American Journal of Botany 97(7): 1156–1167. 2010.

HYBRIDIZATION AND POLYPLOIDY OF AN AQUATIC PLANT,

RUPPIA (RUPPIACEAE), INFERRED FROM PLASTID AND
NUCLEAR DNA PHYLOGENIES1

Yu Ito2, Tetsuo Ohi-Toma2, Jin Murata2, and Norio Tanaka3,4

2Botanical Gardens, Graduate School of Science, The University of Tokyo, Tokyo, 112-0001, Japan; and 3Tsukuba Botanical
Garden, National Museum of Nature and Science, Tsukuba, 305-0005, Japan

• Premise of the study: The monogeneric family Ruppiaceae is found primarily in brackish water and is widely distributed on all
continents, many islands, and from subartic to tropical zones. Ruppia taxonomy has been confusing because of its simplified
morphology yet high phenotypic plasticity and the existence of polyploidy and putative hybrids. This study addresses the cur-
rent classification of species in the genus, the origin of putative hybrids and polyploids, and the distribution of Ruppia
species.
• Methods: Separate molecular phylogenetic analyses using plastid DNA and nuclear-encoded PHYB data sets were performed
after chromosome observations.
• Key results: The resultant trees were largely congruent between genomes, but were incongruent in two respects: the first incon-
gruence may be caused by long outgroup branches and their effect on ingroup rooting, and the second is caused by the existence
of heterogeneous PHYB sequences for several accessions that may reflect several independent hybridization events. Several
morphological species recognized in previous taxonomic revisions appear paraphyletic in plastid DNA and PHYB trees.
• Conclusions: Given the molecular phylogenies, and considering chromosome number and morphology, three species and one
species complex comprising six lineages were discerned. A putative allotriploid, an allotetraploid, and a lineage of hybrid ori-
gin were identified within the species complex, and a hybrid was found outside the species complex, and their respective puta-
tive parental taxa were inferred. With respect to biogeography, a remarkably discontinuous distribution was identified in two
cases, for which bird-mediated seed dispersal may be a reasonable explanation.

Key words: hybridization; matK; PHYB; phylogeny; polyploidy; rbcL; rpoB; rpoC1; Ruppia; Ruppiaceae.

About 1–2% of all vascular plant species (87 families: Cook, known that many genera and species that are specialized in this
1996) have adapted morphologically and physiologically to way are distributed worldwide (e.g., Najas marina L., Pota-
aquatic environments. Aquatic plants are usually categorized mogeton pectinatus L., Ruppia maritima L., and Zannichellia
into emergent, floating, or submerged types (Sculthorpe, palustris L.: Sculthorpe, 1967; Les et al., 2003). Sculthorpe
1967). In addition, aquatic plants may be adapted to freshwa- (1967) suggested that dispersal of propagules (seeds, turions,
ter, saltwater, or brackish water habitats. Because fresh and and tubers) by water birds may contribute to such wide geo-
brackish water areas are restricted to lakes, rivers, and estuar- graphic distributions.
ies, populations of species adapted to these environments are The genus Ruppia, the only genus of the family Ruppiaceae,
highly geographically isolated. Nevertheless, it has long been is representative of submerged aquatic plants adapted to brackish
waters. The genus is generally limited to estuaries and brackish
lakes as well as inland saline and alkaline lakes (Verhoeven,
1 Manuscript received 14 June 2009; revision accepted 19 May 2010. 1979). Unlike most aquatic plants in fresh or brackish water,
The authors thank H. Freitag, P. García-Murillo, J. Hansen, K. Nonaka, the genus often occurs on oceanic islands, including Hawaii
H. Taneda, H. Ohba, H. Kato, H. R. Na, D. Perleberg, H. J. Cho, L. Rozas, (St. John and Fosberg, 1939), Vanuatu (Hashimoto et al., 2002),
R. Upson, A. R. Ramirez, D. Bryon, and J. J. Orth for providing materials; and Ogasawara (Ono and Okutomi, 1985). As an example of an
S. W. L. Jacobs, A. Skriptsova, H.-K. Choi, Y.-P. Yang, S. R. Yadav, K. Povidisa,
M. Delefosse, T. Hammer, K. Torn, G. Izzo, R. Ishikawa, and S. Stern for
extreme case, R. maritima occurs from subarctic to tropical
help with field research; K. Sasamura for technical advice on chromosome zones in both hemispheres (Den Hartog, 1971). The fruit of R.
observation; and S. Gale, S. Stern, and T. Hammer for improving an earlier maritima is recognized as an important element in the diet of
draft of the manuscript and two anonymous reviewers for valuable comments several species of water birds, and the germination rate of def-
on the manuscript. This study is based in part on the thesis of Y.I. at the ecated seeds is enhanced (Figuerola and Green, 2004). Hence,
University of Tokyo, Japan. The authors are thankful for grants to N.T. it is reasonable to assume that the widespread distribution of
from the Ministry of Education, Culture, Sports, Science, and Technology, R. maritima was caused and is maintained by bird-mediated
Japan (17710194 and 21710248) and from a study project of the National seed dispersal.
Museum of Nature and Science: Integration of Systematics and Molecular Ruppia species are characterized by a simplified morphol-
Phylogenetics of All Groups of Organisms and to Y.I. from the Academic
Research Grant Program (International), The University of Tokyo, Japan
ogy of a slender body, linear leaves, and a pedunculate, spicate
and Global COE Program (Integrative Life Science Based on the Study of inflorescence with a pair of bractless, perianthless flowers, of
Biosignaling Mechanisms), MEXT, Japan. which the peduncle and leaf morphologies have been mainly
4 Author for correspondence (e-mail: ntanaka@kahaku.go.jp) used for taxonomy. These morphological characters often
show high phenotypic plasticity among taxa, among populations
doi:10.3732/ajb.0900168 within a taxon, and even within populations, often leading to

