Pollen morphology of Titanotricheae, Beslerieae, and Coronanthereae (Gesnerioideae, Gesneriaceae): the importance of qualitative and quantitative data

Boraginaceae s.l., the single family of Boraginales, are extremely diverse in terms of morphology, their species are well distributed in South America especially in Brazilian ecosystems. The pollen morphology of 11 Brazilian native species and four genera (Cordia L., Varronia P. Browne, Heliotropium L. and Myriopus Small) of Boraginaceae s.l. was investigated in order to expand the morphological knowledge of their species, contributing with new data for the palynology, taxonomy, and conservation of the family. The pollen grains were acetolyzed, measured, photographed using light microscopy (LM) and scanning electron microscopy (SEM), and described qualitatively. The analyzed species present differences in size, shape, type and number of apertures (3-porate, 3-colpate, 3-colporate and heterocolpate) and exine ornamentation (psilate, echinate, microechinate-verrucate, reticulate and rugulate) of the pollen grains, allowing the distinction of species studied. Multivariate analysis of measurements confirms the qualitative description and pollen diversity in Cordiaceae species. Smaller diameter values and similarities in aperture and exine ornamentation characterize the Heliotropiaceae species.Our data reinforce the taxonomic importance of pollen morphology and Boraginaceae s.l. as eurypalynous.

Background and aims – Pollen grain morphology is an important morphological character for aiding the systematics of flowering plants. For Malpighiaceae, only a single unpublished palynological study has comprehensively sampled ca 60 of this family’s 75 currently accepted genera. To test the systematic relevance of pollen morphology in Amorimia and allies, we characterised the pollen morphology of these lineages. We scored, coded, and mapped 12 characters onto the most recent molecular phylogeny of Amorimia and allies. Material and methods – We sampled 13 species of Amorimia as ingroup and two species of Mascagnia and Ectopopterys soejartoi as outgroup. Pollen grains were acetolised, characterised, and measured using light microscopy and scanning electron microscopy. Pollen quantitative measurements were submitted to a PCA multivariate analysis. Additionally, quantitative and qualitative characters were scored and coded into 12 characters and mapped onto the molecular phylogeny of Amorimia and allies. Key results – Amorimia and allies are stenopalynous due to all species showing the same pollen type, with some subtle differences between the pollen grains, such as details of ornamentation, shape, size, and thickness of the pollen exine. However, the patterns of pollen grain evolution showed that few qualitative and apomorphic characters are informative for intrageneric distinction (i.e. type and number of apertures), and almost all quantitative and homoplastic characters analysed were informative at infrageneric levels within Malpighiaceae. Conclusion – Our results demonstrate that even though the pollen morphology characters of Amorimia and allies show subtle variation, both qualitative and quantitative apomorphic and/or homoplastic characters are highly informative for intra- and infrageneric levels in Malpighiaceae when analysed in a phylogenetic context.

We analyzed the pollen morphology of 13 Brazilian native species of Malvaceae s.l. from forest fragments of Cerrado belonging to nine genera [Bastardiopsis (K. Schum.) Hassl., Byttneria Loefl., Ceiba Mill., Eriotheca Schott & Endl., Guazuma Adans., Helicteres L., Heliocarpus L., Luehea Willd. and Pseudobombax Dugand] in search for characters that can expand the knowledge on the pollen morphology of species of the family that occur in areas of fragments. The pollen grains were acetolyzed, measured, described and photographed under light microscopy and scanning electron microscopy. The species studied were grouped into two pollen types based mainly on the type of aperture [simple aperture (3-porate or 4-colpate) and compound aperture (3-pororate or 3-colporate)], and the quantitative data were analyzed using descriptive and multivariate statistics. Details of apertures (planaperturate, angulaperturate, type of endoaperture, costa, aspis and atrium) and exine ornamentation (verrucate, microreticulate, microreticulate-echinate, reticulate and bireticulate) contributed as secondary data to the segregation of species in an artificial pollen key. The metric characters examined by multivariate analysis allow the distinction of groups of species, based on the length of the apertures and the diameters of the pollen grains. Our results allow the identification among genera and species of Malvaceae s.l. in Brazilian forest fragments.

