Leaf Micromorphology and Anatomical Traits of Leaves as Potential Taxonomic Markers for Infrageneric Classification of Cinnamomum (Lauraceae)

: This study aimed to examine the leaf anatomy of the eight species of Cinnamomum (family Lauraceae) that occur in Sri Lanka to evaluate its potential utility in taxonomic delimitation. Leaf surface micromorphology and internal anatomy were studied using standard light and scanning electron microscopy. Infrageneric and interspecific variation in micromorphology of cuticular materials was observed in both abaxial and adaxial surfaces. Both abaxial and adaxial surfaces had undulating, thick cuticles with different patterns of wax deposition. Leaves were hypostomatic with no special arrangement for epidermal pavement cells. Simple, unicellular, unbranched, solitary and non-glandular trichomes were observed in different species on both surfaces of the leaves. Both adaxial and abaxial surfaces in C. dubium and C. capparu-coronde were densely covered with simple, unicellular/ falcate, long and thin trichomes. Transverse sections (TS) of the leaves were different in shape (symmetrical, asymmetrical, irregular, saucer). The midrib contained one open arch central vascular bundle that was different in shape (oval, elongated, irregular, ‘V’shaped, partially dissected into 2 or 3 segments) in different species. The number of collateral vessels was dissimilar between the species and varied in number (±10 - ±17). Isolated or aggregated crystals were observed in different parts of the leaf and oval-to-round schizogenous secretory cells were present in C. capparu-coronde, C. citriodorum and C. dubium. Leaf cuticular features together with the presence/absence of secretory cavities, trichome shape and density, midrib cross-section outline and shape of vascular bundles are taxonomically informative characteristics that can be used to differentiate the eight species of Cinnamomum that occur in Sri Lanka.


INTRODUCTION
The angiosperm family, Lauraceae Juss.(Order: Laurales) is represented by approximately 2,500 to 3,000 species within approximately 41 species and eight genera (Nishida & van der Werff, 2007).In Sri Lanka, there are 41 species and eight genera in the family Lauraceae (MOE, 2012).Members of this economically important family are used for different purposes such as food, spices and medicine (Khan et al., 2003;Saied et al., 2022;Smerq & Sharma 2011;Pugazhenthi and Suganthi (2013), Wang et al., 2013;Nwokwa et al., 2014).The Sri Lankan species of Cinnamomum (C. capparu-coronde, C. citriodorum, C. dubium, C. litsiaefolium, C. ovalifolium, C. rivulorum, C. Sinharajensis, and C. zeylanicum) are highly variable in leaf morphology in the field within the species (intraspecific) and between the species (interspecific) and often have overlapping and intermediate characters.Environmental plasticity may influence the morphology of the plants largely, making it difficult to rely on leaf morphology to differentiate the eight species.Flowers are rarely found, and flowering time varies among the species (Abeysinghe et al., 2020).In the absence of flowering material, species identification is difficult (Sritharan 1984).However, in the absence of floral or fruiting material, it is possible to use anatomical characters to differentiate the species.Some studies have investigated chemical compounds and biochemical activity in Cinnamomum species (Khan et al., 2003;Saied et al., 2022;2008;Smerq & Sharma 2011), but the reliance on chemical differences is not practical in the field.In some Cinnamomum species, petiole and leaf anatomy have been carried out and valuable anatomical data have been obtained to differentiate the species (Kumar, 2013;Abeysinghe & Scharaschkin, 2019).
Although the morphology of Cinnamomum species found in Sri Lanka species has been studied (Wijesinghe et al., 2004;Azad et al., 2016), no comprehensive information is available regarding their anatomy and very few anatomical studies have been conducted for these species in the world (Kumar, 2013;Abeysinghe & Scharaschkin, 2019).Out of eight species present in the genus Cinnamomum, C. zeylanicum is one of the most economically important spice crops in Sri Lanka and is the world reference for the quality of cinnamon (Barjolle, 2013).Considering the conservation status of the Cinnamomum species, C. capparu-coronde, C. litseaefolium, C. ovalifolium, C. sinharajaense and C. zeylanicum are considered vulnerable species and C. citriodorum, C. rivulorum are endangered.Although earlier C. dubium was declared a vulnerable species (MOE, 2012) in 2020 it has been declared a near threatened (NT) species (The National Red List 2020, 2020).Vegetative and floral morphological characters and molecular characterization of the genus of Cinnamomum have been used to study the diversity studies of Cinnamomum species (Abeysinghe et al., 2020;Bandaranayaka & Pushpakumara, 2020).Morphological characters are affected by environmental conditions.Very few anatomical studies have been undertaken to study the diversity studies of this genus.Accurate identification is essential for conservation and breeding purposes and a comprehensive study of the genus is needed.Since limited anatomical research has been undertaken to study the variation among the Cinnamomum species, the objective of this study was to carry on research on leaf anatomy using light microscopy (LM) and scanning electron microscopy (SEM) to examine whether any taxonomically informative characters of the leaves exist to fill the gap of leaf anatomical characters and to identify and delimit the eight Cinnamomum species found in Sri Lanka.

