Pollen morphology of six species representing three genera of tribe Aptosimeae and six species representing two genera of tribe Myoporeae (Scrophulariaceae) is described and illustrated using light microscopy (LM) and scanning electron microscopy (SEM). Pollen grains in Aptosimeae are 3-syncolporate, prolate, sometimes oblate-spheroidal in shape; mainly medium-sized, occasionally in some taxa small; exine sculpture striate, rarely with microperforations and small granules. Pollen grains in Myoporeae are 3-colpate-diorate, spheroidal, rarely oblate-spheroidal or prolate in shape; mainly medium-sized, occasionally in some taxa small; exine sculpture foveolate, microreticulate, rugulate-microperforate, rugulate, sometimes with small granules. Two major pollen types, 3-syncolporate (Aptosimeae) and 3-colpate-diorate (Myoporeae) are recognized by aperture types. Within these pollen types, eight subtypes are distinguished (three in Aptosimeae, five in Myoporeae) based on pollen size, exine sculpture and details of colpi and endoapertures. Our analysis of palynomorphological data (both published earlier and newly reported here) outlines the same main clades as those revealed by molecular phylogenetic studies, corresponding to Aptosimeae, Leucophylleae Myoporeae and Androya, which differ by their pollen aperture types: 3-syncolporate, 3-colpate-diorate, and 3-colporate, respectively. There are also some differences in exine sculpture patterns. Thus, palynomorphological data are mainly consistent with recent results of molecular phylogenetic studies. Considerations on ancestral pollen character states in early-branching Scrophulariaceae are provided. It is assumed that the colporate type was probably ancestral in Scrophulariaceae; however, the ancestral status of the colpate type cannot be excluded as well.
Introduction
Current opinions on taxonomy and phylogeny of early-branching clades of Scrophulariaceae
Judging from the phylogenetic pattern revealed by recent molecular phylogenetic studies (Olmsted & al. 2001; Oxelman & al. 2005; Tank & al. 2006), the first early-branching lineage of the re-circumscribed Scrophulariaceae Juss. (s.str.) is represented by the tribe Hemimerideae, which is sister to the clade containing all other members of Scrophulariaceae. That large clade, in turn, comprises two subclades, one containing representatives of three tribes, Aptosimeae, Leucophylleae and Myoporeae in their current updated circumscriptions, and another grouping the remaining representatives of Scrophulariaceae.
The tribe Aptosimeae, as outlined in recent studies (Olmsted & Reeves 1995; Olmsted & al. 2001; Fischer 2004; Oxelman & al. 2005; Tank & al. 2006; Takhtajan 2009), is a group represented by three genera (Anticharis Endl., Aptosimum Burch. ex Benth., and Peliostomum E. Mey. ex Benth.) and c. 40 species geographically restricted to tropical and southern Africa and southern Asia from the Arabian Peninsula to Malesia. Two genera, Aptosimum with c. 20 species and Peliostomum with seven species, occur only in tropical and southern Africa, and representatives of Anticharis (with c. 14 species) also occur in southern Asia. South African taxa of Aptosimeae have a distinctive pollination syndrome. These species are reported to be pollinated by several taxa of a specialized group of pollen wasps (Hymenoptera, tribe Masarini), which are behaviourally distinct from all other aculeate wasps, approaching in that respect the true bees. Widespread association of Aptosimeae with these pollen wasps and floral specialization of species of wasp genera Celonites and Masarina have been established (Gess & Gess 2010). The tribe is also of special interest because one of its genera, Anticharis, contains the only known group of species of Scrophulariaceae having C4 photosynthesis (Sage & al. 2011; Khosravesh & al. 2012), which occurs very rarely in Lamiales (also registered in Blepharis Juss. in the Acanthaceae).
The Aptosimeae clade is sister to the clade Androya+Leucophylleae+Myoporeae (Oxelman & al. 2005; Tank & al. 2006). In the working version of the system of Lamiales (Olmstead 2012), Aptosimeae also contain Stemodiopsis Engl. (perhaps erroneously). Oxelman & al. (2005) placed Stemodiopsis in Lindernieae, “[a] novel strongly supported clade of taxa earlier assigned to Scrophulariaceae…” with “unclear relationships to the rest of Lamiales.” Later, Rahmanzadeh & al. (2005) placed this genus in Gratiolaceae (tribe Stemodieae, validly published in Reveal 2012). In publications that followed (Tank & al. 2006; Schäferhoff & al. 2010) Stemodiopsis was placed in Linderniaceae. The newest molecular phylogenetic data by Fisher & al. (2013) confirmed the close relationships of Stemodiopsis with other genera of Linderniaceae.
The Myoporeae clade in a strict sense currently includes the genera Eremophila R. Br. (more than 200 species, Australia, mostly arid regions in the west), Myoporum Sol. ex G. Forst, (c. 30 species, mostly Australian, with some extending to southeastern Asia, islands of the Pacific and Indian oceans), Bontia L. (monotypic, the Caribbean area), Pentacoelium Zucc. (monotypic, from southern Japan through the Ryukyu Islands and Taiwan to southeastern mainland China), and three recently described genera endemic to a small southwestern region in Western Australia: Diocirea Chinnock (four species), and monotypic Calamphoreus Chinnock and Glycocystis Chinnock (Chinnock 2007; Olmstead 2012). In his detailed taxonomic revision of the group, Chinnock (2007) preferred to treat it at the family rank (as Myoporaceae) but admitted that combining Myoporaceae and Leucophylleae is a reasonable option and that the combined “Myoporaceous clade” is “…possibly best treated at subfamily level within the Scrophulariaceae s.str.” (Chinnock 2007: 84). Considering generic relationships, Chinnock (l.c.) indicated that “Diocirea and Calamphoreus can be considered derivatives of Eremophila” and assumed that Glycosystis is closely related to one of the endemic Australian sections of Myoporum.
