A novel approach to study the morphology and chemistry of pollen in a phylogenetic context, applied to the halophytic taxon Nitraria L.(Nitrariaceae)

The pollen morphology of many collections of taxa of the tribe Nigelleae from the family Ranunculaceae which occur worldwide is presented in this study. A total of 88 specimens from 21 taxa, some of which were recently proposed, belonging to the genera Komaroffia, Garidella, and Nigella of Nigelleae were examined using light microscopy (LM) and scanning electron microscopy (SEM). In the tribe, the pollen type is mostly trizonocolpate, but in many taxa and specimens, both trizonocolpate and non-trizonocolpate types occur together. The pollen grains are small to medium (25–53.75 μm × 20–55 μm) in size and oblate to prolate in shape. The exine pattern at the mesocolpium in all the taxa investigated is similar: micro-echinate in LM and micro-echinate-punctate in SEM. The colpus membrane in Komaroffia and Nigella is micro-echinate in both LM and SEM. In Garidella, it is micro-echinate in LM but echinate (spinulose) in SEM. In this study, multivariate analyses, principal component analysis (PCA), and unweighted pair group method with arithmetic mean (UPGMA), were used to evaluate relationships between the genera and species within the tribe with respect to pollen morphology. PCA results show three main groups in the tribe: Garidella, Komaroffia, and Nigella. Moreover, the UPGMA tree also chiefly supports generic segregation into the smaller genera. An overall synthesis of the pollen characteristics of the three genera is provided and discussed.

The pollen morphology of 17 taxa of Gundelia, of which 13 taxa are endemic to Turkey, was investigated by light microscopy (LM) and scanning electron microscopy (SEM). In this study, the pollen morphology of 16 taxa except G. tournefortii was investigated for the first time. The quantitative data were analyzed by descriptive statistics and multivariate statistics. The pollen grains are oblate spheroidal and prolate spheroidal in shape with the polar diameter of 37.01–61.2 μm and the equatorial diameter of 31.2–63.4 μm. The smallest pollen grains were observed in G. anatolica, G. rosea and G. tournefortii, while the largest pollen grains were observed in G. vitekii, G. komagenensis and G. colemerikensis. Pollen ornamentation is echinate in LM and echinate-microperforate in SEM. Statistically, the relationships between pollen characters of the examined taxa were identified using Principal Component Analysis (PCA) and Clustering Analyzis. In addition, a dendrogram was constructed by applying UPGMA based on the determined 15 pollen morphological characters, and the degree of their relationship was discussed. Polar axis, equatorial diameter and colpus length were the major explanations of the pollen morphological characters’ total variation in PCA. Pair group method was used to divide the main pollen taxa into three groups based on the pollen morphology by using arithmetic averages analysis (PCA scatterplot and cluster analysis). As a result, the pollen shape, polar axis, equatorial diameter and spine length were important characters distingushing the taxa from each other. We found some matches and mismatches between pollen and morphological characters, whose significance requires further study.

