Materials and methods

Species sampled. The fruit and seed samples used in this study were collected between 2015 and 2019 from 15 localities in Turkey. The collection data is given in Table 1. The plant specimens of the studied species were housed in the herbarium of Department of Biology, University of Selcuk (KNYA).

Microscopic investigations. The macro- and micro characters of at least 20 or more fruits and seeds per taxon were analysed using a stereo microscope. For the SEM mature and dry fruits and seeds were placed on the double sided tape and then sputter coated with gold for five minutes, and observed under a Zeiss Evo LS10 SEM (Carl Zeiss Microscopy GmbH, Jena, Germany).

Terminology. The fruit and seed terminology used here is adapted from ^{Koch et al. (2009)}, ^{Pinar et al. (2009)}, ^{Mühlhausen et al. (2013)} and ^{Ghaempanah et al. (2013)}.

Statistical analysis. The qualitative and quantitative characters were coded for the numerical analysis to evaluate the similarity relationships of the Turkish Iberis species. Characters are listed in Table 2. For the multivariate analysis, a primary matrix was created for the eight taxa using twelve characters. The clustering analysis was based on ^{Gower’s (1971)} general coefficient similarity, which can be used directly with a mixture of character types (binary, qualitative, and quantitative characters). The UPGMA was selected because it produces an accurate reflection similarity matrix, as measured by the cophenetic correlation coefficient of ^{Sokal & Rohlf (1962)} and symmetrical hierarchical structure (^{Sokal & Rohlf 1962}, ^{McNeill 1979}), and has congruence with the classification derived by traditional methods (^{Ward 1993}). Untransformed, not centered and standardised data were used to create a covariance matrix (^{Baldemir et al. 2018}). MVSP 3.22 software was used for all of the computations.

Results

Fruit micromorphology. The fruit and seed exomorphological characters (colour, shape, size, and surface) were evaluated. The fruit colours were observed as green, purple and yellow (Figure 1, Table 2). The fruit shapes were ovate and ovate-orbicular without wing in examined species. The end of the wing varied from acute to obtuse. The greatest wing width was observed in I. odorata (Figure 1 G), while the least was in I. simplex (Figure 1 F). The smallest fruit was observed in I. saxatilis subsp. saxatilis (mean value: 4.69 mm; Figure 1 C) and the greatest was in I. sempervirens (mean value: 8.03 mm; Figure 1A, Table 2). Fruit surface are determined as striated and smooth among the examined species (Table 2, Figures 2, 3). The fruit of I. halophila, I. carnosa and I. carica have a distinct striated ornamentation (Figures 2 B, E-H). The remaining species have smooth ornamentation (Figures 2 A, C, D, F, G).

Seed micromophology. With regards to the shapes of the seeds of the Iberis species, four types were determined: orbicular, broadly ovate, ovate, and elliptic (Table 2). Orbicular is characteristic only in I. sempervirens (Figure 3 A) and ovate only in I. saxatilis subsp. saxatilis (Figure 3 C). I. halophila, I. saxatilis subsp. magnesiana, I. carnosa and I. simplex have broadly-ovate seeds. Otherwise, I. carica and I. odorata have elliptic shaped seeds. The seed size is ranged from 2.08 mm to 3.69 mm in length and 1.37 mm to 2.52 mm in width (Table 2). The biggest seeds were observed in I. sempervirens, while the smallest were observed in I. carica (Figure 3 H). The seed colour was yellow in I. saxatilis subsp. magnesiana (Figure 3 D) and I. carica. Otherwise, I. saxatilis subsp. saxatilis, I. simplex and I. odorata have light brown-yellow coloured seeds. Additionally, I. sempervirens has light brown and I. carnosa has brownish-black coloured seeds and I. halophila brown seeds. The surfaces of the Iberis species were determined as reticulate-papillate in I. simplex (Figure 4 K, L), reticulate-rugose in I. odorata (Figure 4 M, N) and reticulate in remaining species (Table 2). Epidermal cell varied among the species, as tetragonal-elongated in I. halophila (Figure 4 D), I. saxatilis subsp. saxatilis (Figure 4 F), I. carica (Figure 4 P), tetragonal-polygonal in I. simplex (Figure 4 L), tetragonal and polygonal in I. carnosa (Figure 4 J) and I. odorata (Figure 4 N), tetragonal-elongated and polygonal in I. saxatilis subsp. magnesiana (Figure 4 H), tetragonal in I. sempervirens (Figure 4 B).The anticlinal cell walls were determined as distinct, straight and exposed in I. halophila, I. saxatilis subsp. saxatilis, I. carnosa, and I. carica (Figure 4 D, F, I, P), distinct, straight and sunken in I. saxatilis subsp. magnesiana, I. simplex, and I. odorata (Figure 4 H, L, M) and not distinct in I. sempervirens (Figure 4 A). I. saxatilis subsp. magnesiana had a wax plate that differ from other examined Iberis species (Figure 4 H).The outer periclinal cell walls were observed as convex in I. halophila, I. saxatilis subsp. magnesiana, I. simplex, and I. odorata, concave in I. saxatilis subsp. saxatilis, I. carnosa I. carica and I. sempervirens.

Numerical analysis of the fruit and seed morphology. The dendrogram is presented in Figure 5. This dendrogram revealed two main groups plus I. sempervirens. Group A comprised five taxa with 71 % similarity and Group B included I. carica and I. simplex with 78 % similarity. Notably, I. saxatilis subsp. saxatilis and I. saxatilis subsp. magnesiana belong to different subgroups. I. saxatilis subsp. magnesiana and I. carnosa had the highest value of similarity (87 % similarity).

