Pollen of Melodinus (Apocynaceae): Monads and tetrads

Abstract

Melodinus pollen has been studied by light, scanning electron and transmission electron microscopy. The pollen grains are usually colporate, medium‐sized monads, and their shape varies from suboblate to oblate spheroidal. They are mostly 3‐aperturate, while four species have pollen with both three and four apertures, and one species has exclusively 4‐aperturate pollen. The ecto‐ and endoapertures show large size ranges. Melodinus coriaceus has its pollen in tetrahedral tetrads, while M. orientalis is polymorphic as to dispersal unit (mostly tetrads, sometimes monads). The monotypic genus Craspidospermum, which is the closest known relative of Melodinus, has its pollen also in tetrads, but these are very different (non‐tetrahedral, irregularly pantoporate). On the basis of pollen morphology, it is hypothesised that tetrads evolved independently in Melodinus and Craspidospermum

Keywords:

• Apocynaceae
• Craspidospermum
• Melodinus
• pollen
• tetrads

The Apocynaceae sensu lato make up a large family with more than 400 genera. Endress & Bruyns (2000) divided it into five subfamilies: 1. Rauvolfioideae, 2. Apocynoideae, 3. Periplocoideae, 4. Secamonoideae and 5. Asclepiadoideae. They included eight genera in the Old World tribe Melodineae of the Rauvolfioideae: Craspidospermum (1 sp.), Diplorhynchus (1 sp.), Dyera (2 spp.), Gonioma (2 spp.), Kamettia (1 sp.), Melodinus, Pycnobotrya (1 sp.) and Stephanostegia (2 spp.), of which Kamettia, Melodinus and Pycnobotrya are lianas, while the others are trees (Pichon, 1948b ; Plaizier, 1980; Van der Ploeg, 1983; Leeuwenberg, 1997; Middleton, 2003). The genus Melodinus consists of 19 species, which occur in Southeast Asia, Australia and New Caledonia (Boiteau et al. 1976; Ly, 1981, 1985; Huang, 1986; Forster, 1992; Leeuwenberg, 2003). The monotypic genus Craspidospermum from Madagascar (Leeuwenberg, 1997) is the closest relative of Melodinus (Sennblad & Bremer, 2002).

The pollen of Melodinus has scarcely been studied. Pichon (1948a) was the first to describe its morphology. He also observed the occurrence of tetrads in addition to monads. Craspidospermum also has its pollen in tetrads (Pichon, 1948b ), but their morphology is very different from that of Melodinus tetrads. The aim of the present paper is to describe the pollen of Melodinus and to explore the distribution of tetrads and determine their relationship to those of sister group Craspidospermum.

Material and methods

All 19 Melodinus species recognised by Leeuwenberg (2003) and Craspidospermum verticillatum were available for sampling (see Specimens investigated). When possible, at least two samples per species were taken. All species were studied with light microscopy and scanning electron microscopy, and three Melodinus species (M. cochinchinensis, M. coriaceus and M. cumingii) and C. verticillatum with transmission electron microscopy as well. For light microscopy (LM) the pollen material was acetolysed, mounted in glycerin jelly and sealed with paraffin. Ten pollen grains were measured for the polar and equatorial dimensions and five grains for the ecto‐ and endoaperture sizes. For scanning electron microscopy (SEM) acetolysed pollen was coated with gold and examined in a JSM 5300 microscope. The scanning electron micrographs of C. verticillatum pollen were provided by Laure Civeyrel (Toulouse). For transmission electron microscopy (TEM), whole anthers were embedded in Spurr resin, sectioned with an LKB Ultrotome III, post stained with uranylacetate and lead citrate and examined with a JEOL‐1010. As far as possible the terminology follows Punt et al. (1994).

