MORPHOMETRICS OF THE TRIBE MERREMIEAE AUSTIN ( CONVOLVULACAE ) FROM INDIA

Sixteen species of the tribe Merremieae Austin (Convolvulaceae) from India were morphometrically analyzed with the help of Principal component analysis (PCA) and cluster analysis to explain the relationship between them. PCA showed that quantitative characters like corolla breadth, fruit breadth and length of paracot leaf play important role in bringing together all the species in the same tribe while the characters like leaf length, leaf breadth, petiole length, pedicel length, calyx length and calyx breadth play vital role in the delimitation of taxa within the tribe Merremieae. Cluster analysis and dendrogram revealed that, the genus Operculina S. Manso is segregated from Merremia Denns. ex Endlich.; genus Hewittia Wight. & Arn. is very close to Merremia, probably originated from M. aegyptia (L.) Urban and M. dissecta (Jacq.) Hall. f. cluster and genus Xenostegia Austin & Staples is isolated in the separate cluster. Introduction The Convolvulaceae is one of the largest families of angiosperms consist of 52 genera and 1650 species (Mabberley, 2008) and mainly distributed in the tropical and subtropical regions of the world. Some members of the family Convolvulaceae are medicinally as well as economically important (Austin, 1982; Chopra et al., 1995; Kamalutheen et al., 2009; Sahu and Gupta, 2014). The tribe Merremieae Austin is a diverse tribe in the family Convolvulaceae. Due to many overlapping characters in the members of tribe Merremieae, their proper identification is confusing, which may be due to the lack of sound morphological characters to define it and increase in the number of published species in this tribe. The tribe possesses four genera, viz., Hewittia R. Wight & Arnott, Merremia Dennst. ex Endl., Operculina Silva Manso and Xenostegia Austin & Staples. The genus Hewittia is represented by a single species, Merremia by 70 species, Operculina by 15 species and Xenostegia by only two species in the world (Mabberley, 2008). The present study reveals that, in India the genus Hewittia is represented by a single species, while the genera Operculina and Xenostegia consist of two species each, and the genus Merremia is represented by 11 species. Numerical taxonomy plays important role in segregation of taxa. Cluster analysis (CA) and Principal component analysis (PCA) techniques are commonly used in determining the phylogenetic relationships among different taxa. Applying various methods of numerical taxonomy many authors, e.g. Sneath and Sokal (1973), Chiapella (2000), Gomez-Campo et al. (2001), Sonibare et al. (2004), Henderson (2006), Soladoye et al. (2010), Rahman et al. (2013) and many others have interpreted interrelationship among different genera and families of flowering plants. According to Sonibare et al. (2004) cluster analysis provides a hierarchical classification of entities (taxa) based on the similarity matrix, while PCA is a second method used for reducing the dimensions of the original data which allows visual interpretation of the relationships. Corresponding author. Email: shimpale@yahoo.com 122 DESHMUKH AND SHIMPALE Using macromorphological data and seedling character variations the present study aims to delimit the following 16 species of the tribe Merremieae and to evaluate systematic relationships within the tribe. Materials and Methods Plant specimens: Sixteen species of the tribe Merremieae are listed in Table 1. Freshly collected as well as herbarium specimens deposited in BSI, BAMU, PBL, SUK and herbarium of the The New College, Kolhapur were used in this study. Fresh specimens were collected from different localities of India during 2005 to 2011. Some morphological characters are based on herbarium specimens (Table 1). The species were identified and authenticated with the help of relevant literature (Hooker, 1885; Cooke, 1958; Austin and Staples, 1980; Johari, 1983; Gamble, 1986; Bhandari, 1995; Biju, 1997; Almeida and Almeida, 2001; Singh et al., 2001; Bhagat et al., 2009). All the specimens are deposited in the herbarium of The New