Biosystematic study of Phalaris L. species (Poaceae) in Iran

Introduction

The genus Phalaris L. has had a complicated taxonomic and nomenclatural history (Baldini, 1993, 1995). It comprises 22 species of annual and perennial grasses in temperate regions throughout the world. These are commonly adventives species of open habitats. There are 4 species and 5 taxa of Phalaris in Iran (Bor, 1970): P. minor Retz., P. brachystachys Link.,
P. paradoxa L. (with 2 varieties) and P. arundinacea L.. These species are distributed in various regions of Iran. They are among important forage plants. Members of the genus Phalaris display many variations on the standard structure of the inflorescence (Bor, 1968). There has been no report of systematic study on Phalaris species of Iran.

Many authors demonstrated that sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of proteins extracted from seeds produces a composite pattern for the phenotypes in the analyzed population (Yusaf et al., 2006; Sheidai et al., 2008). SDS-PAGE can be used to characterize the seed protein banding profiles of species and cultivars in several grass genera, compare the cultivars of different geographical origin and provide taxonomically useful descriptors that are substantially free from environmental influence. This procedure has provided useful data for many grasses as Lolium L. and Festuca L. complex (Aiken et al., 1992), Dactylis L. and Leucopoa Griseb. (Aiken et al., 1998). There was no report of SDS-PAGE in Phalaris species but limited variation was found out by means of Isozymes in annual Phalaris species (Hucle and Matus, 1999).

Seed protein banding profiles have proved to provide informative supplementary data for morphological features in resolving problems in the grass taxonomy. In this research, we tried to resolve Phalaris species relationship by using SDS-PAGE as supplementary data for morphology in Iran. The objective of this study was to assess the level of seed electrophoretic patterns of Phalaris taxa in Iran. We tried to reveal the degree of coincidence between morphological variations and SDS-PAGE profiles for Phalaris native to Iran for the first time.

Materials and Methods

In this study, five populations were chosen to study electrophoretic pattern of seed storage proteins. Seed samples were obtained from the sources indicated in Table 1. 1 gr of seed was used from each accession. Voucher specimens for this study were collected from the wild and all have been deposited at the Herbarium of Alzahra University, Tehran.

Table 1. Origin of seed samples of Phalaris species native to Iran

The final extract was loaded on SDS PAGE and stained by coomassic brilliant blue (Lammeli, 1970). We used Jaccard similarity coefficient.In statistical analysis, presence or absence of each band was considered as a qualitative feature. Then, the dendrogram was constructed using WARD hierarchical and UPGMA clustering by SPSS software ver. 11. In order to find the most variable protein band in studied taxa, principal component analysis was done.Standard proteins (b-galactosisase, Ovalbumin, Lactate dehydrogenase, lactoglobulin-b, Lysozyme and Bovine serum albumin) were used to evaluate the molecular weight of the unknown proteins. The protein density was determined by Bradford Protocol. Banding patterns were studied and R.F. values were measured. We used Jaccard similarity coefficient.

Results and Discussion

SDS-Page electrophoretic data were analyzed (Table 2). Jaccard similarity index was evaluated (Table 3). Totally 25 bands were observed for these taxa. The 4^th, 5^th, 6^th, 20^th and 25^th bands were common in studied taxa. While band numbers 11, 13, 14, 15, 17, 21 and 24 were only observed in P. paradoxa var. paradoxa and P. paradoxa. Band number 9 was exclusively observed in P. paradoxa var. praemorsa. Merely in P. arundinacea the band number 12 was shown. Band number 19 was found only in P. minor. All of the studied taxa had band number one but not P. brachystachys. The highest numbers of bands were observed in P. paradoxa var. paradoxa and the least one in P. brachystachys (Figure 1).

In order to find most variable protein band in the studied taxa, principal component analysis was implemented. Primitive analysis showed that three first factors were responsible for the 95% of total studied variation in taxa. In the first factor with almost 61% of the total variation, bands number 1, 7, 10, 11, 14, 15, 16, 17, 18, 21 and 24 had the highest positive correlations. Bands number 8, 12 and 22 had the highest negative correlation. In the second factor with near 20% of observed variation, band number 8, had the highest positive correlation and band number 19 had the highest negative one. In the third factor with 14.16% of total variation, bands number one to three had the highest positive correlations (Table 4).

