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. 2014 Jul 3;4:5553. doi: 10.1038/srep05553

Molecular phylogeny of Orthetrum dragonflies reveals cryptic species of Orthetrum pruinosum

Hoi Sen Yong 1, Phaik-Eem Lim 1,2,a, Ji Tan 1,2, Yong Foo Ng 3, Praphathip Eamsobhana 4, I Wayan Suana 5
PMCID: PMC5381552  PMID: 24989852

Abstract

Dragonflies of the genus Orthetrum are members of the suborder Anisoptera, family Libellulidae. There are species pairs whose members are not easily separated from each other by morphological characters. In the present study, the DNA nucleotide sequences of mitochondrial and nuclear genes were employed to elucidate the phylogeny and systematics of Orthetrum dragonflies. Phylogenetic analyses could not resolve the various subfamilies of the family Libellulidae unequivocally. The nuclear 28S rRNA gene is highly conserved and could not resolve congeneric species of Orthetrum. Individual mitochondrial genes (COI, COII, and 16S rRNA) and combination of these genes as well as the nuclear ITS1&2 genes clearly differentiate morphologically similar species, such as the reddish species pairs O. chrysis and O. testaceum, and the bluish-coloured species O. glaucum and O. luzonicum. This study also reveals distinct genetic lineages between O. pruinosum schneideri (occurring in Malaysia) and O. pruinosum neglectum (occurring north of Peninsular Malaysia from India to Japan), indicating these taxa are cryptic species.

Dragonflies of the genus Orthetrum Newman, 1833 are members of the suborder Anisoptera, family Libellulidae. The genus contains some 61 species spread across the Old World1. Among these Orthetrum dragonflies, there are species pairs whose members are not easily separated from each other by morphological characters, e.g. the reddish-coloured species O. chrysis and O. testaceum, and the bluish-coloured species O. glaucum and O. luzonicum.

The Crimson-tailed Marsh Hawk Orthetrum pruinosum (Burmeister, 1839) is a widespread species occurring from west India to Japan and south to Malaysia and the Sunda Islands. The subspecies in Malaysia is O. p. schneideri Förster, 1903 and that north of Peninsular Malaysia (India to Japan) is O. p. neglectum (Rambur, 1842).

The DNA nucleotide sequences of mitochondrial and nuclear genes have been employed to elucidate the phylogeny and systematics of Orthetrum dragonflies2,3. To-date the most comprehensive phylogenetic study of Orthetrum dragonflies involves all the nine Japanese species2. In the present study, the DNA nucleotide sequences of mitochondrial and nuclear genes were employed to elucidate the phylogeny and systematics of Orthetrum dragonflies. This study, covering a more extensive taxon sampling, provides a new insight to the evolutionary relationships of Orthetrum dragonflies. The molecular phylogeny based on ITS1&2, COI, COII and 16S nucleotide sequences, reveals the occurrence of cryptic species in O. pruinosum.

Results

Aligned sequences and genetic divergence

The total length for each aligned sequences for various molecular markers and their parsimony information are sumarised in Supplementary Table 1. The uncorrected ‘p'-distance between Orthetrum species based on 16S rDNA, COI, combined COI + 16S rDNA, combined COI + COII + 16S rDNA, ITS1&2, and combined COI + COII + 16S rDNA + 28S rDNA + ITS1&2 nucleotide sequences are summarized in supplementary Tables 2–6 respectively. The interspecific ‘p' distance was many folds larger than intraspecific ‘p' distance. For COI, the intraspecific p-distance ranged from 0.00–3.99% (highest in O. melania), while interspecific p-distance ranged from 3.33% (O. melania and O. triangulare) to 17.29% (O. chrysis and O. sabina) (Supplementary Table 2). For 16S rDNA, the intraspecific p-distance ranged from 0.00–2.10% (highest in O. glaucum); the interspecific p-distance ranged from 0.60% (O. melania and O. triangulare) to 9.92% (O. abbotti and O. poecilops) (Supplementary Table 2).

