Grana ISSN: 0017-3134 (Print) 1651-2049 (Online) Journal homepage: https://www.tandfonline.com/loi/sgra20 Pollen morphology of the genus Omphalodes Mill. (Cynoglosseae, Boraginaceae) António Pereira Coutinho , Sílvia Castro , Rodrigo Carbajal , Santiago Ortiz & Miguel Serrano To cite this article: António Pereira Coutinho , Sílvia Castro , Rodrigo Carbajal , Santiago Ortiz & Miguel Serrano (2012) Pollen morphology of the genus Omphalodes Mill. (Cynoglosseae, Boraginaceae), Grana, 51:3, 194-205, DOI: 10.1080/00173134.2012.665943 To link to this article: https://doi.org/10.1080/00173134.2012.665943 Published online: 18 Jun 2012. Submit your article to this journal Article views: 553 View related articles Citing articles: 4 View citing articles Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=sgra20 Grana, 2012; 51: 194–205 Pollen morphology of the genus Omphalodes Mill. (Cynoglosseae, Boraginaceae) ANTÓNIO PEREIRA COUTINHO1 , SÍLVIA CASTRO1, RODRIGO CARBAJAL2, SANTIAGO ORTIZ2 & MIGUEL SERRANO2 1 CFE, Centre for Functional Ecology, Department of Life Sciences, University of Coimbra, Coimbra, Portugal, 2 Department of Botany, Faculty of Pharmacy, University of Santiago de Compostela, Galicia, Spain Abstract To better understand the taxonomy and phylogeny of the genus Omphalodes (Boraginaceae), the pollen morphology of 23 species encompassing all major geographical and morphological groups was studied from acetolysed pollen grains using light and scanning electron microscopy. Three pollen types were distinguishable through apertural morphology and sculpture. A dichotomous key to the pollen types and pollen descriptions are provided. Despite being fairly homogenous, palynological data provided useful information to understand the relationships of Omphalodes, considering the uselessness of most floral and fruit characters, karyological homogeneity and striking disjunct geographical distribution. At the suprageneric level, pollen morphology supports the traditional tribal classification of Omphalodes within Cynoglosseae, in contrast to published molecular phylogenetic analyses. At the infrageneric level, pollen morphology agrees with phylogenetic analyses suggesting that Omphalodes could be a polyphyletic group. Palynological data would support the segregation of a monophyletic group comprising the Japanese species, corroborating recent phylogenetic results and define two different pollen types for the Omphalodes s. str. group. Biogeographical considerations are also discussed. Keywords: Acetolysis, dichotomous key, disjunct biogeographic pattern, ring-like aperture, phylogenetic relationships, pollen types, taxonomy Omphalodes (Boraginaceae, Boraginoideae) comprises 28 species of herbs from temperate habitats, including mostly narrow endemics but also some ornamental species like O. verna. The genus has a striking disjunct distribution in the Northern Hemisphere, occurring in three distant areas: Europe and adjacent areas of western Asia, northern Mexico and Texas, and Japan, being an unusual variant of the north temperate disjunct biogeographic pattern (sensu Raven, 1972). The genus shows great variation in habit (perennial, biennial and obligate annual species), ecology (ranging from coastal dunes to mountain summits or mesic habitats) and morphology (e.g. fruit characters). Still, some species seem to form natural subgroups as, for example the Japanese species or the annual species from south-western Europe. Several sections have been described for the western Euro-Asiatic taxa (de Candolle, 1846; Brand, 1921; Popov, 1953), but their evolutionary significance has not been assessed. It has been suggested that the Japanese species should be considered a subgenus (Popov, 1953) and that O. scorpioides could even deserve an independent genus (Johnston, 1924), albeit no formal proposals have been made so far. Despite its biogeographic singularity and the evidence for a certain infrageneric structure (Popov, 1953), no attempts have been made to find morphological characters that could clarify the internal relationships of the genus. The subfamily Boraginoideae has been divided into several tribes that vary from four to eight, depending on the author (de Candolle, 1846; Riedl, 1997). Omphalodes has been traditionally ascribed to the tribe Cynoglosseae (Brand, 1921), mainly Correspondence: António Pereira Coutinho, CFE, Centre for Functional Ecology, Department of Life Sciences, University of Coimbra, PO Box 3046, 3001-401 Coimbra, Portugal. E-mail: cafe@bot.uc.pt (Received 16 November 2011; accepted 16 February 2012) ISSN 0017-3134 print / ISSN 1651-2049 online © 2012 Collegium Palynologicum Scandinavicum http://dx.doi.org/10.1080/00173134.2012.665943 Pollen morphology of Omphalodes 195 because of the nutlet apical attachment to the gynobase (Johnston, 1924). Indeed, the internal systematics of the Boraginoideae, and in particular of the Cynoglosseae (Johnston, 1924; Riedl, 1997), heavily relies on fruit traits. However, this classification has been considered weak (Al-Shehbaz, 1991) mainly because fruits from this group are suggested to be under evolutionary selective pressures, thus, being labile characters (Johnston, 1924; Selvi et al., 2011) unsuitable for taxonomic purposes. Molecular analyses of Boraginaceae including all Boraginoideae tribes (Långström & Chase, 2002) and a representative of Omphalodes (the type species O. verna) have been performed to shed light on the phylogenetic relationships of the family. Interestingly, the phylogeny recovered merged the tribes Cynoglosseae, Eritrichieae, Myosotideae and Trigotideae