Paton et al. [
Leaf micromorphology may provide insights into how plant species evolve in terms of leaf anatomy and respond to different climatic conditions. Morphology of epidermal cells, stomata, and trichome types have been used as systematic characters and a combination of some of these features is relevant especially to the identification of species within the tribe Mentheae (subfamily Nepetoideae) in Labiatae family [
With regard to leaf anatomy, members within the Labiatae family have diacytic and anomocytic stomata both on the abaxial and adaxial surfaces although tetracytic stomata have been observed in the family as well [
With regard to organization of the leaf chlorenchyma, isobilateral, centric, or dorsiventral mesophyll has occasionally been reported in Lamiaceae family [
Description of the ten
Species | Succulent leaf | Height (m) | No. of inflorescence Flowers | Cymes | Pedicels length (mm) | Calyx length (mm) | Corolla length (mm) | Petiole length (mm) | Leaf apex | Bracts | Nutlet color | Staminal filaments | Leaf blade | Succulent stem | Eglandular hairs |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
|
+ | 1–5 | 10–14 | 5–7 | 3–7 | 3–5 | 8–26 | 3–50 | Ob/rd | ov/api | Brown/black | Fused | El/ov | + | An/pt |
|
+ | 0.1–0.6 | 6 | 3 | 2 | 3–5 | 6–14 | 3–10 | Ob/rd | ov | Grey/black | Fused | El/ov | − | Rt/pt |
|
+ | 0.4–2.5 | 10–40 | 5–20 | 2-3 | 1.5 | 3–5 | 0 | Ob/rd | El/ov | Brown/red | Free | El/obv | + | Rt/pt |
|
+ | 0.3–0.5 | 6 | 3 | 6-7 | 5 | 11–15 | 3 | Ob/rd | Obv/El | Brown/red | Fused | Ov/El/Obv | − | pt |
|
+ | 0.3–1 | 6–12 | 3–6 | 3–6 | 3-4 | 8–16 | 5–30 | Ob | EL | Brown/black | Fused | Triangular | − | pt |
|
+ | 1 | 10–14 | 5–7 | 1–3 | 2 | 5–9 | 1–15 | Ob | El | Brown/red | Free | Rhombic | + | Rt/pt |
|
− | 0.3–1.2 | 6–10 | 3–5 | 3–5 | 2-3 | 6–8 | 1–20 | Ob/rd | ov | Brown/black | Fused | Ov | − | Unknown |
|
+ | 0.4–1.5 | 6 | 3 | 3 | 5-6 | 14–22 | 2–10 | Ob/rd | ov | Grey/black | Fused | El/ov | − | An/pt |
|
+ | 0.5–2.5 | 6–24 | 3–12 | 3–10 | 2-3 | 8–15 | 5–20 | Ac/rd | ov | Brown/red | Fused | Triangular | − | An/pt |
|
+ | 0.3–0.5 | 10–20 | 5–10 | 3 | 3 | 4–15 | 2–13 | Ob/rd | Ov/api | Brown/red | Fused | Ov | + | rt/pt |
Morphological characters obtained from the latest
Leaves and voucher specimens of ten
DNA was extracted from fresh leaves of the collected
All the
One centimeter cubed of leaf tissue was removed from the middle section of the leaves of three individuals of each
To study leaf anatomy, portions of leaf lamina from three individuals of each species were cut into 4–10 mm2 sections prior to rehydration. Tissues were then rehydrated in a series of 25% alcohol, 10% alcohol, and distilled water followed by staining in saturated aqueous safranin O. The stained tissues were then dehydrated in an alcohol series, 50% alcohol and 100% Xylene. The dehydrated tissues were then infiltrated in a series of xylene : paraffin oil (50% : 50%) and 100% paraffin oil followed by a second series of molten paraplast. The infiltrated tissues were later embedded in paraffin in casting boats. Paraffin embedded blocks were then sectioned transversely at 2
