Cypselar morphology of Spilanthes Jacq. (Asteraceae) and their taxonomic significance

INT J CURR SCI 2015, 17: E 1-11 RESEARCH ARTICLE ISSN 2250-1770 Abstract Cypselar morphology of Spilanthes Jacq. (Asteraceae) and their taxonomic significance Reshmi GR* and R. Rajalakshmi Department of Botany, University of Kerala, Karyavattom PO, Kerala, India *Corresponding author: grrechugr@gmail.com; Phone: 0091-9048088397 The Spilanthes is known as toothache plant and is accredited with the local anesthetic action due to the Spilanthol content. From a taxonomic point of view, this species is confused with other genera and species due to its similar morphological features, and require accurate studies to clarify the controversial aspects in the botanical classification of this plant. In the course of detailed cypselar morphological diversity study in the genus, Spilanthes (including 8 species), we found variations in the presence of marginal cilia, arrangement of marginal cilia, presence and nature of pappus bristle and nature of surface striations. In Spilanthes the achene morphology is the most striking factor for species separation. Several macro- and micro-morphological characters were observed using stereoscope and scanning electron microscope. The results showed that seeds were heteromorphic or homomorphic with trigonous or laterally compressed and showed morphological variations in size, presence of marginal cilia, arrangement of marginal cilia, presence and nature of pappus bristle, nature of surface striations. Cypsela characters can be used as an additional taxonomic criterion at specific level for this genus. Exomorphic characters of cypselas serve as reliable taxonomic marker in systematic study. A key is prepared for the identification of the investigated species based on cypsela characters. Keywords: Spilanthes; heteromorphic; homomorphic; taxonomic marker Received: 14 th April 2015; Revised: 04 th May; Accepted: 18 th May; IJCS New Liberty Group 2015 Introduction Seed morphology has long been considered an important source of character information for taxonomic purposes and has been proposed as a feature that reflects the evolutionary history of plants (Barthlott, 1984). Data on features of seed micromorphology have been reported to be useful for taxonomy in numerous plant groups (Juan et al., 2000; Metzing and Thiede, 2001; Segarra and Mateu, 2001; Plaza et al., 2004; Adams et al., 2005; Song et al., 2005). Seed size, shape, and epidermal surface features have figured prominently in the characterization of seed morphological diversity (Barthlott, 1981; Barthlott and Hunt, 2000), and it has been argued that these seed characters may provide data for circumscribing taxa at different taxonomic levels (Barthlott, 1981; Barthlott, 1984). While some studies support this hypothesis (Gamarra et al. 2012), other investigations show that the systematic and taxonomic value of seed micromorphology may be limited (Hsiao and Chuang, 1981; Davitashvili and Karrer, 2010) Seed morphological features have been used in the past for circumscribing a number of infrafamilial taxa (Don, 1823; Cogniaux, 1891). Seed