Results

Melodinus (Figs  1 , 2 A–H , 3 A–F )

Figure 1 Melodinus pollen, SEM. A–C. M. cochinchinensis: (A) ±Polar view of 3‐aperturate grain (Hu 11931); (B) Equatorial view of 3‐aperturate grain (Hu 7668); (C) Polar view of 4‐aperturate grain (Hu 7668). D–F. M. orientalis (Schmutz 3168a): (D) Polar view of 3‐aperturate monad; (E) ±Equatorial view of 3‐aperturate monad; (F) Polar/equatorial view of 3‐aperturate monad with apertures and mesocolpium centres folded inwards. G–I. M. orientalis (Winckel 1856b): (G) Upper view of tetrahedral tetrad; (H) Lower view of tetrahedral tetrad; (I) Oblique view of tetrahedral tetrad. J–L. M. phylliraeoides (Compton 1666): (J) Polar view of 3‐aperturate grain; (K) ±Equatorial view of 3‐aperturate grain; (L) Polar view of 4‐aperturate grain. M–O. M. reticulatus (Mackee 29524): (M) Polar view of 3‐aperturate grain; (N) Equatorial view of 3‐aperturate grain with mesocolpium centres folded inwards; (O) Detail of Fig. 1 N, showing scabrate mesocolpium centre. Scale bar – 5 µm (A–N), 1 µm (O).

Figure 2 A–H. Melodinus pollen, LM (A) Polar view of 3‐aperturate grain (M. phylliraeoides, Balansa 215); (B) Equatorial view of 3‐aperturate grain (M. cumingii, McGregor, 1267); (C) Polar view of 4‐aperturate grain (M. aeneus, Mackee 21599); (D) Equatorial view of 4‐aperturate grain (M. aeneus, Mackee 26345); (E) Polar view of 3‐aperturate grain (M. forbesii, Floyd 5396); (F) Equatorial view of 3‐aperturate grain (M. vitiensis, Gillespie 3190); (G) Upper view of tetrahedral tetrad (M. orientalis, De Vogel 5249); (H) Lower view of tetrahedral tetrad (M. orientalis, Winckel 1856b). I–P. Craspidospermum verticillatum pollen, I–L. SEM (Civeyrel 1234) and M–P. LM (Nilsson 26‐9‐1994). (I) ±Tetragonal tetrad; (J) Rhomboidal tetrad; (K) Detail of rhomboidal tetrad with irregularly situated pores; (L) Detail of rhomboidal tetrad with irregularly situated pores; (M–P) ±decussate tetrad in four subsequent foci, showing several porate apertures. Scale bar – 10 µm (A–P).

Figure 3 A–F. Melodinus pollen, TEM. A, B. M. cochinchinensis (Hu 11931): (A) Cross‐section of colpus and adjacent mesocolpia; (B) Cross‐section of pollen wall. C, D. M. cumingii, (BS 48894): (C) Length‐section through endoaperture; (D) Cross‐section of pollen wall. E, F. M. coriaceus (SF 1519): (E) Cross‐section near aperture; (F) Cross‐section of common wall of two units within tetrad. G, H. Craspidospermum verticillatum pollen, TEM (Civeyrel 1234); (G) Cross‐section through aperture; (H) Cross‐section of common wall of two units within tetrad. Scale bar – 1 µm (A–H).

Melodinus pollen grains are medium sized [P  =  21.8 (26.9)35.3 µm; E  =  25.2(29.6)38.6 µm]. The shape of the grains is mostly suboblate to oblate spheroidal [P/E  =  0.73(0.88)0.97]. The outline of 3‐aperturate grains in polar view is circular to obtusely triangular and of 4‐aperturate grains obtusely quadrangular. The outline in equatorial view is elliptic to circular. The centres of the mesocolpia are often more or less folded inwards. The pollen grains are mostly shed as monads; sometimes (M. coriaceus and most samples of M. orientalis), they are united in (a)calymmate tetrahedral tetrads (Figs 1 G–I, 2 G, H).

The pollen grains are usually 3‐aperturate. M. angustifolius, M. cochinchinesis, M. cumingii and M. phylliraeoides have pollen with three and pollen with four (<5% per sample) apertures (Fig. 1 A–C, J–L). M. aeneus has exclusively 4‐aperturate pollen (Fig. 2 C, D).

