Two different genetic strains of stalked-Ulva (Ulvales, Chlorophyta) grow on intertidal rocky shores in Ebisujima, central Japan

Stalked‐Ulva has been recognized as an ecologic form of Ulva pertusa Kjellman with narrow and extensive stipes that grows on rocky shores with strong wave action. However, it is possible that stalked‐Ulva includes more than two taxa, because it has been shown previously that some isolates of stalked‐Ulva did not cross with U. pertusa. Therefore, further crossing tests, observations of swarmer behavior, culture studies and comparison of DNA sequences were made to clarify whether or not all stalked‐Ulva are included within U. pertusa. Crossing tests showed that stalked‐Ulva contained two different types, one that crossed with U. pertusa (Up strain) and one (N strain) that did not. The biflagellate swarmers of N strains showed negative phototaxis and were slightly larger than male and female gametes of Up strains. Thalli cultured from the swarmers of N strains released the same type of swarmers again. The N strains have an asexual life history, reproducing solely by biflagellate swarmers. Internal transcriber spacer sequence analysis indicated that these two entities are U. pertusa (Up strain) and U. fasciata Delile (N strain). In culture both strains differ morphologically from wild thalli with stipes; it seems that the two different taxa both show a petiolate morphology when growing under the same environmental conditions.     

Resolving the identity of commercially cultivated Ulva (Ulvaceae,Chlorophyta) in integrated seaweed-abalone aquaculture farms in South Africa

Species of Ulva have a wide range of commercial applications and are increasingly being recognized as promising candidates for integrated aquaculture. In South Africa, Ulva has been commercially cultivated in integrated seaweed-abalone aquaculture farms since 2002, with more than 2000 tonnes of biomass cultivated per annum in land-based paddle raceways. However, the identity of the species of Ulva grown on these farms remains uncertain. We therefore characterized samples of Ulva cultivated in five integrated multi-trophic aquaculture farms (IMTA) across a wide geographical range and compared them with foliose Ulva specimens from neighboring seashores. The molecular markers employed for this study were the chloroplast-encoded Ribulose-1,5-bisphosphate carboxylase oxygenase (rbcL), the Internal Transcribed Spacer (ITS) of the nuclear, and the chloroplast elongation factor tufA. All currently cultivated specimens of Ulva were molecularly resolved as a single species, U. lacinulata. The same species has been cultivated for over a decade, although a few specimens of two other species were also present in early South African IMTA systems. The name Ulva uncialis is adopted for the Ulva “Species A” by Fort et al. (2021), Molecular Ecology Resources, 22, 86) significantly extending the distribution range for this species. A comparison with wild Ulva on seashores close to the farms resulted in five new distribution records for South Africa (U. lacinulata, U. ohnoi, U. australis, U. stenophylloides, and U. aragoënsis), the first report of a foliose form of U. compressa in the region, and one new distribution record for Namibia (U. australis). This study reiterates the need for DNA confirmation, especially when identifying morphologically simple macroalgae with potential commercial applications.     

Methods for the Induction of Reproduction in a Tropical Species of Filamentous Ulva

The green seaweed Ulva is a major fouling organism but also an edible aquaculture product in Asia. This study quantified for the first time the effect of key factors on the reproduction of a tropical species of filamentous Ulva (Ulva sp. 3). The controlled timing of release of swarmers (motile reproductive bodies) was achieved when experiments were initiated in the early afternoon by exposing the thalli to a temperature shock (4°C) for 10 min and subsequently placing them into autoclaved filtered seawater under a 12 h light: 12 h dark photoperiod at 25°C. The release of swarmers then peaked two days after initiation. In contrast, segmentation, dehydration, salinity or time of initiation of experiments had no effect of any magnitude on reproduction. The released swarmers were predominantly biflagellate (95%), negatively phototactic and germinated without complementary gametes. This indicates that Ulva sp. 3 has a simple asexual life history dominated by biflagellate zoids.     

MOLECULAR ASSESSMENT OF ULVA SPP. (ULVOPHYCEAE,CHLOROPHYTA) IN THE HAWAIIAN ISLANDS1

Sequences of the nuclear internal transcribed spacer 1 (ITS1) region and the chloroplast rbcL gene were obtained from 86 specimens of Ulva (including “Enteromorpha”) from five of the main Hawaiian Islands. These 86 specimens were divided into 11 operational taxonomic units (OTUs) based on analyses of primary sequence data and comparisons of ITS1 secondary structure. Of the 11 OTUs, six have not previously been reported from anywhere in the world. Only three represented exact sequence matches to named species (Ulva lactuca L., syn. U. fasciata Delile; U. ohnoi Hiraoka et Shimada); two others represented exact sequence matches to unnamed species from Japan and New Zealand. Of the 12 species names currently in use for Hawaiian Ulva, only one, U. lactuca (as U. fasciata), was substantiated. General morphology of the specimens did not always correspond with molecular OTUs; for example, reticulate thallus morphology, previously considered diagnostic for the species U. reticulata Forssk., was expressed in thalli assigned to U. ohnoi and to one of the novel OTUs. This finding confirms a number of recent studies and provides further support for a molecular species concept for Ulva. These results suggest that Ulva populations in tropical and subtropical regions consist of species that are largely unique to these areas, for which the application of names based on types from temperate and boreal European and North American waters is inappropriate. Ulva ohnoi, a “green tide” species, is reported from Hawaii for the first time.     