Table 2. Seed storage protein banding profiles of seed samples of Phalaris species native to Iran (1- band is present in the seed sample, 0- band is absent in the seed sample).

Table 3. Jaccard similarity index based on electrophoretic data of seed storage protein in Phalaris taxa native to Iran

Figure 1: Seed protein banding profile of wild Phalaris species of Iran. A) P. paradoxa var. paradoxa;
B) P. paradoxa var. praemorsa; C) P. brachystachys; D) P. arundinacea; D) P. minor; M) marker.

Table 4. Factor analysis results based on SDS-PAGE electrophoretic characters for Phalaris species of Iran

Cluster analysis result is shown in WARD dendrogram (Figure 2). UPGMA dendrogram was similar to WARD one. The taxa are clearly separated based on electrophoretic data of seed storage proteins. Results revealed that two varieties of P. paradoxa as var. praemorsa and var. paradoxa were closely related. High similarity index is a reflex of genomic identity (J=0.857). Dendrogram showed close relationship and high protein similarity (J=0.583) between P. arundinacea and P. brachystachys. On the other hand, P. minor comprised a separate cluster itself. Ordination of studied taxa based on PCA (Figure 3), showed that there was a concordance with cluster analysis.

Figure 2. Dendrogram depicting clustering by WARD method of Taxa of Phalaris, by cluster analysis of seed storage protein

Figure 3. PCA ordination of the Phalaris species based on SDS-PAGE characters

Authors are convinced of existence of two centers of variation for this taxon in the whole world: Mediterranean region with 11 species and South West of USA with 4 species. There are four species of this genus in Iran, all belonging to the Old World species group (Baldini, 1993). P. minor has a vast distribution region in Irano-Touranian and Saharo-Sindian phytogeographic region (Keshavarzi et al., 2007). P. paradoxa has a high morphological variability. Morphological studies in Iran revealed that two variations of P. paradoxa (P. paradoxa var. praemorsa and P. paradoxa var. paradoxa)make a closely related group also with P. minor. P. brachystachys is related to these taxa at the level 10. P. arundinacea which is the only perennial species of these taxa in Iran which makes a separate cluster and it is far from the other species.

Phalaris species of Iran showed differences in leaf anatomical structure and leaf dorsal epidermis (Keshavarzi et al., 2009). Main differences were observed in hair type and frequency, stomata number in dorsal leaf area, stomata size and general outline of leaf cross sections. These were of diagnostic value and an identification key was made based on these features. Anatomical studies revealed that two varieties of P. paradoxa were distingusihed from each other while morphological studies (Keshavarzi et al., 2011) indicated a close relationship between these two. Main morphological diagnostic features for these varities, which are used in identification keys, are the shape of rudimentary spikelets and ligule surface. In P. paradoxa var. praemorsa rudimentary spikelets were club like and ligule surface lacked hair while P. paradoxa var. paradoxa showed no club like rudimentaries and had hairy ligule.

In the meantime, anatomical and morphological studies both confirmed the separation of the studied taxa and the present result of SDS-PAGE was in accordance with previous results although there were some differences in clustering patterns.

Electrophoretic data also confirmed a close relationship and identity between the two variations of P. paradoxa. P. arundinacea was very different from the other species of this genus morphologically, but it was located near them according SDS-PAGE data. Hucle and Matus (1999) stated that P. minor, being an auto-tetraploid taxon, is a highly variable species within this genus according results of studying its enzyme electrophoretic patterns. We found out high morphological variability in different accessions of this species as well (Keshavarzi et al., 2007). As this species is tetraploid with 2n=28 (Baldini, 1995) it is not surprising. Sometimes this variability is related to soil conditions. Rich or poor soils cause difference of morphological features of P. minor individuals. Despite high variation of this species P. brachystachys (2n=12) and P. paradoxa (2n=14) are diploids with more similar populations (Baldini, 1995). As P. arundinacea is out of selection it was supposed to show more variability. We thought that low variability could be due to insufficient sampling from localities. A vast field study and new collections from different species of Phalaris is recommended. As enzyme electrophoresis are capable presenting inter- and intra- specific variations and also some morpho-geographic subspecies could be separated.