Sampling and Fixation
The fully expanded leaf samples (about twenty from each) were taken from a similar age, same size and healthy three individuals of eight species of Cinnamomum from the Cinnamon Research Station, Department of Export Agriculture, Palolpitiya, Matara, Sri Lanka to eliminate variation that might be caused by sampling.Materials for anatomical study were fixed in FAA (Formaldehyde: Ethanol: Acetic Acid in 10%:50%:5% + 35% water) for 24-48 hrs depending upon the thickness and subsequently stored in 70% ethanol (Huang & Yeung, 2015;Retamales et al., 2015).Samples were sent to Queensland Herbarium, Brisbane Botanic Gardens, Mt Coot-tha, Australia following relevant quarantine protocols.All anatomical observations were carried out at Queensland University of Technology, Brisbane, Australia.Before using plant materials, (whenever necessary) preserved samples in 70% alcohol were rehydrated in graded series.

Sectioning, Staining, and Light Microscopy (LM)
Three or five leaf samples per species were selected for anatomical examination.Free-hand transverse sections of the median portion of the midrib and leaf lamina were made and stained with toluidine blue.Stained sections were observed under the Nikon Eclipse 50i compound microscope and images were captured using the Nikon NIS-Elements imaging software (Nikon Instruments Inc., Amsterdam, Netherlands).

Preparing Epidermal Peels and Scanning Electron Microscopy (SEM)
Epidermal peels were taken from five or more leaves from each species and at least three slides were prepared from different parts of a single leaf for each species.Both adaxial and abaxial surfaces were examined and data on stomatal distribution, trichome morphology and surface ornamentation were recorded and images were taken as stated above.1x1 cm leaf samples were dehydrated using a graded ethanol series and then a critical point dried (Anderson, 1951) in an Autosamdri-815 automatic critical point drier (Tousimis, Rockville, USA).Samples were mounted on stubs with self-adhesive double-sided carbon discs.Leaf samples were mounted to have abaxial and adaxial faces and sputter-coated with gold palladium for the 70s using a Leica EM SCD005 Gold Coater (Leica Microsystems, Macquarie Park, NSW, Australia).Examination and documentation of images were conducted using an FEI Quanta 200 SEM/ESEM (FEI, Hillsboro, Oregon, USA) or TM 3000 operated at 10 kV.Cluster analysis of the species was carried out using the Minitab 17 software package using the Euclidean distance treating with the complete method and unweighted pair-group method with arithmetic averages (UPGMA) as the sorting strategy.Principal components analysis (PCA) was used to analyze the 18 variables to reduce the dimension of factors.