Pollen morphology of Ligeriinae Hanst. (Gesneriaceae): Diagnostic features and their systematic importance

Gloxiniinae comprises herbs and subshrubs usually growing on rocky outcrops, rocky fields or gallery forests. For Gesneriaceae in some cases variations in pollen morphology allow the differentiation of genera and species. We studied the pollen grains of ten Brazilian species of Gloxiniinae (Gesneriaceae), including the genera Chautemsia, Gloxinia, Goyazia, Mandirola and Seemannia with the purposes of describing the pollen morphology of these genera, and contributing to the taxonomic characterization of the group and a better delimitation of genera and species of Gesneriaceae. The pollen grains were acetolyzed, measured, photographed and described qualitatively. Quantitative data were analyzed through descriptive and multivariate statistics. Non-acetolyzed pollen grains were observed in SEM and TEM in order to provide more details of the ornamentation and the structure of the exine. The analyzed species differ as to the pollen amb, shape, length, width and ends of colpi, presence or absence of margo, type of endoaperture and exine ornamentation. We found circular, subcircular and subtriangular amb, oblate spheroidal and subprolate shape, long or very long colpi, narrow, wide or very wide, with or without margo, lalongate or lolongate endoaperture in microreticulate or reticulate pollen grains. The reticulate ornamentation appears in the Gloxinia species’ pollen grains, and lolongate endoapertures represent the pattern for the subtribe pollen grains except in Chautemsia. The pollen grain metrics examined by principal component analysis allow generic distinction. We conclude that qualitative and quantitative pollen data help to delimit the genera and species of Gloxiniinae.

Rutaceae (Sapindales) is represented by approximately 2040 species and 150–170 genera. Conchocarpus is the genus with the highest number of species and is circumscribed by a combination of character states, making it a polymorphic group. A recent study proposed a new genus, Dryades, formed by species formerly positioned in Conchocarpus. This study aims to describe the pollen morphology of Dryades species, comparing it with the palynology of phylogenetically related species, to present data that can characterize the species of Dryades. We analyzed the pollen grains of Dryades species and eight species of the genera Andreadoxa, Angostura, Erythrochiton, Galipea, Rauia and Conchocarpus. The pollen grains were acetolyzed, measured, described qualitatively, and analyzed quantitatively. Illustrations using light microscopy (LM) and scanning electron microscopy (SEM) are presented. Multivariate analysis was performed. The pollen grains are monads, isopolar, medium to very large in size, with subcircular to subtriangular amb, and oblate-spheroidal to prolate shape. The apertures are 3- or 5-colporate, varying in size (very small to very large colpi). The endoapertures are usually lalongate, rarely circular. The pollen grains analyzed are microreticulate, reticulate, macroreticulate, reticulate-baculate, baculate or verrucate-baculate. Both qualitative and quantitative characteristics of pollen grains proved to be important in distinguishing genera and grouping species within genera (in particular, the number of apertures, the length of the ectoaperture, the exine ornamentation and the size of the pollen grains). Dryades species show similarities in pollen morphology to species of related genera such as Andreodoxa, Galipea and especially Rauia. The shape of pollen grains and the number of apertures in Dryades are distinct from those found in Conchocarpus macrophyllus. The pollen data observed in this study confirm the pollen diversity of Rutaceae.

