The pollen morphology of seven species representing four genera of the tribe Teedieae (Scrophulariaceae) is described and illustrated using light microscopy (LM) and scanning electron microscopy (SEM). Two major pollen types, 3-colporate and 4-colporate, are recognized by aperture types. Within these pollen types, subtypes are distinguished based on exine sculpture, colpi, colpus membrane, and endoaperture characters. Within type I (3-colporate), three subtypes are recognized: Ia — sculpture psilate, rarely with microperforations; Ib — sculpture microperforate and microreticulate; and Ic — sculpture macroreticulate. Type II (4-colporate) is represented by only one subtype IIa — sculpture psilate, rough, and foveolate. The revealed characteristics of pollen grains are taxonomically significant at the generic and specific levels. Our palynomorphological data are consistent with the results of molecular phylogenetic studies. Pollen grains in Teedieae are typically characterized by the colporate aperture type (probably ancestral in Scrophulariaceae) and rather primitive characters of surface and sculpture of colpi membranes. Similar palynomorphological patterns in Teedieae and Buddlejeae can be viewed as ancestral for pollen characters observed in more advanced clades of Scrophulariaceae.
The tribe Teedieae, in its present circumscription, belongs to the family Scrophulariaceae, and its placement in this family in the strict sense is confirmed by recent molecular phylogenetic studies (Olmstead & Reeves 1995; Olmstead & al. 2001; Oxelman & al. 2005; Tank & al. 2006). As currently understood, the tribe is a rather compact group represented by (6 or)7(or 8) genera and 14 or 15 species, geographically restricted to S and SW Africa and Madagascar (Fischer 2004; Olmstead 2012). However, this group is important and interesting from a phylogenetic viewpoint, since its members have been revealed to be among the early branching lineages of the large clade containing also the crown clades of Scrophulariaceae, corresponding to tribes Buddlejeae, Limoselleae, and Scrophularieae (Oxelman & al. 2005). According to Oxelman & al. (2005), “Teedieae, Buddlejeae, Camptoloma, Phygelius, Manuleeae, and Scrophularieae form a very strongly supported monophyletic group based on chloroplast DNA sequences.” In this article we informally refer to this group as “crown Scrophulariaceae”.
The circumscription of Teedieae was rather contradictory, and some taxonomic problems still remain, even after recent molecular studies. Some researchers placed Oftia Adans. in the family Myoporaceae (Takhtajan 1966; Cronquist 1981; and others), Spielmaniaceae (Takhtajan 1987) or even in its own family, Oftiaceae (Takhtajan 1997, 2009).
According to molecular phylogenetic studies (Oxelman & al. 2005; Tank & al. 2006), the Teedieae include the genera Dermatobotrys Bolus, Freylinia Colla, Oftia, Teedia Rudolphi and probably some others, and are strongly supported as a monophyletic group. Takhtajan (2009) included in Teedieae only the genera Ranopisoa J.-F. Leroy and Teedia. He placed Dermatobotrys in the tribe Russelieae, and separated Freylinia and Phygelius E. Mey. ex Benth. in the tribe Freylinieae. According to recent data, the Freylinieae (Barringer 1993) probably do not merit recognition and this name should be considered a synonym of Teedieae. Barringer (1993) included in his Freylinieae the genera Antherothamnus N. E. Br., Freylinia, Manuleopsis Thell. and Phygelius; such circumscription has not been supported by molecular phylogenetic studies (Oxelman & al. 2005). Reveal (2012) placed Buddlejeae and Teedieae in a separate subfamily, Buddlejoideae. Further discussion of relationships of and within Teedieae is provided by Oxelman & al. (2005).
According to a working version of the synoptical classification of Lamiales (Olmstead 2012), the Teedieae include Dermatobotrys, Freylinia, Manuleopsis, Oftia, Phygelius, Ranopisoa (probably better placed in Oftia s.l.) and Teedia. In this system, Manuleopsis is also placed in Limoselleae (most probably a technical error), and Antherothamnus belongs to Scrophularieae (which is confirmed by molecular data).
Thus, easily observed morphological characters of the mentioned genera seem to be contradictory or even misleading, allowing various options of placement of these taxa in a phylogenetic system. In view of that, it is advisable to add to the analysis also some additional micromorphological characters, including palynomorphological ones. Our recent studies of pollen morphology of Scrophulariaceae and related families of Lamiales (Tsymbalyuk & Mosyakin 2009, 2010, 2012, 2013) and comparison of their results with recent molecular phylogenetic data revealed some important trends in pollen character evolution within the group. However, some phylogenetically important groups of Scrophulariaceae remained poorly studied palynomorphologically, or not studied at all. It is especially true for some little-known and geographically restricted genera, especially those occupying phylogenetically crucial early-branching positions in available cladograms.