1. Background Stroke, i.e. loss of brain function(s) due to disturbance in the blood supply to the brain, is the main cause of adult disability (e.g. paralysis) in the world, leaving more than half of the patients dependent on daily assistance. In Qatar, stroke is a major health problem with an estimated incidence of 238/100,000 per year for the population over 45 years old [1]. Stroke patients are often hospitalized and/or subjected to intensive rehabilitation programs for long periods of time, and their quality of life is severely affected socially and economically. Around 10% of the hospital beds in Qatar are occupied by stroke patients [1]. Thus, without major advances to improve prevention, treatment and rehabilitation of stroke, the social and economic costs of this disease will increase dramatically. There are pathological and physiological changes on the cellular and molecular levels associated with stroke. The objective of this work is to determine the molecular and structural changes occurring in the tissue of rat's brain. Vibrational spectroscopy, i.e. Fourier transform infrared (FTIR) imaging spectroscopy, was used as rapid and objective diagnostic platform to investigate the pathological and pathological changes in the rat's brain sections three weeks after stroke. FTIR spectroscopy was also used to differentiate between the biochemical makeup of the white and grey matters of a healthy control brain samples. Also, in the current study, scanning electron (SEM), energy dispersive X-ray spectroscopy (EDX), and atomic force microscopic (AFM) techniques were assessed to study the structural changes in the rat's brain tissues after experiencing an induced stroke. 2. Experimental 2.1. Sample preparation Rats were anesthetized using 2–3% isoflurane. Experimental stroke was induced in rats by 90-min occlusion of the right middle cerebral artery with an intraluminal filament. Rats were euthanized with a lethal dose of sodium pentobarbital and transcardially perfused with 4% paraformaldehyde. Rat's brains were extracted, embedded in paraffin and then serially sliced, using semi-automated rotary microtome, into 5 μm thickness sections for the FTIR imaging and AFM analysis and 35 μm thickness for the SEM and EDX analysis. The brain sections were mounted on MirrIR CFR, Low-e microscope slides for the FTIR imaging analysis, and on aluminum metal for the SEM analysis and EDX analysis. The paraffin was removed from the samples by using xylene and isopropanol. 2.2. Instrumentation 2.2.1. FTIR Imaging Measurements The FTIR images were obtained using FTIR spectrometer (Agilent Technology) at a reflection mode within the range of 4000–700 cm –1 . Spectral images were analyzed using Metlab software (The Mathworks Inc.). Origin 2015 software was used for graph drawing. Principal component analysis (PCA) was performed to study the spectral data variations between the FTIR spectra and images. 2.2.2. Scanning Electron Microscopy (SEM) Rat's brain sections of 35 μm thickness were mounted on aluminum slides for SEM analysis. All the samples were viewed with a FEI Quanta 200, USA scanning electron microscope at 10 kV. SEM micrographs of the brain stroke and healthy rat's sections were compared. Elemental distribution in both healthy and induced stroke brain sections were investigated by using energy dispersive X-ray spectroscopy (EDX) equipped with SEM. The spectra provided a semi-quantitative view of the elemental composition of both weight and atomic percent. 2.2.3. Atomic Force Microscopy (AFM) Bruker atomic force microscopy (AFM) was used for imaging and quantitatively determining the local elastic properties of healthy and induced stroke rat's brain sections. A controllable and constant force was applied at each data point and using the resulting force-distant curve for the formation the AFM images. Brain sections were scanned at 10 μm by 10 μm. About 100 force-distance curve were collected for each healthy and induced stroke brain sections and two random scan lines of force-distance curves was recorded. 3. Results and Discussions The FTIR spectroscopy results indicated that the white matter is richer in lipid content than the grey matter as shown in Figs. 1 and 2. The infrared spectrum images showed a decrease in the lipid content of the white matter associated with the induced stroke brain sections. FTIR bands assigned to the bio-chemical makeup such as proteins, lipids and ester varied in positions, line-shape, and intensity between control and induced stroke brain samples. The spectral images showed that there is a configuration changes is associated with the lipid bands in the rat's brain white matter that experienced stroke. The FTIR spectral images of the white matter in the induced stroke brain sections indicated that amide I and ester bands experienced a bio-chemical changes as shown in Fig. 3 and 4. Figure 5 shows the second derivative of the collected FTIR spectra from induced stroke brain sections. In Fig. 5, there are spectral differences that assigned to ester and protein regions. Figure 6a represents the loading spectra of the first three principal component analysis (PC1, PC2 and PC3). The variations principally were located in the regions of amide I band at (∼1695-1637 cm –1 ) and small variation in the amide II band at (1543 cm –1 ). Figure 6b represents the loading spectra of the PC4, PC5 and PC6. The variations principally were located in the protein region, mainly amide I band at (∼1695-1637 cm –1 ) and ester band at about 1730 cm –1 . The use of FTIR imaging and chemometric analyses such as principal component analysis (PCA) of spectral data allows to investigate and differentiate spectral images pattern collected from control and stroke rat's brain samples. The scanning electron microscope results showed that lesion region in the induced stroke brain sections are enriched by the selected elements such as Fe and Ca as shown in Fig. 7 (a & b). Scanning electron microscope (SEM) micrographs indicated that there is structure change in the induced stroke brain section. The structure of stroked brain sample in the nanometer scale appeared to be significantly rough compared to the control brain sample (Fig. 8 a & b). Atomic force microscope (AFM) images showed that the stroke brain section is swollen compared to healthy brain sections. The AFM images of the induced stroke brain sections appeared more stretched when compared to the control brain section image as shown in Fig. 9 (a & b). AFM results also showed that the force-distance curves in Fig. 10, recorded using control (healthy) brain sections (blue) and induced stroke brain sections (red). The force-distance showed that the AFM cantilelver deflection of the healthy brain samples is higher than the induced stroke brain section. This indicate that the healthy brain section are softer and elastic than the induced stroke brain sections. 4. Conclusion FTIR imaging spectroscopy, scanning electron and atomic force microscopy techniques were able to analyze and differentiate between the healthy and induced stroke rat's brain sections on the molecular, structural and global levels making them valuable tools to investigate, diagnose and study the structural plasticity of the stroke induced brain. FTIR imaging spectroscopy in combination with multivariate analysis such as principal component analysis (PCA) is a non-destructive technique that proves to be rapid, accurate and straightforward to be performed. It constitutes a powerful approach to be used as a medical diagnosis tool to investigate the pathological changes associated with stroke in the brain tissues. Keywords Fourier Transform Infrared (FTIR) imaging spectroscopy, Scanning Electron Microscopy (SEM), Atomic Force Microscopy (AFM), Brain tissue, Stroke, Chemometric Analysis Acknowledgments This article was made possible by a NPRP award [5 - 381 - 3 - 10] from the Qatar National Research Fund (a member of The Qatar Foundation). The statements made herein are solely the responsibility of the authors. We would like to acknowledge the Center for Advanced Materials at Qatar University for performing the AFM analysis, and Central Laboratories Unit at Qatar University for performing the SEM and EDX analysis. Reference [1] Hamad, A., Sokrab, T.E., Momeni, S., Mesraoua, B., and Lingren, A., Stroke in Qatar: a one-year, hospital-based study. J Stroke Cerebrovasc Dis, 2001. 10(5): p. 236–41.

