Different levels of inbreeding depression between outcrossing and selfing Serapias species

We quantified inbreeding depression for fruit production, embryo vitality and seed germination in three deceptive orchids, Serapias vomeracea, S. cordigera and S. parviflora, which do not provide any reward to their pollinators, and are predicted to experience high outcrossing. Of the three species examined only S. parviflora was autonomously selfing. Both S. vomeracea and S. cordigera showed highly significant differences in fitness between selfed and outcrossed progenies, resulting in high levels of inbreeding depression, which increased in magnitude from seed set to seed germination. Inbreeding depression may promote outcrossing in Serapias by acting as a post-pollination barrier to selfing. Cumulative inbreeding depression across three stages in S. parviflora was lower that in both outcrossing species. The large difference in germination between selfed and outcrossed seeds is an important issue in conservation biology.     

Genetic diversity, population structure and phylogenetic inference among Italian Orchids of the Serapias genus assessed by AFLP molecular markers

The means by which new species arise remain a fundamental question in the Orchid family evolution. Even if the Orchid-pollinator interactions are highly specific, events of cross-hybridization in Serapias are possible and represent a central issue of the plant's evolutionary biology. Italian Orchids have a wide variety of pollination systems and highly diverse floral traits, considered as some of the main tools for activating the speciation processes. The assess of diversity in Serapias taxa and eventual phylogenetic relationships among Serapias lingua, S. parviflora, S. vomeracea, S. apulica, S. nurrica, S. neglecta, S. cordigera, S. politisii, and some putative inter-specific hybrids were investigated at the DNA level by AFLP markers. The present molecular study on Serapias populations (1) confirms some previous finding, (2) opens new hypotheses on the possible evolutionary and taxonomic relationships existing among the Serapias species, and (3) gives indications about the real consistence of hybrid individuals in a genus characterized by a relevant gene flow.     

Morphometric and molecular (RAPD) analysis of six <Emphasis Type="Italic">Serapias</Emphasis> taxa from Croatia

The morphometric analyses and genetic variability assessed by RAPD markers have been used to analyse relations among six Serapias taxa from Croatia (S. istriaca, S. pulae originally described as hybrid, S. ionica, S. vomeracea, S. lingua and S. cordigera). S. istriaca distributed in southern Istria and the island of Lošinj and S. pulae stenoendemic taxon distributed only in southern Istria S. ionica is endemic to the Ionian and Dalmatian islands, while the remaining taxa are more widely distributed. The obtained results shows that the endemic S. istriaca is a well characterised taxon, that S. pulae is a hybrid between S. istriaca and S. lingua and that the hybrid is morphologically and genetically more similar to S. lingua than the second parental species S. istriaca. The division into the subsections Steno-, Medio- and Platypetalae is founded based on the floral morphology while the division into the sections Serapias and Bilamellaria is not evident in the quantitative morphological and genetic analyses. Furthermore, considerable genetic resemblance between S. vomeracea and S. ionica was established.     

Comparative analysis of floral scents in four sympatric species of Serapias L. (Orchidaceae): clues on their pollination strategies

Orchid species of Mediterranean genus Serapias often live in sympatry, exhibit similar floral morphology, bloom in the same period and share the same pollinators. Previous studies on Serapias species have ascertained that reproductive isolation is based on pre-pollination barriers, that secretory cells and trichomes are typically distributed on the floral labellum and that flowers produce aliphatic compounds. In this study we compare the floral scent composition of four widespread, co-occurring Serapias species, namely Serapias lingua, Serapias parviflora, Serapias vomeracea and Serapias cordigera. Our goals are to assess if differences in floral scent may act as interspecific pre-pollination barriers and if these olfactory signals may be involved in the pollination strategy of Serapias. We find that all the selected species produce C20–C29 alkanes and alkenes and, in addition, have detected the presence in S.?cordigera of large amounts of oleate and stearate ethyl ester. Our findings help to clarify that the sympatric Serapias species have slightly different floral scent signatures that may account for their relevant role as pre-pollination barriers. Therefore, the pollination strategy of Serapias relies not only on the tubular shape of their floral corolla but also on the production of olfactory signals that may lure potential pollinators and even assure a sufficient degree of pollinator fidelity.     

