Taxonomic Revision of Genus Ephedra Tourn. ex L. in Egypt with Intra-Gender Diversity in Morphometric Traits and Fatty Acid Composition

The genus Ephedra Tourn. ex L. (Ephedraceae) still exhibits taxonomic complexity that has not yet been resolved. This study aimed to determine the taxonomic identity of the Ephedra species in Egypt and identify the fatty acid profile and its diversity at the gender level as a taxonomic tool for specimens lacking reproductive cones. The current study provides a pioneering approach that distinguishes Ephedra species at the gender level. A total of 120 fresh individuals were collected from 20 locations representing different habitats where Ephedra plants grow in Egypt. In addition, herbarium specimens were deposited in Egyptian herbaria. The studied morphological traits included 30 vegetative characteristics and 72 traits of the reproductive organs of both genders. The fatty acid content was measured using gas–mass chromatography (GC-Mass). The taxonomic revision revealed that the Genus Ephedra was represented in the Egyptian flora by five species, Ephedra alata in section Alatae and E. aphylla, E. ciliata, E. foemina, and E. pachyclada in section Ephedra. South Sinai hosts these five species and represents the center of diversity for this genus in Egypt. The vegetative characteristics were subjected to principal component analysis (PCA), which revealed a distinct separation of the five studied species. Similarly, the cone traits treated by hierarchical clustering revealed intra-gender variations. The taxonomic key was developed based on the morphological traits to distinguish the studied species at the gender level. In total, 51 fatty acids were identified from the studied species and grouped as 18 saturated, 16 monounsaturated, and 17 polyunsaturated fatty acids. In the absence of reproductive cones, the lipid content and fatty acid composition of the vegetative parts displayed significant interspecific and intra-gender variations. Therefore, fatty acids can be used to efficiently identify the studied species when they lack reproductive cones. This study proved the efficacy of a multidisciplinary approach to identify Ephedra species at the gender level and recommends this trend for future studies of this genus.


