A Systematic Revision of the Genus Plectranthus L . ( Lamiaceae ) in Saudi Arabia Based on Morphological , Palynological , and Micromorphological Characters of Trichomes

This study aims to investigate the morphology and ultrastructure characters of pollen grains and trichomes in order to evaluate their systematic value of these characters in specific and intraspecific separation of the Saudi Arabian Plectranthus species. A critical systematic revision of 7 species of Plectranthus (Lamiaceae) in Saudi Arabia was conducted by means of numerical analyses based on thirty-one morphological characters, including vegetative parts, seeds, pollen grains, and trichomes. Macroand micro-morphological characters, including seed and pollen shape, size, coat sculpture, trichome structure, were studied. It reveals the presence of seven species, including two endemic species. The pollen grains were zonocolpate, hexacolpate, prolate to subprolate. Three types of exine ornamentation were recognized. Also, two trichome types could be distinguished and classified into glandular and non-glandular. The glandular trichomes could be distinguished as peltate, capitate and digitiform. The eglandular trichomes were single, uniseriate, multicellular. Pollen and trichome characters were found to be valuable, while seed characters presented only minor taxonomic value. On the basis of UPGMA clustering analysis four branches and clusters were distinguished. The results offer useful data for evaluating the taxonomy of Plectranthus both at subgeneric and sectional levels. Our results indicated some degree of similarity among the species of subgenus Burnatastrum. Plectranthus arabicus is considered as a separate group and may be treated as separate subgenus. Furthermore, the endemic species Plectranthus asirensis and


Introduction
Lamiaceae is a large family that is widely spread and in nearly all habitats and altitudes its species can be found.The genus Plectranthus L'Hér. is one of the largest genera of Lamiaceae, belonging to the subfamily Nepetoideae, tribe Ocimeae, subtribe Plectranthinae.It comprises about 300 species distributed in both tropical and warm regions of the Old World [1] [2].A phylogenetic study of the tribe Ocimeae, based on plastid genes to which Plectranthus belongs, showed that Plectranthus was paraphyletic [3].[4] [5] monographed the species of Plectranthus, and divided Plectranthus into seven sections: Germanea (Lam.)Benth., Coleoides Benth., Heterocylix Benth., Melissoides Benth., Isodon Schrad.ex Benth., Pyramidium Benth., and Amethvstoides Benth.However, in [6], Bentham latter revised this arrangement, recognizing two primary groups: Sect.Germanea and sect.Isodon.In sect.Germanea, with Germanea and Coleoides as subsections (in which the great majority of conventional Plectranthus spp.are placed), the calyx is 2-lipped with the upper lip consisting of a single broad tooth and the lower lip of four narrower acute or acuminate teeth: the cymes are usually sessile with the pedicels arising from the axis of the inflorescence.Sect.Isodon, with Isodon, Pyramidium, Amethvstoides and Melissoides as subsections, was distinguished by the calyx being equally 5-toothed, in some groups more or less 2-lipped with the upper lip composed of 3 teeth and the lower lip of 2 teeth: the cymes are pedunculate and branched.A similar classification was accepted by [7], however, Germanea and Isodon were treated as subgenera.In each subgenus the largest sections, Coleoides and Isodon, respectively, were subdivided into a number of series.[8] revised the genus in South Africa and based on the inflorescence characters he divided Plectranthus into five subgenera: Nodiflorus Codd, Burnatastrum (Briq) Codd, Coleus (Lour.)Codd, Calceolanthus Codd and Plectranthus based on the inflorescence characters.[9] reviewed the genus Plectranthus and its ethnobotanical used broadly, providing a complete understanding of the global ethno-botany of Plectranthus.Their review touches on the horticultural uses of the genus.In Saudi Arabia, Plectranthus species are used of economic and traditional medicine and have potential for development towards their use in the primary health care system.In Saudi Arabia, Plectranthus barbatus Andr. is the most important species of the genus, and is used as a remedy for stomach, intestine, liver disorders, heart problems, respiratory diseases and it is also resistant to insect attack [10].Also, P. tenuiflorus (Vatke) Agnew is cultivated as ornamental.The leaf extract of P. tenuiflorus is also used to treat ear infections, and the leaves of P. asirensis Wood J. R. I. and P. cylindraceus Benth.are used as antiseptic and deodorant dressing for wounds [11] [12].Some species of Plectranthus are problematic to identify because of a lack of clear cut morphological criteria to differentiate not only among species within the genus but also among the closely related genera.This has resulted in many taxonomic difficulties in the naming of species with the result that species have often been placed in some closely related genera such as Coleus Lour.[3].In the Flora of Saudi Arabia, [13] reported Plectranthus by seven species: P. arabicus Bruce E. A., P. cylindraceus, P. tenuiflorus, P. comosus Sims, P. barbatus, P. pseudomarrubioides Willemse R.H. and P. asirensis, but [14] accepted only 6 species viz.P. arabicus, P. cylindraceus, P. tenuiflorus, P. lanuginosus (Benth.)Agnew, P. barbatus, and P. asirensis.
Pollen morphology provide a number of characteristics that are potentially useful for species identification, phylogenetic implication, and character-state evolution [15]- [22].However, there are no works dealing with seed and pollen morphology of Plectranthus.Moreover, trichomes are broadly dispersed over the aerial reproductive and vegetative parts of the members of Lamiaceae and are generally distinguished as glandular and non-glandular trichomes.[17] found that trichome micromorphology was useful for systematics and reconstructing the phylogeny of Lamiaceae.In some genera of Lamiaceae, the trichome morphology is helpful in infrageneric classification [23] [24].Unfortunately, there is not much information on trichome morphology in Plectranthus.Only a few studies have reported on the structure and meaning of the trichomes occurring in Plectranthus and include the species Plectranthus madagascariensis Benth.[25], Plectranthus ornatus Codd [26], Plectranthus neochilus schltr.[27], P. laxiflorus Benth.[28] and P. tenuiflorus [29].Therefore, this study aims to investigate the morphology and ultrastructure characters of pollen grains and trichomes in order to evaluate their systematic value of these characters in specific and intraspecific separation of the Saudi Arabian Plectranthus species.