and located Omphalodes outside of the Cynoglosseae s. l. clade at an unresolved position among all tribes of the subfamily Boraginoideae. Recent molecular phylogenetic analyses of Omphalodes species (Serrano et al., unpublished data) recovered five main clades: the American species, the annual European species, the western Eurasian perennial species, the Japanese species, and O. scorpioides. Two clades, the Japanese group and O. scorpioides, are separately nested among other genera of Cynoglosseae, therefore making the genus polyphyletic as currently circumscribed. However, morphological and karyological support for the inferred relationships is scarce. Floral characters are relatively homogeneous throughout the genus and fruit characters, albeit very variable, hardly show discontinuities that could be clearly correlated with the inferred phylogenetic groups. Karyological information is also relatively useless to clarify the internal evolutionary relationships of the genus, since the base number of the Boraginaceae (x = 12; Lewis, 1980) is much conserved within the Cynoglosseae (Luque & Valdés, 1986; Coppi et al., 2006; Selvi et al., 2011) and is present as 2n = 24 in both western Eurasian (Grau, 1967) and Japanese (Kadota, 2009) species of Omphalodes. The exceptions are the European annual taxa, having a derived chromosome number 2n = 28, (Grau, 1967; Valdés, 1987) and O. caucasica Brand with 2n = 22 (Grau, 1967). The Boraginaceae show great diversity in pollen ornamentation, size, shape and number of apertures and the family is considered one of the most eurypalynous among the angiosperms (Díez & Valdés, 1991). Several palynologists have demonstrated the importance of the palynological characters to understand the taxonomy, ecology and evolution of Boraginaceae (e.g. Díez et al., 1986; Ning et al., 1992; Scheel et al., 1996; Retief & Van Wyk, 1997; Liu et al., 2010). Pollen morphology has been used as a valuable tool in delimiting genera within Cynoglosseae (Barbier & Mathez, 1973) and in suggesting several evolutionary hypotheses (Clarke et al., 1979; Bigazzi et al., 2006). A few authors published some significant studies of the pollen morphology of Omphalodes using light microscopy (LM) and, in some cases, scanning electron microscopy (SEM) (Barbier & Mathez, 1973; Díez, 1984; Ahn & Lee, 1986; Díez & Valdés, 1991). Still, these are partial studies including only a few species and, thus, pollen morphology of Omphalodes is insufficiently known. The goal of this work was to investigate for the first time the variability of pollen morphology in Omphalodes, studying the majority of the species recognised and encompassing all major geographical and morphological groups, through light and scanning electron microscopy of acetolysed material. Since pollen is highly variable in Boraginaceae, it was hypothesised that the extremely disjunct distribution range of Omphalodes spp. suggests an ancient origin of the genus that may have allowed enough evolutionary time for accumulation of informative morphological changes. Palynological information was used to address two main questions in order to shed light to the complex internal systematics and the striking geographical pattern of Omphalodes. First, considering that molecular phylogenetic analyses question the position of Omphalodes within the Cynoglosseae (Långström & Chase, 2002), palynological data was studied to test whether pollen morphology supports the traditional tribal affiliation of the genus. Second, considering the homogeneity and continuum of variation of plant morphology, palynological data was explored to test if there is correlation between intrageneric variation in pollen morphology and the recent phylogenetic results that singularise, among others, the Japanese group. Material and methods Pollen samples were collected from a total of 34 herbarium specimens of the genus Omphalodes (see ‘Specimens investigated’) from the following institutions: COI, IEB, K, MA, P, SALAF, SANT, TEX, TI and TNS (abbreviations follow Holmgren et al., 1990). The samples included 26 Omphalodes taxa with representatives from all the geographic regions. All pollen samples were subjected to acetolysis according to the method proposed by Erdtman (1960). The terminology used for pollen descriptions follows Punt et al. (2007) and Hesse et al. (2009). For LM, pollen grains were pre-treated with t-butanol, mounted in silicone oil (Andersen, 1960) and observed using a Motic BA 310 light 196 A. P. Coutinho et al. Figure 1. SEM micrographs of pollen grains of Omphalodes. A–G. Omphalodes japonica pollen type: A. Omphalodes akiensis, equatorial view; B. Omphalodes japonica, equatorial view; C. Omphalodes krameri, equatorial view; D. Omphalodes akiensis, detail of apertural system and granula in the margins of colporus and pseudocolpus (arrow); E. Omphalodes krameri, detail of apertural system and granula in the margins of colporus and pseudocolpus (arrow); F, G. Omphalodes japonica, detail of apertural system and granula in the margins of colporus and pseudocolpus (F, arrow) and detail of the exine showing the perforations (G, arrow). ra – ring-like aperture. Scale bars – 2 µm (A–C); 1 µm (D–F); 500 nm (G). Pollen morphology of Omphalodes 197 microscope with an oil immersion objective lens (magnification = 1000×). The exine thickness was measured in 15 pollen grains from each taxon using a micrometer. For SEM, the acetolysed pollen grains were air dried (Ahn & Lee, 1986), mounted on aluminium stubs and coated with a 30 nm layer of gold/paladium for eight minutes at high vacuum in a sputtering