Comparative pollen and foliar micromorphological studies using light microscopy and scanning electron microscopy of some selected species of Lamiaceae from Alpine Zone of Deosai Plateau,Western Himalayas

The study was conducted to highlight a detailed account of morphology of pollen chosen species of Lamiaceae through scanning electron microscopy, and the anatomical characteristics of leaf epidermis of seven species using simple light microscopy. In results, Anisomeles indica and Otostegia aucheri belong to subfamily Lamioideae because it has tricolpate pollen while the rest eight species belong to subfamily Nepetoideae (hexacolpate pollen). The exine sculpturing of pollen of studied species was found to be reticulate. In the family Lamiaceae, four kinds of stomata were found anomocytic, anisocytic, diacytic, and actinocytic, respectively. The cell wall patterns of epidermal cells were irregular or polygonal with straight or undulate walls. It was noted that the variety of the epidermal trichomes seems of taxonomically important for the identification of species of Lamiaceae. Both nonglandular and glandular trichomes were analyzed. The nonglandular trichomes were characterized with long, thin, and pointed apical unicellular cells. The nonglandular trichomes were A‐shaped in Thymus linearis. In Perovskia abrotanoides, stellate glandular trichomes were observed whereas in A. indica and Mentha royleana both glandular and nonglandular trichomes were found. In A. indica, the nonglandular trichomes were sessile and peltate in M. royleana. For the first time in this study, pollen and foliar micromorphological features of selected species of this area are carried out. These taxonomic characters were found to be important in discrimination of species from each other. In future, the detailed study with comprehensive morphology coupled with other important characters is required for delimitation of taxa at various levels.     

Pollen morphological variation of Berberis L. from Pakistan and its systematic importance

Due to overlapping and diverse morphological characters, Berberis is among the most taxonomically complex genera. Palynology is one of the taxonomic tools for delimitation and identification of complex species. In this study, pollens of 10 Berberis species were analyzed through light and scanning electron microscopy. Qualitative as well as quantitative features (pollen shape, size, presence or absence of colpi, colpi length and width, exine thickness, ornamentation, pollen class, aperture, and polar–equatorial ratio) were measured. Five species were observed to have colpate (pantocolpate) with elongated ends, radially symmetrical, isopolar, monads, and psilate‐regulate pollens. In polar view, six pollen were spheroidal, two were ovoid, one spherical, and one oblate. Similarly, variation in pollen length was prominent and the largest pollen on polar view was recorded for B. psodoumbellata 60–65 μm (62.4 ± 0.9), while the smallest one was observed for B. lycium 29–35 μm (32.2 ± 1). The observed variation in both quantitative and qualitative features were important in taxonomic identification. This shows that palynological characters are helpful in identification of Berberis genus at the species level.     

Pollen morphology of the genus Sophora (Fabaceae) and its taxonomic implications

The genus Sophora (Fabaceae) is one of the taxonomically challenging genera with high economic and medical values. In this study, the pollen morphology of 43 samples of 27 species, 4 subspecies, and 4 varieties of the genus Sophora and 3 closely related genera was examined using scanning electron microscopy to evaluate the pollen diversity of the genus and its taxonomic significance. Pollen grains of the studied species were tricolporate (rarely six-aperture), and pollen shape varied from suboblate, spheroidal, subprolate to prolate. Echinate external ornamentation was reported for the first time in some species of the genus. Aperture membrane ornamentation and outline in a polar/equatorial view were described for the first time in the genus. Principal component analysis was used to understand the relationship and discrimination between the species and the genera, with six components accounting for 79.92% of the total variance. Taxonomic keys based on pollen morphology were also constructed to easily identify the taxa of the genus through palynological characteristics. Results showed that pollen morphology alone is not sufficient to elucidate or reconstruct taxonomic relationships within the genus Sophora, but palynological assessments can provide some useful information for identifying taxonomically problematic taxa.     