Phenetic analysis (cluster and principal components) is commonly used as a tool to better understand the trends of morphological variations, by indicating relationships among taxa inform of dendrograms or cladograms [
After DNA sequencing,
GenBank accession numbers for genes from the ten
|
Gene | GenBank accession number | Gene | GenBank accession number |
---|---|---|---|---|
|
|
MF495684 |
|
MF495698 |
|
|
MF495685 |
|
MF495696 |
|
|
MF495686 |
|
MF495700 |
|
|
MF495687 |
|
MF495701 |
|
|
MF495688 |
|
MF495699 |
|
|
MF495689 |
|
MF495703 |
|
|
MF495690 |
|
MF495695 |
|
|
MF495691 |
|
MF495702 |
|
|
MF495692 |
|
MF495697 |
|
|
MF495693 |
|
MF495694 |
Aligned sequences were used in the creation of phylogenetic trees following maximum composite likelihood model and employing UPGMA and Bootstrap resampling as a statistical method and as a test of phylogeny, respectively. The most likely phylogenetic trees were constructed using gene sequences from the ten
A bootstrap consensus phylogenetic tree based on
The numbers at branch nodes are bootstrap values which indicate percentage number of bootstrap iterations which support the tree at that particular point of divergence/at each node of the phylogenetic tree. The higher the bootstrap value, the more the topology of the phylogenetic tree is supported. Species close to each other in the phylogenetic tree are closely related. Based
Based on the
Both
Overall variations in leaf anatomical characters observed in three individuals of each of the ten
Leaf micromorphology in the ten
Species | Nature of the leaf | Stomata occurrence | Stomata type | Epidermal cell type | Trichome type | Glandular trichomes | Mesophyll | Palisade layer | Average number of stomata on the abaxial surface | Average number of stomata on the adaxial surface |
---|---|---|---|---|---|---|---|---|---|---|
|
Succulent | Ab/Ad | Anomocytic | Polygonal | Long- stalked | Present | Dorsiventral | Long columnar cells | 35 | 25 |
|
Succulent | Ab/Ad | Anomocytic | Sinuous | Short-stalked | Present | Homogenous | Inconspicuous | 50 | 36 |
|
Nonsucculent | Ab/Ad | Anomocytic | Polygonal | Short-stalked | Absent | Dorsiventral | Long columnar cells | 47 | 35 |
|
Succulent | Ab/Ad | Anomocytic | Sinuous | Short-stalked | Present | Homogenous | Inconspicuous | 36 | 25 |
|
Succulent | Ab/Ad | Anomocytic | Polygonal | Capitate | Present | Dorsiventral | Long columnar cells | 45 | 39 |
|
Succulent | Ab/Ad | Anomocytic | Polygonal | Capitate | Present | Homogenous | Inconspicuous | 58 | 49 |
|
Succulent | Ab/Ad | Anomocytic | Sinuous | Short-stalked | Present | Homogenous | Inconspicuous | 28 | 20 |
|
Succulent | Ab/Ad | Anomocytic | Sinuous | Short-stalked | Present | Homogenous | Inconspicuous | 28 | 23 |
|
Succulent | Ab/Ad | Anomocytic | Polygonal | long-stalked | Present | Dorsiventral | Long columnar cells | 35 | 27 |
|
Succulent | Ab/Ad | Anomocytic | Sinuous | Short-stalked | Absent | Homogenous | Inconspicuous | 45 | 33 |
