Resolving taxonomic problems in the genus Ceropegia L. (Apocynaceae: Asclepiadoideae) with vegetative micromorphology

The genus Ceropegia L. of Family Apocynaceae, subfamily Asclepiadoideae comprises 213 accepted taxa distributed in tropical and sub-tropical regions of Africa, India, Australia and neighbouring regions. The taxa are mainly identified on the basis of flower morphology. A study was undertaken to reveal micromorphology of 26 taxa distributed in Western Ghats of India to solve the identity problems. The micromorphology is studied with standard microscopic methods in five replicates of each character and taxa to reveal the parameters, distribution of stomata, stomatal index, stomatal density, epidermal cell wall pattern and stomatal measurements. The data obtained was subjected to ANOVA to find out the experimental mean, standard deviation and standard error. A consensus phylogeny tree is constructed using the PAST on the basis of Jaccard similarity coefficient. Results of study revealed that, micro morphological characters, viz., type of stomata, number and characters of subsidiaries, anticlinal cell wall pattern, and stomatal index are very significant in delimitation of closely allied taxa. Beside ‘paracytic’ stomata, ‘tetracytic’, ‘isotricytic’, & ‘anomocytic’ stomatal types, and amphistomatic distribution are recorded for first time in Ceropegia. The taxa are separated easily from each other using vegetative micromorphology and can be identified even in absence of flowers. An identification key and phylogenetic tree is derived on the basis of vegetative micromorphology and gross morphological characters.


INTRODUCTION
The genus Ceropegia L. of family Apocynaceae, subfamily Asclepiadoideae comprises many narrow endemic and threatened species distributed only in tropical and sub-tropical regions especially, Africa, India, Australia, and neighbouring region. The genus comprises of 213 accepted species, eight unplaced, and 36 unassessed taxa of species and infra-specific ranks (Plant list 2019; Plant of the World Online 2020). Most of the taxa are endemic to the regions or locations from where they are described. In India, it is represented by about 68 taxa comprising of 61 species, two subspecies and five varieties with recent descriptions by Diwakar & Singh (2011), Rahangdale & Rahangdale (2012), Kamble et al. (2012), Sujanapal et al. (2013), and Kumar et al. (2018). Of these taxa, 40 species and six varieties are endemic to India, while 34 species and two varieties are endemic to Western Ghats (Jagtap & Singh 1999;Singh et al. 2015).
The taxa under this genus are mainly described on the basis of floral characters, while many vegetative characteristics are similar in many taxa. The taxonomists in the world have tried to resolve systematic crises among these species or taxa below rank of species. Since last decade many new taxa at specific and infra-specific rank are described (Singh et al. 2015). Still, many of the Indian taxa are kept under unresolved categories by international databases (such as The Plant List (2019) as well as POWO (2020)) due to insufficient data related to allied species and varieties. In India, there are two major regions of distribution of this genus, viz., Himalaya and peninsular India. Most species found in Western Ghats or peninsular India possess tubers, while those found in the Himalayan region do not have tubers and are nonsucculent. In general, all these taxa are described on the basis of morphological characters; especially on floral morphology as the allied taxa do have many similarities in vegetative characters. The vegetative micro-morphology was neglected in most of the cases, may be because of morpho-similarities among the taxa in vegetative condition. There is negligible literature available on microscopic characters of vegetative organs; and Ceropegia species are generally identified on the basis of habit and flower characters. Therefore, it is a difficult task to identify the species without flowers. So, there is need of micro-morphological as well as anatomical characters that would be helpful in identification of taxa at species and infra-specific level in vegetative state. Considering these facts and lacunae in the literature a study was undertaken to reveal micro-morphological characters of taxa of this genus and provide identification key on the basis of vegetative and micro-morphological characters.

Plant Materials
Materials for the present study comprise a total 26 taxa of the genus Ceropegia L. from Western Ghats. Fresh materials were collected from wild plants as well as plants maintained in the nursery for study. The locations of taxa included in the study range from Nashik in Maharashtra to Coimbatore in Tamil Nadu through Wayanad in Kerala. A list of taxa under study with their status, distribution, and locations of collection for the present study is given in the Table 1.
In are retained because these taxa are merged without any solid reasons and without comparing the characters said in original protologues of above mentioned taxa. As the present study is aimed to find out more stable characters for correct distinction between the taxa, the above mentioned taxa are accepted.

Vegetative morphology
Total 14 vegetative morphological characters of each taxon are recorded by observation of living materials. They are mentioned in the Table 4 to avoid repetition. Only newly observed characters are mentioned in detail.

