Comparative Taxonomic Study of Launaea Cass. (Asteraceae, Cichorioideae) in Egypt

: Launaea is a polymorphic genus with inter- and intraspeciﬁc inconsistencies. The study aimed to revise the classiﬁcation and identiﬁcation of Launaea species in Egypt. The study also aimed to assess the inter- and intraspeciﬁc variation among taxa using macro- and micromorphological characters. Numerical analysis was carried out on 156 specimens, which were collected from 63 sites in Egypt. The results revealed that L. arabica , L. capitata , L. intybacea and L. spinosa were signiﬁcantly ( p -value < 2.2 × 10 − 16 *** ) discriminated from the other species at a high dissimilarity level. Launaea acanthodes was correctly identiﬁed as Lactuca orientalis . The study supported the inclusion of L. intybacea , L. massauensis and L. nudicaulis within section Microrhynchus, the separation of L. angustifolia and L. arabica in two distinct species. Two biotypes of L. nudicaulis were morphologically differentiated. Launaea fragilis subsp. fragilis showed considerable plasticity and variability in various characters; therefore, L. tenuiloba was considered to be merely an ecotype of L. fragilis . Launaea mucronata comprises two subspecies; cassiniana and mucronata , differentiated by their pappus type, conspicuous secondary ribs of the inner achenes, and the number of spines on the polar area of pollen grains.

Taxonomy 2021, 1 193 The genus was subjected to various taxonomic demarcations and allocations of its species in Egypt, which have been summarized in the Supplementary Materials (Table S1).
The current study aimed to review the classification and identification of Launaea species based on an extensive array of macro-and micromorphological characters. The study also aimed to assess the inter-and intraspecific variation among taxa.

Macromorphological Characters and Numerical Analysis
For the vegetative and floral attributes, three replicas were measured for each specimen. Specimens were examined using Olympus stereomicroscope and measured using ImageJ (1.51J8) [15]. All leaf characters were studied for the rosette leaves, except the leaf attachment and the auricle margin were studied in the cauline leaves. For the achene macromorphology, measurements were carried out on nine replicas for each specimen. The studied achenes were selected from three different positions in the fruiting capitulum, marginal, submarginal and inner, to investigate the achenes variation. The submarginal achenes represented a transition state between the marginal and the inner ones; therefore, their measurements were omitted. The achenes measurements were conducted using Olympus stereomicroscope supported with 1 cm ocular micrometer. The terminology of macromorphological characters follows De Vogel [16] Kilian [7], Simpson [17] and Roque et al. [18].
One hundred and fifty-six specimens were subjected to numerical analysis by using 82 macromorphological characters; 16 vegetative, 37 floral and 29 achene characters. The complete list of the assessed quantitative and qualitative macromorphological characters and their states is presented in the Supplementary Materials (Table S3). The agglomerative cluster analysis was performed through the PAST program for mixed data set [19]. The dendrogram was constructed by the UPGMA method of sorting based on the Manhattan dissimilarity coefficient.

Micromorphological Characters (SEM) and Numerical Analysis
The achene and pollen micromorphological characters were investigated for 17 samples obtained from 17 hypothetical groups. These individuals were selected based on the cluster analysis resulting from the study of 82 macromorphological characters performed to assess the inter-and intraspecific variation among taxa. Marginal and inner achenes were preliminarily examined using a stereomicroscope to ensure that they were of normal size, shape and maturity. The achenes were mounted directly on stubs using double-sided adhesive tape and then coated with gold, up to a thickness of 400 A in a sputter-coating unit (JFC-1100 E). The samples were examined and photographed with a JEOL JSM-5300 scanning electron microscope. A total of 11 micromorphological attributes were assessed. The terminology follows Barthlott [20], Simpson [17], Zhang et al. [21] and Hussein and Eldemerdash [22].
The pollen grains were mounted directly on a glass cover and coated with gold up to a thickness of 400 A in a sputter-coating unit (JFC-1100 E). An average of ten pollen grains were examined and photographed for each specimen with a JEOL JSM-5300 scanning electron microscope. The number of spines on the polar area (excluding ridges), the ratio of polar area to pollen area and the apocolpium index were investigated. The latter was calculated according to the following equation: Apocolpium index = (apocolpium diameter/equatorial diameter) × 100. Fifteen morphological characters were evaluated according to Punt et al. [23] terminology. The complete list of the assessed quantitative and qualitative micromorphological characters and their states were presented in the Supplementary Materials (Table S4).
The cluster analysis was achieved based on 26 micromorphological characters. The similarity among specimens was determined through the Hamming dissimilarity coefficient, and the dendrogram was constructed by the UPGMA method of sorting by using the PAST program of Hammer et al. [19]. Also, a Principal Coordinate Analysis (PCoA) was conducted to visualize similarities and dissimilarities among the 17 groups. The PCoA was performed by using the R-software (Vienna, Austria), with the required packages installed [24].

