Phylogeny of Potamogeton (Potamogetonaceae) Revisited: Implications for Hybridization and Introgression in Argentina

Home , Alismatales, Potamogeton, Potamogetonaceae, Potamogeton pusillus

Bull. Natl. Mus. Nat. Sci., Ser. B, 42(4), pp. 131–141, November 22, 2016

Yu Ito1,2, Gerardo Lucio Robledo3, Laura Iharlegui4 and Norio Tanaka5,*

1 School of Biological Sciences, University of Canterbury, Christchurch, Canterbury 8020, New Zealand 2 Present address: Xishuangbanna Tropical Botanical Garden, The Chinese Academy of Sciences, Kunming 650223, P. R. China 3 Laboratorio de Micología, Instituto Multidisciplinario de Biología Vegetal, CONICET-Universidad Nacional de Córdoba, Argentina 4 Museo de La Plata, Paseo del Bosque s/n, 1900 La Plata, Buenos Aires, Argentina 5 Department of Botany, National Museum of Nature and Science, Amakubo 4–1–1, Tsukuba, Ibaraki 305–0005, Japan * E-mail: [email protected]

(Received 28 July 2016; accepted 28 September 2016)

Abstract Potamogeton is a cosmopolitan genus of 90–95 species in which numerous hybridization events have occurred worldwide. A plant recently collected from Argentina exhibited ambiguous morphology that does not match any species of the genus. We aimed to assess if the plant coexisting with another Potamogeton species is a product of reticulate evolution. A concatenated plastid DNA (psbA-trnH, trnL intron, and trnL-trnF) and nuclear ribosomal DNA (5S-NTS) data sets, primarily based on previous studies sample set mainly consisting of American and Asian species, were analyzed using Bayesian inference. Nuclear ribosomal ITS sequences were also obtained from ﬁve Argentina materials. We recovered similar topologies from both the plastid DNA and nuclear ribosomal 5S-NTS analyses in which most specimens are consistently placed. The specimen of primal interest from Argentina strongly clustered with co-occurring linear-leaved species in the 5S-NTS tree, but was genetically identical to broad-leaved ones in the plastid DNA analysis. The ITS sequence of the specimen was the same as that of the linear-leaved species and no polymorphisms were observed. Considering the discrepant phylogenetic positions between the trees and lack of ITS infra-individual variations, the origin of the specimen from Argentina is better explained by hybridization and subsequent introgression.

Key words: Alismatales, aquatic plants, Bayesian inference, hybridization, introgression, molecular phylogeny, plastid DNA, Potamogeton, topological conﬂicts, 5S-NTS.

Introduction center of species diversity in temperate regions of the northern hemisphere (Kaplan et al., 2013), Potamogeton L. is one of the three genera of with nearly 70% of the world’s species occurring the aquatic plant family Potamogetonaceae. The in either Asia, Europe, or North America (Wieg- number of the species varies depending on litera- leb and Kaplan, 1998). In contrast, the species in ture, but 90–95 species are in general accepted in Southern South America, comprising Argentina, the world (Haynes and Holm-Nielsen, 2003; Chile, and Uruguay, has been scarcely studied. WCSP, 2014). This cosmopolitan genus has its Following Tur (1982), Wiegleb and Kaplan 132 Yu Ito et al.

(1998), and Haynes and Holm-Nielsen (2003), hybridization events have occurred; therefore, it nine well-recognized species were recorded in is recognized as a “classic” example of hybridiza- southern South America: i) P. crispus L. (cosmo- tion in aquatic plants (Les and Philbrick, 1993). politan); ii) P. ferrugineus Hagstr. (South Amer- In their morphology-based monographic work, ica); iii) P. gayi A.Benn. (South America); iv) P. Wiegleb and Kaplan (1998) reported the number illinoensis Morong (North and South America); of hybrids that are approximately the same as the v) P. linguatus Hagstr. (South America); vi) P. number of non-hybrid species. Subsequent montevidensis A.Benn. (South America); vii) P. molecular studies have detected and documented polygonus Cham. (South America); viii) P. pusil- further cases of hybridization in Potamogeton, lus L. (cosmopolitan); and ix) P. spirilliformis including ones that do not exhibit obvious mor- Hagstr. (South America). As reported in other phological characteristics (Les et al., 2009). A regions, difﬁculties of morphological identiﬁca- case of triple hybridization (a hybrid arisen from tion of Potamogeton species are known in South crosses of a primary hybrid with a third species) America. A plant recently collected from Argen- has been also reported in Potamogeton (Kaplan tina (Y. Ito YI1992 & al.; Fig 1; Appendix 1) and Fehrer, 2007), implying that the genus has exhibited ambiguous morphology that does not undergone rather complicated evolution. The match any above-mentioned species of the genus aforementioned morphologically ambiguous (Fig. 1; Table 1). plant from Argentina may be a product of hybrid- Potamogeton is a genus in which numerous ization, because it coexists with P. pusillus.