The intraspecific p-distance for ITS1&2 sequences ranged from 0.00–5.05% (highest in O. luzonicum); the interspecific p-distance ranged from 1.14% (O. pruinosum neglectum and O. testaceum) to 21.12% (O. sabina and O. chrysostigma) (Supplementary Table 3).

The intraspecific p-distance for the combined COI + 16S rDNA sequences ranged from 0.00–1.78% (highest in O. sabina); the interspecific p-distance ranged from 1.15% (O. pruinosum neglectum and O. testaceum) to 12.23% (O. chrysis and O. Sabina; O. japonicum and O. Sabina) (Supplementary Table 4). For the combined mitochondrial markers (COI + COII + 16S rDNA) the intraspecific p-distance ranged from 0.00–1.94% (highest in O. pruinosum schneideri); the interspecific p-distance ranged from 7.32% (O. chrysis and O. pruinosum schneideri) to 12.58% (O. chrysis and O. sabina) (Supplementary Table 5).

For the combined five markers (COI + COII + 16S rDNA + 28S rDNA + ITS1&2) the intraspecific p-distance ranged from 0.00–1.55% (highest in O. pruinosum schneideri); the interspecific p-distance ranged from 4.20% (O. chrysis and O. sabina) to 9.51% (O. chrysis and O. sabina) (Supplementary Table 6).

Phylogenetic relationships based on 28S rDNA nucleotide sequences

There were no distinct nucleotide sequence divergence among the congeners of Orthetrum (supplementary Fig. 1). The various subfamilies of the family Libellulidae were not resolved unequivocally.

Phylogenetic relationships based on 16S rDNA nucleotide sequences

Orthetrum pruinosum schneideri clustered with O. chrysis and both were distinctly separated from O. testaceum and O. pruinosum neglectum (Fig. 1). O. sabina from Peninsular Malaysia was not grouped together with O. sabina of India, Japan and Fiji. Additionally, O. luzonicum from Peninsular Malaysia was distinct from O. luzonicum of China and Japan.

Figure 1. BI tree based on 16S rDNA nucleotide sequences.

Figure 1

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Numeric values at the nodes are Bayesian posterior probabilities/ML bootstrap.

Phylogenetic relationships based on COI nucleotide sequences

Orthetrum pruinosum schneideri clustered with O. chrysis and both were distinctly separated from O. testaceum and O. pruinosum neglectum (Fig. 2). The peninsular Malaysian taxon of O. luzonicum clustered with those of China and Japan. Likewise, O. sabina from Peninsular Malaysia clustered with O. sabina of India, Japan and Fiji.

Figure 2. BI tree based on COI nucleotide sequences.

Figure 2

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Numeric values at the nodes are Bayesian posterior probabilities/ML bootstrap.

Phylogenetic relationships based on COII nucleotide sequences

There were two major clusters of Orthetrum species (supplementary Fig. 2): (I) [O. pruinosum schneideri, O. chrysis], O. testaceum, O. melania, O. luzonicum, O. glaucum, O. albistylum with weak support posterior probability (PP = 0.51) values and no support from maximum likelihood (ML); and (II) O. sabina.

Phylogenetic relationships based on ITS1 and ITS2 nucleotide sequences

The ITS nuDNA nucleotide sequences clearly separated O. pruinosum schneideri and O. pruinosum neglectum (Fig. 3) indicating distinct genetic lineages. O. pruinosum schneideri nested with O. chrysis while O. pruinosum neglectum nested with O. testaceum. The component taxa of Orthetrum were grouped in two distinct clades separated by a clade of other Libellulid genera. O. sabina was not nested with other Orthetrum taxa. The genus Orthetrum and the Libellulid subfamilies were not monophyletic.

Figure 3. BI tree based on ITS1&2 nucleotide sequences.

Figure 3

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Numeric values at the nodes are Bayesian posterior probabilities/ML bootstrap.