Androya H. Perrier, the monotypic genus represented by A. decaryi H. Perrier, is sometimes placed in Myoporeae or treated as a genus unassigned to any tribe (see Olmstead 2012). In earlier versions of his system, Takhtajan (1987, 1997) placed Androya in Buddlejaceae. Takhtajan (2009) later moved it to Myoporaceae, together with Bontia, Eremophila (incl. Pholidia R. Br. and Stenochilus R. Br.) and Myoporum.
The Leucophylleae clade contains Capraria L. (four species), Eremogeton Standl. & L. O. Williams (monotypic) and Leucophyllum Bonpl. (c. 16 species, including Faxonanthus Greenm.; see Henrickson & Flyr 1985). Geographically this group is restricted to Central America and the Caribbean, southernmost regions of North America, and some parts of South America (see the map in Gándara & Sosa 2013). In all recent versions of Takhtajan's system (Takhtajan 1987, 1997, 2009), Leucophylleae contained just two genera, Eremogeton and Leucophyllum, while Capraria was placed in the tribe Gratioleae of Scrophulariaceae s.l. Rahmanzadeh & al. (2005) included Capraria in Gratiolaceae tribe Gratioleae, which contradicts the placement suggested by Oxelman & al. (2005) and Gándara & Sosa (2013). The generic status of Faxonanthus and the phylogenetic relationships of Eremogeton remained unresolved until recently, due to the lack of molecular data (Oxelman & al. 2005). Gándara & Sosa (2013) filled that gap and included in their molecular study representatives of all three genera, paying special attention to Leucophyllum. They demonstrated that this genus in its traditional circumscription is not monophyletic, since both Capraria and Eremogeton are rooted within it. The alternative taxonomic options they suggested are (1) to submerge all three entities in one genus or (2) to split Leucophyllum into three genera, preserving the generic rank for Capraria and Eremogeton.
Molecular studies (Oxelman & al. 2005; Tank & al. 2006; Gándara & Sosa 2013) convincingly revealed that the sister clades corresponding to re-circumscribed tribes Leucophylleae and Myoporeae are together, in turn, sister to Androya. Reveal (2012) united tribes Aptosimeae, Leucophylleae and Myoporeae in Scrophulariaceae sub-fam. Myoporoideae.
Published data on pollen morphology
Scarce information is available on pollen grains of representatives of Aptosimeae. As far as we know, only pollen grains of Aptosimum depressum Burch. were studied by Erdtman (1952) using light microscopy. The situation with data on pollen morphology of Myoporeae and Leucophylleae is better. In particular, Erdtman (1952, light microscopy only) studied pollen of Bontia daphnoides L., Myoporum laetum G. Forst., M. sandwicense A. Gray, Pholidia adenotricha F. Muell. ex Benth. (now Eremophila adenotricha (F. Muell. ex Benth.) F. Muell.), P. alternifolia (R. Br.) Wettst. (now E. alternifolia R. Br.) and Capraria biflora L. and mentioned some affinity of pollen grains of C. biflora to those in some Myoporaceae (recognized then as a separate family). Punt & Leenhouts (1967; light microscopy only) studied pollen grains of the monotypic genus Androya restricted to Madagascar. Minkin & Eshbaugh (1989) provided information on pollen grains of Leucophyllum texanum Benth. (light and scanning electron microscopy). Niezgoda & Tomb (1975) studied pollen grains of 14 species of Scrophulariaceae (tribe Leucophylleae and C. biflora) and 14 species of Myoporaceae using light, scanning and transmission electron microscopy. The most extensive palynomorphological study of Myoporeae (Australian taxa) was recently carried out by Chinnock (2007), who studied pollen grains of 28 species, including four species of Diocirea, two species of the monotypic Calamphoreus and Glycocystis, 19 species of Eremophila and three species of Myoporum. Pollen grains of Bontia daphnoides and Pentacoelium bontioides Siebold & Zucc. were also studied for comparison.
Considering new data on phylogenetic relationships of early-branching (basal) lineages of Scrophulariaceae and the virtual absence of information on pollen grains of Aptosimeae, we decided to partially fill this gap. In order to obtain additional data for comparison, we also studied some species of Myoporeae. The main objective of the study was to analyse and compare data on pollen morphology of early-branching lineages of Scrophulariaceae with molecular phylogenetic evidence for revealing possible ways of morphological evolution and hypothetical ancestral character states of pollen in this family.
Material and methods
Pollen from six species belonging to all three genera of Aptosimeae was sampled in the herbarium of the Missouri Botanical Garden, St Louis, Missouri, U.S.A. (MO). Pollen from six species representing two genera (Eremophila and Myoporum) of Myoporeae was sampled in the herbarium of the M.G. Kholodny Institute of Botany, the National Academy of Sciences of Ukraine, Kyiv (Kiev), Ukraine (KW); herbarium codes according to Thiers (2015+). Investigated specimens are cited according to the label information.