characters are very helpful for identification of a large number of species or genera. The fruits of Asteraceae are technically termed as achene or cypsela which is developed from bicarpellary unilocular ovary (Marzinek et al., 2008). The fruit is basically non endospermous but it has one to two layers of endosperm at mature state. In mature cypsela fruit wall or pericarp is either add pressed with seed wall or secondarily separated from seed wall. In spite of the global occurrence of the family, the fruits of the Asteraceae have not been intensively studied, as can be confirmed by the discordance seen in the names attributed to its fruits. The genus, Spilanthes Jacq. (Asteraceae) consists of about 30-40 species which are distributed along the new and old world tropics (Jansen, 1981). The genus is traditionally divided into two sections mainly based on the nature of head; sect. Spilanthes (-sect. Salivaria DC.). Having discoid heads and sect. Acmella with radiate heads (Cassini, 1822; De Candolle, 1836; Moore, 1907). Study by Jansen (1981) has revealed that this character alone does not hold good for the separation of the two. Following Moore (1907) considering Spilanthes as a genus including Acmella Rich. as a section, only 8 species are now distributed all over Kerala (Reshmi and Rajalakshmi, 2014). These include 4 rayed head species and 4 non-rayed head species. S. ciliata, S. vazhachalensis, S. tetralobata and S. uliginosa are rayed head species whereas; S. calva, S. paniculata, S. ghoshinis and S. radicans are non-rayed head species (Reshmi and Rajalakshmi, 2014). Extreme morphological similarity between the species of Spilanthes creates a challenge for taxonomic classification and it forms controversies in authentic pharmacological and phytochemical outcomes. Cypsela and capitullum morphological diversity studies usually solve this major taxonomical ambiguity. Cypsela microsculpture analysis has been considered more and more a taxonomic tool, being also important for higher and medium level classification within the family (Anderberg, 1991; Bremer, 1994). The characteristics of the seeds have been little used in taxonomic study for the classification of most seeds. The orientation, colour, shapes, size, thickness and measurements which make up the morphometrics differ considerably from taxa-taxa, so that rather few of the accumulated data bear directly on the problem of tribal and genetic relationship. Brief cypselar external features have usually been included by different floristic workers during their preparation of floristic accounts. In many cases, morphological characteristics, such as seed shape and testa ornamentation, can be used to distinguish species and varieties. Fruits and seeds tend to show less phenotypic plasticity, in comparison with other organs. Seed characters are less affected by environmental conditions, and often reflect genetic differences. The purpose of this study is to describe and compare cypsela morphological characteristics of Spilanthes species in Kerala and to provide a basis for constructing a more meaningful taxonomic treatment for the better understanding of the different species in the genus Spilanthes and to evaluate the possible use of cypsela morphometrics in taxonomic classification. Materials and Methods The present work based on the seeds of 8 species of Spilanthes Jacq. collected from different localities of Kerala. Comparative study of cypselas was done in both stereo dissecting binocular and compound microscope.

For morphological study fresh, mature, dried and healthy achenes were collected from different localities of Kerala and kept in vials. For each taxon collection locally and altitude is given (Table 1). When possible, up to four specimens were analyzed to confirm the obtained results. Morphological characters were examined under light and scanning electron microscope. Light microscopy has been carried out with the help of Stereozoom (Olympus DP11) microscope. For scanning electron microscopy dry achenes were mounted directly on graphite tape and sputter coated for 45 seconds with gold in fine coater (JFC-1200, Jeol, Japan), analyzed and photomicrographed by SEM (JSM-5600 LV, Jeol, Japan JEOL Model JSM - 6390LV) at a voltage ranging 10-20 KV. Data analysis was performed using SPSS 7.0 and MVSP 3.22. Results Cypsela characteristics A summary of the distribution of the seed characters (seed size, shape, colour, surface pattern) are given in the Table 2. The fruits of the Spilanthes denominated as cypselae, are dry, indehiscent, unilocular, originating from an inferior ovary. The pappus, a modified calyx, is inserted in the apical region of the cypsela. Basally, an abscission region is located in relation to the inflorescence axis (clinanthium) called the carpopodium. Basal abscission zone of the fruit, formed by one or more rows of cells generally different from the Fig. 1. Stereozoom microscopic images of Spilanthes Jacq. e a. S. calva b. S. ciliata c. S. uliginosa d. S. vazhachalensis e. S. radicans f. S. tetralobata g. S. paniculata h. S. ghoshinis

Fig. 2A. SEM images of cypsela of Spilanthes Jacq. a) Cypsela of S. calva b) portion enlarged c) Cypsela of S. ciliata d. portion enlarged e) Cypsela of S. uliginosa f) portion enlarged g) Cypsela of S. paniculata h) portion enlarged Fig. 2B. SEM images of cypsela of Spilanthes Jacq. i) Cypsela of S. radicans j) portion enlarged k) Cypsela of S. tetralobata l) portion enlarged m) Cypsela of S. vazhachalensis n) portion enlarged o) Cypsela of S. ghoshinis p) portion enlarged

