International Research Journal of Biological Sciences ______E-ISSN 2278-3202 Vol. 5(2), 30-35, February (201 6) Int. Res. J. Biological Sci.

A preliminary study of Retinacular shape Variation in Sensu Scotland and Vollesen () and its Phylogenetic significance

Saikat Naskar Department of Botany, Barasat Govt. College, Barasat, Kolkata, 700124, [email protected]

Available online at: www.isca.in, www.isca.me Received 31 st December 2015, revised 14 th January 2016, accepted 1st February 201 6

Abstract Retinacula are synapomorphic for Acanthoideae clade. Morphometric study of retinacula from the members of Acanthoideae has been done to understand the significance of its shape variation in phylogentic context. The monophyly of the sub -tribes Andrographina e and Barleriinae of Acanthoideae are supported by their unique kinds of retinacular shape. The overlapping distribution of the species of Ruellinae and Justiciinae in 2D scattered plot based on retinacular shape is also supported th e link between these two sub-tribes which was established by molecular phylogenetic studies. Therefore retinacular shape variations within Acanthoideae are important for its phylogenetic interpretation.

Keywords: Reticaula, Shape variation, Acanthoideae.

Introduction propel seeds away from the parent when fruit dehisce 8. The structural or shape variation of retinacula within the The sub-family Acanthoideae sensu Scotland and Vollesen members of Acanthoideae and its significance in phylogeny are (Acanthaceae) is characterized by the presence of Retinacula. not studied yet even though it is considered as sole 1 Lindau classified Acanthaceae into four subfamilies of which morphological synapomorphic character in Acanthoideae clade. Acanthoideae contains more than 95 percent of total species. Therefore the present stu dy is intended to find out the 2 Barmekamp excluded the non-retinaculate members from significance of shape variation within some common members Acanthaceae and divided the family into two subfamilies. of Acanthaceae. It is a preliminary study due to selection of a Molecular phylogenetic study did not support the exclusion of limited number of specimens. non-retinaculate members from Acanthaceae. Therefore in more 3 recent classification of Acanthaceae by Scotland and Vollesen The shape variation is generally studied by obtaining a series of the family is classified into three subfamilies where non - linea r measurements and/or angles from shapes followed by retinaculate members are retained. The g enus Avicennia is now multivariate analysis, viz., Principal Component Analysis 4,5 included as a member of broadly circumscribe Acanthaceae . (PCA), Discriminate Analysis (DA), Canonical Variate Analysis (CVA) etc., of these quantitative data. Collectively this process Although various morphological traits are frequently used by is known as morph ometric analysis. In the present study the taxonomists for characterization and identification of the family retinacula morphometric analysis is performed. Acanthaceae, none is eligible to prove synap omorphy in the basal clade of Acanthaceae in molecular phylogenetic Materials and Methods viewpoint. But few morphological traits support the clades within Acanthaceae. For example, reduced ovule number in A total of 17 species of Acanthoideae sensu Scotland and ovary for Nelsonioideae+ Thunbergioideae clade; presence of Vollesen (Table-1) were collected from three southern districts retinacula f or Acanthoideae clade; presence of cystolith for (North 24 Pgs., Sout h 24 Pgs. and Howrah) of West Bengal, Ruellieae+ Justicieae+ Andrographideae+ Barlarieae clade 5; India. The identity of collected taxa was confirmed following presence of quincuncial aestivation for Barlerieae clade 3,6 are the literatures (Bengal Plants by D. Prain, 1903 and Flora of synapomorphic characters. Scotland and Vollesen 3 also showed British India, Vol. IV, 1885) and virtual herbaria (Herbarium of some specific types of pollens are synapomorphic for some IIIM, Jammu; Kew Herbarium et c.). Herbarium sheets of each clades within Acanthoideae. Except pollen characters other collected specimens were prepared and kept in Barasat Govt. synapomorphic characters within Acanthaceae have been poorly College Herbarium for future references. Voucher information resolved, especially the retinacula character. Retinacula are was also maintained with the herbarium sheets. The 17 studied uni versal within Acanthoideae. Retinacula are a hook -shaped species were distributed under 11 genera. F our genera contain structure which subtends each seed within fruit. It is actually a more than one species. This type of selection was done to study modified funiculus that persists within mature fruits 7. Retinacula inter-speci fic retinacula shape variation.

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Table-1 Taxa under the subfamily Acanthoideae for present study Sub-Tribe (sensu Species (Abbreviation) Scotland and Vollesen, Collection No. Date and Place of Collection 2000) paniculata (Burm.f.) Nees 26.04.15; Domjur, Andrographinae PK0065 [Ap] Howrah

04.04.15; Indian Botanic Garden, gangetica (L.) T.Anderson [Ag] Justiciinae PK0011 Sibpur, Howrah

26.04.15; Domjur, cristata L. [Bc] Barleriinae PK0045 Howrah

05.03.15; Maslandapur, Barleria prionitis L. [Bp] Barleriinae PD0003 North 24-Pgs.