College, Kolhapur (NCK), India. Table 1. List of species of the tribe Merremieae along with voucher specimens. No. Species Specimens examined 1 Hewittia malabarica (L.) Suresh Maharashtra: Ratnagiri, Bhatia beach, 2.11.2011, Shimpale 9835 (NCK) 2 Merremia aegyptia (L.) Urban Maharashtra: Sangali, Miraj, 8.11.2006, Shimpale 3998 (NCK) 3 M. dissecta (Jacq.) Hall. f. Maharashtra: Osmanabad, Kasabe Tadawale, 25.1.2009, Shimpale 4098 (NCK) 4 M. gangetica (L.) Cuford. Maharashtra: Kolhapur, Rankala, 12.12.2012, Shimpale 5620 (NCK) 5 M. hederacea (Burm. f.) Hall. f. Maharashtra: Pune, Baramati, 12.11.2008, Shimpale 2209 (NCK) 6 M. peltata (L.) Merr. Great Nicobar Island: Dwivedi 17024 without date (PBL) 7 M. quinquefolia (L.) Hall. f. Maharashtra: Kolhapur, Sarnobatwadi, 9.2.2010, Shimpale 5629 (NCK); Karnataka Belgaum district, Nipani, 23.3.2010, VBS 63 (SUK) 8 M. rajsthanensis Bhandari Rajsthan: Jodhpur, Sardarsamand, 29.8.1975, Bhandari 1976 (CAL) 9 M. rhyncorhiza (Dalz.) Hall. f. Karnataka: Belgaum, Kankumbi, 17.8.2008, Shimpale 3342 (NCK) 10 M. tuberosa (L.) Rendle Maharashtra: Kolhapur, Tarabai Park, 29.12.2009, Shimpale 2629 (NCK) 11 M. umbellata (L.) Hall. f. Maharashtra: Kolhapur, Dajipur, 22.3.2006, Shimpale 269 (NCK) 12 M. vitifolia (Burm.f.) Hall. f. Maharashtra: Kolhapur, Dajipur, 22.3.2006 Shimpale 276 (NCK) 13 Operculina tansaensis Sant. & Patel Maharashtra: Thane, Thansa lake, 28.11.2010, Shimpale 3229 (NCK) 14 O. turpethum (L.) S. Manso Maharashtra: Ratnagiri, Dapoli, 16.1.2008, Shimpale 998 (NCK) 15 Xenostegia filiformis (Thunb.) Almeida Karnataka: Khanapur, 2.1.2001, Shimpale 794 (NCK) 16 X. tridentata (L.) Hall. f. Maharashtra: Sindhudurg, Malvan, 12.11.2009, Shimpale 754 (NCK); Nanded District, Dharmabad, 12.12.1993 Madhukar 6716 (BAMU) MORPHOMETRICS OF THE TRIBE MERREMIEAE 123 Morphometric studies: Morphometric studies were carried out on freshly collected as well as herbarium specimens that are deposited in BSI, BAMU, PBL, SUK and The New College, Kolhapur (NCK). Measurements were taken for 19 selected quantitative characters e.g. leaf length, leaf breadth, petiole length, pedicel length, calyx length, calyx breadth, corolla length, corolla breadth, stamen length, filament length, anther length, ovary length, style length, fruit length, fruit breadth, seed length, seed breadth, apical notch length, paracot length, and recorded on record sheets using as many as numbers of specimens were available for each taxonomic operational unit (OTU). The mean and standard deviation values for all the 19 quantitative characters were calculated and processed for PCA and CA (Kovach, 1999) by keeping data standardized at similarity matrix and tolerance of Eigen analysis set at 1E-010. Results and Discussion Nineteen parameters of 16 species of the tribe Merremieae from India were examined using numerical methods. The morphological features employed for delimitation of the 16 species with their means and standard deviations are shown in Table 2. Similarity matrix based on correlation of Merremieae species (Table 3) shows that close resemblance of species could be observed when certain characters are employed. It is observed that, there is significant correlation between leaf length and corolla breadth, leaf length and corolla length, leaf breadth and petiole length, leaf breadth and calyx length, petiole length and calyx length, petiole length and corolla breadth, pedicel length and paracot length, calyx length and calyx breadth, calyx breadth and fruit breadth, corolla length and corolla breadth, corolla length and style length, corolla breadth and style length, stamen length and paracot length, filament length and paracot length, style length and fruit breadth, style length and seed length, fruit length and seed length, fruit length and fruit breadth. Cluster analysis shows that Xenostegia tridentata distinctly differs from Hewittia malabarica and Merremia aegyptia while both the species of the genus Xenostegia, distinctly differs from M. dissecta, M. hederacea, M. peltata, M. quenquifolia, M. rajsthanensis, M. rhyncorhiza, M. tuberosa, M. vitifolia, Operculina