chamber (Jeol JFC-1100 Ion Sputter). Pollen grains were then observed with a Zeiss FESEM ULTRA plus scanning electron microscope (operating at 15 kV) and micrographs of at least 15 pollen grains were taken using ImageTool (v.3.0 for Windows, University of Texas Health Science Centre, San Antonio, TX, USA). Due to the small pollen size, micrographs were then used for morphometric analysis. The following characters were measured in 15 pollen grains from each taxon: polar axis (P), equatorial axis (E), maximum width of the grain, pseudocolpi length, colpori ectoaperture length and endoaperture width. The P:E ratio was then calculated. The general pollen morphology and particularly the type of sculpture of the margins of the pseudocolpi and colpori and the presence/absence of a ring-like aperture were studied also from the SEM micrographs. The ring-like aperture is an outer circumferential aperture at the equator of the pollen grain (following Hesse et al., 2009). Descriptive statistics (mean and standard deviation of the mean) of the quantitative variables were calculated for each species (subspecies were not considered due to the low variability observed within species). Multivariate analyses were performed to investigate the structural organisation of the species studied based on all the quantitative and qualitative palynological characters combined. Principal component analysis (PCA) was performed using the specimen mean values for and all the measurements, except exine thickness. Palynological description of pollen types were made based on the quantitative and qualitative morphologic results. Results The morphology of the pollen belonging to 23 species of Omphalodes (covering 82% of the genus) was studied using LM and SEM (Figures 1, 2; Table I). This study constitutes the first survey of the pollen morphology for 19 of the species studied. Overall, pollen morphological features, including polarity, symmetry, shape, size and exine thickness of the species of Omphalodes studied were revealed to be fairly constant (Figures 1, 2). The most important characters for pollen type delimitation were qualitative: the sculpture of the margins of the colpori and pseudocolpi and the presence/absence of a ring-like aperture. Despite the continuous gradient of transition observed among the species for all the quantitative characters (Table I), it was possible to recognise three pollen types based on the presence/absence of a ring-like aperture and on the presence/absence of conspicuous granula in the apertural margins: O. verna pollen type, O. japonica pollen type and O. chiangii pollen type (see descriptions later). Interestingly, the abundance of these sculpture elements seems to be positively correlated with annual habits and negatively with perennial American species, and the presence of a ringlike aperture is restricted to the Japanese perennial species (Figures 1, 2; Table I). The first three axes accounted for 86.1% of the total variation (55.1, 17.2 and 13.8% of variation explained by the first, second and third components, respectively) as obtained via PCA of the specimens studied (Figure 3). The first component had a high negative loading for all the palynological characters except P:E ratio, while the second component had negative loadings for all the characters except E, pseudocolpi length and the presence/absence of granula in the ectoaperture margins, for which positive loadings were obtained (Figure 3). The multivariate analysis enabled us to separate the three pollen types across the two first components (Figure 3). Clearly, a group including the Japanese species (Omphalodes japonica pollen type) separates from the remaining species in both axes due to their greater pollen size (reflected in the characters measured) and qualitative characters (presence of a ring-like aperture and granula in the apertural margins; Figure 3). The remaining two groups (O. verna and O. chiangii pollen types) show a continuum, but can be separated from each other across the 씮 Figure 2. SEM micrographs of pollen grains of Omphalodes. A–D. Omphalodes chiangii pollen type: A. Omphalodes cardiophylla, equatorial view; B. Omphalodes chiangii, equatorial view; C. Omphalodes cardiophylla, detail of apertural system and margins of colporus and pseudocolpus without granula; D. Omphalodes chiangii, detail of apertural system and margins of colporus and pseudocolpus without granula. E–K. Omphalodes verna pollen type: E. Omphalodes linifolia, equatorial view; F, G. Omphalodes littoralis, equatorial view (F) and detail of apertural system and granula in the margins of colporus and pseudocolpus (G, arrow); H. Omphalodes commutata, detail of apertural system and granula in the margins of colporus and pseudocolpus (arrow); I. Omphalodes luciliae, equatorial view with visible perforations (arrow); J. Omphalodes verna, equatorial view; K. Omphalodes nitida, detail of apertural system and granula in the margins of colporus and pseudocolpus (arrow). Scale bars – 1.5 µm (A, B, E, F, I, J); 500 nm (C, D, G, H, K). 