Numerical Analysis of Macromorphological Characters
Eighty-two macromorphological characters were studied for 156 specimens; 18 vegetative and 37 floral traits (Figure 1), and 29 achenes macromorphological traits (Figures 2  and 3). The performed numerical analysis was illustrated in Figure 4.
Reichardia tingitana was segregated at the highest dissimilarity level (1.420), whereas the taxa of Launaea were divided into four groups representing the four corresponding sections: Zollikoferia at 1.253, Acanthosonchus at 1.134, and both Launaea and Microrhynchus at 1.013. A total of 17 hypothetical groups were proposed: R. tingitana, Acanthosonchus (L. spinosa), Launaea (L. capitata), four groups in Microrhynchus (L. intybacea, L. massauensis and two forms of L. nudicaulis) and ten groups within Zollikoferia (L. angustifolia subsp. angustifolia, L. angustifolia subsp. arabica, L. mucronata subsp. mucronata, L. mucronata subsp. cassiniana and six forms of L. fragilis subsp. fragilis). The morphological distinction between these groups is summarized in the Supplementary Materials (Table S5). achenes were preliminarily examined using a stereomicroscope to ensure that they were of normal size, shape and maturity. The achenes were mounted directly on stubs using double-sided adhesive tape and then coated with gold, up to a thickness of 400 A in a sputter-coating unit (JFC-1100 E). The samples were examined and photographed with a JEOL JSM-5300 scanning electron microscope. A total of 11 micromorphological attributes were assessed. The terminology follows Barthlott [20], Simpson [17], Zhang et al. [21] and Hussein and Eldemerdash [22]. The pollen grains were mounted directly on a glass cover and coated with gold up to a thickness of 400 A in a sputter-coating unit (JFC-1100 E). An average of ten pollen grains were examined and photographed for each specimen with a JEOL JSM-5300 scanning electron microscope. The number of spines on the polar area (excluding ridges), the ratio of polar area to pollen area and the apocolpium index were investigated. The latter was calculated according to the following equation: Apocolpium index = (apocolpium diameter/equatorial diameter) × 100. Fifteen morphological characters were evaluated according to Punt et al. [23] terminology. The complete list of the assessed quantitative and qualitative micromorphological characters and their states were presented in the Supplementary Materials (Table S4).
The cluster analysis was achieved based on 26 micromorphological characters. The similarity among specimens was determined through the Hamming dissimilarity coefficient, and the dendrogram was constructed by the UPGMA method of sorting by using the PAST program of Hammer et al. [19]. Also, a Principal Coordinate Analysis (PCoA) was conducted to visualize similarities and dissimilarities among the 17 groups. The PCoA was performed by using the R-software (Vienna, Austria), with the required packages installed [24].

Numerical Analysis of Macromorphological Characters
Eighty-two macromorphological characters were studied for 156 specimens; 18 vegetative and 37 floral traits (Figure 1), and 29 achenes macromorphological traits (Figures 2 and 3). The performed numerical analysis was illustrated in Figure 4.
Section Zollikoferia was separated into four clusters at 0.50 dissimilarity level. Cluster "3" comprised the two subspecies of L. mucronata; cassiniana and mucronata. While, cluster "4" aggregated groups 13 and 10 of L. fragilis subsp. fragilis, and the other specimens of the latter subspecies were assembled below cluster "5". Finally, cluster "6" isolated L. angustifolia subsp. angustifolia.
The distinction among the seventeen hypothetical groups is performed also by the Principle Coordinate Analysis (PCoA) (Figure 10). The first and second axis explained 31.56% and 28% of the variance, respectively, which emphasize a moderate variation for the data. The taxa representing each section were assembled together. The widest range of variation was noticed in section Zollikoferia, especially among the taxa of L. fragilis subsp. fragilis and between the two subspecies of L. angustifolia.
Section Zollikoferia was separated into four clusters at 0.50 dissimilarity level. Cluster "3" comprised the two subspecies of L. mucronata; cassiniana and mucronata. While, cluster "4" aggregated groups 13 and 10 of L. fragilis subsp. fragilis, and the other specimens of the latter subspecies were assembled below cluster "5". Finally, cluster "6" isolated L. angustifolia subsp. angustifolia.
The distinction among the seventeen hypothetical groups is performed also by the Principle Coordinate Analysis (PCoA) (Figure 10). The first and second axis explained 31.56% and 28% of the variance, respectively, which emphasize a moderate variation for the data. The taxa representing each section were assembled together. The widest range of variation was noticed in section Zollikoferia, especially among the taxa of L. fragilis subsp. fragilis and between the two subspecies of L. angustifolia. Taxonomy 2021, 1, FOR PEER REVIEW 8