The apertures are always colporate. The ectoapertures are colpi of 9.8–23.1×1.9–5.0 µm; sometimes, they are much reduced (Fig. 1 G–I). The colpus ends are acute to obtuse. The endoapertures are circular to lalongate pori or colpi of 1.5–5.4×7.7–15.0 µm, and nearly always provided with moderately thick polar costae (Figs 2 B, D, F, 3 C).

The exine is psilate‐punctate, but the centres of the mesocolpia may have a more or less deviating, rugulate to (micro‐) fossulate or scabrate ornamentation (e.g. Fig. 1 A, N).

The values and states of a number of selected characters (LM, SEM) of all Melodinus species are provided in Table I.

Table I. Values and states of selected pollen characters of Melodinus species.

Taxon	N	Disp. unit	P	E	P/E	Outline polar view	Apert. no	Ectoapert. length	Ectoapert. width	Endoapert. length	Endoapert. width	Mesocolp. centres
Melodinus acutifloris	2	m	26.3	31.3	0.84	c/t	3	20.8	4.8	5.4	15.0	d
M. aeneus	3	m	26.4	31.6	0.84	c	4	11.0	5.0	5.4	11.0	n
M. angustifolius	2	m	23.5	31.6	0.74	c/t	3/4	22.5	4.0	4.4	8.3	n
M. axillaris	1	m	27.8	30.4	0.92	t	3	18.7	3.5	5.0	13.9	n
M. balansae	3	m	26.1	28.4	0.93	t	3	19.3	3.1	2.7	14.8	d
M. baueri	2	m	25.9	27.8	0.93	t	3	18.5	2.1	2.9	8.9	d
M. cochinchinensis	2	m	26.5	30.6	0.86	t/q	3/4	18.7	3.1	1.7	8.5	d
M. coriaceus	2	t	23.7	32.5	0.73	t	3					d
M. cumingii	2	m	27.9	30.2	0.93	t/q	3/4	17.5	2.9	1.5	8.7	d
M. forbesii	2	m	21.8	25.2	0.87	t	3	15.0	2.9	1.7	4.8	d
M. fusiformus	2	m	26.2	29.3	0.90	t	3	14.4	2.5	1.5	7.7	d
M. glaber	2	m	26.2	27.6	0.94	t	3	21.0	2.9	2.9	10.4	d
M. honbaensis	1	m	35.3	38.6	0.81	t	3	9.8	3.5	1.7	9.4	n
M. orientalis	8	m/t	23.8	29.7	0.80	t	3	17.5	2.7	3.7	11.0	d
M. philippinensis	1	m	26.5	30.0	0.88	t	3	12.1	1.9	1.7	7.7	d
M. phylliraeoides	2	m	27.7	28.8	0.97	c/t	3/4	23.1	2.3	3.3	11.2	d
M. reticulatus	3	m	29.4	30.8	0.96	t	3	17.9	2.7	4.6	9.4	d
M. scandens	4	m	26.5	28.9	0.91	t	3	17.3	2.1	2.3	9.6	n
M. vitiensis	2	m	21.8	25.8	0.95	t	3	13.8	1.9	2.1	11.0	n
N – number of samples; Dispersal unit: m – monad, t – tetrad; P – length polar axis; E – equatorial diameter; Outline polar view: c – ±circular, q – quadrangular, t – triangular; Mesocolpium centres: d – ornamentation ±deviating, n – ornamentation not deviating. All measurements (P and E, length and width of ecto/endoapertures) are averages in µm. A slash (/) between two states means that both states are present.

TEM of M. cochinchinensis and M. cumingii (monads; Fig. 3 A–D). Exine thickness is 0.8–1.0 µm; nexine ±1/2 of the exine, completely (?) ectexinous; infratectum 1/4 to 1/3 of the exine, granular and with sparse irregular columellae; tectum 1/4 to 1/3 of the exine.

TEM of M. coriaceus (tetrads; Fig. 3 E, F). Internal walls±straight, fused into a 0.7–1.2 µm thick common wall, which consists of a 0.3–0.7 µm thick common tectum (with large perforations and extensive narrow median separations) and a similar infratectum and nexine as in the external walls. External walls convex, 0.6–0.7 µm thick, nexine ±1/5 of the exine, with an indistinct endexine and smooth inner surface; infratectum ±1/4 of the exine, granular or irregularly columellate; tectum 1/2 to 2/3 of the exine.

