J.Natn.Sci.Foundation Sri Lanka 2017 45 (3): 307 - 318 DOI: http://dx.doi.org/10.4038/jnsfsr.v45i3.8194 RESEARCH ARTICLE A taxonomic synopsis of Nymphaea nouchali Burm. f. and LQIUDVSHFL¿FWD[D Shashika Guruge 1,2, Deepthi Yakandawala 2* and Kapila Yakandawala 3 1 Postgraduate Institute of Science, University of Peradeniya, Peradeniya. 2 Department of Botany, Faculty of Science, University of Peradeniya, Peradeniya. 3 Department of Horticulture and Landscape Gardening, Faculty of Agriculture and Plantation Management, Wayamba University of Sri Lanka, Makandura, Gonawila. Revised: 03 January 2017; Accepted: 16 February 2017 Abstract: Nymphaea nouchali H[KLELWVDUDQJHRIÀRZHUFRORXUV INTRODUCTION ZKHUHVRPHWD[RQRPLFFLUFXPVFULSWLRQVFRQVLGHUWKHFKDUDFWHU as of WD[RQRPLF VLJQL¿FDQFH LQ UHFRJQLVLQJ LQIUDVSHFL¿F taxa. The revision of the Sri Lankan Nymphaeaceae has not Water-lilies ( Nymphaea / DUH DPRQJ WKH VKRZLHVW UHFRJQLVHG DQ\ LQIUDVSHFL¿F WD[D N. nouchali populations aquatic plants and have attracted the attention of H[KLELWLQJ PRUSKRORJLFDO YDULDWLRQV ZLWKLQ D UDQJH RI ÀRUDO botanists, horticulturists and plant enthusiasts FRORXUVZHUHHQFRXQWHUHGGXULQJWKH¿HOGVWXGLHV conducted in .DEDWRYD et al ., 2014). The genus Nymphaea exhibits GLIIHUHQWSDUWVRIWKHFRXQWU\7KHUHIRUHWKHSUHVHQWVWXG\ZDV D ZRUOGZLGH GLVWULEXWLRQ DQG FRPSULVHV PRUH WKDQ FRQGXFWHGZLWKWKHREMHFWLYHRIHYDOXDWLQJWKHPRUSKRORJLFDO 50 extant species (Borsch et al 7KH ZDW er-lily variations of N. nouchali populations using multivariate family, Nymphaeaceae, is descended from an old and analyses of morphological and molecular sequence data. Both morphometric analysis using 59 characters and matK and evolutionarily early group, that as fossil evidence trnH-psbA VHTXHQFHGDWDVXSSRUWHGWZRVXEJURXSVZLWKLQ N. suggests, has occupied the earth from the early Cretaceous nouchali, FOHDUO\ FRUUHVSRQGLQJ WR WKH GLIIHUHQFHV LQ ÀRZHU period 125 – 115 million years ago (Friis et al ., 2001). FRORXURQHJURXSZLWKEOXHÀRZHUVDQGWKHRWKHUZLWKZKLWH )XUWKHU ZDWHUOLOLHV DUH OHJHQGDU\ LQ WKH KLVWRU\ DQG DQG SLQN ÀRZHUV ,Q DGGLWLRQ FRORXU RI WKH SHWLROH DQG WKH traditions of native groups in countries such as Egypt, leaf abaxial surface also supported the grouping. The study China, Thailand and India, including Sri Lanka. UHFRJQLVHGWZRLQIUDVSHFL¿FWD[DIRU N. nouchali in Sri Lanka, N. nouchali var. nouchali ZLWKEOXHÀRZHUV>µ1LOPDQHO¶ 