The genus Wittmackia has 44 species distributed in two centers of diversity: the Brazilian clade and the Caribbean clade. The Brazilian clade includes 29 species, with geographic distribution concentrated in the Northeast of Brazil. This study reports the morphology, ultrastructure, pollen viability and stigma receptivity by different microscopy techniques of 23 species of the genus Wittmackia endemic to Brazil and occurring in Atlantic Forest areas. Pollen morphology was evaluated by scanning electron microscopy and light microscopy by the weak lactic acetolysis method. Histochemical tests were performed with Alexander's solution and fluorescein diacetate. For invitro germination of pollen grains, two culture media were evaluated. Stigma receptivity was evaluated with hydrogen peroxide and α-naphthyl acetate. The Wittmackia genus presented biporate pollen grains, monads to tetrads, with oblate, suboblate or oblate-spheroidal shape, circular or elliptical scope, and heteropolar or sub-isopolar morphology. The stigmas are of the conduplicate-spiral type, with differences in color and size. In both histochemical tests, high viability was observed, mainly at anthesis, coinciding with invitro germination. The species presented high enzymatic activity during receptivity, with a very strong positive response, mainly at anthesis. Studies of pollen morphology and viability provide important information for the conservation and genetic improvement of Wittmackia species, also assisting in the taxonomy of the genus and the reproductive success of future controlled crosses.

Pollen morphology of 28 taxa belonging to the genus Sabulina Rchb. (Caryophyllaceae) from Turkey was examined and compared using light microscopy and scanning electron microscopy. The current study aimed to describe the palynological characteristics of the Sabulina taxa and to assess the diagnostic value of these features for taxonomic implications. The pollen grains of Sabulina are radially symmetrical, apolar, rarely isopolar, and pantopolyporate with 8-28 pores. Their shape is prolate-spheroidal with the A axes (long axes) 19.5 μm and the B axes (short axes) 19-39.5 μm. Exine sculpturing is microechinate-punctate in all taxa. Principal component analysis was used to evaluate the significant pollen morphological characters for distinguishing the taxa, with three components accounting for 95.112% of the total variance. Numerical analysis revealed that pollen sizes, pore diameters, pore numbers, distance between pores, spinule density, and punctum numbers are particularly the most useful variables for separating the Sabulina taxa. The results also display a partial relationship between the Sabulina taxa clusters based on pollen morphology and the systematics of these taxa based on general macromorphology.