Sinuous: containing many curved edges,
Leaf anatomical characters which contributed to most of the differences among the ten
Leaf micromorphology data was subjected to hierarchical cluster analysis. Character states were assigned values and scored in a matrix, the matrix (Table
Leaf micromorphology character states matrix of the ten
|
Leaf micromorphology characters from the ten | ||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|
Succulent leaf | Stomata distribution | Stomata type | Epidermal Cell type | Trichome type | Glandular Trichomes | Nature of mesophyll | Palisade layer | Stomata no. (adaxial) | Stomata no. (abaxial) | ||
|
1.00 | 3.00 | 1.00 | 1.00 | 2.00 | 1.00 | 1.00 | 1.00 | 2.00 | 3.00 | Character state scores/values |
|
1.00 | 3.00 | 1.00 | 2.00 | 2.00 | 1.00 | 2.00 | 2.00 | 3.00 | 4.00 | |
|
2.00 | 3.00 | 1.00 | 1.00 | 2.00 | 2.00 | 1.00 | 1.00 | 3.00 | 4.00 | |
|
1.00 | 3.00 | 1.00 | 2.00 | 2.00 | 1.00 | 2.00 | 2.00 | 3.00 | 3.00 | |
|
1.00 | 3.00 | 1.00 | 1.00 | 1.00 & 3.00 | 1.00 | 1.00 | 1.00 | 3.00 | 4.00 | |
|
1.00 | 3.00 | 1.00 | 1.00 | 1.00 & 3.00 | 1.00 | 2.00 | 1.00 | 4.00 | 5.00 | |
|
1.00 | 3.00 | 1.00 | 2.00 | 2.00 | 1.00 | 2.00 | 2.00 | 2.00 | 2.00 | |
|
1.00 | 3.00 | 1.00 | 2.00 | 2.00 | 1.00 | 2.00 | 2.00 | 2.00 | 2.00 | |
|
1.00 | 3.00 | 1.00 | 1.00 | 1.00 & 3.00 | 3.00 | 1.00 | 1.00 | 2.00 | 3.00 | |
|
1.00 | 3.00 | 1.00 | 2.00 | 2.00 | 2.00 | 2.00 | 2.00 | 3.00 | 4.00 |
Succulent leaf: 1 (present), 2 (absent), stomata occurrence: 1 (adaxial surface), 2 (Abaxial surface), and 3 (both surfaces), stomata type: 1 (anomocytic), epidermal cell type: 1 (sinuous), 2 (polygonal), trichome type: 1 (long-stalked), 2 (short-stalked), and 3 (capitate), glandulartrichomes: 1 (present), 2 (absent), and 3 (both glandular and nonglandular trichomes present), mesophyll: 1 (homogenous), 2 (dorsiventral), palisade layer: 1 (normal long columnar cells), 2 (inconspicuous), stomata number on the adaxial surface: 1 (<20 stomata), 2 (20–30 stomata), 3 (30–40 stomata), 4 (40–50 stomata), and 5 (>50 stomata), stomata number on the abaxial surface: 1 (<20 stomata), 2 (20–30 stomata), 3 (30–40 stomata), 4 (40–50 stomata), and 5 (>50 stomata).
Hierarchical cluster analysis of the leaf micromorphology character state matrix in Table
Using gross morphological characters of the ten
Gross morphology character state matrix of the ten
|
Gross morphological characters from the ten | |||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Succulent leaf | Height | Inflorescence | Cymes | Pedicel length | Calyx length | Calyx nature | Corolla length | Corolla colour | Leaf apex | Bracts | Nutlets | Staminal filaments | Leaf blade | Petiole | Stems | Stems colour | Eglandular hairs | Stems shape | ||
|
1.00 | 4.00 | 2.00 | 2.00 | 3.00 | 3.00 | 1.00 | 3.00 | 4.00 | 1.00 | 1.00 | 2.00 | 1.00 | 1.00 | 3.00 | 1.00 | 1.00 | 1.00 | 1.00 | Character state scores/values |
|
1.00 | 2.00 | 1.00 | 1.00 | 1.00 | 3.00 | 1.00 | 2.00 | 1.00 | 1.00 | 2.00 | 3.00 | 1.00 | 1.00 | 1.00 | 2.00 | 3.00 | 2.00 | 1.00 | |