Investigations on the adaptive significance of pollen morphology were conducted on 17 variables of pollen, floral and anthecological characters in 100 species of angiosperms. Three close interrelationships among the characters were established by factor analysis: (1) positive correlation between grain size, exine thickness, and aperture number; (2) strong association of grain shapes with aperture types, i.e. the prolate grain with colpus and the spherical and the oblate shapes with pore; and (3) a positive correlation among the pollinator's body size, the floral size, the style length, and the pollen grain size. Based on the above results, the following suggestions on the adaptive aspects of pollen can be made: (1) exine thickness is important for mechanical support with respect to grain size and apertur-e number; (2) larger grains require more apertures for rapid water absorption from the stigmatic surface; (3) the association of grain shape and aperture type plays a role in accommodation of volume change when the grains absorb or lose water; and (4) grain size is related to the capacity of stored nutrients which determine the potential length of a pollen tube. Diversity of the angiosperm pollen grain has been recognized since the last century (Wodehouse, 1935; Davis & Heywood, 1963; Faegri & Iversen, 1964; Erdtman, 1966), but relatively little attention has been paid to the functional and adaptive aspects of the highly durable exine. Kerner appreciated the functional role of the grooves (previously folded inwards) when the grain is inflated by water uptake prior to germination (in Payne, 1972) and Wodehouse (1936) speculated on the evolution of harmomegathy in the accommodation of volumetric changes. More recently numerous anecdotal observations on various functional aspects of pollen have been noted (e.g. Larson & Lewis, 1961; Henrickson, 1967; Muller, 1969, 1973; Dulberger, 1975; Taylor & Levin, 1975; Kress, Stone & Sellers, 1978) and a few workers (e.g. Payne, 1972; Chaloner, 1976) have met with limited success in showing correlative features that link pollen structure to function. Perhaps the most intriguing new findings are those of J. Heslop-Harrison and colleagues (Heslop-Harrison, 1976). The chambered exine that is characteristic of so many angiosperms serves as the repository for tapetum-derived incompatibility proteins. The functional corollary to these findings implies that pollen lacking chambered exine (e.g. Cana, Helicornia, Musa, parasitic Gentianaceae, etc.) would not be capable of sporophytic incompatibility. Confirmation of corollary has yet to be established. In spite of these provocative new findings, the adaptive significance of most pollen features is not known (Cronquist, 1968; Ehrendorfer, 1973; Lewis, 1977). As Stebbins (1974, I Botany, Duke University, Durham, NC 27706. Pr-esent address: Biology, jeonbuk National University, Jeonju, Korea. This content downloaded from 157.55.39.116 on Sun, 18 Sep 2016 06:02:05 UTC All use subject to http://about.jstor.org/terms 2 SYSTEMATIC BOTANY [Volume 3 p. 292) has pointed out, this lack of understanding is due largely to the complexity of the problem and the paucity of information on a comparative basis. The complex variables of pollen diversity and pollination systems might usefully be analyzed by multivariate statistics. Factor analysis is one such statistical technique for reducing a large number of variables to a small number of hypothetical factors. These factors are often biologically interpretable because they summarize the predominant trends of correlated character variation and thus identify subgroups of functionally related characters (Seal, 1968). Employment of factor analysis seems appropriate for study in which I have attempted to isolate subgroups of inter-related pollen and anthecological characters and to discover possible causal factors responsible for these correlations (Sokal & Rohlf, 1969). MATERIALS AND METHODS Pollen and anthecological characters from a total of 100 species from various angiosperm families (Table 1) were observed and scored. Sample size for each species was generally 3-10 pollen grains or flowers. Selection of the species was based on the availability of limited data for a given species from pollen slides, herbarium specimens, and information about the pollination mechanism. In some cases where no slides of pollen or flowers were available, I was able to substitute data from Stuchlik's (1967) pollen descriptions and the floral descriptions by Grant and Grant (1965). The observed correlations among the various characters associated with the floral biology have led to some very positive statements. I wish to stress, however, that sample sizes are small and fewer than 0.04% of the angiosperms have been examined. Pollen from the collection of Dr. D. A. Livingstone in the Department of Zoology, Duke University, was examined with a brightfield microscope; flowers were measured under a stereo-microscope from the specimens housed in the Duke University Herbarium. The raw data of the taxa examined are presented in Table 1. Characters Examined.-The total of 17 characters examined consists of 8 pollen and 9 anthecological features. Characters exhibiting continuous variation were measured in conventional metric units. Discrete states for characters 4, 6, 9, 10, 11, 12, 13 were numerically coded on criteria ordinal scales representing each trend of morphological variation. The characters and their states (noted in parentheses) or units, in cases of measured characters, are as follows. Pollen characters 1) Grain length, the length of polar axis in gm. 2) Grain width, the equatorial diameter in ,um. 3) Equatorial shape (width/length ratio). 4) Aperture number (0-100). This content downloaded from 157.55.39.116 on Sun, 18 Sep 2016 06:02:05 UTC All use subject to http://about.jstor.org/terms 1978] LEE: FUNCTIONAL SIGNIFICANCE OF ANGIOSPERM POLLEN 3 5) Exine thickness in ,gm. 6) Sculpture pattern [psilate, scabrate, ver-rucate, gemmate, clavate, psilate, baculate, echinate (1); rugulate, striate (3); reticulate, areolate, insulate (5)]. 7) Pore width, diameter perpendicular to the polar axis in ,um. 8) Colpus length in gum. Anthecological characters The anthecological characters and the reasons for including each character are described below. 9) Vector size [abiotic (1); insects (3); birds, bats (5)]. The relationships between plants and their pollinators are maintained by the balance of a mutual need for energy, i.e. pollinators maximizing food intake and plants minimizing energy devoted to providing food for pollinators while maximizing seed set (Stiles, 1971; Lyon & Chadek, 1971; Heinrich & Raven, 1972). The value of pollen as a food source may be expressed not only by the quantity of pollen, which is a function of size, and the amount of pollen per anther, flower, or plant, but by the pollinator's specific nutritional requirements (e.g. bat-flower pollen which contains higher concentrations of certain amino acids, Howell, 1974). Since study did not assess related size features such as nutrition, data for pollen size per se may be expected to show only weak correlations with data for pollinator size. However, the inclusion of character is worthwhile in order to see how well it correlates with flowersize characters (Taylor & Levin, 1975). 10) Floral type [primitive, simple (1); actinomorphic (2); reduced, protected (3); zygomorphic (4)]. Leppik (1957, 1958, 1968) classified floral shape into the above six types and discussed the evolutionary trends and significance in relation to pollinators. The floral types as well as other morphological aspects of the flower would have resulted from the adaptation to the pollinators and seed dispersal vectors (Grant, 1963). It is not known, however, what relationships are involved between the evolutionary trends in the various floral characters and between floral and vector characters. Although some relationships of the floral types to the sensory capability and food searching activity of pollinating insects have been revealed by Leppik's efforts, it may be difficult to relate the floral characters to the pollen characters. It is interesting, however, to look for relationships between pollen and flower morphology and to investigate the functional significance of such relationships. The primitive and simple and reduced and protected types were lumped in the same categories due to the difficulty of separating them. 11) Temperature parameter [arctic (1); temperate (3); tropical (5)]. Temperature is an important environmental variable (Kurtz & Liverman, 1958). Most organisms flourish within a limited temperature range and the tolerance for flowering is even more restricted. This content downloaded from 157.55.39.116 on Sun, 18 Sep 2016 06:02:05 UTC All use subject to http://about.jstor.org/terms 4 ~~~~~~~~~~~~~~~~~SYSTEMATIC BOTANY [Volume 3 (Ll) AS1X0 O O o O O O0O O n O) 00 O1 Oco 0 0 G11 x O GO OO (91) -1-d O vb n O O0 dHn tG = OC) r0' 0' t0 tu00 GQ O C) O) C t 00 t MG4roG sH g, .ANIVMD N~~c n c c c 't n cq cq cq LC N4 N4 n 4tc\1 n c c Q0 c n n C I cVM n cq cq N n t-N N qn n n c Lc c 0 c GN c ; a=;t W ;= 2

