The chloroplastic trnL intron and the nuclear internal transcribed spacer (ITS) region were sequenced for 11 Nepenthes species recorded in Peninsular Malaysia to examine their phylogenetic relationship and to evaluate the usage of trnL intron and ITS sequences for phylogenetic reconstruction of this genus.
Results
Phylogeny reconstruction was carried out using neighbor-joining, maximum parsimony and Bayesian analyses. All the trees revealed two major clusters, a lowland group consisting of N. ampullaria, N. mirabilis, N. gracilis and N. rafflesiana, and another containing both intermediately distributed species (N. albomarginata and N. benstonei) and four highland species (N. sanguinea, N. macfarlanei, N. ramispina and N. alba).
Conclusions
The trnL intron and ITS sequences proved to provide phylogenetic informative characters for deriving a phylogeny of Nepenthes species in Peninsular Malaysia. To our knowledge, this is the first molecular phylogenetic study of Nepenthes species occurring along an altitudinal gradient in Peninsular Malaysia.
Keywords
ITSNepenthesPeninsular MalaysiaPitcher plant
Background
Seven genera of pitcher plants namely Cephalotus, Heliamphora, Darlingtonia, Brocchinia, Nepenthes, Sarracenia and Catopsis. Pitcher plants have a worldwide distribution [1]. Nepenthes is the largest genus belonging to the family Nepenthaceae and consists of 120 species and five incompletely described taxa in seven geographical groupings: Nepenthes of Peninsular Malaysia and Indochina, Nepenthes of Borneo, Nepenthes of Sulawesi, Nepenthes of Sumatra and Java, Nepenthes of the Philippines, Nepenthes of New Guinea and Maluku Islands, and lastly Nepenthes of the Outlying Areas [1]. The endemic species of Nepenthes occur throughout Southeast Asia, particularly in the Sunda region, which includes Borneo, Sumatra, the Malay Peninsula, Java and some of the southern islands of the Philippines [2].
The Nepenthes flora in Peninsular Malaysia is relatively poor with only 11 species presently recorded (N. alba Ridl., N. albomarginata T. Lobb ex Lindl., N. gracillima Ridl., N. ampullaria Jack, N. benstonei C. Clarke, N. gracilis Korth., N. macfarlanei Hemsl., N. rafflesiana Jack, N. mirabilis (Lour.), Druce, N. ramispina Ridl. and N. sanguinea Lindl.). In comparison, there are 36 species recognized in Borneo, representing the greatest diversity of Nepenthes species in Southeast Asia [1, 3]. Clarke [2] reported only ten species of Nepenthes in Peninsular Malaysia, classifying N. alba as a heterotypic synonym of N. gracillima. Four of these species are restricted to montane habitats (N. macfarlanei, N. gracillima, N. sanguinea, N. ramispina), two species are found mainly on low hills (N. albomarginata and N. benstonei) and another four are lowland species (N. ampullaria, N. gracilis, N. mirabilis and N. rafflesiana). McPherson [1] described both N. alba and N. gracillima as distinct species.
The taxonomy of Nepenthes is based primarily on morphology (shape, color, size and ornamentation) [1–6]. Jebb and Cheek [7] recognized a single species, N. vieillardii, in New Caledonia into a single species, even though several species were recognized based on morphological variation. Kurata et al. [8] clarified the morphological diversity and verified the species classification of N. vieillardii, and they tentatively supported the taxonomic classification based on the pitcher morphology by Jebb and Cheek [7]. The current classification of Nepenthes in Peninsular Malaysia is also based on morphological characteristics, with distinct differences apparent between species found at high, intermediate and low altitudes.
Previous molecular phylogenetic studies of Nepenthes based on chloroplast (trnK intron and matK gene) and nuclear (PRT1 and a translocated copy of trnK) sequences have provided a well-supported phylogeny of many species [9–11]. Using the plastid trnK intron in phylogenetic reconstruction, the three lineages can be separated according to habitat [9]. The first lineage included all species found in Sumatra, the Malay Peninsula and the Southeast mainland; the second lineage consisted of the species from Sulawesi, Borneo and the Philippines; and the third lineage comprised Nepenthes from New Guinea and Sulawesi [9, 11]. Meimberg and Heubl [11] also suggested that biogeographic “outlier species” occurring in Seychelles (N. pervillei Blume.), Sri Lanka (N. distillatoria L.), Madagascar (N. madagascarensis Poir. and N. masoalensis Schmid-Hollinger) and India (N. khasiana Hook. F.) are related to the three lineages consisting of all taxa from the Indo-Malay region.