Craspidospermum (Figs  2 I–P , 3 G, H )

Craspidospermum pollen is shed as (a)calymmate non‐tetrahedral tetrads of diverse shape (mostly decussate or ±flat, rarely T‐shaped). The constituent units are diversely shaped (orientation polar axis and equatorial plane unknown), 25–30 µm, irregularly 5–10‐porate. Most pores are situated near the sutures, those of neighbouring grains often in ±adjacent positions. The pores are ±circular, each consisting of a relatively small (2–4 µm) ectoporus and a heavily costate endoporus; the costa (endoannulus) is ±granular towards the endoporus. The exine is psilate‐imperforate.

TEM (Fig. 3 G, H). Internal walls±straight, fused into a 1.0–1.2 µm thick common wall, which consists of a 0.5 µm thick common tectum (without perforations, with sparse narrow median separations) and a similar infratectum and nexine as in the external walls. External walls convex, 0.9–1.3 µm thick, nexine 1/3–1/2 of the exine, ectexinous, with an irregularly undulate smooth inner surface; infratectum ±1/10 of the exine, granular (short columellae?); tectum 1/2 to 2/3 of the exine.

Discussion

The tribe Melodineae consists of eight genera. Six of them, Diplorhynchus, Dyera, Gonioma, Kamettia, Pycnobotrya and Stephanostegia, shed their pollen exclusively as 3‐aperturate monads (Pichon, 1948b , 1950; Nilsson, 1986; Van der Ham, 1998). Most species of Melodinus also have monads, while M. coriaceus (both samples) and seven of the eight samples of M. orientalis have tetrahedral tetrads. One sample of M. orientalis has 3‐colporate monads like those found in most other Melodinus species. All Melodinus material has been identified by Leeuwenberg (2003), the last monographer of the genus. The monotypic genus Craspidospermum also has tetrads (Pichon, 1948b ), but their morphology is very different from that of Melodinus tetrads: non‐tetrahedral and irregularly pantoporate.

Tetrads are not common in the subfamily Rauvolfioideae, occurring in only five of the 84 genera: Callichilia (tribe Tabernaemontanae; Pichon, 1948b ), Condylocarpon, including Rhipidia (tribe Alyxieae; Van der Ham et al. 2001), Craspidospermum, Melodinus and Pagiantha (tribe Tabernaemontanae; Pichon, 1948b ). Further, they are found in Apocynum, including Poacynum and Trachomitum (subfamily Apocynoideae; Nilsson, 1986, 1990; Endress et al. 1996), and in most of the 40 genera of the subfamily Periplocoideae (Nilsson et al. 1993; Endress & Bruyns, 2000). Those of Callichilia and Pagiantha resemble Melodinus tetrads, being tetrahedral and colporate. In contrast, the tetrads of all other groups, including Condylocarpon and Craspidospermum, are non‐tetrahedral (decussate, rhomboidal, tetragonal, T‐shaped), irregularly tetrahedral or rarely tetrahedral (Nilsson et al. 1993, fig. 9H), and irregularly (panto)porate or inaperturate. As far as known, Melodinus and Pagiantha are the only Apocynaceae genera in which monads and tetrads occur together. Melodinus orientalis is the only species in which monads as well as tetrads are found.

Further, this discussion focuses on the tetrads of Melodinus and its closest relative, Craspidospermum. The tetrads of both Melodinus species are very alike, and the constituent units are similar to the monads occurring in the genus. However, the Melodinus tetrads are very dissimilar to those of Craspidospermum. Melodinus tetrads are tetrahedral, while the constituent units are 3‐colporate and have moderate polar costae and an infratectum measuring 1/3–1/4 of the exine. Craspidospermum tetrads are diversely non‐tetrahedral, while the units are irregularly 5–10‐porate and have heavy annular costae and an infratectum measuring ±1/10 of the exine.