Micro-morphology
For epidermal study, five leaves per sample were picked at 4-6 th nodes from base. Epidermal peelings of suitable size were made from both upper and lower surfaces of each leaf. Peelings were treated with 5% Sodium Hypochlorite for clearing. Then epidermal debris was brushed off under observation using Lawrence and Mayo stereo-zoom microscope NSZ-606 and the peel was finally mounted in 50% Glycerine. Thereafter, slides were observed under research microscope (Lawrence & Mayo Model No. XSZ-N107T) for epidermal details. For this, five leaves were taken and from each leaf five different fields were observed and data was recorded, making total observation size of 25 sample areas. The observed parameters are, type of stomata, distribution of stomata on leaf surface (hypostomatic/amphistomatic), stomatal density/mm 2 , stomatal Length (µ), Width (µ), pore length J TT (µ), characters and number of subsidiaries and epidermal cell wall characters as per the techniques described by Dilcher (1974) and Kotresha & Seetharam (2000). The classification of stomatal types given by Prabhakar (2004) was followed to recognize stomatal complex, with number and characteristics of subsidiaries. For this, number of stomata in a unit area is recorded for total sample size as that for stomatal index observations.

Parameters
3. Stomatal length (μ): The length of stomata is measured as the longitudinal end to end distance of guard cells, using ocular meter scale standardized for each magnification with the stage micrometer.
4. Stomatal width (μ): This is the maximum length in the middle transect of stoma with the ocular meter scale.

J TT
This is length of stomatal opening observed with the help of ocular meter scale.
Statistical analysis comprising of mean, variance, standard deviation, and standard error are applied for each parameter as per method given by Singh & Chaudhary (1985).

Phylogenetic analysis
Phylogenetic analysis was done on the basis of 42 vegetative morphological and micro-morphological characters ( Table 4). For phylogenetic analysis PAST ver.03 was used as per Harper (1999), Hammer et al. (2001), Hammer & Harper (2006). In this, the characters were represented in the binary format (Table 5), which was used to generate a phylogenetic tree by Jaccard similarity coefficient model and data replication method -bootstrap method at 1,000 replications on the basis of a matrix of characters.

RESULTS
The observations regarding stomatal distribution, type, subsidiaries character & number, and the anticlinal wall characters of epidermal cells are presented in the Table 2 and Image 1. The results are described under the subheads as mentioned below.

Distribution and type of stomata
The stomata are distributed only on lower surface of the leaf (hypostomatic) in all the taxa studied except in C. bulbosa. In both varieties, C. bulbosa var. bulbosa and C. bulbosa var. lushii, the leaves are amphistomatic, i.e., stomata are present on both surfaces of the leaves. Four types of stomata, i.e., paracytic, anomocytic, tetracytic, and isotricytic are recorded in the taxa under study. Among these types, the paracytic stomata are most common and found in 16 taxa, the anomocytic are less common and recorded in four taxa; while the tetracytic stomata are observed in only C. elegans. Five taxa have mixed stomata; of which, four taxa viz., C. anantii, C. attenuata var. attenuata, C. maharashtrensis, and C. sahyadrica have isotricytic and tetracytic intermixed with each other, while in C. rollae paracytic stomata were found along with anomocytic stomata.

Characters and number of subsidiaries
The subsidiaries in studied taxa are either distinct (different than epidermal cells) or indistinct (same as epidermal cells). The stomatal complexes in 12 taxa have distinct subsidiaries while 13 taxa have indistinct ones. Among the 16 taxa having paracytic stomata, 12 taxa have distinct subsidiaries while remaining four have indistinct ones. In C. rollae, where stomata are of mixed type, i.e., paracytic and anomocytic, the subsidiaries were distinct along the paracytic stomata while with anomocytic stomata there were indistinct subsidiaries. The number of subsidiaries is also variable from two to five, but generally showed a fixed number or a range in all the specimens of same taxon. The number is fixed to two subsidiaries in nine taxa and four subsidiaries in two taxa. In other 15 taxa, the number of subsidiaries ranges 2-3 in three taxa, 2-4 in three taxa, 3-4 in seven taxa, and 4-5 subsidiaries in two taxa with anomocytic stomata. The taxa having mixed stomata with isotricytic and tetracytic types have indistinct subsidiaries.

