Biodiversity Data Journal : Taxonomic paper
DNA barcoding reveals a new record of Potamogeton distinctus (Potamogetonaceae) and its natural hybrids, P. distinctus × P. nodosus and P. distinctus × P. wrightii (P. ×malainoides) from Myanmar
Corresponding author: Yu Ito (firstname.lastname@example.org)
Academic editor: Luis Cayuela
Received: 24 Feb 2014 | Accepted: 25 Feb 2014 | Published: 28 Feb 2014
© 2014 Yu Ito, Norio Tanaka, Rachun Pooma, Nobuyuki Tanaka.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0) which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation: Ito Y, Tanaka N, Pooma R, Tanaka N (2014) DNA barcoding reveals a new record of Potamogeton distinctus (Potamogetonaceae) and its natural hybrids, P. distinctus × P. nodosus and P. distinctus × P. wrightii (P. ×malainoides) from Myanmar. Biodiversity Data Journal 2: e1073. doi: 10.3897/BDJ.2.e1073
Indo-China floristic region is among the 34 richest floristic regions of the world, and its plant diversity is still under investigation. Here we report a new record of an aquatic plant, Potamogeton distinctus, from Myanmar, a part of the region, that is detected by means of DNA barcoding method. The molecular method further identified the other specimens as hybrids of Potamogeton: one is P. ×malainoides (P. distinctus × P. wrightii), and the other is P. distinctus × P. nodosus. The first of these was thus far genetically confirmed in China, but the parental combination of the hybrid in Myanmar was reciprocal to those reported from China. The second hybrid was also recorded from China, but the maternal lineage was revealed for the first time, in this case it was P. distinctus. The present study showed that 1) nrITS is useful to distinguish closely related Potamogeton species as well as hybrids among them and 2) atpB-rbcL has higher utility than other frequently used plastid DNA markers. We thus propose nrITS and atpB-rbcL as DNA barcoding markers for future Potamogeton studies.
DNA barcoding, flora, Myanmar, new record, Potamogeton
With many Southeast Asian countries included, Indo-China is among the 34 richest floristic regions of the world (
Potamogeton distinctus is among the broad-leaved long-petioled Potamogeton species that is widely distributed in East Asia and Southeast Asia, including the southern part of China and Thailand (
Potamogeton is known to have aneuploidy, polyploidy, and hybridization (
In such cases, analysis of plant DNA sequence data can provide an effective method, that is known as DNA barcoding (e.g.,
The present study aimed to assess the potential occurrence of Potamogeton distinctus and its inter-specific hybrids, if any are present, in Myanmar. To do so, we applied a taxon-specific DNA barcoding method. First, in order to evaluate the utility of selected DNA barcoding markers, we performed simultaneous molecular phylogenetic analyses based on a sample set of precisely identified broad-leaved long-petioled Potamogeton specimens, occasionally suplimented with some GenBank accessions. Then, using the DNA barcoding markers, we assigned broad-leaved long-petioled Potamogeton specimens from Myanmar, which could not be identified by morphology due to either the lack of diagnostic floral characters or intermediate vegetative morphology or both. The resulting molecular insights of broad-leaved long-petioled Potamogeton species in Myanmar will be used to document a new record of Potamogeton species for the flora of Myanmar, to discuss the origin and the evolution of hybrids of Potamogeton in Myanmar, and to propose DNA barcoding markers for future Potamogeton studies.
We carried out a field expedition to Myanmar in 2008 and collected four relevant specimens, i.e., broad-leaved long-petioled Potamogeton specimens, including three non-flowering and one flowering ones in Shan state (
Morphological comparison among the four Potamogeton specimens collected in Myanmar and three broad-leaved long-petioled Potamogeton species potentially distributed in Myanmar (a:
|taxon||P. distinctus||P. nodosus||P. wrightii||Potamogeton sp. (N. Tanaka & al. 080061)||Potamogeton sp. (N. Tanaka & al. 080657)||Potamogeton sp. (N. Tanaka & al. 080631)||Potamogeton sp. (N. Tanaka & al. 080662)|
|Floating leaf veinc,d||11-21||11-21||9-13||13-18||10-12||14||9-13|
|Petiole length (Submerged leaves)a||Petioled||Unpetioled||N/A||Petioled||Petioled||Petioled||Petioled|
|Petiole length (Submerged leaves)b||1-200 mm||1-200 mm||2-70 mm||25-30 mm||40-50 mm||80-100 mm||25-30 mm|
|Petiole length (Submerged leaves)d||1.5-2.3 x length of blade||0.2-1.5 x length of blade||N/A||0.2-0.3 x length of blade||0.7-0.8 x length of blade||0.8-1.0 x length of blade||0.2-0.4 x length of blade|
|Petiole length (Floating leaves)b||80-260 mm||18-210 mm||N/A||45-85 mm||15-30 mm||85-120 mm||75-120 mm|
|Petiole length (Floating leaves)c||up to 400 mm||up to 200 mm||up to 200 mm|
To evaluate the utility of selected DNA barcoding markers through performing molecular phylogenetic analyses, comparative materials of Potamogeton distinctus, P. nodosus, and P. wrightii were collected in Japan, Mexico, and Thailand (
List of the GenBank accessions of atpB–rbcL, rpl20–rps12, trnT–trnL, trnL, trnL–trnF, and nrITS for ingroup and outgroup of Potamogeton species used in the phylogenetic analyses. Sequences obtained in the present study are shown in underline. Note that four Myanmar specimens are identified by DNA barcoding (see Discussion). Herbaria abbreviations: Forest Herbarium, Bangkok, Thailand = BKF; Wuhan Institute of Botany, Hubei, People's Republic of China = HIB; Kochi Prefectual Makino Botanical Garden, Kochi, Japan = MBK; Forest Research Institute, Pyinmana, Myanmar = RAF, The University of Tokyo Herbarium, Tokyo, Japan = TI; National Museum of Nature and Science, Tsukuba, Japan = TNS.