A light and scanning electron microscopy technique was conducted to investigate the palynological features of 19 species belonging to 15 genera of the family Asteraceae. The species under investigation yielded pollen in a range of shapes, including spheroidal, prolate, and subprolate. Trizoncolporate, Tricolporate, and Tetracolporate are the three types of pollen apertures observed in examined species. Except for Gazania rigens, which has reticulate ornamentation under SEM, the exine pattern in all studied species is echinate. The majority of the species had isopolar polarity, whereas some members were apolar and heteropolar. The quantitative parameters that are polar to equatorial diameter, P/E ratio, length of colpi, width of colpi, length of the spine, width of spine, and exine thickness was measured by using light microscopy. The Coreopsis tinctoria had the lowest mean polar 19.75 μm to mean equatorial diameter 18.25 μm while the Silybum marianum had the highest polar 44.7μm to equatorial 48.2μm value. The value for colpi length/width was highest in Cirsium arvensis and lowest in C. tinctoria, measuring 9.7/13.2μm and 2.7/4.7μm, respectively. The spine length varied from 0.5μm in Sonchus arvensis to 5.5μm in Calendula officinalis. Verbesina encelioides had the highest exine thickness measurement (3.3μm), whereas S. arvensis had the lowest (0.3μm). The pollen surface of Tagetes erectus, has the highest number (65) whereas the lowest spines number (20) is found in S. arvensis. A taxonomic key based on pollen traits is provided for quick identification of the species. Significant implications for the systematics of the Asteraceae family can be drawn from the reported pollen's quantitative and qualitative data. RESEARCH HIGHLIGHTS: SEM and LM technique was used to study the palynological features of pollen grains. Quantitative and qualitative pollen features of 19 species of family Asteraceae are described. Morphological traits of pollen grains are important for the systematics of family Asteraceae.

