Micromorphological variation forage crops of Loliinae subtribe (Poaceae)

Background: Loliinae subtribe (Poaceae) composed of famous temperate grasses with forage and ornamental importance worldwide. Some elements as Lolium, Festuca and Vulpia are widely used as fodder and ornamental in gardens. In present project micromorphological characters of lemmas and paleas using scanning electron microscopy for 56 taxa of 32 species of Loliinae, have been investigated. In present project micromorphological characters of lemmas and paleas using scanning electron microscopy for 56 taxa of 32 species of Loliinae, have been investigated. By evaluating these set of features in abaxial lemma and palea, we aimed to clarify the taxonomic value in this subtribe for the first time. Other purpose of present study was to assess the impact of silica bodies and epicuticular wax in species for livestock palatability. Results: Features studied including long cells length, long cells shape, periclinal wall of long cells, short cells, silica cells, prickles, prostration of crown cells thin layer of crown cells, macroharis and wax shape. Two new types of lemma and palea micromorphological traits were introduced here. Multivariate data analysis was obtained using PAST software. Conclusions: Lolium temulentum is a toxic weed and recorded as dangerous for livestock because of having increased amount of wax and silica bodies at the vegetative parts. Taxa relationships were discussed based on micromorphological characters and the results were in concordance with the previous molecular findings. Although some genera studied, were not grouped properly. Description of micromorphological features of lemma and palea in subtribe

Loliinae, as one of the largest subtribes of temperate grasses, is distributed almost worldwide (Minaya et al., 2017). Among the genera of Loliinae, some are more important and widespread as Lolium (8 sp.), Festuca (500 sp.) and Vulpia (25-30 sp.).These annual and perennial grasses are of fodder and weed importance with limited medicinal applications.
Although some of these genera are easily distinguished but some show morphological similarities and overlaps in features. The Festuca-Lolium complex can be considered as group of taxa belonging to Festuca and Lolium with closely related. Even there is hybridization between two latter genera (Lolium and Festuca) and the xFestulolium Aschers. & Graebner. is the result that can be fertile (Clatyon & Renvoize 1986, Jauhar 1993, Wipff 2002. By an inflorescene transformation from panicle to spike, Lolium is thought to be derived from Festuca (Bulińska-Radomska & Lester 1988). Species of Vulpia have been shown to hybridize with species of Festuca (Ainscough et al. 1986). Phylogenetic studies of Vulpia based on nuclear internal transcribed spacer (ITS) and chloroplast DNA (trnL-F, RFLP) showed that Vulpia and Festuca were not monophyletic (Darbyshire & Warwick 1992, Catalan et al. 2007). Torrecilla & Catalan (2002) determined two separate evolutionary lineages as fine-leaved and broadleaved in Festuca. The affinity of Lolium to the broad-leaved Festuca and that of Vulpia to the fineleaved Festuca is confirmed (Aincough et al. 1986).
Silica bodies are in fact anti-feedant agent as the grasses with high amount of silica show reduced palatability (Hodkinson 2018). Mainly silica are deposited in vegetative parts but they are also present in reproductive parts as glume, lemma and palea surface. The deposition of silicon caused enhanced strength and rigidity so the water loss via cuticle is decreased. It is especially very functional in tolerance to the lodging, fluctuation in temperature, radiation and drought stresses (Ma and Yamaji 2006).
Silicon is also found in the leaf, glume, lemma and palea margins as silicaceous trichomes. Control of temperature along drought periods and higher temperature periods with high evaporation rate is a proposed role for the silica bodies.
Epicuticular wax which was thoroughly reviewed and classified Barthlott et al. (1998), is a functional tool to overcome aridity of the environment by decreasing the water loss via epidermis surface and stomata. They pointed to 23 different types of epicuticular wax. In the Poaceae family, diketonetubules, platelets, longitudinally aggregated rodlets are mainly observed (Barthlott et al. 1998).
Epidermal micromorphology of lemma and palea has not been widely studied in the Pooideae, especially in the Loliinae. Bodoux (1971) considered Lolium multiflorum Lam., F. pratensis Hudon, Festuca arundinacea Schreber, and their hybrids, and found that two classes, based on epidermal traits in the intercostal regions, could be separated based on the shape of cell wall and the existence of cork cells. Aiken and Lefkovitch (1984) studied the Canadian rough fescue complex (F. campestris, F. hallii, Festuca altaica, F. scabrella), and Dube´ & Morisset (1996a, b) studied in micromorphology of F. rubra s.l.. Indumentum and shape of the lemma in Vulpia have been studied (Brullo et al. 2003).  Scanning electron microscopy For scanning electron microscopy (SEM), the samples were transferred directly to aluminum stubs, coated with gold under high vacuum, and examined with VEGA3 TESCAN scanning electron microscopes (SEM) at Alzahra University, Tehran, Iran. Ten micromorphological characters (nine Qualitative and one quantitative) were examined for both lemma and palea for separation of taxa (Table 2). The quantitative character (length of the long cells) was measured too. Table 2 Lemma and palea micromorphological qualitative characters and character states in subtribe Loliinae No.

Statistical analysis
In total, two quantitative and 18 qualitative micromorphological features of both lemma and palea surfaces were performed for multivariate analysis containing ( information about general relationships and similarities between them. The data were standardized (mean = 0, variance = 1) for these analyses. Euclidean and taxonomic distance are used among the species (Podani 2000).

