Taxonomic Revisiting and Phylogenetic Placement of Two Endangered Plant Species: Silene leucophylla Boiss. and Silene schimperiana Boiss. (Caryophyllaceae)

The genus Silene L. is one of the largest genera in Caryophyllaceae, and is distributed in the Northern Hemisphere and South America. The endemic species Silene leucophylla and the near-endemic S. schimperiana are native to the Sinai Peninsula, Egypt. They have reduced population size and are endangered on national and international scales. These two species have typically been disregarded in most studies of the genus Silene. This research integrates the Scanning Electron Microscope (SEM), species micromorphology, and the phylogenetic analysis of four DNA markers: ITS, matK, rbcL and psb-A/trn-H. Trichomes were observed on the stem of Silene leucophylla, while the S. schimperiana has a glabrous stem. Irregular epicuticle platelets with sinuate margin were found in S. schimperiana. Oblong, bone-shaped, and irregularly arranged epidermal cells were present on the leaf of S. leucophylla, while Silene schimperiana leaf has “tetra-, penta-, hexa-, and polygonal” epidermal cells. Silene leucophylla and S. schimperiana have amphistomatic stomata. The Bayesian phylogenetic analysis of each marker individually or in combination represented the first phylogenetic study to reveal the generic and sectional classification of S. leucophylla and S. schimperiana. Two Silene complexes are proposed based on morphological and phylogenetic data. The Leucophylla complex was allied to section Siphonomorpha and the Schimperiana complex was related to section Sclerocalycinae. However, these two complexes need further investigation and more exhaustive sampling to infer their complex phylogenetic relationships.


Introduction
Caryophyllaceae contain 70-86 genera and 2200 species, which are distributed all over the world [1]. The family is divided into four subfamilies: Alsinoideae, Caryophylloideae, Paronychioideae, and Polycarpoideae "Polycarpaoideae" [2]. Within the Caryophylloideae, the tribe Sileneae DC. is regarded as the largest tribe in the family [3].
Silene L. is one of the largest genera in Caryophyllaceae, with about 850 species, distributed throughout Eurasia, from temperate regions of the Mediterranean basin to central and western Asia [4,5]. The genus Silene L. is divided into three subgenera, Lychnis (L.), Behenantha (Otth) Torr. & A. Gray, and Silene (Rohrbach) [6], as well as 34 sections, based on morphological and phylogenetic analyses [4].
Egypt's Sinai Peninsula is a central area between Africa and Asia with a unique ecosystem [7,8]. South Sinai is home to 14 endemic and threatened plant species [5]. Twenty-nine Silene taxa are native and well recognized in Egypt; two are endemic, Silene leucophylla Boiss. and S. oreosinaica Chowdhuri, while Silene schimperiana Boiss. is nearendemic [9][10][11].
Silene leucophylla and S. schimperiana are two perennial hemicryptophytes. They are isolated at high elevations (1775-2099 m) and grow in the Saint Katherine Protectorate Mountains' rocky habitats in Southern Sinai.
Silene leucophylla Boiss. and S. schimperiana are accepted names. These names were derived from World Checklist of Selected Plant Families (WCSP). No synonyms are recorded for either taxa [12]. Silene leucophylla is a critically endangered species with a reduced population size, i.e., 109 individuals [13], whereas, S. schimperiana has been evaluated as endangered at a national scale [14]. The relatively small populations are susceptible to threats such as overgrazing and environmental changes [15]. The chromosome count and karyotype study was performed for S. schimperiana, it was (2n = 2x = 24) [16]. Therefore, the wild population of Silene species could be under severe ecological pressures that would lead to their extinction [14].
Rohrbach [6] classified S. leucophylla and S. schimperiana in the same section, "Botryosilene". While he considered S. leucophylla in the Nutantes series, S. schimperiana was classified by him into series Sclerocalycinae. Classification of the genus Silene by [10,11] considered S. leucophylla a member of section Siphonomorpha, while S. schimperiana was allied to section Sclerocalycinae (Subsection Chlorifoliae).
The infraspecific classification of the genus Silene based on morphological description showed that Silene leucophylla and S. schimperiana allied to subgenus Silene sections Siphonomorpha Otth and section Sclerocalycinae Boiss., respectively [9][10][11]. A table of morphological differences between Silene leucophylla and Silene schimperiana and the nearest elements of the genus Silene is represented in the Supplementary (Table S1).
Phylogenetic analysis is essential for explaining structural, ecological, taxonomical, and functional biodiversity characteristics in an evolutionary background [26,27]. The critical species Silene leucophylla and S. schimperiana have been disregarded in recent phylogenetic and taxonomic studies of the genus Silene [4,28].
The present study explores the micromorphological characterization and molecular phylogeny of S. leucophylla and S. schimperiana, revealing the phylogenetic placement of these species and helping to resolve sectional classification within the whole genus.
The type of stomatal complex was anomocytic in both species (Figure 1). Stem qualitative and quantitative traits are summarized in Table 1 and Supplementary (Table S2). surface. A thin layer of epicuticular wax was found in S. leucophylla. Simultaneously, S. schimperiana was covered with irregular crustose platelets of <1 µm height, with sinuate margin.
The type of stomatal complex was anomocytic in both species (Figure 1). Stem qualitative and quantitative traits are summarized in Table 1 and Supplementary (Table S2).  Geom-boxplot and ANOVA indicate a significant variation of stomatal traits among the two species with p-value (p = 0.0054, R-squared = 0.9487). Higher median stem stomata of S. leucophylla than S. schimperiana was also recorded in ( Figure 2).