6 The genus is represented in Sri Lanka by three %OXH ZDWHUOLO\ ( @ DQG N. nouchali var. versicolour (Sims) native taxa, N. nouchali Burm. f. (‘Nil manel’;‘Manel’), *XUXJH DQG<DNDQGDZDOD ZLWK HLWKHU ZKLWH RU SLQN ÀRZHUV 5HFRJQLVLQJLQIUDVSHFL¿FWD[DRI N. nouchali in Sri Lanka is N. pubescens Willd. (‘Olu’) and N. rubra Roxb. ex VLJQL¿FDQWWRWKHFRXQWU\QRWRQO\WRDFNQRZOHGJHLWVQDWXUDO $QGUHZV µ5DWKX 2OX¶ µ5DWK EHUDOL\D¶ 'DVVDQD\DNH diversity, but in addition most importantly, as N. nouchali *XUXJH et al. , 2014). In addition to naturally LV WKH GHFODUHG QDWLRQDO ÀRZHU RI 6UL /DQND In this context, RFFXUULQJZDWHUOLOLHVD IHZ H[RWLFRUQDPHQWDOVSHFLHV WKH UHFRJQLWLRQ RI WKH EOXHÀRZHUHG JURXS DV N. nouchali have been introduced in the past and one violet- var. nouchali ZRXOG VWUHQJWKHQ LWV LGHQWLW\ RYHU WKH RWKHU ÀRZHUHGVSHFLHV µ'DPPDQHO¶ KDVEHFRPHQDWXUDOLVHG ÀRZHUFRORXUJURXSVRI N. nouchali. A taxonomic key for the WKURXJKRXW WKH ORZODQGV LQ Sri Lanka. The erroneous Sri Lankan Nymphaea DQGGHVFULSWLRQVIRUWKHWZRLQIUDVSHFL¿F taxa are also produced. LGHQWL¿FDWLRQRIWKLVVSHFLHVDV N. nouchali has caused FRQIXVLRQ DV LW ZDV DOVR XVHG WR GHSLFW µ1LO PDQHO¶ Keywords: ,QIUDVSHFL¿F WD[D matK and trnH-psbA, WKH QDWLRQDO ÀRZHU RI 6UL /DQND <DNDQGDZDOD morphometric analysis, Nymphaea nouchali . <DNDQGDZDOD * Corresponding author ([email protected]) 308 Shashika Guruge et al. 7KHÀRZHUFRORXURI N. nouchaliH[KLELWVDUDQJHZKHUH WKHÀRZHUOHDIDED[LDODQGDGD[LDOVXUIDFHVDQGSHWLROH different taxonomic treatments describe the colour as a ZDV GHWHUPLQHG XVLQJ WKH 5R\DO +RUWLFXOWXUDO 6RFLHW\ combination of either ‘blue’, ‘pale blue’, ‘blue-violet’, colour chart (RHS Colour Chart, 2001). Special attention µYLROHW¶ µSLQN¶ µSXUSOHUHG¶ RU µZKLWH¶ 7ULPHQ ZDVSDLGWRWKHFKDUDFWHUVZLWKGLVWLQFWYDULDWLRQV7KHOLVW Dassanayake, 1996; La-ongsri et al ZLWKRXW RIFKDUDFWHUVWRJHWKHUZLWKWKHFKDUDFWHUVWDWHVHPSOR\HG emphasis on the character as being of taxonomic in the morphometric analyses is given in Table 1. VLJQL¿FDQFH2QWKHRWKHUKDQGVRPHFLUFXPVFULSWLRQV KDYHFRQVLGHUHGÀRZHUFRORXUDVDQLPSRUWDQWIHDWXUHLQ Data analysis UHFRJQLVLQJ LQIUDVSHFL¿F WD[D +RRNHU &RQDUG 1905; Slocum et al 5HFRJQLVLQJ LQIUDVSHFL¿F 0RUSKRORJLFDO GDWD ZHUH VXEMHFWHG WR SULQFLSDO taxa is important in terms of documenting the coordinate analysis (PCoA) and hierarchical cluster biodiversity richness of a country. Field studies carried DQDO\VLV &$ XVLQJWKHVWDWLVWLFDOVRIWZDUH3$67 YHU out throughout Sri Lanka have revealed N. nouchali 2.15) (Hammer et al 7KH FOXVWHU VROXWLRQ ZDV SRSXODWLRQVH[KLELWLQJPRUSKRORJLFDOYDULDWLRQZLWKLQD VHOHFWHG IURP WKH EHVW VXLWDEOH DOJRULWKP ZKHUH WKH UDQJHRIÀRUDOFRORXUVEOXHSLQNDQGZKLWHDVUHFRUGHG *RZHUތV GLVWDQFH ZDV XVHG WR FDOFXODWH WKH VLPLODULW\ LQOLWHUDWXUHZDUUDQWLQJDUHYDOXDWLRQRIWKHYDOLGLW\RI PHDVXUHVZLWKWKHSDLUHGJURXS>XQZHLJKWHGSDLUJURXS the description of LQIUDVSHFL¿FWD[D7KHEOXHÀRZHUHG PHWKRG ZLWK DULWKPHWLF PHDQ 83*0$ @ RSWLRQ DQG forms of N. nouchali are locally FDOOHGµ1LOPDQHO¶ZKLOH WKHVLQJOHOLQNDJHDOJRULWKPZLWKWKHKLJKHVWFRSKHQHWLF ZKLWHÀRZHUHGIRUPVDUHJHQHUDOO\NQRZQDVµ7HOROX¶ FRUUHODWLRQYDOXH7KHRUGLQDWLRQDQDO\VLVZDVSHUIRUPHG although they are more related to ‘Manel’ than ‘Olu’ ZLWK WKH *RZHUތV GLVWDQFH WUDQVIRUPDWLRQ H[SRQHQW <DNDQGDZDOD 3HDERWXZDJH +HQFHWKHSUHVHQW C = 2) to generate a distance matrix to use in the PCoA. VWXG\ ZDV FRQGXFWHG ZLWK WKH REMHFWLYH RI HYDOXDWLQJ )ROORZLQJ WKH UHVXOWV RI WKHVH DQDO\VHV HDFK PDMRU morphological variations of N. nouchali populations FRQVLVWHQWO\UHFRYHUHGFOXVWHUZDVLGHQWL¿HG using multivariate analyses of morphological characters and a phylogenetic analysis using molecular sequence Molecular studies data. *HQRPLF '1$ ZDV H[WUDFWHG IURP IUHVK SHWDOV RI N. nouchali representing DOOWKUHHÀRZHUFRORXUVEOXH METHODOLOGY WKUHH GLIIHUHQW SRSXODWLRQV ZKLWH WKUHH GLIIHUHQW SRSXODWLRQV DQG SLQN WZR GLIIHUHQW SRSXODWLRQV Morphological studies DQG GULHG ZLWK VLOLFD JHO XVLQJ WKH 4LDJHQ SODQW '1$ H[WUDFWLRQ NLW 4LDJHQ 9DOHQFLD &$ 7KH Sample collection WZR FKORURSODVW JHQH UHJLRQV matK ( matK -390f &*$7&7$77&$77&$$7$777& matK - )LHOGYLVLWVZHUHPDGHFRYHULQJ all three major climatic U 7&7$*&$&$&*$$$*7&*$$*7 ]RQHVRIWKHLVODQG viz ., Wet, Intermediate and Dry Zones (Cuenoud et al ., 2002) and psbA-trnH [ psbA -F5'– IURP± )LJXUH ,QGLYLGXDOVZHUHFROOHFWHG *77$7*&$7*$$&*7$$7*&7& 6DQJ et al ., 1997), IURPSRSXODWLRQVZLWKHDFKGHQRWHGE\DQDFURQ\P trnH-R &*&*&$7**7**$77&$&$$7&& WR IDFLOLWDWH WKHLU UHIHUHQFH 0RUSKRORJLFDO GDWD ZHUH 7DWH 6LPSVRQ @ ZHUH DPSOL¿HG XVLQJ WKH studied in detail at the Plant Systematics Laboratory, polymerase chain reaction (PCR) technique. For all the Department of Botany, University of Peradeniya. DPSOL¿FDWLRQV3URPHJD*R7DT ® Flexi DNA polymerase 9RXFKHUVSHFLPHQVDQGOLYHFROOHFWLRQVZHUHPDLQWDLQHG (Madison, WI, USA) ZDV