmate polyads occur in some species of Dichrostachys, Gagnebina, and some species of Xylia; acalymmate tetrads occur in some species of Dichrostachys, one species of Leucaena, one species of Desmanthus, some species of Xylia and Schleinitzia; acalymmate monads occur in both Dichrostachys and Leucaena; eumonads occur in most species of Desmanthus, Neptunia, Calliandropsis, Kanaloa and most species of Leucaena.

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Pollen wall architecture Many terms associated with pollen wall architecture, and particularly categories used to describe tectal ornamentation, are arbitrary and have not been consistently applied. Terms used here to categorize tectal ornamentation follow Walker & Doyle (1975) (their figures 7–9). The exine in Leucaena has well developed columellae (Fig. 37) and is tectate, or occasionally semi-tectate. In Leucaena, the external surface of the tectum is devoid of raised ornamentation and ranges from tectate, smooth, almost psilate (without perforations) in a few species (Fig. 32) to smooth, perforate, punctate or finely foveolate (randomly distributed isodiametric channels <1 lm in diameter) in the majority of species (Figs 33–35), to semi-tectate, finely reticulate with small lumina (>1 lm in diameter) (Fig. 36) in two species. This variation appears to be quantitative and continuous (Figs 32–36); the division between tectate perforate with large perforations and semi-tectate reticulate with small lumina, is arbitrary and difficult to define (Moore, Webb & Collinson, 1991). Although variation in tectal ornamentation within Leucaena is continuous and therefore cannot be readily divided into discrete character states (Stevens, 1991), discrete variation is encountered between genera and tectal ornamentation may provide a useful character at generic level. Across the genera of the Dichrostachys, Xylia and Leucaena groups sensu Lewis & Elias (1981), only a few species of Desmanthus share the psilate/punctate/finely foveolate tectal ornamentation found in Leucaena with considerable variation in tectal ornamentation across the remaining genera. This variation was used by Luckow (1993 and 1995) in her cladistic analyses of both Desmanthus and the Dichrostachys group, and by Herna´ndez & Guinet (1990) to assess the generic affinities of Calliandropsis. Coding of tectal ornamentation into discrete character states for phylogenetic analysis within these groups is complicated by a number of difficulties. Firstly, as with the variation described above within Leucaena, several of the categories commonly used to describe tectal ornamentation represent arbitrary limits imposed on a continuum. For example it is difficult to separate punctate or perforate from foveolate, foveolate from fossulate, scabrate from verrucate or striate from rugulate tectal patterns. Secondly, variation in tectal ornamentation has been reported within species and even within individuals. For example, Luckow (1993) reported the occurrence of both fossulate and striate pollen within a single individual of Desmanthus pubescens B.L. Turner, showing that this is at least partly related to differences between male and hermaphrodite flowers. Finally, tectal ornamentation can vary dependent on position on the pollen grain, particularly in the case of monad units derived from polyads, where tectal ornamentation varies between proximal and distal faces of the monad units. For example, in Schleinitzia novoguineensis, the tectum is coarsely rugulate on the poles (Fig. 30), but fossulate on the equatorial region. These difficulties led Luckow (1995) to code verrucate and foveolate tectal ornamentation as a single character state from which she was able to separate reticulate and striate states. I have added two more character states, ‘rugulate’ (Fig. 30) and ‘psilate/ punctate’ to incorporate the variation observed in the genera not included by Luckow (1995), namely Kanaloa, Schleinitzia, Xylia and Leucaena. DISCUSSION

Although characters of the pollen and anthers are found to be quite conservative in many groups of angiosperms and were considered to be so in the mimosoid

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Figures 32–37. Continuous variation in tectal ornamentation within Leucaena. Fig. 32. L. collinsii subsp. collinsii, psilate tectum. Fig. 33. L. leucocephala, punctate tectum with micro-perforations. Fig. 34. L. peblana, punctate/ perforate tectum. Fig. 35. L. pallida, foveolate tectum. Fig. 36. L. trichandra, finely reticulate tectum. Fig. 37. L. leucocephala, approximate transverse section through ectexine showing columellae. Scale bars in Figs 32–36=3 lm; Fig. 37=1 lm.