The pollen morphology of 11 Anacardiaceae species from the Mexican State of Veracruz was studied. Pollen grains were acetolyzed and examined under light and scanning electron microscopy to determine whether their morphological characteristics are useful to differentiate taxa in this family. Photomicrographs were obtained with both types of microscopes and used as the basis for describing the pollen. Pollen descriptions included traits such as shape, the type and number of apertures, exine ornamentation, size of the polar and equatorial axes, and diameter in polar view. Most species have isopolar pollen grains, with tricolpate or tricolporate apertures. Only Pistacia mexicana has pentaporate pollen. Pollen grains exhibit a triradial or radial symmetry in polar view; their shape in equatorial view can be prolate-spheroidal, subprolate, or prolate. The size of axes in equatorial view, diameter in polar view, and the exine ornamentation show distinctive characteristics in each species. However, the most common ornamentation types are microreticulate and microstriate. Ten pollen types were identified based on combinations of traits such as the type and number of apertures, shape in equatorial and polar view, and exine ornamentation. The results confirm that the eurypollinic pollen of Anacardiaceae is valuable for differentiating taxa, highlights the importance of studying pollen morphology and contributes to increasing the knowledge about the pollen morphology of this family in Mexico.

The present study aimed to characterise the pollen morphology of 18 native species of Papilionoideae (Fabaceae), analysing and describing the qualitative and quantitative data of pollen grains of native species in remaining forest fragments of the Brazilian Cerrado biome. Using pollen morphological traits may contribute to characterising genera and species and a better understanding of biodiversity. And identify the morphological variation in the pollen grains of these species. The analyses were carried out with herbal materials, with the pollen grains being acetolysed and measured using light microscopy. Details of the exine were investigated using scanning electron microscopy. Qualitative data were described, and quantitative data were subjected to descriptive statistical analysis and multivariate analysis. The Papilionoideae taxa analysed present 3-colporate pollen grains (except Cochliasanthus caracalla). Variations in size, amb, shape, length, and width of the apertures, type of endoaperture, ornamentation, and thickness of the exine layers contribute to the distinction of species and confirm the pollen diversity of the subfamily. Details such as bridge, margo, and fastigium help identify the studied pollen. Pollen grains in 3-colporate monads, with mainly microreticulate or reticulate ornamentation, characterise the palynology of Papilionoideae; however, the pollen has diagnostic characteristics (especially regarding the apertures – bridge, margo and fastigium) that confirm the subfamily as a eurypalynous group.

Pollen morphology of Rubiaceae from Cerrado forest fragments: Pollen unit, polarity and diversity of the types of apertures

Pollen grains of 58 wetland species of angiosperms belonging to 17 families, growing in different wetlands of Sub-Himalayan biodiversity hotspots, Northern Bengal and lower Gangetic plains, India were investigated during a 4-year span of 2015-2019. Freshly collected anthers were processed through the acetolysis method and the obtained pollen grains were accurately studied under LM and SEM and properly photographed. Different attributes viz. shape, size, aperture type and exine ornamentation of pollen grains served as consistent features were used for authentic identification of the studied species. To study the phylogeny of pollen grains, characters like polarity, symmetry, aperture and exine sculpturing were found most significant for species segregation. Investigated pollen grains were mostly prolate-spheroidal, spheroidal or triangular, rarely prolate or elongated, exine psilate, granulate, reticulate or microechinate type. Most of the observable aperture of pollen grains of studied species were colporate, colpate and porate, while ulcerate and inaperturate grains were also observed in the studied aquatic members of the families Cyperaceae and Potamogetonaceae. All the gathered data were further statistically analyzed through ANOVA, PCA and Pearson Correlation Matrix to understand the interrelationship among the species. The permanent slides of identified pollen grains were deposited at the herbarium of North Bengal University [NBU] for future references. HIGHLIGHTS: Study of Pollen morphology of 58 wetland species under light & scanning electron microscopy. Unique apertures and exine ornamentation used to understand taxonomic evaluation of studied species. Pollen character supports interrelationship among the studied species. Relationship between exine thickness and the habitat of the wetland plants.