Fig. 3. UPGMA clustering Fig. 4. PCO case scores of statistical data ones in the achene wall, more or less hardened. It is the anchor point of the inferior ovary of the floret to the inflorescence or head. It also constitutes the abscission zone of the achene. It is the scar which is attached with the receptacle. Stylopodium is a composite structure of style base and associated nectaries, situated at the apical part of cypsela. Eight taxa studied in the present study showed significant differences in the cypsela characteristics (Fig. 1). Each taxa can be differentiated by the use of cypsela characteristics. The shape is varied from oblong- obovate and colour is brown-black. Obovate cypsela seen in S. uliginosa and S. calva and all others has oblong shape. Size of cypsela is also varied. SEM study showed peculiar differences among the cypsela of all the species (Fig. 2A and Fig. 2B). Major differences were noticed in the arrangement of lateral bristles and marginal cilia. Stylopodial structures present in all the species, but carpopodium present in all the species except S. uliginosa and S. radicans. Two types of achenes, trigonous and laterally compressed achenes present in all the species except S. tetralobata and S. ghoshinis. In S. ciliata margins sparsely to moderately ciliate with straight tipped hairs, with evident cork-like margins, truncate/ slightly retuse apex and acute base, pappus usually absent or sometimes of very short bristles less

Table 1. Place of collection and altitude of all the taxa collected for analysis Species name Place of collection Altitude (m) S. ciliata Mulakuzha, Thiruvalla 30.448 S. ciliata Mundakkayam 67.182 S. ciliata Manarkulanji 173.486 S. ciliata Lakkidi 784.473 S. calva Munnar 1461.557 S. calva Ponmudi 914.603 S. calva Chemmunji 1280.54 S.calva Kilivayal, Wayanad 746.686 S. radicans Vandiperiyar 820.746 S. radicans Mukkam, Calicut 21.529 S. radicans Ponmudi 914.603 S. radicans Vellikulangara, Thrissur 28.838 S. vazhachalensis Mananthavadi 763.137 S. vazhachalensis Kumily 892.255 S. vazhachalensis Koothattukulam 34.859 S. vazhachalensis Mailapra 37.905 S. paniculata Vagamon 1003.375 S. paniculata Vandiperiyar 820.746 S. tetralobata Koothattukulam 34.859 S. tetralobata Kaipattoor 27.230 S. tetralobata Cheppad, Alapuzha 10.322 S. tetralobata Erumeli 49.659 S. ghoshinis Pala 23.798 S. ghoshinis Koothattukulam 34.859 S. ghoshinis Puthoorvayal 758.434 S. ghoshinis Idukki 788.063 S. uliginosa Panagod, Thiruvananthapuram 52.659 S. uliginosa Neyyattinkara 26.165 S. uliginosa Kuruva 42.061 S. uliginosa Mailapra 37.905

Table 2. Quantitative and qualitative characteristics of Cypsela Species CL (mm) SCIL 2.242± 0.32 CW (mm) LPL (mm) 0.99±1.0 0.53 ±0.12 SPL (mm) 0 Brownish black Colour Shape Carpo podium Stylo podium oblong present Present Truncate/ slightly retuse Apex Base No. of pappus bristles Surface ornamentation Acute 1 Rectangular celled SU 1.55±0.113 0.72±0.4 0.33 ±0.32 0.11 ±0.34 Black obovate absent Present Truncate Obtuse 2 Rectangular celled SV 2.21 ±0.54 0.94±0.7 0.94 ±0.56 0.42 ±2.8 Brownish black oblong Minutely present Present Truncate/ slightly retuse Acute 2 Deeply grooved round celled SR 2.092± 0.63 0.85±1.2 0.66 ±0.89 0.62 ±0.11 Brownish black oblong absent present U shaped Acute 2 Rectangular celled SC 1.888± 0.92 0.65±2.8 0 0 Brownish black obovate present present Truncate Obtuse 0 Deeply grooved round celled SG 1.698± 0.33 0.75±0.9 0.75 ±0.67 0.27 ±0.43 Brown oblong present Present Truncate Acumin ate 2 Rectangular celled ST 2.11±0.123 1.03 ±0.54 0.49 ±0.98 0 Brownish black oblong Minutely present Present Truncate Obtuse 1 Rectangular celled Note: SCIL-S. ciliata, SU-S. uliginosa, SV- S. vazhachalensis, SR-S. radicans, SC-S. calva, SG-S. ghoshinis, ST-S. tetralobata, SRCL-Cypsela length, CW-Cypsela width, LPL-Long pappus length, SPL-Small pappus length (p±0.05)

