Pollen Dimorphism of Several Members of Nymphaeaceae and Nelumbonaceae : An Index of Geographical and Ecological Variation
Somnath BHOWMIK*, Badal Kumar DATTA
Tripura University, Plant Taxonomy and Biodiversity Laboratory, Department of Botany, Suryamaninagar, 799022, India; [email protected] (*corresponding author)
Abstract
Pollen morphology of five Nymphaea (Nymphaeaceae) species, growing in Tripura, India were analysed using Scanning Electron Microscopy. Pollen grains of Nymphaea are dimorphic (ellipsoidal and spheroidal). The exine pattern also varies among the species. The variation as reported in the present study in terms of exine pattern of the studied species suggests the feasibility of applying the data in the identification of the genus of Nymphaea. The difference in exine patterns with the earlier reports may be interpreted as reflections of genetic variations possibly due to mutational changes effected by ecological conditions. The present pollen dimorphism may be attributed by introgression of populations. The variability in pollen morphology, including size variation and morphological differences, is often associated with hybrids among angiosperm groups. The examinations of percentages of aborted grains, generally considered a good indicator of hybridity. The occurrence of monosulcate pollens in Nelumbo nucifera along with dominant tricolpate pollens may be considered as aberrant pollens because of very low percentage of occurrence of monosulcate pollens. The ecological and geographical variations in pollen morphology could be an index of the genetic impact of the environment on the plant. Thus the present difference in terms of exine pattern could be useful to separate them at varietal level.
Keywords: Nymphaea Linn., Nelumbo nucifera Gaertner, pollen dimorphism, pollen morphology, scanning electron microscopy
Introduction
Palynology is unique, while one can obtain tremen- dous amount of information from a little material in a short time (Walker and Doyle, 1975). The constant fea- tures and the sculpturing of the exine make pollen grains a highly recognizable object by which parent genera or even species may be recognized (Harris, 1955; Moore and Webb, 1978). Application of pollen morphology in plant taxonomy is best evidenced in flowering plants, especially in the angiosperms. The largest variety of pollen morph types occurs among the angiosperm plants (Nair, 1964).
Lindley (1830) was probably the first person to make use of pollen character in the classification of Orchidaceae, and later the significance of pollen morphology in plant taxon- omy has been stressed by several workers (Cranwell, 1952;
Erdtman, 1952, 1957; Selling, 1947; Wodehouse, 1935) and is under use for the taxa of variable ranks (Chanda et al., 1988; Erdtman, 1952; Faegrie and Iversen, 1964).
Angiospermous pollens are divided into two fundamen- tal type’s viz., monosulcate or its derivatives and tricolpate pollen or its derivatives. Colpate pollen is essentially re- stricted to dicotyledons, while sulctae pollens are found in gymnosperms, monocotyledons and some Ranalean dicot’s (Walker and Doyle, 1975). The field of palynology has a tremendous contribution to the systematic and phy- logeny of angiosperms because of the evolutionary trends in pollen wall architecture which provides an important
source of phylogenetic information of major importance.
The Nymphaeaceae family is placed among the primitive families of dicotyledonous angiosperms by most taxono- mists. There has been an increased focus on Nymphaeales in recent years as evidence from broad spectrum of phylo- genetic studies that water lilies are most primitive among the flowering plants (Taylor and Osborn, 2006). Erdt- man (1952), Shiga and Kadono (2007) had described the pollen grains of Nymphaeaceae. Pollens of the cultivated Nymphaea variety were also studied by Singh et al. (1969).
Further information on pollen grains of the Nymphaeaceae members was added by Jones and Clarke (1981). More- over, Murthy (2000) had described the palynological fea- tures of six species of Nymphaea of India. The light mi- croscopic studies provide the structural profile of pollen;
the scanning electron microscopy (SEM) studies are of paramount importance in gaining knowledge on the fine morphology of exine surface for application at microtaxa levels and gene tic resources, as influenced by ecosystem.
Materials and methods
Five species of Nymphaea Linn. [Nymphaea micrantha Guillemin et Perrottet, Nymphaea pubescens Willdenow, Nymphaea rubra Roxburgh ex Andrew , Nymphaea stel- lata var major F. Mueller, Nymphaea stellata F. Mueller]
(Nymphaeaceae) and Nelumbo nucifera Gaertner (Nelum- bonaceae) were collected from Tripura, India. All collected Received 09 April 2012; accepted 13 July 2012
specimens were identified and deposited at the herbarium of Botany Department (TUBH), Tripura University, In- dia. Pollen grains for Light Microscopy were prepared fol- lowing the standard acetolysis method of Erdtman (1952).
For SEM the pollen grains are preserved in Formalin Ace- tic Alcohol (FAA) at 4ºC temperature. The pollen grains were prepared for light and scanning microscopy (SEM) by the standard methods described by Erdtman (1952).