198 A. P. Coutinho et al. Pollen morphology of Omphalodes 199 second component (Figure 3). These observations corroborate the pollen types defined later. General palynological description of Omphalodes Pollen grains are radially symmetrical, isopolar, rectangular-elliptic and more or less constricted at the equator (dumbbell-shaped), more or less rounded at the poles, sub-circular to subhexagonal in polar view; prolate to perprolate; P:E = 1.3 – 2.8 (2.3 ± 0.4), P = 6.4 – 10.9 (8.6 ± 0.9) µm, E = 2.2 – 6.2 (3.8 ± 0.2) µm, maximum width of the grains = 2.2 – 7.5 (4.8 ± 0.6) µm. Apertural system is 6-zono-heterocolpate: three pseudocolpi 3.6 – 9.0 (6.3 ± 0.9) µm long, three colpori elongated-rhomboidal, operculate with ectoaperture 2.7 – 6.6 (4.5 ± 0.8) µm long and endoaperture lalongate, 0.9 – 3.8 (1.7 ± 0.5) µm wide; margins of the pseudocolpi and of the colpi without granula or granulate; colpal and pseudocolpal membranes psilate; ring-like aperture absent or present; opercula granulate. Exine is 0.5–1.0 µm thick, costae slightly developed. Sculpture finely granulate, perforate. Dichotomous key to pollen types 1 2 Margins of the pseudocolpi and colpori conspicuously granulate 2 Margins of the pseudocolpi and colpori not granulate Omphalodes chiangii pollen type Ring-like aperture present Omphalodes japonica pollen type Ring-like aperture absent Omphalodes verna pollen type Pollen type descriptions Omphalodes japonica pollen type. — Species included: Omphalodes akiensis, O. japonica, O. krameri and O. laevisperma. Pollen grains radially symmetrical; isopolar; rectangular-elliptic and more or less constricted at the equator, more or less rounded at the poles; sub-circular to subhexagonal in polar view; prolate to perprolate, P:E = 1.8 – 2.5 (2.2 ± 0.3), P = 8.1 – 10.7 (9.7 ± 0.5) µm, E = 3.2 – 5.6 (4.4 ± 0.6) µm, maximum width of the grains = 4.4 – 6.0 (5.4 ± 0.4) µm. Apertural system 6-zono-heterocolpate: three pseudocolpi 4.7 – 8.5 (7.0 ± 0.8) µm long, three colpori elongated-rhomboidal with the ectoaperture 2.7 – 6.5 (4.9 ± 0.9) µm long and the endoaperture lalongate, 0.9 – 3.3 (2.2 ± 0.7) µm wide; margins of the pseudocolpi and colpori conspicuously granulate; colpal and pseudocolpal membranes psilate; ring-like aperture present. Exine 0.5–1.0 µm thick, costae slightly Figure 3. Principal component analysis (PCA) performed with the palynological characters and the specimens of Omphalodes. Specimens are represented in the first and second PCA components and were marked according to the pollen type: Omphalodes chiangii pollen type (), O. japonica pollen type (◦) and O. verna pollen type ( ). Percentage of variance explained by the component factors is also provided in parenthesis in each axis. Inset gives the projection of the variables on the same factor axes with the following variables in a clockwise direction: P:E, colporus; P, maximum width of the grain, ring-like aperture, pseudocolpus; E and margin ornamentation. developed. Sculpture finely granulate, imperforate (Figure 1). Omphalodes chiangii pollen type. — Species included: Omphalodes acuminata, O. cardiophylla, O. chiangii, O. erecta, O. lojkae and O. mexicana. Pollen grains radially symmetrical; isopolar; rectangularelliptic and more or less constricted at the equator, more or less rounded at the poles; sub-circular to sub-hexagonal in polar view; prolate to perprolate, P:E = 31.2 – 3.8 (2.4 ± 0.4), P = 7.0 – 10.7 (8.5 ± 0.7) µm, E = 2.3 – 6.2 (3.7 ± 0.8) µm, maximum width of the grains = 3.7 – 6.3 (4.9 ± 0.6) µm. Apertural system 6-zono-heterocolpate: three pseudocolpi 4.6 – 8.4 (6.4 ± 0.8) µm long, three colpori elongated-rhomboidal, operculate, with the ectoaperture 2.7 – 6.1 (4.3 ± 0.7) µm long and the endoaperture lalongate, 1.1 – 2.7 (1.6 ± 0.3) µm wide; margins of the pseudocolpi and colpori not granulate; colpal and pseudocolpal membranes psilate; ring-like aperture absent; opercula granulate. Exine 0.5–1.0 µm thick, costae slightly developed. Sculpture finely granulate, perforate (Figure 2A–D). Omphalodes verna pollen type. — Species included: Omphalodes aliena, O. commutata, O. kuzinskyanae, O. linifolia, O. littoralis, O. luciliae, O. brassicifolia, O. nitida, O. richardsonii, O. ripleyana, O. rupestris, Pseudocolpus (µm) Colporus (µm) Endo– aperture (µm) E (µm) P:E Maximum grain width (µm) Omphalodes japonica pollen type O. akiensis 9.2 – 9.6 (9.4 ± 0.2) O. japonica 9.1 – 10.7 (9.8 ± 0.4) O. krameri 9.1 – 10.6 (9.9 ± 0.6) O. laevisperma 8.7 – 10.9 (9.8 ± 0.4) 3.9 – 4.8 (4.4 ± 0.4) 4.1 – 5.6 (4.7 ± 0.5) 4.2 – 4.9 (4.6 ± 0.2) 3.6 – 5.8 (4.6 ± 0.6) 1.9 – 2.4 (2.2 ± 0.3) 1.8 – 2.5 (2.1 ± 0.2) 2.0 – 2.4 (2.1 ± 0.2) 1.7 – 2.6 (2.2 ± 0.2) 4.9 – 5.2 (5.0 ± 0.1) 4.8 – 6.0 (5.4 ± 0.3) 5.2 – 6.0 (5.6 ± 0.3) 4.8 – 5.9 (5.4 ± 0.4) 7.05 – 7.06 (7.1 ± 0.0) 6.1 – 7.8 (7.1 ± 0.6) 6.9 – 8.5 (7.6 ± 0.5) 6.8 – 9.0 (7.6 ± 0.5) 5.8 – 6.1 (6.0 ± 0.1) 4.7 – 5.9 (5.4 ± 0.5) 3.6 – 6.5 (4.7 ± 0.9) 4.4 – 7 – 1 (5.4 ± 0.7) 2.9 – 3.1 (3.0 ± 0.1) 1.5 – 3.3 (2.3 ± 0.5) 1.1 – 1.3 (1.2 ± 0.1) 0.7 – 2.1 (1.5 ± 0.4) Omphalodes chiangii pollen type O. acuminata 7.0 – 8.7 (7.8 ± 0.4) O. cardiophylla 7.6 – 8.9 (8.1 ± 0.4) O. chiangii 7.3 – 9.5 (8.6 ± 0.7) O. erecta 8.4 – 9.9 (9.0 ± 0.4) O. lojkae 8.1 – 9.8 (8.9 ± 0.5) O. mexicana 7.9 – 10.7 (8.5 ± 0.8) 2.2 – 3.5 (3.0 ± 0.3) 2.3 – 3.6 (3.0 ± 0.4) 4.9 – 6.2 (4.7 ± 0.8) 3.0 – 4.4 (3.9 ± 0.4) 3.3 – 4.4 (3.9 ± 0.3) 2.8 – 4.2 (3.6 ± 0.4) 2.2 – 3.7 (2.7 ± 0.4) 2.1 – 3.8 (2.8 ± 0.4) 3.6 – 6.2 (4.7 ± 0.8) 2.1 – 2.9 (2.4 ± 0.2) 2.0 – 2.7 (2.3 ± 0.2) 1.9 – 3.0 (2.4 ± 0.3) 3.7 – 4.9 (4.4 ± 0.3) 3.9 – 5.2 (4.4 ± 0.4) 1.3 – 2.4 (1.9 ± 0.3) 4.1 – 5.4 (4.9 ± 0.4) 4.5 – 6.1 (5.5 ± 0.4) 4.9 – 5.6 (5.1 ± 0.2) 5.2 – 7.1 (6.0 ± 0.6) 5.1 – 7.8 (6.2 ± 0.5) 4.1 – 6.3 (5.2 ± 0.5) 5.2 – 7.8 (6.6 ± 0.7) 5.8 – 8.0 (6.7 ± 0.6) 5.7 – 8.4 (6.9 ± 0.7) 2.9 – 5.0 (3.9 ± 0.6) 3.6 – 6.1 (4.4 ± 0.6) 4.7 – 7.5 (6.0 ± 0.9) 4.3 – 5.6 (4.7 ± 0.4) 4.0 – 5.1 (4.3 ± 0.4) 3.8 – 5.5 (4.6 ± 0.5) 1.1 – 2.3 (1.4 ± 0.3) 1.1 – 1.6 (1.3 ± 0.2) 1.4 – 2.1 (1.8 ± 0.3) 1.1 – 2.1 (1.8 ± 0.5) 1.1 – 2.1 (1.6 ± 0.4) 1.3 – 1.7 (1.5 ± 0.1) Omphalodes verna pollen type O. aliena 7.8 – 10.8 (8.8 ± 0.6) O. comuttata 8.0 – 9.7 (8.1 ± 