Phylogenetic relationships based on combined nucleotide sequences

The combined COI and COII sequences yielded three major clusters (Fig. 4): (I) [O. pruinosum schneideri, O. chrysis], O. testaceum, O. triangulare, O. luzonicum with PP supoprt of 0.92 and no support from ML; (II) O. glaucum; and (III) O. sabina. Similar topology resulted from the combined COI + COII + 16S rDNA nucleotide sequences (supplementary Fig. 3). The combined 5 markers (supplementary Fig. 4) showed three clades: (I) O. chrysis, O. pruinosum schneideri, O. testaceum; (II) O. glaucum, O. sabina; and (III) O. luzonicum.

Figure 4. BI tree based on COI + COII nucleotide sequences.

Figure 4

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Numeric values at the nodes are Bayesian posterior probabilities/ML bootstrap.

The combined COI + 16S rDNA sequences of Orthetrum taxa formed five major clusters (Fig 5): (I) [O. pruinosum schneideri, O. chrysis], O. testaceum, O. pruinosum neglectum, O. melania; (II) [O. internum, O. japonicum], O. poecilops, O. albistylum; (III) O. luzonicum; (IV) O. glaucum; and (V) O. sabina. The first four clusters (I–IV) had full PP and high ML support except cluster V with moderate support of PP = 0.79 and ML = 79%.

Figure 5. BI tree based on COI + 16S rDNA nucleotide sequences.

Figure 5

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Numeric values at the nodes are Bayesian posterior probabilities/ML bootstrap.

Discussion

The phylogeny of the dragonflies (suborder Anisoptera) has been extensively studied4,5,6,7,8,9,10. Nine genera of Libellulidae have been reported to be monophyletic11. In the present study with more extensive taxon sampling, the various subfamilies of the family Libellulidae as well as the component taxa of the genus Orthetrum were not resolved unequivocally as monophyletic by the 28S rDNA (supplementary Fig. 1), 16S rDNA (Fig. 1), COI (Fig. 2), and ITS1&2 (Fig. 3) nucleotide sequences.

Species complexes in the genus Orthetrum have been uncovered by DNA sequence analyses. Based on molecular phylogeny and morphological characteristics, Orthetrum internum McLachlan, 1894 (previously regarded as O. japonicum internum McLachlan, 1894) is resolved as a genuine/distinct species from O. japonicum japonicum (Uhler, 1858)2,12. Likewise, O. triangulare and the allied taxon O. melania are well separated by the nuclear (ITS1 and ITS2) and mitochondrial (COI and 16S rRNA) genes3. Additionally, O. melania is separated into four subgroups: O. m. melania (mainland Japan), O. m. continentale (China, Korea and Taiwan), O. m. yaeyamense (Yaeyama Island, Japan), and O. m. ryukyuense (Amami, Kerama, Okinawa and Tokara, Japan).

In the present study, the nuclear 28S rDNA nucleotide sequences were highly conserved and could not resolve congeneric species of Orthetrum (supplementary Fig. 1). The 28S rRNA gene has been found to be better for resolving deep branching in the Odonata13. However, the mitochondrial genes (COI, COII and 16S) and the nuclear ITS1&2 genes unequivocally separated morphologically similar species, such as the reddish-coloured O. chrysis and O. testaceum and the bluish-coloued species O. glaucum and O. luzonicum (Figs. 1,2,3,4, Supplementary Fig. 2). Additionally, the 16S rDNA sequences revealed distinct genetic lineages of (1) O. luzonicum from Peninsular Malaysia and China-Japan, and (2) O. sabina of Peninsular Malaysia and India-Japan-Fiji (Fig. 1).

In the phylogeny based on nine Japanese Orthetrum species, O. pruinosum neglectum clusters with O. melania2. The present study based on the ITS1&2 (Fig. 3), COI (Fig. 2), 16S rDNA (Fig. 1) and combined COI + 16S rDNA (Fig. 5) nucleotide sequences and with more extensive taxon sampling indicates that O. pruinosum neglectum clusters nearer to O. testaceum than O. melania. The allied/sibling taxon O. pruinosum schneideri is grouped with O. chrysis (Figs. 1,2,3,4,5, Supplementary Figs. 2–4). It is distinctly separated from O. pruinosum neglectum. The two taxa are, without reasonable doubt, cryptic species of a species complex. In the African dragonfly genus Trithemis, COI and ND1 genes reveal three distinct genetic clusters of T. stricta but these taxa could not be identified by using classical taxonomic characters14.