RESULTS AND DISCUSSION
In this study, leaf micromorphology (using SEM) and anatomy (epidermis, stomata, trichomes, mesophyll, vascular system, crystals, and secretory cavities using LM) of eight extant species of Sri Lankan Cinnamomum were examined and compared using standard protocols for histology to differentiate Cinnamomum species found in Sri Lanka

Cuticle and Cuticular waxes
In this study, the different patterns in the deposition of cuticle material on both adaxial and abaxial surfaces of the leaves of Cinnamomum species were examined and displayed different patterns in the deposition of cuticle material (Table 1, Figure 1 -16).Although both the adaxial and abaxial surfaces of the leaves were covered with a thick film of epicuticular waxes, each species showed distinct ultrastructural characteristics, ridge prominence, and continuity of wax.Each species displays a unique pattern in the structure of its waxy cuticle.This is due to the differences in the arrangement and morphology of wax crystals, the presence of grooves, and the density of the wax layer.
Both adaxial and abaxial surfaces of the leaves of all species have a thick continuous or discontinuous (undulating) thick film of epicuticular waxes.The waxy cuticle of each species showed a unique ultrastructure on both leaf surfaces and the structure of the waxy cuticles differed between the adaxial and abaxial surfaces of the same species as well as the between species (Figure 1 -16).The thickness of the film of cuticle waxes may also appear to vary in different places of the leaf (Figure 1-16).The wax deposition pattern of each species differed in the ultrastructure, prominence of grooves and continuity of cuticular wax (Figure 1 -16).The cuticle was either smooth in one area or undulated in another area of the same leaf surface (abaxial or adaxial) or both patterns can be found on both surfaces in all species (Figure 1-16).This variation within a single surface suggests a high degree of adaptability or specialization in response to micro-environmental conditions and more undulated surfaces may have adapted to maximize water retention.The abaxial surface had more ornamentation than the adaxial surface.The comparison of the cuticle deposition of all species is presented in Table 1.The adaxial surface of all species appeared severely undulated and was densely covered with cuticular wax.More ornamentation was observed on the abaxial surface.This might be due to this surface facing less direct sunlight and thus having more complex structures to cope with other environmental factors like humidity, pest attacks, and physical damages etc. Pushpa Damayanthi Abeysinghe On the adaxial surface of C. capparu-coronde, cuticular wax deposition had irregular and undulate different patterns (Figure 1, 9 and Table.1).C. citriodorum was characterized by the high density of cuticular wax clusters on both the adaxial and abaxial surfaces (Figure 2, 10).The adaxial surface of C. dubium had a wavy structure (Figure 3) while on the abaxial surface, waxes were deposited as large flakes and intermingled with other platelets (Figure 11).The adaxial surface of C. ovalifolium had undulated cuticles (Figure 5) with small crusts and larger size flake deposits on the abaxial surface (Figure 13).On the adaxial surface of C. rivulorum, the cuticular waxes seemed to protrude from the leaf surface and form angular granular/ rodlets with a tubular shape at the base (Figure 6) while on the abaxial surface, different shapes of unevenly distributed wax granules were observed (Figure 14).
In C. sinharajaense wavy and undulated structures with an irregular shape, wax deposition was observed on the adaxial surface (Figure 7) and on top of the wax layer, different shapes (rod, club, branched) of wax particles were observed on the abaxial surface (Figure 15).In some areas of the adaxial surface of C. litseafolium and C. zeylanicum deep grooves were present (Figure 4 and Figure 8 respectively).In C. zeylanicum adaxial surface had wax secretion forming an undulating pattern without distinct shapes (Figure 8).Irregular shape wax particles were observed on the leaf abaxial surface of C. capparucoronde (Figure 9), C. litseaefolium (Figure 12) and C. zeylanicum (Figure 16).
Different shapes of flakes of C. rivulorum (Figure 6) were observed.Granular wax particles were observed on the abaxial surface of C. sinharajaense (Figure 15).Densely deposited crusts or platelets of waxes were characterized by the abaxial surface of C. zeylanicum (Figure 16).The wax deposition pattern of the cuticle in different species on adaxial and abaxial surfaces seemed to be a species-specific characteristic feature of the studied species.Therefore, the pattern of epicuticle wax deposition can be considered as a diagnostic character to identify species.These results showed that cuticle features are very important in the identification of Cinnamomum species and useful for the taxonomy of Lauraceae Cuticle characters have been studied in genera of Aspidostemon, Beilschmiedia, and Cryptocarya and are a promising tool to solve the proper generic placement of poorly known species (Nishida & Christophel, 1999;Nishida & van der Werff, 2007;Nishida et al., 2016).
These results show insights into how different species adapt their wax layer to their specific environmental needs.Within the same species, the wax structure differs between the adaxial and abaxial surfaces.This indicates specialized adaptations to different functional or environmental requirements on each side of the leaf.The differences in cuticular deposition among Cinnamomum species may have diverse adaptations of these plants to their environments.The dense and undulated wax on the adaxial surface could be crucial for protection against intense sunlight and for reducing water loss through transpiration and the ornamentation on the abaxial surface could enhance gas exchange or protect against herbivores and pathogens.
Inter-species variation of the wax deposition of different species may be linked with the evolutionary adaptations to different climates, habitats, or ecological niches.