American Journal of Botany 97(7): 1156–1167, 2010; http://www.amjbot.org/ © 2010 Botanical Society of America
1156
July 2010] Ito et al.—Phylogeny of RUPPIA 1157

taxonomic confusion (Van Vierssen et al., 1981; Hara, 1983; et al. [2008]; Bryonia: Volz and Renner [2008]; Arabidopsis:
Aedo and Fernandez Casado, 1988). Nevertheless, several re- Shimizu-Inatsugi et al. [2009]).
gional taxa have been described based on a comparison of lim- To address the taxonomic inconsistencies in Ruppia, we ex-
ited samples from areas such as Asia (Hara, 1983), the Pacific amined the phylogenetic relationship of Ruppia species within
region (St. John and Fosberg, 1939), North America (Fernald the context of the recent classification proposed by Zhao and
and Wiegand, 1914), and Europe (Triest and Symoens, 1991). Wu (2008) and investigated the role of hybridization and poly-
Van Vierssen et al. (1981) suggested that an adequate comparison ploidization in the evolution of this genus. We performed mo-
using plant material from around the world would be needed to lecular phylogenetic analyses of the genus covering a broad
derive a more robust classification of Ruppia. Recently, Zhao taxonomic sample and representing a wide geographic range. In
and Wu (2008) reviewed the classification of Ruppia and rec- addition, chromosome numbers were determined for certain
ognized five species: R. maritima, R. cirrhosa (Petagna) representatives, and the morphology was reevaluated in light of
Grande, R. megacarpa R. Mason, R. polycarpa R. Mason, and the phylogenetic data. We used these new lines of evidence to
R. tuberosa J. S. Davis & Toml. (Table 1). Of these, R. marit- address the present classification of species in the genus, the
ima and R. cirrhosa were treated as widespread species, origin of putative hybrids and polyploids, and the distribution
whereas the other three were deemed endemic to Oceania. pattern of Ruppia species.
Most of the regional taxa in this classification were treated as
synonyms of the two widespread species (e.g., R. rostellata as
a synonym of R. maritima and R. drepanensis and R. spiralis MATERIALS AND METHODS
as synonyms of R. cirrhosa). However, the taxonomic criteria
of Zhao and Wu (2008) are not always applicable in identify- Taxon sampling—In total, 46 accessions from 44 localities of Ruppia spe-
ing herbarium collections and live specimens (Y. Ito and cies were collected from Asia, Europe, North Africa, North America, Oceania,
Pacific, and South America (Appendix 1). One widespread species, R. marit-
N. Tanaka, personal observation). In addition, morphological ima, was also reported in coastal areas of Africa (Verhoeven, 1979), but no
intermediates with sterile flowers have been reported in local high-quality specimens that would have allowed DNA extraction and analysis
populations (Kadono, 1994), suggesting that intragenus hy- were available. Using only the morphological key of Zhao and Wu (2008), each
bridization may have occurred, further complicating its taxo- sample was tentatively identified as one of five species. Leaf blade width, which
nomic classification. was recognized as a valuable character for distinguishing between R. cirrhosa,
Polyploidy occurs widely in plants, and polyploidization (al- R. megacarpa, and R. polycarpa by Zhao and Wu (2008), was excluded be-
cause of its continuous variation across the remainder of the accessions. In ad-
lopolyplodization) following hybridization plays an important dition, chromosome numbers noted by Zhao and Wu (2008) were not considered
role in speciation (Arnold, 1997; Soltis and Soltis, 2009). In for species assignment because they only cited previous studies, some of which
Ruppia, diploid (2n = 20) and tetraploid (2n = 40) races have were incorrect, (e.g., 2n = 10 for R. maritima; Table 1). Two accessions of R.
been reported from several regions of the world, whereas trip- polycarpa from Asia and North America and one accession of R. megacarpa
loids (2n = 30) and hexaploids (2n = 60) are only occasionally from Asia were identified, although both species were treated as endemic to
found (reviewed in Talavera et al., 1993; Table 1). In past clas- Oceania by Zhao and Wu (2008). Positive identification of accessions collected
without fruit was problematic for all taxa, except R. tuberosa, and these acces-
sifications, some researchers have recognized the two wide- sions were treated as Ruppia A to C. Potamogeton maackianus A. Benn. (Pota-
spread species, R. maritima and R. cirrhosa, as diploid and mogetonaceae) and Syringodium isoetifolium (Asch.) Dandy (Cymodoceaceae)
polyploid, respectively (Reese, 1962). However, others have were used as outgroups following the phylogeny of the core alismatid families
recognized the former as both diploid and tetraploid and the presented by Les et al. (1997).
latter as tetraploid (Van Vierssen et al., 1981). Still others have
described the former as tetraploid and the latter as diploid DNA extraction, amplification, and sequencing—Total genomic DNA
(Cirujano, 1986), or recognized diploid and tetraploid entities was extracted from silica gel-dried leaf tissue using the method of Doyle
in both species (Triest and Symoens, 1991). This confusion and Doyle (1987) after pretreatment with HEPES buffer (pH 8.0) (Setoguchi
and Ohba, 1995). Sequences determined in the current study were regis-
may have been caused by the paucity of material available for tered with the DNA Data Bank of Japan (DDBJ), which is linked to
study, reflecting the ambiguity of classification for the genus GenBank, and their accession numbers are given with the sample informa-
based on morphology alone. As such, a molecular phylogenetic tion in Appendix 1.
analysis based on a combined data set of plastid and nuclear Fragments of the plastid genes matK, rbcL, rpoB, and rpoC1 (collectively re-
DNA (ptDNA and nDNA) corresponding to uniparental and bi- ferred to as ptDNA) were amplified by polymerase chain reaction (PCR) using
parental inheritance would be particularly useful in elucidating the following primer pairs. The primers for rbcL, RM_F (5′-TATTTGCA-
AGGGAATTAGGA-3′) and RM_R (5′-AAGCTTCACGGATAATTTCA-3′),
the existence of hybrids and the origin of polyploids (e.g., Doyle which amplify fragments of the gene (542 bp), were newly designed based on the
and Doyle, 1999), as well as in differentiating taxa and their nucleotide sequence of R. maritima (accession number U03729) from GenBank.
phylogenetic relationships. Furthermore, low- or single-copy For other genes, published or modified primers were used: RM_749F (5′-TTGA-
genes have been used as nuclear markers for many plant groups GCGAACACATTTCTATG-3′) modified from Whitten et al. (2000) and 8R
because they are less prone to mistaken orthological assessment (Ooi et al., 1995) for the matK gene (550 bp), 2f and 4r for rpoB (508 bp), and 1f
than those in large gene families and have been used for many and 3r for rpoC1 (508 bp) (Royal Botanic Gardens, Kew: http://www.rbgkew.
org.uk/barcoding/update.html). PCR amplification was conducted using TaKaRa
plant groups (e.g., Madia/Raillardiopsis group: Barrier et al. Ex Taq polymerase (TaKaRa Bio, Shiga, Japan), and PCR cycling conditions
[1999]; Glycine tomentella polyploidy complex: Doyle et al. were 94°C for 60 s; then 30 cycles of 94°C for 45 s, 52°C for 30 s, 72°C for 60 s;
[2002]; Spartina: Ainouche et al. [2004]; Eragrostis tef: Ingram and finally 72°C for 5 min. The PCR products were digested using ExoSAP-IT
and Doyle [2003]; Geinae: Smedmark et al. [2003]; Paeonia: (GE HealthCare, Piscataway, New Jersey, USA) and amplified using the ABI
Sang et al. [2004]; Viburnum: Winkworth and Donoghue PRISM Big Dye Terminator v3.1 (Applied Biosystems, Foster City, California,
[2004]; Vandenboschia radicans complex: Ebihara et al. [2005]; USA) with the same primers as those used for PCR. DNA sequencing was per-
formed using an ABI PRISM 377 DNA Sequencer (Applied Biosystems). Com-
Silene: Popp et al. [2005]; Rosa carolina complex: Joly et al. plementary electropherograms were compared by eye using the software
[2006]; Cardamine: Lihová et al. [2006]; Capsella: Slotte et al. Genetyx-Win Version 3 (Software Development Co., Tokyo, Japan).
[2006]; Paniceae: Doust et al. [2007]; Spartina: Fortune The PHYB gene (a distinct member of the phytochrome gene family) was
et al. [2007]; Brumus: Fortune et al. [2008]; Persicaria: Kim selected as a nuclear marker, based on its phylogenetic utility as a single
 1158

Table 1. (A) Past classifications with emphasis on chromosome numbers and (B) taxa proposed in the present study in Ruppia and morphological comparison of taxa proposed in the
present study.