Fig. 1. Habitat of Potamogeton sp. YI1992_TNS in Córdoba, Argentina. Phylogeny of Potamogeton (Potamogetonaceae) 133 ﬂower no. ＜ 12 2–7 numerous Inﬂorescences carpel no. 4 (3–)4(–7) (2–)4 present leaves Floating ?absent absent absent/ apex acute/ subob - tuse acuminate acuminate/ mucronate 17(–19) no. veins (3–)5 (1–)3(–5) (7–)9– margins denticulate/ entire entire entire minutely 60(–90) length– 8–16 (15–)20– 3.5–7 width ratio width (mm) width 2.5(–3.1) 40(–53) sp. (obtained in this study) and its two inferred parental species (adapted from Wiegleb and Kaplan, Wiegleb from (adapted species parental this study) and its two inferred in sp. (obtained 2.5–9 (0.3–)0.8– (4–)15– Submerged leaves Submerged 85(110) 180(–220) length (mm) 20–85 (15–)20– (50–)70– sessile petiolate (mm) sessile sessile short petiolate/ shape oblong/ oblanceolate/ elliptical linear–lanceolate linear narrowly sp. 1998) Table Table 1. of Potamogeton morphology and reproductive Vegetative Potamogeton P. pusillus P. P. illinoensis P. 134 Yu Ito et al.

Molecular phylogenetic analyses on Potamogeton 2008). The data set of Lindqvist et al. (2006) is have been independently conducted by different particularly relevant for our purpose because it research groups based on their own unique sam- mainly consisted of North American species that ple and data sets that scarcely overlapped each either are distributed in South America or appar- other (e.g., Iida et al., 2004; Lindqvist et al., ently have South American relatives (Wiegleb 2006; Wang et al., 2007; Zhang et al., 2008; Les and Kaplan, 1998; Haynes and Holm-Nielsen, et al., 2009; Ito and Tanaka, 2013; Kaplan et al., 2003). Topological incongruence between recon- 2013). Therefore, it remains unclear which phy- structed phylogenetic trees were compared, with logeny is the most reliable one, as significant a particular focus on the taxa from Argentina. In topological incongruences have occasionally addition, DNA sequences of the internal tran- been found among the studies (Ito and Tanaka, scribed spacer (ITS) region of nrDNA were gen- 2013). Lindqvist et al. (2006) provided a rela- erated to seek another line of evidence of hybrid- tively well-resolved phylogeny of the genus, ization. comprising broad-leaved and linear-leaved lineages based on the 5S non-transcribed spacer Materials and Methods [hereinafter called 5S-NTS of the nuclear ribosomal DNA (nrDNA)], followed by Kaplan et Taxon sampling al.’s (2013) 5S-NTS tree. Still, a question The data set for molecular phylogenetic analy- remains as to whether the same or similar topol- ses mainly consists of sequences deposited in ogy can be recovered with a plastid DNA (here- GenBank: 41 samples (Lindqvist et al., 2006); 26 inafter called ptDNA) data set because Lindqvist (Zhang et al., 2008, unpublished data); four et al. (2006) failed to reconstruct a resolved phy- (Kaplan and Fehrer, 2007; Kaplan et al., 2013). logeny in their ptDNA analysis using two fast- Seventeen out of samples used in Lindqvist et al. evolving markers, psbA-trnH and trnL intron, (2006) that lack either ptDNA or 5S-NTS were and no analyses using ptDNA sequences were not included in this study. Five samples from performed by Kaplan et al. (2013). However, Argentina were added, which were P. gayi (one Lindqvist et al.’s (2006) data set itself appears to specimen), P. pusillus (three), and the unidenti- be useful if 19 outgroups from the other genera fied Potamogeton sp. YI1992_TNS (one). The of Potamogetonaceae and other distantly related data set included 76 samples, which were equiva- monocots and non-monocot families, e.g., Ara- lent to 46 species including one putative hybrid, ceae, Alismataceae, Juncaginaceae, and Magnoli- P.