We believe that a better understanding of phylogenetic distribution of pollen morphology patterns in Teedieae, a lineage branching between the early-branching (“basal”) and crown Scrophulariaceae, is therefore important for reconstructing the evolution of pollen character in the group.
The objectives of our research were to identify the main morphological characters of pollen grains in representatives of Teedieae and their correlation with molecular phylogenetic data, to assess their taxonomic significance, and to explore possibilities of their use for solving taxonomic problems.
The available information about pollen grains of representatives of Teedieae is rather scarce. Erdtman (1952) studied pollen grains of Oftia africana (L.) Bocq. using light microscopy. Niezgoda & Tomb (1975) studied pollen grains of Scrophulariaceae (tribe Leucophylleae) and Myoporaceae using light, scanning and transmission electron microscopy. They included in their research only one species of Oftia (O. africana) and reported that this species has 4-colpate pollen grains with foveolate sculpture of the exine. They stated that in these features it differs from other studied representatives of Scrophulariaceae and Myoporaceae, which usually have 3-colpatediorate pollen grains and reticulate and rugulate sculpture of the exine (Niezgoda & Tomb 1975). In our opinion, this interpretation is inaccurate, since O. africana has 4-colporate pollen grains.
In conclusion, published results of previous palynomorphological investigations reported little information concerning peculiarities of pollen grains in Teedieae. Six species, of which pollen morphology is reported here, have never been studied palynologically before.
Material and methods
Pollen from seven species belonging to four genera (Freylinia, Oftia, Phygelius, Teedia) of Teedieae was sampled in the herbarium of the Missouri Botanical Garden, St Louis, Missouri, U.S.A. (MO).
Pollen morphology was studied using light microscopy (LM) and scanning electron microscopy (SEM). For light microscopy studies (LM, Biolar, ×700), the pollen was acetolysed following Erdtman (1952). For size determinations, 20 measurements were taken along the polar and equatorial axes for each species.
For scanning electron microscopy (SEM, JSM-6060LA), pollen grains were treated with 96 % ethanol, then these samples were sputter-coated with gold at the Center of Electron Microscopy of the M. G. Kholodny Institute of Botany (Kyiv, National Academy of Sciences of Ukraine). The micrographs were minimally edited with Adobe Photoshop 5.5 to enhance the images.
General description of pollen grains
Pollen grains in monads, radially symmetrical, isopolar, 3-colporate or 4-colporate, oblate-spheroidal, spheroidal, and prolate; mainly medium-sized, occasionally in some taxa small; polar axis (P) 18.6–31.9 µm, equatorial diameter (E) 18.6–31.9 µm. Outline in polar view (amb) 3-lobate, slightly 3-lobate, 4-lobate, or slightly 4-lobate. Colpi of the following types: (1) long, narrow, 1.3–2.7 µm wide, ends acute; (2) long, medium-width, 2.4–4 µm wide, ends rounded; (3) long, wide, 4–5.3 µm wide, ends acute; (4) short, narrow, 1.1–1.6 µm wide, ends rounded; (5) short, medium-width, 1.6–2.4 µm wide, ends rounded; (6) short, wide, 2–4 µm wide, ends rounded. Colpus membrane with psilate or granulate surface.
Summary of pollen types and subtypes.
Endoapertures distinct or indistinct, 3.3–9.3 µm long, 2.4–9.3 µm wide, of the following types: (1) circular (in Freylinia lanceolata, Phygelius aequalis), (2) elliptic (F. tropica, P. capensis), (3) rectangular (Oftia africana, O. revoluta), (4) rectangular and square (Teedia lucida).
Exine thin, 1.1–2 µm thick. Tectum nearly equal to infratectum (columellae layer) or 2× thinner than lower layers. Columellae distinct or indistinct, thick, short, arranged regularly. Exine sculpture psilate, rough, microperforate, foveolate, microreticulate, or macroreticulate.
Pollen types and subtypes
Pollen grains in the studied taxa can be subdivided into two basic types, based on their aperture types. Each type contains one to three subtypes, separated mainly according to the exine sculpture but also partly using details of the colpi and endoapertures (Table 1).
Type I: 3-colporate. This type includes two genera: Freylinia and Phygelius.
Subtype Ia: Sculpture psilate, rarely with microperforations; colpi long, medium-width, 2.4–4 µm wide, ends rounded; colpus membrane psilate; endoapertures circular: Freylinia lanceolata.