In summary, phylogenetic analyses of a more extensive taxon sampling based on nucleotide sequences of mitochondrial and nuclear genes indicate that the various subfamilies of the family Libellulidae and the genus Orthetrum are not resolved unequivocally as monophyletic. The nuclear 28S rRNA gene is highly conserved and could not resolve congeneric species of Orthetrum. Individual mitochondrial genes (COI, COII, and 16S rRNA) and combination of these genes as well as the nuclear ITS1&2 genes clearly differentiate morphologically similar species, such as the reddish species pairs O. chrysis and O. testaceum, and the bluish-coloured species O. glaucum and O. luzonicum. This study also reveals distinct genetic lineages between O. pruinosum schneideri (occurring in Malaysia) and O. pruinosum neglectum (occurring north of Peninsular Malaysia from India to Japan), indicating these taxa are cryptic species. The finding of O. pruinosum occurring as a species complex paves the way for an in-depth phylogeographical study to determine the systematic status of the component taxa. Likewise, phylogeographical studies are needed for O. luzonicum and O. sabina.

Methods

Ethics statement

No specific permits were required for the described field studies. The dragonflies were collected in disturbed habitats such as open ditches and ponds. No specific permissions were required and the dragonflies are not endangered or protected species.

Specimens

Specimens of the Orthetrum dragonflies for the present study were collected using sweep net or plastic bag. They were identified with established literature15,16. In addition, Ictinogomphus decoratus (Anisoptera, Gomphidae) was included for comparison. Two species of Ceriagrion (Zygoptera, Coenagrionidae) were used as outgroup. Details of the species studied are listed in Table 1.

Table 1. Nucleotide sequences of COI, COII, 16S rRNA, 28S rRNA, ITS1 and/or ITS2 sequences for the taxa of Orthetrum of the family Libellulidae used in the present study. Ictinogomphus decoratus (family Gomphidae), Ceriagrion chaoi and C. cerinorubellum (suborder Zygoptera) were used as outgroups. NA, not available.