A) Past classification Taxa (Chromosome numbers [2n])

Reese (1962) R. spiralis (40, 60) R. maritima var. brevirostris (20)
Van Vierrsen et al. (1981) R. cirrhosa (40) R. maritima var. maritima (20, 40)
R. maritima var. brevirostris (40)
Jacobs & Brock (1982) — R. maritima (20) R. polycarpa (18, 20) R. megacarpa (20) R. tuberosa (20, 30)
Cirujano (1986) R. drepanensis (20) R. maritima (40)
Triest & R. cirrhosa var. cirrhosa (40) R. maritima var. maritima (20, 40)
Symoens (1991) R. cirrhosa var. drepanensis (20) R. maritima var. brevirostris (20, 40)
Zhao & Wu (2008) R. cirrhosa (40) R. maritima (10) R. polycarpa (18, 20) R. megacarpa (20) R. tuberosa (n.a.)

B) Present study Proposed taxa

R. maritima complex R. polycarpa R. megacarpa Hybrid R. tuberosa
“Tetraploid” “Diploid” “Triploid” “Filifolia” “Utahian” “Occidentalis”
Characters
Chromosome numbers (2n) 40 20 30 unk. unk. 20 18 20 unk. 20
Distribution Eur./Asia/ Eur./Asia/ Asia S. Am. Inl. Am. Asia/Inl. Am. Ocea. Asia/Ocea. Asia Ocea.
Ocea. Paci./N. Am.
Habitats Brackish/ Brackish Brackish Brackish Saline Brackish/ Brackish Brackish Brackish Hypersaline
American Journal of Botany

fresh Fresh alkaline

Turions Absent Absent Absent Absent Absent Absent Absent Absent Absent Present
Leaf length Short Short Short Short Short Long Short Long Long Short
Leaf tips Sharp Sharp Sharp Sharp Sharp Sharp Sharp Flat Flat Sharp
Peduncles Short/curved Short/curved Intermediate Short/curved Short/curved Long/coiled Long/coiled Long/coiled n.a. Long/coiled
Stalks Present Present n.a. Present Present Present Present Present n.a. Absent
Fruits Small Small n.a. Small Small Small Small Large n.a. Small
Carpels Four Four Four Four Four Eight Eight Four Four Eight

Abbreviations: Eur., Europe plus North Africa; Inl. Am., Inland North America; Ocea., Oceania; Paci., Pacific; N. Am., North America; S. Am., South America; n.a., not applicable; unk., unknown
[Vol. 97
July 2010] Ito et al.—Phylogeny of RUPPIA 1159

or low copy nuclear locus (Mathews et al., 2000; Simmons et al., 2001). Data analysis—Multiple sequences of each gene were manually aligned
Fragments of a part of exon 1 of PHYB were initially amplified by PCR using because no length mutation was detected. Phylogenetic analyses were indepen-
the published primers of B-up and B-down (Mathews et al., 2000) for 12 dently performed for ptDNA and PHYB data sets because we detected some
representative samples. The PCR cycling conditions were 94°C for 90 s; well-supported incongruence between these genomes (see below). The incon-
then 35 cycles of 94°C for 45 s, 60°C for 30 s, 72°C for 90 s; and finally 72°C gruence length difference test (Farris et al., 1994) was conducted to test congru-
for 10 min. The fragments obtained were digested with ExoSAP-IT and di- ence between four ptDNA genes using a partition homogeneity test with 1000
rectly sequenced. After checking the homology of the obtained sequences by replicates in the program PAUP* 4.0b10 (Swofford, 2002). There was no sig-
conducting a BLAST search (National Center for Biotechnology Information), nificant heterogeneity indicated by this test (P value > 0.05 for all six pairs), and
three specific forward and reverse primers for Ruppia were newly designed, so the four ptDNA data sets were combined. One representative sequence was
as follows: phyB_38F (5′-CTCGCTGTTCGTGCTATCTCG-3′), phyB_ used for accessions having the identical combined sequence.
ruppiaF (5′-CCATACTTCTCCCAGATGCATTCC-3′) and phyB_ruppiaR Phylogenetic inference was performed using maximum parsimony (MP)
(5′-CCATACTTCTCCCAGATGCATTCC-3′). For PCR amplification of all and maximum likelihood (ML) in PAUP*, as well as Bayesian inference (BI)
materials, either phyB_38F or phyB_ruppiaF was used together with B- in the program MrBayes 3.1.2 (Ronquist and Huelsenbeck, 2003). In the MP
down to obtain 562 and 1050 bp fragments, respectively, under the follow- analysis, a heuristic search was performed with 100 random addition replicates
ing condition: 94°C for 60 s; then 25 cycles of 94°C for 45 s, 60°C for 60 s, involving tree-bisection-reconnection (TBR) branch swapping, with the Mul-
72°C for 90 s; and finally 72°C for 5 min. The PCR products were purified Trees option in effect. The MaxTrees option was set at no limits for the
using GeneClean (BIO 101, Carlsbad, California, USA). On direct sequenc- analysis. Bootstrap analyses (Felsenstein, 1985) were performed using 1000
ing of 22 accessions, overlapping double peaks were found at the same sites replicates with TBR branch swapping and the simple addition sequences. In the ML
for complementary strands in the electropherograms. These products were analysis, heuristic searches were performed with 10 random addition replicates
cloned using a TOPO TA Cloning kit for Sequencing (Invitrogen, Carlsbad, using a best-fit model (TVM+G for PtDNA and K80+G for PHYB). Parameter
California, USA). At least 16 clones per sample were chosen and their values were estimated by a hierarchical likelihood ratio test in the program
sequences were determined using the same procedure as that used in the Modeltest 3.7 (Posada and Crandall, 1998). Bootstrap analyses were performed
first PCR followed by direct sequencing. For the cloned sequences, nucle- using 100 replicates with TBR branch swapping and as-is addition sequences
otides that were not detected by direct sequencing were regarded as PCR under the same ML models. In the BI analysis, hierarchical likelihood ratio
errors. tests implemented in the program MrModeltest 3.7 (Nylander, 2002) were used