Light and scanning electron microscopy-based foliar morpho-anatomical comparison of selected family Rosaceae members distributed in District Lahore,Punjab, Pakistan

In the present study morpho-anatomical characterization of selected Rosaceae members distributed in District Lahore was performed. Light and scanning electron microscopy was used for systematic characterization of the selected 19 species. Distinguished morpho-anatomical features such as size and shape of epidermal cells, size and type of stomatal cells, size and shape of trichomes, oil droplets, and silica bodies were contrasted. Results reported remarkable variations which could be taxonomically useful in identification of these members. Polygonal epidermal cells were observed in Eriyobotraya japonica, Potentilla bifurca, Potentilla supina, and Prunus amygdalus. However, Prunus cerasus possessed irregular-shaped epidermal cells that can be distinguished from hexagonal epidermal cells of Prunus persica. Similarly, stomatal type varied among some members. Paracytic or perisocytic stomata were observed in E. japonica whereas P. bifurca observed paracytic and anisocytic stomata. Lengths of guard cells were also of variable sizes. The average length of guard cells ranged from 53 (52–54) μm to 74 (73–75) μm in abaxial view. Potentilla supina had biggest, while Rosmarinus officinalis had tiny guard cells. Trichomes were tubular, stellate, cylindrical, ribbon-like, glandular, and nonglandular. Silica bodies in the present investigation were bilobed, rounded, and oval-shaped. It is inferred that diverse anatomical features proved to be valuable taxonomic tools that could be fruitfully helpful in identification of plants at specific as well as generic level.     

Detailed macro and micromorphology of grasses caryopsis using scanning electron and light microscopy

Micromorphology of 15 grass caryopsis was investigated through scanning electron microscopy for its implications in the identification of grasses. Both macro and micromorphological characters especially caryopsis shape and surface morphology were found significant in the taxonomic diagnosis. Shape morphology of caryopsis varied among the species and lanceolate, fusiform, narrow ellipsoidal, ovate, elliptical, plano-convex, fusiform, linear ovate, broadly elliptical, broadly pllipsoidal, plano-convex, oblique ovate, and subglobose shapes were recorded. Digitaria nodosa (3.31 × 0.7 mm) and Echinochloa crus-galli (3.26 × 1.44 mm) had the largest caryopsis size, while Acrachne racemosa (0.76 × 0.54 mm) and Eragrostis minor (0.62 × 0.38 mm) had the smallest caryopsis size. Macro and micromorphological characters were useful for the diagnosis of genus and species. Utilizing both qualitative and quantitative character taxonomic key at generic and species level were made.     

Light microscopy of Pakistani Berberis leaf cuticles and its taxonomic implications

Taxonomy of the genus Berberis is quite complex, due to overlapping morphological characters, making it very difficult to differentiate the species within the genus. In order to resolve this taxonomic complexity, the foliar anatomy of 10 Berberis L. species was carried out, for the first time from Pakistan, using light microscopy (LM). Significant variation in terms of epidermal cells shape, size, cell wall pattern, and stomata type was observed. B. baluchistanica has the largest epidermal cells, Adaxial: length = 45–(53.9 ± 3.6)–62.5 μm; and width = 22.5–(26.3 ± 1.3)–30 μm; Abaxial: length = 37.5–(43.25 ± 2.5)–50 μm; and width = 20–(22.6 ± 0.8)–25. The highest number of stomata was observed in B. glaucocarpa as 62 on the abaxial surface while the lowest number of stomata was recorded in B. baluchistanica as 8 on the adaxial surface. Of 10 investigated species, 6 possess anomocytic type stomata, while 2 species that is, B. aitchisonii and B. parkeriana have both anomocytic and anisocytic stomata while B. baluchistanica and B. calliobotrys have only paracytic type stomata. The highest number of cells per unit area was present on the adaxial surface of B. calliobotrys ranging from 245–(252.4)–260 followed by B. parkeriana with 209–(227.8)–250 on the abaxial surface. Stomatal index (SI) also varied considerably and was the lowest (2.6) percentage in B. baluchistanica and highest (31.9) percentage in B. kunawurensis. A taxonomic key based on micro‐morphological characters is provided for species identification.     