Plant Material
Plants were collected during the flowering stages from different places in Saudi Arabia (Appendix 1).The identification was simplified according to [13] [14], and the voucher specimens are deposited at the Herbaria of King Saud University, Kew and Edinburgh (KSU, K and E).

Pollen Grain and Seed Characters
Pollen samples of each studied species were composed from herbarium specimens.All investigations were carried out on acetolysed pollen grains according to [30].For light microscopy (LM), the pollen grains were observed by an Olympus type BH-2.Photomicrographs were taken with an Olympus photomicroscope.The measurements are based on 20 readings from each slide.The polar axis (P), equatorial diameter (E) and P/E ratio were calculated.For scanning electron microscopy (SEM), acetolysed pollen grains were dehydrated in ethanol sequences and mounted on a metallic stub in few drops of ethanol.The specimens were coated with gold in Apolaron E1100 ion sputtering Device, then viewed at 25 -30 KV in a JOEL JSM 5300 scanning electron microscope of the Central Laboratory Faculty of Science, Sohag University, Egypt.The terminology and main morphological ideas are based on [31]- [33].
Only mature seeds were taken for investigation.The dried seeds were examined by light microscope (Olympus type BH-2), and 10 -15 seeds for each taxon were chosen to cover the range of variation.The terminology used here follows authors such as [15] [16] [34].

Trichome Morphology
For trichomes morphology, dried specimens were used.Small portions of leaves or stems were taken and mounted on a stub using double adhesive tape.The specimens were processed as described above.The terminology used follows [25] [26] with some modification.

Morphological Characters Observations
Table 3 shows the characters and character states scored for plant, pollen, trichome and seed morphology, averaged for each OTU.In total 31 characters were taken into consideration, comprising 5 quantitative and 26 qualitative characters.Fifteen of the qualitative characters were scored as binary and the remaining were scored as multi-state characters.