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legumes (Dnyansagar, 1955), they are in fact highly variable and plastic within Leucaena and more generally in the Leucaena and Dichrostachys groups of the Mimoseae. The data presented provide the basis for designation of a number of putative homologies for cladistic analysis. These include anthers hairy or glabrous, presence or absence and type of connective protrusion, type of pollen aperture, pollen in monads or polyads, type of polyad and tectal ornamentation. Full anther and pollen data for all species of Leucaena is summarized in Table 1. The full systematic implications of these data will be explored in a subsequent paper and in a forthcoming systematic monograph of Leucaena. Given the diversity of anther and pollen morphology at species and generic level, further survey of these characters across all closely related genera is likely to yield useful data. The observation of apparently persistent intact tapetal membranes in some species of Leucaena and potentially also within Schleinitzia and Alantsilodendron requires confirmation through further field investigation of pollen dispersal and cryopreservation of fresh anthers. The biological and systematic significance of the tapetal membrane remains to be understood. The occurrence and significance of transverse septa each with a tapetal layer that partition the anther locules in Parkia and occasionally in Dichrostachys, as observed by Dnyansagar (1954, 1955) also remain to be fully investigated, adding further impetus for new work in this area. It is notable that within Leucaena, the occurrence of a persistent tapetal membrane is correlated with occurrence of pollen in loosely aggregated polyads composed of 26–28 acalymmate monads and that the length of a single polyad of this type is comparable to the length of the intact tapeta, suggesting that there is a single polyad in each. Although this correlation applies within Leucaena, elsewhere in the Mimoseae similar structures are associated with other types of polyad, e.g. Schleinitzia, which has polyads composed of acalymmate tetrads (Fig. 31) which are loosely associated into larger and very variable polyads. Again some of the large aggregated polyads of Schleinitzia novoguineensis observed after acetolysis are comparable in size to the intact tapetal membrane sacs observed for that species. It is possible that the tapetal membrane in the Mimoseae plays a role in the initial cohesion of loosely aggregated acalymmate polyads, but again, further work will be required to investigate this hypothesis. Kenrick & Knox (1989) described a similar “culture sac” surrounding the locules of anthers of Acacia subulata, outside the closely packed spherical orbicules, in which a single compound pollen grain develops, speculating that this may function as a cement to strengthen the bonds linking the grains of the polyad together. Of immediate taxonomic significance is the confirmation of L. multicapitula as a species distinct from L. trichodes and its close allies L. macrophylla and L. nelsonii, despite their close similarity in leaf and pod morphology which led previous authors to treat L. multicapitula as conspecific with L. trichodes. Five characters were discovered during this study which separate L. multicapitula from L. trichodes and L. macrophylla: pollen in acalymmate tetrads vs acalymmate monads, colporate vs porate pollen apertures, introrse vs latrorse orientation of thecae on the anther, absence vs presence of an apiculum on the anther and absence vs presence of a persistent tapetal membrane. Evidence from analysis of cpDNA of Leucaena (Harris et al., 1994) also supported the distinction of L. multicapitula from these species; morphological data presented here support these results.

absent absent absent absent absent broad, flattened

absent absent small, rounded small, rounded absent absent absent absent broad, flattened

hairy

glabrous hairy hairy hairy hairy

hairy

hairy

hairy hairy glabrous glabrous hairy hairy

hairy

hairy hairy

hairy

L. multicapitula

L. nelsonii

L. pallida L. pueblana L. pulverulenta L. retusa L. salvadorensis L. shannonii subsp. shannonii L. shannonii subsp. magnifica L. trichandra L. trichodes

L. sp. nov.

absent

broad, flattened

absent

absent absent small, pointed absent absent

hairy hairy sparsely hairy hairy sparsely hairy

L. collinsii L. confertiflora L. cuspidata L. diversifolia L. esculenta subsp. esculenta L. esculenta subsp. matudae L. greggii L. involucrata L. lanceolata L. leucocephala L. macrophylla