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 the samples were sputter-coated with gold at the Center of Electron Microscopy of the M.G. Kholodny Institute of Botany (National Academy of Sciences of Ukraine, Kyiv). The micrographs were minimally edited with Adobe Photoshop 6.0 to enhance the images. Terminology used in descriptions of pollen grains mainly follows the glossaries by Punt & al. (1994) and Tokarev (2002), with minor adjustments.
Results
General description of pollen grains
Aptosimeae
Pollen grains in monads, radially symmetrical, isopolar, 3-syncolporate, prolate, sometimes oblate-spheroidal; mainly medium-sized, occasionally in some taxa small: P[olar axis]=25.3-45.2 µm, E[quatorial diameter]=15.9-33.2 µm. Outline in polar view 3-lobate, in equatorial view elliptic, sometimes circular. Colpi 2.4–6.6 µm wide, fused on apocolpia, with distinct, strict and slightly thickened margins; colpus membrane smooth.
Endoapertures mainly distinct, rarely indistinct, lalongate or sometimes lolongate, elliptic or circular, 4–7.9 µm long, 2.7–9.3 µm wide, sometimes covered by margins of colpi.
Exine thin, 0.7–2 µm thick. Exine layers invisible, sometimes tectum two times thinner than lower layers. Exine sculpture striate, rarely with microperforations and small granules; apertures membrane psilate, granulate and granulate-verrucate.
Myoporeae
Pollen grains in monads, radially symmetrical, isopolar, 3-colpate-diorate, spheroidal, rarely oblate-spheroidal or prolate; mainly medium-sized, occasionally in some taxa small: P=17.3-35.9 µm, E=14.6-33.2 µm. Outline in polar view 3-lobate, in equatorial view elliptic or circular. Colpi 2.7–6.6 µm wide, with distinct ± strict thickenings, or with slight to indistinct thickenings; margins uneven, slightly tapering to pointed or rounded ends; colpus membrane smooth.
Endoapertures distinct or indistinct, 4–13.3 µm long, 1.3–5.3 µm wide, lalongate, elliptic, margins uneven.
Exine 1.3–2.7 µm thick. Tectum two times thinner than infratectum. Columellae indistinct or distinct, short, arranged sparsely. Exine sculpture foveolate, microreticulate, rugulate-microperforate, rugulate, sometimes with small granules. Colpus membrane smooth, sometimes granulate.
Pollen types and subtypes
Pollen grains in the studied taxa can be subdivided into two basic types, based on their aperture types, with three and five subtypes, respectively. The subtypes are separated mainly according to the pollen size, exine sculpture, and details of the colpi and endoapertures.
Type I: 3-syncolporate.
This type includes all three genera of Aptosimeae.
Subtype Ia: P=25.3-33.2 µm, E=15.9-22.6 µm. Sculpture striate, rarely with microperforations. Colpi 2.7–4 µm wide. Anticharis.
1. Endoapertures distinct, elliptic, 4–6.6 µm long, 2.7–5.3 µm wide. Colpus membrane smooth. Anticharis imbricata.
2. Endoapertures indistinct, mainly circular, rarely elliptic, 4–6.6 µm long, 4–6.6 µm wide. Colpus membrane smooth and granulate. Anticharis linearis.
Subtype Ib: P=25.3-34.6 µm, E= 18.6-23.9 µm. Sculpture striate, rarely with microperforations and small granule. Colpi 2.4–4 µm wide. Peliostomum.
1. Endoapertures distinct, circular or elliptic, 4–5.3 µm long, 2.7–6.6 µm wide, covered by margins of colpi. Colpus membrane smooth, rarely granulate-verrucate. Peliostomum leucorrhizum.
2. Endoapertures distinct, circular or elliptic, 4–6.6 µm long, 2.7–4 µm wide, covered by margins of colpi. Colpus membrane smooth, rarely granulate. Peliostomum virgatum.
Subtype Ic: P=34.6-45.2 µm, E=22.6-33.2 µm. Sculpture striate. Aptosimum.
1. Colpi 2.4–2.7 µm wide. Endoapertures distinct, lolongate, elliptic, 5.3–7.9 µm long, 7.9–9.3 µm wide. Colpus membrane smooth or granulate-verrucate. Aptosimum spinescens.
2. Colpi 2.7–6.6 µm wide. Endoapertures distinct, circular, 6.6–7.9 µm long, 6.6–7.9 µm wide. Colpus membrane granulate and granulate-verrucate. Aptosimum indivisum.
Type II: 3-colpate-diorate.
This type includes all studied members of Myoporeae.
Subtype IIa: P=17.3-23.9 µm, E= 18.6-23.9 µm. Sculpture foveolate. Colpi 2.7–5.3 µm wide, with pointed ends. Endoapertures indistinct, elliptic, 5.3–7.9 µm long, 1.3–2.4 µm wide. Eremophila rotundifolia, E. sturtii.
Subtype IIb: P=18.6-22.6 µm, E=18.6-21.3 µm. Sculpture microreticulate. Colpi 2.7–3.3 µm wide, with pointed ends. Endoapertures distinct or indistinct, elliptic, 4–6.6 µm long, 2.7–5.3 µm wide. Myoporum oppositifolium.