tansaensis and O. turpethum (Table 4). Merremia tuberosa distinctly differs with H. malabarica, M. aegyptia, M. gangetica, M. quenquifolia, M. rajsthanensis and M. umbellata. It was also determined that Operculina turpethum is closely related with the O. tansaensis, M. vitifolia, M. rhyncorhiza, M. dissecta and M. aegyptia, while M. vitifolia closely relate with M. umbellata, M. rajsthanensis, M. quenquifolia, M. dissecta and H. malabarica. The relationships among the species of the tribe Merremieae is shown in Figure 1. Fig. 1. Dendrogram showing relationship among the species of tribe Merremieae 124 DESHMUKH AND SHIMPALE MORPHOMETRICS OF THE TRIBE MERREMIEAE 125 126 DESHMUKH AND SHIMPALE MORPHOMETRICS OF THE TRIBE MERREMIEAE 127 UPGMA dendrogram based on cluster analysis, mean character difference and constrained clustering strategy reveals that, greater affinity exists in between Operculina tansaensis and O. turpethum (Fig. 1). It is also pointed out that, in dendrogram, 16 species clearly forms distinct two clades i.e. I and II. Clade I is divided into subclade A and subclade B. Subclade A comprises Hewittia malabarica, M. aegyptia, M. dissecta, M. gangetica and M. hederacea, while subclade B comprises M. peltata, M. quenquifolia, M. rajsthanensis, M. rhyncorhiza, M. tuberosa, M. umbellata, M. vitifolia, O. tansaensis and O. turpethum. Clade II comprises Xenostegia filiformis and X. tridentata. On the basis of dendrogram it is clear that both the Xenostegia species distantly differs with the remaining species of tribe Merremieae, which supports Austin and Staples (1980) segregation of the genus Xenostegia from Merremia. The present study is the first report depicting correlation between the members of the tribe Merremieae from India and also predicts the phylogenetic relationship 


Introduction
The Convolvulaceae is one of the largest families of angiosperms consist of 52 genera and 1650 species (Mabberley, 2008) and mainly distributed in the tropical and subtropical regions of the world.Some members of the family Convolvulaceae are medicinally as well as economically important (Austin, 1982;Chopra et al., 1995;Kamalutheen et al., 2009;Sahu and Gupta, 2014).
The tribe Merremieae Austin is a diverse tribe in the family Convolvulaceae.Due to many overlapping characters in the members of tribe Merremieae, their proper identification is confusing, which may be due to the lack of sound morphological characters to define it and increase in the number of published species in this tribe.The tribe possesses four genera, viz., Hewittia R. Wight & Arnott, Merremia Dennst.ex Endl., Operculina Silva Manso and Xenostegia Austin & Staples.The genus Hewittia is represented by a single species, Merremia by 70 species, Operculina by 15 species and Xenostegia by only two species in the world (Mabberley, 2008).The present study reveals that, in India the genus Hewittia is represented by a single species, while the genera Operculina and Xenostegia consist of two species each, and the genus Merremia is represented by 11 species.
Numerical taxonomy plays important role in segregation of taxa.Cluster analysis (CA) and Principal component analysis (PCA) techniques are commonly used in determining the phylogenetic relationships among different taxa.Applying various methods of numerical taxonomy many authors, e.g.Sneath and Sokal (1973), Chiapella (2000), Gomez-Campo et al. (2001), Sonibare et al. (2004), Henderson (2006), Soladoye et al. (2010), Rahman et al. (2013) and many others have interpreted interrelationship among different genera and families of flowering plants.According to Sonibare et al. (2004) cluster analysis provides a hierarchical classification of entities (taxa) based on the similarity matrix, while PCA is a second method used for reducing the dimensions of the original data which allows visual interpretation of the relationships.
Using macromorphological data and seedling character variations the present study aims to delimit the following 16 species of the tribe Merremieae and to evaluate systematic relationships within the tribe.