Apart from molecular phylogenetic studies of Nepenthes using nuclear PRT and plastid matK DNA sequences [9–11], there has been little development in the molecular systematics of Nepenthes. We report here the potential of the plastid trnL intron and nuclear ITS DNA sequences for the phylogenetic inference of Nepenthes in Malaysia. We also report here the phylogeographics of the Nepenthes species found in Peninsular Malaysia based on the DNA sequence data.
Methods
Sample collection
All the plant materials were collected from 11 sites (one individual from each of these localities) in Peninsular Malaysia, which covered nearly the complete natural range of the species except for N. gracillima Ridl (Table 1). Herbarium specimens were identified by Ruzi Rahman and deposited at the Universiti Kebangsaan Malaysia Herbarium (UKMB), Universiti Kebangsaan Malaysia.
Table 1
Sample location of Nepenthes species in Peninsular Malaysia and the outgroup species (Sarracenia flava)
Name
Accessions
State
Altitude (m)
GenBank (trnL intron)
GenBank (ITS)
N. ampullaria
INBIOSIS-N:AMP-2011
Johor–Ulu Tiram–Mersing
100–150
JX042566
JX042554
N. gracilis
INBIOSIS-N:GRA-2011
Selangor–Bukit Putri–UKM Bangi
100–150
JX042567
JX042555
N. mirabilis
INBIOSIS-N:MIR-2011
Selangor–Rawang/Batu Arang
50–100
JX042568
JX042556
N. rafflesiana var. elongata
INBIOSIS-N:RAF-E-2011
Johor–Ulu Tiram–Mersing
150–300
JX042569
JX042557
N. rafflesiana var. nivea
INBIOSIS-N:RAF-N-2011
Johor–Ulu Tiram–Mersing
150–300
JX042570
JX042558
N. albomarginata
INBIOSIS-N:ALBO-2011
Pahang–Cameron Highland
600–1300
JX042571
JX042559
N. benstonei
INBIOSIS-N:BEN-2011
Kelantan–Bukit Bakar
600–1000
JX042572
JX042560
N. sanguinea
INBIOSIS-N:SAN-2011
Pahang–Cameron Highland
1000–1500
JX042573
JX042561
N. macfarlanei
INBIOSIS-N:MAC-2011
Kelantan–Cameron Highland
800–1800
JX042574
JX042562
N. ramispina
INBIOSIS-N:RAM-2011
Pahang–Cameron Highland
600–1700
JX042575
JX042563
N. alba
INBIOSIS-N:ALBA-2011
Pahang–Cameron Highland
800–1800
JX042576
JX042564
Sarracenia flava
INBIOSIS-S:FLAVA-2011
Selangor
300 (Sarracenia originated from North America–grown in Selangor, lowland condition)
JX042577
JX042565
Molecular procedures
DNeasy Plant Mini Kit (Qiagen, Germany) was used in extraction of total genomic DNA from fresh leaf tissue. The plastid trnL intron and the nuclear internal transcribed spacer (ITS) region consisting of ITS1, ITS2 and the 5.8S were PCR amplified. The amplification of the trnL intron was done using primers c (5′-CGA AAT CGG TAG ACG CTA CG-3′) and d (5′-GGG GAT AGA GGG ACT TGA AC-3′) [12], and the amplification of the ITS region was done using primers P17F (5′-CTA CCG ATT GAA TGG TCC GGT GAA-3′) and 26S-82R (5′-TCC CGG TTC GCT CGC CGT TAC TA-3′) [13]. A 100 µL PCR reaction was used in target gene amplification and consisted of the following components: 55 μL dH2O, 20 μL 10 × buffer, 10 μL of 25 mM MgCl2, 2.0 μL of 20 mM dNTPs, 1.0 μL each forward and reverse primers, 100 mM, 1.0 μL of Taq DNA polymerase (Promega) and 5 μL genomic DNA (20 ng/uL). The PCR was carried out in thermocycler (Applied Biosystem, USA) under the following cycling conditions: preliminary denaturation at 95 °C for 2 min; 35 cycles at 95 °C for 1 min, 45/66 °C for 1 min, and 72 °C for 1 min; final elongation at 72 °C for 5 min. The resultant PCR products were cleaned with PureLink™ PCR Purification Kit (Invitrogen) according to the manufacturer’s protocol.