×haynesii Hellq. & G.E.Crow (Appendix 1). aceae, are excluded from the analysis. Compara- tive data are available by Zhang et al. (2008, DNA extraction, PCR amplification, and unpublished data), which using trnT-trnL, trnL sequencing intron, and trnL-trnF, recovered roughly the Total genomic DNA was extracted from the same broad-leaved and linear-leaved lineages five newly collected Potamogeton specimens (Zhang et al., 2008). from Argentina following the method outlined in To assess whether the unidentified Potamogeton Ito et al. (2010) and their sequences of ptDNA (Y. Ito YI1992 & al.) with ambiguous morphol- and nrDNA regions were determined by PCR ogy from Argentina is a product of hybridization, amplification and direct sequencing. The follow- we employed simultaneous molecular phyloge- ing primer pairs were used for PCR and sequenc- netic analyses of nrDNA and ptDNA based on ing: psbAF and trnHR (Sang et al., 1997) for data sets of Lindqvist et al. (2006) and Zhang et psbA-trnH; “c” and “d” (Taberlet et al., 1991) for al. (2008) and comparable data from GenBank, trnL intron; “e” and “f” (Taberlet et al., 1991) for which occasionally contains trnL-trnF from trnL-trnF; and PI and PII (Cox et al., 1992) for ptDNA (Kaplan and Fehrer, 2007; Zhang et al., 5S-NTS, and ITS-4 and ITS-5 for nrITS (Bald- Phylogeny of Potamogeton (Potamogetonaceae) 135 win, 1992). The PCR amplification was con- (Miller et al., 2010) after the best models had ducted using TaKaRa Ex Taq polymerase been determined in MrModeltest ver. 3.7 (Nyl- (TaKaRa Bio, Japan), and PCR cycling condi- ander, 2002); these models were GTR＋I＋G for tions were 94°C for 60 s; then 30 cycles of 94°C ptDNA and GTR＋G for 5S-NTS. For gap char- for 45 s, 52°C for 30 s, 72°C for 60 s; and finally acters, the datatype＝standard option of 72°C for 5 min. The PCR products were cleaned MrBayes was used. Analyses were run for using illustra ExoProStar (GE Healthcare, Piscat- 8,150,000 million and 460,000 million genera- away, USA) and then reacted using ABI Big Dye tions for the ptDNA and 5S-NTS data sets, Terminator ver. 3.1 (Applied Biosystems, USA) respectively, until the average standard deviation with the same primers as those used for the PCR of split frequencies dropped below 0.01, sam- amplifications. DNA sequencing was performed pling every 1,000 generations and discarding the with an ABI PRISM 3130xl DNA sequencer first 25% as burn-in. The convergence and effec- (Applied Biosystems). Automatic base calling tive sampling sizes (ESS) of all parameters were was checked by eye using Genetyx-Win ver. 3 checked in Tracer ver. 1.6 (Rambaut et al., (Software Development Co., Japan). All sequences 2014). All trees were visualized using FigTree generated in the present study have been submit- ver. 1.3.1 (Rambaut, 2009). The data matrices ted to the DNA Data Bank of Japan (DDBJ), and the MrBayes trees are available at Treebase which is linked to GenBank, and their accession (TB2:S18639). numbers and voucher specimen information are presented (Appendix 1). Results