|Myanmar||N. Tanaka & al. 080061 (RAF, TI, MBK)||AB871483||AB871491||AB871501||AB871506||AB871512||AB871518|
|Myanmar||N. Tanaka & al. 080657 (RAF, TI, MBK)||AB871485||AB871493||AB871503||AB871508||AB871514||AB871519|
|Myanmar||N. Tanaka & al. 080631 (RAF, TI, MBK)||AB871484||AB871492||AB871502||AB871507||AB871513||AB871520|
|P. distinctus × P. nodosus|
|Myanmar||N. Tanaka & al. 080662 (RAF, TI, MBK)||AB871486||AB871494||N/A||AB871509||AB871515||AB871522|
|P. perfoliatus||Japan||YI01687 (TNS)||AB871489||AB871499||AB744026||AB744014||AB744020||AB744008|
|P. lucens||Japan||YI00049 (TNS)||AB206987||AB871496||AB695137||AB695129||AB695133||AB206990|
The voucher specimens are retained in either of the following herbaria: BKF; MBK; RAF; TI; TNS. Those of
For the newly obtained samples, total genomic DNA was extracted and sequencing of five plastid regions was performed using the procedure outlined by
Sequences of the nrITS were obtained using primers ITS-4 and ITS-5 (
Sequences of the atpB-rbcL,rpl20-rps12, trnT-trnL, trnL, trnL-trnF, and nrITS regions were manually aligned using the simple indel coding method of
Phylogenetic analyses were independently performed for data sets of ptDNA (atpB-rbcL, rpl20-rps12, trnT-trnL, trnL, trnL-trnF) and nrITS, respectively. Phylogenetic inference was performed using maximum parsimony (MP) in PAUP* 4.0b10 (
?Bhutan, China (nationwide), Korea, Japan, Myanmar, Nepal, ?Philippines, Thailand, ?Vietnam (modified from
Potamogeton distinctus shows a wide range of phenotypic plasticity, especially in leaf morphology. It seems to be that the two-carpellate flower, the diagnostic character of the species, is essential for precise morphological identification in the field; identification with vegetative morphology alone is to be avoided (see Discussion).
The length of the combined five ptDNA regions alignment containing ten accessions totaled 3456 bp, of which two characters were parsimony-informative. Based on this data set, one MP tree (tree length = 27 steps; consistency index = 1.0; retention index = 1.0) and a BI 50% consensus tree were obtained. These trees showed congruent phylogenetic relationships and thus only the MP tree is presented here (
The most parsimonious trees of Potamogeton based on (A) the combined plastid DNA (atpB–rbcL, rpl20–rps12, trnT–trnL, trnL, trnL–trnF) sequences and (B) nrITS sequences. Each one of the two outgroups is trimmed to clarify ingroup phylogeny. ACCTRAN optimisation is used for branch length measures; terminals are aligned with dotted lines. Numbers above the branches indicate bootstrap support (BP) calculated in the maximum parsimony, and those below indicate Bayesian posterior probabilities (PP). Samples in regular and bold face indicate comparative ones and those from Myanmar, respectively. Some accessions in each tree represent multiple identical accessions. Note that some samples have heterogeneous nrITS copies; for these, the sequence pairs are named #1 and #2, respectively, and colored in red.