Plastome sequences of two New World bamboos—Arundinaria gigantea and Cryptochloa strictiflora (Poaceae)—extend phylogenomic understanding of Bambusoideae

• Premise of the study: Two New World species of Bambusoideae, Arundinaria gigantea and Crytpochloa strictiflora, were investigated in a phylogenomic context. Complete plastome sequences have been previously determined and analyzed for nine bambusoid species that exclusively represent Old World lineages. The addition of New World species provides more complete information on relationships within Bambusoideae. • Methods: Plastomes from A. gigantea and C. strictiflora were sequenced using Sanger methods. Phylogenomic and divergence estimate analyses were conducted on both species with 23 other Poaceae. • Key Results: Phylogenomic and divergence analyses suggested that A. gigantea diverged from within Arundinarieae between 1.94–3.92 mya and that C. strictiflora diverged as the sister to tropical woody species between 24.83 and 40.22 mya. These results are correlated with modern relative diversities in the two lineages. • Conclusions: The two New World bamboos show unique plastome features accumulated and maintained in biogeographic isolation from Old World taxa. The overall evidence for A. gigantea is consistent with recent dispersal, and that for C. strictiflora is consistent with vicariance.     

Reproductive biology and conservation genetics ofSerapias vomerace (Orchidaceae)

Serapias vomeracea is considered to be a particular example of the deceptive strategy of terrestrial orchids. Information on its reproductive biology and population genetic variation is needed to develop conservation strategies. The pollination biology and breeding system were investigated in four populations ofS. vomeracea. Hand-pollination experiments showed thatS. vomeracea is self-compatible. Bagged inflorescence produced no fruits; artificial pollination resulted in 89.7–100% fruit set through induced autogamy, 91.1–100% with geitonogamous, and 89.3–94.4% following xenogamous pollination. Fruit set in the open-pollinated control ranged from 13.4 to 20.9%. We evaluated the spatial genetic structure of 13 populations using five nuclear microsatellite loci. Italian and isle of Corfu populations ofS. vomeracea are characterized by a low mean allele number, ranging from 2.4 to 3.8, by intermediate to high heterozygosity levels, ranging from 0.50 to 0.71, and by frequent deviations from the Hardy-Weinberg equilibrium (9 out 13 populations). Most of theS. vomeracea populations exhibited significant heterozygote excess. The population size reduction is most probably the cause of the low genetic variation observed in the populations ofS. vomeracea. For the genetic conservation and management ofS. vomeracea suitable strategies should be developed based on these data.     

MONOPHYLY OF ANEUPLOID ASTRAGALUS (FABACEAE): EVIDENCE FROM NUCLEAR RIBOSOMAL DNA INTERNAL TRANSCRIBED SPACER SEQUENCES

Evolutionary relationships within Astragalus L. (Fabaceae) were inferred from nucleotide sequence variation in nuclear ribosomal DNA of both New World and Old World species. The internal transcribed spacer regions (ITS) of 18S–26S nuclear ribosomal DNA from representatives of 26 species of Astragalus, three species of Oxytropis DC., and two outgroup taxa were analyzed by polymerase chain reaction amplification and direct DNA sequencing. The length of the ITS 1 region within these taxa varied from 221 to 231 bp, while ITS 2 varied in length from 207 to 217 bp. Of the aligned, unambiguous positions, approximately 34% were variable in each spacer region. In pairwise comparisons among Astragalus species and outgroup taxa, sequence divergence at these sites ranged from 0 to 18.8% in ITS 1 and from 0 to 21.7% in ITS 2. Parsimony analyses of these sequences resulted in a well-resolved phylogeny that is highly concordant with previous cytogenetic and chloroplast DNA evidence for a major phylogenetic division in the genus. These data suggest that the New World aneuploid species of Astragalus form a monophyletic but morphologically cryptic group derived from euploid species of Old World (Eurasian) origin, which are consequently paraphyletic.     