Data analysis Molecular phylogeny We assembled two datasets from the 76 sam- The ptDNA data set of Potamogeton included ples, which included the aforementioned ﬁve 1,391 aligned characters (psbA-trnH: 364 bp; samples from Argentina: i) ptDNA (psbA-trnH, trnL intron: 591 bp; trnL-trnF: 436 bp) and 17 trnL intron, and trnL-trnF) and ii) nuclear indels, of which 146 characters including the 5S-NTS. Missing data found in ptDNA were binary-coded indels are polymorphic. The topol- retained because “it should generally be possible ogy is resolved, yet the support values are mostly to accurately place incomplete taxa in phyloge- low (Fig. 2a). nies, if enough informative characters are sam- The 5S-NTS data set of Potamogeton included pled” (Wiens and Morrill, 2011); those are used 310 aligned characters and six indels, of which as equivocal characters (N). Following Kaplan et 275 characters including the binary-coded indels al. (2013), one of the basal-most Potamogeton are polymorphic. We obtained a well-resolved taxa in their ITS tree, namely P. spirillus, were tree, which topology followed that of the ptDNA chosen as an outgroup for addressing the intrage- tree (Fig. 2b). neric relationships, because this region was too Respective four groups, that belonged to lin- variable to allow a reliable alignment with the ear-leaved and broad-leaved lineages detected in other two genera. Sequences were aligned using Lindqvist et al. (2006) and Kaplan et al. (2013), MAFFT ver. 7.058 (Katoh and Standley, 2013) were recovered in either or both ptDNA and using “leave gappy regions” option and then 5S-NTS trees; those are numbered from groups inspected manually. L1–L4 and groups B1–B4, respectively: group Phylogenetic inference was performed using L1 (P. diversifolius–P. spirilus); group L2 (P. Bayesian inference (BI; Yang and Rannala, compressus–P. gayi–P. obtusifolius–P. subsibiricus– 1997). Analyses were conducted with MrBayes P. trichoides]; group L3 [P. clystocarpus–P. foliosus– ver. 3.2.2 (Ronquist and Huelsenbeck, 2003; P. friesii–P.×haynesii–P. pusillus (China)–P. Ronquist et al., 2012) run on the CIPRES portal strictifolius]; group L4 [P. oxyphyllus–P. pusillus 136 Yu Ito et al. - - ) and b) the nuclear 5S-NTS data sets. Letters sets. 5S-NTS data nuclear the b) and trnL - trnF ) intron, and intron, trnL based on a) the concatenated plastid DNA ( psbA - trnH , DNA plastid concatenated the a) on based Potamogeton refer to groups noted in the text. The six accessions of which phylogenetic positions are inconsistent between the data sets emphasized in bold, for which cor The six accessions of which phylogenetic refer to groups noted in the text. by dotted arrows. Bayesian posterior probabili responding phylogenetic positions in the other analysis are indicated Numbers above or below the branches indicate ＜ 0.7 are indicated by hyphens. ties (PP). Clades with PP Fig. 2. for trees MrBayes Phylogeny of Potamogeton (Potamogetonaceae) 137