The length of nrITS alignment composed of 20 accessions totaled 645 bp, of which six characters were parsimony-informative. In the phylogenetic analysis of nrITS data set, one MP tree (tree length = 43 steps; consistency index = 1.0; retention index = 1.0) and a BI 50% consensus tree were obtained. These trees showed congruent phylogenetic relationships and thus only the MP tree is presented here (
In both ptDNA and nrITS trees, the three morphologically closely related species were well differentiated from one another. With Potamogeton lucens and P. perfoliatus as outgroup, P. wrightii and the clade of P. distinctus and P. nodosus were clustered (63 MP bootstrap (BS), 1.0 BI posterior probability (PP) in ptDNA; 87 MP BS, 1.0 PP in nrITS). Potamogeton nodosus from Mexico and P. nodosus-related nrITS sequence of P. distinctus × P. nodosus HDZY5-7 showed variation, yet the two sequences were clustered each other (62 MP BS, 0.99 BI PP). GenBank accessions of P. ×malainoides and P. distinctus × P. nodosus (
Of the four broad-leaved long-petioled Potamogeton specimens from Myanmar, two were genetically identical to P. distinctus from Japan and Thailand (N. Tanaka & al. 080061, N. Tanaka & al. 080657;
The combined five ptDNA regions were separately analyzed to facilitate the utility as individual DNA markers. The comparison included nrITS. Between the closely related species, Potamogeton distinctus and P. nodosus, where two nucleotide substitutions were observed in nrITS, atpB-rbcL exhibited one nucleotide substitution, while trnT-trnL showed a difference in mononucleotide repeat unit (
Comparison of the ITS sequences of the three broad-leaved long-petioled Potamogeton species and hybrids used in the phylogenetic analysis. Note that substitutions observed at 571 bp and 579 bp are due to apparent infra-specific variation in P. nodosus.
|P. distinctus × P. nodosus||T||C||T||C||A||G||T||G||G||T|
|P. nodosus (Mexico)||T||C||T||G||C||G||T||G||A||C|
Comparison of the atpB-rbcL and trnT-trnL sequences of the three broad-leaved long-petioled Potamogeton species and hybrids used in the phylogenetic analysis.
|P. distinctus||ATTT||A||G||T (3)||A (8)|
|P. nodosus||ATTT||A||C||T (2)||A (8)|
|P. wrightii||------||C||G||T (2)||A (7)|
In order to assess the potential occurrence of Potamogeton distinctus and its hybrids, if any are present, in Myanmar, the present study applied a taxon-specific DNA barcoding method. The simultaneous molecular phylogenetic analyses successfully distinguished broad-leaved long-petioled Potamogeton species, P. distinctus, P. nodosus, and P. wrightii, as well as hybrids among them, P. ×malainoides (P. distinctus × P. wrightii) and P. distinctus × P. nodosus (
Applying the comparative samples’ sequence data as DNA barcodes, the broad-leaved long-petioled Potamogeton specimens from Myanmar were genetically assigned. As a result, two out of four specimens were identified as P. distinctus, a widely distributed species in East Asia, Southeast Asia and the Pacific, including southern part of China and Thailand, but not in Myanmar (
The taxon-specific DNA barcoding also revealed two hybrids of Potamogeton in Myanmar, and among which was P. ×malainoides (P. distinctus × P. wrightii). This hybrid is known from China (
The other hybrid of Potamogeton identified in Myanmar is P. distinctus × P. nodosus. This hybrid is also known from China, yet no maternal lineage was conclusively identified in the previous study (
The taxon-specific DNA barcoding method presented here will be applicable in elucidating further diversity of Potamogeton in other floras. With some modification on marker selection, this method will be also applicable for floras that focus on other taxa.
Thanks to the following personnel in Myanmar and Thailand for their help in arranging our field work: U Htun Paw Oo, Ex-director of Nature and Wildlife Conservation Division; U Soe Win Hlaing, Ex-director General, Forest Department, Ministry of Environmental Conservation and Forestry, S. Saengrit, and N. Suphuntee (BKF). We also thank Prof. Tetsuo Koyama, director of the Kochi Prefectural Makino Botanical Garden (Japan), who initiated the Myanmar-Japanese cooperative program to inventory the plants of Myanmar, and gave us the opportunities to study the Myanmar plants, Prof. J. Murata of the University of Tokyo (Japan), who principally organises the Myanmar-Japanese inventory project, Dr. T. Ohi-Toma, assistant professor of the University of Tokyo (Japan), who helps the first author to use the facilities. Thanks also to Dr. T. Kajita and Dr. Y. Kajita for providing Potamogeton nodosus from Mexico and Dr. D. Potter (USA) and Mrs. V. Gray (New Zealand) for editing draft.
Y. Ito participated in the design of the study, in the collection of samples, in the analysis and interpretation of the data, and in the writing of the manuscript; Nr. Tanaka participated in the design of the study, in the analysis and interpretation of the data, and assisted in the writing of the manuscript; R. Pooma participated in the analysis and interpretation of the data and assisted in the writing of the manuscript; and Nb. Tanaka participated in the design of the study, in the analysis and interpretation of the data and assisted in the writing of the manuscript.