0.4) O. kuzinskyanae 7.4 – 9.1 (8.4 ± 0.5) 2.9 – 4.6 (3.4 ± 0.4) 3.1 – 4.5 (3.7 ± 0.4) 3.0 – 4.6 (4.0 ± 0.5) 2.2 – 3.0 (2.6 ± 0.2) 2.0 – 2.6 (2.4 ± 0.2) 1.8 – 2.4 (2.1 ± 0.3) 4.2 – 6.3 (4.8 ± 0.4) 4.1 – 5.9 (4.6 ± 0.5) 4.0 – 5.2 (4.6 ± 0.4) 5.2 – 7.8 (6.2 ± 0.6) 5.9 – 7.2 (6.5 ± 0.4) 5.5 – 7.8 (6.2 ± 0.7) 3.7 – 5.5 (4.4 ± 0.59 3.6 – 5.5 (4.6 ± 0.5) 3.1 – 5.9 (5.0 ± 0.7) 0.9 – 2.0 (1.4 ± 0.3) 1.4 – 2.1 (1.7 ± 0.2) 1.1 – 1.7 (1.4 ± 0.2) Taxa P (µm) Habit and geographical distribution Margines ornamentation Ring–like aperture Yes Yes Perennial, Japan Yes Yes Perennial, Japan Yes Yes Perennial, Japan Yes Yes Perennial, Japan No No Perennial, Mexico No No Perennial, Mexico No No No No Perennial, America Perennial, Mexico No No No No Yes No Yes No Yes No Perennial, Caucasus Perennial, Mexico Annual/perennial, Mexico, Texas Annual, Iberian Peninsula Annual, Iberian Peninsula (Continued) 200 A. P. Coutinho et al. Table I. Summary of the quantitative and qualitative pollen morphological characters for the Omphalodes species examined. Table I. (Continued). Taxa O. linifolia O. littoralis subsp. gallaecica O. littoralis subsp. littoralis O. lucilieae O. brassicifolia O. nitida O. richardsonii O. ripleyana O. rupestris O. verna E (µm) P:E 8.1 – 10.7 (9.1 ± 0.7) 7.8 – 10.0 (8.9 ± 0.7) 8.1 – 10.2 (9.4 ± 0.5) 6.4 – 7.7 (7.1 ± 0.4) 7.4 – 9.8 (8.3 ± 0.6) 7.4 – 9.3 (8.4 ± 0.5) 7.3 – 8.7 (7.7 ± 0.4) 6.4 – 8.6 (6.8 ± 0.7) 6.5 – 8.9 (7.6 ± 0.8) 7.4 – 10.5 (8.4 ± 0.7) 8.4 – 9.3 (8.9 ± 0.3) 2.7 – 4.8 (3.5 ± 0.5) 3.4 – 4.5 (3.8 ± 0.3) 3.2 – 4.3 (3.8 ± 0.3) 2.8 – 4.0 (3.5 ± 0.4) 3.3 – 4.3 (4.0 ± 0.3) 3.0 – 4.2 (3.5 ± 0.3) 2.6 – 3.2 (2.9 ± 0.2) 2.7 – 4.0 (3.4 ± 0.4) 3.7 – 4.8 (4.7 ± 0.3) 3.7 – 4.9 (4.4 ± 0.3) 4.2 – 4.9 (4.5 ± 0.3) 1.9 – 3.5 (2.6 ± 0.3) 2.1 – 2.6 (2.3 ± 0.1) 2.2 – 3.0 (2.5 ± 0.2) 1.8 – 2.8 (2.1 ± 0.3) 1.7 – 2.5 (2.1 ± 0.2) 1.9 – 2.9 (2.4 ± 0.2) 2.4 – 3.1 (2.7 ± 0.2) 2.1 – 2.5 (2.3 ± 0.1) 1.5 – 2.1 (1.8 ± 0.2) 1.7 – 2.4 (1.9 ± 0.2) 1.8 – 2.2 (2.0 ± 0.2) 2.2 – 5.0 (4.6 ± 0.6) 4.1 – 5.0 (4.5 ± 0.3) 4.4 – 5.4 (5.0 ± 0.3) 3.4 – 4.9 (4.0 ± 0.3) 4.3 – 5.3 (4.7 ± 0.4) 4.3 – 5.7 (4.7 ± 0.4) 3.8 – 4.4 (4.1 ± 0.2) 3.4 – 4.5 (3.9 ± 0.3) 4.2 – 5.2 (4.6 ± 0.4) 4.4 – 5.7 (4.8 ± 0.4) 4.5 – 7.5 (5.3 ± 0.8) Pseudocolpus (µm) Colporus (µm) Endo– aperture (µm) 5.4 – 8.1 (6.6 ± 0.6) 5.1 – 7.4 (6.1 ± 0.6) 4.7 – 7.4 (6.2 ± 0.7) 3.6 – 6.2 (5.0 ± 0.7) 4.8 – 8.0 (6.2 ± 0.8) 6.5 – 8.5 (7.0 ± 0.7) 4.8 – 6.3 (5.3 ± 0.5) 4.2 – 5.8 (5.2 ± 0.4) 5.5 – 7.8 (6.4 ± 0.7) 5.4 – 7.3 (6.2 ± 0.6) 4.7 – 7.3 (5.8 ± 0.9) 2.9 – 6.0 (4.6 ± 1.0) 4.0 – 6.6 (5.4 ± 0.8) 2.7 – 5.1 (3.9 ± 0.7) 2.9 – 4.1 (3.4 ± 0.4) 2.8 – 5.2 (4.2 ± 0.7) 3.2 – 4.7 (4.1 ± 0.4) 3.3 – 5.4 (4.0 ± 0.6) 3.8 – 6.1 (4.8 ± 0.6) 4.1 – 6.5 (5.0 ± 0.6) 4.0 – 4.9 (4.4 ± 0.3) 4.1 – 6.5 (5.2 ± 0.7) 1.1 – 2.3 (1.6 ± 0.3) 1.5 – 1.9 (1.6 ± 0.2) 0.9 – 1.6 (1.3 ± 0.2) 1.3 – 2.2 (1.6 ± 0.2) 1.1 – 1.7 (1.4 ± 0.2) 2.4 – 3.8 (2.9 ± 0.5) 1.0 – 1.7 (1.3 ± 0.2) 1.1 – 1.7 (1.4 ± 0.2) 1.5 – 2.5 (1.9 ± 0.3) 1.1 – 2.4 (2.6 ± 0.5) 2.1 – 3.1 (2.7 ± 0.4) Habit and geographical distribution Margines ornamentation Ring–like aperture Yes No Yes No Yes No Yes No Yes No Yes No Yes No Yes No Yes No Yes No Perennial, Anatolia Perennial, Caucasus Biennial, Europe Yes No Perennial, Europe Annual, Iberian Peninsula Annual, Iberian Peninsula Annual, West France Perennial, Greece, West Asia Annual, Iberian Peninsula Perennial, Iberian Peninsula Perennial, Mexico Notes: P, polar axis; E, equatorial axis, P:E, ratio between polar and equatorial axes; margines ornamentation refers to the presence/absence of conspicuous granula in pseudocolpi and colpi margines. For each taxon and character the following information is provided: minimum and maximum, followed by mean ± standard deviation of the mean in parenthesis. Quantitative variables are provided in micrometres, except P:E. Dichotomous key for the pollen types is provided in the ‘Results’ section. Pollen morphology of Omphalodes 201 O. scorpioides P (µm) Maximum grain width (µm) 202 A. P. Coutinho et al. O. scorpioides and O. verna. Pollen grains radially symmetrical; isopolar; rectangular-elliptic and more or less constricted at the equator, more or less rounded at the poles; sub-circular to subhexagonal in polar view; prolate to perprolate, P:E = 1.5 – 3.5 (2.3 ± 0.4), P = 6.4 – 10.9 (8.5 ± 0.9) µm, E = 2.6 – 5.8 (3.8 ± 0.6) µm, maximum width of the grains = 2.2 – 7.5 (4.6 ± 0.6) µm. Apertural system 6-zono-heterocolpate: three pseudocolpi 3.6 – 9.0 (6.2 ± 0.9) µm long, three colpori elongated-rhomboidal, operculate, with ectoaperture 2.8 – 6.6 (4.6 ± 0.8) µm long and endoaperture lalongate, 0.9 – 3.8 (1.7 ± 0.5) µm wide; margins of the pseudocolpi and colpori conspicuously granulate; colpal and pseudocolpal membranes psilate; ring-like aperture absent; opercula granulate. Exine 0.5–1.0 µm thick, costae slightly developed. Sculpture finely granulate, perforate (Figure 2E–K). Discussion The results of this study revealed that most of the pollen morphological features (including polarity, symmetry, shape, size and exine thickness) of Omphalodes species are quite constant, though the sculpture of the aperture margins and presence/absence of a ring-like aperture varies among the taxa. Overall, the results obtained agreed with previous descriptions (Liu et al., 2001; Hargrove & Simpson, 2003; Bigazzi et al., 2006), despite some varied values, most probably due to different methodologies (e.g. Barbier & Mathez, 1973; Díez & Valdés, 1991; Buchner & Halbritter, 2011). Regardless of the fairly continuous gradient observed across the species studied for all the quantitative characters, it was possible to