For light microscopy, the pollen grains were mounted in glycerine-jelly and observations were made using an Olym- pus Microscope using a 10x eye piece. For SEM studies, pollen grains were first dried and then directly transferred with a fine needle to a metallic stub using double-sided adhesive tape and coated with gold in an IB2 ion coater.
The SEM examination was carried out on a S530 Hitachi Scanning Electron Microscope, Japan at Burdwan Univer- sity (USIC). The terminology used is in accordance with Erdtman (1952), Faegrie and Iversen (1964), Walker and Doyle (1975), Nair (1964).
Results
The pollen grains were mostly isopolar or heteropolar.
The shape was commonly sub-prolate, elliptic, spheroi- dal or sub-spheroidal. The pollen grains were united in monads in all cases. Apertures were monocolpate in all the studied species. However the exine sculpturing varied ex- tremely within the studied taxa.
Nelumbo nucifera Gaertner (Herb No: Bhowmik &
Datta, 431; Fig. 1, 2, 3)
Pollen grains isopolar, trilobed. Polar axis (50.61- ) 57.44 ± 3.88 (-61.62 ) µm and Equatorial diameter (48.44-) 55.24 ± 4.18 (59.42) µm. Pollen grains Sub- prolate, rounded -trilobed, tricolpate (dominant, 96.47%) and occasional monosulcate (3.53%), colpi (52.19-) 55.63
± 2.26 (-56.37) µm. Exine ( 3.52-) 3.86 ± 0.93 (-4.35 ) µm thick. Exine regulates.
Fig. 1. Flowering twig of Nelumbo nucifera Gaertner (Nelumbo- naceae)
Fig. 2. X600, showing tricolpate pollens of Nelumbo nucifera Gaertner
Fig. 3. X1500, showing monosulcate pollen of Nelumbo nucifera Gaertner
Fig. 4. Flowering twig of Nymphaea micrantha Guillemin et Per- rottet
Nymphaea micrantha Guillemin et Perrottet (Herb No: Bhowmik & Datta, 452; Fig. 4, 5, 6)
Pollen grains, heteropolar, bilateral, boat shaped.
Length (17.6-) 21.12 ± 1.76 (22.0) µm and the breadth (30.2-) 33.38 ± 2.23 (-35.25) µm. Pollen grains dimorphic, ellipsoidal (dominant, 78.96%) and spheroidal (21.04%), monocolpate, colpi (29.56-) 32.16 ± 1.27 (-33.56) µm.
Exine (1.13-) 1.56 ± 0.18 (-2.21) µm. Exine gemmate.
Nymphaea pubescens Willdenow (Herb No:
Bhowmik & Datta, 458; Fig. 7, 8, 9,10)
Pollen grains, heteropolar, bilateral, boat shaped.
Length (18.93-) 22.47 ± 1.86 (-23.65) µm and breadth (31.21-) 34.58 ± 2.36 (-35.67) µm. Pollen grains di- morphic ellipsoidal (dominant, 85.47 %) and spheroi- dal (14.53%), monocolpate, colpi (28.63-) 33.84 ± 1.38 (-34.52) µm. Exine (1.25-) 1.63 ± 0.33 (-2.27) µm. Exine striate.
Fig. 5. X4000, showing dimorphic pollen grains of Nymphaea micrantha Guillemin et Perrottet
Fig. 6. X3000, showing exine pattern of Nymphaea micrantha Guillemin et Perrottet
Fig. 7. Flowering twig of Nymphaea pubescens Willdenow
Fig. 8. X3000, showing monocolpate ellipsoidal pollens of Nymphaea pubescens Willdenow
Fig. 9. X3000, showing spheroidal pollens of Nymphaea pubes- cens Willdenow
Nymphaea rubra Roxburgh ex Andrews (Herb No:
Bhowmik & Datta, 453; Fig. 11, 12, 13)
Pollen grains heteropolar, bilateral, boat shaped.
Length (13.2-) 16.83 ± 2.04 (-17.6) µm and breadth (26.4-) 29.7 ± 1.86 (-30.8) µm. Pollen grains dimorphic ellipsoidal (dominant, 78.52%) and spheroidal (21.48%), monocolpate, colpi (24.27-) 28.36 ± 1.75 (-29.67) µm.
Exine (1.07-) 1.23 ± 0.22 (-1.93) µm. Exine fossulate.
Fig. 10. X3000, exine pattern in Nymphaea pubescens Willde- now
Fig. 11. Flowering twig of Nymphaea rubra Roxburgh ex An- drews
Fig. 12. X3000, monocolpate spheroidal pollens of Nymphaea rubra Roxburgh ex Andrews
Fig. 13. X3000, exine surface of Nymphaea rubra Roxburgh ex Andrews
Fig. 14. Flowering twig of Nymphaea stellata var. major F. Muel- ler
Nymphaea stellata var. major F. Mueller (Herb. No.
Bhowmik & Datta, 460; Fig.14, 15, 16)
Pollen grains bilateral, heteropolar, boat-shaped.