Subtype Ib: Sculpture microperforate and microreticulate.
1. Colpi long, narrow, 1.3–2.7 µm wide, ends acute; colpus membrane psilate; endoapertures elliptic: Freylinia tropica.
2. Colpi long, wide, 4–5.3 µm wide, ends acute; colpus membrane psilate; endoapertures elliptic: Phygelius capensis.
Subtype Ic: Sculpture macroreticulate; colpi long, wide, 4 µm wide, ends acute; colpus membrane psilate; endoapertures circular: Phygelius aequalis.
Type II: 4-colporate. This type includes two genera: Oftia and Teedia.
Subtype IIa: Sculpture psilate, rough, and foveolate.
1. Colpi short, narrow, 1.1–1.6 µm wide, ends rounded; colpus membrane psilate; endoapertures rectangular: Oftia africana.
2. Colpi short, medium-width, 1.6–2.4 µm wide, ends rounded; colpus membrane psilate and granulate; endoapertures rectangular: Oftia revoluta.
3. Colpi short, wide, 2–4 µm wide, ends rounded; colpus membrane psilate and granulate; endoapertures rectangular or square: Teedia lucida.
Descriptions of pollen grains
LM — Pollen grains 3-colporate, oblate-spheroidal and occasionally spheroidal. Amb (polar view) slightly 3-lobed. P = 23.9-30.6 µm, E = 23.9-31.9 µm. Colpi long, 2.4–4 µm wide, with indistinct, ± strict margins, slightly thickening near endoapertures, tapering to rounded ends; colpus membrane psilate. Endoapertures distinct, circular, 5.3–9.3 µm long, 4–9.3 µm wide. Exine 1.1–1.6 µm thick. Exine layers indistinct, sometimes tectum 2× thinner than lower layers. Columellae distinct, thick, short, arranged regularly. Exine sculpture psilate.
SEM — Sculpture psilate, rarely with microperforations.
Specimen investigated — South Africa: Western Cape province: S Cape (Quarter degree Grid Ref. 3321 AD, Ladismith), Sevenweekspoort, streamsides, 14 Mar 1981, L. Hugo 2576 (MO).
LM — Pollen grains 3-colporate, prolate, spheroidal and occasionally oblate-spheroidal. Amb (polar view) 3-lobed. P = 18.6-30.6 µm, E = 18.6-25.3 µm. Colpi long, 1.3–2.7 µm wide, with distinct, ± level margins, slightly tapering to pointed ends; colpus membrane psilate, sometimes with granules over endoapertures. Endoapertures indistinct, elliptic, 4–7.9 µm long, 2.7 µm wide, covered by margins of colpi. Exine 1.3–1.6 µm thick. Tectum nearly as thick as infratectum. Columellae distinct, thick, short, arranged regularly. Exine sculpture microreticulate.
SEM — Sculpture microperforate and microreticulate. Cells of reticulum small, rounded or elongated, with broad walls. Colpus membrane psilate.
Specimen investigated — South Africa: N Province [Limpopo province]: (Grid Ref. 2427CB), Thabazimbi-Warmbaths road, roadside, dry stream bank, purple form, 25 Sep 1988, A. Fabian 1197 (MO).
LM — Pollen grains 3-colporate, prolate. Amb (polar view) 3-lobed. P = 26.6-31.9 µm, E = 21.3-25.3 µm. Colpi long, 4 µm wide, with distinct level margins, ends acute; colpus membrane psilate. Endoapertures indistinct, ± circular, 5.3–6.6 µm long, 4–7.9 µm wide. Exine 1.1–2 µm thick. Tectum 2× thinner than infratectum. Columellae distinct, short, arranged regularly. Exine sculpture reticulate.
SEM — Sculpture macroreticulate. Cells of reticulum large, rounded, elongated, rounded-triangular, or rounded-angular. Colpus membrane psilate.
Specimen investigated — South Africa: OVS [Free State province]: (Grid Ref. 2828DA), Kestell, Golden Gate Hoogland Nas. Park, naby klein waterval suid van Ruskamp, 2000 m, Jan 1974, L. C. C. Liebenberg 8245 (MO).
LM — Pollen grains 3-colporate, prolate and occasionally spheroidal or oblate-spheroidal. Amb (polar view) 3-lobed. P = 23.9-29.3 µm, E = 22.6-26.6 µm. Colpi long, 4–5.3 µm wide, with indistinct margins, ends acute; colpus membrane psilate. Endoapertures distinct or indistinct, elliptic, 6.6–7.9 µm long, 4 µm wide, sometimes covered by margins of colpi. Exine 1.1–1.3 µm thick. Tectum 2× thinner than infratectum. Columellae distinct or indistinct, arranged regularly. Exine sculpture reticulate.