Anticlinal wall pattern
The patterns of anticlinal walls of epidermal cells are observed and recorded in all the taxa under study. There are three patterns, 'straight', 'rounded (curved)', and 'undulate (wavy)'. Out of all 26 taxa, 10 have undulate anticlinal walls, eight taxa have rounded walls, and remaining eight have straight anticlinal walls. The results showed that, anticlinal wall pattern is not found to be associated with the other characters studied. This character is independent of stomatal type, size or number and type of subsidiaries. But, it is important for the differentiation between different taxa.

Stomatal characteristics
The experimental results about stomatal measurements are presented in Table 3 and described below.

Stomatal Density (SD)
The mean value for stomatal density/mm 2 of leaf area is 252.31±8.50 with the highest value of 403.64±10.60 in C. maharashtrensis followed by C. anantii (390.91), and C. jainii (370.91), while the lowest SD 98.18±3.40 in C. juncea subtended by C. bulbosa var. lushii (105.45), C. bulbosa var. bulbosa (127.27), and C. hirsuta (152.73). C. juncea is a succulent taxon with highly reduced leaves J TT and also have lowest value of SI.

Stomatal Size
Regarding the stomatal dimensions, the largest stomata are recorded in Ceropegia juncea, while the smallest stomata in the C. attenuata var. mookambikae. The experimental mean for stomatal length is 29.54±0.73µ, stomatal width 19.68±0.67µ and the pore length 19.68±0.64 microns. The stomatal length is highest in C. juncea (39.0µ), followed by C. vincifolia (32.5µ), C. bulbosa (32.25µ), C. oculata (32.0µ), and C. candelabrum (31.5µ). The succulent species C. juncea have largest stomata with respect to length 39.0±1.5µ, width 26.5±1.7µ, and pore length 27.0±0.97µ; while those values for C. attenuata var. mookambikae are 23.0±0.5µ, 15.0±0.79µ, and 12.0±0.5µ, respectively having the smallest stomata. As per the ratio of length x width, the stomatal size is ranging from the smallest one of 345µ 2 in C. attenuata var. mookambikae to 1033.5µ 2 in C. juncea has largest stomata with the experimental mean value of 587.3µ 2 .

Phylogeny
A consensus phylogenetic tree based on 42 morphological and micro-morphological characters obtained after 1,000 replications revealed that the dendrogram is divided into three clusters ( Figure  1). Cluster I comprises seven taxa: C. maccannii, C. panchganiensis, C. rollae, C. attenuata var. attenuata, C. sahyadrica, C. maharashtrensis, and C. anantii. All these taxa are clustered with unit distance of 0.2-0.8.
Cluster II comprises total 15 taxa with unit distance of