Trichomes
Leaves of all species were pubescent and trichomes were present on both surfaces.All trichomes were unicellular, unbranched, non-glandular, solitary and had an acute apex .The density of the trichomes differed on both surfaces of the same species, as well as in different species.In general, on the adaxial surface, the trichome density is generally higher compared to the abaxial surface.Trichomes varied in length (Figure 17 -32), curvature and density, for example straight (Figure 17 -20), curved (Figure 18, 27, 28, 29), short (Figure 17,20,22,24,25,32), long (Figure 18,26,29,30,31), conical (Figure 17 -C.capparu-coronde), 24, 25), slightly wavy (Figure 22), dense (Figure 17,19,27) and sparse or low density (Figure 20,22,32).The presence or absence of trichomes, abundance, and distribution over both leaf surfaces, on midrib and leaf lamina, were not consistent within a species and varied between species.However, on the adaxial surface in all species, the density of the trichomes was higher than on the abaxial surface (e.g. Figure 19, and 27 (C.dubium).The different densities of trichomes of different species could be an adaptive feature for protection from herbivores, reduction of water loss, or protection from excessive sunlight.

Stomata
In SEM of species, it was impossible to identify epidermal cells and stomata, because waxes covered the epidermal cells and stomata.Moreover, cuticular wax may act as a protective layer covering the stomata and normal epidermal cells.Under the light microscope, the leaf upper epidermis of all species contained no stomata as a rule.Mostly round-shape stomata occurred on the lower epidermis (hypostomatic) and were randomly distributed and irregularly oriented (Figure 30,33,34).As in other species of genera of Lauraceae (Cinnamomum pauciflorum, Baruah & Nath, 2006; Actinodaphne cupularis, Beilschmiedia robusta, Caryodaphnopsis sp. and some other genera, Zeng et al., 2014), studied Cinnamomum species consistently had hypostomatic leaves.Anomocytic stomata (Figure 30) with no special arrangement of epidermal cells, so subsidiary cells and all the cells were normal epidermal cells with different shapes such as rectangular, irregular or round (Figure 34).Infrageneric levels variations, paracytic stomatal complex, round shape stomata (Ocotea nutans),   (Betim et al., 2020).
Hypo-stomatic leaves may have a significant evolutionary strategy enhancing a plant's ability to survive in various environmental conditions by minimizing water loss and optimizing gas exchange.Therefore, these leaves may have a crucial role to play in the adaptability of plants in harsh conditions.