Introduction
Globally, Gymnospermae include 873 species in 14 families [1].Gymnospermae are represented in the Egyptian flora by two families, Cupressaceae and Ephedraceae [2].Ephedraceae is a monogeneric family represented by a single genus, Ephedra Tourn.ex L. Worldwide, this genus includes 68 species [3,4], or 69 species [5], or 73 accepted species (https://powo.science.kew.org/accessed on 1 July 2024).It is distributed across both the New World and Old World [6,7].Ephedra species are typically perennial dioecious shrubs or under-shrubs [5] with xeromorphic features, assimilating branches, and opposing or Plants 2024, 13, 2442 2 of 20 whorled leaves that are frequently reduced to membranous sheaths [4,8].The flowering of Ephedra species occurs between February and March.They have flowers in small cones: male flowers are subtended by a bract, two-lipped perianth, and staminal column with 2-9 anthers; female flowers are solitary or in groups of 2-3, subtended by 2-4-(6) pairs of bracts; and ovules have scarious or fleshy bracts.
Amongst Gymnosperms, the genus Ephedra is remarkably notorious for its research scarceness [9].Taxonomically, it is classified into three sections based on the female cone bracts, which appear thickened, fleshy, and colorful in section Ephedra, dry-membranous, free, and winged in section Alatae Stapf, and free and coriaceous-dry in section Asarca Stapf [10].
The genus Ephedra is represented in Egyptian flora by two sections, Alatae and Ephedra.Section Alatae is represented by E. alata (tribe Tropidolepides).Section Ephedra includes two tribes, namely, tribe Scandentes, which includes three species (E.aphylla, E. foliata, and E. foemina), and tribe Pachyclada, which includes E. pachyclada subsp.sinaica [4,11].Out of these five species, E. alata and E. pachyclada are among the "Least Concern Species ′′ according to the IUCN.
The fatty acid composition of Gymnospermae can be used as a powerful taxonomic tool [1].For example, the 5-olefinic acid is a characteristic fatty acid of Gymnospermae that Angiospermae does not synthesize, except in the family Rananculaceae [1].Fatty acids from the leaves of 50 Gymnospermae species belonging to 14 families were studied earlier [1,[18][19][20].The later authors studied the fatty acids of the photosynthetic parts of 137 species belonging to 14 families, including five Ephedra species.Unfortunately, none of them was a Mediterranean species.
Most studies that have investigated the fatty acid composition in Gymnospermae do not specify the gender level (male and female individuals), and such data are still lacking.There are very scarce investigations into the fatty acid profile at the gender level in Gymnospermae.Hierro et al. [21] investigated the fatty acids of male and female Ginkgo biloba, but there is no sufficient work on Gymnosperms that can be generalized.Concerning the genus Ephedra, Nokhsorov et al. [22] investigated the role of lipids in Ephedra monosperma as an adaptive response to low temperature, but they did not make any reference to the gender level.Despite the importance of the fatty acid composition as an adaptation strategy, the lipid profile of Ephedra species at the gender level is still lacking.
During our field observations, we noticed confusion in the identification of the different Ephedra species because Ephedra ′ s morphological characteristics were reduced, and there are only a few characters, so the taxonomy of the genus Ephedra Tourn.ex L. has always been dubious and has been partially studied.Because the majority of the genus ′ s categorization has been based on restricted vegetative features, including the leaf length, female cone bracts, number of seeds per female cone, and plant habit, the infrageneric relationships among Ephedra have remained unclear.There is still a gap in the previous studies that enables taxonomists to distinguish between the genders in specimens lacking reproductive cones.Fortunately, the lipid profile and fatty acid profile may also play a role in discriminating the different Ephedra species and may differ between both genders of the same species.Therefore, fatty acids and the lipid content could be an efficient tool in the identification of species when specimens lack reproductive organs.This study was applied to the Egyptian Ephedra species and aimed to (1) use morphological traits for both species and gender identification, (2) apply the fatty acid composition for both species and gender identification, and (3) use the fatty acid composition to differentiate between male and female specimens.Erect shrub up to 1.0 m tall.Stem circular, moderately branched at base.Branches grooved, papillose surface, stout, opposite, and scabrous.Leaves elliptical, 1.8-3 × 0.8-1.5 mm, opposite, sparsely ciliate, united for more than half their length (Figure 1).Cone bracts broad and woody with free scarious margins.Female cones grouped in axillary clusters carried on the straight peduncle.Cone bracts 4-5(-6) free pairs, each less than 1/3 cone length, orbicular, rounded apex, shortly lacerate margin.Outer bracts of strobilus 4-5 × 2-3 mm, equal in length.Inner bracts of strobilus 6-7.5 × 3.5-4 mm, less than twice as long as next bract.Fruit dry, creamy, cone-like, carried on erect peduncle.Seeds 6.5-8 × 2.5-4 mm, (1)-2 ovules/cone, flattened ovoid, brownish, 1-2 mm longer than inner female bract; micropylar tubule straight, 2-3 mm long (Figure 2 and Supplementary Table S1).Male cones grouped in axillary clusters, bracts 11-17 free pairs, each 2.5-3.2 × 1.2-1.5 mm, elliptical-obovoid, entire, acute-rounded apex, sub-equal.Strobilus bracts 6-12 pairs, each 1.5-3 × 1-2 mm, obovate, entire, retuse-rounded apex, obtuse-rounded base, sub-equal.Antherophore 2.5-5 mm branched above, anthers 3-6/strobilus, umbellate form (Figure 3 and Supplementary Table S1).

Species and Gender Key Based on Morpho-Taxonomic Traits
This constructed key was based on the current investigation and is supported by earlier works, among them [4,15,16,25].