recognise three pollen types (each one including several species) that differed mostly in qualitative characters related with the presence/absence of a ring-like aperture and of conspicuous granula in the apertural margins. The need to include several con-generic species in one pollen type has been already described for the Boraginaceae (e.g. Barbier & Mathez, 1973; Clarke, 1977; Díez, 1984, Díez & Valdés, 1991; Scheel et al., 1996) and some palynologists have even considered the genus, more than the species, as the basic palynosystematic unit (e.g. Pons, 1970). This view is valid especially when studying taxonomic groups with low sculptural variability such as Cynoglosseae. In addition to the three pollen types described, two main patterns arose from the palynological data obtained in this study. First, the Japanese taxa form a group strongly characterised by its greater size, presence of a ring-like aperture and presence of granula in the apertural margins, supporting recent phylogenetic analyses. Second, though the perennial species from both the Old and the New World could belong either to the O. chiangii or O. verna pollen types, the pollen of all annual species belongs exclusively to the O. verna pollen type. It is also interesting to note that within the O. verna pollen type, the pollen grains of O. scorpioides are more compact and globose than the pollen grains of the other species. Among the pollen characters studied, the occurrence of a ring-like aperture was of major importance in the discrimination of pollen types, namely the Japanese taxa. The existence of a ring-like aperture has already been described for some genera of Cynoglosseae (e.g. Cryptantha, Paracaryum, Rindera and Solenanthus; Hargrove & Simpson, 2003; Bigazzi et al., 2006), but was never reported for Omphalodes up to now (e.g. Barbier & Mathez, 1973). The heterocolpate condition was constant in all the species of Omphalodes studied, as well as in other taxa of Cynoglosseae (Anh & Lee, 1986; Valdés, 1987; Díez & Valdés, 1991; Hargrove & Simpson, 2003; Coutinho et al., unpublished results) being, most probably, an apomorphic condition for Omphalodes and Cynoglosseae (Hargrove & Simpson, 2003). Three functions have been proposed for the heterocolpate condition: Points for pollen tube growth, points for the exchange of physiologically active substances and harmomegathy (Muller, 1979), the latter being solely associated with pseudocolpi (Hargrove & Simpson, 2003). In Omphalodes, harmomegatic reactions are indeed facilitated by the presence of pseudocolpi that enable the accommodation of volume changes after (de)hydration, and by a thin exine that increases the flexibility of the pollen grain (Bolick, 1981; Payne, 1981). The perforations and opercula observed in Omphalodes pollen grains may also constitute important regulatory structures for the exchange of physiologically active substances (Bolick, 1981; Payne, 1981). Phylogenetic analysis based on the atpB plastid marker places Omphalodes in an isolated position within Boraginoideae and suggests no relation with the Cynoglosseae (Långström & Chase, 2002). However, the pollen morphological data collected in this study does not support this molecular inference; instead, palynological data corroborates the classical inclusion of the genus within Cynoglosseae. In fact, Cynoglosseae s. l. is the only tribe of the subfamily Boraginoideae that possess heterocolpate pollen, with colporate pollen being restricted to Boragineae and Lithospermeae. Still, independent evolution of heterocolpate pollen in Omphalodes and Cynoglosseae should be tested in a phylogenetic context using more molecular markers. It is also interesting to observe that the O. japonica pollen type is similar to the Cynoglossum officinale L. pollen type Pollen morphology of Omphalodes 203 (Clarke, 1977) and the C. creticum Mill. pollen type (Díez, 1984), while the O. verna pollen type resembles the Myosotis arvensis (L.) Hill and M. stolonifera J. Gay pollen types (Clarke, 1977; Díez, 1984). Based on palynological data, it was possible to clearly discriminate the Japanese species from the remaining species of Omphalodes. This corroborates the segregation of the monophyletic group of Japanese species from Omphalodes proposed by recent molecular phylogenetic analyses (Serrano et al., unpublished data). Our results revealed that Omphalodes is a quite stenopalynous taxon (except for the aperture margin sculpture and presence of a ring-like aperture), in contrast to other genera of the Cynoglosseae, in which the high variability of pollen morphology significantly assisted taxonomic delimitations within the tribe (Barbier & Mathez, 1973). The observed low variability within Omphalodes does not support the hypothesis of a relatively ancient diversification of the genus, as suggested by the disjunct distribution. Alternatively, a possible trend to palynological stasis in the genus needs to be tested in a dated phylogenetic framework. Finally, the results obtained have also significant biogeographic implications. Pollen morphology gives support to a reduced concept of Omphalodes that would include only species with O. chiangii or O. verna pollen types. These taxa display a disjunct distribution range between West Eurasia and North America that fits in the well-known MadreanTethyan Flora pattern (Raven & Axelrod, 1974; Wen & Ickert-Bond, 2009). However, whether the Omphalodes disjuction originated in the first half of the Tertiary, as proposed by the Madrean-Tethyan hypothesis (Axelrod, 1975), or resulted from more recent long distance dispersal