Morphology of pollen grains of Weigela floribunda ( Caprifoliaceae ) : application in taxonomy

The aim of this research was to provide detailed quantitative and qualitative characteristics on pollen grains of Weigela floribunda (Siebold & Zucc.) K. Koch (Caprifoliaceae) as applied for taxonomy. Pollen morphology of W. floribunda was studied using both light microscopy and scanning electron microscopy. Pollen grains are 3-porate, rarely 4-porate, suboblate to spheroidal (P/E=0.80–1.05); in equatorial view elliptic or circular, in polar view circular or subcircular; mediumor largesized (P=42.56–61.18 μm, E=42.56–66.50 μm). Pores are circular with distinct or indistinct margins and distinct annuli. Exine sculpture is echinatemicroechinate-nanoechinate. Pollen grains of W. floribunda were analyzed for the first time in the present study.

It is well known that morphological characters of pollen grains as additional diagnostic features are often efficiently used for taxonomy (Jacobs et al., 2011;Mosyakin, Tsymbalyuk, 2015, 2017Tsymbalyuk et al., 2018Tsymbalyuk et al., , 2019aTsymbalyuk et al., , b, 2020Tsymbalyuk et al., , 2021Dirmenci et al., 2019, etc.). However, the available information about pollen grains of representatives of the genus Weigela is limited. Donoghue (1985) investigated pollen grains of W. florida (Bunge) A. DC. using both light microscopy and scanning electron microscopy. Jacobs et al. (2011) investigated pollen grains of three species (W. coraeensis Thunb., W. decora (Nakai) Nakai, W. Maximowiczii (S. Moore) Rehder of the genus Weigela using scanning electron microscopy; however, the photomicrographs are absent in that publication. The PalDat online database contains some information on pollen grains and photomicrographs of W. floribunda using light microscopy (Auer, 2021).
The aim of the present research was to provide detailed quantitative and qualitative characteristics of the pollen grains of W. floribunda and to evaluate the importance of these data for taxonomy.
Materials and methods. Pollen grains of W. floribunda were sampled at the National Herbarium of Ukraine (KW -Herbarium of the M. G. Kholodny Institute of Botany, National Academy of Sciences of Ukraine, Kyiv, Ukraine). Herbarium acronyms are given following Index Herbariorum (Thiers, 2008-onward, continuously updated). Abbreviations of names of taxon authors mainly follow Brummitt, Powell (1992), with additions and updates available from IPNI (2021).
In general, the methods used in the present study were the same as those we used earlier (Tsymbalyuk et al., 2020(Tsymbalyuk et al., , 2021. Pollen grains were studied using both light microscopy (LM) and scanning electron microscopy (SEM). For LM studies (Biolar, ×700), the pollen was acetolyzed following the method by Erdtman (1952), mounted on slides with glycerinated gelatin, and analyzed and photomicrographed using a light microscope. For size determinations, 30 measurements were taken along the polar axis (P) and equatorial diameter (E); 20 measurements of the exine and annulus thickness, width and length of pores were performed. The P/E ratio was calculated in order to determine the pollen shape. For all quantitative characters, descriptive statistics was applied and the range (minimum and maximum values), arithmetic mean and standard deviation were calculated. The number of echini/microechini/nanoechini per unit area (100 µm²) was determined. The slides are deposited in the Palynotheca (reference pollen collection) at the National Herbarium of Ukraine (Bezusko, Tsymbalyuk, 2011).
For SEM (JEOL JSM-6060LA), dry pollen grains were treated with 96%-ethanol, then samples were sputtercoated with gold and investigated at the Center of Electron Microscopy of the M. G. Kholodny Institute of Botany. The measurements of the echini, microechini and nanoechini were taken on five pollen grains from SEM micrographs and made using the program AxioVision Rel. 4.8.2. Terminology used in descriptions of pollen grains follows the glossaries by Punt et al. (2007) and Halbritter et al. (2018).
Results and discussion. The original data on quantitative pollen characters are summarized in Table. LM and SEM photomicrographs of pollen grains are shown in Figures 1 and 2. Description of pollen grains of W. floribunda.

SEM.
Exine sculpture echinate-microechinate-nanoechinate. Echini cylindrical, 1.32-2.64 µm high, 0.96-1.62 µm wide at base, with straight sides and acute apices, echini higher than basal width, (2-5/100 µm²). Echini with convex sides, acute or obtuse apices, 1.02-1.61 µm high, 0.95-1.48 µm wide at base, echini higher than basal width, (2-3/100 µm²); microechini with convex sides and obtuse apices, 0.76-0.82 µm high, 0.74-0.98 µm wide at base, about as broad as height (3-5/100 µm²); nanoechini with convex sides and obtuse apices, 0.38-0.59 µm high, 0.67-0.81 µm wide at base, broader than high (1-5/100 µm²); tectum psilate-granulate in area between echini. Pore membranes granulate.  The obtained data demonstrated that pollen grains of W. floribunda have a porate type of apertures. A characteristic feature of pollen grains of this species is also a very thin exine undifferentiated into layers, as well as the absence of columellae. The exine sculpture is represented by echini of two types: (1) echini with a cylindrical, parallel-sided basal part with a conical acute apex; the basal part is normally longer than the apex, height of echini exceeding their basal width; and (2) echini with a more or less circular basal part, with an obtuse or rarely acute apex; height of echini exceeding their basal width. Microechini about as broad as high, nanoechini broader than high. The pore type of apertures and echinate sculpture are characteristic of pollen grains of the genera Knautia L., Dipsacus L., Cephalaria Schrad. (Tsymbalyuk et al., 2018(Tsymbalyuk et al., , 2019(Tsymbalyuk et al., a, b, 2020. However, pollen grains of W. floribunda are morphologically different from pollen grains of other studied species. The pollen characteristics described here may be used in future studies aiming at completing the knowledge on all Weigela species and at understanding the evolution of pollen morphology in Caprifoliaceae in general.