(North and South America)]; group B1 (P. collected by ourselves. crispus–P. maackianus–P. robbinsii); group B2 Potamogeton sp. (Y. Ito YI1992 & al.) has [P. alpinus–P. distinctus–P. malaianus–P. ptDNA close to those of broad-leaved species, malainoides–P. nodosus (RRH6256 UNA)–P. such as P. illinoensis and P. lucens (group B3: perfoliatus–P. richardsonii–P. wrightii]; group Fig. 2a) and nrDNA identical or closely related B3 [P. gramineus–P. illinoensis (VEM87816 to that of the co-occurring linear-leaved P. pusil- UNA)–P. lucens]; group B4 (P. amplifolius–P. lus (group L3: Fig. 2b). Considering the incon- floridanus–P. oakesianus–P. natans–P. pulcher). gruence of phylogenetic position and the lack of Of samples from Argentina, Potamogeton sp. ITS intra-individual polymorphisms, which has YI1992_TNS was positioned significantly differ- been used as a line of evidence to identify Pota- ently between ptDNA and 5S-NTS trees (group mogeton hybrids (e.g. Kaplan and Fehrer 2007, B3 in ptDNA; group L4 in 5S-NTS; Fig. 2). Du et al., 2010), not simple hybridization but introgression following multiple hybridizations Nuclear DNA (ITS) sequence comparisons between linear-leaved and broad-leaved species ITS sequences obtained from the five speci- better explain the origin of Potamogeton sp. mens from Argentina were 713 bp in length. No YI1992_TNS. With the empirically confirmed intra-individual variation were found in any of maternal inheritance of chloroplast DNA in Pota- the specimens, except the 1-bp intra-individual mogeton (Kaplan and Fehrer, 2006), hybridiza- variation observed in Potamogeton pusillus tion between a paternal P. pusillus and a maternal YI1997. No sequence differences were found broad-leaved Potamogeton species is likely. The between P. pusillus and Potamogeton sp. maternal parent is most probably P. illinoensis Y1992_TNS, from which P. gayi is distinguish- because this American species is also distributed able by two nucleotide substitutions. in Argentina (Haynes and Holm-Nielsen, 2003), where it once may have occurred with P. pusillus (which yet co-exists in the river in Córdoba, Discussion Argentina) and have repeatedly hybridized with The present study reconstructed phylogenies its pollen, termed “cytoplasmic introgression” of Potamogeton based on ptDNA and nuclear (Rieseberg, 1997). 5S-NTS of nrDNA data sets, in which six accessions show significant inconsistency between the Conclusions trees (Fig. 2). Such topological incongruences We performed simultaneous molecular phylo- resulting from ptDNA and nrDNA markers are genetic analyses of Potamogeton based on previ- often reported in phylogenetic studies (Wendel ous studies’ data sets and our newly collected and Doyle, 1998, Degnan and Rosenberg, 2009). samples. The topological comparison between Although some causes of phylogenetic incongru- ptDNA and 5S-NTS clearly exhibited significant ence, e.g. gene choice and incomplete lineage incongruences. A single accessions from Argen- sorting, are suggested, hybridization and intro- tina that was inconsistently positioned between gression are likely to be attributed to the topolog- the trees may be a product of hybridization or ical conflicts in Potamogeton, as is concluded by introgression. Future phylogenetic researches Hamzeh and Dayanandan (2004), Fehrer et al. may aim at i) improving the support values by (2007), Tippery and Les (2011), Ito et al. (2013), adding more valuable ptDNA regions, ii) seeking Ren et al. (2015), Soto-Trejo et al. (2015). The alternative nrDNA markers, such as low-copy six specimens discrepantly resolved between the nuclear DNA loci, and iii) applying alternative trees may indicate such reticulate evolution. methods such as AFLP and RADseq. Here, out of the six accessions, we discussed only for Potamogeton sp. YI1992_TNS, that was 138 Yu Ito et al.