Apiculum

Vestiture

Species

Anthers

normal tapetal membrane persists normal

normal

tapetal membrane persists normal normal normal normal normal normal

normal normal normal normal tapetal membrane persists normal

normal

normal normal normal normal normal

Dehiscence

monads polyads (acalymmate monads) monads

monads

monads monads monads monads polyads (acalymmate monads) polyads (acalymmate tetrads) polyads (acalymmate monads) monads monads monads monads monads monads

monads

monads monads monads monads monads

Monads/polyads

T 1. Summary of anther and pollen data for Leucaena.

tricolporate

tricolporate pantoporate

tricolporate

tricolporate tricolporate tricolporate tricolporate tricolporate tricolporate

pantoporate

tricolporate

tricolporate tricolporate tricolporate tricolporate pantoporate

tricolporate

tricolporate tricolporate tricolporate tricolporate tricolporate

Apertures

Pollen

punctate punctate punctate punctate

perforate, punctate

finely reticulate sparsely punctate

psilate

finely reticulate perforate, punctate perforate, punctate perforate, punctate perforate, punctate perforate, punctate

sparsely punctate

perforate, punctate

sparsely punctate perforate, punctate psilate perforate, punctate finely punctate

perforate, punctate

psilate perforate, perforate, perforate, perforate,

Tectum

ANTHERS AND POLLEN OF LEUCAENA 193

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COLIN E. HUGHES ACKNOWLEDGEMENTS

I would like to thank Robert Scotland for help and advice on pollen analysis and SEM as well as for comments on several drafts of this paper. Sue Barnes, Louisa Jones and Chris Jones of the microscopy unit at the Natural History Museum, London, are thanked for help with SEM and Cledwyn Merrimen and John Baker in Oxford for help with critical point drying and photographic work respectively. I also thank Melissa Luckow and David Mabberley for comments on the manuscript, Dave Du Puy for making available un-mounted material of Alantsilodendron and the curators at K and FHO for facilitating material on loan. This paper was written while working on ODA Forestry Research Programme project R.4524 investigating the genetic resources of Leucaena.