XVHG according to the at the Department of Botany, University of Peradeniya, manufacturer’s recommendations. PCR DPSOL¿FDWLRQV Sri Lanka and the Faculty of Agriculture and Plantation ZHUH FDUULHG RXW LQ ȝ/ UHDFWLRQ VROXWLRQV WKDW Management, Wayamba University of Sri Lanka during FRQWDLQHG î 3&5 UHDFWLRQ EXIIHU P0 0J&O 2, the study. P0GHR[\QXFOHRWLGHWULSKRVSKDWH G173V ȝ0 RI HDFK IRUZDUG DQG UHYHUVH SULPHU 8 RI7DT '1$ Character coding SRO\PHUDVH DQG ± ȝ/ XQTXDQWL¿HG '1$ H[WUDFW7KH3&5SURJUDPPHZDVUXQRQDQ(SSHQGRUI 'DWD ZHUH REWDLQHG IURP WKUHH LQGLYLGXDOV UDQGRPO\ Mastercycler thermal cycler (Hauppauge, NY, USA). selected from each population, and qualitative and The programme consisted of 3 min of initial denaturation TXDQWLWDWLYHFKDUDFWHUVZHUHH[DPLQHGLQWKHODERUDWRU\ DW&F\FOHVRIVGHQDWXUDWLRQDW&DQG either by the naked eye or under a dissecting or VDQQHDOLQJDW&&IRU matK and psbA-trnH , VWHUHRPLFURVFRSH /HLFD;:' &RORXURI UHVSHFWLYHO\PLQSULPHUH[WHQVLRQDW&IROORZHG September 2017 Journal of the National Science Foundation of Sri Lanka 45(3) ,QIUDVSHFL¿FWD[DRI1\PSKDHDQRXFKDOL E\D¿QDOH[WHQVLRQIRUPLQDW&3&5SURGXFWV genetic analyser (Department of Molecular Biology ZHUH UXQ RQ D DJDURVH JHO VWDLQHG ZLWK HWKLGLXP and Biotechnology, Faculty of Science, University of bromide, and visualised on a UV illuminator. The 3HUDGHQL\D 6UL /DQND 5DZ VHTXHQFHV ZHUH VFUHHQHG PROHFXODUPDVVRIWKHUHVXOWHGEDQGVZDVHVWLPDWHGZLWK and assembled and consensus for resulted sequences DNE'1$ODGGHUDQGFRQ¿UPHGWKHDPSOL¿FDWLRQRI RI IRUZDUG DQG UHYHUVH SULPHUV ZHUH FRPSLOHG XVLQJ WKHSULPHU2EWDLQHG3&5SURGXFWVZHUHVXEPLWWHGIRU BioEdit version 7.1.11 (Tom Hall, Ibis Biosciences, sequencing reactions using Applied Biosystems, 3500 California, USA). Basic Local Alignment Search Tool or Figure 1: Sample collection locations of Nymphaea nouchali for the present study Journal of the National Science Foundation of Sri Lanka 45(3) September 2017 310 Shashika Guruge et al. the BLAST (Altschul et al ZDV XVHG WR VHDUFK VHTXHQFHOLVWZDVFKHFNHGIRUDJLYHQVDPSOH¶VLGHQWLW\ WKH PRVW VLPLODU VHTXHQFH GHSRVLWHG LQ WKH *HQ%DQN to a particular species. CLUSTALW ZDV used to perform WKDWPDWFKHGZLWKWKHREWDLQHGVHTXHQFH7KHUHVXOWLQJ multiple sequence alignment (Thompson et al ., 1997). Table 1: List of characters and their character states used in the morphological studies Character Character state/unit 6KDSHRIWKHÀRZHUEXG