Fig. 1. (A) One of two most parsimonious trees of combined plastid DNA (matK, rbcL, rpoB, and rpoC1) gene sequences. (B) One of four most par-
simonious trees of nuclear encoded PHYB sequences. Accessions were tentatively identified by morphology following the taxonomic criteria of Zhao and
Wu (2008). DELTRAN optimization was used for branch length measures of the phylograms. Numbers above the branches indicate bootstrap support (BP)
calculated in maximum parsimony (bold) and maximum likelihood (italic) analyses and those below indicate Bayesian prior probabilities (PP). BP <70 and
PP <0.9 are indicated by asterisks. Boldfaced accessions have heterogeneous PHYB sequences; for these, sequence pairs are connected by a dotted line and
named #1 and #2, respectively. Note that some accessions in each tree represent multiple identical accessions.
1160 American Journal of Botany [Vol. 97

for substitution model selection (GTR+G for ptDNA and K80+G for PHYB). PB-II (which includes identical sequences derived from R.
The Bayesian Markov chain Monte Carlo algorithm was run for 110 000 gen- megacarpa from northern Japan and Australia, Ruppia A from
erations in the ptDNA analysis and for 200 000 generations in the PHYB analy-
sis until the average standard deviation of split frequency was below 0.01, with
northern Japan and Ruppia B from Russian Far East) is the sister
four incrementally heated chains starting from random trees and sampling one group of the remaining taxa, with a single accession of
out of every 100 generations. Of the generations, the first 25% of trees (27 500 R. polycarpa A from Australia (branch PB-III), the next suc-
and 50 000 generations for each data set, respectively) were discarded as cessive sister group. The remaining accessions are divided into
burn-in, and the remaining trees were used to calculate a 50% majority-rule three major lineages: (1) PB-IV, comprising two weakly to
consensus tree and to determine posterior probabilities for branches. The data moderately supported subclades: PB-IV-1 (57%, 56%, and 0.68
matrices and the MP and ML trees are available from the TreeBASE database
(http://www.treebase.org, study accession S10485).
for MP, ML bootstrap supports, and Bayesian posterior proba-
bility) and PB-IV-2 (83%, 84%, and 1.00), (2) clade PB-V, with
Chromosome observations—Somatic chromosome number of a subset of
two sequence types, and (3) PB-VI with a single accession. The
samples from 15 localities was obtained by light microscopic examination. latter two lineages (clade PB-V and branch PB-VI) are weakly
Root tips collected in the field were pretreated with 0.002 M 8-hydroxyquino- supported as sister taxa (63%, 65%, and 0.86). Ruppia A from
line at 4°C overnight, and fixed with freshly mixed Carnoy’s fixative (3 : 1 ethyl northern Japan had two sequences, one each belonging to
alcohol : acetic acid) for at least 30 min, and then preserved at 4°C. For micro- branch PB-II and clade PB-IV, respectively; the latter sequence
scopic observation, root tips were soaked in 1 N HCl for 1 h followed by 10 min was identical to that of R. polycarpa B from northern Japan and
at 60°C. After being immersed in tap water, the materials were stained in a drop
of 1.5% orcein acetate solution on a slide glass, and then squashed.
inland North America. Similarly, R. cirrhosa from Europe,
R. maritima E and F from Europe, North Africa, Asia, and
South America, and Ruppia C from northern Japan each com-
RESULTS prised two sequences that fell in distinct positions on the tree
(i.e., clades PB-IV and PB-V, or PB-IV and PB-VI).
Phylogenetic inferences based on ptDNA— Of all recovered
accessions, those with identical sequence are represented by a Chromosome numbers— Four cytotypes, 2n = 18, 20, 30,
single name as operational taxonomic units (OTUs), e.g., R. and 40, were observed across the 15 samples (Fig. 2). Based on
maritima A to G (Fig. 1A). Based on the combined data set these observations and those of previous studies (reviewed in
consisting of the four genes (2108 bp), two MP trees (tree length = Talavera et al., 1993), the basic chromosome number of Ruppia
279 steps, consistency index = 0.9498, retention index = 0.8955) is predicted to be x = 10. The lowest number, 2n = 18 observed
and one ML tree with -lnL = 4434.9927 were obtained. We in only R. polycarpa A from Australia is likely to be of aneu-
refer to single-accession lineages, or clades comprising identical ploid origin. Diploids (2n = 20) were observed from Asian,
sequences as “branches” here, and label clades of interest Australian, and North American material. Only one examined
with the prefix “Pt” (for plastid-based lineages) or “PB” (for sample of Ruppia C from northern Japan, was triploid (2n =
PHYB-based lineages). Because MP, ML, and BI analyses recov- 30). Six accessions from Asia and Europe were found to be
ered largely congruent topologies, one of the two MP trees is tetraploids (2n = 40) (Appendix 1).
shown (Fig. 1A). In the ptDNA tree, the branch Pt-I (comprising
R. megacarpa from northern Japan and Australia, Ruppia A from
northern Japan, and Ruppia B from Russian Far East) is the sister DISCUSSION
group of everything else. Two distinct haplotypes of R. tuberosa
form a well-supported clade (clade Pt-II) that is the sister group Comparison of plastid and PHYB phylogenetic infer-
of the remaining accessions. In one of the two MP trees, branch ences— The separate molecular phylogenetic analyses of Rup-
Pt-III (R. polycarpa A from Australia) is the sister group of a pia based on ptDNA and PHYB sequence data sets provide
large clade with a basal polytomy: the latter clade includes one valuable new insights. Of these, topological incongruencies be-
branch (Pt-VI) and two well-supported clades (Pt-IV and Pt-V). tween the ptDNA and PHYB trees are especially notable. One
Clade Pt-IV consists of subclades Pt-IV-1 (which includes ac- topological incongruence involves distinctly placing PHYB se-
cessions of R. maritima A from North America, R. polycarpa quences from individual accessions (clade PB-IV, that includes
B from northern Japan and inland North America, and Ruppia several accessions whose plastid sequences belong to branches
C from northern Japan) and subclade Pt-IV-2 (which includes Pt-I and Pt-VI, and clades Pt-IV and Pt-V). Similar topological
R. maritima B to D from Asia, Europe North America, and incongruences between ptDNA and nDNA trees have been ob-
Pacific). Clade Pt-V consists of R. cirrhosa from Europe and served elsewhere and have been inferred to represent the ef-
R. maritima E and F from Asia and Europe; branch Pt-VI is the fects of hybridization and/or polyploidization (e.g., Winkworth
accession of R. maritima G from the Falkland Islands (South and Donoghue, 2004; Obbard et al., 2006; Ohi-Toma et al.,
America). 2006; Kim et al., 2008). The details of the inferred hybridiza-
tion and polyploidization events in Ruppia are discussed later.
Phylogenetic inferences based on PHYB— Of 45 Ruppia The alternative scenario, though it seems less likely, is the pos-
accessions, 22 have two different sequences (the remainders sibility of gene losses following the duplication of the PHYB
have one). In the phylogenetic analyses, four MP trees (tree locus, which occurs singly in genomes of grasses (Mathews
length = 516 steps, consistency index = 0.7946, retention index = and Sharrock, 1996), except in maize (Childs et al., 1997; Sheehan
0.8188) and one ML tree with -lnL = 4001.3640 were obtained. et al., 2004). If this is the case, however, the resultant tree may
One of the four MP trees that has the same branching pattern as be expected to have two clear lineages, as is the case of GB-
those recovered from the ML and BI analyses is shown (Fig. SSI-1 and -2 in family Rosaceae (Evans et al., 2000), Adh1 and
1B). The names of individual accessions correspond to those in Adh2 in Paeonia (Sang et al., 2004), and WaxyA and WaxyB in
Fig. 1A. In the PHYB tree, the clade PB-I (which contains R. Spartina (Fortune et al., 2008). That is apparently not the case
tuberosa A and B, each of which yielded two distinct sequence in the present study; the plastid and nuclear trees are otherwise
types) shows evidence of moderate rate elevation. The branch mostly congruent.
July 2010] Ito et al.—Phylogeny of RUPPIA 1161