Plant specimens:
Sixteen species of the tribe Merremieae are listed in Table 1.Freshly collected as well as herbarium specimens deposited in BSI, BAMU, PBL, SUK and herbarium of the The New College, Kolhapur were used in this study.Fresh specimens were collected from different localities of India during 2005 to 2011.Some morphological characters are based on herbarium specimens (Table 1).The species were identified and authenticated with the help of relevant literature (Hooker, 1885;Cooke, 1958;Austin and Staples, 1980;Johari, 1983;Gamble, 1986;Bhandari, 1995;Biju, 1997;Almeida and Almeida, 2001;Singh et al., 2001;Bhagat et al., 2009).All the specimens are deposited in the herbarium of The New College, Kolhapur (NCK), India.

Morphometric studies:
Morphometric studies were carried out on freshly collected as well as herbarium specimens that are deposited in BSI, BAMU, PBL, SUK and The New College, Kolhapur (NCK).Measurements were taken for 19 selected quantitative characters e.g.leaf length, leaf breadth, petiole length, pedicel length, calyx length, calyx breadth, corolla length, corolla breadth, stamen length, filament length, anther length, ovary length, style length, fruit length, fruit breadth, seed length, seed breadth, apical notch length, paracot length, and recorded on record sheets using as many as numbers of specimens were available for each taxonomic operational unit (OTU).The mean and standard deviation values for all the 19 quantitative characters were calculated and processed for PCA and CA (Kovach, 1999) by keeping data standardized at similarity matrix and tolerance of Eigen analysis set at 1E-010.

Results and Discussion
Nineteen parameters of 16 species of the tribe Merremieae from India were examined using numerical methods.The morphological features employed for delimitation of the 16 species with their means and standard deviations are shown in Table 2.
Similarity matrix based on correlation of Merremieae species (Table 3) shows that close resemblance of species could be observed when certain characters are employed.It is observed that, there is significant correlation between leaf length and corolla breadth, leaf length and corolla length, leaf breadth and petiole length, leaf breadth and calyx length, petiole length and calyx length, petiole length and corolla breadth, pedicel length and paracot length, calyx length and calyx breadth, calyx breadth and fruit breadth, corolla length and corolla breadth, corolla length and style length, corolla breadth and style length, stamen length and paracot length, filament length and paracot length, style length and fruit breadth, style length and seed length, fruit length and seed length, fruit length and fruit breadth.
Cluster analysis shows that Xenostegia tridentata distinctly differs from Hewittia malabarica and Merremia aegyptia while both the species of the genus Xenostegia, distinctly differs from M. dissecta, M. hederacea, M. peltata, M. quenquifolia, M. rajsthanensis, M. rhyncorhiza, M. tuberosa, M. vitifolia, Operculina tansaensis and O. turpethum (Table 4).Merremia tuberosa distinctly differs with H. malabarica, M. aegyptia, M. gangetica, M. quenquifolia, M. rajsthanensis and M. umbellata.It was also determined that Operculina turpethum is closely related with the O. tansaensis, M. vitifolia, M. rhyncorhiza, M. dissecta and M. aegyptia, while M. vitifolia closely relate with M. umbellata, M. rajsthanensis, M. quenquifolia, M. dissecta and H. malabarica.The relationships among the species of the tribe Merremieae is shown in Figure 1.UPGMA dendrogram based on cluster analysis, mean character difference and constrained clustering strategy reveals that, greater affinity exists in between Operculina tansaensis and O. turpethum (Fig. 1).It is also pointed out that, in dendrogram, 16 species clearly forms distinct two clades i.e.I and II.Clade I is divided into subclade A and subclade B. Subclade A comprises Hewittia malabarica, M. aegyptia, M. dissecta, M. gangetica and M. hederacea, while subclade B comprises M. peltata, M. quenquifolia, M. rajsthanensis, M. rhyncorhiza, M. tuberosa, M. umbellata, M. vitifolia, O. tansaensis and O. turpethum.Clade II comprises Xenostegia filiformis and X. tridentata.On the basis of dendrogram it is clear that both the Xenostegia species distantly differs with the remaining species of tribe Merremieae, which supports Austin and Staples (1980) segregation of the genus Xenostegia from Merremia.The present study is the first report depicting correlation between the members of the tribe Merremieae from India and also predicts the phylogenetic relationship within the members of the tribe.