|
1.00 | 1.00 | 3.00 | 3.00 | 1.00 | 1.00 | 2.00 | 1.00 | 1.00 | 1.00 | 3.00 | 1.00 | 2.00 | 1.00 | 4.00 | 1.00 | 2.00 | 3.00 | 1.00 | |
|
1.00 | 2.00 | 1.00 | 1.00 | 3.00 | 3.00 | 1.00 | 2.00 | 2.00 | 1.00 | 4.00 | 1.00 | 1.00 | 1.00 | 1.00 | 2.00 | 3.00 | 2.00 | 1.00 | |
|
1.00 | 3.00 | 2.00 | 1.00 | 2.00 | 2.00 | 3.00 | 1.00 | 3.00 | 2.00 | 1.00 | 2.00 | 1.00 | 2.00 | 3.00 | 2.00 | 3.00 | 2.00 | 1.00 | |
|
1.00 | 3.00 | 2.00 | 2.00 | 1.00 | 1.00 | 3.00 | 2.00 | 4.00 | 2.00 | 5.00 | 1.00 | 2.00 | 3.00 | 2.00 | 1.00 | 1.00 | 3.00 | 1.00 | |
|
2.00 | 4.00 | 1.00 | 1.00 | 2.00 | 2.00 | 1.00 | 1.00 | 4.00 | 1.00 | 1.00 | 2.00 | 1.00 | 1.00 | 2.00 | 2.00 | 3.00 | 4.00 | 1.00 | |
|
1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 3.00 | 4.00 | 1.00 | 2.00 | 3.00 | 1.00 | 1.00 | 1.00 | 2.00 | 3.00 | 1.00 | 1.00 | |
|
1.00 | 1.00 | 3.00 | 3.00 | 3.00 | 2.00 | 3.00 | 2.00 | 2.00 | 3.00 | 2.00 | 1.00 | 1.00 | 2.00 | 2.00 | 2.00 | 1.00 | 1.00 | 1.00 | |
|
1.00 | 2.00 | 2.00 | 2.00 | 1.00 | 2.00 | 4.00 | 2.00 | 2.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 2.00 | 1.00 | 1.00 | 3.00 | 1.00 |
Succulent leaf: 1 (present), 2 (absent), height: 1 (<0.3 m), 2 (0.3–0.5 m), 3 (0.5–1 m), and 4 (>1 m), inflorescence: 1 (with <10 flowered verticils), 2 (with 10–20 flowered verticils), and 3 (with >20 flowered verticils), cymes: 1 (<5 flowered), 2 (5–10 flowered), and 3 (>10 flowered), pedicel length: 1 (<3 mm long), 2 (3–6 mm long), and 3 (>6 mm long), calyx length: 1 (<2 mm long), 2 (2–4 mm long), and 3 (>4 mm long), nature of calyx: 1 (pubescent), 2 (tomentose), 3 (villose), and 4 (pubescent to villose), corolla length: 1 (<10 mm long), 2 (10–20 mm long), and 3 (>30 mm long), corolla colour: 1 (blue to purple with white marks), 2 (white with white marks), 3 (pale blue to lilac), and 4 (blue with purple marks), leaf apex: 1 (obtuse to rounded), 2 (obtuse), and 3 (acute to rounded), bracts: 1 (ovate, apiculate), 2 (ovate), 3 (ovate to elliptic), 4 (obovate to elliptic), and 5 (broadly elliptic), nutlets: 1 (brown with reddish dots), 2 (brown with dark dots), and 3 (dark grey with dark dots), staminal filaments: 1 (fused), 2 (free), Leaf blade: 1 (elliptic, ovate, obovate), 2 (triangular), and 3 (trullate, rhombic), petiole length: 1 (<10 mm long), 2 (10–20 mm long), 3 (20–50 mm long), and 4 (epetiolate/sessile), stem: 1 (succulent), 2 (nonsucculent/soft wooded), stem colour: 1 (sometimes purplish above), 2 (pinkish to dark red), and 3 (unknown), eglandular hairs: 1 (antrose and patent), 2 (patent), 3 (retrose and patent), and 4 (unknown), stem shape: 1 (rounded and quadrangular).
Hierarchical cluster analysis of the above gross morphology character state matrix (Table
Two main clusters/groups are evident from the dendrogram above which occur at about the same horizontal distance. One cluster (H) contains
Based on the current study, the
A bootstrap consensus phylogenetic tree based on
(a) Transverse section of
Transverse section of
Trichomes identified in
Leaf micromorphology average linkage dendrogram.