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Aquatic Botany 89: 34–42. hybrid origin of Nymphoides montana Aston (Menyan- 140 Yu Ito et al. 5S-NTS DQ786472 DQ786471 DQ786480 DQ786501 DQ786465 FJ495513 DQ786499 KF270967 FJ495464 FJ495465 DQ786510 DQ786479 FJ495503 DQ786473 DQ786492 FJ495491 FJ495492 FJ495494 DQ786457 DQ786461 FJ495487 DQ786498 FJ495471 KF270960 DQ786486 DQ786458 DQ786476 DQ786477 FJ495473 FJ495474 DQ786467 FJ495472 KF270963 FJ495481 FJ495482 FJ495484 DQ786466 DQ786474 DQ786489 DQ786488 DQ786478 DQ786494 DQ786495 DQ786496 KF270961 KT634288 trnL - trnF EF432090 EF432067 EF432091 EF432084 EF432079 EF432071 EF432072 EF432094 EF432093 EF174576 EF432077 EF432098 EF432100 EF174578 EF174575 KT634283 trnL intron DQ786435 EF428400 EF428377 EF428401 DQ786445 DQ786436 EF428394 EF428389 EF428414 EF428415 DQ786423 EF428411 DQ786431 EF428381 DQ786425 DQ786426 EF428382 DQ786424 EF428404 DQ786432 EF428403 EF174576 EF428387 EF428408 EF428410 DQ786433 DQ786434 EF174578 DQ786427 DQ786428 DQ786429 DQ786430 EF174575 KT634278 psbA - trnH DQ786565 DQ786541 DQ786540 DQ786543 DQ786539 DQ786542 DQ786556 DQ786562 DQ786526 DQ786535 DQ786534 DQ786528 DQ786558 DQ786527 DQ786563 DQ786536 EF174574 DQ786564 DQ786538 EF174573 DQ786537 DQ786561 DQ786559 DQ786533 DQ786530 DQ786529 DQ786560 KT634263 used in the present study. Also provided are: used in the present study. (2013) (2013) (2013) et al. et al. et al. Reference (2006) (2006) (2006) (2006) (2006) (2006) (2006) (2006) (2006) (2006) (2006) (2006) (2006) (2006) (2006) (2006) (2006) (2006) (2006) (2006) (2006) (2006) (2006) (2006) (2006) (2006) (2008; Unpubl.) (2008; Unpubl.) (2008; Unpubl.) (2008; Unpubl.) (2008; Unpubl.) (2008; Unpubl.) (2008; Unpubl.) (2008; Unpubl.) (2008; Unpubl.) (2008; Unpubl.) (2008; Unpubl.) (2008; Unpubl.) (2008; Unpubl.) (2008; Unpubl.) (2008; Unpubl.) et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. Lindqvist Lindqvist Zhang et al. Lindqvist Lindqvist Zhang et al. Zhang et al. Lindqvist Lindqvist Zhang et al. Lindqvist Lindqvist Zhang et al. Zhang et al. Zhang et al. Zhang et al. Lindqvist Lindqvist Zhang et al. Lindqvist Lindqvist Zhang et al. Lindqvist Lindqvist Zhang et al. Lindqvist Lindqvist Lindqvist Zhang et al. Zhang et al. Zhang et al. Zhang et al. Kaplan & Fehrer (2007); Lindqvist Lindqvist Kaplan & Fehrer (2007) Lindqvist Lindqvist Lindqvist Lindqvist Lindqvist Kaplan & Fehrer (2007); Lindqvist Lindqvist this study Kaplan & Fehrer (2007); Locality USA: Texas USA: Michigan China USA: Oregon Australia: NSW China China Papua New Guinea USA: Montana China USA: Michigan USA: Michigan China China China China USA: Alaska USA: New York State York USA: New China USA: Michigan USA: Alabama China USA: New Jersey USA: Texas China USA: Nevada USA: Alabama USA: Minnesota China China China China Czech Republic USA: Ohio Denmark The Netherlands USA: Pennsylvania USA: Florida USA: Alabama Bolivia USA: Florida France USA: Alabama USA: Michigan Argentina Czech Republic trnL intron, - trnF , 5S-NTS, and ITS) of Potamogeton Code CBH12906_UNA RRH6256_UNA W.Z0526_WH JSW26June1981_ UNA RRH8471_UNA W.Z0502_WH W.Z0528_WH GL7805_UNA