Exploring the history of Serapias politisii (Orchidaceae), a narrow endemic on the opposite coasts of the Otranto channel: insights from molecular investigations

Knowledge of genetic spatial structure may provide insights into the causes of population disjunctions in plants. Serapias politisii is a narrow endemic with only a few populations scattered along the opposite coasts of the Otranto strait (southern Adriatic Sea). It was originally considered to be of hybrid origin between S. vomeracea ssp. laxiflora and S. parviflora, a possibility suggested also by a DNA phylogenetic study that grouped Italian and Greek populations in two distinct clades. In this study we have carried out additional plastid DNA sequencing and an AFLP analysis of the three taxa. Whereas the geographical distribution of four plastid DNA haplotypes supports the likelihood of a double hybrid origin or of a plastid capture, AFLP data do not support such a hypothesis, because S. politisii shows several private alleles, some of which are shared by Italian and Greek populations. In light of the floristic specificity of the coasts bordering the Otranto strait, we consider that the present‐day disjunction of S. politisii could have originated either by a long‐distance seed dispersal or by a fragmentation of an old polymorphic population. The pairwise mismatch distribution excludes a recent expansion of the populations examined. In light of recent evidence concerning the Mediterranean Lago‐Mare period and the westward haplotype evolution detected in some Euro‐Mediterranean trees, we argue that fragmentation of a previously continuous population could be an intriguing possibility. © 2010 The Linnean Society of London, Botanical Journal of the Linnean Society, 2010, 162 , 572–580.     

Functional differentiation in pollination processes among floral traits in Serapias species (Orchidaceae)

Floral displays, influencing attractiveness to insects, increase the number of pollinator visits and the efficiency of each visit in terms of pollen exchange and thus affect the plant reproductive success. Here, we conducted an in situ manipulation experiment to investigate whether the floral modifications affect reproductive success in natural orchid populations of Serapias lingua and Serapias vomeracea. We estimated male and female reproductive success of three treatment groups, disassembly of floral tube, cutting of lip, and painting of the callus surface, in terms of pollinaria removed/deposited and fruit production. Results revealed that phenotypic modification had opposite effects on reproductive success of two examine species. Indeed, reproductive success was significantly increased by the detached of the petals and sepals, and decreased, due to callus painting and lip removal, in S. lingua. On the contrary, unmanipulated plants of S. vomeracea showed significantly higher value of pollinaria removed and deposited and fruit set than manipulated ones. The differences between S. lingua and S. vomeracea agree to the different pollination strategy of examined species. S. vomeracea shows shelter imitation strategy, and thus, the disassembly of tunnel‐like corolla does not allow the insects to use the flower as a refuge, while S. lingua is a sexually deceptive orchid and therefore the opening of the flower made more visible callus (visible at a greater distance) increasing the pollinators attraction. This study provides evidence that pollinators were largely sensitive to the experimental modification of the flower phenotype, which is consistent with the presence of significant selection on individual floral characters. Our experimental investigations of the effects of variation in display on pollinator visitation provide insights into the evolution of floral morphology in orchid with shelter imitation strategy.     

DNA CONTENT OF SEVEN SPECIES OF ASTEREAE AND ITS SIGNIFICANCE TO THEORIES OF CHROMOSOME EVOLUTION IN THE TRIBE

Relative amounts of nuclear DNA were determined in root tip cells of seven species of Astereae: Aster hydrophilus Greene, A. oblongifolius Nutt., A. riparius H.B.K., Machaeranthera boltoniae (Greene) Turner and Home, M. brevilingulata (Sch-Bip.) Turner and Home, M. parviflora Gray, and M. tenuis (S. Wats.) Turner and Home. The results show that A. hydrophilus and M. brevilingulata, with a chromosome number of n = 9, have less nuclear DNA than other closely related species which are either n = 4 or n = 5. Cytological analyses of meiosis in the intergeneric hybrid M. parviflora X A. hydrophilus showed cells with two or more small chromosomes of the latter species pairing with single large chromosomes of the former. Pachytene cells of the hybrids M. parviflora X A. hydrophilus, M. parviflora X A. riparius, and M. boltoniae X M. tenuis showed some unpaired chromosome segments. The significance of these results to chromosome evolution in the tribe Astereae is discussed.     

A review of cross‐backed grasshoppers of the genus Dociostaurus Fieber (Orthoptera: Acrididae) from the western Mediterranean: insights from phylogenetic analyses and DNA‐based species delimitation