Data Analysis
One type of analyses was performed with NTSYS-pc 2.02k software (Applied Biostatistics Inc., Setauket, New York, USA).I performed a cluster analysis using average taxonomic distance and UPGMA clustering (procedures SIMINT, SAHN, and TREE).To decrease the effects of different scales of measurement for different characters, the values for each character were standardized with procedure STAND, according to the formula: yI, STD = (yi − AVGyi)/STDyi), Where the default value in NTSYS-pc (STAND) for yi = the value to be standardized, AVGyi = the average of all values for the character, and STDyi = the standard deviation.The cophenetic relationship coefficient between the distance matrix and the tree matrix was calculated to examine the goodness of fit of the cluster analysis to the distance matrix (procedures COPH and MXCOMP) [35].

Seed Characters
The seed morphological characters of the studied taxa of the genus Plectranthus as shown by light microscopy are presented in (Table 1).

Seed Shape
The seed shape varies from ovoid in P. asirensis, P. barbatus and P. hijazensis Abdel Khalik, to sub-spherical in the P. arabicus, P. cylindraceus, P. pseudomarrubioides and P. tenuiflorus.

Seed Color
The colors of the seeds are of low diagnostic and systematic value among species.The colors vary from reddish-brown in P. arabicus, P. asirensis P. hijazensis and P. tenuiflorus, yellowish-brown in P. cylindraceus and P. pseudomarrubioides, to black in P. barbatus.

Pollen Grain Characters
The main features of the investigated pollen grains are summarized in (Table 1).Selected LM and SEM micrographs of pollen grains studied are presented in (Figures 1-3).

Pollen Shape
The ratio between the mean polar axis (P) and the mean equatorial diameter (E) can be used to assign the pollen grains to shape classes.
In the examined species, there is no wide variation in pollen shape.Pollen grains are commonly sub-prolate or prolate.It is prolate in P. tenuiflorus (Figure 2(A7), Figure 2(B7)) and subprolate in the rest of the taxa (Figures 1-3).

Pollen Apertures
Apertures are simple.All species investigated are zonocolpate.The colpi are narrow to slit-like after acetolysis, they are usually widest at the equatorial view and gradually narrow towards the poles.The number of colpi is hexacolpate.

Exine Sculpture
The exine ornamentation has a complex structure.Generally, the sexine is thicker than nexine.The tectum of the pollen grains in almost all species examined is double-reticulate.The lumina of the main reticulum are in filled by a substratum that is either perforate or reticulate.The main reticulum is referred to as the primary reticulum  Type (B): Granulate-perforate.The primary lumina are reticulate and the secondary one is granulate-perforate in P. barbatus (Figure 1

Basic Types of Trichomes
Observations with Light and Scanning Electron Microscope (LM and SEM) revealed that non-glandular and glandular trichomes on the plant surfaces (Figure 4).

Non-Glandular Trichomes
The eglandular trichomes are single, uniseriate, multicellular, pointed, erect or leaning towards the leaf apex.It consists of two to eight cells.Each non-glandular trichome appears to be supported by a basal cellular pedestal cells.SEM observation revealed the presence of two types of non-glandular trichomes-branched with two fids in P. arabicus (Figure 4(5)) and unbranched trichomes which is consisted of 2 -8 cells in all the species of Plectranthus (Figure 4(1-4)).

Glandular Trichomes
The glandular trichomes are of three main types: 1) Peltate trichomes This type consists of a basal epidermal cell, a very short stalk cell and a large round head in P. cylindraceus and P. pseudomarrubioides (Figure 4(8)). 2

) Capitate trichomes
This type with long stalked possesses one basal cell, along two to three celled stalk variable length and a unicellular bulb-shaped head in P. hijazensis (Figure 4(7)).
3) Digitiform trichomes These consist of two to three cells, in line of similar diameter and approximately equal length.There is no clear distinction between head and stalk cells.Therefore, these glandular trichomes are made up of one basal cell, one or two stalk cells and one apical secretory (head-like) cell in P. tenuiflorus (Figure 4(6)).