Fig. 2. Somatic chromosomes in representatives of Ruppia. (A) 2n = 18 (Australia). (B) 2n = 20 (China). (C) 2n = 30 (Japan). (D) 2n = 40 (Japan).
Bar indicates 10 μm.

Another instance of topological incongruence was detected Systematics of Ruppia— The phylogenetic relationships in-
concerning the earliest evolutionary splits in the genus. Such ferred from molecular data of ptDNA and PHYB were com-
incongruence between gene trees may have a number of bio- pared to the classification of Zhao and Wu (2008) (summarized
logical or methodological causes, e.g., mistaken orthology of in Fig. 3). In the classification, only R. tuberosa corresponds to
duplicated genes, plastid capture via hybridization, and incom- the circumscription indicated by clades Pt-II and PB-I in the
plete lineage sorting (Wendel and Doyle, 1998; Sang, 2002). molecular phylogenies. This species is morphologically unique
Although members of the branches, clades, and subclades de- in possessing sessile fruits and swollen turions at the end of its
tected in each of the ptDNA and PHYB trees mostly correspond shoots, and it is ecologically unusual because it occurs in hyper-
(R. megacarpa and Ruppia B in Pt-I and PB-II, Pt-II and PB-I, saline lakes in Australia (Davis and Tomlinson, 1974; Jacobs
Pt-III and PB-III, R. maritima A and R. polycarpa B in Pt-IV-1 and Brock, 1982). Thus, R. tuberosa is phylogenetically as well
and PB-IV-1, R. maritima B to D in Pt-IV-2 and PB-IV-2, R. as morphologically and ecologically distinct (Table 1). Al-
cirrhosa and R. maritima E and F in Pt-V and PB-V, and Pt-VI though diploid (2n = 20) and triploid (2n = 30) individuals have
and PB-VI), the branching patterns at the basal position in both been reported for the species (Snoeijs and Van Der Ster, 1983),
trees clearly differed. In the ptDNA tree, the branch Pt-I defines only diploids (2n = 20) were confirmed in the current study.
the root of the tree, and the clade Pt-II was at the next. In con- Other species in Zhao and Wu (2008) were not supported by
trast, clade PB-I in the PHYB tree (which corresponds to clade ptDNA and PHYB branches, clades, or subclades. Given these
Pt-II) defines the root of Ruppiaceae. Given that use of a dis- molecular phylogenies, chromosome numbers, and reevaluated
tantly related outgroup can affect the root of an ingroup (e.g., morphological characters, we discuss species recognition within
Graham et al., 2002; Shavit et al., 2007; Graham and Iles, 2009), Ruppia and propose a revised classification (Fig. 3, Table 1).
this may be a function of long outgroup branches being attracted The pair of branches Pt-I and PB-II includes R. megacarpa
to relatively long branches within the ingroup, a possible in- from northern Japan and Australia, Ruppia A from northern Ja-
stance of long-branch attraction (Felsenstein, 1978). pan, and Ruppia B from Russian Far East (Fig. 3). Of these,
1162 American Journal of Botany [Vol. 97

Fig. 3. Taxonomic grouping proposed in this study based on the trees presented in Fig. 1. The root positions are shown as arrowheads (outgroups
trimmed for clarity). Clade PB-I is modified to fit clade Pt-II. The three well-defined Ruppia species are represented by closed bars, and the six entities of
the R. maritima complex are represented by shaded bars. Four cases of putative hybrids/polyploids are represented by open or open-shaded bars. The col-
lecting area of each accession is given as Asia, Euro, Inl. Am, N. Am, Ocea, Paci, and S. Am (respectively referring to Asia, Europe plus North Africa, in-
land North America, North America, Oceania, Pacific, and South America). Boldfaced accessions have heterogeneous PHYB sequences. Dotted lines
connect corresponding branches, clades, and subclades between plastid DNA and PHYB trees. Less-supported branches are shown as dotted lines. Chromo-
some counts are given in brackets beside accessions (uncounted accessions are shown as asterisks). Note that some accessions in each tree represent mul-
tiple identical accessions.