Molecular and morphological diversity of Narragansett Bay (RI,USA) Ulva (Ulvales,Chlorophyta) populations

Macroalgal bloom‐forming species occur in coastal systems worldwide. However, due to overlapping morphologies in some taxa, accurate taxonomic assessment and classification of these species can be quite challenging. We investigated the molecular and morphological characteristics of 153 specimens of bloom‐forming Ulva located in and around Narragansett Bay, RI, USA. We analyzed sequences of the nuclear internal transcribed spacer 1 region (ITS1) and the chloroplast‐encoded rbcL; based on the ITS1 data, we grouped the specimens into nine operational taxonomic units (OTUs). Eight of these OTUs have been previously reported to exist, while one is novel. Of the eight OTUs, all shared sequence identity with previously published sequences or differed by less than 1.5% sequence divergence for two molecular markers. Previously, 10 species names were reported for Ulva in Rhode Island (one blade and nine tube‐forming species) based upon morphological classification alone. Of our nine OTUs, three contained blade‐forming specimens (U. lactuca, U. compressa, U. rigida), one OTU had a blade with a tubular stipe, and six contained unbranched and/or branched tubular morphologies (one of these six, U. compressa, had both a blade and a tube morphology). While the three blade‐forming OTUs in Narragansett Bay can frequently be distinguished by careful observations of morphological characteristics, and spatial/temporal distribution, it is much more difficult to distinguish among the tube‐forming specimens based upon morphology or distribution alone. Our data support the molecular species concept for Ulva, and indicate that molecular‐based classifications of Ulva species are critical for proper species identification, and subsequent ecological assessment or mitigation of Ulva blooms.     

Systematics of green algae resembling Ulva conglobata,with a description of Ulva adhaerens sp. nov. (Ulvales,Ulvophyceae)

A phylogenetic and morphological study of green algae resembling Ulva conglobata from Japan was undertaken, along with morphological observations of the original material of U. conglobata Kjellman. The samples resembling U. conglobata included five genetically distinct species: U. fasciata, U. pertusa, U. tanneri, Ulva sp. 1 and Ulva sp. 2. The discovery of marginal denticulations in some of the original material of U. conglobata, made it possible to distinguish those species without denticulations: U. pertusa, U. tanneri and Ulva sp. 2. The morphological characteristics of Ulva sp. 1 matched those of U. conglobata, but Ulva sp. 1 was not clearly identified as U. conglobata owing to the lack of DNA sequence data of the original material. Ulva sp. 2 had lobes adhering to each other by rhizoids. This morphological feature is stable in Ulva sp. 2 and unique among Ulva species. In conjunction with the molecular data, Ulva sp. 2 was described as a new species, U. adhaerens sp. nov. This species features rhizoidal extensions in regions other than the base and an elaborate arrangement of the extensions used for adhesion. It thereby expands our knowledge of the morphogenesis of the morphologically simple genus Ulva.     

Ulva (Chlorophyta,Ulvales) Biodiversity in the North Adriatic Sea (Mediterranean,Italy): Cryptic Species and New Introductions

Ulva Linnaeus (Ulvophyceae, Ulvales) is a genus of green algae widespread in different aquatic environments. Members of this genus show a very simple morphology and a certain degree of phenotypic plasticity, heavily influenced by environmental conditions, making difficult the delineation of species by morphological features alone. Most studies dealing with Ulva biodiversity in Mediterranean waters have been based only on morphological characters and a modern taxonomic revision of this genus in the Mediterranean is not available. We report here the results of an investigation on the diversity of Ulva in the North Adriatic Sea based on molecular analyses. Collections from three areas, two of which subject to intense shipping traffic, were examined, as well as historical collections of Ulva stored in the Herbarium Patavinum of the University of Padova, Italy. Molecular analyses based on partial sequences of the rbcL and tufA genes revealed the presence of six different species, often with overlapping morphologies: U. californica Wille, U. flexuosa Wulfen, U. rigida C. Agardh, U. compressa Linnaeus, U. pertusa Kjellman, and one probable new taxon. U. californica is a new record for the Mediterranean and U. pertusa is a new record for the Adriatic. Partial sequences obtained from historical collections show that most of the old specimens are referable to U. rigida. No specimens referable to the two alien species were found among the old herbarium specimens. The results indicate that the number of introduced seaweed species and their impact on Mediterranean communities have been underestimated, due to the difficulties in species identification of morphologically simple taxa as Ulva.     