The pollen grains of 53 species and 57 taxa of two genera (Cistanche, Orobanche s. l.) of tribe Orobancheae were studied with light microscopy (LM). Thirty-five of these species were also studied with scanning electron microscopy (SEM). Two pollen types (Orobanche and Phelipanche) and six pollen groups (I–VI) were described. A principal component analysis was performed to help clarify some taxonomic problems at generic, specific and infraspecific levels. Some transition forms exist between the two pollen types. We consider that these data support the elevation of Trionychon to the rank of subgenus (Phelipanche). The pollen morphology of the subgenus Boulardia and of the genus Cistanche is quite similar to that of subgenera Orobanche and Phelipanche, respectively. Our results support the position of Boulardia as the sister taxon to Orobanche s. s. The contribution of pollen morphology to the resolution of several taxonomic problems related to specific and infra-specific taxa was discussed. Within the Orobancheae, the tricolpate, isopolar, microreticulate-scabrate pollen grains are plesiomorphic and the apolar, granulate, inaperturate pollen grains are apomorphic. Some taxa (e.g. O. cernua, O. cumana, O. lavandulacea and O. mutelii) present pollen heteromorphism, which could be related to the existence of geographical clines. The polymorphic (inaperturate, colpate), scabrate-perforate, tectate-columellate pollen grains present in some species (O. cernua, O. cumana) possibly represent an intermediate evolutionary type. Considering the separation of specific and infraspecific taxa in the Orobancheae, LM is apparently more useful than SEM, although information from SEM is clearly relevant as a complement for LM.

The family Euphorbiaceae is one of the most taxonomically complex and diverse family among the angiosperm. Therefore, this study aims to document the pollen morphology of the Euphorbiaceae taxa collected from different geographical regions of the Hainan Island South China, to strengthen the taxonomy and support the subfamilial classification. The Euphorbiaceae includes 23 species and 10 genera of the bi-ovulate subfamily Phyllanthoideae and tri-ovulate Crotonoideae, Euphorbioideae, and Acalyphoideae were examined under light and scanning electron microscopy. The principal component analysis was performed by using six variables to show variation and relationships between the species. A significant variation was found in the pollen morphology (exine and colpus sculpturing). The croton-type pattern of the pollen was found the diagnostic trait of the Crotonoideae. The Euhphorboideae taxa were mostly homogenous however, variation was found in the muri pattern. The Acalyphoideae, have fossulate scabrate and verrucate exine sculpturing that can delimit from the rest of sub-families. The Phyllanthoideae, showed reticulate exine, tri to tetra-colporate having various colpus morphology. The maximum pollen size was found in Croton hirtus (59.49 × 60.86 μm) and minimum in Acalypha indica (10.86 × 73 μm). Similarly, the maximum colpus width was observed in Euphorbia pulcherrima (8.3μm). Hence pollen traits have relevance in the taxonomy and phylogenetic of the family to define species boundaries at various taxonomic levels. This study can support and strengthen the taxonomy of subfamilies and fill the palynological gap of these taxa in Hainan Island and will contribute to future systematic studies of the Euphorbiaceae.

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.

The tribe Inuleae is widely distributed within Asteraceae and exhibits considerable morphological variation, which complicates species classification. Pollen morphology provides relatively stable features for species delimitation, yet comprehensive palynological data for many species remain limited. In this study, the pollen morphology of 19 species from eight genera of Inuleae was investigated using light microscopy (LM) and scanning electron microscopy (SEM), with a focus on 10 quantitative traits. Multivariate analyses, including principal component analysis (PCA) and hierarchical clustering analysis (HCA), showed that both pollen size and exine ornamentation contribute to interspecific differentiation. Differences were especially notable between Blumea and Carpesium, with the latter showing larger pollen grains and more slender, scattered spines. This research also presents first-time palynological descriptions of Blumea, Carpesium, Inula, Laggera, Pentanema, and Pterocaulon. Overall, the findings indicate that pollen morphological traits are informative for species differentiation and lay a foundation for further palynological classification within Inuleae.

In this study, we report the current palynological characteristics of Iberis taxa naturally distributed in Turkey. The pollen morphology of 10 native Iberis taxa was investigated across 35 populations. In the light of our taxonomic review of Turkish Iberis taxa, a detailed palynological description is provided for the genus. The palynological characteristics of Iberis taxa were examined using scanning electron microscopy (SEM) and light microscopy (LM) to confirm their diagnostic importance at a specific and/or generic level. We evaluated different characteristics including polar axis, equatorial axis, colpus length and width, amb diameter, apocolpium, exine structure, and sculpture. Pollen characteristics were measured on 30 pollen grains from each specimen under LM. Details of the exine sculpture were also analyzed under SEM. Generally, the pollen grains of the genus Iberis are small to medium sized, radially symmetrical, isopolar, spheroidal, almost tricolpate, and rarely syncolpate or tetracolpate. The exine sculpture is reticulate, and the structure is semitectate. Two pollen types for the genus were identified based on the colpus surface feature: Iberis type I (with non-ornamented colpus surface) and Iberis type II (with ornamented colpus surface). Principal component analysis and cluster analysis were performed to reveal patterns and groupings within the related taxa concerning pollen morphology. It was found that pollen size may provide a limited but valuable metric for distinguishing Iberis species. However, due to the wide variation in some populations, it would be useful to have palynological data alongside chromosomal, molecular and macro-micromorphological data to resolve the complexity in the taxonomy of the genus.