J TT DISCUSSION
As the genus Ceropegia is considered to be xerophytic because of its escape mechanism against the hot period by perennial tubers. It should be preferably hypostomatic and the results are in corroboration with the concept, but the exceptions are both the varieties of C. bulbosa as they have amphistomatic leaves. Both the varieties of this *-the data of amphistomatic taxa are taken from mean of both the surfaces and then the experimental mean is calculated | ˄-upper surface of leaf | ˅-lower surface of leaf.
taxon are usually distributed in dry rain-shadowed area of the Western Ghats, even though they are amphistomatic; this may be due to more succulent leaves than other taxa. Metcalfe & Chalk (1950) reported isobilateral leaves in the species of Ceropegia and Hoya R.Br. having fleshy leaves. Therefore, present results also confirm the amphistomatic nature of some taxa in Ceropegia which have fleshy leaves. The presence of stomata only on lower surface of leaf is an adaptation to reduce the rate of transpiration. It is interesting to note that except C. candelabrum, C. elegans, and C. juncea, all the taxa have tubers and their vegetative growth is confined to the rainy season only, but still they have hypostomatic leaves. This is an interesting fact and raises further curiosities towards the taxa. The taxa C. candelabrum, C. elegans, and C. juncea generally remain in their active growth during summer season also and therefore their hypostomatic nature is justified, but for other taxa which grow in high rainfall area and escape the dry spell, especially about 8-9 months of year except rainy season and still have the hypostomatic leaves. This fact is interesting and the hypostomatic leaves must be a qualitative character governed by genes only; because in the present study beside C. bulbosa three more taxa, viz., C. candelabrum, C. elegans and C. juncea, have succulent leaves but have only hypostomatic nature unlike C. bulbosa. Stoma tetracytic: Absent (0), Present (1) 23 Stoma isotricytic & tetracytic: Absent (0), Present (1) 24 Stoma paracytic & anomocytic: Absent (0), Present (1)
The results revealed that, mean value of stomatal index for the taxa under study, i.e., experimental mean is 14.75±0.35. The highest SI is found in C. attenuata var. attenuata while the lowest value for SI is observed in C. juncea (8.18±0.37). The mean value for stomatal density/ mm 2 of leaf area is 252.31±8.50 with the highest value of 403.64±10.60 in C. maharashtrensis and the lowest 98.18±3.40 in C. juncea. There is no correlation observed among the taxa with respect to SI values, but for SD the taxa occurring in the dry climates and with succulent habits have relatively lower values for the parameter. The SI remain unchanged even in different seasons in soybean cultivars (Rahangdale 2003) suggesting true genetic nature of this parameter. The SD is the lowest in C. juncea a succulent taxon with highly reduced leaves and increasing to some extent in C. bulbosa var. lushii, C. bulbosa var. bulbosa, and C. hirsuta. Low SD helps these taxa to retain more water in plant body and survive during dry spell of the year. On the other hand, taxa growing under high rainfall conditions, viz., C. attenuata, C. anantii, C. jainii, C. media, C. mahabalei, C. anjanerica, and C. maharashtrensis, have high stomatal density ranging from 325 to 403 stomata/mm 2 , thereby adapting to the high annual rainfall of about 2,000-4,000 mm. It is interesting to note that, C. concanensis found in the Konkan region, on lateritic rock plateaux with very negligible soil have intermediate value of stomatal density. This is in accordance with it's relatively broader leaves and habitat because the rain water never remain on the sloppy plateaux. Such plateaux show relatively dry conditions as compared to the little more soil rich habitats where C. anantii and C. attenuata occur.
Present study revealed that on the basis of micromorphological characters, the taxa with overlapping vegetative characters can be easily identified. The type of stomata, number and nature of subsidiary cells and anticlinal wall pattern are important characters to differentiate between very closely allied taxa at species as well as infraspecific ranks. This has been shown in an artificial key as well as the morphological phylogeny J TT   tree derived in the present study. These results are in corroboration of the previous studies in different angiosperm taxa. Metcalfe & Chalk (1988) reported that stomatal index is independent of the changes in epidermal cell size brought by the environmental factors, and therefore, the SI is generally considered as a taxonomic character along with the others. Many of the works have considered SI as a strong taxonomic character to delimit the allied taxa of family Vitaceae (Patil & Patil 1983, 1984, in Bauhinia L. (Kotresha & Seetharam 1995), and Cassia L. species (Kotresha & Seetharam 2000). The leaf micromorphological characters are very significant to compare the fossil taxa with the allied species of extant taxa (Dilcher 1974). Doyle & Endress (2000) observed that, the morphological evidences overcome the weak molecular evidences while assigning the phylogeny of angiosperms. Kotresha & Seetharam (2000) studied epidermal micromorphology of Cassia L. species and delimited the species within the genus on the basis of stomatal index, size, and epidermal cell wall structure (especially the anticlinal wall pattern) and the type of stomata. Thus, stomatal characters and micromorphology can play a very significant role in resolving the taxonomic ambiguities. Present study is also showing similar outcomes giving significant information in delimitation of Ceropegia species and varieties on the basis of micromorphological characters. For example, two varieties of C. mahabalei, viz., var. mahabalei and var. hemalatae, are different with respect to many characters including stem scandant, undulate leaf margins, 2-4 indistinct subsidiaries and rounded anticlinal walls of epidermal cells in latter, whereas var. mahabalei have erect stout stem, straight leaf margins, two distinct subsidiaries, and undulate epidermal anticlinal walls. These characters are other than the characters which are provided in original protologue. Variety hemalatae does not match in any characters with C. oculata. Similar