Leaf Epidermal Cells
The leaf epidermal cells of the adaxial (AD) and abaxial (AB) surface of S. leucophylla were oblong, bone-shaped, and irregularly arranged. On the other hand, in S. schimperi-

Leaf Epidermal Cells
The leaf epidermal cells of the adaxial (AD) and abaxial (AB) surface of S. leucophylla were oblong, bone-shaped, and irregularly arranged. On the other hand, in S. schimperiana, the AD and AB surfaces' epidermal cells were "tetra-, penta-, hexa-, and polygonal" parallelly arranged (Figure 3).  The anticlinal walls (AW) were sunken, irregularly channeled and curved in S. leucophylla. At the same time, it was sunken but straight in S. schimperiana. The fine relief of the epidermal cell wall was highly ribbed on the A.B. surface and moderately ribbed on the AD surface in S. leucophylla. In contrast, it was covered by irregular epicuticular crustose platelets in S. schimperiana ( Figure 3). Significant variations in the size of leaf epidermal cells were recorded by ANOVA analysis for the two species (p < 2.2 × 10 −16 , R-squared = 0.9708). The smallest stomatal area was recorded on the AB surface of S. leucophylla (46.44-74.64 = 61.42 ± 8.56 µm 2 ), whereas the largest area was recorded on the AD surface of S. schimperiana (102.20-253.50 = 152.30 ± 37.92 µm 2 ). Those measurements were confirmed by grouped boxplot for abaxial and adaxial leaf ( Figure 4).

Stomatal Complex
Leaves of the studied Silene taxa were amphistomatic. They have raised diacytic stomata with smooth guard cells observed in S. leucophylla, while S. schimperiana attained sunken diacytic stomata with irregular epicuticular crustose platelets on guard cells (

