View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Estudo Geral Pl Syst Evol 269: 159–170 (2007) Plant Systematics DOI 10.1007/s00606-007-0585-3 and Evolution Printed in The Netherlands A contribution to the ultrastructural knowledge of the pollen exine in subtribe Inulinae (Inuleae, Asteraceae) A. Pereira Coutinho, A. M. Dinis Department of Botany (Laboratory of Electron Microscopy and Palynology), Faculty of Sciences and Technology, University of Coimbra, Coimbra, Portugal Received 21 December 2006; Accepted 12 June 2007; Published online 2 November 2007 Ó Springer-Verlag 2007 Abstract. To better understand the relationships The Inulinae sensu Anderberg et al. (2005) within the Asteroideae and Inuleae, the structure comprise 38 genera and 480 species mainly with of the pollen exine was investigated in seven a Eurasian and Mediterranean distribution. genera and nine species of the subtribe Inulinae Roughly, it corresponds to the union of the using LM, TEM and SEM. All taxa have a subtribes Euinuleae and Buphtalmeae, created senecioid pattern of exine. The tectal complex by Bentham in 1873, to the union of the consists of three main layers that differ in thick- subtribes Inulinae and Buphtalminae, created ness and morphology: a tectum, a columellar layer, and a layer consisting of the basal region by Hoffmann in 1890, and to the union of the of the columellae. The absence or the vestigial groups Inula, Geigeria and Carpesium of the condition of the foramina is considered as a subtribe Inulinae sensu amplo, proposed by plesiomorphy within the Asteroideae. All taxa Merxmu¨ller et al. in 1977. The Inulinae (sensu have a complex apertural system that consists of Anderberg et al. 2005) were previously included an ecto-, a meso-, and an endoaperture. These in the Inuleae s. l., which also comprised the apertures intersect respectively the tectal complex, Gnaphalieae and the Plucheinae (as Plucheae), the foot layer and the upper part of the endexine, and proved to be polyphyletic (Bremer 1987, and the inner layer of the endexine. A continuous Anderberg 1988, Karis 1993, Bayer and Starr transition among the different species of Inulinae 1998, Panero and Funk 2002, Wagstaff and was found for all the quantitative characters Breitwieser 2002, Goertzen et al. 2003). examined. This relative homogeneity of the pollen The most modern concept of the Inuleae, morphological characters enhances the naturality of the subtribe Inulinae. proposed by Anderberg et al. (2005), consider the tribe as including the Inuleae (sensu Ander- Keywords: Pollen; exine; Inulinae; Inuleae; taxo- berg 1991a) and Plucheae (sensu Anderberg nomy; cladistics; electron microscopy 1991b). Within the Asteroideae, the Inuleae Correspondence: Anto´nio Pereira Coutinho, Department of Botany, Faculty of Sciences and Technology, University of Coimbra, 3001-455 Coimbra, Portugal e-mail: [email protected] 160 A. Pereira Coutinho, A. M. Dinis: Pollen exine in Inulinae (sensu Anderberg et al. 2005) was placed on structure is of great importance to understand different positions in the various morphological the evolution, systematic and ecology of the and molecular cladograms of the Asteraceae. Asteraceae. Using light microscope (LM), Stix This tribe was considered as sister group to the (1960) and Leins (1968, 1971) examined the rest of the Asteroideae taking into account the structure of the pollen exine in, respectively, 5 morphological studies performed by Bremer genera and 11 species of Inuleae (which were (1987, 1994) and Karis (1993) and the two non- grouped under 1 pollen type) and 32 genera coding chloroplast sequences investigation of and 137 species (which were grouped under 20 Bayer and Starr (1998). In the ndhF tree made pollen types). So far, the only transmission by Kim and Jansen (1995) the tribe Senecioneae electron microscopic (TEM) studies concern- was placed in a polytomy together with two ing the pollen exine of the Inuleae (sensu other clades: one including the Inuleae and the Anderberg et al. 2005) are those in Inula Heliantheae s. l., and the other including the britannica (Skvarla and Turner 1966), Alago- Anthemideae, Astereae, Calenduleae and pappus dichotomous and Blumea mollis Gnaphalieae. Based on comparative chloro- (Skvarla et al. 1977). The pollen wall architec- plastidial DNA sequence data, Panero and ture of the Inuleae was not investigated using Funk (2002) considered the Inuleae as sister the scanning electron microscope (SEM), mak- group of a large clade comprising 13 different ing the pollen exine of this tribe one of the less tribes and the Senecioneae as sister group of the studied within the Asteraceae. other Asteroideae. ITS investigations carried In the present work we investigated the out by Wagstaff and Breitwieser (2002) placed structure of the pollen exine in Asteriscus the Inuleae as the sister group of a clade aquaticus (L.) Less., Dittrichia viscosa (L.) comprising the Heliantheae s. l. and the