Implications of foliar epidermal micromorphology using light and scanning electron microscopy: A useful tool in taxonomy of Korean irises

The genus Iris L., comprising approximately 210 species, is one of the most species-rich genera in the family Iridaceae. In this study, the first comprehensive leaf micromorphological characters of Korean irises were studied using light and scanning electron microscopy. Our objective was to evaluate the foliar micromorphological characteristics (namely epidermal cells, stomata types, and guard cell size) of Korean Iris taxa in a systematic context. All the investigated Korean Iris taxa had amphistomatic or hypostomatic leaves with anomocytic stomatal complexes. Guard cell length varied among species, ranging from 24.8 μm (I. rossii) to 56.0 μm (I. domestica). Although the presence of papillae on the outer periclinal wall is not of taxonomic significance, leaf margin pattern, guard cell size, and sunken stomata type were useful for species-level identification of Korean Iris species. The occurrence of polymorphic stomatal types was reported here for the first time, and the correlation between genome size and epidermal guard cell length was discussed.     

Histomicroscopy and normal anatomy of the adult killifish Aphanius hormuzensis (Teleostei; Aphaniidae) from the Persian Gulf coastal environment

The histomicroscopy and normal anatomy of the major body organ systems were investigated in the adult killifish, Aphanius hormuzensis using histological examination, X‐ray imaging, double staining, light microscopy and scanning electron microscopy (SEM). Based on the histomicroscopic observations, the kidney, liver and swim bladder in the studied species were comparable to other fish models. The anterior portion of the kidney is bulbous, while the posterior portion is narrow and elongated; the liver has a single lobe and the swim bladder is a single‐chambered organ with no connection to the digestive tract (physoclistous). X‐ray imaging and double staining examination showed 12 abdominal and 15 caudal vertebrae and a single hypural plate in the caudal skeleton. According to light microscopy, the scales were rounded to pentagonal in shape with three types of radii (primary, secondary and tertiary), and the urohyal bone was elongated. SEM microscopy showed a single row of tricuspid teeth on the upper and lower jaw, respectively, each tooth has two lateral cusps that are shorter than the middle one. The number of teeth was 17–18 in the upper jaw and 19–20 in the lower jaw. The saccular otoliths were rounded‐trapezoid in shape with a moderately incised and V‐shaped excisura. The members of killifishes are an important group for biologists because of their evolutionary properties, regeneration capacity and usefulness as biological control and also for the ecotoxicological assessment of environmental pollution. The outcomes of this study may provide a useful basis for future research on the genus Aphanius.     

Identification of monocot flora using pollen features through scanning electron microscopy

Pollen used to track structural and functional evolution in plants as well as to investigate the problems relative to plant classification. Pollen characters including ornamentation, shape, apertural pattern, pollen symmetry, colpus length, width, and margins used to detect the similarities and dissimilarities between genera and also species of the same genus. In this study pollen features of 20 monocot species belonging to 15 genera of the Amaryllidaceae, Asparagaceae, Iridaceae, Ixioliriaceae, Liliaceae, and Xanthorrhoeaceae were studied using scanning electron microscopy (SEM) and light microscopy (LM). In this study two species that is Zephyranthes citrina and Tulbaghia violacea were reported for the first time from Pakistan. Pollen grains were visualized with LM. Non‐acetolyzed and acetolyzed pollen were examined using SEM. A taxonomic key was developed to highlight the variation in pollen features in order to make their systematic application for correct species identification.     