Stomatal Complex
Leaves of the studied Silene taxa were amphistomatic. They have raised diacytic stomata with smooth guard cells observed in S. leucophylla, while S. schimperiana attained sunken diacytic stomata with irregular epicuticular crustose platelets on guard cells ( Figure 3).
ANOVA of the length, width and area for the stomatal pore, stomatal complex and subsidiary cells of both AB and AD were p = 5.638 × 10 −11 , R-squared = 0.9304. Grouped boxplot for those measurements were represented in ( Figure 5).
The lowest stomatal index (SI%) was recorded in S. leucophylla (11.76-12.12 = 11.94 ± 0.25), whereas the highest SI (12.90-20.69 = 15.10 ± 3.76) was noticed for the AD surface in S. schimperiana. The pheatmap in (Figure 6) represents the overall variations between the investigated taxa. S. leucophylla representatives grouped in a separate cluster diverting from representatives of S. schimperiana. The correlogram correlation analysis showed a significant relationship among numerous traits; (Figure 7). ANOVA of the length, width and area for the stomatal pore, stomatal complex and subsidiary cells of both AB and AD were p = 5.638 × 10 −11 , R-squared = 0.9304. Grouped boxplot for those measurements were represented in ( Figure 5).
The lowest stomatal index (SI%) was recorded in S. leucophylla (11.76-12.12 = 11.94 ± 0.25), whereas the highest SI (12.90-20.69 = 15.10 ± 3.76) was noticed for the AD surface in S. schimperiana. The pheatmap in (Figure 6) represents the overall variations between the investigated taxa. S. leucophylla representatives grouped in a separate cluster diverting from representatives of S. schimperiana. The correlogram correlation analysis showed a significant relationship among numerous traits; (Figure 7).

Phylogeny
The phylogenetic placement of the Egyptian species S. leucophylla and S. schimperiana within the whole genus was conducted using novel DNA sequences. The used markers were: a nuclear marker "nrDNA" Internal Transcribed Spacer (ITS), and three plastid markers "cpDNA": matK; psbA-trnH, and rbcL.
The matrices of the DNA sequences of ITS, matK, psb-A/trn-H, and rbcL consisted of 48, 34, 36, and 23 Silene taxa, respectively. The outgroups were Petrocoptis glaucifolia, P. viscosa and P. pyrenaica in the ITS tree. Agrostemma githago and Petrocoptis pyrenaica were used as outgroups in matK. Bufonia multiceps was used in both psb-A/trn-H and and rbcL.
The results showed that section Sclerocalycinae represented a well-supported monophyletic clade (PP = 1) composed of 22 Silene taxa in the ITS analysis ( Figure 8) and four taxa in the matK (Figure 9). The Egyptian species S. schimperiana was related to this section.
Section Siphonomorpha was also retrieved successfully. It consisted of 20 Silene including the Egyptian endemic species S. leucophylla. This section was moderately supported in both phylogenies PP = 0.72 in the ITS (Figure 8), and in the matk PP = 0.8 (Figure 9).