Cluster Analysis
The results of the cluster analyses are presented in (Figure 5, Table 2).In the UPGMA dendrogram, 4 major branches and clusters (A-D) with approximately 97% similarity are distinguished: 1) branch A includes only Plectranthus arabicus; 2) branch B contains only P. barbatus; 3) group C comprises P. cylindraceus and P. pseudomarrubioides; and 4) group D includes group of P. asirensis, P. hijazensis and branch of P. tenuiflorus.The subgenera and sections show intra variability among themselves.In general, UPGMA indicates that our study follows the currently applied subgenera sectional classification of Plectranthus by [3]

Discussion
Many authors have tried to provide an accepted system to split the genus Plectranthus into subgenera, sections and series [3]- [8], see Table 2).These studies were based on 1 or 2 characters from these morphological characters such as calyx forms, flower, and inflorescence structure.In the present study a large number of morphological, palynological, seed and trichomes characters were used.In general, the results show that different patterns of the studied characters are helpful in distinguishing various species (Table 3); they do not confirm the subgenera and sectional classification of the genus Plectranthus proposed by Bentham's classifications [4]- [7] but confirm somewhat the subgenera and sectional classification of Plectranthus by [3] [8].
In branch A (P. arabicus) Within branch A, [6] treated P. arabicus and P. asirensis as a separate section Isodon.[7] treated these species as subgenus Isodon.Our results do not support the placement of P. arabicus with P. asirensis in the subgenus (section) Isodon.This is due to the placement of P. arabicus within a separate branch with high genetic similarities and distinguishing from the rest of the species by having annual life form; plant length (15 cm); watery-succulent; sessile leaves; Branched and glandular multicellular hairs.
[37] investigated anatomical characters of the stem and leaf in Plectranthus and distinguished P. arabicus from the rest of the species by having terete stem, narrow cortex with only parenchyma, 6 bundles, and covered with bi-multicellular and branched non glandular hairs.Generally, these results disagree with those of [6] [7] regarding placement of P. arabicus and P. asirensis in an enlarged concept of subgenus Isodon section Isodon and agree with [37] to suggest that P. arabicus should be treated as separate subgenus.

