DNA barcoding reveals a new record of Potamogeton distinctus (Potamogetonaceae) and its natural hybrids, Potamogeton distinctus × Potamogeton nodosus and Potamogeton distinctus × Potamogeton wrightii (Potamogeton ×malainoides) from Myanmar

Abstract 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 Potamogeton ×malainoides (Potamogeton distinctus × Potamogeton wrightii), and the other is Potamogeton distinctus × Potamogeton 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 Potamogeton 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.


Introduction
With many Southeast Asian countries included, Indo-China is among the 34 richest floristic regions of the world (Van Dijk et al. 2004), and its plant diversity is still under investigation. In the region, Myanmar is one of the countries where the floristic work has been insufficiently carried out, so many new species or noteworthy plant collections are still being reported from that country (Tanaka 2005). In order to explore further diversity of the flora, the present study targeted plant species that have not been recorded from Myanmar but are widely recognized in its neighbouring countries, such as southern part of China and Thailand. A member of an large aquatic genus, Potamogeton L., P. distinctus A. Benn. is one of these species.
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 (Wiegleb 1990). Both the only floristic checklist of Myanmar and the first aquatic plants checklist of Myanmar do not include this species but lists morphologically similar other broad-leaved long-petioled species, P. nodosus Poir. and P. wrightii Morong (Kress et al. 2003, Ito andBarfod 2014). Whereas the other reported Potamogeton species from Myanmar can be easily distinguished from P. distinctus, e.g., by the shape of submerged or floating leaves, the two broad-leaved long-petioled Potamogeton can only be recognized with floral morphology because the reliable diagnostic character of P. distinctus is the flower with two carpels, which is four-carpellate in the other species (Wiegleb 1990); this characteristic, of course, could not be applied to non-flowering specimens, which many of Potamogeton collections from Myanmar are. This indicates that P. distinctus might be misidentifed as one of the other broad-leaved longpetioled Potamogeton species and thus overlooked in the flora.
Potamogeton is known to have aneuploidy, polyploidy, and hybridization (Les 1983). The different cytotypes, i.e., aneuploids and polyploids, are phylogenetically well clustered ; hence no inter-specific taxonomic confusions occur by aneuploidy and polyploidy. On the other hand, the known numerous inter-specific hybrids may cause a confusion, because the hybrids are in most cases difficult to recognize solely based on morphological investigation (Les et al. 2009). In Myanmar, although no natural Potamogeton hybrids have been reported, among the listed nine species and two synomyous ones by Kress et al. (2003) or six species by Ito and Barfod (2014) are P. nodosus and P. wrightii, both of which are known to hybridize with P. distinctus in China (Du et al. 2010). Hence, taxonomic confusion might have occurred in the inventory of P. distinctus in Myanmar with apparent hybridiｚations with the other broad-leaved longpetioled Potamogeton species.
In such cases, analysis of plant DNA sequence data can provide an effective method, that is known as DNA barcoding (e.g., Chase et al. 2005, Kress et al. 2005, CBOL Plant Working Group 2009). This method was initially launched to target diverse plant groups with universal DNA markers, e.g., flowering plants (trnH-psbA and the multi-copy internal transcribed spacer of nuclear ribosomal DNA ( (Liu et al. 2001, Nitta 2008. In Potamogeton, four candidate DNA markers were tested and of these nrITS was proposed as the most useful DNA barcoding marker (Du et al. 2011). The nuclear DNA marker would be applicable for any purposes because almost all Potamogeton species as well as hybrids were distinguishable with this marker (e.g., Du et al. 2010, Ito and Tanaka 2013, Kaplan and Fehrer 2011, Les et al. 2009). Meanwhile, in order to understand precisely the apparent hybridization events, plastid DNA (ptDNA) markers should be simultaneously applied, so that maternal phylogenetic information would be available (Kaplan and Fehrer 2006). The candidate markers included atpB-rbcL ), rpl20-rps12 (Kaplan and Fehrer 2011), and trnT-trnL, trnL, trnL-trnF (Ito and Tanaka 2013).
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 taxonspecific 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.

Plant material
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 nonflowering and one flowering ones in Shan state ( Table 1). None of the specimens could be morphologically identified as any of three broad-leaved long-petioled Potamogeton species potentially distributed in Myanmar (P. distinctus, P. nodosus, and P. wrightii) due to either the lack of diagnostic floral characters or intermediate vegetative morphology or both. The morphological characters of the unidentified specimens were summarized to facilitate comparison with the three Potamogeton species ( 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 ( Table 2). As we failed to collect hybrids of P. distinctus, the nrITS data sets of two Potamogeton hybrids were obtained from GenBank: P. ×malainoides Miki ( P. distinctus × P. wrightii) and P. distinctus × P. nodosus (Du et al. 2010). Besides, two outgroup species were selected following Lindqvist et al. (2006) and included into the sample set; those were P. lucens L. and P. perfoliatus L. Note that four out of the six comparative materials were previously used for molecular phylogenetic analyses (Ito and Tanaka    The voucher specimens are retained in either of the following herbaria: BKF; MBK; RAF; TI; TNS. Those of Du et al. (2010) are kept in HIB. Sequences were deposited at the DNA Data Bank of Japan (DDBJ) and their accession numbers and voucher information are given in Table 2.