Subtype IIc: P=21.3-25.3 µm, E=22.6-23.9 µm. Sculpture microreticulate. Colpi 3.3–5.3 µm wide, with pointed ends. Endoapertures distinct, elliptic, 5.3–9.3 µm long, 2.7–3.3 µm wide. Eremophila elderi.
Subtype IId: P=27.9-35.9 µm, E=26.6-31.9 µm. Sculpture rugulate-microperforate. Colpi 4–5.3 µm wide, with pointed ends. Endoapertures distinct, elliptic, 7.9–10.6 µm long, 2–2.4 µm wide. Eremophila glabra.
Subtype IIe: P=27.9-34.6 µm, E=25.3-33.2 µm. Sculpture rugulate. Colpi 2.7–6.6 µm wide, with rounded ends. Endoapertures distinct or indistinct, elliptic, 4–13.3 µm long, 2–4 µm wide. Eremophila debilis.
Descriptions of pollen grains
Tribes, genera within tribes and species within genera are listed alphabetically. The summary of pollen measurements for all studied taxa is provided in Table 1. Main pollen morphology characters are additionally summarized in Table 2.
Aptosimeae
Anticharis
Endl.
Anticharis imbricata
Schinz (Fig. 1A, G; 3A–D).
LM — Pollen grains 3-syncolporate, prolate, in polar view 3-lobed, in equatorial view elliptic. P=25.3-33.2 µm, E=15.9-22.6 µm. Colpi 2.7-4 µm wide, with distinct, strict margins, slightly thickened, fused on apocolpia; colpus membrane smooth, sometimes with granules over endoaperture. Endoapertures distinct, elliptic, 4–6.6 µm long, 2.7–5.3 µm wide. Exine 0.7–1.3 µm thick. Exine layers invisible. Exine sculpture invisible.
SEM — Sculpture striate, rarely with microperforations. Colpus membrane smooth, sometimes granular over endoaperture.
Specimen investigated — Namibia: grid ref. 2114 AA, flats SW of Brandberg West Mine, sandy stony open flats with scattered Welwitschia plants, 14 May 1976, Oliver & Müller 6666 (MO).
Anticharis linearis Hochst. ex Asch. (Fig. 1B, H; 3E–H).
LM — Pollen grains 3-syncolporate, prolate, in polar view 3-lobed, in equatorial view elliptic. P=26.6-30.6 µm, E=19.9-22.6 µm. Colpi 2.7–4 µm wide, with distinct, strict margins, slightly thickened, fused on apocolpia; colpus membrane smooth, sometimes with granules over endoaperture. Endoapertures indistinct, mainly circular, rarely elliptic, 4–6.6 µm long, 4–6.6 µm wide. Exine 0.7–1.3 µm thick. Exine layers invisible. Exine sculpture invisible.
SEM — Sculpture striate, rarely with microperforations. Colpus membrane smooth and granulate, sometimes granular over endoaperture.
Specimen investigated — Namibia: grid ref. 2618 AA, low bush plains c. 45 km N of Keetmanshoop, 13 Mar 1988, P. Goldblatt & J. Manning 8748 (MO).
Aptosimum
Burch, ex Benth.
Aptosimum indivisum
Burch, ex Benth. (Fig. 1C, I; 3I–L).
LM — Pollen grains 3-syncolporate, prolate, in polar view 3-lobed, in equatorial view elliptic. P=37.2-45.2 µm, E=27.9-31.9 µm. Colpi 2.7–6.6 µm wide, with distinct, strict margins, slightly thickened, fused on apocolpia; colpus membrane smooth, sometimes with granules over endoaperture. Endoapertures distinct, circular, 6.6–7.9 µm long, 6.6–7.9 µm wide. Exine 1.1–2 µm thick. Exine layers invisible. Exine sculpture indistinct.
SEM — Sculpture striate. Colpus membrane granulate and granulate-verrucate, sometimes granular over endoaperture.
Specimen investigated — South Africa: Northern Cape: grid ref. 3119 (Calvinia) BD, along rail tracks near Downes Siding, 31°29′03″S, 19°57′04″E, 3606 ft, 12 Sep 2004, Peter Goldblatt & L. J. Porter 12415 (MO).
Fig. 1.
Pollen grains of Aptosimeae (SEM) — A, G: Anticharis imbricata; B, H: A. linearis; C, I: Aptosimum indivisum; D, J: A. spinescens; E, K: Peliostomum leucorrhizum; F, L: P. virgatum; A–B, D–F: equatorial view; C: equatorial and polar view; exine sculpture: G, J, L: striate; H, K: striate with microperforations; I: granulate and granulate-verrucate aperture membranes. — Scale bars: A-F = 5 µm; G-L = 1 µm.
Aptosimum spinescens (Thunb.) Weber (Fig. 1D, J; 3M–P).
LM — Pollen grains 3-syncolporate, prolate, rarely oblate-spheroidal, in polar view 3-lobed, in equatorial view elliptic, sometimes circular. P=34.6-39.9 µm, E=22.6-33.2 µm. Colpi 2.4–2.7 µm wide, with distinct, strict margins, slightly thickened, fused on apocolpia; colpus membrane smooth, sometimes with granules over endoaperture. Endoapertures distinct, elliptic, 5.3–7.9 µm long, 7.9–9.3 µm wide. Exine 1.1–1.3 µm thick. Exine layers invisible. Exine sculpture indistinct.