The phylogenetic tree based on the DNA sequences of the psb-A/trn-H showed that section Sclerocalycinae is not monophyletic. Silene schimperiana represented a sister clade to other Silene taxa belonging to section Siphonomorpha Otth: S. acaulis and S. jenissensis, and S. vulgaris related to the subgenus Behenanthae. Similarly, section Siphonomorpha was polyphyletic composed of 30 taxa, including the Egyptian species S. leucophylla ( Figure 10).
The matrices of the DNA sequences of ITS, matK, psb-A/trn-H, and rbcL consisted of 48, 34, 36, and 23 Silene taxa, respectively. The outgroups were Petrocoptis glaucifolia, P. viscosa and P. pyrenaica in the ITS tree. Agrostemma githago and Petrocoptis pyrenaica were used as outgroups in matK. Bufonia multiceps was used in both psb-A/trn-H and and rbcL.
The results showed that section Sclerocalycinae represented a well-supported monophyletic clade (PP = 1) composed of 22 Silene taxa in the ITS analysis ( Figure 8) and four taxa in the matK (Figure 9). The Egyptian species S. schimperiana was related to this section. Section Siphonomorpha was also retrieved successfully. It consisted of 20 Silene including the Egyptian endemic species S. leucophylla. This section was moderately supported in both phylogenies PP = 0.72 in the ITS (Figure 8), and in the matk PP = 0.8 (Figure 9). The phylogenetic tree of the rbcL section Sclerocalycinae including S. schimperiana, represented a robust but unresolved clade (PP = 0.99). Simultaneously, Section Siphonomorpha including S. leucophylla was retrieved with a moderately supporting value PP = 0.88 ( Figure 11).
In the four markers' combined phylogenetic tree, the ingroup consisted of 102 taxa with 97 Silene taxa and five taxa from other genera ( Figure 12). While Bufonia multiceps and Petrocoptis pyrenaica constituted the outgroup clades, Agrostemma githago was imbedded in the ingroup taxa. The phylogenetic tree of the rbcL section Sclerocalycinae including S. schimperiana, represented a robust but unresolved clade (PP = 0.99). Simultaneously, Section Siphonomorpha including S. leucophylla was retrieved with a moderately supporting value PP = 0.88 (Figure 11).  The phylogenetic tree of the rbcL section Sclerocalycinae including S. schimperiana, represented a robust but unresolved clade (PP = 0.99). Simultaneously, Section Siphonomorpha including S. leucophylla was retrieved with a moderately supporting value PP = 0.88 (Figure 11).  The 97 ingroups of Silene taxa were divided into two subgenera: Silene subg. Silene Rohrbach, and Silene subg. Behenantha (Otth) Torr. & A. Gray, and neither of these two subgenera was monophyletic in our reconstruction. The subgenus Silene was composed of six sections: Sclerocalycinae, Siphonomorpha, Pulvinatae, Portenses, Auriculatae, and Atocion, all of which were not monophyletic. Silene schimperiana and S. leucophylla clustered into their related sections as proposed by single markers analyses, Sect. Sclerocalycinae and Sect. Siphonomorpha, respectively. In the four markers' combined phylogenetic tree, the ingroup consisted of 102 taxa with 97 Silene taxa and five taxa from other genera (Figure 12). While Bufonia multiceps and Petrocoptis pyrenaica constituted the outgroup clades, Agrostemma githago was imbedded in the ingroup taxa.