Discussion
The current study is the first comprehensive taxonomic study on Launaea in Egypt based on morphological data. R. tingitana was used as an outgroup. The outgroup can be defined as a more distantly related group of taxa that serves as a reference group when determining the phenetic relationships of the in-group [25]. According to Kilian et al. [14], Reichardia and Launaea are the most closely related genera within Hyoseridinae. The

Discussion
The current study is the first comprehensive taxonomic study on Launaea in Egypt based on morphological data. R. tingitana was used as an outgroup. The outgroup can be defined as a more distantly related group of taxa that serves as a reference group when determining the phenetic relationships of the in-group [25]. According to Kilian et al. [14], Reichardia and Launaea are the most closely related genera within Hyoseridinae. The

Discussion
The current study is the first comprehensive taxonomic study on Launaea in Egypt based on morphological data. R. tingitana was used as an outgroup. The outgroup can be defined as a more distantly related group of taxa that serves as a reference group when determining the phenetic relationships of the in-group [25]. According to Kilian  [14], Reichardia and Launaea are the most closely related genera within Hyoseridinae. The detailed characterization of Launaea sections and the species in each one, as well as the taxonomic status for each species, will be summarized in the following part.

Section Acanthosonchus
It is a monospecific section containing L. spinosa, although Zareh et al. [26] have added L. acanthodes (Boiss.) Kuntze as a new record, based on one herbarium specimen (kept in ASTU herbarium) from Wadi Telah in Sinai (7.10.1983). In the current study, this specimen is correctly identified as Lactuca orientalis (Boiss.) Boiss. (Syn. Scariola orientalis Boiss.), which is characterized by spinescent shrub and woolly-floccose white stems of green stripes, as well as the sessile heads with up to 5 florets per capitulum and the achene of 8 main ribs on each face with scabridulous pappus [4,5,27]. That is in agreement with Kilian [7], as Lactuca orientalis is confusing and similar to L. acanthodes.