Length (25.13-) 28.07 ± 0.58 (-32.31) μm, and breadth (32.80-) 37.61±3.71 (-41.04) μm. Pollen grains dimor- phic sub-spheroidal (dominant,83.95%) and ellipsoidal (16.05%) , monocolpate, colpi (32.31-) 35.51 ± 0.89 (-39.40)μm long. Exine (2.21-) 2.96 ± 0.35 (-3.15) µm thick. Exine surface foveolate.
Nymphaea stellata F. Mueller (Herb No. Bhowmik
& Datta, 461; Fig.17, 18, 19)
Pollen grains bilateral, heteropolar, boat-shaped.
Length (25.13-) 28.07 ± 0.58 (-32.31) μm and breadth (32.80-) 37.61±3.71 (-41.04) μm. Pollen grains dimorphic ellipsoidal (78.59%) and spheroidal (21.41%), monocol- pate, colpi (32.31-) 35.51 ± 0.89 (-39.40) μm long. Exine (2.21-) 2.96 ± 0.35 (-3.15) µm thick. Exine psilate.
Fig. 15. X3000, showing monocolpate pollen with exine pattern in Nymphaea stellata var. major F. Mueller
Fig. 16. X1000, showing dimorphic pollen grains of Nymphaea stellata var. major F. Mueller
Fig. 17. Flowering twig of Nymphaea stellata F. Mueller
Fig. 18. X1000, showing dimorphic pollens of Nymphaea stel- lata F. Mueller
Fig. 19. X3000, showing exine pattern of Nymphaea stellata F.
Mueller
ies, for example examinations of percentages of aborted grains, generally considered a good indicator of hybridity, are needed to concern these assumptions. The ecological and geographical variations in pollen morphology could be an index of the genetic impact of the environment on the plant. Thus the present difference in terms of exine pattern could be useful to separate them at varietal level.
Acknowledgement
The first author is thankful to Tripura University, In- dia, for providing RET fellowship in order to carry out the research work. The first author also greets his warm regards to Dr. Srikanta Chakraborty, Senior Teacher, University Science Instrumentation Centre and Central Instrumen- tation Facility, Burdwan University for his constant coop- eration during the work.
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Discussion
Pollen morphology is presently a global accepted tool in consideration of plant taxonomy and evolution, and with the SEM providing information on finer architecture, the application of pollen in comparative morphology has be- come possible in gaining new knowledge at varietal and even ecosystem levels. Pollen grains of Nelumbo are dominantly (96.47%) tricolpate, but some monosulcate pollen grains are also reported in the present study (3.53%). Bank et al.
(2007) also reported small percentage of aberrant pollens in Nelumbo with one aperture. Kupriyanova (1976) found a small percentage of monosulcate grains among the oth- erwise largely tricolpate pollen produced by Nelumbo. She suggested that this pollen variation provided evidence of the origin of tricolpate from monosulcate pollen. In earlier knowledge, the pollen of Nymphaea is basically described as boat shaped (ellipsoidal). The presence of most primi- tive types of pollen grains viz. boat shaped , monocolpate grains clearly indicate the primitive nature of this family the dimorphic situation (ellipsoidal and spheroidal/sub- spheroidal) in all the studied taxa includes a new findings to the pollen morphology of Nymphaea. The exine surface as revealed by SEM is also varying greatly among the stud- ied five taxa. The exine surface of Nymphaea micrantha earlier reported as aerolate from Kerala, India (Ansari et al., 2005), however in the present study the gemmate ex- ine pattern is reported. Similarly Murthy (2000) reported granulate exine for Nymphaea rubra. Nymphaea pubescens and Nymphaea rubra is often treated as synonym (Cock, 1996). The different exine sculpturing between the spe- cie could useful for to treat both as different species. The exine pattern in Nymphaea stellata as observed in present study is of foveolate. Khan (1995) reported baculate exine from Nymphaea stellata. The difference in exine patterns with the earlier reports may be interpreted as reflections of genetic variations possibly due to mutational changes effected by ecological conditions. Nymphaea stellata var.
major and Nymphaea stellata could differentiate only by their flower colour and flower size. The different exine sculpturing could be useful to identify both at varietal lev- el. The variation as reported in the present study in terms of exine pattern of the studied species suggests the feasibil- ity of applying the data in the identification of the genus of Nymphaea. El-Ghazaly and Rowley (1997) showed that considerable variability existed in both pollen shape and ridge morphology within a single microsporangium. Pol- len dimorphism has been well studied in heterostylous angiosperms (Dulberger, 1975; Kohler, 1976; Ganders, 1979; Goldblatt and Manning, 1989). Variability in pollen morphology, including size variation and morphological differences, is often associated with hybrids among angio- sperm groups (Chaturvedi et al., 2000). It may be that the observed pollen dimorphism is the result of introgression of populations (Ickert-Bond et al., 2003). Further stud-
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