Phylogenetic analyses and species delimitation methods are powerful tools for understanding patterns of species diversity. Given the current biodiversity crisis, such approaches are invaluable for urgent assessment and delimitation of truthful species, particularly of endangered and morphologically cryptic taxa from vulnerable areas submitted to strong climate change and progressive human intervention such as the M editerranean region. In this study, we applied two DNA ‐based species delimitation methods and performed a B ayesian phylogenetic reconstruction using three mitochondrial gene fragments (12S , 16S and COI) to solve several taxonomic uncertainties among species of cross‐backed grasshoppers (genus Dociostaurus F ieber) from the western M editerranean. P hylogenetic analyses demonstrate the polyphyletic character of subgenera Dociostaurus , Kazakia B ey‐B ienko and Stauronotulus T arbinsky and, thus, the necessity of revising the currently accepted taxonomic subgenera within the genus Dociostaurus . We propose the split of closely related taxa with allopatric distributions such as D. (S.) kraussi and D. (S.) crassiusculus , considering the later a distinct species limited to the I berian P eninsula and excluding the name crassiusculus from other forms of D. (S.) kraussi from E ast E urope and A sia. Estimates of divergence times indicate that diversification of Dociostaurus probably happened during the M iocene–P liocene (3–7 Ma), and the split of the studied pairs of sister taxa took place during the middle and late P leistocene (1–2 Ma). This study highlights the need for more molecular studies on the genus and their different species for a better understanding of their evolution, genetic variation and population dynamics in order to prioritize strategies for their adequate conservation and management.     

Systematics of the Saguinus oedipus group of the bare-face tamarins: Evidence from facial morphology

The systematics of the Saguinus oedipus group within the bare-face tamarins remains open to question. Hershkovitz (Living New World Monkeys (Platyrrhini), Vol. 1. Chicago: University of Chicago Press, 1977) places the cotton-top and rufus-naped tamarins as subspecies of Saguinus oedipus (S. o. oedipus and S. o. geoffroyi, respectively). In contrast, several other authors have argued that these two taxa should be considered separate species (S. oedipus and S. geoffroyi). Phylogenetic relationships within the group are also disputed. Resolving these different interpretations has been difficult in part because no study of this group has included an objective measure of expected levels of specific vs. subspecific variation. We used facial measurements from 179 adult crania to address the systematics of this group and included a related species that is known to include multiple subspecies. Our sample included three taxa from the S. oedipus group of the bare-face tamarins (S. oedipus, S. geoffroyi, and S. leucopus) and six subspecies from the related hairy-face tamarin species S. fuscicollis. Comparisons to S. leucopus provided a relative measure of species-level differences. Analyses that included S. fuscicollis provided a measure of subspecific variation. There was no evidence of facial sexual dimorphism in any of these taxa. A variety of multivariate statistical analyses including discriminant function and cluster analysis suggest that S. oedipus and S. geoffroyi differ morphologically at a level consistent with species-level distinctions. The extent of differences between these taxa is large. The differences in their facial morphology was on the order of differences between S. oedipus or S. geoffroyi and S. leucopus rather than the extent of variation among S. fuscicollis subspecies. Furthermore, a comparison of collecting localities revealed that the variation we observed among S. oedipus and S. geoffroyi was not clinal but presented a large morphological discontinuity at the boundary between taxa. Our analyses also suggested that S. leucopus is more similar to S. oedipus than is either to S. geoffroyi. Finally, it may be that there are some distinct species within the S. fuscicollis group. However, this hypothesis, along with other phylogenetic relationships suggested by this study, will require more data and further study. © 1992 Wiley-Liss, Inc.     

Molecular systematics of threadfin breams and relatives (Teleostei,Nemipteridae)

Threadfin breams and relatives of the family Nemipteridae comprise 69 currently recognized species in five genera. They are found in the tropical and subtropical Indo‐West Pacific and most are commercially important. Using recently developed molecule‐based approaches exploiting DNA sequence variation among species/specimens, this study reconstructed a comprehensive phylogeny of the Nemipteridae, examined the validity of species and explored the cryptic diversity of the family, and tested previous phylogenetic hypotheses. A combined data set (105 taxa from 41 morphospecies) with newly determined sequences from two nuclear genes (RAG1 and RH) and one mitochondrial gene (COI), and a data set with only COI gene sequences (329 newly obtained plus 328 from public databases from a total of 53 morphospecies) were used in the phylogenetic analysis. The latter was further used for species delimitation analyses with two different tools to explore species diversity. Our phylogenetic results showed that all the currently recognized genera were monophyletic. The monotypic genus Scaevius is the sister group of Pentapodus and they together are sister to Nemipterus. These three genera combined to form the sister group of the clade comprising Parascolopsis and Scolopsis. The validity of most of the examined species was confirmed except in some cases. The combined evidence from the results of different analyses revealed a gap in our existing knowledge of species diversity in the Nemipteridae. We found several currently recognized species contain multiple separately evolving metapopulation lineages within species; some lineages should be considered as new species for further assignment. Finally, some problematic sequences deposited in public databases (probably due to misidentification) were also revised in this study to improve the accuracy for prospective DNA barcoding work on nemipterid fishes.     