AES5147_UNA W.Z0520_WH RRH3800_UNA LJD1414_UNA W.Z0514_WH W.Z0645_WH W.Z0646_WH W.Z0641_WH SST21_UNA CBH15409_UNA W.Z0506_WH RRH5068_UNA RRH10212_UNA W.Z0507_WH CBH11328_UNA CBH16574_UNA W.Z0532_WH AT3814_UNA MB694_UNA VEM87816_UNA W.Z0531_WH W.Z0512_WH W.Z0536_WH W.Z0540_WH ZK97.829_PRA JKB1977:109_UNA RRH8736_UNA Denny_PRA JKB1985:254_UNA RRH10166_UNA RRH10216_UNA NR3432_UNA RRH10165_UNA Wolff_PRA LJD1255_UNA RRH6217_UNA Banifacio1855_SI ZK96.638_PRA 3432 (UNA) Voucher et al. C. B. Hellquist 12906 (UNA) R. Haynes 6256 (UNA) Wang & Zhang 0526 (WH) Wang s.n., 26 June Williams J. S. 1981 (UNA) R. Haynes 8471 (UNA) Wang & Zhang 0502 (WH) Wang & Zhang 0528 (WH) Wang G. Leach 7805 (UNA) A. E. Schuyler 5147 (UNA) Wang & Zhang 0520 (WH) Wang R. R.Haynes3800 (UNA) L. J. Davenport 1414 (UNA) Wang & Zhang 0514 (WH) Wang & Zhang 0645 (WH) Wang & Zhang 0646 (WH) Wang Wang & Zhang 0641 (WH) Wang S. S. Talbot 21 (UNA) Talbot S. C. B. Hellquist 15409 (UNA) Wang & Zhang 0506 (WH) Wang R. Haynes 5068 (UNA) R. Haynes 10212 (UNA) & Zhang 0507 (WH) Wang C. B. Hellquist 11328 (UNA) C. B. Hellquist 11328 C. B. Hellquist 16574 (UNA) & Zhang 0532 (WH) Wang 3814 (UNA) Tiehm A. M. Birk 694 (UNA) V. E. McNeilus 87-816 (UNA) V. Wang & Zhang 0531 (WH) Wang Wang & Zhang 0512 (WH) Wang & Zhang 0536 (WH) Wang & Zhang 0540 (WH) Wang Z. Kaplan 97/829 (PRA) J. K. Bissell 1977:109 (UNA) R. Haynes 8736 (UNA) Denny s. n. (PRA) P. J. K. Bissell 1985:254 (UNA) R. Haynes 10166 (UNA) R. Haynes 10216 (UNA) R. Haynes 10165 (UNA) P. Wolff x. n. (PRA) Wolff P. L. J. Davenport 1255 (UNA) R. Haynes 6217 (UNA) N. Ritter Banifacio 1855 (SI) Z. Kaplan 96/638 (PRA) Reichb. Raf. Raf. Tuckerm. Tuckerm. Taxon Morong Morong A. Benn. A. Benn. Voucher (Herbaria in parentheses); Code; Locality; Reference Voucher Rupr. A.Benn. A.Benn. Morong haynesii Hellq. & G. E. Crow Appendix 1. List of the GenBank accessions ( psbA - trnH , × P. nodosus Poir. P. P. nodosus Poir. P. P. malainoides P. natans L. P. Raoul ochreatus P. P. maackianus A.Benn. P. maackianus A.Benn. P. malaianus Miq. P. P. cristatus Regel & Maack P. P. A. Gray oakesianus Robbins ex. P. P. hillii P. obtusifolius Mert. & Koch. P. P. distinctus P. L. compressus P. L. compressus P. L. compressus P. confervoides P. P. alpinus Balb P. amplifolius P. P. alpinus Balb. P. P. illinoensis P. P. distinctus P. P. gramineus L. P. P. crispus L. P. crispus L. P. P. amplifolius P. clystocarpus Fernald P. crispus L. P. crispus L. P. P. illinoensis P. P. diversifolius P. P. gramineus L. P. gramineus L. P. gramineus L. P. gramineus L. P. P. lucens L. P. lucens L. P. P. diversifolius P. floridanus Small P. foliosus Raf. P. friesii P. gayii P. gayii P. P. gramineus L. P. P. gramineus L. P. Phylogeny of Potamogeton (Potamogetonaceae) 141 5S-NTS FJ495495 FJ495497 DQ786491 FJ495488 FJ495489 FJ495475 FJ495477 FJ495478 DQ786462 FJ495479 DQ786475 KT634284 KT634285 KT634286 DQ786505 FJ495499 FJ495500 DQ786504 DQ786463 DQ786464 DQ786460 DQ786490 DQ786507 DQ786502 DQ786483 DQ786508 DQ786509 FJ495466 DQ786511 KT634287 trnL-trnF EF432080 EF432107 EF432078 EF432081 EF432073 EF432095 EF432082 KT634279 KT634280 KT634281 EF432096 EF432099 EF432069 KT634282 trnL intron EF428390 EF428417 EF428388 EF428391 EF428383 EF428405 EF428419 EF428392 KT634274 KT634275 KT634276 DQ786439 EF428406 EF428409 DQ786438 DQ786440 DQ786442 DQ786443 