Table 3. Principal component analysis showing three principal clusters Character PC1 PC2 PC3 Seed length 0.511 0.461 0.431 Seed width 0.300 0.219 0.243 Long pappus length 0.187 0.000 0.243 Small pappus length 0.000 0.000 0.107 Surface pattern 0.000 0.301 0.000 Seed colour 0.000 0.301 0.000 Seed shape 0.000 0.301 0.000 Carpopodium 0.301 0.301 0.301 Stylopodium 0.000 0.000 0.000 Seed apex 0.000 0.301 0.301 Seed base 0.000 0.301 0.477 No. of pappus Bristles 0.301 0.000 0.477 Eigen values 1.147 0.069 0.056 Percentage variation 84.464 5.080 4.116 Cum. Percentage 84.464 89.544 93.660 than 1 mm long. S. radicans have corky and densely ciliate margins, with 0.6-0.9 mm long cilia facing upwards, U shaped apex and acute base, pappus of 2 unequal/sub-equal bristles present. In S. paniculata margins enormously ciliate with straight-tipped hairs, c shaped apex and obtuse base, pappus of 2 unequal/subequal bristles present. S. uliginosa have moderately ciliate margins with straight-tipped hairs, lacking an evident cork-like margin, truncate at apex and obtuse at base, pappus of 2 unequal bristles present. S. vazhachalensis, possess densely ciliate margins with cilia 0.4-0.5 mm long, cilia facing upwards, truncate or slightly retuse apex and acute base, pappus of 2 subequal bristles present whereas, S. calva is completely epappose or eciliate with truncate at apex and obtuse base. S. tetralobata have minutely ciliate margin having cilia with 0.025-0.035 mm length, cilia facing upwards, truncate at apex and base obtuse, one pappus bristle present. In S. ghoshinis minutely ciliate along margin, with 0.4-0.5 mm long cilia and facing upwards, truncate at apex and base acuminate, pappus of 2-unequal bristles present. The surface ornamentation is also shows peculiar differences. Deeply grooved round celled surface pattern present in S. calva and S. vazhachalensis whereas others have rectangular celled surface ornamentation. Glossy phytomelanin layer is present in all the species. Data analysis Statistical data show significant variations in the cypsela characteristics. Principal component analysis shows 3 principal axes and highly loaded characters in each axis were seed length, Surface pattern, Seed colour,

Seed shape, Carpopodium, seed apex, seed base and no. of pappus bristles (Table 3). Percentage variation recorded was 84.46% and cumulative percentage is 93.66%. The dendrogram shows two principal clusters. The S. calva lies more divergent with a Euclidean distance of 0.779. S. ghoshinis and S. tetralobata are more similar, lies with minimum Euclidean distance of 0.344 (Fig. 3). PCO case scores are concordant with the dendrogram (Fig. 4). Discussion The term cypsela adopted here for the fruits of Spilanthes, was based on the terminology used by Marzinek et al. (2008), who define cypsela as a complex fruit, dry, indehiscent, unilocular, with a single seed not adnate to the pericarp (linked only by the funicle) and originating from an inferior ovary; the achene is similar to the cypsela but originating from a superior ovary. It is important to emphasize that many botanists have ignored this distinction and continue to use the term achene (Spjut 1994). From the systematic point of view, the exomorphology of cypselas is of great value and these features may be employed for better understanding of the taxa. The taxonomist has usually neglected comparative cypselar morphology. The lack of authentic cypselar morphological data has hindered the identification of isolated fruits or cypselas, particularly when floral stages are not available. The science of seed identification has become increasingly important in modern scientific agriculture and without it, there would be little use in perfecting the methods of cultivating useful plants. Structure and orientation of pappus have diacritical value for differentiation of taxa. The pappus has a dual function (Stuessy and Garver, 1996), it is a defensive structure against predators of the head, blocking the space between florets; and it facilitates dispersal of the achenes. Pappus elements may serve in epizoochory through attachment to fur or feathers and in anemochorous dispersal of the achenes by increasing the resistance between the air and the achene and hence prolonging the time of fall. According to Sheldon and Burrows (1973), effectiveness of the pappus in disseminating achenes by anemochory is determined not only by the shape and size of the pappus, but also by the size of the achenes and ecological factors. In the genus Spilanthes, it has almost similar morphology so it creates ambiguity in identification of actual species and it creates challenges in taxonomy. From the present study it seems clear that all the eight species studied were shown significant differences in the cypsela morphology. The species can be separated based on the nature of cypsela, presence of pappus bristles, marginal cilia and carpopodium. Cypselae were monomorphic in S. tetralobata and in S. ghoshinis but all others have dimorphic achenes. Cypselae are epappose or eciliate in S. calva, 1-pappus bristle in S. ciliata and S. tetralobata and 2-pappus bristles in S. ghoshinis, S. paniculata, S. vazhachalensis, S. uliginosa and S. radicans. From the above data cypsela morphometrics can be utilized for the taxonomic classification of genus Spilanthes. A taxonomic key is prepared based on the cypsela characteristics, which is described as follows. Key to the species 1. Cypsela monomorphic (with laterally compressed) a. Brownish black, 2.1-2.2x1-1.03 mm long, margin minutely ciliate having cilia with 0.025-0.035 mm length, cilia facing upwards, truncate at apex and