I.
Margins  [4,13] mentioned that the nomenclature and history of the Mediterranean Ephedra species are chiefly complicated.Moreover, Ephedra alte C. Meyer of P. Forsskål was identified later as female E. foliata Boiss ex Meyer and male E. aphylla; this confusion was enhanced by the close morphology, ecology, and distribution range [26,27].The current investigation of the five Mediterranean Ephedra species revealed several distinguishable features.The leaf length in the studied Ephedra species is notably variable among species as well as between the genders of each species.The total leaf length and the length of the free part of E. ciliata female and male individuals were notably longer than those in the other species, while the length of the united part of the leaf and leaf sheath of E. alata was longer than in the other individuals (Figure 1).Leaf sheath and cone bracts are glabrous in E. foemina and E. pachyclada, while both are ciliated in E. alata, E. aphylla, and E. ciliata.These data were reported earlier by Danin and Hedge and Faried et al. [4,25].E. foemina is the only creeping and insect-pollinated species, while the rest are wind-pollinated and erect species [24].
Figure 4 shows the PCA based on 30 vegetative characteristics of the studied species, at dimensions 1 and 2, with a cumulative variance of 49.2% for dimension 1, declaring the clear separation of the studied species into two groups along this dimension: the right group with three species, E. alata, E. aphylla, and E. ciliata, and the left group representing E. foemina and E. pachyclada.This grouping depends mainly on the absence of trichomes in the species of the left group.The higher width of the node up to 5 mm and clear stem grooves distinguish E. alata, the triangular shape of the leaf distinguishes E. aphylla, while the longer leaf length up to 15 mm and the high density of trichomes distinguish E. ciliata.While E. foemina is distinguished from the species of the right group by its climbing and prostrate habit as well as its branching all over the stem, E. pachyclada is distinguished from other species by the absence of a free part of the leaf and the dense branches at the node (up to 13/node).The achieved PCA grouping is analogous to the taxonomic identity of these species reported in previous studies [4,8,16].
Figure 5 outlines the hierarchical clustering analysis based on the 72 morphological characteristics of the cones in the studied species, revealing the separation of each gender (male or female) into a separate group.The main separating characteristics for the male gender were the numbers of both male cones and anthers/strobili, while the length of both the female cones and the inner bracts were the efficient traits in the female gender grouping (Figure 6).The characteristic of staminate cones in Ephedra is a distinctive feature, especially for the E. alata characterized by branched antherophore and stalked anthers, while other species are characterized by unbranched antherophore and sessile anthers [29].
The bract length of the female cone is not only a distinctive characteristic (Figure 6), but its size also showed more significant dissimilarity between the studied species than the male cone (Figure 6).The bract size of the female cone in female E. alata was significantly longer than that in the other individuals.The female cone traits are also the main distinctive characteristics for the Chinese Ephedra species [10].Figure 5 outlines the hierarchical clustering analysis based on the 72 morphological characteristics of the cones in the studied species, revealing the separation of each gender (male or female) into a separate group.The main separating characteristics for the male gender were the numbers of both male cones and anthers/strobili, while the length of both the female cones and the inner bracts were the efficient traits in the female gender grouping (Figure 6).The characteristic of staminate cones in Ephedra is a distinctive feature, especially for the E. alata characterized by branched antherophore and stalked anthers, while other species are characterized by unbranched antherophore and sessile anthers [29].
The bract length of the female cone is not only a distinctive characteristic (Figure 6), but its size also showed more significant dissimilarity between the studied species than the male cone (Figure 6).The bract size of the female cone in female E. alata was significantly longer than that in the other individuals.The female cone traits are also the main distinctive characteristics for the Chinese Ephedra species [10].

Species Distribution and Habitats in Egypt
Ephedra alata, E. aphylla, and E. ciliata are distributed in the Egyptian deserts, including Sinai, in addition to the Mediterranean strip; E. ciliata is the least prevalent species.Each species possesses a characteristic habitat, desert sandy plains for E. alata, calcareous slopes and wadi beds for E. aphylla, while E. ciliata inhabits rocky slopes.E. foemina and E. pachyclada are very rare in prevalence with a restricted distribution range, as both grow hanging on rocky cliffs.E. foemina was traced in a few individuals in Sinai and the Mediterranean strip, while E. pachyclada was restricted to South Sinai.The distribution range and prevalence status recorded in our study were in harmony with the earlier floristic surveys [2,4,15,16].Individuals were collected from 20 localities (indicated by stars in Table 1).

Species Distribution and Habitats in Egypt
Ephedra alata, E. aphylla, and E. ciliata are distributed in the Egyptian deserts, including Sinai, in addition to the Mediterranean strip; E. ciliata is the least prevalent species.Each species possesses a characteristic habitat, desert sandy plains for E. alata, calcareous slopes and wadi beds for E. aphylla, while E. ciliata inhabits rocky slopes.E. foemina and E. pachyclada are very rare in prevalence with a restricted distribution range, as both grow hanging on rocky cliffs.E. foemina was traced in a few individuals in Sinai and the Mediterranean strip, while E. pachyclada was restricted to South Sinai.The distribution range and prevalence status recorded in our study were in harmony with the earlier floristic surveys [2,4,15,16].Individuals were collected from 20 localities (indicated by stars in Table 1).