SEM — Sculpture striate. Colpus membrane smooth or granulate-verrucate, sometimes granular over endoaperture.
Specimen investigated — Namibia: grid ref. 2416 DB, Maltehöhe district, N of Maltehöhe, about 45 km on road to Walvis Bay, 24°40′495″S, 16°49′513″E, 1341 m, dry grassland, sandy loam soil, dryland (veld), level flat slope, 11 Mar 1995, P. M. Burgoyne 3472 (MO).
Peliostomum
E. Mey. ex Benth.
Peliostomum leucorrhizum
E. Mey. ex Benth. (Fig. 1E, K; 3Q–T).
LM — Pollen grains 3-syncolporate, prolate, in polar view 3-lobed, in equatorial view elliptic. P=25.3-31.9 µm, E=18.6-22.6 µm. Colpi 2.7–4 µm wide, with distinct, strict margins, slightly thickened, fused on apocolpia; colpus membrane smooth, with granules over endoaperture. Endoapertures distinct, circular or elliptic, sometimes lolongate, 4–5.3 µm long, 2.7–6.6 µm wide, covered by margins of colpi. Exine 1.1–1.3 µm thick. Exine layers usually invisible; sometimes visible, with tectum two times thinner than lower layers. Exine sculpture invisible.
SEM — Sculpture striate, rarely with microperforations and small granules. Colpus membrane smooth, rarely granulate-verrucate.
Specimen investigated — South Africa: Northern Cape Province: 31 km N of Britstown (on N 13), karroid veld and sandy lax shrub steppe, 29 Sep 1974, B. Nordenstam & J. Lundgren 2106 (MO).
Peliostomum virgatum E. Mey. ex Benth. (Fig. 1F, L; 3U–X).
LM — Pollen grains 3-syncolporate, prolate, in polar view 3-lobed, in equatorial view elliptic. P=27.9-34.6 µm, E=19.9-23.9 µm. Colpi 2.4–4 µm wide, with distinct, strict margins, slightly thickening, fused on apocolpia; colpus membrane smooth, with granules over endoaperture. Endoapertures distinct, circular or elliptic, 4–6.6 µm long, 2.7–4 µm wide, covered by margins of colpi. Exine 0.7–1.3 µm thick. Exine layers usually invisible; sometimes visible, with tectum two times thinner than lower layers. Exine sculpture invisible.
SEM — Sculpture striate, rarely with microperforations and small granules. Colpus membrane smooth, rarely granulate.
Specimen investigated — South Africa: Northern Cape: grid ref. 2917 DC, Namaqua National Park, Keerom, Keurbos homestead, 29°59′39″S, 17°39′38.5″E, 464 m, level succulent karroo, well-drained sand soil, granite, full sun, 12 Aug 2009, Bester S. P. 9438 (MO), det. J. Ready, Sep 2009.
Myoporeae
Eremophila
R. Br.
Eremophila debilis
(Andrews) Chinnock (= Myoporum debile (Andrews) R. Br.) (Fig. 2E, K; 4Q–T).
LM — Pollen grains 3-colpate-diorate, prolate, oblate-spheroidal, rarely spheroidal, in polar view 3-lobed, in equatorial view elliptic or circular. P=27.9-34.6 µm, E=25.3-33.2 µm. Colpi 2.7–6.6 µm wide, with distinct, strict and thickened, or indistinct margins, slightly tapering to rounded ends; aperture membranes smooth. Endoapertures distinct or indistinct, elliptic, 4–13.3 µm long, 2–4 µm wide. Exine 2–2.7 µm thick. Tectum two times thinner than infratectum. Columellae distinct, short, sparsely arranged. Exine sculpture distinct, microreticulate.
SEM — Sculpture rugulate, sometimes with small granules. Colpus membrane smooth, sometimes also with small granules.
Specimen investigated — Australia: Queensland: Darling Downs district, Yelarbon, around cemetery, c. 1 km from P.O., 28°34′S, 150°45′E, 240 m, topography flat, brown loamy soil, damaged Eucalyptus woodland, 10 Oct 1983, E. M. Canning 5820 & B. Rimes (KW).
Eremophila elderi F. Muell. (Fig. 2A, G; 4A–D).
LM — Pollen grains 3-colpate-diorate, spheroidal, rarely oblate-spheroidal and prolate, in polar view 3-lobed, in equatorial view circular. P=21.3-25.3 µm, E=22.6-23.9 µm. Colpi 3.3–5.3 µm wide, with distinct margins, slightly tapering to pointed ends; aperture membranes smooth. Endoapertures distinct, elliptic, 5.3–9.3 µm long, 2.7–3.3 µm wide. Exine 2–2.4 µm thick. Tectum two times thinner than infratectum. Columellae distinct or indistinct, short, arranged sparsely. Exine sculpture distinct, reticulate.
SEM — Sculpture microreticulate, rarely with small granules near colpi. Lumina of reticulum small, rounded, elongated or rounded-angular. Colpus membrane smooth, sometimes with small granules.
Specimen investigated — Australia: South Australia: NW region, N-facing slopes of N-most extremity of groups of prominent inselbergs between Deering Hills and Mann Ranges, c. 18 km NE of Mt Cooperinna, 26°15′00″S, 130°05′30″E), common between rocks on hillside, 8 Nov 1978, N. N. Donner 6619 (KW).