base obtuse, one pappus bristle present with 0.483-0.5 mm long... S. tetralobata b. Brownish black,1.5-1.6x0.6-0.75 mm long, minutely ciliate along margin, with 0.4-0.5 mm long cilia and facing upwards, truncate at apex and base acuminate, pappus of 2-unequal bristles. S. ghoshinis 2. Cypsela dimorphic (Both trigonous and laterally compressed) a. Brownish black, 1.5-1.8x0.5-0.7 mm long, completely epappose or eciliate, Truncate at apex and obtuse base....s. calva b. Brownish black, 1.4-2.2 0.5-1 mm long, margins sparsely to moderately ciliate with straight tipped hairs, with evident cork-like margins, truncate/ slightly retuse apex and acute base, pappus of 2 subequal bristles..... S. vazhachalensis f. Brownish-black, 2.05-2.1x 0.6-0.9 mm long, corky and densely ciliate at margins, with 0.6-0.9 mm long cilia facing upwards, U shaped apex and acute base, pappus of 2 unequal/subequal bristles.. S. radicans Acknowledgement The first author thanks Kerala State Council for Science, Technology and Environment (KSCSTE), Government of Kerala, Thiruvananthapuram, India for its financial support to carry out the research programme. References Adams CA, Baskin JM, Baskin CC (2005). Trait stasis versus adaptation in disjunct relict species: slightly retuse apex and acute base, pappus evolutionary changes in seed dormancy-breaking usually absent or sometimes of very short bristles less than 1 mm long...... S. ciliata c. Brownish black, 1.2-1.8x0.4-0.7 mm long, moderately ciliate with straight-tipped hairs having length less than 0.1 mm, lacking an evident corklike margin, truncate at apex and obtuse at base, pappus of 2 unequal bristles longer bristle 0.2-0.7 mm long, shorter 0.1-0.5 mm long.... S. uliginosa d. Black, 2.2-2.9 0.8-1 mm, evident cork-like margins, margins enormously ciliate with straighttipped hairs, c shaped apex and obtuse base, pappus of 2 unequal/subequal bristles, longer bristle 0.5-1.1 long, shorter bristle 0.4 0.9 mm long......s. paniculata e. Black, 2.1-2.5x0.8-1 mm long, densely ciliate, cilia 0.4-0.5 mm long, cilia facing upwards, truncate or and germination requirements in a subclade of Aristolochia subgenus Siphisia (Piperales). Seed Science Research 15: 161-173. Anderberg AA (1991). Taxonomy and phylogeny of the tribe Gnaphalieae (Asteraceae). Opera Botanica 104: 43. Barthlott W (1981). Epidermal seed surface characters of plants: systematic applicability and some evolutionary aspects. Nord J Bot 1: 345 355. Barthlott W (1984). Microstructural features of seed surfaces. In: Heywood VH, Moore DM, editors. Current Concepts in Plant Taxonomy vol. 25 Academic Press, London, UK, 95-104. Barthlott W, Hunt D (2000). Seed diversity in the Cactaceae, subfamily Cactoideae (Succulent Plant Research vol. 5). Sherborne: David Hunt 173. Bremer K (1994). Asteraceae: cladistics and classification. Nordic Journal of Botany 14: 462.

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