Ruppia A also harbored a second, distantly related PHYB se- sequence divergence in ptDNA and PHYB among accessions
quence in the subclade PB-IV-1. Because the chromosome from northern East Asia and southern Oceania, geographical
numbers of some accessions of R. megacarpa were diploids isolation in both hemispheres in R. megacarpa may have oc-
with 2n = 20, R. megacarpa and Ruppia B are considered to be curred recently, possibly as a result of seed dispersal by birds.
diploid R. megacarpa, with larger fruits (if any) and flat leaf Accessions that key to R. polycarpa according to Zhao and
tips (Table 1), and Ruppia A is probably of hybrid origin (dis- Wu (2008) occur at two different positions in the ptDNA and
cussed later). With the exception of whether fruits are actually PHYB trees (Fig. 3): R. polycarpa A from Australia in branches
set, there are no differences in morphology between R. mega- Pt-III and PB-III, and R. polycarpa B from northern Japan and
carpa and the hybrid (Table 1). Although R. megacarpa was inland North America in subclades Pt-IV-1 and PB-IV-1. Zhao
formerly recognized as a species endemic to Oceania (Mason, and Wu (2008) recognized R. polycarpa based on 4–16 (usually
1967), R. megacarpa as described here exhibits a largely dis- 6–8) carpels and coiled, long peduncles, following the conclu-
continuous distribution in northern East Asia and southern Oce- sions of Jacobs and Brock (1982), but they did not mention leaf
ania (Fig. 4, Table 1). No R. megacarpa-like herbarium specimens characteristics. The accession of R. polycarpa A has short leaves
have been observed from any of the areas between these two (up to 150 mm) and leaf sheaths (ca. 10 mm), whereas the acces-
distribution centers. Although there are no sufficiently old fossil sion of R. polycarpa B has conspicuously longer leaves (up to
records of Ruppia, except those from the Pliocene (Zhao et al., 220 mm) and leaf sheaths (ca. 65 mm). The latter is similar to R.
2004), given the divergence time of the basal lineage of Rup- occidentalis Watson with 4–9 carpels and long leaf sheaths (14–
piaceae (ca. 65 Myr: Janssen and Bremer, 2004) and the lack of 57 mm) and has often been recognized in inland alkaline lakes
July 2010] Ito et al.—Phylogeny of RUPPIA 1163

Fig. 4. Geographic distribution of Ruppia based on the accessions used in the current study. Hybrid: black; R. megacarpa: blue; R. tuberosa: yellow;
R. polycarpa: red; R. maritima complex: purple.

in North America (Kaul, 1992). Hence, at least R. polycarpa A maritima E to G have two distantly related PHYB sequences
from Australia with its unique phylogenetic position and mor- (Fig. 3). Given their respective chromosome numbers, R. marit-
phological distinctiveness should be treated as R. polycarpa, ima B to D, Ruppia C, and R. cirrhosa plus R. maritima E and F
and R. polycarpa B should be treated separately along with its are defined as “Diploid”, “Triploid”, and “Tetraploid” entities,
related accessions (see later). With respect to chromosome num- respectively, and the second PHYB sequences could possibly be
bers, the accession of R. polycarpa A from Australia is 2n = 18, the result of polyploidization (see below). Consequently, the
which is the same as one of two cytotypes reported (2n = 18 and “Tetraploid” entity includes both R. cirrhosa and R. maritima,
2n = 20) for R. polycarpa (Mason, 1967; Brock, 1982). indicating a large variation in peduncle length. Ruppia maritima
Ruppia maritima identified by its noncoiled or few-coiled G, whose cytotype is unknown, is defined here as the “Filifolia”
short peduncles (<50 mm) was divided among four lineages in entity, considering the taxonomic history of Ruppia in South
the ptDNA and PHYB trees together with Ruppia C, R. cir- America, where R. filifolia was recognized by Skottsberg (1916)
rhosa, and R. polycarpa B (Fig. 3): (1) R. maritima A from in- on the basis of its many-branched filiform stems. Ruppia filifolia
land North America in subclades Pt-IV-1 and PB-IV-1, includes plant materials from the Falkland Islands (Moore,
including R. polycarpa B from northern Japan and inland North 1973); its second PHYB sequence appears to be of hybrid origin
America and Ruppia C from northern Japan; (2) R. maritima B (see below). From an ecological viewpoint, R. maritima A,
to D from Asia, Europe, North America, and Pacific in sub- whose cytotype is unknown, is characterized by its special habi-
clades Pt-IV-2 and PB-IV-2; (3) R. maritima E and F from Asia, tat, namely an inland salt lake in Utah, USA, and so we refer to
Australia, and Europe in clades Pt-V and PB-V and subclade it as the “Utahian” entity. Most other accessions of R. maritima
PB-IV-2 including R. cirrhosa from Europe with long pedun- inhabit brackish water areas along coastlines. As mentioned, R.
cles (ca. 160 mm); and (4) R. maritima G from South America polycarpa B seems to correspond morphologically to R. occiden-
in branches Pt-VI and PB-VI and subclade PB-IV-1. The acces- talis and is therefore referred to as the diploid “Occidentalis”
sions of R. maritima in four lineages could not be distinguished entity with 2n = 20 (Table 1). On the basis of past studies and
by any morphological characters; thus, accessions in clade herbarium specimens, the “Occidentalis” entity is distributed
Pt-IV to branch Pt-VI and clade PB-IV to branch PB-VI, except continuously from inland North America to Alaska, the Kuril
those of Ruppia A, are treated as part of the R. maritima com- Islands (Takahashi and Kuwahara, 1998), Sakhalin, and Hokkaido,
plex here. Japan (Fig. 4). Consequently, we provisionally name “Diploid”,
In the complex, R. maritima A to D and R. polycarpa B have “Triploid”, “Tetraploid”, “Filifolia”, “Utahian”, and “Occidentalis”
one PHYB sequence, whereas Ruppia C, R. cirrhosa, and R. entities within the broader R. maritima complex.
1164 American Journal of Botany [Vol. 97