Eremophila glabra (R. Br.) Ostenf. (Fig. 2B, H; 4E–H).
LM — Pollen grains 3-colpate-diorate, prolate, rarely spheroidal and oblate-spheroidal, in polar view 3-lobed, in equatorial view elliptic, rarely circular. P=27.9-35.9 µm, E=26.6-31.9 µm. Colpi 4–5.3 µm wide, with distinct, ± strict, thickened margins, slightly tapering to pointed ends; aperture membranes smooth. Endoapertures distinct, elliptic, with uneven margins, 7.9–10.6 µm long, 2–2.4 µm wide. Exine 2.4–2.7 µm thick. Tectum two times thinner than infratectum. Columellae mainly indistinct or distinct, short, sparsely arranged. Exine sculpture distinct, microreticulate.
SEM — Sculpture rugulate-microperforate, rarely with small granules near colpi. Colpus membrane smooth, sometimes with small granules.
Specimen investigated—Australia: South Australia: N Eyre Peninsula, foot of Iron Duke, at S end of Middleback Range, c. 50 km SW of Whyalla, growing in deep red sand, 29 Dec 1970, A. E. Orchard 2942 (KW).
Fig. 2.
Pollen grains of Eremophila and Myoporum (SEM) — A, G: E. elderi; B, H: E. glabra; C, I: E. rotundifolia; D, J: E. sturtii; E, K: E. debilis; F, L: M. oppositifolium; A–C, E: equatorial view; D, F: polar view; exine sculpture: G, L: microreticulate; H: rugulate-microperforate; I–J: foveolate; K: rugulate. — Scale bars: A-F = 5 µm ; G-L = 1 µm.
Eremophila rotundifolia F. Muell. (Fig. 2C, I; Fig. 4I–L).
LM—Pollen grains 3-colpate-diorate, prolate and oblate-spheroidal, rarely spheroidal, in polar view 3-lobed, in equatorial view elliptic or circular. P=18.6-23.9 µm, E=18.6-23.9 µm. Colpi 4–5.3 µm wide, with indistinct, ± strict margins, slightly tapering to pointed ends; aperture membranes smooth. Endoapertures indistinct, elliptic, with uneven margins, 5.3–6.6 µm long, 1.3–2.4 µm wide. Exine 1.1–1.6 µm thick. Tectum two times thinner than infratectum. Columellae distinct, short, sparsely arranged. Exine sculpture distinct, microreticulate.
SEM — Sculpture foveolate. Colpus membrane smooth.
Specimen investigated — Australia: South Australia: Lake Eyre Basin, Beresford Hill, 29°16′S, 136°40′E, 80–100 m, extinct spring mound, limestone formation, 4 Oct 1978, K. Chorney 1020 (KW).
Eremophila sturtii R. Br. (Fig. 2D, J; 4M–P).
LM — Pollen grains 3-colpate-diorate, prolate, rarely spheroidal, in polar view 3-lobed, in equatorial view elliptic, rarely circular. P=17.3-21.3 µm, E=14.6-18.6 µm. Colpi 2.7–5.3 µm wide, with indistinct, uneven margins, slightly tapering to pointed ends; aperture membranes smooth. Endoapertures indistinct, elliptic, with uneven margins, 5.3–7.9 µm long, 1.3–2.4 µm wide. Exine 1.1–1.6 µm thick. Tectum two times thinner than infratectum. Columellae indistinct. Exine sculpture mainly indistinct, microreticulate.
SEM—Sculpture foveolate, rarely with granules near colpi. Colpus membrane smooth, sometimes with small granules.
Specimen investigated —Australia: Queensland: Warrego district, c. 25 km SW of Eulo on Hungerford road, flood plain of Paroo River, 28°20′S, 144°54′E, grey sandy clay soil, Eucalyptus largiflorens open woodland with well-developed shrubby layer of Eremophila mitchellii and E. sturtii, 13 Sep 1973, R. J. Henderson H2048 & D. E. Boyland (KW).
Myoporum
Sol. ex G. Forst.
Myoporum oppositifolium
R. Br. (Fig. 2F, L; 4U–X).
LM — Pollen grains 3-colpate-diorate, spheroidal, rarely oblate-spheroidal and prolate, in polar view 3-lobed, in equatorial view circular. P=18.6-22.6 µm, E=18.6-21.3 µm. Colpi 2.7–3.3 µm wide, with distinct, slightly thickening or indistinct margins, slightly tapering to ± pointed ends; aperture membranes smooth. Endoapertures distinct or indistinct, elliptic, 4–6.6 µm long, 2.7–5.3 µm wide. Exine 2–2.7 µm thick. Tectum two times thinner than infratectum. Columellae indistinct, short, sparsely arranged. Exine sculpture distinct, reticulate.
SEM — Sculpture microreticulate, sometimes with small granules. Lumina of reticulum small, rounded, elongated or rounded-angular. Colpus membrane smooth, sometimes with small granules.
Specimen investigated — Australia: Western Australia: SW division, Augusta c. 115 km SSW of Bunbury, near golf links, 12 Oct 1967, A. M. Ashby 2377 (KW).
Discussion
Comparative pollen morphology of Aptosimeae
The palynomorphological data showed that pollen grains of the studied taxa of Aptosimeae have similar aperture types and sculpture; however, they differ in their size and some features of apertures (Table 1, 2). Thus, species of Aptosimum have the largest pollen grains and endoapertures as compared to two other genera, Anticharis and Peliostomum (Table 1). Pollen grains of the two studied species of Peliostomum have distinctive features in the structure of their apertures: endoaperture edges are covered with edges of colpi, a feature not observed in Anticharis and Aptosimum.