26.04.15; Domjur, viride (Forssk.) Alston [Ev] Justiciinae PK0049 Howrah

Hemigraphis hirta (Vahl) T.Anderson 26.04.15; Domjur, Ruelliinae PK0061 [Hh] Howrah

Hygrophila triflora (Roxb.) Fosberg and 15.04.15 ; Sonarpur, Ruelliinae PB0026 Sachet [Ht] South 24-Pgs.

15.04.15 ; Sonarpur, phlomoides Nees [Hp] Ruelliinae PB0031 South 24-Pgs.

15.04.15 ; Sonarpur, adhatoda L. [Ja] Justiciinae PB0029 South 24-Pgs.

15.04.15 ; Sonarpur, Justicia gendarussa Burm.f. [Jg] Justiciinae PB0030 South 24-Pgs.

04.04.15; Indian Botanic Garden, Justicia simplex D. Don [Js] Justiciinae PK0019 Sibpur, Howrah

Peristrophe bicalyculata (Retz.) Nees 26.04.15; Domjur, Justiciinae PK0057 [Pb] Howrah

05.03.15; Maslandapur, imbricata (Forssk.) Sweet [Pi] Ruelliinae PD0005 North 24-Pgs.

26.04.15; Domjur, prostrata Poir. [Rp] Ruelliinae PK0061 Howrah

12.05.15 ; Madhyamgram, Ruellia suffruticosa Roxb. [Rs] Ruelliinae PK0153 North 24-Pgs.

26.04.15; Domjur, Ruellia tuberosa L. [Rt] Ruelliinae PK0073 Howrah

26.04.15; Domjur, pectinata (L.) Nees [Rp] Justiciinae PK0051 Howrah

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Sectioning of mature fruits for retinacula morphometric scattered plot for better understanding about retinacula study: Retinacula morphology was studied from mature fruits. morphological variation among the species of Acanthoideae. Longitudinal sections of fruits were obtained by free hand sectioning. Images of sections were captured under a Magnus Results and Discussion (Olympus) Brand Stereoscopic Microscope fitted with photographic attachment. At least three sections from three Retinacula shapes are highly variable in Acanthoideae (Figure- different fruits of each individual species were captured. 2). It ranges from much curved to slightly curved or pointed end to blunt end structures. In Andrographis the curvature in Digitalization of retinacula morphology and morphometric retinacula is highest while in Rungia the curvature is lesser. The measurements: All jpg format digital images were converted apexes of retinacula of Hygrophila triflora are blunt whereas in into a single tps format file using tpsUtil Software 9. This file other studied species they are pointed. The number of retinacula was opened in tpsDig2 software. A series of linear per fruit is highest in Andrographis and Hygrophila , while measurements and an angle were taken for morphometric study Rungia and Phaulopsis have shown only two retinacula per by this software. The selected length of each retinacula were- fruit. McDae et al. 5 suggested that the increased number of basal breadth; length; middle breadth; sub-middle breadth; sub- ovule, each of which is subtended by a retinaculum, is a apex breadth; and angle between retinacula apex and placental secondary increase in Andrographideae (= Andrographinae axis (Figure-1). A total of six lengths and one angle were sensu Scotland and Vollesen) and in some Rullieae (=Rullinae measured. All measurements were noted in mm scale. sensu Scotnald and Vollesen). Because reduced ovule number may be synapomorphic for Acanthaceae except Nelsonioideae Principal Component Analysis (PCA) of morphometric where six to numerous ovules are found. measurements: PCA was performed based on the multivariate data generated in tpsDig2 software. Before PCA, mean of each The spatula like head of retinacula with maximum curvature measurement was calculated, because at least three distinguish Andrographis from other studied taxa. measurements for each length per species were taken. Andrographinae is a monophyletic group 5. The morphological STATISTICA 8.0 software was used for PCA. Species were synapomorphy in this clade is a special kind of pollens which considered as variable during PCA. The result of statistical are characterized by three compound apertures and highly analysis was presented in a 2D scattered plot, where each point ornamented thick exine 10 . Along with this unique kind of represented a species. 95% confidence ellipses were drawn pollens the unique kind of retinaculum shape may be around the distribution of the species of same sub-tribes of synapomorphic for Andrographinae clade. Acanthoideae. Some retinacular images were added in 2D