DQ786444 DQ786437 EF428379 KT634277 psbA-trnH DQ786544 DQ786545 DQ786566 KT634259 KT634260 KT634261 DQ786548 DQ786547 DQ786549 DQ786550 DQ786552 DQ786531 DQ786553 DQ786546 DQ786532 DQ786554 DQ786555 DQ786557 KT634262 Reference (2006) (2006) (2006) (2006) (2006) (2006) (2006) (2006) (2006) (2006) (2006) (2006) (2006) (2006) (2006) (2008; Unpubl.) (Unpubl.) (2008; Unpubl.) (2008; Unpubl.) (2008; Unpubl.) (2008; Unpubl.) (2008; Unpubl.) (2008; Unpubl.) (2008; Unpubl.) (2008; Unpubl.) (2008; Unpubl.) et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. Zhang et al. Zhang et al. Lindqvist Zhang et al. Zhang et al. Zhang et al. Zhang et al. Zhang et al. Zhang et al. Lindqvist Lindqvist this study this study this study Lindqvist Zhang et al. Zhang et al. Lindqvist Lindqvist Lindqvist Lindqvist Lindqvist Lindqvist Lindqvist Lindqvist Lindqvist Lindqvist Zhang et al. Lindqvist this study Locality Appendix 1. Continued China China Botswana China China China China China China Denmark USA: Tennessee Argentina Argentina Argentina USA: Alabama China China USA: Montana USA: Alabama USA: Alabama Canada USA: Ohio USA: Michigan USA: Alabama USA: Tennessee Spain USA: Pennsylvania China USA: New York State York USA: New Argentina Code W.Z0516_WH W.Z0648_WH GEGR.HMB1485_ UNA W.Z0513_WH W.Z0517_WH W.Z0508_WH W.Z0533_WH W.Z0650_WH W.Z0518_WH RRH8739_UNA VEM87840_UNA YI1993_TNS YI1997_TNS YI2011_TNS RRH10213_UNA W.Z0534_WH W.Z0538_WH BM.AES4980_UNA SST079_UNA SST951351_UNA JSP1726_UNA JKB1982:203_UNA RRH5286_UNA SST262_UNA VEM87843_UNA MB.PG11April1987_ UNA JKB1988:106_UNA W.Z0504_WH RRH3344_UNA YI1992_TNS (2008) but is re-identified (Ito and Tanaka 2013). Tanaka (2008) but is re-identified (Ito and Voucher Wang & Zhang 0516 (WH) Wang Wang & Zhang 0648 (WH) Wang G. E. Gibbs Russell & H. M. Biegel 1485 (UNA) Wang & Zhang 0513 (WH) Wang Wang & Zhang 0517 (WH) Wang Wang & Zhang 0508 (WH) Wang Wang & Zhang 0533 (WH) Wang Wang & Zhang 0650 (WH) Wang Wang & Zhang 0518 (WH) Wang R. Haynes 8739 (UNA) V. E. McNeilus 87-840 (UNA) V. YI1993 & al. (TNS) Ito Y. Y. Ito YI1997 & al. (TNS) Ito Y. Y. Ito YI2011 & al. (TNS) YI2011 Ito Y. R. Haynes 10213 (UNA) Wang & Zhang 0534 (WH) Wang Wang & Zhang 0538 (WH) Wang B. McCune & A. E. Schuyler B. McCune & 4980 (UNA) S. & S. Talbot 079 (UNA) Talbot S. & S. & S. Talbot 95-1351 (UNA) Talbot S. & J. S. Pringle 1726 (UNA) J. K. Bissell 1982:203 (UNA) R. Haynes 5286 (UNA) S. & S. Talbot 262 (UNA) Talbot S. & V. E. McNeilus 87-843 (UNA) V. Garcia s. n., M. Bernues & P. April 1987 (UNA) 11 J. K. Bissell 1988:106 (UNA) Wang & Zhang 0504 (WH) Wang R. Haynes 3344 (UNA) YI1992 & al. (TNS) Ito Y. P. praelongus in Zhang et al. P. * A.Benn. L. L. L. sp. L. Miq. Miq. Cham. & Taxon L. Originally named as * Schlecht. P. octandrus Poir. P. P. octandrus Poir. P. P. octandrus Poir. P. oxyphyllus P. P. oxyphyllus P. P. perfoliatus P. P. perfoliatus P. P. perfoliatus P. P. perfoliatus P. P. praelongus ” “ P. P. pulcher Tuckerm. P. pusillus L. P. P. pusillus L. P. P. pusillus L. P. P. pusillus L. P. P. pusillus L. P. P. pusillus L. P. var. pusillus pusillus var. P. Rydb. richardsonii P. Rydb. richardsonii P. Oakes robbinsii P. P. spirillus Tuckerm. P. P. strictifolius P. P. subsibiricus Hagstr. P. P. tennesseensis Fernald P. trichoides P. Robbins vaseyi J. W. P. wrightii P. P. zosteriformis Fern. P. Potamogeton