DNA extraction, amplification and sequencing
For the newly obtained samples, total genomic DNA was extracted and sequencing of five plastid regions was performed using the procedure outlined by . For the sequencing, previously used accessions were occasionally involved. We selected the following DNA regions that were used in previous molecular studies of Potamogeton as DNA barcoding markers: atpB-rbcL ), rpl20-rps12 ( Sequences of the nrITS were obtained using primers ITS-4 and ITS-5 (Baldwin 1992) under the same conditions used for the phyB amplification in . The total length was 713 bp. On direct sequencing of ten samples, overlapping double peaks were found at the same sites for complementary strands in the electropherograms. These products were cloned using a TOPO TA Cloning kit for Sequencing (Invitrogen, Carlsbad, California, USA). At least 16 clones per sample were chosen and their sequences were determined using the same procedure as that used in the first PCR followed by direct sequencing. For the cloned sequences, nucleotides that were not detected by direct sequencing were regarded as PCR errors.

Data analysis
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 Simmons and Ochoterena (2000). Gaps associated with mononucleotide repeat units were removed from consideration in the phylogenetic analysis because of problems related to homology assessment (Kelchner 2000) and because technical artifacts might be responsible for the variation (Clarke 2001). One representative sequence was used for accessions having the identical combined sequence.
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 (Swofford 2002) and Bayesian inference (BI; Yang and Rannala 1997) in MrBayes 3.1.2 (Ronquist and Huelsenbeck 2003) as described by Ito and Tanaka (2013); the only differences were the best-fit model for BI analysis on ptDNA (F81) and nrITS (HKY). The Bayesian Markov Chain Monte Carlo algorithm was run for 1 million generations for both ptDNA and nrITS data sets. Four incrementally heated chains were used that started from random trees and sampled one out of every 100 generations. The first 25% of the sampled generations (250,000 generations for each data set, respectively) were discarded as burn-in, and the remaining trees were used to calculate a 50% majority-rule consensus tree and to determine posterior probabilities for branches. The data matrices and the MP trees are available from the TreeBASE (S14928).

Taxon discussion
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).

Molecular phylogenetic analyses based on ptDNA and nrITS
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 (Fig. 1).
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 (Fig. 1).
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 (  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.

DNA barcoding for broad-leaved long-petioled Potamogeton specimens from Myanmar
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;Figs 2,3). Another specimen had P. wrightii haplotype and both of the heterogeneous nrITS sequences of P. ×malainoides (N. Tanaka

Utility of DNA barcoding markers for Potamogeton species
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 (Tables 3, 4). Among the three species, in which ten nucleotide substitutions were found in nrITS, atpB-rbcL included one length variation (indel) and two nucleotide substitutions; trnT-trnL region had two mononucleotide repeat units, in which repeat numbers are differed.
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.

Discussion
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 (Fig. 1). The obtained phylogeny is congruent with the nuclear 5S-NTS phylogeny of Lindqvist et al. (2006), the only molecular phylogeny that resolves the three Potamogeton species relationships. Below we will document a new record of Potamogeton species for the flora of Myanmar, discuss the origin and the evolution of hybrids of Potamogeton in Myanmar, and propose DNA barcoding markers for future Potamogeton studies.

Potamogeton nodosus, a new record for the flora of Myanmar
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 (Wiegleb 1990). Here we document a new record for the flora of Myanmar.

Hybridization among broad-leaved long-petioled Potamogeton species 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 (Du et al. 2010), yet a difference is found between the Chinese and Myanmar cases in maternal lineage: Potamogeton ×malainoides from China has P. distinctus as a maternal parent (Du et al. 2010), but that from Myanmar has P. wrightii as a maternal parent. This kind of reciprocal hybridizations occasionally occur in Potamogeton, i.e., P. ×anguillanus, P. ×fluitans, P. ×inbaensis, P. ×lanceolatifolius, P. ×sudermanicus, and P. 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.
×suecicus (reviewed in Ito and Tanaka 2013). In terms of morphology, P. ×malainoides in Myanmar showed both P. distinctus character, i.e., larger number of leaf veins, and that of P. wrightii, i.e., the acute to acuminate leaf tip (Table 1), and no major differences are found between the reciprocal hybrids (Du et al. 2010). In other cases of Potamogeton hybrids, reciprocal hybrids are partly distinguishable, e.g., reciprocal P. ×anguillanus shows no differences in morphology but exhibited differences in drought tolerance (Iida et al. 2007); P. ×inbaensis with different maternal lines is roughly distinguishable by leaf morphology (Amano et al. 2008).
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 (Du et al. 2010). The present study successfully identified P. distinctus as the maternal lineage of this hybrid for the first time. From the morphological point of view, it is difficult to evaluate the morphological intermediacy between the parental species as both species show large phenotypic plasticity in quantitative morphology, e.g., leaf petiole length. Du et al. (2011) reported that nrITS is the most useful marker for DNA barcoding of Potamogeton. The present study verified its utility by distinguishing three closely related species, P. distinctus, P. nodosus, and P. wrightii, as well as hybrids among them (Fig. 1, Table 3). Meanwhile, in order to understand hybridization events precisely, we simultaneously used plastid DNA markers, including those used in previous molecular studies, i.e., atpB-rbcL ), rpl20-rps12 (Kaplan and Fehrer 2011), and trnT-trnL, trnL, trnL-trnF Tanaka 2013, Zhang et al. 2008). Given that atpB-rbcL showed higher utility than the others (Table 4), here we propose nrITS and atpB-rbcL as DNA barcoding markers for Potamogeton species. Note that trnT-trnL has similar resolution to distinguish closely related Potamogeton species, yet the differences are found only in mononucleotide repear units, which technical artifacts might be responsible for the variation (Clarke 2001).

Utility of DNA barcoding markers for Potamogeton species
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. 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.

Author contributions
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.