Section Microrhynchus
Launaeaintybacea, L. massauensis and L. nudicaulis belong to section Microrhynchus. Launaea intybacea was translocated from Lactuca to Launaea as Launaea fallax or Launaea goraeensis [4,30]. It is a very rare species, and it was not recorded in the Egyptian collection of Kilian [7] and Boulos [5]. The present study includes a unique sample deposited in the CAI Herbarium. The sample was collected from Wadi Laseitit (Halayeb Triangle) by Täckholm in 1962. For Launaea massauensis, the results support involving it with L. intybacea and L. nudicaulis in the same section. This finding corresponds to Kilian [7] for assorting L. massauensis in Microrhynchus instead of Brachyrrhampus DC, as was classified by Boissier [31].
Launaea nudicaulis is a common polymorphic species in Egypt [4,5,30]. The investigation of the vegetative and floral characters reveals significant variation resulting from many intermediate forms. The most distinguished characters are the leaf size and shape, capitulescence type, floret dimensions, achene size, color and the degree of wrinkles that decrease in the inward direction. The description coincides with the studies of Amin [6], Kilian [7] and Alavi [32], where the achenes are variable in width, color and degree of wrinkles and achieve a gradual decrease towards the head center.
Two biotypes are distinguished morphologically: biotype 1 (group 6) and biotype 2 (group 7). For biotype 1, the capitulescence is either divaricate or divaricate with a cluster. The marginal achenes are subcuneate of 2.05-3.35 × 0.30-0.70 mm, with long incurved or recurved wrinkles of acute apex, and epidermal cells among wrinkles with short acute projections. The inner achenes are cylindrical of 2.05-3.40 × 0.25-0.60 mm. They are either compressed or uncompressed with elongated adjusted epidermal cells. The pappus is relatively short, of 5.85 to 9.95 mm, and the bristles reveal four different thicknesses with sparse protuberance density. The size of the pollen grains is 13.19-13.99 × 14.28-14.92 µm. The equatorial ridge varies from 4.89 to 5.56 µm, and the apocolpium diameter from 4.10 to 4.48 µm. The depth of abporal lacunae ranges from 2.31 to 2.71 µm, while that of paraporal lacuna is 1.97 to 2.50 µm. The paraporal ridge ornamentation is either spiny-perforate or spiny-granulate, and the number of spinules on the polar area ranges from 13 to 17.
For biotype 2, the capitulescence is divaricated. The marginal achenes are subfusiform of 2.40-3.90 × 0.30-0.65 mm, with long squarrose wrinkles of acute apex, and long acuminate projections of the epidermal cells among wrinkles. The inner achenes are compressed and columnar of 2.15-3.30 × 0.30-0.50 mm, and their epidermal cells are elongated nearly adjusted. The pappus ranges from 7.15 to 10.30 mm, and its bristles possess five different thicknesses of moderate protuberance density. The size of the pollen grains is 11.74-12.34 × 11.52-13.12 µm, the equatorial ridge is from 5.46 to 5.69 µm, and the apocolpium diameter is from 3.21 to 4.39 µm. The depth of abporal lacunae varies from 2.29 to 2.40 µm, whereas the depth of paraporal lacuna ranges from 1.97 to 2.04 µm. The paraporal ridge ornamentation is spiny, and the number of spinules on the polar area ranges from 13 to 15. The achene macromorphological characters of biotype 2 are congruent with the description of L. procumbens, mentioned by Alavi [32], Kilian [7], Boulos [5] and Qureshi et al. [33], excluding the deciduous homomorphic pappus.
Therefore, the present study suggests two biotypes of L. nudicaulis, one of which shares some macromorphological characters with L. procumbens, such as the subfusiform marginal achenes, the columnar inner achenes and the dimorphic pappus [34]. Determining the taxonomic rank of these biotypes requires freshly collected specimens for L. procumbens and more specialized molecular techniques, such as DNA barcoding, amplified fragment length polymorphism (AFLP), or microsatellites.
In the first subgroup, L. amal-aminiae N.Kilian was recognized by Amin [35] as a distinct and undescribed species. Kilian [7] identified this species according to its leafless flowering stems, slender capitula, and the different indumentum of the achenes. This description was based on five old specimens collected from Sinai, El-Arish, and Wadi Tundebar in Ras Benass (Red Sea) in 1849, 1855 and 1864, respectively. Besides, two specimens were collected from Suez since 1886 and 1908. Through personal communication with Prof. Amal Amin, she clarified that there were two voucher specimens representing this species, kept at the CAI Herbarium. These two specimens were collected from Suakin el Qadim, and from Halayeb Triangle. Nowadays, the two specimens no longer exist at the CAI Herbarium; also Suakin el Qadim became a part of Sudan. As a result, this species was not involved in the present study. The second subgroup, angustifolia, was represented in Egypt by L. angustifolia Muschl. with two subspecies: angustifolia and arabica. Launaea angustifolia subsp. angustifolia was first recorded in Egypt by Zareh et al. [26,36], whereas Kilian [7] declared that the species is restricted to a small area in NW Africa, and it had not been found in Egypt. Our study characterizes the subspecies with its papillose marginal achenes that showed long unconnate papillae in the form of two wings, glabrous inner, and achenes dimorphic pappus. An extensive field survey must be done to address the distribution of this species in Egypt.
