Pollen morphological analysis of the genus Lallemantia ( Lamiaceae ) of Iran

Lallemantia is a small genus of Lamiaceae with five herbaceous annual or biennial species. Some of these species are of medicinal value, while others have been used as the source of food. All five Lallemantia species occur in Iran. Although, there are many discussions about infra-generic variations in the genus, very scarce comprehensive studies are found about its taxonomy. Therefore, in the current study, pollen grain morphology was investigated using scanning electron microscopy. We used SPSS ver. 15 and PAST ver. 2.17 for multivariate statistical analyses of data. In total, we studied eleven qualitative and quantitative traits. Pollen equatorial and polar views were similar among the species and were prolate and circular, respectively. Pollen ornamentations differed as bireticulate (L. royleana and L. iberica), microreticulatm (L. peltata and L. Baldshuanica) and reticulatefaveolate (L. canescens). PCA-biplot showed some traits such as pori length/width ratio were important in identifications of species. The studied species created two groups in the WARD method tree of palynological data. L. royleana and L. baldshuanica were grouped together, and in the other group L. canescens and L. iberica placed nearly and L. peltata placed far from them. Our obtained results were similar to previous morphological study of the genus. The present study revealed taxonomic implication of the micromorphological study in the interspecific classification of the genus.


Introduction
Lallemantia Fisch.& C. A. Mey. is a genus of subfamily Nepetoideae, tribe Mentheae of family Lamiaceae.It is a small genus with 5 species that are herbaceous annual or biennial plants.These species characterized by simple leaves; a thyrsoid, spike-like or oblong, often interrupted inflorescence; ovate to rotund or sometimes linear, aristate-toothed bracteoles; and oblong, trigonous, smooth and mucilaginous nutlets (Harley et al., 2004).
Lallemantia species are widely distributed in different parts of the world such as Afghanistan, China, India, Kazakhstan, Kyrgyzstan, Pakistan, Iran, Russia, Tajikistan, Turkmenistan, Uzbekistan, SW Asia and Europe, with the Caucasian region as the center of its origin.All five Lallemantia species occur in Iran (Rechinger, 1982).
Some of Lallemantia species are of medicinal value (Mahmood et al., 2013), while others have been used as the source of food (Rivera Nunez, 1992).For example, Lallemantia royleana (Benth. in Wall.)Benth., is commonly called as Balangu, Tukhm-e-balanga or Balangu-e-shirazi, is an important medicinal plant in Iranian folk medicine.In addition, this aromatic herb is cultivated in various parts of the world such as North India for the sake of its highly medicinal and mucilaginous seeds that are widely used as remedial agents in different disorders.Different studies (Naghibi et al., 2005;Razavi, Karazhiyan, 2009;Razavi, Moghaddam, 2011) showed that nutlets of these plants are more common part of traditional medicine, although there are very limited data about its nutritive components.It seems that seed contains carbohydrates, oil, fiber, tannins and protein.
Several studies have been conducted using light microscopy and scanning electron microscopy in order to describe pollen grain features and to examine the available classification of Lamiaceae family (Abu-Asab, Cantino, 1994;Oybak-Dönmez et al., 1999;Dinç, Öztürk, 2008).Kamrani and Riahi (2017), examined phylogenetic relationships among the Lallemantia species and its close allies (Lamiaceae, Mentheae) using nuclear (ITS) and plastid (trnL, trnL/F, trnS/G, rpl32, and rpl32-trnL) DNA sequences.Their findings from bayesian and parsimony analyses proved that Lallemantia is monophyletic.Erdtman (1945) based on different palynological variables, such as aperture number and number of nuclei in shed pollen, divided the Lamiaceae family into two main subfamilies: Lamioideae and Nepetoideae.In Lallemantia the pollen grains are generally symmetrical, isopolar, zonohexacolpate and prolate to prolate-spheroidal in shape (Dinç et al., 2009).
Very scares studies are found about the infrageneric classification of the genus in the world and most of them were not comprehensive.Because, all five species of the genus are only found in Iran and in the other countries we have up to three species.For example, Dinç et al. (2009) could differentiate three species, namely Lallemantia iberica, L. canescens, and L. peltata occurring in Turkey by using micro-morphological features related to the pollen, nutlet and trichome.
At yet, we have no report on pollen grain morphology of the genus in Iran.Therefore the aims of the present study are to describe pollen grains characters in five species of Lallemantia and evaluate the taxonomic importance of palynological data as well as species relationship in this genus.Furthermore, pollen morphological traits of L. royleana and L. baldshuanica Gontsch.were described by the first time for the world.

Plant materials
Extensive field visits and collections were undertaken during 2014-2015.We selected one population of each species, and per each population five flowering stems (in total 25 plant specimens) were randomly collected and used for pollen grain morphological studies.The plant samples were identified based on the descriptions provided in Flora Iranica (Rechinger, 1982) and Flora of Iran (Jamzad, 2012).The voucher specimens are deposited in the herbarium of Shahid Beheshti University (HSBU) (Table1and Fig. 1).In total, we examined eleven qualitative and quantitative palynological variables.The studied palynological features were: polar and equatorial axes view, exine ornamentation type, polar axis length (P), equatorial axis length (E), polar / equatorial (P/E) length ratio, mesocolpium length (LC), pori length, pori width, apocolpium length and width.

Samples preparation
For SEM micrographs, pollen grain samples mounted on the stubs using double-sided adhesive tape and coated with gold in spotter coater.The specimens were examined and photographed using a Phillips × L20 Scanning Electron Microscopy.From each species twenty intact pollen grains were selected.UTHSCSA Image Tool Version 3.0 was used to carry out required measurements.

Statistical descriptions of the quantitative and qualitative palynological features of Lallemantia species growing in
Iran are given in Table 2. Pollen terminology follows Faegri andIversen (1989) andPunt et al. (1994).

Data analyses
The quantitative palynological variables were first standardized (Mean = 0, Variance = 1) and used to the establish Euclidean distance among pairs of species.For grouping of the plant species, the WARD (Minimum spherical characters) tree with PAST program was used.PCA (Principal components analysis) biplot was used to identify the most variable characters among the studied species (Podani, 2000).PAST version 2.17 and SPSS ver. 15 (Hammer et al. 2012) were used for statistical analyses of data.

Results
The pollen grains in all studied species were monad, isopolar, symmetrical, zonohexacolpate and prolate in shape.Selected electron micrographs of pollen grains were presented in Fig. 2.
The polar and equatorial views were similar in all species and were circular and prolate, respectively.However, the pollen ornamentations differed among the species and were observed as bireticulate (L.royleana and L. iberica), microreticulate (L.peltata and L. baldschuanica) and reticulate-faveolate (L.canescens).
Significant positive correlations were found between some quantitative palynological variables.For example, polar axis length had a significant positive correlation (p ≤ 0.05) with pori length (r =0.88) and pori width (r= 0.89).A significant positive correlation (p ≤ 0.05, r= 0.88) was recorded between mesocolpium length with equatorial axis length.Apocolpium length significantly correlated (p ≤ 0.05, r = 0.88) with apocolpium width.

Table 2 .
Details of pollen morphology in Lallemantia species (all values are in µm).