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Lewis GP, Elias TS. 1981. Mimoseae. In: Polhill RM, Raven PH, eds. Advances in Legume Systematics. Part 1. Kew: Royal Botanic Gardens, 155–168. Lewis GP, Guinet P. 1985. Notes on Gagnebina (Mimosoideae-Leguminosae) in Madagascar and neighbouring islands. Kew Bulletin 41: 463–470. Lorence DH, Wood KR. 1994. Kanaloa, a new genus of Fabaceae (Mimosoideae) from Hawaii. Novon 4: 137–145. Luckow M. 1993. Monograph of Desmanthus (Leguminosae-Mimosoideae). Systematic Botany Monographs 38: 1–166. Luckow M. 1995. A phylogenetic analysis of the Dichrostachys group (Mimosoideae: Mimoseae). In: Crisp MD, Doyle JJ, eds. Phylogeny. Advances in Legume Systematics. Part 7. Kew: Royal Botanic Gardens, 63–75. Luckow M, Grimes J. 1997. A survey of anther glands in the Mimosoid legume tribes Parkieae and Mimoseae. American Journal of Botany 84: in press. Miller P. 1834. Dictionary of Gardening, Botany and Agriculture. 9th Ed. London: Orr & Smith. Moore PD, Webb JA, Collinson ME, 1991. Pollen Analysis. 2nd Ed. Oxford: Blackwell Scientific Publications. Nevling LI, Niezgoda CJ. 1978. On the genus Schleinitzia (Leguminosae-Mimosoideae). Adansonia ser. 2 18: 345–363. Parkin J. 1951. The protrusion of the connective beyond the anther and its bearing on the evolution of the stamen. Phytomorphology 1: 1–8. Patterson C. 1982. Morphological characters and homology. In: Joysey KA, Friday AE. eds. Problems in Phylogenetic Reconstructions. Academic Press, 21–74. Pettit JM. 1966. A new interpretation of the structure of the megaspore membrane in some gymnosperm ovules. Journal of the Linnean Society (Botany) 59: 253–262. Rudall PJ, Myers G, Lewis GP. 1994. Floral secretory structures in Caesalpinia sensu lato and related genera. In: Ferguson IK, Tucker SC, eds. Structural Botany. Advances in Legume Systematics. Part 6. Kew: Royal Botanic Gardens, 41–57. Sorensson CT. 1988. Pollinating and emasculating techniques for Leucaena species. Leucaena Research Reports 9: 127–130. Stevens PF. 1991. Character states, morphological variation and phylogenetic analysis: a review. Systematic Botany 16: 553–583. Tucker SC. 1987. Floral initiation and development in legumes. In: Stirton CH, ed. Advances in Legume Systematics. Part 3. Kew: Royal Botanic Gardens, 183–239. Villiers JF. 1994. Alantsilodendron Villiers, a new genus of Leguminosae-Mimosoideae from Madagascar. Bulletin Muse´um National d’Histoire Naturelle. B. Adansonia 16(1): 65–70. Walker JW, Doyle JA. 1975. The basis of Angiosperm phylogeny: palynology. Annals Missouri Botanical Garden 62: 664–723. Wodehouse RP. 1935. Pollen Grains. Vol. 1. London: McGraw-Hill. Za´rate S. 1994. Revisio´n del ge´nero Leucaena Benth. en Me´xico. Annales del Instituto de Biologı´a, Universidad Nacional Auto´noma de Me´xico, Serie Bota´nica 65(2): 83–162. APPENDIX Anthers: specimens examined Leucaena collinsii Britton & Rose subsp. collinsii, C.E. Hughes 1760, Mexico, 6.xi.1993, FHO. Leucaena confertiflora Za´rate, C.E. Hughes 1152, Mexico, 26.iii.1992, FHO. Leucaena cuspidata Standl., C.E. Hughes 1856, Mexico, 29.xi.1993, FHO. Leucaena diversifolia (Schltdl.) Benth., C.E. Hughes 1867, Mexico, 5.xii.1993, FHO. Leucaena esculenta (Moc¸. & Sesse´ ex DC.) Benth. subsp. esculenta, C.E. Hughes 1779, Mexico, 10.xi.1993, FHO. Leucaena greggii S. Watson, C.E. Hughes 695, Mexico, 27.iv.1985, FHO. Leucaena involucrata Za´rate, C.E. Hughes 1522, Mexico, 19.viii.1992, FHO. Leucaena lanceolata S. Watson, C.E. Hughes 1767, Mexico, 7.xi.1993, FHO. Leucaena leucocephala (Lam.) de Wit, C.E. Hughes 639, Mexico, 23.iii.1985, FHO. Leucaena macrophylla Benth., C.E. Hughes 1830, Mexico, 22.xi.1993, FHO. Leucaena multicapitula Schery, C.E. Hughes 795, Panama, 23.iii.1986, FHO.

Leucaena nelsonii Britton & Rose, C.E. Hughes 386, Mexico, 12.xi.1983, FHO. Leucaena pallida Britton & Rose, C.E. Hughes 1775, Mexico, 8.xi.1993. FHO. Leucaena pulverulenta (Schltdl.) Benth., C.E. Hughes 1859, Mexico, 30.xi.1993, FHO. Leucaena retusa Benth., C.E. Hughes 1361, Honduras, 27.vii.1990, FHO. Leucaena shannonii J.D. Sm. subsp. magnifica C.E. Hughes, C.E. Hughes 1748, Guatemala, 1.xi.1993, FHO. Leucaena trichodes ( Jacq.) Benth., C.E. Hughes 1017, Ecuador, 5.vi.1987, FHO. Alantsilodendron alluaudiana R. Vig., D. Du Puy 134, Madagascar, K. Alantsilodendron sp, D. Du Puy 40, Madagascar, K. Gagnebina commersoniana (Baill.) R. Vig., Renvoize 1365, K. Schleinitzia novoguineensis (Warb.) Verdc. A. Gillison 25310, Papua New Guinea, 4.x.1966, K. Schleinitzia insularum (Guill.) Burkart, J.F. McCormick s.n., Tong, xii.1934, K. Xylia torreana Brenan, D.H. Eccles 181, Zimbabwe, 20.x.1968, K.