Midrib Shape
Transverse sections of leaves along the midrib showed different outline shapes and according to the outline of the sections along the midrib, leaves can be categorized into six ( ).The main differences in the six types were due to the shape, symmetry, and raised or non-raised above and below the vascular bundle.
Although there were some features unique to each species, basically all the species had  39) had a highly raised (pointed hump) above the vascular bundle.C. ovalifolium (Figure 40) had a less raised structure below and above the vascular bundle.Thick-walled collenchyma cells were located underneath the upper epidermis and above the vascular bundle, above the lower epidermis and below the vascular bundle of the midrib (Figure 36 -47).
In the form of a cluster of sclenchyma, cells were observed mainly around the vascular tissues.However, semicircular to arch shape bundles in midrib with a belt of continuous sclerenchymatous sheath had been observed in O. duckei (Coutinho et al., 2006) Laurus nobilis (Serebrynaya et al., 2017), Endlicheria paniculate, Nectandra barbellate and O. indecora (Goncalves et al., 2018).
A raised upper surface, pointed hump above and saucer or round shape below the vascular bundle might facilitate efficient water runoff, minimizing water pooling on the leaf surface, preventing microbial infections and maintaining a clean surface for optimal light absorption.
A transverse section along the blade in all the species just below the cuticle layer, the blade had both upper and lower one-cell-thick epidermal layers (Figure 44).Unspecialized epidermal pavement cells generally show the entire range from straight to undulating anticlinal walls, rectangular, isodiametric, tetragonal to polygonal or tabloid with different sizes (Figure 26, 28, 29,30, 31, 32 -34) or sinuous In Lauraceae members, anticlinal cell walls have been categorized as straight, curved, wavy, undulate, sinuate and Ώ-shaped (Coutinho et al., 2006;Shi et al., 2014;Goncalves et al., 2018).Between the epidermal layers, one layer of compactly arranged palisade parenchyma cells, and between the palisade cells and lower epidermal cells, several layers of loosely arranged mesophyll cells (Figure 44) which are dorsiventral with intercellular spaces known as spongy parenchyma were observed.Between the palisade cells and the lower epidermis, spongy parenchyma cells were present.In some species (C.capparu-coronde Leaf micromorphology had been studied in some species of the family Lauraceae and the leaf morphoanatomy of Cinnamomum species was very much similar (hypostomatic stomata, rectangular cells of the periclinal wall, uniseriate epidermis, one central vascular bundle, sclerenchymatous sheath around the vascular bundle etc.) to O. duckei, a member of the family Lauraceae (Coutinho et al., 2006).
As observed in this study similar results (typical leaf anatomical characters, mucilage cells, thick cuticle) had been obtained for leaf epidermis and thick-walled erect hairs on the abaxial surface of the leaf of Cinnamomum species (Bakker et al., 1992), straight or undulated epidermal cells of Endlicheriach alisea, Ocoteah elicterifol (Nishida & van der Werff, 2007), minute to strongly sinuous, straight lines to strong sinuous epidermal cells (Ceolin et al., 2009) were observed.Different shapes of anticlinal walls (smooth and angular, irregularly thickened, beaded buttressed, branched, rounded.undulate.sinuous) of epidermal cells of Syndiclis (Lauraceae) and its allies, Cryptocarya were observed (Yang et al., 2012;Zeng et al., 2014;Nishida et al., 2016).Moreover, some studies have been conducted using cuticular features of extant Lauraceae in delimiting lauraceous species (Shi et al., 2014).

Vascular Bundle
The structure and arrangement of the vascular bundle within the Cinnamomum species provide significant understandings for species differentiation.The arrangement of the tissues in a transverse section of the laminar along which were 1-5 cells parallelly arranged vertically and they were spaced each other by fibers in the vascular bundle.
However, the number of collateral vessels differs among the species (Figure 48 1.The vascular system has received the most attention in the study of leaf anatomy (Kocsis & Borhidi, 2003;Noraini et al., 2016;Talip et al., 2017).A similar tissue arrangement: fundamental parenchyma cells, discontinuous layers of sclerenchyma cells surrounding the vascular bundle, open arch vascular bundle, and collateral type xylem tissues had been observed in O. duckei (Coutinho et al., 2006).In this study, pigmented cells were observed in all the species in ground tissues (Figure 36 -42) which had been observed in worldwide genera of the family Lauraceae (Betim et al., 2020).Therefore, the research on the leaf anatomy of Cinnamomum species allowed us to reveal species differences based on the shapes of vascular bundles.These figures illustrate the transverse sections of the leaf midrib and provide visual confirmation of the different vascular bundle shapes and the presence of sclerenchyma, pigmented cells, and collateral vessels.Different shapes of crystals of calcium oxalate in the form of prismatic, acicular, raphides, and rosette have been observed in C. malabatrum Preesr in India (Kumar, 2013).Parenchyma cells embedded with different colours (orange, yellow, brown, black, or dark red) chemicals/ pigments were located on both dorsal and ventral sides of the vascular bundle.Schizogenous secretory cells oval to the round were present with spaced distribution.Secretory and oil cells were observed in O. duckei (Coutinho et al., 2006).They play an important role in plant defense and are often used as diagnostic features in plant identification.These characteristics could help in identifying species and understanding their evolutionary relationships.