ABI Prism Dye Terminator Cycle Sequencing Ready Reaction kit and ABI PRISM 3100 Genetic Analyzer (Perkin-Elmer, Foster City, CA) was used in DNA sequencing, following the manufacturer’s instructions. Direct sequencing was applied on both DNA regions in forward and reverse directions. The sequencing primers for the trnL intron were the same as the PCR primers. Primers P16F (5′-TCA CTG AAC CTT ATC ATT TAG AGG-3′) and P25R (5′-GGG TAG TCC CGC CTG ACC TG-3′) [13] were used to sequence the ITS region.
Sequence analysis
ClustalX Multiple Sequence Alignment programme was used to align DNA sequences using the default settings. Sarracenia flava was designated as the outgroup in the phylogenetic analyses. The DNA sequences of the ITS region and trnL intron were combined in phylogenetic analyses. The evolutionary history was analyzed by neighbor-joining (NJ) and maximum parsimony (MP) methods using PAUP* 4.0b10 [14]. All positions containing gaps and missing data were removed from the dataset using the ‘complete deletion’ option. The pairwise distances generated using the uncorrected “p” model were used to construct the NJ tree. For the MP analysis, the cladograms were constructed utilizing unordered parsimony with equal weight. A heuristic search was conducted with the tree bisection–reconnection (TBR) branch swapping algorithm, random stepwise addition and ‘Mul-Trees’ option set on all the characters were weighted equally. The internal branch supports for the NJ and MP trees were assessed with Bootstrap analyses using 500 replicates. Bayesian inferences were carried out [15, 16] using MrBayes 3.12 [17]. The general reversible model with Gamma distributed rate heterogeneity (GTR + G) was selected by AIC using MrModelTest version 2.2 [18]. Two simultaneous metropolis-coupled Monte-Carlo Markov chains for >1,000,000 generations, a sample frequency of 100 generations and average standard deviation of split frequencies 0.009311 was used in Bayesian inference.
Results
The PCR amplification and subsequent sequencing produced the ITS and trnL intron ITS fragments with size ranges of 555–559 and 673–705 bp, respectively. A total of 1311 characters were involved in the phylogenetic analyses of which 1012 were constant and 42 were parsimony-informative, while 257 variable characters were parsimony-uninformative. The pairwise genetic distances among the Nepenthes taxa ranged from 0 to 0.0342 (Table 2). The analyses of the combined trnL and ITS sequence data produced the NJ, MP and Bayesian trees with same topology. The NJ tree is shown in Fig. 1a. The MP analysis produced two most parsimonious trees with a tree length of 327 steps, CI = 0.9755 and RI = 0.9424. One of the parsimonious trees was similar to the topology of the Bayesian inference tree (Fig. 1b). The bootstrap values obtained from the MP analysis are shown above the branches.
Table 2
Pairwise distances of Nepenthes species generated from the combined trnL intron and ITS sequences based on uncorrected “p” model
1
2
3
4
5
6
7
8
9
10
11
12
N. gracilis
–
N. rafflesiana var. elongata
0.00405
–
N. ampullaria
0.02427
0.02346
–
N. mirabilis
0.00488
0.00569
0.02522
–
N. rafflesiana var. nivea
0.00405
0.00000
0.02346
0.00569
–
N. albomarginata
0.03078
0.02673
0.02876
0.03169
0.02673
–
N. benstonei
0.03079
0.02673
0.02796
0.0317
0.02673
0.00238
–
N. sanguinea
0.03324
0.02919
0.03119
0.03415
0.02919
0.00318
0.00477
–
N. macfarlanei
0.03243
0.02837
0.03038
0.03333
0.02837
0.00159
0.00397
0.00158
–
N. ramispina
0.03324
0.02919
0.03119
0.03415
0.02919
0.00238
0.00477
0.00237
0.00079
–
N. alba
0.03159
0.02753
0.02955
0.03249
0.02753
0.00079
0.00317
0.00238
0.00079
0.00159
–
Sarracenia flava
0.2265
0.22316
0.22292
0.22296
0.22316
0.21805
0.21710
0.2185
0.21783
0.21783
0.21721
–
Fig. 1
a Neighbor-joining tree generated based on the combined trnL and ITS sequence data with yellow pitcher plant (Sarracenia flava) as outgroup, a carnivorous plant native to the New World. The numbers above the branches represent the bootstrap percentage. b Majority-rule consensus tree resulted from the Bayesian analysis of trnL and ITS sequence data. Numbers at below branches are posterior probabilities. The bootstrap values from the MP analysis are shown above the branches
All the trees indicated the formation of two major genetic lineages: (1) Clade A (N. albomarginata, N. benstonei, N. sanguinea, N. macfarlanei, N. ramispina and N. alba) and (2) Clade B (N. ampullaria, N. mirabilis, N. rafflesiana and N. gracilis). These two major clades were supported by high bootstrap values and good posterior probability values (Fig. 1).