Lipid Content and Fatty Acid Composition at Interspecific and Intra-Generic Levels
The lipid content of the assimilating parts demonstrates significant interspecific variations.Figure 7 shows that the greater total lipid content was recorded in E. ciliata males (66 mg/g), followed by E. alata females (64 mg/g), while the lowest concentrations were reported in the female of E. foemina and E. ciliata (30 and 39 mg/g, respectively).Moreover, the intra-gender variation was notable for each species; nevertheless, the genders of each species were collected from the same population.Supplementary Table S2 shows that the males of E. ciliata, E. pachyclada, and E. foemina have greater total lipid contents (66, 56, and 51 mg/g, respectively) than the female gender.By contrast, male E. alata and E. aphylla contained lower lipid contents (45 and 40 mg/g, respectively) than the female gender.
The alternation in lipid content of the cell membrane is the chief response to environmental stresses [22].Accordingly, the detected higher lipid content of the studied genders (male E. ciliata and female E. alata; Figure 7) may reflect their better response to environmental stresses than other genders.High lipid contents help plants overcome climatic stresses, such as drought and higher temperatures [35,36].
contained lower lipid contents (45 and 40 mg/g, respectively) than the female gender.
The alternation in lipid content of the cell membrane is the chief response to environmental stresses [22].Accordingly, the detected higher lipid content of the studied genders (male E. ciliata and female E. alata; Figure 7) may reflect their better response to environmental stresses than other genders.High lipid contents help plants overcome climatic stresses, such as drought and higher temperatures [35,36].

Fatty Acid Composition at Interspecific and Intra-Gender Levels
The heat map (Figure 8) presents the common fatty acids in the studied Ephedra species visualized by color intensity into three clusters.The first cluster grouped male individuals of E. alata, E. aphylla, and E. foemina in addition to female E. aphylla based on their higher percentages of palmitic, oleic, and linoleic acids.The second cluster grouped the females of E. pachyclada and E. alata with higher percentages of linoleic and rumanic acids, followed by palmitic acid.The third cluster comprised male E. pachyclada and female E.