The two studied species of Aptosimum differ in their pollen size and structure of apertures. Pollen grains of A. indivisum are larger, with wider colpi and circular endoapertures, while pollen grains of A. spinescens are smaller, with narrower colpi and elliptic endoapertures.
The studied species of Anticharis differ in the shape of endoapertures (Table 2).
Species of Peliostomum have pollen grains similar in size, colpi structure and exine sculpture. In both P. leucorrhizum and P. virgatum we observed both circular and elliptic endoapertures; however, in addition to that, lolongate endoapertures also sometimes occur in P. leucorrhizum.
Probably the specialized pollination by pollen wasps (Gess & Gess 2010) mentioned above is partly responsible for at least some peculiar pollen morphology features of Aptosimeae revealed in the present study.
Comparative pollen morphology of Myoporeae
Our data showed that pollen grains in the studied species of tribe Myoporeae are similar by their 3-colpate-diorate type of apertures but differ in their size, exine sculpture and structural features of apertures (Table 1, 2).
The studied species of Eremophila differ in their pollen size, exine sculpture and structural features of apertures. The smallest pollen grains are characteristic of Eremophila elderi, E. rotundifolia and E. sturtii. Pollen grains of E. rotundifolia and E. sturtii are similar in having narrow endoapertures and foveolate sculpture; in contrast, pollen grains of E. elderi have wider endoapertures and microreticulate exine sculpture. Pollen grains of E. debilis and E. glabra are larger than those in the other three species. These two species differ by their colpi structure: colpi in E. debilis have rounded ends, whereas in E. glabra the ends are pointed. Pollen grains of these two taxa also differ from those in all other species by their exine sculpture (Table 2).
Pollen of Myoporum oppositifolium is similar to pollen of Eremophila rotundifolia and E. sturtii in size and colpi structure; however, it differs by exine sculpture and endoaperture structure (Table 1, 2).
In general, our data are comparable to data provided by Niezgoda & Tomb (1975), with some additional features revealed. In particular, we found in members of Eremophila some exine sculpture types that were not reported earlier: foveolate, rugulate-microperforate and rugulate. Niezgoda & Tomb (1975) mentioned that rugulate exine sculpture occurs only in two studied species, Capraria biflora and Leucophyllum pruinosum I. M. Johnst. Our studies demonstrated that this sculpture pattern also occurs in some representatives of Eremophila.
Fig. 3.
Pollen grains of Aptosimeae (LM) — A–D: Anticharis imbricata; E–H: A. linearis; I–L: Aptosimum indivisum; M–P: A. spinescens; Q–T: Peliostomum leucorrhizum; U–X: P. virgatum; A, B, E, F, I, J, M, N, Q, R, U, V: equatorial view; C, D, G, H, K, L, O, P, S, T, W, X: polar view. — Scale bars: A-X = 10 µm.
Fig. 4.
Pollen grains of Eremophila and Myoporum (LM) — A–D: E. elderi; E–H: E. glabra; I–L: E. rotundifolia; M–P: E. sturtii; Q–T: E. debilis; U–X: M. oppositifolium; A, B, E, F, I, J, M, N, Q, R, U, V: equatorial view; C, D, G, H, K, L, O, P, S, T, W, X: polar view. — Scale bars: A-X = 10 µm.
Table 1.
Summary of pollen measurements. All dimensions are in µm.
Chinnock (2007) reported that pollen grains of almost all genera (selected representatives of Calamphoreus, Eremophila, Glycocystis and Myoporum) have reticulate exine (coarsely and finely reticulate), and only all four species of Diocirea have punctitegillate (perforate) exine sculpture. However, our new data indicated that an exine sculpture pattern very similar to punctitegillate (perforate) also occurs in some species of Eremophila, in particular, E. rotundifolia and E. sturtii. Because of that, we believe that this peculiar exine pattern cannot be regarded as a reliable character distinguishing Diocirea and Eremophila.
Species of Eremophila studied by us belong to different sections (following the system proposed by Chinnock 2007) and differ by their pollen morphology. However, at present we have insufficient palynological data to analyse whether these characters support the sections, and pollen studies of additional taxa of that genus are needed.
Comparison of palynomorphological and molecular phylogenetic evidence
According to molecular phylogenetic data (Oxelman & al. 2005; Khosravesh & al. 2012), Anticharis and Peliostomum are sister groups within Aptosimeae. Palynomorphological data confirm the close relationship between these two genera, as shown above. It has been also shown that within Anticharis three clades can be outlined reflecting the gradual transition to C4 photosynthesis (Khosravesh & al. 2012). Aptosimum is sister to the Anticharis+Peliostomum clade, and pollen morphology data also support that pattern, since the morphological differences between pollen grains of Anticharis and Peliostomum are exceeded by the differences between those two genera and Aptosimum.
According to molecular phylogenetic data (Oxelman & al. 2005), the genera Bontia, Eremophila and Myoporum (Myoporeae in the strict sense) form a clade sister to the Capraria+Leucophyllum clade (Leucophylleae). Our palynomorphological data, as well as data of other authors (Niezgoda & Tomb 1975), are compatible with the molecular phylogenetic data. Pollen grains of all studied representatives of these genera are 3-colpate-diorate, with foveolate, microreticulate, reticulate, rugulate-perforate and/or rugulate exine sculpture.