Numerical analyses
The numerical analysis of micromorphological qualitative and quantitative features by use of clustering (UPGMA) and ordination (PCA) methods were produced ( Figs. 1 and 2). These analyses of the micromorphological characters studied did not support the genera classification and separation of subtribe Loliinae. The genera of subtribe Loliinae were approximately intermixed and they did not delimit properly. Therefore, the genera have high affinity by micromorphological characters. UPGMA tree of micromorphological features revealed that species of the Lolium separated from other genera and were placed close to Festuca. Festuca tuberclosa was placed close to Festuca sulcata. The species of the Vulpia and Loliolum approximately separated and showed high affinity to Festuca.
Festololium-loliaceum taxa were grouped with Festuca and Lolium species.

Micromorphological variation of the lemma and palea epidermis
Description of micromorphological features of lemma and palea in subtribe Loliinae

Lemma
The lemma surface was homogeneous and showed epicuticular waxes, with various types and densities. The genera studied illustrated six types of waxes that have been observed in various genera of the subtribe Loliinae (Table 2). Lolium temulentum had a high density of wax at the lemma and pales surface.
Two different types of short cells were observed. In first form short cells were filled by suberin (shot cells) and in the second type by silicon (silica bodies).

Discussion
In the subtribe Loliinae, the lemma and palea micromorphological features have shown to be of taxonomic significance. The lemma and palea epidermal characteristics have demonstrated to be to be taxonomically important and useful. The micromorphological traits among examined taxa, were useful for taxa identification. Results of the UPGMA tree and PCA plot based on the lemma and palea features, revealed that the genera of the subtribe Loliinae were placed close to each other (Figs. 1 and 2).
Results of the UPGMA clustering and PCA plot based on the characteristic of lemma and palea abaxial surface, showed that the clusters of Lolium and Festuca species were closely arranged due to the shape of silica bodies in palea surface. A controversy in taxonomy relationships between Lolium and Festuca species have long been remained (Chehrazi et al. 2014).
In these analyses, the clusters of Festuca, Loliolum, Psilurus, Vulpia and Leucopoa were closely related and their species were not separated efficiently. Soreng et al. (2017) proposed that three genera as Loliolum, Psilurus and Vulpia, should be considered as synonyms of Festuca according to molecular data. Findings of present study are in concordance with Soreng et al. (2017).
Castellia tuberclosa was considered as a separate taxon (Soreng et al. 2017) but our findings support the close relationship of this taxon to the Festuca body (Festuca tuberclosa).
Lolium species were separated from other genera and placed close to the Festuca ones. Same result was achieved by phylogenetic classification using matK and ndhF plastid DNA markers (Soreng et al. 2017). xFestulolium taxa were placed among Lolium and Festuca species. Also, introgression is possible between these (Wipff 2002).
Lolium temulentum and L. persicum (in-breeding species) were placed closed to each other while the cross-breeding species composed separate clusters based on micromorphological characters. Lolium loliaceum is considered by Terell (1968) as synonym of Lolium rigidum, but in Flora of Iraq and Flora Iranica (Bor 1968, Bor 1970) is reported as a separate species. Although it is now considered as a synonyme for L. rigidum but in accessions studied, L. loliacum and L. rigidum were composed separate clusters probably because of different shape of long cells, protrusion of crown cells and thin layer of crown cells in lemma; shape of long cells, anticlinal walls of long cells, short cells, protrusion of crown cells and thin layer of crown cells in palea.
A close relationship was found among the three cross-breeding species (L. rigidum, L. perenne, and L. multiflorum). Although these species are three separate species, however, nowadays it is evident that, these are evolutionary related and show a recent divergence (Abbas et al. 2006).

Species of Festuca have been separated to broad-leaved and fine leaved fescues by vegetative and
reproductive features (Kar et al. 2019). At the subgeneric level, Festuca sulcata and F. rubra (Fineleaved) belonged to the Festuca subgenus, grouped as a common main cluster (Fig. 1)  Based on present findings, species of Vulpia were placed in fine-leaved Festuca species cluster.
Results showed that Lolium temulentum which is toxic weed, had a high mass of wax and silica bodies at the lemma and palea surface, this makes it dangerous for livestock. Also, some species such as L.
incurvus had a high density of silica bodies at the reproductive parts. But in other species, a low density of silica bodies observed increased their palatability for livestock. It is believed that in the grass family these are antifeedant agents (Massey & Hartley 2006, 2009).
Silicon-rich species as Lolium perenne and Festuca ovina were significantly less consumed by locusts than species with low silica content (Massey & Hartley 2009). Hodkinson (2018) reported that silica production in grasses could be an effect of grazing pressure. Herbivores avoid using rough leaves with the high density of silica owing to the harms of feeding. In the Poaceae, reproductive parts (lemma and palea) showed different shape and density in epicuticular waxes. There are other defensive roles for the epicuticular wax in insect invasion, bacterial influence, ultraviolet radiation and frost (Yang et al. 2015 Conclusions In summary, micromorphological features of lemma and palea surfaces were studied using SEM; revealed that the genera of Loliinae subtribe were not separated properly based on these features. At the species level of inbreeder Lolium (L. persicum and L. temulentum) were placed far from crossbreeder ones (L. perenne , L. multiflorum and L. rigidum). Broad-leaved species of Festuca have been separated from fine leaved fescues. Also, Loliolum, Psilurus, Vulpia and Leucopoa were closely related.
The existence of silica and wax in different parts as lemma and palea, was important to adapt to harsh conditions such as endurance to the habitat, alteration in temperature, drought stresses and feeding deterrent to herbivores.

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