Subgenus Calceolanthus Codd (P. barbatus) (branch B)
Within branch B, P. barbatus corresponds to previously recognized position within subgenus Germanea section Germanea [6] [7].However, [8] reviewed the genus and he put this species within subgenus Calceolanthus.Furthermore, [3] presented a phylogenetic analysis of basils and allies using the DNA sequence of the chloroplast trnL, trnL-trnF inter gene spacer and rps 16.They indicated that Plectranthus is paraphyletic, and within Plectranthus, they show that P. barbatus clade is well supported (98% support).Moreover, [38] analyzed the interspecific diversity among four species of Plectranthus: P. grandis (Cramer L. H.) Willemse R. H., P. barbatus, P. neochilus Schltr., and P. amboinicus (Lour.)Spreng., and the intraspecific diversity of P. barbatus in southern Brazil, by means of the RAPD technique.They observed a higher genetic similarity between P. grandis and P. barbatus (77%), and they showed P. barbatus genotypes from different locations in Brazil showed a genetic similarity 96% -100%.The results do not support the situation of P. barbatus with other taxa in the subgenus (section) Germanea.This is due to the placement of P. barbatus within a distinct branch with high genetic similarities and split from the rest of the subgenera on the basis of the species with quadrangular stem, not succulent leaf, terminal inflorescence, black seed, exine sculpture of pollen grain with primary lumina is reticulate and secondary is granulate-perforate.These results disagree with those of [6] [7], but agree with [3] [8] [38].
Plectranthus cylindraceus and P. pseudomarrubioides match to the previously recognized position within subgenus Germanea section Germanea [6] [7].Furthermore, [8] treated these species as subgenus Calceolan-thus.[3] presented a phylogenetic investigation of basils and allies using the DNA sequence of the chloroplast trnL, trnL-trnF, rps 16 and augmented by morphological data.They illustrated that P. cylindraceus and P. buchananii Baker was separated from the rest of the species of Plectranthus with 98% support.[9] studied the phylogeny of 62 species of Plectranthus and its ethno-botanical uses extensively.They provided an informal classification that separated the species into two main clades.Clade 1, the Coleus clade of [3], broadly corresponding to the formally recognized genus Coleus, is divided into two subclades, clades 1a and b.Clade 2 is recognized as the Plectranthus clade.Within subclade 1b, they proved that P. cylindraceus and P. pseudo-marrubioides are well separated (group 7) from the rest of taxa.
Moreover, [10] surveyed 34 species of Plectranthus for exudate flavonoids to see whether the scattering of these compounds would support a recent classification of the genus based on molecular and morphological characters.They identified two major groups the Coleus and Plectranthus clades.They found that flavanones were restricted to only five species of the Plectranthus, and flavonols were only found in two species of the Coleus clade, P. cylindraceus and P. pseudomarrubioides.Generally, these data are support and congruent with those of [3] [6]- [10].
Subgenus Coleus (Lour.)Codd (group D) Within group D, one major cluster and branch with 0.97 similarities were identified.The first cluster includes the endemic P. asirensis and P. hijazensis.However, the second branch includes P. tenuiflorus treated as section Aromaria Benth.that belongs to the genus Coleus, as subgenus Germanea, and subgenus Coleus that belong to Plecranthus.[9] treated P. tenuiflorus, P. asirensis and other connected within one group (group 8) but in two separated sub-clades (1a and 1b).
Inside the cluster of the endemic P. asirensis and P. hijazensis have been recognized with 0.45 morphological similarities.These species can be clearly defined on the basis of various structures: sub-shrubs, not succulent, erect, woody stem at the base, terete to quadrangular stem outline, petiolate leaves, serrate leaf margins, bracts deciduous, ovoid seed, seed size (1.2 -1.5 × 0.8 -1.2).Moreover, [37] showed that the anatomical characters of the stem and leaf might be useful in distinguishing P. asirensis and P. hijazensis from the rest of the species by their shared terete to undulate stem, wide cortex with angular collenchyma and parenchyma, numerous bundles.
An another branch of the species represented by P. tenuiflorus shares the same densely hairs surface, woody stem, terete to quadrangular stem outline, petiolate leaves, terminal inflorescence, bract deciduous, hairy with glandular sepal surface, reddish-brown seed.
Current observations confirmed the possibility that P. asirensis and P. hijazensis should be a monophyletic group and there are close relationships between these species and P. tenuiflorus subgenus Germanea, and subgenus Coleus and these results are in agreement with the results of [8] [9] [37].

Conclusion
A taxonomic revision of seven Plectranthus species (Lamiaceae) in Saudi Arabia was conducted by means of numerical analysis based on thirty-one morphological characters, including vegetative parts, seeds, pollen grains, and trichomes.Macro-and micro-morphological characters, including seed and pollen shape, size, coat sculpture, trichome structure, were studied.Two endemic species exist.The data presented here considerably underline the importance of seed, pollen and trichome characters within the genus Plectranthus both on subgeneric and sectional levels.Some species of Plectranthus have similar trichome shapes, pollen grains and seed characters, but are quite different in gross morphology and can easily be recognized as distinct species.The molecular systematic study on Plectranthus [3] [9] including some species distributed in Saudi Arabia, allows an interpretation of the usefulness of pollen, trichome and seed characters in Plectranthus, these can be considered as phylogenetic informative characters.Our results indicate some degree of similarity among the species of subgenus Burnatastrum (Briq) Codd and support the monophyly of the subgenus as suggested by [8] [9].A remarkable result from this study was identifying P. arabicus as a separate group and suggesting that it should be treated as a separate subgenus.Furthermore, the endemic P. asirensis and P. hijazensis appear to be a homogeneous group and there are close relationships between this group and P. tenuiflorus.A key for the identification of the investigated taxa based on studied characters is provided.

Figure 5 .
Figure 5. Dendrogram illustrating the relationships of the investigated species of Plectranthus based on morphological characters.

Table 1 .
Seed, pollen and trichome characters of the investigated taxa of Plectranthus in Saudi Arabia.

Table 3 .
Characters and character states used in morphometric analysis of the Plectranthus.