The monotypic Madagascan genus Androya has been revealed by molecular phylogenetic analysis (Oxelman & al. 2005) as the most early-branching clade, which is sister to the clade containing all other genera of Myoporeae and Leucophylleae. According to Punt & Leenhouts (1967), pollen grains of Androya are 3-colporate, with smooth exine sculpture, thus differing considerably from pollen grains of other taxa of Myoporeae and Leucophylleae.
Oxelman & al. (2005) indicated that members of Leucophylleae (Capraria and Leucophyllum) are closely related to Myoporeae according to anatomical and palynomorphological evidence. Considering possible relationships of Aptosimeae, they mentioned that “[m]onophyly of Aptosimeae is strongly supported by our chloroplast DNA sequences, and a sister group relation with Androya, Leucophylleae, and Myoporeae is moderately supported. All have 3-colpate, diporate [sic!] pollen (Erdtman 1952; Niezgoda & Tomb 1975; Punt 1980)” (Oxelman & al. 2005: 419). The word “diporate” in their text is most probably an error for “diorate”.
In fact, our data indicate that pollen grains in the clades Aptosimeae, Leucophylleae+Myoporeae and Androya evidently differ by their aperture types: 3-syncolporate, 3-colpate-diorate, and 3-colporate, respectively. There are also some differences in exine sculpture patterns: striate in Aptosimeae; rather diverse (but not striate nor smooth) in Leucophylleae+Myoporeae, a more diverse and species-rich group; and smooth in Androya (Table 2).
Table 2.
Summary of pollen morphological characters: original data compared with literature data. “—” = no data reported.
Continued.
Our palynomorphological data, as well as other palynomorphological (Niezgoda & Tomb 1975) and molecular (Oxelman & al. 2005) evidence, indicate that the Androya clade probably merits recognition as a separate tribe, if Leucophylleae and Myoporeae are also recognized as separate tribes. An alternative logical solution would be an extended Myoporeae, re-circumscribed to include Leucophylleae and Androya.
Possible ancestral pollen character states in Scrophulariaceae
Pollen grains of representatives of all these tribes are characterized by the complex colporate type (combining ectoapertures and endoapertures). It may be assumed that this type is ancestral in the family Scrophulariaceae; however, the ancestral status of the colpate type cannot be excluded as well. If we postulate the colporate type as being ancestral in the family, then the 3-colporate type with smooth exine sculpture is probably the most ancient pattern, being also typical for many representatives of Buddlejeae and Teedieae (Tsymbalyuk & Mosyakin 2013; Mosyakin & Tsymbalyuk 2015), as well as for Androya. Further evolutionary changes of this type could have led to the formation of more advanced 3-colpatediorate pollen with rugulate and reticulate exine sculpture (peculiar to Leucophylleae+Myoporeae) and 3-syncolporate pollen with mainly striate sculpture (typical for Aptosimeae, as shown in this article), either in parallel or consecutively.
Our original data on pollen morphology of Hemimerideae, most probably the basalmost clade of Scrophulariaceae, will be published later. However, it is already evident from our preliminary data that two subclades of that tribe have colpate and colporate pollen, respectively, which is consistent with our hypothesis on pollen evolution patterns in early-branching Scrophulariaceae.
Conclusions
Our analysis of available palynomorphological data (both published earlier and newly reported here) clearly outlines the same main clades as those revealed by molecular phylogenetic studies (Oxelman & al. 2005; Gándara & Sosa 2013): Aptosimeae (3-syncolporate pollen grains, striate exine sculpture); Leucophylleae+Myoporeae (3-colpate-diorate pollen grains, foveolate, microreticulate, rugulate-microperforate, rugulate and/or reticulate exine sculpture); and Androya (3-colporate pollen grains, smooth exine sculpture). The 3-colporate pollen type with smooth exine sculpture, typical for Androya and occurring also in many representatives of Buddlejeae and Teedieae (Tsymbalyuk & Mosyakin 2013; Mosyakin & Tsymbalyuk 2015), is hypothesized to be ancestral in Scrophulariaceae. The clades Leucophylleae+Myoporeae and Aptosimeae have more advanced pollen types: 3-colpate-diorate with mainly rugulate and reticulate exine in the former, and 3-syncolporate with mainly striate sculpture in the latter.
Acknowledgements
The authors express their gratitude to James C. Solomon, Head Curator of the Missouri Botanical Garden herbarium (MO), Tatyana V. Shulkina, and staff members of MO, and to Nataliya M. Shyian, Head Curator of the National Herbarium of Ukraine (KW; herbarium of the M. G. Kholodny Institute of Botany, National Academy of Sciences of Ukraine), for their cooperation and assistance in extracting pollen samples from herbarium specimens. Special thanks are due to Peter H. Raven for his long-term support and encouragement of the senior author's research at the MO herbarium. Kind help and cooperation of Dmytro O. Klymchuk, Head of the Center of Electron Microscopy (M. G. Kholodny Institute of Botany), is greatly appreciated. The authors are grateful to two anonymous reviewers for their detailed review of the manuscript, valuable comments and suggestions, and to Nicholas Turland for his skilful editorial comments.
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