Discussion
The current research represents the first detailed study of the micromorphological and molecular phylogenetic placement of the endemic S. leucophylla and the near-endemic S. schimperiana native to the Sinai Peninsula, Egypt.
Leaf tomentum and the stem wax secretion of Silene species have significant ecological and systematic importance concerning the interaction between plants and their environment [29]. The current study confirms the hairy leaf texture of S. leucophylla, whereas S. schimperiana was glabrous. In contrast, irregular epicuticular crustose wax platelets were observed on the stem and leaf of S. schimperiana only. The obtained results were consistent with [23], who indicated that where species grow in drier habitats, they can attain straight to curved anticlinal walls. Silene leucophylla and S. schimperiana have amphistomatic leaves. This is also a characteristic feature of species occupying xerophytic habitats [30].
Guard cells integrate multiple environmental signals and control the aperture width to ensure appropriate stomatal function for plant survival [31]. Therefore, guard cells have been studied extensively as a model system for scrutinizing environment sensing dynamics and mechanisms [25]. In S. leucophylla, the stem stomatal complex area measured 1.2 times larger than S. schimperiana. Quantitative characteristics of the leaf stomatal complex and the epidermal cells on the leaf's AD were more extensive than those on the A.B. in both species. These indicate great integrity between the cell geometry and size of the epidermal cells and genetic constraints, and environmental factors [32,33].
The cell dimensions of the stomatal complex of S. schimperiana were 2.6-2.9 times larger than those of S. leucophylla. The stomatal pore and its area were 24.4-33.5 times larger S. schimperiana than in S. leucophylla. Additionally, the subsidiary cell area was 2.69-3.13 times larger in S. schimperiana than S. leucophylla.
As suggested by [34][35][36], plant species located in high-elevation and shady areas similar to the growing habitats of both S. leucophylla and S. schimperiana in the mountains of the Sinai Peninsula are usually characterized by a low average Stomatal Index (SI%). This critical trait (SI%) was 12.42% in S. leucophylla and 14.44% in S. schimperiana. The SI% for both species were considerably low compared to the high SI% (≥95.58%) reported in plant species growing in low-elevation and sunny habitats [22].
Rohrbach [6] classified S. leucophylla and S. schimperiana in the same section, "Botryosilene". While he considered S. leucophylla in the Nutantes series, S. schimperiana was classified by him into the series Sclerocalycinae. Classification of the genus Silene by [10,11] considered S. leucophylla a member of section Siphonomorpha, while S. schimperiana was allied to section Sclerocalycinae (Subsection Chlorifoliae).
The Sileneae taxonomy and systematics were recently studied by [4,9,37], who integrated morphological and molecular phylogeny to reveal the phylogenetic relationships within the tribe Sileneae. According to [4,9], S. leucophylla belongs to the subsection Brachypodae (Boiss.) Gürke allied to section Siphonomorpha, while S. schimperiana belongs to subsection Sclerocalycinae.
In the current study, the phylogenetic analysis of each single and the four combined markers confirmed the generic, sectional and phylogenetic placement of the Egyptian species under investigation-with both S. leucophylla and S. schimperiana related to the subgenus Silene. At the same time, the sectional placement shows that S. leucophylla can be placed into the S. sect. Sclerocalycinae and S. leucophylla into S. sect. Siphonomorpha.
The phylogenetic trees' topologies were generally consistent with the generic and sections of the Silene classification [4]. Most Silene sections were not monophyletic and the support values for clades were either weak or moderate. The clade support shows low support of section Siphonomorpha in the ITS analysis PP = 0.72 (Figure 8). In contrast, it was moderately supported in the rbcL analysis PP = 0.88 (Figure 1), and strongly supported (PP = 1) in both matK ( Figure 9) and psb-A/trn-H ( Figure 10).
The results of Bayesian analysis of the ITS, matK, and rbcL data sets supported the monophyly of the sections Siphonomorpha and Sclerocalycinae. However, psb-A/trn-H and the combined analysis failed to retrieve the monophyly of those sections.
According to [37], S. sect. Siphonomorpha s.str. was monophyletic and should be considered a separate section. The current study evaluated that this section should incorporates two complexes: the Leucophylla complex and the Schimperiana complex (Figures 8 and 9).
The Leucophylla complex (Figure 8) was considered to be allied to the widely distributed Nutans group [38]. However, the current study reveals that the Leucophylla complex is confined to narrowly distributed taxa native to the South Mediterranean region, S. flavescens: Bulgaria, Greece, Hungary, Romania, and Yugoslavia; S. leptoclada: East Aegean Island, Turkey, and North Africa; S. leucophylla: endemic to Egypt; S. yemensis: Eritrea, Ethiopia, Saudi Arabia, and Yemen. Moreover, S. saxifraga, a native and widely distributed species in Europe, constitutes a sister group of the Leucophylla complex. In addition to the geographical distribution pattern, the current study confirms that members of the Leucophylla complex shared several morphological traits. Those traits were perennial life form, plant texture, stem woody base, leaf type, flowers number and inflorescence type, calyx shape, and capsule features [39][40][41][42].