Systematics and Evolution of the Dasyurid Marsupial Genus Sminthopsis: I. The Macroura Species Group

Genetic variation within the macroura species group, which includes Sminthopsis macroura, S. virginiae, S. douglasi, and S. bindi, was examined through analyses of complete mitochondrial 12S rRNA gene sequences, partial control-region DNA sequences, and allozymes. Divergent genetic lineages appear to be present within S. macroura and S. virginiae, and it is likely that this genetic divergence equates to currently unrecognized taxonomic diversity. Specimens of S. macroura (as currently recognized) belong to three genetically distinct lineages that are highly divergent from one another. Two of these lineages may be synonymous with two previously recognized dunnart species—S. froggatti and S. stalkeri. The third appears to represent "true" S. macroura and is itself genetically heterogeneous, with a number of subgroups present within it that may also represent currently unrecognized taxa. The mitochondrial DNA sequence divergences observed between S. virginiae nitela and the two other S. virginiaesubspecies are equivalent to, or greater than, those noted between other dunnart species. Allozyme divergences between these subspecies were however slightly lower, and determination on whether S. virginiae nitela should be returned to full species status (S. nitela) may require further evidence. Phylogenetic relationships between species in the macroura group appear to have been partially resolved, with individual 12S rRNA and combined mitochondrial DNA analyses recovering S. bindi as the earliest diverging taxon. Other relationships between species in the group were either not consistently recovered or lacked strong support.     

DNA barcoding reveals taxonomic uncertainty in Salminus (Characiformes)

Salminus is a genus composed of four species of migratory fishes and top predators. Although this group has great economic and ecological importance, the species level diversity of Salminus is not yet completely clarified. Our goal was to detect if this taxonomic problem is the consequence of lineage divergence within species, and, if so, whether these divergences are sufficient to flag potentially undescribed taxa. We employed the standard DNA barcoding analyses and a generalized mixed Yule-coalescent model (GMYC) using one mitochondrial (COI) marker and Bayesian Inference (BI) reconstruction for one nuclear (RAG2) marker for all currently recognized species of Salminus, sampled across different hydrographic basins. Eight MOTUs (Molecular Operational Taxonomic Units) were determined by distance and model-based analyses, and recovered with BI analyses for COI. Only Salminus affinis and Salminus franciscanus formed monophyletic haplogroups. Salminus brasiliensis and Salminus hilarii had two and four distinct mitochondrial lineages, respectively, and higher intraspecific K2P distances than the adopted optimum threshold. The RAG2 gene tree supported two lineages of S. hilarii (S. hilarii Amazon and S. hilarii Araguaia), while the other mitochondrial lineages of S. hilarii and S. brasiliensis were not supported. All lineages of both species, corresponded to morphological variation described in previous studies. We suggest, based on the DNA barcoding analysis, a new taxonomic scenario and conservation polices for Salminus in the Brazilian territory.     

Systematics of North American Froelichia (Amaranthaceae subfam. Gomphrenoideae) II: Phylogeny and biogeographic speciation patterns inferred from nrITS sequence data

Molecular phylogenetic analyses of the nuclear ribosomal DNA internal transcribed spacer (ITS 1 and ITS 2) and the 5.8S gene were used to infer a phylogeny among the ten recognized taxa of Froelichia in North America. Analyses using both maximum parsimony (MP) and maximum-likelihood (ML) depicted a low level of sequence divergence though it was sufficient in most cases to differentiate taxa. Froelichia xantusii, a species restricted to southern Baja California was shown to be the basalmost member of the group subtending three clades. Two of the clades received good bootstrap support in the MP analysis and corresponded to a genetically homogeneous F. interrupta, and a clade comprising the two species F. latifolia and F. texana. A third clade receiving low bootstrap support contained F. floridana, F. gracilis, F. arizonica, and F. drummondii. Species diversity within the genus was centered within the Tamaulipan Brushland region of north-east Mexico and the southern portion of the US state of Texas where taxa from two of the three principal clades occurred, indicating a region of high speciation and diversification within the genus.     