Figure-1 Moprhometric mesurements of retinacula

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Andrographis and the two species of Barleria have been plotted Taxonomists are always searching the suitable non-molecular in 2D scattered plot (Figure-3) in isolated positions, which characters to describe a group. Since the retinaculum is the indicates their uniqueness. Unlike Andrographis the retinacula only synapomorphic character for the entire Acanthoideae clade, shapes of Barleria are apparently not very distinguishable from it demands a special attention. In this regard this preliminary the species of Justiciinae and Ruellinae. Barleriinae is also a study on retinacular variation within Acanthoideae indicants its monophyletic group with only non-molecular synapomorphic significance in phylogenetic context. character is its quincuncial aestivation 3,6 . Critical study on retinacula shape considering more species from Barleriinae may Acknowledgment be given better retinacular shape oriented syapomorphy. I would like to thanks Principal, Barasat Govt. College and Overlapping retinacula shapes are observed among the members Head, Department of Botany, Barasat Govt. College, Kolkata of Ruelliinae and Justiciinae. The shape variation is highest in for providing me space and facilities to complete the present Justiciinae followed by Rueliinae. Interestingly being the research work. I would also like to thanks my students who help members of same genus the retinacula shape of Hygrophila me to collect the specimens for present study. phlomoides and H. triflora is completely different. In H. phlomoides the retinacula tips are pointed whereas H. triflora References shows a blunt tip. Another interesting fact is that, being the member of two different sub-tribes the retinacula shape 1. Lindau G. (1895). Acanthaceae, In: Engler A. and Prantl, similarity is observed between Phaulopsis and Rungia . Both K. (eds.). Die Natfirlichen Pflanzenfamilien , 4(3b), 274– have two retinaculate and close in 2D scattered plot. Naskar 11 353. also showed that closeness between these two taxa in a phenetic 2. Bremekamp C.E.B. (1965). Delimitation and subdivision study based on gross morphology and pollen morphology. of the Acanthaceae. Bulletin of Botanical Survey of India , Justiciinae-Ruelliinae relationship has already been established 7, 21-30. in molecular phylogeny. They are sister taxa and they have a 3. Scotland R.W. and Vollesen K. (2000). Classification of strong support for monophyletic group 5. Retinacula shape Acanthaceae. Kew Bulletin , 55, 513-589. similarity is also supporting their closeness and their overlapping distribution indicates their link. Daniel et al. 12 4. Schwarzbach A.E. and McDade L.A. (2002). suggested that Justiciinae-Ruellinae clade has no non-molecular Phylogenetic relationships of the mangrove family synapomorphic support. In this regard the retinacula shape is a Avicenniaceae based on chloroplast and nuclear non-molecular synapomorphy for this clade, but critical study ribosomal DNA sequences. Systematic Botany , 27, 84– considering numerous sampling is needed. 98. 5. McDade L.A., Daniel T.F. and Kiel C.A. (2008). Toward Conclusion a comprehensive understanding of phylogenetic relationships among lineages of Acanthaceae s.l. This study indicates that the retinacular shape variation within (). American Journal of Botany , 95, 1136–1152. the clades of Acanthoideae is important to explain the molecular phylogenetic schemes on Acanthoideae (Acanthaceae).

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Figure-2 Retinacula in Acanthoideae. a- Andrographis paniculata ; b- Barleria cristata ; c- Barleria prionitis ; d- Hygrophila triflora ; e- Hygrophila phlomoide ; f- ; g- Asystasia gangetica ; h- Ruellia tuberose ; i- Ruellia suffruticosa ; j- Ruellia prostrate ; k- bicalyculata ; l- Justicia simplex ; m- Rungia pectinata ; n- Phaulopsis imbricate

Figure-3 Species of Acanthoideae are plotted against their morphometric data score against PC 1 and PC 2. These two PCs are responsible for about 74% and 17% of total variation

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6. Scotland R.W., Sweere J.A., Reeves P.A. and Olmstead 11. Naskar S. (2015). Numerical of some R.G. (1995). Higher-level systematics of Acanthaceae members of Acanthoideae sensu Scotland and Vollesen determined by chloroplast DNA sequences. American (Acanthaceae) from North 24 Parganas District, West Journal of Botany , 82, 266–275. Bengal, India based on Gross Morphology and Pollen Characters. International Research Journal of Biological 7. Judd W.S., Campbell C.S., Kellogg E.A. and Stevens Sciences , 4, 48-55. P.F. (1999). Plant Systematics: A Phylogenetic Approach . Sinauer Associates, Massachusetts, USA. 12. Daniel T.F., McDade L.A., Manktelow M. and Kiel C.A. (2008). The “ lineage” (Acanthaceae: 8. Witztum A. and Schulgasser K. (1995). The mechanics Acanthoideae: Justicieae): Delimitation and intra-lineage of seed expulsion in Acanthaceae. Journal of Theoretical relationships based on cp and nrITS sequence data. Biology , 176, 531–542. Systematic Botany , 33, 416 – 436. 9. Rohlf F.J. (2012). tps series of software. Retrieved from http://life.bio.sunysb.edu/morph/morphmet.html. 10. Scotland R.W. (1992). Pollen morphology of Andrographideae (Acanthaceae). Review of Palaeobotany and Palynology , 72, 229–243.

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