On the basis of pollen morphology, it is hypothesised here that tetrads evolved in Melodinus independently from those of Craspidospermum. Possibly, this happened only once, because M. coriaceus and M. orientalis might be closely related, both being included by Pichon (1948a) in the section Pleurophacelus. According to Leeuwenberg (2003), most other Melodinus species placed by Pichon in this section belong to M. orientalis. M. baueri was included by Pichon in this section with a question mark, while M. australis Maiden & Betche non (F.Muell.) Pierre appeared to be a younger synonym of M. acutifloris (section Dichostemma). Further, there is no correlation between pollen character states and the subdivision into sections made by Pichon (1948a ; 60 species accommodated in seven sections). Leeuwenberg (2003; 19 species) did not propose any subdivision of Melodinus.

Craspidospermum tetrads are deviating within the Rauvolfioideae. They are not even comparable to the non‐tetrahedral tetrads of the rauvolfioid Condylocarpon, which are inaperturate, have a very thin wall, and are considered as neotenous (Van der Ham et al. 2001). Actually, Craspidospermum tetrads are similar, also in exine ultrastructure, to the tetrads of Apocynum of the subfamily Apocynoideae (Nilsson, 1990; Nilsson et al. 1993), but this genus is very remote from Craspidospermum in the molecular tree presented by Sennblad & Bremer (2002). If this tree is a good reflection of the actual relationships within the family Apocynaceae, then the tetrads of Craspidospermum and Apocynum represent a remarkable case of parallel or convergent evolution.

Acknowledgements

We would like to thank Bertie‐Joan van Heuven, Ben Kieft and Wim Star for their technical assistance, Laure Civeyrel (Toulouse) for the scanning electron micrographs of the pollen of C. verticillatum, Mary Endress (Zürich) for her advise concerning the systematics of the Apocynaceae and for reviewing the manuscript, and Toon Leeuwenberg (Wageningen) for material.

Specimens investigated

Craspidospermum verticillatum Madagascar: Civeyrel 1234 (Z), Nilsson 26‐9‐1994 (TAN), 30‐9‐1994 (S)

Melodinus acutifloris New Guinea: Pullen 384 (A), Veldkamp & Stevens 5985 (US)

M. aeneus New Caledonia: Mackee 14432 (K), 21599 (L), 26345 (Z)

M. angustifolius Taiwan: Kramer, Zogg & Gassner 7899 (WAG) Vietnam; Tonkin: Poilane 12780 (P)

M. axillaries China: Mao 04087 (KUN)

M. balansae New Caledonia: Baumann & Bodenheim 5951 (Z), 5964a (Z), Mackee 4826 (E)

M. baueri Norfolk Island: Maiden & Boorman anno 1902 (BM), Robinson anno 1884 (MEL)

M. cochinchinensis Hong Kong: Hu 7668 (A), 11931 (A)

M. coriaceus Malaysia: SF 1519 (L), Teo & David 868 (L)

M. cumingii Philippines: BS 48894 (BR), McGregor 1267 (US)

M. forbesii New Guinea: Floyd 5396 (L), Craven & Schodde 883 (L)

M. fusiformus Indochina: Pételot 908 (AHUC) China: Ching 5410 (A)

M. glaber Fiji: Gillespie 3720 (AHUC) New Hebrides: Kajewski 729 (A)

M. honbaensis Indochina: Chevalier 38813 (WAG)

M. orientalis Indonesia, Borneo: Hallier 3072 (L) u.s., Celebes: De Vogel 5249 (AHUC) Indonesia, Flores: Schmutz 3168a (L) Indonesia, Java: Winckel 1856b (L) New Guinea: Gideon 57473 (L) Philippines: Wenzel 2605 (AHUC), Ramos 1536 (L) Indonesia, Sumatra: De Wilde & Duyfjes 19970 (L)

M. philippinensis Philippines: Cuming 1574 (M)

M. phylliraeoides New Caledonia: Balansa 215 (P), Compton 1666 (BM)

M. reticulates New Caledonia: Mackee 29524 (P), McPherson 5351(M), Veillon 6188 (P)

M. scandens New Caledonia: Däniker 1121a (Z), Mackee 26316 (Z), 32762 (L), 32800 (L)

M. vitiensis Fiji: Degener 15269 (A), Gillespie 3190 (US)