Average linkage dendrogram showing taxonomic relationships of the ten
Comparison of the two trees based on the
A dendrogram produced from the cluster analysis of morphological characters described in the Flora of Tropical East Africa (Figure
Grayer investigated the distribution of exudate flavonoids in
Leaf micromorphology in the present study involved investigation of the internal leaf anatomy and stomatal distribution of the ten
Epidermal cell type, type of stomata, type of trichome, and the nature of the mesophyll have been used before to differentiate
Organisms grouped together in a dendrogram imply that they are more similar morphologically. In most cases similarity based on morphology is related to genetic similarity because traits are determined by genes and a similar character in different plants strongly suggests that there is a shared gene by those species possessing the morphological character. Hence, it can be assumed that, the more close the species are grouped, the more recently they diverged from a common ancestor. Morphological characters are often affected by environmental factors and morphological variation seen in species can either be environmentally induced or genetically based [
As mentioned before, a dendrogram produced from cluster analysis of gross morphological characters described in the Flora of Tropical East Africa (Figure
Findings of the leaf micromorphology study have been supported by previous classification based on their ethnobotanical uses where most of the species belonging to
Morphological characters have been employed widely in the identification and classification of plants. In closely related species, these morphological characters may differ by a small margin and it may not be easy to identify and classify closely related species. Studies on leaf micromorphology are important sources of taxonomic characters for classification of plants which are difficult to delimit. Some of these characters are of diagnostic value and have been used successfully in infrageneric and interspecific classification in plants. Most morphological characters overlap when we look at species at the lower taxonomic ranks especially in genus and subgenus levels making it hard to classify species based on the general morphological characters. As a result diagnostic morphological characters are vital when it comes to morphology based phylogenetic studies. Micromorphological characters are examples of such diagnostic characters which have proved vital in the delimitation of species below the genus level [
Based on the current leaf micromorphology study epidermal cell type, type and nature of trichomes, mesophyll type, and palisade layer have proved to be vital diagnostic characters for differentiating the ten species investigated, while characters such as the presence or absence of a succulent leaf, type of stomata, number of stomata, and occurrence of stomata have been shown to be less important in differentiating the ten species. Dendrogram produced from the leaf micromorphology characters classifies the ten
With regard to molecular phylogeny, phylogenetic trees reconstructed based on the
From the leaf micromorphology study,
Analysis of morphological data through cluster analysis groups species together based on their overall similarity and this grouping can be presented in form of cladograms, dendrograms, or even phylogenetic trees. Morphological characters have various shortcomings especially when used to delimit species at the genus level. Most of these characters overlap and it’s hard to identify the diagnostic characters which can be used to differentiate one species from the rest. It has been argued that the most suitable method for classifying species based on morphological data has not yet been decided and different methods applied on the same data can result in different classifications [
Although molecular and biochemical data have many advantages and their increase is being used in systematic studies, it is important that morphological systematics should not be entirely abandoned because they can aid in the classification of poorly known groups as well as study of fossil data [
Further research involving more
Morphological characters are affected by environmental factors and the same morphological character within the same species can be pronounced or decreased depending on the ecological zone where the species is thriving. Hence, it is very important for one to establish whether differences among species observed from morphological characters are due to actual genetic differences or environmental factors. Species classification using morphological data is usually based on diagnostic characters rather than phylogenetic analysis and the classification produced may not give the correct evolutionary relationships of the species involved. For one to make accurate phylogenetic inferences from morphological data dendrograms, it is important to compare such dendrograms with phylogenetic trees based on molecular or biochemical data.
An earlier version of this research work was presented in XXI AETFAT Congress: Systematics, biogeography and Conservation of African plants and fungi, held at the Catholic University of Eastern Africa, May 2017 [
The authors have not declared any conflicts of interest.
The authors are very grateful to the University of Nairobi and the German Academic Exchange Service (DAAD) for funding the above study.