Ulva diversity in the Yellow Sea during the large‐scale green algal blooms in 2008–2009

In the Yellow Sea of China, large‐scale green tides have broken out for three consecutive years from 2007 to 2009. As part of the efforts to localize the algal source, two cruises were conducted in the early stage and the outbreak stage of the bloom in 2009. We analyzed the morphological and genetic diversity of drifting Ulva specimens and culture‐derived isolates from seawater sampled in different localities. For phylogenetic analyses, the nuclear encoded ribosomal DNA internal transcribed spacer region (ITS nrDNA) and the plastid encoded large subunit of ribulose‐1, 5‐bisphosphate carboxylase/oxgenase gene (rbcL) were used. Our molecular and morphological data indicate that the dominant free‐floating Ulva species in 2008 and 2009 possibly belonged to a single strain of the U. linza‐procera‐prolifera (LPP) clade. The ITS sequences from bloom‐forming algal samples with dense branches were identical to those from U. linza‐like specimens without branches derived from the Yellow Sea. Microscopic individuals of the dominant Ulva strain were detected in eight stations, revealing that spore dispersal in the water helped to enlarge biomass in the water during the outbreak stage of green tide in the Yellow Sea.     

Free-floating Ulva in the southwest Netherlands: species or morphotypes? a morphological,molecular and ecological comparison

Free-floating Ulva L. biomass in the eutrophic brackish ‘Veerse Meer’ lagoon (southwest Netherlands) consists of four morphologically identified species: U. curvata (Kützing) De Toni, U. lactuca L., U. rigida C. Agardh and U. scandinavica Bliding. U. curvata could be recognized easily because of the characteristic central cavity in the holdfast of the attached plants, the arrangement of cells in rows and the single pyrenoid in each cell. U. rigida was distinguished by the thick thallus and the large number of pyrenoids. The Veerse Meer isolate, however, was slightly different from the isolate from the Oosterschelde estuary (the Netherlands). U. lactuca and U. scandinavica showed a high degree of overlap in thallus thickness and cell size, but U. scandinavica usually possessed more pyrenoids. However, doubts have frequently been expressed about the use of some morphological characters in Ulva taxonomy. To determine the validity of such characters in the identification of Ulva species, the morphological variation within and between morphological species was recorded and a molecular data set generated. To detect possible ecophysiological differences between species, optimum temperatures and salinities for growth were determined experimentally. The sequences of the nuclear ribosomal DNA internal transcribed spacer 2 (ITS2) and flanking regions of U. lactuca, U. rigida and U. scandinavica from the Veerse Meer were all identical, but differed from that of U. rigida from the Oosterschelde estuary. Ulva species from the Veerse Meer were most closely related to U. armoricana and U. rigida from Brittany (2.9% and 3.5% divergence respectively); the difference between U. rigida from the Veerse Meer and from the Oosterschelde estuary was 7.5%. Rooted trees, based on a comparison of these sequences with sequences of other Ulva and Enteromorpha species obtained from the literature, using Monostroma arcticum as outgroup, suggested that Ulva is paraphyletic with respect to Enteromorpha. The optimum temperature for growth of U. curvata was 25?°C; for all other species it was 10?°C. The optimum salinity for growth was 30?°C for all isolates. It is concluded that U. lactuca, U rigida and U. scandinavica from the Veerse Meer are all members of one highly polymorphic species.     

Cryptic diversity of Ulva (Ulvales,Chlorophyta) in the Great Bay Estuarine System (Atlantic USA): introduced and indigenous distromatic species

Distromatic foliose blades of the algal genus Ulva are notoriously difficult to identify due to their simple morphologies and few diagnostic characteristics that often exhibit intraspecific variation and interspecific overlap. Hence, species differentiation is difficult and diversity estimates are often inaccurate. Two major goals of this study were to assess the diversity of distromatic Ulva spp. in the Great Bay Estuarine System (GBES) of New Hampshire and Maine, USA, and to compare historical and present day records of these species. Molecular analysis (using ITS sequences) of field-collected specimens revealed four distinct taxa: Ulva lactuca, U. rigida, U. compressa, and U. pertusa. Prior to molecular screening, Ulva lactuca was the only distromatic Ulva species reported for the GBES. Ulva pertusa and the foliose form of U. compressa are newly recorded for the Northwest Atlantic, and the range of U. rigida has been extended. Molecular analysis of historical herbarium voucher specimens indicates that U. rigida, U. pertusa, and the foliose form of U. compressa have been present in the GBES since at least 1966, 1967, and 1972, respectively. The distromatic morphotype of U. compressa is found only in low salinity areas, which suggests that salinity may influence its morphological development. Molecular and morphological evaluations are critical if we are to distinguish between cryptic taxa, accurately assess biodiversity, and effectively monitor the spread of non-indigenous macroalgae.     

