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 (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 (Table 1).

Table 1. Download as CSV

Morphological comparison among the four Potamogeton specimens collected in Myanmar and three broad-leaved long-petioled Potamogeton species potentially distributed in Myanmar (a: Wiegleb 1990; b: Wiegleb and Kaplan 1998; c: Wiegleb 2002; d: Guo et al. 2010).

	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)
Characters
Carpel numbera,b,c,d		2	4	4	N/A	N/A	4	N/A
Leaf tipa,b,c,d		Round	Round	Acute	Round	Round	Acute	Round
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 (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 2013).

Table 2. Download as CSV

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.

Accession	Locality	Voucher	atpB-rbcL	rpl20-rps12	trnT-trnL	trnL	trnL-trnF	nrITS
INGROUP
P. distinctus
	Japan	YI01686 (TNS)	AB871488	AB871498	AB744025	AB744013	AB744019	AB744007
	Thailand	YI01729 (BKF)	AB871490	AB871500	AB871505	AB871511	AB871517	AB871525
	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
P. nodosus
	Mexico	YI01195 (TNS)	AB871487	AB871497	AB871504	AB871510	AB871516	AB871524
P. wrightii
	Japan	YI00048 (TNS)	AB206988	AB871495	AB695139	AB695131	AB695135	AB206991
P. ×malainoides
	China	HDZY8 (HIB)	N/A	N/A	N/A	N/A	N/A	FJ956881
								FJ956882
	Myanmar	N. Tanaka & al. 080631 (RAF, TI, MBK)	AB871484	AB871492	AB871502	AB871507	AB871513	AB871520
								AB871521
P. distinctus × P. nodosus
	China	HDZY5 (HIB)	N/A	N/A	N/A	N/A	N/A	FJ956875
								FJ956876
	China	HDZY6 (HIB)	N/A	N/A	N/A	N/A	N/A	FJ956877
								FJ956878
	China	HDZY7 (HIB)	N/A	N/A	N/A	N/A	N/A	FJ956879
								FJ956880
	Myanmar	N. Tanaka & al. 080662 (RAF, TI, MBK)	AB871486	AB871494	N/A	AB871509	AB871515	AB871522
								AB871523
OUTGROUP
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 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.

For the newly obtained samples, total genomic DNA was extracted and sequencing of five plastid regions was performed using the procedure outlined by Ito et al. (2010). 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 (Ito et al. 2007), rpl20-rps12 (Kaplan and Fehrer 2011), trnT-trnL, trnL, trnL-trnF (Ito and Tanaka 2013, Zhang et al. 2008), and nrITS (e.g., Du et al. 2010, Ito and Tanaka 2013, Kaplan and Fehrer 2011, Les et al. 2009). The atpB-rbcL (seven samples), rpl20-rps12 (nine), trnT-trnL (four), trnL (five), and trnL-trnF (five) regions of chloroplast DNA were amplified and directly sequenced using primers atpB-2F and rbcL-2R (Manen et al. 1994) for atpB-rbcL (779–787 bp), rpl-20 and 5'-rps-12 (Hamilton 1999) for rpl20-rps12 (794 or 813 bp), and Po-trnT2F (Ito and Tanaka 2013) and “b” (Taberlet et al. 1991) for trnT-trnL (807–809 bp), “c” and “d” (Taberlet et al. 1991) for trnL intron (593 bp), and “e” and “f” (Taberlet et al. 1991) for trnL-trnF (403 bp). Note that trnT-trnL was missing from Potamogeton sp. (N. Tanaka & al. 080662).

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 Ito et al. (2010). 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.

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).

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).

Figure 1.

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 (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 (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 (Du et al. 2010) have diverged heterogeneous nrITS sequences, and non-hybrid species have homogenous nrITS sequences (Fig. 1).

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 & al. 080631; Fig. 4); the other of the remaining two exhibited P. distinctus haplotype and both of the heterogeneous nrITS sequences of P. distinctus × P. nodosus (N. Tanaka & al. 080662; Fig. 5).

Figure 2.

A voucher specimen of Potamogeton distinctus (N. Tanaka & al. 080061).

Figure 3.

A voucher specimen of Potamogeton distinctus (N. Tanaka & al. 080657).

Figure 4.

A voucher specimen of Potamogeton ×malainoides (N. Tanaka & al. 080631).

Figure 5.

A voucher specimen of Potamogeton distinctus × P. nodosus (N. Tanaka & al. 080662).

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.

Table 3. Download as CSV

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.

Taxon	nrITS
	14	21	55	426	436	444	480	561	571	579
P. distinctus	T	C	T	C	A	G	T	G	G	T
P. distinctus × P. nodosus	T	C	T	C	A	G	T	G	G	T
	T	C	T	G	C	G	T	G	G	T
P. nodosus (Mexico)	T	C	T	G	C	G	T	G	A	C
P. wrightii	G	A	A	C	C	A	A	A	G	T
P. ×malainoides	T	C	T	C	C	A	T	G	G	T
	G	A	A	C	C	A	A	A	G	T

Table 4. Download as CSV

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.

Taxon	atpB-rbcL			trnT-trnL
	380-383	547	563	403-405	507-514
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)

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.

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. ×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 (Ito et al. 2007), rpl20-rps12 (Kaplan and Fehrer 2011), and trnT-trnL, trnL, trnL-trnF (Ito and 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).

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.