Utilization of foliar cuticle morphology for the identification of weedy grasses

For the robust identification of weeds of taxonomically complex families like Poaceae, scanning electron microscopy (SEM) and light microscopy (LM) are of significant implications. In this study, SEM and LM methods were used by investigating 12 Pakistani weedy grass species as an example for foliar epidermal micromorphological characters for its role in the identification of grasses species. Qualitative and quantitative foliar micromorphological variations in stomatal type, number, guard cells, subsidiary cells, silica bodies and trichomes were studied. Significant variation was observed in both qualitative and quantitative features and based on this diversity in epidermal characters, a key was drawn to delimitate the species. Variations in stomata, silica bodies (SiO₂·nH₂O), macrohairs, microhairs, hooks, papillae, prickles, and other epidermal structures were used in establishing the taxonomic key. The data were also statistically evaluated by determining its mean, standard deviation, variance, the coefficient of variance, and standard error. Foliar epidermal characters such as silica bodies shape and trichomes were taxonomically more useful than stomatal types, subsidiary, and guard cells. SEM and LM micromorphological characters were of substantial importance in the delimitation of closely related species.     

Palyno‐morphological investigations of subtropical endangered flora of Capparidaceae through light and scanning electron microscopy

The research was performed to investigate pollen morphology of endangered species of Capparidaceae in subtropical regions of Pakistan. The distinguishing characters were investigated by using light microscope and scanning electron microscope. Palynological study is comprised of pollen shape, pollen type, exine sculpturing, polar and equatorial diameter, length and width of colpi, mesocolpium, and exine thickness. In polar view, Cleome viscosa exhibited the highest pollen size 26.4 (32.7–24.5 μm) ±0.776 whereas Capparis spinosa appeared to be the lowest 12.6 (14.5?10.7 μm) ±0.400. In equatorial view, Cleome viscosa had the largest pollen size 17.1 (20.0–15.0 μm) ±0.606 and Capparis spinosa had the smallest pollen size 9.7 (12.50–8.00 μm) ±0.394. The maximum fertility percentage has been observed in Capparis spinosa, that is, 98.96% and minimum in Cleome viscosa, that is, 82.93%. Diagnostic key has been constructed to state the essential diagnostic features by means of which the taxa can be identified. Remarkable variations have been observed in pollen size, shape, and exine sculpturing. All the selected species were tricolporate. Prolate to subprolate pollen were observed. There is a great variation existed in exine sculpturing such as in Capparis decidua and C. sp. nova sculpturing is reticulate, in Capparis himalayensis sculpturing is Scabrate granulate, in Capparis spinosa sculpturing is Psilate, in Cleome viscosa sculpturing is regulate‐reticulate, in Dipterygium glaucum sculpturing is regulate and in Gynandropsis gynandra sculpturing is striate‐regulate. On the basis of overall characteristics of pollen it seems that palynology of this family is helpful at the generic and specific level.     

Pollen morphology of subfamily Caryophylloideae (Caryophyllaceae) and its taxonomic significance

The Pollen morphology of subfamily Caryophylloideae (Caryophyllaceae) from different phytogeographical region of Pakistan has been evaluated. In this research, 16 species belong to 6 genera of subfamily Caryophylloideae have been studied using light (LM) and scanning electron microscopy (SEM) for both qualitative and quantitative characters. Different palyno‐morphological features were observed including; pollen ornamentation, pore ornamentation, echini arrangement, echinidensity, number of pori, size of polar and equatorial diameter, P/E ratio, exine thickness, and size of pore were studied. The palyno‐morphological characters of subfamily Caryophylloideae have taxonomically significant in identification and delimitation of species. Two pollen types, i.e., subspheroidal (15 species) and prolate (one species Vaccaria hispanica) were observed. Microechinate‐punctate and microechinate‐perforate pollen ornamentation were examined. Two pollen types of pori, i.e., prominent (5 species) and sunken (11 species) ornamentation have been reported. Three types of echini arrangement have been reported irregular, regular and rather regular, while three type of echinidensity (i.e., medium, dense and sparse) were observed. Pori numbers were found different in different species range from 5 in Silene apetala to 19‐35 in Silene vulgaris. Based on different palyno‐morphological characters, taxonomic key was develop for quick and accurate identification. The quantitative data were processed using SPSS software for average, minimum, maximum, and standard error.     