Key to species on the basis of fruit and seed morphology

Discussion

This study examined the utility of using the fruit and seed micromorphology of Iberis species in Turkey. The sculpturing of surfaces has been used for problems in taxonomy to observe relationships of species in Brassicaceae (^{Khalık & van der Maesen 2002}, ^{Khalik et al. 2002}, ^{Pinar et al. 2007}, ^{Atçeken et al. 2016}, ^{Karaismailoğlu & Erol 2018}, ^{Karaismailoğlu 2019}, ^{Gönen et al. 2019}). The outcomes showed that the seed micromorphology was more useful than fruit micromorphology to separate Iberis taxa in Turkey.

The fruit morphology of the Iberis species investigated included characters believed essential for species level distinction. ^{Gabr (2018)} had a detailed study of the fruit micromorphology in the Brassicaceae family. He stated that the fruit characters such as fruit shape, surface, and beak traits separated the family into tribes and groups. In Iberis, the fruit size and colour were good characters (Table 2). The largest fruits were determined in I. sempervirens. The fruit colour of I. halophila, I. saxatilis subsp. saxatilis, I. saxatilis subsp. magnesiana, I. carnosa and I. odorata was observed as purple-green. The fruit colour of I. sempervirens, I. simplex and I. carica was observed as green-yellow. When considering the surface ornamentation of the fruit, two surface sculptures were determined, smooth and striated. Three species, I. halophila, I. carnosa and I. odorata, had striated ornamentation on their fruit surface, while the remaining species had smooth surface sculpturing. The fruit morphological characteristics just mentioned were found to be valuable to distinguish among species. These characters are presented here for the first time for the taxa of Iberis of Turkey and were important to construct the identification key.

The macro- and micromorphology of the seeds were determined as the most distinctive characters for distinguishing the eight Iberis taxa. The seed colour and size, and ornamentation provided great potential for distinguishing seed morphological variations at species level as in previous studies (^{Barthlott 1984}, ^{Khalik & van der Maesen 2002}, ^{Pinar et al. 2007}, ²⁰⁰⁹, ^{Ghaempanah et al. 2013}, ^{Atçeken et al. 2016}, ^{Karaismailoğlu & Erol 2018}, ^{Karaismailoğlu 2019}, ^{Gönen et al. 2019)}. In the current study, the light brown-yellow seed colour in I. saxatilis subsp. saxatilis, I. simplex and I. odorata, light brown in I. sempervirens, brown in I. halophila, brown-black in I. carnosa, yellow in I. saxatilis subsp. magnesiana, and I. carica were observed (Table 2). These seed colour variation was described as a new character for Iberis taxa in the present study.

The features of the seed coat, such as surface sculpturing, anticlinal and periclinal wall cells, and epidermal cell shape have been determined as useful in the delimitation of taxa within some genera in the family Brassicaceae (^{Moazzeni et al. 2007}, ^{Atçeken et al. 2016}, ^{Gabr 2018}, ^{Karaismailoğlu & Erol 2018}, ^{Karaismailoğlu 2019}, ^{Gönen et al. 2019}). In this research, the seed coat ornamentation, which was reticulate, reticulate-rugose and reticulate-papillate can be considered essential diagnostic features at species level. For example, reticulate-rugose seed coat is distinctive in I. odorata, while reticulate-papillate is unique in I. simplex. In Brassicaceae, reticulate surface ornamentation is the most common character at the generic level (^{Zeng et al. 2004}, ^{Moazzeni et al. 2007}, ^{Atçeken et al. 2016}, ^{Karaismailoğlu & Erol 2018}, ^{Karaismailoğlu 2019}, ^{Gönen et al. 2019}) as it was confirmed since six of the eight taxa studied show reticulate seed coat. In taxonomy, epidermal cell shape on the seed surface is important at generic and also subgeneric level in the family Brassicaceae (^{Karaismailoğlu 2019}). These seed characteristics showed great variation in the Iberis species examined. In the examined species, the epidermal cells were tetragonal-elongated with anticlines distinct, straight and exposed in I. halophila, I. saxatilis subsp. saxatilis and I. carica; tetragonal-polygonal in I. carnosa and I. odorata; tetragonal and polygonal in I. simplex; tetragonal with anticlines nondistinct in I. sempervirens; and tetragonal-elongated and polygonal with anticlines distinct, straight and sunken in I. saxatilis subsp. magnesiana. With this study, we confirmed that the epidermal traits of the seed could be a good taxonomic character to separate Iberis species.

The results from cluster analysis show that the examined species of Iberis that fall into two main groups coincide with fruit and seed morphology (Figure 5). According to UPGMA analysis based on fruit and seed morphological data, each species was distinctly separated from each other. Iberis saxatilis subsp. saxatilis and I. saxatilis subsp. magnesiana are in different groups. The two subspecies can be easily separated from each other by the retrorsely setulose stem indumentum in I. saxatilis subsp. magnesiana, while the stem of I. saxatilis subsp. saxatilis is glabrous and now several seed features of the anticlines and the outer periclinal walls also support their recognition. Additionally, Iberis sempervirens is a semi-shrub plant that has no close relatives in Turkish Iberis species and has the most distinct fruits and seeds of the species studied. These results were congruent with the palynomorphological analysis of Turkish Iberis species by ^{Citak (2019)}. Seed length and width, colour of seed, fruit colour, and seed ornamentation are the most valuable variables for separating Iberis species. In further studies we predict that the systematic problems of the genus Iberis will be solved by providing anatomical, morphological and more molecular studies.