events still remains an open question. Conclusion This study investigated for the first time the variability of pollen morphology in Omphalodes, encompassing 23 species (82% of the recognised species) and all major geographical and morphological groups. Palynological data provided useful information to assist the complex systematics of the genus Omphalodes considering the uselessness of most floral and fruit characters, karyological homogeneity and striking geographical distribution. At the suprageneric level, pollen morphology supports the traditional tribal classification of the genus, in contrast to previously published molecular phylogenetic analyses. At the infrageneric level, pollen morphology corroborates the recent phylogenetic results for Omphalodes and clearly separates, among others, the Japanese taxa in a distinct group. Acknowledgements The authors are grateful to the Directors of the COI, IEB, K, MA, P, SALAF, SANT, TEX, TI and TNS herbaria for the loan of herbarium vouchers. The authors also thank Augusto Dinis for allowing the use of the equipment from the Laboratory of Microscopy, Department of Life Sciences (University of Coimbra), and to the staff of the Confocal and Electron Microscopy Unit (University of Santiago de Compostela). The Portuguese Foundation for Science and Technology financed the work of Sílvia Castro (FCT/BPD/41200/2007). The work of Miguel Serrano was partially financed by the CBC Programme Spain-Portugal 2007–2013 (POCTEP), project 479-BIODIV-GNP. Specimens investigated Omphalodes acuminata B. L. Rob. Mexico: Nuevo León, Monterrey. Ford M186 (TEX 218154); Mexico: Nuevo León, Villa Santiago. Johnson & Barkley 1609M (TEX 120476). Omphalodes akiensis Kadota. Japan: Hiroshima pref., Hirami-dani gorge. Kubota s.n. (TNS 737082). Omphalodes aliena A. Gray ex Hemsl. USA: Texas, Presidio. Correll & Rollins 23704. (TEX 35421); Mexico: Nuevo León, Minas Manto Blanco. Johnston, Wendt & Chiang 10248F (TEX); Mexico: Nuevo León, Rayones. Hinton 20173 (TEX 223071). Omphalodes cardiophylla A. Gray ex Hemsl. México: Coahuila, General Cepeda. Encina 228 & Závala. (TEX 223076). Omphalodes chiangii Higgins. Mexico: Sierra del Carmen. Carranza s. n. (IEB 15903). Omphalodes commutata G. López. Spain: Málaga, Serrania de Ronda. Fuertes, Ladero, G. López & Navarro s.n. (SALAF 12451). Omphalodes erecta I. M. Johnst. Mexico: Galeana. Hinton s.n. (IEB 22275); Mexico: Aramberri. Hinton s.n. (IEB 23145). Omphalodes japonica (Thunb.) Maxim. Japan: Kyoto, Yamashiro. Togashi s.n. (TI); Japan: Cultivated in Karuizawa Botanical Garden. Arai s.n. (SANT 59953). Omphalodes krameri Franch et Sav. Japan: Nagano Pref., Kitasakugun. Yahara et al. s.n. (TI). Omphalodes kuzinskyanae Willk. Portugal: Cascais, Quinta do Espírito Santo. Torre s.n. COI; Portugal: Cascais, near Guincho. Carballal & Serrano s.n. (SANT 50016). Omphalodes laevisperma Nakai. Japan: Murakamishi. Togashi s.n. (TI). Omphalodes linifolia (L.) Moench. Spain: Málaga, El Burgo. Perez & Molero s.n. (SANT 12850); Portugal: Serra da Arrábida. Carbajal & Serrano s.n. (SANT 50020). Omphalodes littoralis Lehm. subsp. gallaecica M. Laínz. Spain: Ferrol, Covas. Carbajal & Serrano s.n. (SANT 51847). Omphalodes littoralis Lehm. subsp. littoralis. France: Charente-Inf., La Rochelle. Giraudias s.n. (P 271712). Omphalodes lojkae Sommier et Levier. Georgia: Svanethi. Schethekauzi s.n. (MA 575919). Omphalodes luciliae Boiss. Greece: Parnaso massif, Gorge Gouna. Maire & Petitmengin s.n. (MA 94701). Omphalodes brassicifolia Sweet. Spain: Cáceres, Arroyomolinos de la Vera. Ladero & Amor s.n. (SALAF 23592). Omphalodes mexicana S. Watson. Mexico: Nuevo León, Galeana, Cerro el Gallo. Hinton 21036 (IEB 137190). 204 A. P. Coutinho et al. Omphalodes nitida Hoffmanns et Link. Spain: Corunha, Ames. Fagúndez s.n. (SANT 53295). Omphalodes richardsonii G. L. Nesom. Mexico: Tamaulipas, Gómez Farias. Richardson 1114 (TEX); Mexico: Tamaulipas, Gómez Farias. Woodruff 160 (TEX). Omphalodes ripleyana P. H. Davis. Cultivated in Edinburgh. Davis s.n. (K). Omphalodes rupestris Boiss. Caucasus region. Desoulavy s.n. (B 10 0293510). Omphalodes scorpioides (Haenke) Schrank. Slovakia: Slovensky Kras. Zertova s.n. (MA 219178); Austria: Niederösterreich, Fuss des Rosaliengebirges. Barta s.n. (MA 732115). Omphalodes verna Moench. Italy: Udine, Valli del Natisone-Valle dell’Erbezzo. Feoli & Feoli s.n. (MA 350089). References Ahn, Y. M & Lee, S. (1986). A palynotaxonomic study of the Korean Boraginaceae. Korean Journal of Plant Taxonomy, 16, 199–215. Al-Shehbaz, I. (1991). The genera of Boraginaceae in the southeastern United States. Journal of the Arnold Arboretum, Supplement 1, 1–169. Andersen, S. (1960). Silicon oil as a mounting medium for pollen grains. Danmarks Geologiske Undersøgelse, 4, 116–140. Axelrod, D. I. (1975). Evolution and biogeography of MadreanTethyan sclerophyll vegetation. Annals of the Missouri Botanical Garden, 62, 280–334. Barbier, E. & Mathez, J. (1973). Contribution à létude des Cynoglossées (Boraginacées): Pardoglossum, genre nouveau du Bassin Méditerranéen occidental. Candollea, 28, 281–323. Bigazzi, M., Nardi, E. & Selvi, F. (2006). Palynological contribution to the systematics of Rindera and the allied genera Paracaryum and Solenanthus (Boraginaceae-Cynoglosseae). Willdenowia, 36, 37–46. Bolick, M. R. (1981). Mechanics as an aid to interpreting pollen structure and function. Review of Palaeobotany and Palynology, 35, 61–69. Brand, A. (1921). Boraginaceae-Boraginoideae-Cynoglosseae. In A. Engler (Ed.), Das Pflanzenreich Volume 78, pp. 1–183. Leipzig: Engelmann. Buchner, R. & Halbritter, H. (2011). Omphalodes verna. In R. Buchner & M. Weber (Eds), PalDat – a palynological database: Descriptions, illustrations, identification, and information retrieval. http://www.paldat.org/index.php?module=search&nav=sd&ID =103904&system=1 (accessed 1 November 2011). Clarke, G. C. S. (1977). The Northwest European pollen flora, 10. Boraginaceae. Review of Palaeobotany and Palynology, 24, 59–101. Clarke, G. C. S., Chanda, S. & Sahay, S. (1979). Pollen morphology in the genus Pardoglossum (Boraginaceae) with some observations on heterocolpate pollen. Review of Palaeobotany and Palynology, 28, 301–309. Coppi, A., Selvi, F. & Bigazzi, M. (2006). Chromosome studies in Mediterranean species of Boraginaceae. Flora Mediterranea, 16, 253–274. de Candolle, A. (1846). Borage. In A. de Candolle (Ed.), Prodromus systematis naturalis regni vegetabilis, Pars decima, pp. 1–178. Paris: Sumptibus Victoris Masson. Díez, M. J. (1984). Contribución al Atlas Palinológico de Andalucia Occidental, I. Boraginaceae. Lagascalia, 13, 147–171. Díez, M. J. & Valdés, B. (1991). Pollen morphology of the tribes Eritrichieae and Cynoglosseae (Boraginaceae) in the Iberian Peninsula and its taxonomic significance. Botanical Journal of the Linnean Society, 107, 49–66. Díez, M. J., Valdés, B. & Fernández, I. (1986). Pollen morphology of Spanish Lithospermum s. l. (Boraginaceae) and its taxonomic significance. Grana, 25, 171–176. Erdtman, G. (1960). The acetolysis method. Svensk Botanisk Tidskrift, 54, 561–564. Grau, J. (1967). Primäre und sekundäre Chromosomenbasis zahlen bei Omphalodes. Österreichische Botanische Zeitschrift, 114, 66–72. Hargrove, L. & Simpson, M. G. (2003). Ultrastructure of heterocolpate pollen in Cryptantha (Boraginaceae). International Journal of Plant Sciences, 164, 137–151. Hesse, M., Halbritter, H., Zetter, R., Weber, M., Buchner, R., Frosh-Radivo, A. & Ulrich, S. (2009). Pollen terminology – an illustrated handbook. Wien: Springer. Holmgren, P. K., Holmgren, N. H. & Barnett, L. C. (1990). Index herbariorum, Part I. The herbaria of the World. New York: New York Botanical Garden. Johnston, I. M. (1924). Studies in the Boraginaceae, III. 1, The New World genera of the Boraginoideae. Contributions from the Gray Herbarium of Harvard University, 73, 42–78. Kadota, Y. (2009). Omphalodes akiensis (Boraginaceae), a new species from Hiroshima prefecture, western Japan. Journal of Japanese Botany, 84, 342–349. Lewis, W. H. (1980). Polyploidy in angiosperms: Dicotyledons. In W. H. Lewis (Ed.), Polyploidy: Biological relevance, pp. 241–268. New York: Plenum Press. Liu, J.-X., Li, J.-Y., Zhang, Y.-L. & Ning, J.-C. (2010). Pollen morphology of the tribe Lithospermeae of Boraginoideae in China and its taxonomic significance. Plant Systematics and Evolution, 290, 75–83. Liu, J.-X., Zhang, Y.-L., Ning, J.-C., Zhao, Y.-Y., Li, Y.-X., Zhang, J.-M. & Sun, X.-H. (2001). Pollen morphology of the tribe Cynoglosseae of Boraginoideae (Boraginaceae) in China. Acta Phytotaxaxonomica Sinica, 39, 515–522. Luque, T. & Valdés, B. (1986). Caryological study of Spanish Boraginaceae III. Cynoglossum L. s. str. Willdenowia, 15, 485–496. Långström, E. & Chase, M. W. (2002). Tribes of Boraginoideae (Boraginaceae) and placement of Antiphytum, Echiochilon, Ogastemma and Sericostoma: A phylogenetic analysis based on atpB plastid DNA sequence data. Plant Systematics and Evolution, 234, 137–153. Muller, J. (1979). Form and function in angiosperm pollen. Annals of the Missouri Botanical Garden, 66, 593–632. Ning, J.-C., Xi, Y.-Z. & Zhang, Y.-L. (1992). Pollen morphology of the tribe Anchuseae DC. (Boraginaceae) and its taxonomic significance. Cathaya, 4, 79–90. Payne, W. W. (1981). Structure and function in angiosperm pollen wall evolution. Review of Palaeobotany and Palynology, 35, 39–59. Pons, A. (1970). Le pollen. Paris: Presses Universitaires de France. Popov, M. G. (1953). Boraginaceae. In B. K. Shischkin (Ed.), Flora of the USSR, 19 pp. 97–691. Moscow: Izdatel’stvo Akademii Nauk SSR. Punt, W., Hoen, P. P., Blackmore, S., Nilsson, S. & Le Thomas, A.(2007). Glossary of pollen and spore terminology. Review of Palaeobotany and Palynology, 143, 1–81. Raven, P. H. (1972). Plant species disjunctions: A summary. Annals of the Missouri Botanical Garden, 59, 234–246. Raven, P. H. & Axelrod, D. I. (1974). Biogeography and post continental movements. Annals of the Missouri Botanical Garden, 61, 539–673. Retief, E. & Van Wyk, A. E. (1997). Palynology of southern Africa Boraginaceae: The genera Lobostemon, Echiostachys and Echium. Grana, 36, 271–278. Pollen morphology of Omphalodes 205 Riedl, H. (1997). Boraginaceae. In C. Kalkman, H. P. Noteboom, W. J. de Wilde, D. W. Kirkup & P. F. Stevens (Eds), Flora Malesiana, series 1, Spermatophyta, pp. 43–144. Leiden: Rijksherbarium Publications Department. Scheel, R., Ybert, J.-P. & Barth, O. M. (1996). Pollen morphology of the Boraginaceae from Santa Catarina State (southern Brazil), with comments on the taxonomy of the family. Grana, 35, 138–153. Selvi, F., Coppi, A. & Cecchi, L. (2011). High epizoochorous specialization and low DNA sequence divergence in Mediterranean Cynoglossum (Boraginaceae): Evidence from fruit traits and ITS region. Taxon, 60, 969–985. Valdés, B. (1987). Boraginaceae. In B. Valdés, S. Talavera & E. Fernández-Galiano (Eds), Flora vascular de Andalucía Occidental 2, pp. 373–404. Barcelona: Ketres Editora. Wen, J. & Ickert-Bond, S. (2009). Evolution of the MadreanTethyan disjunctions and the North and South American amphitropical disjunctions in plants. Journal of Systematics and Evolution, 47, 331–348.