is the case of varieties of C. attenuata, viz., var. attenuata and var. mookambikae. The later variety has paracytic stomata with two distinct subsidiaries and SI 15.05, while the variety attenuata has mixed stomata of isotricytic and tetracytic types with 3-4 indistinct subsidiaries stomatal index 21.80. As per the key provided in the present study variety mookambikae is more allied to C. concanensis than with C. attenuata var. attenuata. The consensus phylogenetic tree has three clusters. The type of stomata is defining character for these three clusters and supported by the number & nature of subsidiary cells. Cluster I is having anomocytic and mixed type of stomata with indistinct variable number of (two/three/four/five) subsidiary cells. All the taxa in this cluster are erect ones. Cluster II comprises total 15 taxa having paracytic stomata. This cluster further has two sub-clusters. Sub-cluster IIa comprises eight taxa having two distinct subsidiaries in all. From the dendrogram it is observed that the taxa, C. evansii, C. fantastica, C. oculata, and C. lawii are closely related having unit distance of 0. 2-0.4. Along these C. candelabrum is separated from others on the basis of having fascicled roots and undulate anticlinal epidermal walls. Sub-cluster IIb comprises seven taxa having indistinct and variable subsidiaries. C. mahabalei var. hemalatae is separated from others on the basis of scandant habit, undulate leaf margins and high SI value. The cluster III is an externally linked group has four taxa clustered with unit distance of 0.2-0.8. It comprised two varieties of C. bulbosa, C. elegans, and C. juncea. This is the group of taxa having succulent stems and leaves. Among them C. juncea is placed as distinct taxon as it has highly reduced leaves. Among these C. elegans has tetracytic stomata while others have paracytic ones and therefore, it is placed little distantly. The dendrogram depicts clear differences among related taxa on the basis of micromorphology, especially the type of stomata, anticlinal wall pattern, SI and supported by gross morphology. In the present study, the grouping of C. maccannii, C. panchganiensis, C. sahyadrica, and C. rollae in first cluster, as well as C. anjanerica, C. mahabalei, C. jainii, C. media, and C. odorata in second cluster is in corroboration with the molecular phylogeny done by Surveswaran et al. (2009) where these taxa were placed in two separate clusters.
It is necessary to discuss here the delimitation of some of the taxa in present study. C. mahabalei var. mahabalei and C. mahabalei var. hemalatae are distinct in gross morphology of stem, leaves, inflorescence and the corolla beak, in a similar way they also differ in having indistinct 2-4 subsidiaries, rounded anticlinal walls and higher SI in the later variety. In the dendrogram they are also placed apart from each other on the basis of above characters. Similarly, two varieties of C. attenuata, viz., var. attenuata and var. mookambikae, are also placed distantly in the dendrogram in two different clusters. This is because of the type of stomata, number & characters of subsidiaries, size of stomata and SI. The most confusing taxa in terms of vegetative and floral morphology are C. rollae and C. lawii, as they are morphologically very similar to each other. Results of the present study separated them in the dendrogram as well as in the identification key on the basis of type of stomata, number and feature of subsidiaries, anticlinal wall pattern, and size of stomata. Thus, their identity can be confirmed on the basis of micro-morphology even in vegetative state. Similarly, J TT all the above mentioned taxa are separated on the basis of micro-morphological characters in the artificial key also. Thus, micro-morphological characters when combined with the gross morphological characters give better opportunity for correct identification of the taxa. The inter-specific variations in the stomatal characters such as stomatal index and stomatal frequency in Vitis L., Cissus L., and Leea D. Royen ex L. were studied by Patil & Patil (1984) and the taxa within these three genera were delimited on the basis of the stomatal characters and relationship among them was also established. Stomatal size and frequency was used to delimit between 56 species of genera of family Vitaceae by Patil & Patil (1983) and opined that, the stomatal data is supporting other characters to delimit the species and also to establish phylogenetic relationship to some extent. The results of present study show a similar trend in the genus Ceropegia.
Therefore, on the basis of studied micromorphological characters combined with other vegetative characters 26 taxa of Ceropegia at species and infraspecific level are delimited and an identification key is being provided for easy identification. These characters provide a very easy way of identification even in absence of flowers of these taxa.

CONCLUSION
The micro-morphology of taxa in the genus Ceropegia L. is revealed and is determinative for the identification of taxa even in a vegetative state in combination with the other general morphological characters. The type of stomata, number and nature of subsidiary cells, anticlinal epidermal wall pattern and stomatal index are key characters for differentiation of the taxa. Besides paracytic stomata, anomocytic, tetracytic and mixed, isotricytic + tetracytic and paracytic + anomocytic stomata are found in the genus Ceropegia. These characters are crucial in correct identification of the taxa at species and infraspecific level. Occurrence of tetracytic and mixed stomata is a first report in the genus by this study. It is also confirmed that, C. mahabalei var. hemalatae and C. attenuata var. mookambikae are distinct taxa from their allied taxa, respectively. www.threatenedtaxa.org The Journal of Threatened Taxa (JoTT) is dedicated to building evidence for conservation globally by publishing peer-reviewed articles online every month at a reasonably rapid rate at www.threatenedtaxa.org. All articles published in JoTT are registered under Creative Commons Attribution 4.0 International License unless otherwise mentioned. JoTT allows allows unrestricted use, reproduction, and distribution of articles in any medium by providing adequate credit to the author(s) and the source of publication.