Structure and composition of Unio pictorum shell: arguments for the diversity of the nacroprismatic arrangement in molluscs

Mollusc shells are complex organomineral structures, the arrangement and composition depending on the species. Most studies are dedicated to shells composed of an aragonite nacreous and a calcite prismatic layer, so the nacreous prismatic model based on Pinctada and Atrina–Pinna. Here, we studied the micro‐ and nanostructure, the mineralogy and composition of a nacroprismatic bivalve species: Unio pictorum. The prismatic layer of Unio is aragonite, and the inner structure of the prismatic units strongly differs from those of the calcitic layers. The shape of the prisms varies depending on their growth stage. The first layers of nacre are similar to those of gastropods (columnar nacre), then evolve towards the typical bivalve arrangement (sheet nacre). Na, Sr, Mg, P and S are present in both prisms and nacre. The organic prismatic envelopes are rich in sulphur amino acids, whereas organic sulphate is present within the prisms and the nacreous tablets. P is present as phosphate, probably a mixture of organic and mineral complex. Chemical distribution maps confirm the absence of an organic membrane between the nacre and the prisms. The comparison of the structure, mineralogy and composition of Unio pictorum and different species show the diversity of nacroprismatic shells, and that these features are taxonomically dependent.     

Palynological investigation of some selected species of family Fabaceae from Pakistan,using light and scanning electron microscopy techniques

Pollen morphology of 11 species of family Fabaceae that is, Trifolium alexandrinum, Trifolium resupinatum, Arachis hypogaea, Lathyrus aphaca, Medicago lupulina, Vicia sativa, Lathyrus odoratus, Pongamia pinnata, Melilotus indicus, Medicago polymorpha, Medicago sativa from Pakistan has been investigated by light and scanning electron microscopy. Pollen were generally tricolporate, radially symmetrical, isopolar, elliptic in equatorial view and triangular in polar view under LM. Results showed that pollens were per‐prolate (T. alexandrinum), prolate (T. resupinatum, V. sativa, L. odoratus, Melilotus indicus, M. polymorpha, M. sativa) and sub‐prolate (A. hypogaea, L. aphaca, M. lupulina, P. pinnata). The larger polar/equatorial (P/E) ratio was found in T. alexandrinum (2.26 μm) and the smallest was found in M. lupulina (1.21 μm). The exine of T. resupinatum was 3.00 μm in thickness while others posses smaller exine thickness. The larger pore diameter was found in P. pinnata (16.01 μm) while others have smaller. The length of colpi was larger in Arachis hypogaea (32.24) while others posses smaller. Eight types of surface ornamentation (Psilate, faintly rugulate). Perforate and rugulate to verrucate have been observed under SEM. The pollens were europalynous type. Pollen morphology proved to be useful for the specific delimitation and serve as a tool for the identification and classification of taxa at specific and generic levels and can also be used as a key for the taxonomic features. Diversity in exine sculpture is helpful indicative characters for the isolation of closely related species. Hence, it is clear that both qualitative and quantitative characters of pollen can be useful for differentiating between taxa at specific level.     

Karyological and electrophoretic differences between <i>Pomacea flagellata</i> and <i>P. patula catemacensis</i> (Caenogastropoda: Ampullariidae)

