Cryptic species are morphologically indistinguishable, yet reproductively isolated. Morphological boundaries between species can also be obscured by hybridization and clonality. Determining the roles of reproductive isolation, hybridization, and clonality in morphologically indistinguishable taxa is essential to determining appropriate species-level taxonomic rankings for conservation purposes. The taxonomic status of the endangered Little Aguja pondweed of west Texas, Potamogeton clystocarpus, is uncertain due to a lack of fixed morphological differences between it and two sympatric congeners. Morphology, amplified fragment length polymorphisms (AFLPs), and sequences of the internal transcribed spacer (ITS) region and trnL-F intron and spacer were used to determine the degree of genetic distinctiveness, hybridization and clonality for this rare species. AFLPs indicate that P. clystocarpus is a genetically distinct lineage compared to P. pusillus and P. foliosus. No hybrids involving P. clystocarpus were detected, but two putative hybrids involving P. pusillus and P. foliosus were identified. Clonal growth was only detected in P. pusillus. A combination of morphological and molecular markers was successful in determining the genetic distinctiveness of an endangered cryptic species, Potamogeton clystocarpus. Further sampling in this and adjacent drainages is necessary to assess the degree of endemism of P. clystocarpus and confidently rule out hybridization and clonality in this taxon.

This study represents the first comprehensive analysis of phylogenetic relationships within the Australian water-lilies, Nymphaea subg. Anecphya. Our 51-accession dataset covers all 10 species of the subgenus, except the newly described N. alexii, and includes information from the nuclear ITS as well as from the chloroplast trnT–trnF region. The results show that molecular data are consistent with morphology, because the subdivision of subg. Anecphya into two major clades, a large-seeded and a small-seeded group, could be confirmed. Within the large-seeded group, Nymphaea atrans and N. immutabilis seem to form one clade, whereas samples of N. gigantea, N. georginae, N. macrosperma and N. carpentariae form another. Relationships within the small-seeded group, containing all samples of N. violacea, N. elleniae and N. hastifolia, are less clear, since the trees obtained from the chloroplast and the nuclear marker are incongruent. The samples of N. violacea do not form a monophyletic group in each of the trees, but—at least in the ITS tree—group with either N. elleniae or N. hastifolia/Ondinea, respectively. Polymorphisms among ITS paralogues, i.e. substitutions at single nucleotide positions and length polymorphisms, have been observed in some samples of N. violacea. This fact as well as the incongruent phylogenetic signal obtained from the chloroplast and the nuclear genomes point to recent hybridisation or introgression in this group. Remarkably, Ondinea purpurea is resolved within the small-seeded group by both markers and seems to have a close relationship to N. hastifolia. Although incomplete lineage sorting cannot be fully excluded to explain high variability in N. violacea, molecular data potentially hint to a case of still imperfect taxonomy.

The occurrence and diversity of Potamogeton hybrids was investigated in eastern North America, the region of the highest diversity of Potamogeton species in the world. For various reasons, however, the existence of hybrids in this area has been largely overlooked. ITS direct sequencing and RFLPs revealed four previously unknown hybrids, which are described as Potamogeton × aemulans ( P. bicupulatus × P. epihydrus ), P. × mirabilis ( P. gramineus × P. oakesianus ), P. × versicolor ( P. epihydrus × P. perfoliatus ), and P. × absconditus ( P. perfoliatus × P. richardsonii ). These are the first confirmed hybrids involving P. bicupulatus , P. epihydrus , P. oakesianus , and P. richardsonii . Another hybrid, P. × nitens ( P. gramineus × P. perfoliatus ), is for the first time confirmed for North America with molecular markers. The hybrids' maternal parents were revealed by cpDNA sequencing. Phenotypically, two of the hybrids more closely resemble other taxa than their parents. Hybrid diversity and recognition in North America and in Europe are compared. Morphological versus molecular identification of hybrids, occurrence of hybrids in the absence of their parents, parental species relationships, long-term persistence of hybrid clones, vegetative dispersal, frequency of hybridization events, and hybrid distribution patterns are discussed.

Thorough understanding of biodiversity is a fundamental prerequisite for biological research. A lack of taxonomic knowledge and species misidentifications are particularly critical for conservation. Here we present an example of Potamogeton floridanus, the Florida Pondweed, an endangered taxon endemic to a small area in the Florida panhandle, whose taxonomic status remained controversial for more than a century, and all previous attempts to elucidate its identity have failed. We applied molecular approaches to tackle the origin of the mysterious taxon and supplemented them with morphological and anatomical investigations of both historical herbarium collections and plants recently collected in the type area for a comprehensive taxonomic reassessment. Sequencing of two nuclear ribosomal markers and one chloroplast non-coding spacer resulted in the surprising discovery that P. floridanus is a hybrid of P. pulcher and P. oakesianus, with the former being the maternal parent. The hybrid colony is currently geographically isolated from the distribution range of P. oakesianus. We show that previous molecular analyses have failed to reveal its hybrid identity due to inadequate nuclear DNA sequence editing. This is an example how the uncritical use of automized sequence reads can hamper molecular species identifications and also affect phylogenetic tree construction and interpretation. This unique hybrid taxon, P. ×floridanus, adds another case study to the debate on hybrid protection; consequences for its conservation are discussed.

SEM studies of xylem of stems of Nuphar reveal a novel feature, not previously reported for any angiosperm. Pit membranes of tracheid end walls are composed of coarse fibrils, densest on the distal (outside surface, facing the pit of an adjacent cell) surface of the pit membrane of a tracheid, thinner, and disposed at various levels on the lumen side of a pit membrane. The fibrils tend to be randomly oriented on the distal face of the pit membrane; the innermost fibrils facing the lumen take the form of longitudinally oriented strands. Where most abundantly present, the fibrils tend to be disposed in a spongiform, three-dimensional pattern. Pores that interconnect tracheids are present within the fibrillar meshwork. Pit membranes on lateral walls of stem tracheids bear variously diminished versions of this pattern. Pits of root tracheids are unlike those of stems in that the lumen side of pit membranes bears a reticulum revealed on the outer surface of the tracheid after most of the thickness of a pit membrane is shaved away by the sectioning process. No fibrillar texturing is visible on the root tracheid pits when they are viewed from the inside of a tracheid. Tracheid end walls of roots do contain pores of various sizes in pit membranes. These root and stem patterns were seen in six species representing the two sections of Nuphar, plus one intersectional hybrid, as well as in one collection of Nymphaea, included for purposes of comparison. Differences between root and stem tracheids with respect to microstructure are consistent in all species studied. Microstructural patterns reported here for stem tracheid pits of Nymphaeaceae are not like those of Chloranthaceae, Illiciaceae, or other basal angiosperms. They are not referable to any of the patterns reported for early vascular plants. The adaptational nature of the pit membrane structure in these tracheids is not apparent; microstructure of pit membranes in basal angiosperms is more diverse than thought prior to study with SEM.