No. Sample Name Sampling Location Collection Code GenBank/DDBJ Accession Number    
COI COII 16S 28S ITS1 ITS2        
Samples derived from this study    
Odonata    
Libellulidae    
1 Orthetrum chrysis University Malaya OCHR1 AB860015 AB860042 AB860069 AB860097 KJ802958 KJ802986
2 Orthetrum chrysis University Malaya OCHR3 AB860016 AB860043 AB860070 AB860098 KJ802959 KJ802987
3 Orthetrum chrysis University Malaya OCHR5 AB860017 AB860044 AB860071 AB860099 KJ802960 KJ802988
4 Orthetrum chrysis Lanchang, Pahang OCHR6 AB860018 AB860045 AB860072 AB860100 KJ802961 KJ802989
5 Orthetrum glaucum University Malaya OGLA1 AB860019 AB860046 AB860073 AB860101 KJ802962 KJ802990
6 Orthetrum glaucum University Malaya OGLA2 AB860020 AB860047 AB860074 AB860102 KJ802963 KJ802991
7 Orthetrum glaucum University Malaya OGLA3 AB860021 AB860048 AB860075 AB860103 KJ802964 KJ802992
8 Orthetrum glaucum University Malaya OGLA4 AB860022 AB860049 AB860076 AB860104 KJ802965 KJ802993
9 Orthetrum glaucum University Malaya OGLA5 AB860308 KF248113 KF248140 KF581186 KJ802966 KJ802994
10 Orthetrum glaucum University Malaya OGLA6 AB860023 AB860050 AB860077 AB860106 KJ802967 KJ802995
11 Orthetrum glaucum Lentang, Pahang OLGA7 AB860024 AB860051 AB860078 AB860107 KJ802968 KJ802996
12 Orthetrum testaceum University Malaya OTES1 AB860025 AB860052 AB860079 AB860108 KJ802969 KJ802997
13 Orthetrum testaceum University Malaya OTES2 AB860026 AB860053 AB860080 AB860109 KJ802970 KJ802998
14 Orthetrum testaceum University Malaya OTES3 AB860027 AB860054 AB860081 AB860110 KJ802971 KJ802999
15 Orthetrum testaceum University Malaya OTES4 AB860028 KF248112 KF248139 KF581185 KJ802972 KJ803000
16 Orthetrum testaceum University Malaya OTES5 - - - - KJ802973 KJ803001
17 Orthetrum testaceum University Malaya OTES6 AB860029 AB860056 AB860083 AB860112 KJ802974 KJ803002
18 Orthetrum luzonicum Pahang OLUZ1 AB860037 AB860064 AB860091 AB860118 KJ802980 KJ803008
19 Orthetrum luzonicum Pahang OLUZ2 AB860038 AB860065 AB860092 AB860119 KJ802981 KJ803009
20 Orthetrum pruinosum schneideri Lentang, Pahang OPRU1 AB860032 AB860059 AB860086 AB860115 KJ802977 KJ803005
21 Orthetrum pruinosum schneideri Rengit, Pahang OPRU2 AB860033 AB860060 AB860087 AB860116 KJ802978 KJ803006
22 Orthetrum pruinosum schneideri Lentang, Pahang OPRU3 AB860034 AB860061 AB860088 AB860117 KJ802979 KJ803007
23 Orthetrum pruinosum schneideri Maliau, Sabah OPRU4 AB860035 AB860062 AB860089 - - -
24 Orthetrum pruinosum schneideri Maliau, Sabah OPRU5 AB860036 AB860063 AB860090 - - -
25 Orthetrum sabina Kampar, Perak OSAB1 AB860030 AB860057 AB860084 AB860113 KJ802975 KJ803003
26 Orthetrum sabina Lanchang Pahang OSAB2 AB860031 AB860058 AB860085 AB860114 KJ802976 KJ803004
Odonata    
Gomphidae    
27 Ictinogomphus decoratus Lanchang, Pahang IDEC1 AB860039 AB860066 AB860093 AB860120 KJ802982 KJ803010
28 Ictinogomphus decoratus Lanchang, Pahang IDEC2 AB860040 AB860067 AB860094 AB860121 KJ802983 KJ803011
Odonata    
Coenagrionidae    
29 Ceriagrion chaoi University Malaya CCHA20 AB860041 AB860068 AB860095 AB860122 KJ802984 KJ803012
30 Ceriagrion cerinorubellum University Malaya CCER1 AB860310 AB860307 AB860096 AB860123 KJ802985 KJ803013
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DNA extraction, PCR amplifications and DNA sequencing

The genomic DNA was extracted and PCR amplification was performed as described in Lim et al.17 except with variations in annealing temperature for different primers. The primers and annealing temperature for PCR were: COI –F: 5′- ATAATTGGRGGRTTYGGRAAY TG-3′ and R: 5′- CCAAARAATCAAAATAARTGT TG-3′18, at 50°C; COII: C2-J-3102: 5′-AAATGGCAACATGAGCACAAYT-3′ and TK-N-3773: 5′-GAGACCAGTACTTGCTTTCAGTCATC-3′19 at 50°C; 16S rDNA: 5′-TTGACTGTACAAAGGTAGC-3′ and 5′-GATATTACGCTGTTATCCC-3′20 at 50°C; 28S rDNA: 28sf, 5′-AAGGTAGCCAAATGCCTCATC-3′ and 28sr, 5′-AGTAGGGTAAAACTAACCT-3′ at 52°C13; ITS1: CAS18sF,5′- TACACACCGCCCGTCGCTACTA-3′ and CAS5p8sB1d, 5′- ATGTGCGTTCRAAATGTCGATGTTCA-3′21 at 67°C; and ITS2: CAS5p8sFc, 5′-TGAACATCGACATTTYGAACGCACAT-3′ and CAS28sB1d, 5′-TTCTTTTCCTCCSCTTAYTRATATGCTTAA-3′21 at 55°C.