SEM — Sculpture microreticulate. Cells of reticulum small, rounded, elongated, or rounded-angular. Colpus membrane psilate.
Specimen investigated — Lesotho: (2928 CB), Lesobeng area, between the Lesobeng school and Ha Lephoi village on the way to the airstrip, 2125 m, common on slope directly above stream, 3 Mar 1990, L. Smook 7231 (MO).
LM — Pollen grains 4-colporate, prolate-spheroidal and spheroidal. Amb (polar view) slightly 4-lobed. P = 22.6-27.9 µm, E = 21.3-27.9 µm. Colpi short, 1.1–1.6 µm wide, with distinct slightly thickened margins, ends rounded; colpus membrane psilate. Endoapertures distinct, elongated, rectangular, 6.6–9.3 µm long, 2.4–4 µm wide. Exine 1.1–1.3 µm thick. Columellae indistinct or sometimes visible. Exine sculpture psilate with thin pleats sparsely arranged, rarely microreticulate.
SEM — Sculpture psilate and foveolate. Colpus membrane psilate.
Specimen investigated—South Africa: Western Cape province: Cape Peninsula, Wynberg Hill, Jun 1950, N. S. Pillans 10046 (MO).
LM — Pollen grains 4-colporate, oblate-spheroidal. Amb (polar view) slightly 4-lobed. P = 21.3-25.3 µm, E = 21.3-27.9 µm. Colpi short, 1.6–2.4 µm wide, with indistinct level margin, ends rounded; colpus membrane psilate. Endoapertures distinct, elongated, rectangular, 6.6–9.3 µm long, 2.7 µm wide. Exine 1.1–1.6 (2) µm thick. Exine layers indistinct, columellae invisible. Exine sculpture psilate, with thin sparsely arranged pleats.
SEM — Sculpture psilate and rough, sometimes foveolate. Colpus membrane psilate, sometimes granulate.
Specimen investigated — South Africa: Northern Cape province: Namaqualand, pipeline track, 12 km from Nababiep towards Spektakelberg, clay substrate with many rocks, 15 Oct 1974, P. Goldblatt 3064 (MO).
LM — Pollen grains 4-colporate, oblate-spheroidal. Amb (polar view) 4-lobed. P = 19.9-25.3 µm, E = 22.6-27.9 µm. Colpi short, 2–4 µm wide, with distinct margins, ends rounded; sometimes two colpi fused in one on apocolpia; colpus membrane psilate and granulate. Endoapertures distinct, longitudinally elongated, square or rectangular, 3.3–5.3 µm long, 5.3–6.6 µm wide. Exine 1.1–1.6 µm thick. Exine layers indistinct, columellae occasionally distinct, short, arranged regularly. Exine sculpture perforate.
SEM — Sculpture psilate, rough, occasionally with small perforations or foveolate. Colpus membrane psilate, granulate.
Specimens investigated — South Africa: Eastern Cape province: Mountain Drive, overlooking Monument building, above by-pass, 3326 BC, 33°19′23″S, 26°31′58″E, 600 m, disturbed ground, 4 Nov 2001, P. B. Phillipson 5332 with P. Kornall [Kornhall?] (MO). — Northern Cape province: 3020 (Loeriesfontein) CD, Kubiskou mountain, W of Loeriesfontein, 30°50′04″S, 19°21′16″E, 4502 feet, in dolerite outcrop, 14 Sep 2006, P. Goldblatt & L. J. Porter 12828 (MO). — Western Cape province: 3320 (Montagu) CA, top of Ouberg Pass, sandstone rocks, in rock crevices, 27 Sep 1997, P. Goldblatt & J. Manning 10772 (MO).
The data obtained demonstrated that pollen grains of the studied taxa are different and can be distinguished at the levels of genera and species.
According to their aperture types, pollen grains in species of Freylinia and Phygelius are similar. Here the pollen grains have long colpi and a psilate colpus membrane, but differ in other characters. In particular, pollen grains of the two studied species of Phygelius have wide colpi with acute ends, but their endoapertures differ in shape: circular in P. aequalis and elliptic in P. capensis. Also, pollen grains of these species differ in their exine sculpture: maeroreticulate in P. aequalis and microperforate and microreticulate in P. capensis.
The two species of the genus Freylinia differ in their colpi structure, endoaperture shape, and exine sculpture. On the one hand, the structure of apertures and sculpture of pollen grains of F. tropica are similar to those in Phygelius capensis. But on the other hand, they differ visibly in the width of their colpi.