Athro- Greuter, Inula salicina L., Jasonia tuberosa L., isma group, and the Calenduleae as sister group Limbarda crithmoides (L.) Dumort., Pallenis to the rest of the Asteroideae. Using the same maritima (L.) Greuter, Pallenis spinosa (L.) cladogram approach, Goertzen et al. (2003) Cass., and Pulicaria paludosa Link using LM, placed the Anthemideae and the Inuleae as TEM and SEM. The exine structure of Pulicaria sister groups, respectively, to the remaining of dysenterica (L.) Gaertn. was also investigated the Asteroideae and to a clade comprising the using SEM. These taxa were chosen because Senecioneae and the Calenduleae. Anderberg et they represent various clades in the clado- al. (2005), in their cladograms supported by grams of Eldena¨s et al. (1998) (three clades), ndhF studies, placed the Senecioneae in a Francisco-Ortega et al. (2001) (five clades) and polytomy together with two large clades: Anderberg et al. (2005) (three clades). one comprising the Inuleae-Inulinae, Inuleae- Plucheinae, Heliantheae, and the genera Aniso- Materials and methods pappus, Blepharispermum, Athroisma, Callilepis and Zoutspansbergia, and the other one includ- Pollen grains were collected from herbarium ing the Anthemideae, Astereae, Calenduleae vouchers held at Coimbra (COI) and Oporto and Gnaphalieae. (PO) and then acetolysed according to Erdtman As demonstrated by several authors (1960). Specimens examined and voucher data are (Wagenitz 1955, 1976; Skvarla and Larson given at the end of the paper. The terminology for pollen descriptions follows Punt et al. (1994). 1965; Skvarla and Turner 1966; Skvarla et al. Light microscopy. Pollen grains were mounted 1977; Bolick 1978, 1991; Praglowsky and in silicone oil and observed with a Leitz Laborlux Grafstro¨m 1980; Blackmore 1982; Tormo LM using the X100 oil immersion objective. Each Molina and Ubera Jime´nez 1995; Breitwieser of the following characters: length of polar axis (P), and Sampson 1997; Zavada and Villiers 2000; length of equatorial diameter (E), spine length and Ortiz and Pereira Coutinho 2001; Skvarla et al. spine width, was measured in 30 different pollen 2005), the comprehension of the pollen exine grains. P/E was then established. A. Pereira Coutinho, A. M. Dinis: Pollen exine in Inulinae 161 Scanning electron microscopy. Pollen grains region, internal tectum under the spines, and were treated with ultra-sounds (35 kc/s, 1 h), tectum (Figs. 1D, 2B, 3C). A large number of dehydrated in a graded acetone series (70%– vestigial micro-foramina are seen in the tectal 100%), and critical point dried. They were then complex (Figs. 1C, 1D, 3C). The caveae are mounted on aluminium stubs and sputter coated generally narrow (Figs. 1A, 1C, 2D), despite with a 30 nm layer of gold-palladium prior to the fact that their size varies from species to examination with a JEOL JSM-5400 at 10 kV. species, from grain to grain, and even within Transmission electron microscopy. Pollen grains were fixed in 2% osmium tetroxide in the same pollen exine. In the latter case the 0.1 M sodium cacodilate buffer, pH 7.2, for 24 h, caveae are usually larger under the spines than dehydrated in a graded ethanol series (70%– under the inter-spinular areas (Figs. 1B, 1C, 100%), and embedded in Spurr’s resin. Thin 1F, 3B, 3C). The foot-layer is smooth (Figs. 1 sections were obtained with a LKB Ultrotome F, 2E, 3A) with a thickness of (0.04–) 0.05– NOVA ultramicrotome equipped with a diamond 0.10 (–0.13) lm. The endexine is (0.25–) 0.35– knife, conventionally stained with uranyl acetate 0.59 (–0.80) lm thick, i.e. (3.5–) 5–10 (–13) · and lead citrate, and observed in a JEOL JEM-100 thicker than the foot layer in the inter- SX at 80 kV. Using micrographs of the exine of the apertural areas becoming thicker towards the species studied, 10 measurements were made for apertures (Figs. 1B, 2C, 3F). The upper each of the following characters: thickness of the surface of the endexine is smooth and lamel- exine, endexine, foot layer, columellar layer, tectum lated while the inner surface is disrupted and tectal complex, diameter of both the spinular microperforations and the interspinular microper- (Figs. 1A, 2D, 3E). The ectoapertures (colpi) forations, and distance from the base of each spine are subterminal, broad in the equatorial area in which the spinular microperforations are located and acute at the ends. They intersect the tectal and the spinular columellae end. The values of the complex (Figs. 2C, 3F). The mesoapertures following ratios: E/exine thickness, endexine thick- are lolongate (Fig. 3D) and their limits are ness/foot layer thickness, tectal complex thickness/ more or less covered by those of the ectoap- foot layer thickness + endexine thickness, and ertures making them difficult to observe under spine height/spine width, were then calculated. the SEM. They intersect the foot layer and the upper part of the endexine (Figs. 2C, 3F). The endoapertures are lalongate and either acute Results (Fig.