The widespread Mexican apple snail Pomacea flagellata (Say 1827) and the strictly endemic "tegogolo" P. patula catemacensis (Baker 1922) (restricted to Lake Catemaco), are the only known American Ampullariidae that have haploid complements n=13. Pomacea patula catemacensis has suffered a critical reduction in abundance due to immoderate fishing for human consumption. Chromosome slides were obtained from colchicine-injected Pomacea snails collected from nine locations along the coastal zone of the Gulf of Mexico, including Lake Catemaco, for use in principal component analysis (PCA). Total proteins in foot homogenates were analyzed through isoelectric focusing (IEF) and native-PAGE electrophoresis on polyacrylamide gels. The chromosome number 2n=26 was confirmed for snails from all locations, with a uniform 9 m + 4 sm formula. However, P. patula catemacensis showed significantly larger chromosomes (absolute size) than any population of P. flagellata. Pomacea patula catemacensis also differed from all populations of P. flagellata in a PCA with standardized data, i.e., independently of the absolute size difference between species. Proteins with an acid isoelectric point were dominant in the foot of both species. The electrophoresis analysis showed that P. flagellata has 17 protein bands, with an upper bound at IEF=7.6, while P. patula catemacensis has only 15 bands, with an upper bound at IEF=7 and a more evenly spaced band pattern. Molecular weights ranged from 40 to approximately 130 kDa in both species. Proteins with high values (>94 kDa) were the most abundant. Pomacea patula catemacensis showed a band of 93 kDa, which was absent from all specimens of P. flagellata. Samples of P. flagellata did not cluster according to any geographical pattern in the statistical analyses, nor did they show any taxonomically useful differences in their electrophoretic patterns that merit sub-specific discrimination.     

Comparative authentication of three “snow lotus” herbs by macroscopic and microscopic features

“Snow lotus” is a famous Chinese Materia Medica derived from species of the genus Saussurea (Compositae). To differentiate three representative easily‐confused snow lotus herbs, namely, Saussurea involucrata (Kar. et Kir.) Sch.‐Bip, Saussurea laniceps Hand.‐Mazz., and Saussurea medusa Maxim., macroscopic features of the three herbs were systemically observed, and microscopic features were compared by using ordinary light microscopy, polarized light microscopy and scanning electron microscopy (SEM). The results indicate that, as for macroscopic identification, capitula situation and arrangement, and as for microscopic identification, pollen grains, nonglandular hairs, glandular hairs, and cells of inner surface of the microdiodange can be used to authenticate the three snow lotus herbs. Comprehensive table comparing the characteristics were presented in this study. SEM has been found to provide a number of unique characteristics of pollen grains. Based on the observation of pollen grains, evolution sequence of the three species was speculated. The present method was proven to be efficient, convenient, simple, and reliable, which was successfully applied to the authentication of three snow lotus herbs. Microsc. Res. Tech.1 77:631–641, 2014. © 2014 Wiley Periodicals, Inc.     

Morphofunctional structure of the lingual papillae in three species of South American Camelids: Alpaca,guanaco, and llama

The aim of this study was to compare the anatomical and functional characteristics of the lingual papilla among the Camelidae. For this purpose, tongues of alpaca, guanaco, and llama were used. Numerous long and thin filiform papillae were located in the median groove and none were detected on the rest of the dorsal surface of the lingual apex in alpaca. Secondary papillae originated from the base of some filiform papillae on the ventral surface of alpaca tongue. The bases of some filiform papillae of the lateral surface of the lingual apex were inserted into conspicuous grooves in guanaco and tips of filiform papillae on the dorsal surface of the lingual body were ended by bifurcated apex. On the dorsal surface of the lingual apex of llama, there were no filiform papillae but there were numerous filiform papillae on both the lateral margins of the ventral surface of the lingual apex. Fungiform papillae were distributed randomly on dorsal lingual surface and ventral margins of the tongues of all camelid species. Lenticular papillae were located on the lingual torus and varied in size and topographical distribution for each species. Circumvallate papillae had irregular surfaces in llama and alpaca, and smooth surface in guanaco. In conclusion, llama and alpaca tongues were more similar to each other, and tongues of all camelid species displayed more similarities to those of Bactrian and dromedary camels in comparison with other herbivores and ruminants. Microsc. Res. Tech. 79:61–71, 2016. © 2015 Wiley Periodicals, Inc.