Pollen grains of Oftia and Teedia (phylogenetically sister crown groups, according to Oxelman & al. 2005) are similar in their aperture types, all having short colpi with rounded ends, and psilate, rough, and foveolate exine sculpture. However, there are some differences between these genera: the grains in Teedia have wider colpi and somewhat smaller (shorter but wider) endoapertures than those in Oftia. Moreover, in Teedia two colpi are sometimes fused on apocolpia, a feature never observed in Oftia. The two species of Oftia vary in the width of their colpi.
The patterns of all observed pollen characters seem to be unique for each of the studied species. It means that pollen characters, when looked at in detail, can be used to separate certain species in the tribe.
According to molecular phylogenetic data (Oxelman & al. 2005), Oftia and Teedia are included in the same clade as sister subclades. Palynomorphological data support the hypothesis of close relationships between these two genera, as shown above. The genus Freylinia is a sister group to the Oftia+Teedia clade, more distantly related to either of these genera than they are to each other, as also supported by our data on morphology of pollen grains. The studied members of the crown clade (Oftia+Teedia) have four colpi (probably an advanced character in the tribe), whereas Freylinia and Phygelius have tricolpate pollen grains. The Phygelius branch is sister to the clade containing Freylinia and the Oftia+Teedia clade.
In conclusion, where genera are concerned, although some characters are shared, if all details of palynomorphology are taken into account, they can be used to separate the genera.
The new palynomorphological data are rather consistent with the results of molecular phylogenetic studies. Apparently being a rather early-branching group of Scrophulariaceae, the Teedieae provide insights into the further morphological evolution of pollen grains in the whole family. We believe that in this tribe some ancestral pollen types and characters are preserved from the times of early evolutionary radiation of the family. For instance, representatives of Teedieae usually have the colporate aperture type, which probably should be considered as ancestral in Scrophulariaceae. Species of this tribe demonstrate rather primitive characters of their pollen grains: smooth, rough, perforate, or microreticulate exine sculpture and smooth, rarely granulate, sculpture of colpi membranes. However, some taxa of the tribe (e.g. Phygelius) already demonstrate some advanced pollen traits more peculiar to crown Scrophulariaceae. Pollen grains of P. aequalis have well-developed reticulate sculpture, which is more characteristic of pollen grains of apparently more evolutionarily advanced tribes of the family, such as Limoselleae and Scrophularieae.
Looking beyond the Teedieae, it is interesting to note that pollen grains in the tribe Buddlejeae are also characterized mainly by the colporate pollen type with smooth, rough, perforate, or reticulate surface sculpture and smooth sculpture of colpi membranes (Tsymbalyuk & Mosyakin 2013). In these characters, these two tribes are similar. Some representatives of Buddlejeae (e.g. Gomphostigma virgatum (L. f.) Baill.) also have a well-developed reticulate sculpture (Tsymbalyuk & Mosyakin 2013). Such pollen morphology patterns and character distribution in Buddlejeae are very similar to those found in Teedieae, which probably indicates close phylogenetic links between these clades, or even their parallel evolutionary trends. Moreover, the palynomorphological characters and patterns of their distribution in Teedieae and Buddlejeae can probably be viewed as ancestral (or, at least, close to ancestral) for those in more advanced clades of Scrophulariaceae.
Thus, the study reported here demonstrated rather good correlation of the main morphological characters of pollen grains in Teedieae and related tribes with new molecular phylogenetic data. In order to reveal the pathways of evolution of pollen grains in Scrophulariaceae in more detail, it would be also important to compare the data reported here with similar data on representatives of other early-branching lineages of the family, especially tribes Aptosimeae, Leucophylleae, Myoporeae and Hemimerideae. We already have palynomorphological data on key taxa of these groups. Information on pollen morphology in these tribes, a comparative analysis, and discussion on possible ancestral character states of pollen grains in Scrophulariaceae will be provided in a separate article (in preparation).
The authors express their gratitude to James Solomon and Tatyana Shulkina and other staff members of the Missouri Botanical Garden (MO), for their cooperation and assistance in extracting pollen samples from herbarium specimens. The kind help and cooperation of Dmytro O. Klymchuk, Head of the Center of Electron Microscopy, M. G. Kholodny Institute of Botany, National Academy of Sciences of Ukraine (KW), are greatly appreciated. The authors also thank Olga M. Korniyenko (KW), for her assistance with editing of some SEM and LM images, and two anonymous reviewers for their detailed reviews of the manuscript, valuable comments and suggestions.