Cluster Analysis Using the Micromorphology and Anatomical Data
A dendrogram was constructed using all micromorphology and anatomical data of leaves from Cinnamomum species, employing Minitab software to gain insights into the relationships among the species.Based on the polymorphic anatomical characters (Table 1), a data matrix was prepared, cluster analysis was carried out and resulted in the two main clusters (Figure 56 Figure 57 shows the biplot of Cinnamomum species based on the foliar anatomy and the first and second Principal Components explain 31.76% and 24.84% of total variations respectively.This means PCA showed that the accumulative contribution rate of the two principal components was 56.6%.Thus, the first and second principal components had more than 0.56.The PCA revealed that Wax shape (Wsh), mucilage cells -near to epidermis (Muepi), vascular bundle-dissection (Vbdi), mucilage cells -near to epidermis (Muepi), grooves on the adaxial (Gad), ridges on adaxial (Rad) characters account for first principle component (31.76%) while upper surface shape (Ush), mucilage cell shape (Mush) and abundance  of trichomes on midrib on adaxial (Abuad) account for the second principle component (24.84%).These variables, therefore, are predicted to affect the distribution patterns of foliar anatomy of all species.These data will contribute to enhancing our understanding of the petiole anatomy of Cinnamomum species.C. zeylanicum -Grooves on adaxial surface prominent, abaxial surface observable, partially dissected (2 segments) shape vascular bundle, contour symmetrical, mostly flat above and below vascular bundle, trichomes straight and short, secretory cavities below the palisade parenchyma.

Dichotomous Key for Cinnamomum Species Identification
In constructing the dichotomous key for differentiating Cinnamomum species, detailed observations, results and information from this research were integrated; cuticular wax characteristics, transverse leaf sections along the midrib, the shape and features of the vascular bundle, trichomes length and shape, grooves on the adaxial and abaxial surfaces, the contour, the presence and location of secretory cavities and symmetry of these sections and secretory cells.Such detailed anatomical studies not only aid in species identification but also enhance our understanding of the functional adaptations of these species in their natural habitats.

Grooves on the adaxial surface:
⸰

CONCLUSION
The present study revealed the variations in the foliar anatomy of the genus Cinnamomum and listed the specific characters belonging to each species.Understanding these differences will be useful in selecting and breeding species with desirable traits for agricultural purposes.For example, selecting species with optimal wax properties could enhance drought resistant or pest resistance in crops derived from Cinnamomum.

Table 1 :
Comparison of the polymorphic cuticle and qualitative features of transverse sections of the midrib in all eight Cinnamomum species occurring in Sri Lanka.The character is abbreviated as character code within the parenthesis, and the character states of the same character are assigned serial numbers in parenthesis Characters C. capprucorindo (Cca) Symmetry (SY) Symmetrical (1) Symmetrical (1) Symmetrical (1) Symmetrical (1) Symmetrical (1) Not Symmetrical (2) Symmetrical (1) Symmetrical (1)
Different parts of the leaf blade contained similar or various kinds of crystal idioblasts (crystals, aggregated crystals).Crystals were present as isolated or single crystals were grouped in an irregular arrangement to form aggregated forms.e. g. in mesophyll tissues in the leaf blade in C. cappru-corindo and the transverse section along the midrib.In Cinnamomum species, crystal idioblasts exhibit diverse forms and are found in different parts of the leaf blade.Crystals are commonly found within the spongy or palisade parenchyma cells in the leaf blade.Crystals are often located on both dorsal and ventral sides of the vascular bundle, embedded within parenchyma cells containing various pigments.
); the 'x' consists of C. capparu-corindo and C. citriodorum, C. litseaefolium and C. dubium.The 'y' cluster consists of C. ovalifolium, C. sinharajaense C. rivulorum and C. zeylanicum.Further, C. citriodorum and C. litseaefolium have clustered very closely and clearly show a higher similarity (~ 65%) between the two species and these two species had a close relationship (~ 50%) with C. dubium.These three species clustered with C. capparu-corindo.In the 'y' cluster, C. ovalifolium, and C. sinharajaense had a close relationship with about ~ 45% similarity value (Figure 56).