Fatty Acid Composition at Interspecific and Intra-Gender Levels
The heat map (Figure 8) presents the common fatty acids in the studied Ephedra species visualized by color intensity into three clusters.The first cluster grouped male individuals of E. alata, E. aphylla, and E. foemina in addition to female E. aphylla based on their higher percentages of palmitic, oleic, and linoleic acids.The second cluster grouped the females of E. pachyclada and E. alata with higher percentages of linoleic and rumanic acids, followed by palmitic acid.The third cluster comprised male E. pachyclada and female E. foemina together with female and male E. ciliata, all containing high palmitic acid contents, and the latter species had high contents of oleic and myristic acids.
The studied Ephedra species collected from arid habitats showed the dominance of saturated fatty acids palmitic (C16:0), myristic (C14:0), and stearic (C18:0) in descending concentrations (Figure 8 and Supplementary Table S2).These results align with those by the authors of [1,37], who claimed the dominance of palmitic acid in photosynthetic tissues of the Gymnospermae.
The detected fatty acid composition revealed that fatty acids can distinguish between genders (male and female) of the same species, where valeric, cervonic, timnodonic, myristoleic, pentadecenoic, arachidonic, and docosatetraenoic acids are among the fatty acids present in the female gender only of E. alata, E. aphylla, E. ciliata, E. foemina, and E. pachyclada (Supplementary Table S2).Moreover, male E. ciliata and E. pachyclada lack polyunsaturated fatty acids, which could be used to distinguish males from females in the absence of reproductive organs.The absence of polyunsaturated fatty acids in the males of both species may induce their fragility to environmental stresses [35,36].Our results show that fatty acids in Gymnospermae are not only a powerful taxonomic tool [1] but may extend to distinguish the gender of the species in the genus Ephedra.unsaturated fatty acids, followed by E. alata males (48.30%), and the lowest percentage is recorded for females of E. alata (5.15%).Males of E. ciliata and E. pachyclada are characterized by their lack of polyunsaturated fatty acids.Nokhsorov et al. [22] reported that, in E. monosperma during an extreme reduction in temperature, the concentrations of unsaturated fatty acids (C18:2 and C18:3) increased while the saturated fatty acids, such as C16:0, decreased.The female gender of the studied Ephedra species showed higher percentages of polyunsaturated fatty acids (4.3 and 62.8 in E. foemina and E. alata, respectively) than the male gender (Figure 9 and Supplementary Table S2).This may enable the female gender to adapt better to climatic changes [36].
The male gender had greater percentages of monounsaturated fatty acids than the female gender, except for E. ciliata, which showed a lower percentage (Figure 9 and Supplementary Table S2).However, the fatty acids of Ephedra species collected from cooler habitats showed the dominance (up to 50%) of the polyunsaturated fatty acids linolenic (C18:3) and linoleic (C18:2) in descending order [1].The polyunsaturated fatty acids enhance the species′ adaptability to climatic changes [35,36].In E. monosperma, the concentration of polyunsaturated lipid compounds of phospholipids and fatty acids increased during cold acclimatization [22].
At the date of issue, the fatty acid composition of Gymnospermae species to the gender level has not yet been fully uncovered, except by Hierro et al. [21], who differentiated between male and female Gymnospermae genders in Ginkgo biloba based on fatty acids.The female gender of the studied Ephedra species showed higher percentages of polyunsaturated fatty acids (4.3 and 62.8 in E. foemina and E. alata, respectively) than the male gender (Figure 9 and Supplementary Table S2).This may enable the female gender to adapt better to climatic changes [36].
The male gender had greater percentages of monounsaturated fatty acids than the female gender, except for E. ciliata, which showed a lower percentage (Figure 9 and Supplementary Table S2).However, the fatty acids of Ephedra species collected from cooler habitats showed the dominance (up to 50%) of the polyunsaturated fatty acids linolenic (C18:3) and linoleic (C18:2) in descending order [1].The polyunsaturated fatty acids enhance the species ′ adaptability to climatic changes [35,36].In E. monosperma, the concentration of polyunsaturated lipid compounds of phospholipids and fatty acids increased during cold acclimatization [22].
At the date of issue, the fatty acid composition of Gymnospermae species to the gender level has not yet been fully uncovered, except by Hierro et al. [21], who differentiated between male and female Gymnospermae genders in Ginkgo biloba based on fatty acids.Our work is a pioneer in detecting the fatty acid composition in Ephedra species to the gender level and reveals that fatty acids possess highly distinctive concentrations and types at the intra-gender level.These findings are supported by Hierro et al. [21].

Morphological Investigations
A total of 24 fresh individuals and 30 strobili/species were morphologically investigated in each species.A total of 93 and 74 macro-morphological characteristics were used for female and male individuals, respectively (including shrub features, branches, stems, leaves, cone bracts, male and female strobili, fruits, and seeds) (Supplementary Table S1).

Determination of Crude Lipid
The crude lipid was extracted by methanol and chloroform from one gram of the dried powder plant material using a Soxhlet device for 8-10 h.Following the extraction, the solvent evaporated at low pressure; lipid material was desiccated and determined [38].

Identification of Fatty Acids Using Gas-Mass Chromatography (GC-Mass)
Fatty acids were investigated using a Trace GC1310-ISQ mass spectrometer (Thermo Scientific, Austin, TX, USA) with a direct capillary column, TG-5MS (30 m × 0.25 mm × 0.25 µm film thickness).The temperature of the column oven was adjusted at 50 • C and then raised by 5 • C/min to 230 • C and kept for 2 min.Finally, it was raised by 30 • C/min to 290 • C and held for 2 min.The helium flow rate was 1 mL/min.After a three-minute solvent delay, diluted samples containing one microliter were injected.The temperatures of the injector and MS transfer line were maintained at 250 and 260 • C, respectively.Mass spectra were obtained at 70 eV ionization voltages through the m/z 40-1000 range.Fatty acids were identified by comparing the retention times and mass spectra with mass spectral databases from WILEY 09 and NIST 11 [39,40].