Rosales, a well-supported order of angiosperms encompassing heterogeneous plants, holds significant representation in Brazilian territory, particularly Cerrado regions. These areas are susceptible to human impact. In this context, pollen data is vital in supporting taxonomic and ecological studies for species preservation. We analyzed pollen grains from 15 native Rosales species found in Brazilian Cerrado forest fragments belonging to the Cannabaceae, Moraceae, Rhamnaceae, and Rosaceae families using light and scanning electron microscopy. The pollen grains underwent acetolysis, qualitative description, and measurements, with statistical analysis, including multivariate methods, applied to quantitative data. Qualitative findings were organized for pollen description and multivariate cluster analysis. Our observations revealed pollen grains in monads, isopolar, small to medium, with subcircular, circular, or subtriangular amb, and suboblate, oblate-spheroidal, or prolate-spheroidal. Apertures include 2–3-porate, 3-colpate, or 3-colporate, with long or short ectoapertures, lalongate or circular endoapertures. The exine is thin, microreticulate, psilate-perforate, microgranulate, nanoechinate, or striate ornamentation, with the sexine thicker than the nexine. The key characteristics distinguishing families are number, type of apertures, and exine ornamentation. Quantitative data (length and width of ectoapertures, equatorial diameter in polar view, and polar diameter in equatorial view) contribute to the understanding of the palynology of Rosales from forest fragments, as these characteristics allow us to identify species or groups of species. Our findings confirm Rosales as eurypalinous, while the genera analyzed demonstrate stenopalynous traits. The qualitative and quantitative characteristics of pollen identified here expand the morphological knowledge of the species and can be used to understand the relationship among them.

Chamaecrista is the most diverse genus in the subfamily Caesalpinioideae (Leguminosae), and is one of the richest taxa in terms of species diversity in Brazil. Its monophyly is supported by phylogenetic studies, and associated polyphyletic clades have been identified. Although it is important, its palynological description is limited, and no specific studies on this taxon are available. Thus, the aim of the present study is to characterise the morphology of the pollen grains of some Chamaecrista species to expand the knowledge of the palynology of the group and provide micromorphological data for applied studies. The pollen morphology and ultrastructure of 34 taxa of Chamaecrista, with representatives of the four sections are described based on the results from light, scanning and transmission electron microscopy. The data show that there are morphological differences among the species, making it possible to distinguish them through exine ornamentation and aperture type, including bridge types. The pollen grains are monads, isopolar or apolar, medium to large, and prolate-spheroidal to prolate with a subcircular to triangular shape. There is variation in the apertural type, with 3-colporate, 3-(4)-colporate and 3-(6)-colporate pollen grains, and the 6-colporate pollen grains have a regular tetrahedral shape. The exine is characterised predominantly as perforate, microfossulate, microreticulate, and psilate. Under transmission electron microscopy, the presence of a sexine bridge joining the two edges of the ectoaperture was recorded. The presence of bridge and heteromorphism in the number of apertures and shape of pollen grains are new characteristics for the genus and are described for the first time.

Crotonoideae is a subfamily of Euphorbiaceae, a plant family that is characterized by the presence of laticifers and pollen grains with Croton pattern exine. We analysed 48 species of Crotonoideae from Seasonally Dry Tropical Forests within Caatinga vegetation aiming to contribute to the pollen morphology of this subfamily, focusing on endemics to these forests. Pollen samples were obtained from specimens of the herbarium. Pollen grains were acetolyzed, measured, photographed and described using light and scanning electron microscopy methods. Three pollen types and seven subtypes were recognized based on aperture type, polarity, shape, size and details of the exine sculpture. The exine ornamentation and the type and number of apertures are the most important characteristics for recognizing genera in Crotonoideae. Inaperturate pollen grains with surface pila plicate were observed in representatives of Croton and Jatropha; 4–8 pantoporate pollen grains and a 3-colpate one with psilate pila were observed in Cnidoscolus, and 10–22 pantoporate pollen grains with rugulate pila were observed in Manihot. Due to the high diversity of Crotonoideae pollen morphology, we highlighted its eurypalynous properties, in addition to its taxonomic relevance, for the identification of groups.