Hybridization and polyploidization— As a number of hy- were observed during the fruiting season of Japanese Ruppia,
bridization and polyploidization events have been identified for but many small individuals with rhizomes were observed in the
aquatic monocots (Les and Philbrick, 1993), in Ruppia, four spring (Y. Ito and T. Ohi-Toma, personal observation). The ac-
cases of putative hybridization, including polyploidization (at cession might be a triploid hybrid that reproduces vegetatively
the triploid and tetraploid levels), were inferred from the ptDNA as in triploid hybrids of many other plant groups (e.g., Takano
and PHYB trees. We infer these events based on several acces- and Okada, 2002; Ayres et al., 2008; Gresta et al., 2008). The
sions that harbored heterogeneous sequences, which assigned “Triploid” entity shared the same ptDNA sequence with the
to two different positions in the PHYB phylogeny (Fig. 3): (1) “Occidentalis” entity and harbored the same two PHYB se-
Ruppia A with PHYB sequences in the branch PB-II and the quences as the “Tetraploid” entity from Asia and Australia. Fi-
subclade PB-IV-1; (2) the “Tetraploid” entity of the R. marit- nally, the “Occidentalis” and “Tetraploid” entities in Asia and
ima complex with PHYB sequences in the subclade PB-IV-2 Australia are related to the origin of the “Triploid” entity, based
and the clade PB-V; (3) the “Filifolia” entity of the R. maritima on DNA sequences and chromosome numbers, even though the
complex with PHYB sequences in the subclade PB-IV-1 and the accession does not have a PHYB sequence that is closely related
branch PB-VI; and (4) the “Triploid” entity of the R. maritima to the “Occidentalis” entity. One possible explanation is that
complex with PHYB sequences in the subclade PB-IV-2 and the “Triploid” entity might have subsequently undergone intro-
the clade PB-V (the cytotypes of (1) and (3) are currently un- gression through multiple hybridizations or backcrosses with
known). The origin of these hybridizations is discussed in rela- another putative paternal taxon, the “Tetraploid” entity.
tion to the ptDNA and PHYB trees and cytotypes.
In the first case, Ruppia A from northern Japan was found to Conclusions— To develop a systematic understanding of the
have ptDNA sequences identical to R. megacarpa as well as genus Ruppia, we performed molecular phylogenetic analyses
PHYB sequences identical to R. megacarpa and the “Occiden- using ptDNA and PHYB sequence data sets. Several morpho-
talis” entity of the R. maritima complex. Therefore, it appears logical species recognized in previous taxonomic revisions ap-
that Ruppia A originated through a cross between R. megacarpa pear to be paraphyletic in both trees. Given the molecular
as the maternal parent and the “Occidentalis” entity as the pa- phylogenies, chromosome numbers, and reevaluated morpho-
ternal parent. In one of the two populations of the hybrid, the logical characters, three species—R. megacarpa, R. polycarpa,
“Occidentalis” entity occurs together with Ruppia A, whereas and R. tuberosa—are well defined. The R. maritima complex
R. megacarpa was not found around the hybrid populations but comprise what we provisionally refer to here as the “Diploid”,
occurs nearby in northern Japan, indicating that the hybrid was “Triploid”, “Tetraploid”, “Filifolia”, “Utahian”, and “Occiden-
established there, followed by the disappearance of R. mega- talis” entities. In the complex, three entities of hybrid origin
carpa (Fig. 4). Because only flowering specimens (no fruiting have been detected: the “Tetraploid” entity is an allotetraploid
specimens) were collected, it could be defined as a sterile that is least related to the “Diploid” entity. The “Filifolia” en-
hybrid. tity, in which the cytotype is unknown, is formed by hybridiza-
The second case is the “Tetraploid” entity, which formed dis- tion within the complex. The “Triploid” entity is derived from
tinct clades in the ptDNA and PHYB trees, Pt-V and PB-V, but the “Occidentalis” and “Tetraploid” entities. In addition, one
was also represented in the subclade PB-IV-2 (Fig. 3). Because case of hybridization between distantly related taxa was de-
the heterogeneous PHYB sequences were not closely related, tected, and its respective putative parental taxa were inferred:
the “Tetraploid” entity cannot be considered an autotetraploid. this hybrid derived from R. megacarpa and the “Occidentalis”
Rather, it is an allotetraploid formed by hybridization between entity parentage. Therefore, many hybridization events have
different entities in the complex. In the subclade PB-IV-2, one occurred in Ruppia; however, the number of taxa is relatively
of its PHYB sequences was nested within those of the “Diploid” small. Although R. megacarpa has been generally considered
entity of the R. maritima complex, indicating that subclade PB- to represent one of three species endemic to Oceania (together
IV-2 is a parental lineage, probably paternal, even though no with R. polycarpa and R. tuberosa), a disjunct population of R.
other parental lineage was detected. Recently, it was revealed megacarpa was found in northern Far East Asia. A similar
that tetraploids of the genus Paeonia were derived from crosses Asia–Oceania discontinuous distribution was observed in the
between genetically differentiated geographical diploid races “Tetraploid” entity as well. Bird-mediated seed dispersal is a
(Sang et al., 2004). Therefore, the sampling strategy in this possible explanation for the two cases of the disjunct Asia–
study may not have included the candidate progenitors because Oceania distribution pattern, probably followed by hybridiza-
of the extremely widespread distribution of the genus. On the tion. These results clarify that a taxonomic reappraisal of the
other hand, it is possible that the maternal parent lineage is now complex will be necessary following the examination of addi-
extinct. tional material collected from around the world.
Similarly, the “Filifolia” entity formed distinct branches in
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July 2010] Ito et al.—Phylogeny of RUPPIA 1167

Appendix 1. Population code, GenBank accessions (matK, rbcL, rpoB, rpoC1, and PHYB), Locations, voucher information, entities in the R. maritima complex or
species proposed in the current study, and chromosome numbers. Herbaria abbreviations: The University of Tokyo = TI, Kochi Prefectural Makino Botanical
Garden = MBK, National Museum of Nature and Science = TNS, Hokkaido University = SAP, National Herbarium of New South Wales = NSW.