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Calliandropsis nervosus H.M. Hern & Guinet, C.E. Hughes 1748, Mexico, 11.xi.1993, FHO. Pollen: specimens examined Leucaena collinsii Britton & Rose subsp. zacapana C.E. Hughes, C.E. Hughes 1137, Guatemala, 30.iii.1988, FHO. Leucaena confertiflora Za´rate, C.E. Hughes 1730, Mexico, 26.iii.1992, FHO. Leucaena cuspidata Standl., C.E. Hughes 1580, Mexico, 7.ii.1992, FHO. Leucaena diversifolia (Schltdl.) Benth., C.E. Hughes 1666, Mexico, 24.ii.1992, FHO. Leucaena esculenta (Moc¸. & Sesse´ ex DC.) Benth. subsp. esculenta, C.E. Hughes 1779, Mexico, 10.xi.1993, FHO. Leucaena esculenta (Moc¸. & Sesse´ ex DC.) Benth. subsp. matudae Za´rate, C.E. Hughes 1511, Mexico, 13.viii.1991, FHO. Leucaena greggii S. Watson, C.E. Hughes 696, Mexico, 27.iv.1985, FHO. Leucaena involucrata Za´rate, C.E. Hughes 1522, Mexico, 19.viii.1991, FHO. Leucaena lanceolata S. Watson, C.E. Hughes 567, Mexico, 24.ii.1985, FHO. Leucaena leucocephala (Lam.) de Wit, C.E. Hughes 1547, Mexico, 21.i.1992, FHO. Leucaena macrophylla Benth., C.E. Hughes 1839, Mexico, 23.xi.1993, FHO. Leucaena multicapitula Schery, C.E. Hughes 795, Panama, 23.iii.1986, FHO. Leucaena nelsonii Britton & Rose, D.J. Macqueen 277, Mexico, 26.xi.1991, FHO.

Leucaena pallida Britton & Rose, C.E. Hughes 1506, Mexico, 11.viii.1991, FHO. Leucaena pueblana Britton & Rose, C.E. Hughes 1803, Mexico, 17.xi.1993, FHO. Leucaena pulverulenta (Schltdl.) Benth., C.E. Hughes 1866, Mexico, 2.xii.1993, FHO. Leucaena retusa Benth., C.E. Hughes 1361, Honduras, 27.vii.1990, FHO. Leucaena salvadorensis Standl. ex Britton & Rose, Hughes & Styles 37, Nicaragua, FHO. Leucaena shannonii J.D. Sm. subsp. shannonii, C.E. Hughes 1492, Mexico, 9.viii.1991, FHO. Leucaena shannonii J.D. Sm. subsp. magnifica C.E. Hughes, C.E. Hughes 731, Guatemala, 18.x.1985, FHO. Leucaena trichandra (Zucc.) Urb., C.E. Hughes 1094, Guatemala, 26.ii.1988, FHO. Leucaena trichodes ( Jacq.) Benth., C.E. Hughes 1000. Ecuador, 1.vi.1987, FHO. Leucaena pseudotrichodes (DC.) Britton & Rose, A.H. Liogier 20620, Dominican Republic, 10.xi.1973, US. Leucaena sp nov. C.E. Hughes 1414, Honduras, 25.ii.1991, FHO. Xylia torreana Brenan, D.H. Eccles 181, Zimbabwe, 20.x.1968, K. Xylia evansii Hutch., S.K. Samai 69, Sierra Leone, 5.ii.1964, K. Xylia xylocarpa (Roxb.) Theob., Lace 6110, Burma, 2.iii.1913, K. Schleinitzia novoguineensis (Warb.) Verdc., F.S. Walker & C.T. White 120, Solomon Islands, 6.ix.1945, K. Schleinitzia insularum (Guill.) Burkart, J.F. McCormick s.n., Tonga, xii.1934, K.