Plant Materials
Two herbarium specimens of Silene leucophylla and three of Silene schimperiana were obtained from Assuit University Herbarium (ASTU). Three stem and leaf replicates of each specimen were used in SEM. For molecular analysis, fresh leaf materials were collected from two different localities at Saint Katherine, South Sinai, Egypt. Vouchers of the collected specimens are deposited at the herbarium of Suez Canal University, Ismailia, Egypt (SCUI) under the collection number (SCUI).

Scanning Electron Microscopy (SEM)
The stem, as well as the leaf abaxial (AB) and adaxial (AD) surfaces, were mounted onto stubs with double-sided adhesive tape, coated for 5 min with gold in a polaron JFC-1100E coating unit, and then were examined and photographed with JEOL JSM-IT200 scanning electron microscope unit at Faculty of Science, Alexandria University, Alexandria, Egypt. The epidermal cell characteristics separately described for abaxial (AB) and adaxial (AD) leaf surfaces. The quantitative data of stomatal measurements were recorded in both closed and opened stomata, i.e., length, width, and area. The quantitative characteristics measured by image analysis software [44] followed the terminology in [29].

Statistical Analysis
Sixty-eight traits of the stem and leaf of the examined taxa were analyzed by using the R-software (Vienna, Austria), with the required packages installed [45]. Initially, boxplots were generated using the "ggplot2" library [46], to address the variations in the measured traits of the stem, and the AB and AD of the leaf. Analysis of variance (ANOVA) was performed using the (aov) function. Subsequently, post hoc Tukey Honestly Significant Difference (HSD) was used to figure out which group(s) of the sample differed [47]. The "pheatmap" and "ggplot2" packages [46,48] were used to visualize the similarity and dissimilarity within and among species. The scale of color is relative to the value of the divergence between investigated readings. The red color indicates the high similarity between accessions while the blue color assuming the low similarity [49]. The relationships among the quantitative traits were assessed through correlation analysis which visualized by correlogram using "corrplot2". In the correlogram, the intensity of the colors is an indication for positive correlation, whereas the white box point indicates the insignificant correlation between variables [50].

DNA Extraction, PCR Amplification, Sequencing, and Phylogenetic Analysis
Two fresh leaf materials preserved in silica gel of each species were used for molecular analysis. Total DNA was extracted from silica-gel dried leaves using a silica column method similar to commercially available extraction kits [51]. DNA was extracted using the Cetyltrimethylammonium bromide (CTAB) protocol with some modifications [52]. The PCR amplification were performed in 15 µL volume for ITS, matK, rbcL and psb-A/trn-H containing 5U/µL Taq DNA polymerase with 25 µM MgCl 2 , 10 µM of dNTPs, 10 µM of each primer. Amplifications were conducted using an Applied Biosystems ® -VeritiTM 96well thermal cycler (Thermo Fisher Scientific-Fisher Scientific AS-Postboks 114, Smestad-0309 Oslo, Norway). PCR products were purified with ExoSAP-IT (USB Corporation, Cleveland, OH, USA). PCR products were sent to Macrogen Spain for direct sequencing in both directions with an ABI 3730XL Genetic Analyzer (Life Technologies Corporation 5791 Van Allen Way Carlsbad, CA 92008). For the newly generated sequences, forward and reverse reads were assembled, and the contigs were edited into in GENEIOUS ® v.R9 (Biomatters Ltd., Berkeley, CA 94709-1405 USA https://www.geneious.com accessed 15 January 2021) using a personal license (C.A.). Four data matrices were constructed: ITS, matK, rbcL, and psb-A/trn-H. The ingroup were selected to cover most of the major sections in the genus Silene. Each marker's datasets were initially aligned using MAFFT algorithms, implemented in Geneious, using default alignment parameters, and visually revised to manually correct errors in alignment.
The optimal nucleotide substitution model was estimated using MrModeltest [53] and executed in MrBayes blocks. A 50% majority role consensus tree was constructed to get the posterior probabilities (PP). Posteriori probabilities, values >0.5 at a given branch were considered to support the existence of that branch [54]. All phylogenetic analyses were run on the CIPRES portal [55].

Conclusions
The combined investigations of the morphological data of leaf, stem and the molecular phylogenetic analysis of four molecular markers represented a comprehensive characterization of the endemic S. leucophylla and the near-endemic S. schimperiana native to Egypt, Yemen, and Saudi Arabia. The qualitative and quantitative morphological characters include presence/absence of trichomes, epicuticular wax type, leaf epidermal cells, and complex stomatal traits attributed entirely to well-characterizing endemic endangered species supported the previous taxonomic revisions. The Bayesian phylogenetic inference of S. leucophylla and S. schimperiana using four nuclear markers portrayed the target taxa's phylogenetic position within the whole genus. Both species related to the subgenus Silene and their corresponding sections, where S. leucophylla allied to section Siphonomorpha and S. schimperiana allied to section Sclerocalycinae.
The sectional classification of the Silene species native to the Sinai Peninsula, Egypt has so far always been investigated on the basis of morphological descriptions only. The current research represents the first phylogenetically based study to reveal the sectional classification of S. leucophylla and S. schimperiana. Moreover, two morphologically and phylogenetically supported Silene complexes are proposed by the current research. The Leucophylla complex allied to section Siphonomorpha and the Schimperiana complex related to section Sclerocalycinae. However, these two complexes require further investigation and more exhaustive sampling to infer their complex phylogenetic relationships.