Resolving species boundaries in the critically imperiled freshwater mussel species,Fusconaia mitchelli (Bivalvia: Unionidae)

Species are a fundamental unit of biology, and defining accurate species boundaries is integral to effective conservation and management of imperiled taxa. Freshwater mussels (Bivalvia: Unionidae) are among the most imperiled groups of organisms in North America, yet species boundaries remain uncertain for many taxa. The False Spike, Fusconaia mitchelli (Simpson in Dall, 1895), is a freshwater mussel considered to be endemic to central Texas (Brazos, Colorado, and Guadalupe drainages). Recent research revealed significant intraspecific genetic variation between geographically separated populations of F. mitchelli, which could be indicative of speciation; however, small sample sizes for several of the populations precluded formal taxonomic revision. Here, we increase taxon sampling and use multilocus DNA sequence data and traditional morphometrics to re‐evaluate species boundaries in F. mitchelli. We sequenced three loci: the protein‐coding mitochondrial DNA genes cytochrome c oxidase subunit 1 and NADH dehydrogenase 1, and the nuclear internal transcribed spacer 1. Phylogenetic analyses depicted deep genetic divergence between F. mitchelli in the Guadalupe and those in the Brazos and Colorado drainages, which was further supported by available biogeographic information. Morphometric analyses and coalescent‐based species delimitation models integrating both DNA sequence and morphological data provided strong support for the divergence observed between the two geographically isolated clades of F. mitchelli. Based on these results, we revise taxonomy accordingly by elevating the junior synonym Fusconaia iheringi (Wright, 1898) to represent the Brazos and Colorado populations and restrict the distribution of F. mitchelli to the Guadalupe River drainage. Our findings may impact pending management decisions to protect F. mitchelli under the U.S. Endangered Species Act.     

Molecular phylogeny of animal pathogen Lacazia loboi inferred from rDNA and DNA coding sequences

Lacazia loboi is a geographically restricted, uncultivated fungal pathogen of humans and dolphins. Previous investigations using 18S small unit rDNA, chitin synthase 2 and gp43 DNA sequences positioned L. loboi as a close relative of Paracoccidioides brasiliensis. However, given the few individuals of L. loboi studied and the high degree of genetic variation observed in P. brasiliensis, the existence of L. loboi as an independent species has been questioned. To investigate the phylogenetic position of this species, we conducted a phylogenetic analysis using 20 L. loboi collections (L. loboi was obtained from proven cases of lacaziosis and 14 collections were maintained in mice, the others were analyzed from DNA taken directly from infected human tissue.). L. loboi DNA sequence was compared to that from 17 P. brasiliensis strains that represented the known variation in this species, and outgroup taxa in the Onygenales (Ajellomyces and Coccidioides species). Our analyses used DNA sequence from ITS rRNA, and partial coding sequences of chitin synthase 4, ADP-ribosylation factor, and gp43. Nucleotide variation among strains of L. loboi was minor but numerous nucleotide mismatches and multiple gaps were found for these gene regions among members in the Ajellomycetaceae, including P. brasiliensis. Phylogenies inferred using neighbor-joining, maximum parsimony and Bayesian analyses showed no significant conflict and depicted L. loboi as a well-supported, monophyletic group that was sister to the Paracoccidioides clade. These results argue for maintaining L. loboi as a taxon independent from Paracoccidioides within the Ajellomycetaceae.     

Phenetic studies in Nuphar Sm. (Nymphaeaceae):variation in sect. Nuphar

Previous taxonomic assessments of the hydrophytic genus Nuphar Sm. (Nymphaeaceae) have inadequately considered the range of morphological variation existing in the genus. A comprehensive statistical analysis of morphological features among the recognized taxa of Nuphar is presented here, first using 10 fruit characters to delimit the separation of two infrageneric groups of taxa, and secondly using 31 characters to quantify the morphological variation found within sect. Nuphar. Results from combined phenetic analyses provided empirical support for recognition of two infrageneric groups of species in the genus (largely by fruit, stigmatic disk margin, neck, and stigmatic disk size) and for separation of taxa within sect. Nuphar (by leaf size, and fruit disk and neck size). Statistical analyses demonstrate that extensive variation in ranges of character states exists among sect. Nuphar taxa. However, a suite of qualitative and quantitative characters can be used to recognize five species and three subspecies within this predominantly Old World assemblage.