Discussion
According to Clarke [2], only ten species of Nepenthes are presently recognized in Peninsular Malaysia on the basis of morphology. Four of these species are confined to montane habitats, two are found primarily on low hills and the remainders are lowland species that are common throughout the Sunda region. Based the combined trnL and ITS data, the lowland Nepenthes taxa in Peninsular Malaysia (N. ampullaria, N. rafflesiana, N. mirabilis and N. gracilis) clustered together to form a clade. On the other hand, the two Nepenthes species found on the low hills (N. albomarginata and N. benstonei) and four highland species (N. sanguine, N. macfarlanei, N. ramispina and N. alba) formed another clade. Therefore, this format of clustering corresponds to the altitudinal features of the natural growing habitats of Nepenthes in Peninsular Malaysia, suggesting differences in altitudinal growing environments probably played an important role in driving species radiation in the genus [19].
The four lowland Nepenthes species (N. ampullaria, N. rafflesiana, N. mirabilis and N. gracilis) that occur in Peninsular Malaysia are the same species that form the lowland suite in Sumatra. Two species are primarily found at intermediate altitudes—N. albomarginata and N. benstonei in both Malaysia and Sumatra. Of the four highland species, two (N. sanguinea and N. macfarlanei) have relatively wide distributions, whereas the other two (N. ramispina and N. gracillima) are more restricted. N. albomarginata is sister to one of the major clades, whereas N. benstonei is basal to this entire group of taxa (Fig. 1). This may imply that the highland species have most likely evolved from the lowland species with the transitional form of intermediate altitude species. Long term isolation of each population and limited seed flow, demonstrated in Nepenthes vieillardii of New Caledonia [20], might partly explain the speciation of highland Nepenthes species of Peninsular Malaysia. Climate may also play a role in the speciation of Nepenthes, with changes in vegetation, soil type and nutrient availability permitting a range of distinct ecological niches for Nepenthes to exploit.
Phylogenetic relationships in several carnivorous genera have been inferred using ITS sequences. Use of nrITS2 and nrITS1 in phylogeny reconstruction of 29 species of Pinguicula showed that the molecular phylogeny was congruent with the morphological classification [21].
Conclusions
The trnL intron and ITS sequence data utilised in this work provided informative characters for the molecular phylogenetic inference of Nepenthes species in Peninsular Malaysia. The Nepenthes of Peninsular Malaysia formed two major clusters according to altitudinal distribution. It is reasonable to assume that the highland species have evolved from the lowland species. To our knowledge, this is the first attempt of applying the ITS and trnL intron sequence data as potential markers for Nepenthes species in Peninsular Malaysia. Worldwide, there are over 120 described species of Nepenthes and future work could consider the relationship between these species and those found in Peninsular Malaysia.
Abbreviations
ITS:
internal transcribed spacer
UKMB:
Universiti Kebangsaan Malaysia Herbarium
NJ:
neighbor-joining
MP:
maximum parsimony
Declarations
Authors’ contributions
HB, NMN: Study conception and design; HB, NMN, CYC, SY: Acquisition of data; HB, CYC, SY: Analysis and interpretation of data; HB: Drafting the manuscript; All authors read and approved the final manuscript.
Acknowledgements
We would like to thank Mr. Fauzi and Mr. Saad for their help in collecting the plants.
Competing interests
The authors declare that they have no competing interests.
Availability of data
All the sequences used in this study have been deposited in the GenBank (https://www.ncbi.nlm.nih.gov/genbank/). JX042566; JX042567; JX042568; JX042569; JX042570; JX042571; JX042572; JX042573; JX042574; JX042575; JX042576; JX042577; JX042554; JX042555; JX042556; JX042557; JX042558; JX042559; JX042560; JX042561; JX042562; JX042563; JX042564; JX042565.
Ethics approval and consent to participate
According to the National Policy on Biological Diversity 2016-2025 and Forestry Act 1993 (Malaysia), no approvals or permits were required for the study.