Taxon—Population code, GenBank accessions: matK, rbcL, rpoB, rpoC1, and PHYB (a = partially obtained sequences); Locations; Voucher specimens; Herbaria;
Entities in the R. maritima complex (e.g., “Diploid”) or Lineages; Chromosome number.
Ruppia cirrhosa (Petagna) Grande—SKY; AB507925, AB507885, YI00643; TNS; “Diploid”; 2n =?. TOD; AB507913, AB507873, AB507953,
AB507965, AB508005, AB508058, AB508059; Skye Island, UK; AB507993, aAB508036; Todoroki River, Okinawa, Japan; YI00755; TNS;
YI01299; TNS; “Tetraploid”; 2n = 40. “Diploid”; 2n = 20. UTA; AB507928, AB507888, AB507968, AB508008,
AB508064; Salt Lake City, Utah, USA; YI01274; TNS; “Utahian”;
R. maritima L.—ALB; AB534775, AB534784, AB534795, AB534802,
2n =?. XAO; AB507927, AB507887, AB507967, AB508007, aAB508062,
AB534813; Point Aux Pins, Alabama, USA; YI01247; TNS; “Diploid”; aAB508063; Hasan, Vladivostok, Russia; YI00933; TNS; “Tetraploid”;
2n =?. ALV; AB534777, AB534785, AB534792, AB534801, AB534812;
2n = 40. YHO; AB507914, AB507874, AB507954, AB507994, AB508037;
Laguna de Alvarado, Veracruz, Mexico; YI01387; TNS; “Diploid”;
Yuhong, Sanya city, Hainan Province, China; YI00743; TNS; “Diploid”;
2n =?. ANA; AB507905, AB507865, AB507945, AB507985, AB508028;
2n = 20.
Anatom Island, Vanuatu; TNS9516725; TNS; “Diploid”; 2n = ?.
ART; AB507906, AB507866, AB507946, AB507986, aAB508029; R. megacarpa S. Mason—KAM; AB507929, AB507889, AB507969,
Artern, Germany; YI00876; TNS; “Diploid”; 2n =?. BAR; AB507909, AB508009, aAB508065; Kamoko Lake, Niigata, Japan; YI00173; TNS;
AB507869, AB507949, AB507989, aAB508032; Olong Williams Canal, 2n = 20. MEA; AB507930, AB507890, AB507970, AB508010, aAB508066;
Barataria Bay, Louisiana, USA; YI01226; TNS; “Diploid”; 2n =?. CAP; Small lake, Narracorte, South Australia, Australia; SJ9681; NSW; 2n = 20.
AB507907, AB507867, AB507947, AB507987, aAB508030; Cape Breton MED; AB507931, AB507891, AB507971, AB508011, AB508067; Lakes
Island, Nova Scotia, Canada; CAN521697; SAP; “Diploid”; 2n =?. COR; Entrance, Victoria, Australia; SJ9712; NSW, 2n =?. MEF; AB507932,
AB534778, AB534786, AB534794, AB534800, aAB534810, aAB534811; AB507892, AB507972, AB508012, aAB508068; Corunna Lake, S
Porto Vecchio, Corsica Isl., France; YI01248; TNS; “Tetraploid”; 2n = Narooma, New South Wales, Australia; SJ9717; NSW; 2n =?.
40. DUB; AB507915, AB507875, AB507955, AB507995, AB508038,
AB508039; Dubrovnik, Croatia; YI00878; TNS; “Tetraploid”; 2n =?. FAL; R. polycarpa S. Mason—DEV; AB507934, AB507894, AB507974, AB508014,
AB534779, AB534787, AB534793, AB534799, AB534808, AB534809; AB508070; Devil’s Lake, Minnesota, USA; YI01069; TNS; “Occidentalis”;
Port Stephens, West Falkland, Falklands, U.K,; YI01251; TNS; “Filifolia”; 2n =?. NDA; AB507935, AB507895, AB507975, AB508015, aAB508071;
2n =?. GRA; AB507908, AB507868, AB507948, AB507988, AB508031; Devil’s Lake, North Dakota, USA; YI00960; TNS; “Occidentalis”; 2n =?.
Grand Bay Area, Gulf of Mexico, Jackson, Mississippi, USA; YI01233; POB; AB507938, AB507898, AB507978, AB508018, AB508074; Coila
TNS; “Diploid”; 2n =?. HAS; AB507916, AB507876, AB507956, Creek, S Moruya, New South Wales, Australia; SJ9719; NSW; 2n = 18.
AB507996, aAB508040, aAB508041; Hasunuma Pond, Chiba, Japan; POI; AB507936, AB507896, AB507976, AB508016, AB508072;
YI00822; TNS; “Tetraploid”; 2n = 40. JEJ; AB507917, AB507877, Point Lake, Minnesota, USA; YI01070; TNS; “Occidentalis”; 2n =?
AB507957, AB507997, aAB508042, aAB508043; Seogwi, Jeju, South . RED; AB507937, AB507897, AB507977, AB508017, AB508073;
Korea; YI00879; TNS; “Tetraploid”; 2n =?. LIE; AB507918, AB507878, Redberry Lake, Saskatchewan, Canada; YI01264; TNS; “Occidentalis”;
AB507958, AB507998,aAB508044, aAB508045; Huelva, Spain; YI00877; 2n = 20. TOH_01; AB534775, AB534782, AB534789, AB534796,
aAB534803; Tofutsuko Lake, Hokkaido, Japan; YI01037 ; TNS;
TNS; “Tetraploid”; 2n =?. MAA; AB507919, AB507879, AB507959,
AB507999, aAB508046, aAB508047; Montecollina Bore, c. 218 km “Occidentalis”; 2n =?.
NE Lyndhurst, Strzelecki Track, South Australia, Australia; SJ9694; R. tuberosa Davis & Tomlinson—TUA; AB507939, AB507899, AB507979,
NSW; “Tetraploid”; 2n =?. MAH; AB507910, AB507870, AB507950, AB508019, AB508075, AB508076; c. 3 km N Beachport, South Australia,
AB507990, AB508033; Maharashtra, India; YI01209; TNS; “Diploid”; Australia; SJ9687; NSW; 2n =?. TUE; AB507940, AB507900, AB507980,
2n = 20. MAR; AB507911, AB507871, AB507951, AB507991, AB508034; AB508020, AB508077, AB508078; Golden Beach, Lake Reeve, Victoria,
Chesapeake Bay, Maryland, USA; YI00958; TNS; “Diploid”; 2n =?. MAS; Australia; SJ9706; NSW; 2n = 20.
AB507920, AB507880, AB507960, AB508000, AB508048, AB508049;
Massa, Agadir, Morocco; YI00959; TNS; “Tetraploid”; 2n =?. MIN; Ruppia spp.—KUC; AB507902, AB507862, AB507942, AB507982,
AB507921, AB507881, AB507961, AB508001, aAB508050, aAB508051; AB508023, AB508024; Kuccharoko Lake, Hokkaido, Japan; YI00143;
Minamijima, Ogasawara Isl., Tokyo, Japan; YI00223; TNS; “Tetraploid”; TNS; Hybrid; 2n =?. OGA; AB507904, AB507864, AB507944, AB507984,
2n = 40. ODC; AB507922, AB507882, AB507962, AB508002, AB508027, AB508028; Ogawarako Lake, Aomori, Japan; YI00824;
AB508052, AB508053; Odense, Denmark; YI01304; TNS; “Tetraploid”; TNS; “Triploid”; 2n = 30. TOH_02; AB507903, AB507863, AB507943,
2n =?. OST; AB507923, AB507883, AB507963, AB508003, AB508054, AB507983, AB508025, AB508026; Tofutsuko Lake, Hokkaido, Japan;
AB508055; Sweden; YI01334; TNS; “Tetraploid”; 2n =?. ROM; YI00144; TNS; Hybrid; 2n =?. XAM; AB507933, AB507893, AB507973,
AB534780, AB534783, AB534790, AB534798, AB534806, AB534807; AB508013, AB508069; Hasan, Vladivostok, Russia; YI00939; TNS; R.
Rome, Italy; YI01382; TNS; “Tetraploid”; 2n =?. SKA; AB507924, megacarpa; 2n =?.
AB507884, AB507964, AB508004, AB508056, AB508057; Shiokawa Syringodium isoetifolium (Asch.) Dandy—AB507941, AB507901, AB507981,
River, Okinawa, Japan; YI00754; TNS; “Tetraploid”; 2n = 40. TAK; AB508021, AB508079; Tsukenshima Island, Okinawa, Japan; YI00957;
AB507926, AB507886, AB507966, AB508006, aAB508060, aAB508061; TNS.
Takahoko Lake, Aomori, Japan; YI00579; TNS; “Tetraploid”; 2n =?. TAL;
AB534781, AB534788, AB534791, AB534797, AB534804, AB534805; Potamogeton maackianus A. Benn.—AB559938, AB506769, AB559936,
Tallinn, Estonia; YI01379; TNS; “Tetraploid”; 2n =?. TCH; AB507912, AB559939, AB559939; Yae Aye Kan, Kalaw Township, Shan State,
AB507872, AB507952, AB507992, AB508035; Chigu, Tainan, Taiwan; Myanmar; N. Tanaka & al. 080052; MBK, TI.