Statistical Analysis
The data were analyzed using the software package SPSS, version 20.0 (IBM Corporation, Armonk, NY, USA).The data were first examined for normality and homogeneity of variances using the Kolmogorov-Smirnoff and Levene ′ s tests, respectively.All data exhibit normality, and all statistical comparisons were carried out using one-way analysis of variance (ANOVA) followed by Tukey ′ s post hoc test.Next, the Graph Bad Prism software version 8.4.2 was used to draw histogram plots.R software version 4.3.2(Vienna, Austria) was utilized and loaded with the necessary packages [41].Principal component analysis (PCA) was employed to examine a dataset consisting of continuous variables after installing the "factoextra ′′ and "FactoMineR ′′ packages in R [42].The correlation coefficients for the interaction between variables were obtained and displayed using the "Corrplot ′′ program [43].White with a 0 denotes no association between the two variables, while blue with a 1 shows a strong positive correlation.A red score of −1 indicates a significant negative association.Hierarchical clustering analysis (heat map) was employed to examine continuous variables after installing the "pheatmap ′′ and "RColorBrewer ′′ packages in R [44].Finally, Microsoft Excel 365 was used to draw radar and combo plots (statistical significance was defined as p < 0.05 and non-statistical significance as p > 0.05).

Conclusions
The current study revealed that the genus Ephedra in Egypt includes five species, in two sections: Ephedra alata in section Alatae and E. aphylla, E. ciliata, E. foemina, and E. pachyclada in section Ephedra.The traits of the reproductive cones are more efficient than the vegetative features as a taxonomic tool for identifying Ephedra species at the gender level.Also, the fatty acid composition is a helpful taxonomic tool in identifying Ephedra species when the specimens lack reproductive organs, since fatty acids showed interspecific and intra-generic variations in both type and composition.The study revealed that three species of Ephedra, namely, Ephedra alata, E. aphylla, and E. ciliata (in descending order), possess a wider distribution range and a common prevalence pattern, compared to the

Figure 1 .
Figure 1.Combo plot of the leaf size (L × W mm): a: leaf length (mm); b: length of the free part; c: length of the united part of the leaf; and d: length of the leaf sheath of male and female Ephedra individuals.

Figure 1 . 21 Figure 2 .
Figure 1.Combo plot of the leaf size (L × W mm): a: leaf length (mm); b: length of the free part; c: length of the united part of the leaf; and d: length of the leaf sheath of male and female Ephedra individuals.

Figure 2 .
Figure 2. (A-E): female cones of the studied Ephedra species, (F-J): female strobilus of the studied Ephedra species, and (K-O): seeds of the studied Ephedra species.

Figure 3 .
Figure 3. (A-E): male cones of the studied Ephedra species, (F-J): male strobilus of the studied Ephedra species, and (K-O): antherophore of the studied Ephedra species.

Figure 3 .
Figure 3. (A-E): male cones of the studied Ephedra species, (F-J): male strobilus of the studied Ephedra species, and (K-O): antherophore of the studied Ephedra species.

Figure 4 .
Figure 4. Principal component analysis (PCA) of the 30 morphological characteristics with 49.2% total variance described along the first axis (Dim 1) followed by 30.5% variance exhibited along the second axis (Dim 2).

Figure 4 .
Figure 4. Principal component analysis (PCA) of the 30 morphological characteristics with 49.2% total variance described along the first axis (Dim 1) followed by 30.5% variance exhibited along the second axis (Dim 2).

Figure 5 .
Figure 5. Hierarchical clustering analysis of morphological characteristics of male and female cones based on 72 characteristics.Data are represented by the means of at least 3 replicates.

Figure 5 .
Figure 5. Hierarchical clustering analysis of morphological characteristics of male and female cones based on 72 characteristics.Data are represented by the means of at least 3 replicates.

Figure 9 .
Figure 9. Radar plot of the fatty acids identified in the studied Ephedra species at the gender level.

Figure 9 .
Figure 9. Radar plot of the fatty acids identified in the studied Ephedra species at the gender level.

Table 1 .
Localities for the studied Ephedra species (localities are arranged from north to south).