The PCR products were assayed by electrophoresis on 1.0% agarose mini gels stained with SYBR® Safe DNA gel stain (Invitrogen, USA) and visualised under UV light. The amplicons were isolated and purified using the LaboPassTM PCR purification kit (Cosmo Genetech, South Korea). The purified PCR products were sent to a commercial company for sequencing. The same set of PCR primers were used for DNA sequencing. Samples were sequenced using BigDyeH Terminator v3.1 Sequencing Kit and analysed on an ABI PRISMH 377 Genetic Analyser.

Genetic divergence

To assess the parsimony information of the sequences of the data sets and species level variation of Orthetrum species, selected specimens were used to measure the uncorrected (p) pairwise genetic distances using PAUP* 4.0b10 software22. All individual markers and combined mitochondrial markers (COI + 16S rDNA; COI + COII + 16S rDNA; and COI + COII + 16S rDNA + 28S rDNA) were used to estimate uncorrected (p) pairwise genetic distances.

Phylogenetic analysis

To elucidate the phylogenetic relationship among the different species of Orthetrum species, sequences generated from this study were combined with GenBank sequences (Table 1 and Supplementary Table 7) to construct phylogenetic trees. The generated forward and reverse sequences were manually edited and assembled using ChromasPro v1.5 (Technelysium Pty Ltd., Australia) software. The datasets for all genetic markers were aligned using ClustalX23. In the preliminary alignment for ITS1 and ITS2, the flanking sequences of 18S rDNA and 5.8S rDNA were included as the guide and were only being trimmed off after final alignment before subjected for phylogenetic analysis. For 28S and 16S, the sequences were aligned using MAFFT 624, with Q-INS-i strategy in order to take into account the secondary structure of the RNA. The generated aligned sequences were subjected for the search of the best model to be used for maximum likelihood (ML) and Bayesian Inference (BI) analyses using Kakusan v. 325. Best fit models were evaluated using the corrected Akaike Information Criterion for ML and the Bayesian Information Criterion (BIC) for BI with nonpartitioned on the whole sequence. The selected models for ML and BI of each data set are summarised in Supplementary Table 1. ML analysis was performed via Treefinder version October26 and BI analysis was performed using MrBayes 3.1.227. Bayesian analyses were initiated with a random starting tree and two parallel runs, each of which consisted of running four chains of Markov chain Monte Carlo (MCMC) iterations for 6x106 generations. The trees in each chain were sampled every 200th generation. Likelihood values for all post-analysis trees and parameters were evaluated for convergence and burn-in using the “sump” command in MrBayes and the computer program Tracer ver. 1.5 (http://tree.bio.ed.ac.uk/software/tracer/). The first 30,000 trees were discarded as burn-in (where the likelihood values were stabilized prior before the burn in), and the remaining trees after burn-in were used to calculate posterior probabilities using the “sumt” command.

Supplementary Material

Supplementary Information

Supplementary Tables and Figures

srep05553-s1.doc (746KB, doc)

Acknowledgments

This study was funded in part by MoHE-HIR Grant (H-50001-00-A000025) and the University of Malaya (H-5620009). We thank our institutions for providing various research facilities and other support.

Footnotes

The authors declare no competing financial interests.

Author Contributions H.S.Y. and P.E.L. conceived the research in collaboration with J.T., Y.F.N., P.E. and I.W.S. H.S.Y., Y.F.N. and I.W.S. collected the specimens. H.S.Y. identified the specimens. J.T. conducted the PCR and P.E.L., J.T. and P.E. performed the phylogenetic analyses. H.S.Y. and P.E.L. wrote the paper in collaboration with the co-authors. H.S.Y. and P.E.L. were responsible for the final manuscript version.

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