The JapaneseSocietyJapanese Society for Plant Systematics ISSN1346-7S65 Acta Phytotax, Geobot. 64 (3):ll3-126 (2013) PhylogeneticAnalysis of Nepenthaceae, Based on Internal Transcribed Spacer Nuclear Ribosomal DNA Sequences FiRMAN ALAMsyAHi'2" AND MoToMi IToi JDepartment Studies. Uhiverisity oj'GeneralSystems 7blyo 153-8902,Jopan; 2Department of7btve, ofBiotogicai Science, FZiculty ofAdLithetnatics and Sciences, Ahmad Dahlan *[email protected] Uitivensi(B lanturan, ibgyakarta 55164, lhdonesia. (tiuthorfor correspondeneoj Nepenthaceae, a monotypic family of carnivorous pitcher p]ants comprising Nbpenthes, is widely dis- tributed in Southeast Asia. Tb determine the phytogeography of lVlepenthes in Southeast Asia, and te trace the evolutionary trends of taxonomically important characteristics (i.e., peristomes) of the genus, we analyzed S7 internal transcribed spacer (ITS) nuclear ribosomal DNA (nrDNA) sequences of56 spe- cies of ?Vbpenthes and 1 ITS sequence each ofDionaea muscipula ancl Ancistroeladus robertsoniorerm. To clarify the phylogenetic relationships oflVlepenthes, we examined fbur different methods ofphyloge- netic tree reconstruction. The resulting tree topologies were mostly consistent with one another except for the basal polytomies, Seven monophyletic subclades could be recognized, Similarities and differ- ences in terms of the positions of taxa between the present study and previous studies were ebserved. Judgingfromthephylogenetictreesanddistributionareaofeachspecies,Borneoappearstobeasecond- ary center of diversification fbr Nbpenthes and species ofIVkpenthes may have then radiated within the Sunda Shelfof Southeast Asia. The three character states ofthe peristomes from the upper pitchers were relatively well correlated with the grouping of the species of Nepenthes within seven subclades and showed the limitations ofthe Danser (1928) system for ?Vepenthes, Key words: ITS,IVepenthes, phylegenetic relationship, phytogeography, peristome Nepenthaceae, a monotypic family of carniv- Philippines (21 species, 20 endemic). Although orous pitcher plants comprising ?Vbpenthes, is most extant species oflVepenthes are distributed widely distributed in the Asian tropics, mainly in to the west of the Wallace line, some species, Southeast Asia and the Sunda Shelfregion. The such as IVI ampullaria, IVI gracilis, AL mirabilis, vast majority of species grow in moist regions and AC tentaculata, eccur in both Asia and Wal- throughout the Old World tropics, as far west as lacea (McPherson 2009). The distribution ofthe macingascariensis Madagascar (A4 and A[ maso- genus has been attributed to biogeographic fac- alensis), the Seychelles (NlperviUei), and Sri tors occurring both recently and in the past, in- Lanka (Nl distillatoria) to India (IV] khasiana) in cluding the connectien ofthe Sunda Shelfislands the north, Australia (N tenax and N Towanae) in caused by sea level drop and the isolation of the the south; and New Caledonia (?Vl vieiliardii) in islands caused by increased sea levels, changes in the east (Clarke 2007, Krutzsch 1988, McPherson global climate, and the ability of the species of 2009), IVbpenthes to disperse and colonize new habitats The genus comprises 120 species in Southeast (Clarke 2006, Danser 1928, McPherson 2009, Asia, especially Sumatra (37 species, 29 endem- Meimberg et al. 2001). ic), Borneo (36 species, 29 endemic), and the Meimberg et aL (2001) performeda molecular NII-Electronic Library Service The JapaneseSocietyJapanese Society for Plant Systematics 114 Acta Phytetax. Geobot. Vbl.64 to evaluate phylogenetic analysis of ?Vepenthes based on cosa and N alata havebeen studied DNA sequences of the plastid trnK intron. The the potential value of ITS for phylogenetic recon- trnK intron is more variable than rbcL (Johnsonstruction, This study implies that the ITS regions & Soltis 1995, van den Berg et al. 2005) and is of these species have many variable characteris- widely used for reconstruction of lower taxonom- tics that are potentially infbrmative fbr resolving ic levels and has therefore been the locus of choice the phylogeny of Arapenthes as1ejandro et ai, in molecular phylogenetic analyses of Nepentha- 2008). Ribosomal DNA genes, however, are ceae (Meimberg & Heubl 2006). The results of present in high copy numbers and may therefore their study assumed that colonization of South- be subjected to directional concerted evolution east Asia by AJepenthes was initiated from India, (Wendel et aL 1995a) or intergenomic introgres- since the Indian endemic, IV khasiana, was a sis- sion (Wendel et al. 1995b), ter taxon to all Asian taxa of Nepenthes. A com- The objectives ofthis study were to clarify the parative analysis between the Nepenthes trnK in- phylogenetic relationships ofNepenthes based