Funding
This research was funded by a grant from the Ministry of Higher Education, Malaysia (UKM-RB-06-FRGS0259-2010). This funding source had no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Authors’ Affiliations
(1)
Institute of Systems Biology, Universiti Kebangsaan Malaysia (UKM)
(2)
School of Environmental and Natural Resource Sciences, Faculty of Science and Technology, Universiti Kebangsaan Malaysia (UKM)
References
McPherson SR. Pitcher Plants of the Old World 2 volumes. Poole: Redfern Natural History Productions; 2009.Google Scholar
Clarke CM. Nepenthes of Sumatra and Peninsular Malaysia. Kota Kinabalu: Natural History Publications (Borneo); 2001.Google Scholar
Clarke CM. Nepenthes of Borneo. Vic Carniv Plant Soc J. 2002;65:19.Google Scholar
Phillipps A, Lamb A. Pitcher-plants of borneo. Kota Kinabalu: Natural History Publications (Borneo); 1996.Google Scholar
Danser BH. The Nepenthaceae of the Netherlands Indies. Kota Kinabalu: Natural History Publications (Borneo); 2006.Google Scholar
Jebb MHP, Cheek MR. A skeletal revision of Nepenthes (Nepenthaceae). Blumea. 1997;42(1):1–106.Google Scholar
Kurata K, Jaffré T, Setoguchi H. Variation of pitcher morphology within Nepenthes vieillardii Hook.f. (Nepenthaceae) in New Caledonia. Acta Phytotaxon Geobot. 2004;55:181–97.Google Scholar
Meimberg H, Wistuba A, Dittrich P, Heubl G. Molecular phylogeny of Nepenthaceae based on cladistic analysis of plastid trnK intron sequence data. Plant Biol. 2001;3:164–75.View ArticleGoogle Scholar
Meimberg H, Thalhammer S, Brachmann A, Heubl G. Comparative analysis of a translocated copy of the trnK intron in the carnivorous genus Nepenthes (Nepenthaceae). Mol Phylogenet Evol. 2006;39:478–90.View ArticlePubMedGoogle Scholar
Meimberg H, Heubl G. Introduction of a nuclear marker for phylogenetic analysis of Nepenthaceae. Plant Biol. 2006;8:831–40.View ArticlePubMedGoogle Scholar
Taberlet P, Gieley L, Pautou G, Bouvet J. Universal primers for amplification of three non-coding regions of chloroplast DNA. Plant Syst Evol. 1991;170:97–106.Google Scholar
Popp M, Oxelman B. Inferring the history of the polyploid Silene aegaea (Caryophyllaceae) using plastid and homoeologous nuclear DNA sequences. Mol Phylogenet Evol. 2001;20:474–81.View ArticlePubMedGoogle Scholar
Swofford DL. PAUP: phylogenetic analysis using parsimony (and other methods). Version 4.0. Slunderland: Sinnauer Associates Inc.; 2002.Google Scholar
Yang Z, Rannala B. Bayesian phylogenetic inference using DNA sequences: a Markov chain Monte Carlo method. Mol Biol Evol. 1997;14:717–24.View ArticlePubMedGoogle Scholar
Huelsenbeck JP, Ronquist F, Nielsen R, Bollback JP. Bayesian inference of phylogeny and its impact on evolutionary biology. Science. 2001;294:2310–4.View ArticlePubMedGoogle Scholar
Huelsenbeck JP, Ronquist F. MrBayes: Bayesian inference in phylogenetic trees. Bioinformatics. 2001;17:754–5.View ArticlePubMedGoogle Scholar
Posada D, Crandall KA. Modeltest: testing the model of DNA substitution. Bioinformatics. 1998;1:817–8.View ArticleGoogle Scholar
Thorogood C. The Malaysian Nepenthes: evolutionary and taxonomic perspectives. New York: Nova Science Publishers Inc.; 2010.Google Scholar
Kurata K, Jaffré T, Setoguchi H. Genetic diversity and geographical structure of the pitcher plant Nepenthes vieillardii in New Caledonia: a chloroplast DNA haplotype analysis. Am J Bot. 2008;95(12):1632–44.View ArticlePubMedGoogle Scholar
Degtjareva GV, Casper SJ, Hellwig FH, Schmidt AR, Steiger J, Sokoloff DD. Morphology and nrITS phylogeny of the genus Pinguicula L. (Lentibulariaceae), with special attention to embryo evolution. Plant Biol. 2006;8:778–90.View ArticlePubMedGoogle Scholar