Following Muschler [37], Montasir and Hassib [30], Täckholm [4] and Boulos [5], the description of L. angustifolia in Egypt has been applied to the subspecies arabica. Kilian [7] accepted Launaea arabica (Boiss.) H.Lindb. at the subspecies level; L. angustifolia subsp. arabica (Boiss.) N.Kilian. Whereas the current study considered L. arabica at the species level, that is confirmed through the numerical analyses using all types of data, separately and collectively. Marginal and inner achenes of Launaea arabica display long and linear connate papillae (hyaline papillae), and homomorphic pappus of cottony rays.
The third subgroup mucronata comprises both L. fragilis and L. mucronata. The two species are closely related with relative uniformity and some infraspecific variability [7]. Both taxa were treated as conspecific and were ranked as subspecies or varieties by various authors [32,38,39].
Launaea fragilis is characterized by their auriculated cauline leaves, semiamplexicaul or amplexicaul base, and auricle of either entire or dentate margin. The apices of the outer and middle involucre scales may be acute or acuminate. The inner achenes exhibit conspicuous secondary ribs, and horned base. The pappus is dimorphic with less than 100 setaceous bristles of moderate appressed aristate protuberance, while L. mucronata is specified with auriculated cauline leaves, amplexicaul base, and dissected auricle margin. The apices of the outer and middle involucre scale are acuminate. The inner achenes show ventricose truncate base with either conspicuous or inconspicuous secondary ribs. The pappus is subhomomorphic of setaceous bristles or dimorphic with less than 100 setaceous bristles. The bristles possess inclined aristate protuberance of high density.
The cluster analysis of the micromorphological characters illustrates the separation of groups 10 and 13 of L. fragilis subsp. fragilis with L. mucronata. These groups share some characters, such as the raised straight anticlinal walls of inner achenes, the channelled relief of cell boundary, and the flat outer periclinal walls with striate fine relief. Also, the ratio of polar area to pollen area is less than 40%, and the length spines is more than 1 µm. Some individuals of L. fragilis share characters with L. mucronata, such as the auriculated leaves, the large capitula, and the ventricose base of the inner achenes. These forms were initiated due to the hybridization between the two species, as they were sympatric in the Nile Delta and the Mediterranean coast in Egypt. In these regions, two cytotypes of L. fragilis were identified, diploid (2n = 16) and tetraploid (2n = 32) [7,29,40].
Launaea fragilis subsp. fragilis has demonstrated considerable plasticity and variability in many attributes related to its leaf, capitulum size, number of florets, achene and pappus. These variations were correlated with the ecological amplitude and the wide geographical distribution of the subspecies. Therefore, several names were published at the specific or the infraspecific rank, validly or invalidly, such as L. longiloba (Boiss. and Reut.) Maire, L. resedifolia Druce or L. tenuiloba Muschl. [4,30,41,42].
In Egypt, L. resedifolia was cited as a polymorphic species characterized with linear leaf lobes, the main ribs of the marginal achene are 5 to 6, and a silky pappus with a length up to 12 mm [4,6,32]. González [43] accepted the name L. fragilis for L. resedifolia and pointed out that the basionym of L. resedifolia has belonged to Scorzonera laciniata L. L. longiloba was ranked as an ecotype for L. fragilis and attributed its vigorous plants to the humidity and favorable conditions by Kilian [7]. Täckholm [4] and Feinburn-Dothan [44] classified L. tenuiloba at the species level. They have described this species by its filiform leaves with entire or dentate lobes, pappus length that is equal to the achene, and its inland distribution. Zareh et al. [26] allocated L. tenuiloba as a subspecies: L. fragilis subsp. tenuiloba (Boiss.) Zareh and Mohamed comb. et stat. nov., based on its various leaf lobes, and the pappus that is longer than the achene.
Our findings discriminate six intermediate forms correlated to the environmental factors without specific features. Consequently, the current study consents L. longiloba, L. tenuiloba and the intermediate forms as ecotypes in L. fragilis subsp. fragilis.
Launaea mucronata subsp. cassiniana has lanceolate rosette leaves, and the capitulum (5.70-5.90 × 4.20-5.03 mm) contains about 51 to 56 yellow florets. The compressed inner achenes have inconspicuous secondary ribs, and the epidermal cells have long antrorse projections. The pappus is easily removed and subhomomorphic with the setaceous bristles ranges from 20 to 30 and exhibits three different thicknesses. However, many authors have pointed out the presence of homomorphic pappus rather than the subhomomorphic type [4,5,31,45]. The pollen grain ornamentation on the paraporal ridge is spiny, and the number of spines on the polar area ranges from 3 to 5.

Conclusions
The combined investigations of the macro-and micromorphological traits are used to revise the classification of ten taxa of Launaea and to assess the inter-and intraspecific variation among taxa. The authors suggest the following points: (1) The taxonomic rank of Launaea angustifolia subsp. arabica must be raised to the species level. It is identified in the present study as Launaea arabica. (2) The elimination of L. acanthodes from the Egyptian flora, as it is identified correctly as Lactuca orientalis (Syn. Scariola orientalis). (3) Two biotypes of L. nudicaulis are distinguished morphologically. One of them shares some macromorphological characters with L. procumbens. Launaea procumbens needs further morphological and molecular investigations using freshly collected materials. (4) Launaea fragilis subsp. fragilis displays considerable plasticity and variability in various characters with intermediate forms, so they are considered ecotypes.