on tron and its translocated copy, however, has dem- ITS nucleotide sequences fbr 1) determining the onstratedatopological incengruence. Meimberg phytogeography of Nepenthes in Southeast Asia et al. (2006) and Meimberg & Heubl (2006) and to re-evaluate the scenario suggested by 2) trace the evolu- pointed out the possibility ofintrogression or lin- Meimberg et al. (2001), and eage sorting as the reason fbr the topological in- tionary trends ofperistomes in the genus. Peri- cQngruence ofthe phylegenetic trees, Meimberg stomes are stiff structures comprising an inward- & Heubl (2006) then introduced PTRI (peptidely curved rim surrounding the pitcher opening transferase 1), a nuclear low copy gene as a phy- (McPherson 2009). Peristomes have been used logenetic marker, but the resolution of the phylo- in taxonomic studies (Danser 1928) to distin- & Jebb genetic tree was low and some taxa appeared in guish between related species (Cheek different positions from the previous findings in 2009, Lee et aL 2006, McPherson 2009, Robinson the tree topology. Meimberg et al. (2001) provid- et al, 2009) and to determine new species (Cheek ed two possible interpretations concerning the & Jebb 2009, Lee at al, 2006, Robinson et aL origin ofNepenthes; evolution in the northern [Ib- 2009). thys, or a Gondwanan origin at a time when the Indian plate was separated from Madagascar, Materialsand Methods From the molecular analysis based on matK, they suggested that colonization of Southeast Asia Plantmaterials was from an ancient Indian stock and subse- We analyzed 57 samples from 56 species of quently a new secondary center of diversity de- Nepentkes representing all geographical areas veloped in the Malay Archipelago. Re-eyalua- (Table 1), Ofthe included species, seven (AC cha- tion ofthe origin and diversification of Nbpenthes niana, N, lingulata, Any mindnnaoensis, N naga, using another gene is needed because oftopologi- N platychila, N thai, and N vogelii) were found cal incongruence and low resolution ofthe phylo- within the last 10 years (Akhriadi et al. 2009, genetic trees obtained from previous studies. Cheek & Jebb 2009, Lee et aL 2006, McPherson In our study, we used nucleotide sequences of 2009) and have never been used in phylogenetic the internal transcribed spacer (ITS) to resolve studies. TWo samples from distantly distributed phylogenetic relationships within 7Vepenthes. Nl mirabilis were also examined. The first UVI The characteristics of the ITS region, with its mirabitisl) was from Bengkulu, Sumatra, and the small size, highly conserved fianking regions, second (?Vl mirabilis2) was from West Kaliman- and fast evolutionary rate, have made this nuclear tan, Borneo. The seven species and the two dis- ribosornal DNA (nrDNA) sequence a valuable tantly distributed samples of IVI mirahilis from marker fbr phylogenetic analysis (Baldwin et al, the Malay Archipelago were expected to contrib- 1995). In addition, ITS sequences frem N ventri- ute to a better understanding of the phytogeogra- NII-Electronic Library Service The JapaneseSocietyJapanese Society for Plant Systematics October2013 Al.AMsyAH&ITo-PhylogeneticAnalysisefNepenthaceae 115 phy ofNbpenthes in Southeast Asia. Ancistrocta- sarnple was used for phylogenetic analysis; all se- dus robertsoniorum (Ancistrocladaceae, Gen- quences were deposited in DDBJIEMBLIGen- bank: GQ443551) and Dionaea muscipula (Dro- bank(Table 1), seraceae, Genbank: AB675913) were used as out- groups, because these two fam{lies have been Phytogenetic analysis recognized as sister to Nepenthaceae sequences groups on DNA obtained from the ITS region the of macromolecular characteristics using basis were aligned with ClustalX (Larkin et al. 2007). nuclear 18S rDNA and pTastid rbcL, acpB, and Phylogenetic analysis involving the maximum matK sequences (Albert et al. 1992, Cuenoud et parsimeny (MP) method was performed using 2002, al, Hilu et aL 2003), the PAUP (Phylogenetic Analysis using Parsimo- ny) program, version 4.0blO (Swofford 2002). Amplijication and sequencing Data were analyzed by the heuristic search meth- Total DNA was extracted from silica gel- .od with the tree bisection-reconnection (TBR) dried leaf samples with a Qiagen DNeasy Mini branch swapping and MulTrees options on and Plant Kit (Qiagen, Valencia, CA, USA) fo11owing stepwise addition with simple addition sequences the manufacturer's protocol. Amplification ofthe using one reference taxon (AC thai), All of the ITS region firom Ancistrocladaceae was per- most parsimonious trees (MPTs) were saved. All formed using a set of primers, AITSI characters were equally weighted and unordered (5'-AGAAGTCCACTGAACCTTATC-3;) and (Fitch 1971). Gaps were treated as missing data. AITS4(5'-CGCTTCTCCAGACTACAATTC-3'), Tb evaluate the internal