Parrotia (Hamamelidaceae) pollen morphology and a glimpse into the fossil record and historical biogeography

Histologic Observation of Continuity of Transmural Microvessels Between the Perigraft Vessels and Flow Surface Microostia in a Porous Vascular Prosthesis

Morphological characteristics of pollen grains, including shape, size, pore number, and exine thickness, vary significantly among species and enable the reliable use of palynological data in taxonomic studies. In this context, the present study investigates the pollen morphology of seven Betulaceae taxa (Alnus glutinosa, Betula pendula, Carpinus betulus, Carpinus orientalis, Corylus avellana, Corylus colurna, and Ostrya carpinifolia). Detailed morphometric measurements were carried out using Light Microscopy (LM), and high-resolution images were obtained using Scanning Electron Microscopy (SEM). For each taxon, thirty measurements were taken for the main pollen characters, including polar axis length (P), equatorial diameter (E), pore length (plg), pore width (plt), and exine thickness (Ex). Interspecific differences were evaluated using one-way ANOVA, Tukey's HSD test, and Principal Component Analysis (PCA), and a diagnostic pollen identification key was developed for the investigated species. The results demonstrate statistically significant interspecific variation in pollen size, pore characteristics, and exine thickness. In the PCA ordination, the first principal component (PC1) was mainly associated with pollen size (P and E), clearly separating Carpinus betulus from the remaining taxa. The second principal component (PC2) was primarily related to pore length (plg) and contributed to the separation of Alnus glutinosa from the other small-pollen species. These results show that quantitative pollen morphological characters provide reliable criteria for distinguishing closely related Betulaceae taxa.

Oil-tea belongs to the Camellia genus, an important oil crop in China. However, oil-tea is taxonomically challenging due to its morphological variation, polyploidy, and interspecific hybridization. Therefore, the present study aimed to investigate the flower organs’ morphology and pollen micro-morphology of 18 oil-tea genotypes in detail and discussed their significance for oil-tea taxonomy. The quantitative parameters of flowers were measured using Vernier caliper measurements. Pollen morphology was observed and photographed using scanning electron microscopy (SEM). The results indicated that the flower size varied significantly among the tested oil-tea genotypes, with the corolla diameter ranging from 42.25 μm in C. meiocarpa ‘LP’ to 89.51 μm in C. oleifera ‘ASX09’. The pollen grains of oil-tea are monads and medium grade in pollen size. There were two types of polar views, including triangular or subcircular, with a polar axis length (P) ranging from 27.5 μm in C. oleifera ‘CY67’ to 59.04 μm in C. mairei (H. Lév.) Melch. var. lapidea (Y.C. Wu) Sealy. The equatorial views exhibited oblate, spherical, or oblong shapes, with an equatorial axis length (E) of 21.32 to 41.62 μm. The pollen exine sculpture was perforate, verrucate, and reticulate. The perforation lumina diameter (D) ranged from 0.29 μm in C. magniflora Chang to 1.22 μm in C. yuhsienensis Hu, and the perforation width (W) varied from 0.77 μm in C. osmantha to 1.40 μm in C. gauchowensis ‘HM349’, respectively. Qualitative clustering analysis (Q-type cluster) and principal component analysis (PCA) were conducted using eleven indexes of flower and pollen morphology, and the 18 oil-tea genotypes were classified into three categories. In addition, the correlation analysis showed that there was a significant correlation between pollen size and flower morphology or pollen exine sculpture. These results offer valuable information on the classification and identification of the 18 oil-tea germplasm resources.

A scanning electron microscopy (SEM) study of sediments from Lake Cristol, southern French Alps, with special reference to the identification of Pinus cembra and other Alpine Pinus species based on SEM pollen morphology