Genetic analysis of the Linnaean Ulva lactuca (Ulvales,Chlorophyta) holotype and related type specimens reveals name misapplications,unexpected origins,and new synonymies

Current usage of the name Ulva lactuca, the generitype of Ulva, remains uncertain. Genetic analyses were performed on the U. lactuca Linnaean holotype, the U. fasciata epitype, the U. fenestrata holotype, the U. lobata lectotype, and the U. stipitata lectotype. The U. lactuca holotype is nearly identical in rbcL sequence to the epitype of U. fasciata, a warm temperate to tropical species, rather than the cold temperate species to which the name U. lactuca has generally been applied. We hypothesize that the holotype specimen of U. lactuca came from the Indo‐Pacific rather than northern Europe. Our analyses indicate that U. fasciata and U. lobata are heterotypic synonyms of U. lactuca. Ulva fenestrata is the earliest name for northern hemisphere, cold temperate Atlantic and Pacific species, with U. stipitata a junior synonym. DNA sequencing of type specimens provides an unequivocal method for applying names to Ulva species.     

Molecular assessment of Ulva (Ulvales,Chlorophyta) diversity in Vietnam including the new species U. vietnamensis

Species diversity of Ulva in Vietnam was investigated using three commonly used genetic markers, the nuclear encoded rDNA ITS region and the plastid encoded rbcL and tufA genes. Single locus species delimitation methods, complemented with morphological and ecological information resulted in the delimitation of 19 species. This diversity is largely incongruent with the traditional understanding of Ulva diversity in Vietnam. Only four species identified in this study, U. lactuca, U. reticulata, U. spinulosa, and U. flexuosa, have been previously reported, and seven species, U. ohnoi, U. tepida, U. chaugulii, U. kraftiorum, U. meridionalis, U. limnetica, and U. aragoënsis, are recorded for the first time from Vietnam. Seven genetic clusters could not be associated with species names with certainty. A new species, U. vietnamensis, is described from marine to brackish coastal areas from southern Vietnam based on its morphological and molecular distinctiveness from the currently known Ulva species. A comparison with recent molecular-based studies of Ulva diversity showed that species composition in Vietnam is similar to that of adjacent countries, including Japan, China, as well as Australia. Our study emphasizes the importance of molecular data in the assessment of Ulva diversity, and indicates that a lot of diversity may still remain to be discovered, especially in tropical regions.     

INVESTIGATIONS INTO SOUTHERN AUSTRALIAN ULVA (ULVOPHYCEAE,CHLOROPHYTA) TAXONOMY AND MOLECULAR PHYLOGENY INDICATE BOTH COSMOPOLITANISM AND ENDEMIC CRYPTIC SPECIES1

Appreciation of the true species diversity of the genus Ulva in Australian waters has been blinkered by the unproved assumption that its representatives there are largely cosmopolitan. As species of Ulva are some of the longest‐standing and most widely reported taxa of macroalgae, the presumption that they are worldwide in distribution has led to most Australian members being equated with species originally described from extra‐Australian type localities. Ulva species can be notoriously difficult to identify due to the few and often variable characters on which classical taxonomic studies focus so that names of specimens in hand, as well as names appearing in historical distribution records, are frequently difficult or impossible to verify. The combination of morphological and molecular analyses, the latter involving both nuclear (internal transcribed spacer [ITS]) and plastid (rbcL) markers, is critically important in taxonomic studies of the genus and has here been applied to selected Ulva populations from mostly cool‐temperate southern Australian localities. It has been determined that habit‐ and anatomy‐based keys of standard taxonomic literature are largely adequate for assigning species names based on classical concepts, but they often obscure a number of cryptic and pseudocryptic species that do not conform to extra‐Australian populations of the same designation, as indicated by the corresponding molecular data. Here, we present six species (Ulva australis Aresch., U. compressa Forssk., U. fasciata Delile, U. intestinalis L., U. laetevirens Aresch., U. tanneri H. S. Hayden et J. R. Waaland) for which anatomical and molecular data were congruent with both classical concepts and GenBank accession data and confirm these as cosmopolitan taxa in Australia. We also present six putative species designations based on anatomy [U. clathrata (Roth) C. Agardh, U. flexuosa Wulfen, U. linza L., U. prolifera O. F. Müll., U. stenophylla Setch. et N. L. Gardner, U. brisbanensis sp. nov.] that are inconsistent with molecular data, suggesting novel or cryptic taxa not represented in GenBank.     

Morphological and molecular characterization of free-floating and attached green macroalgae Ulva spp. in the Yellow Sea of China

During the summer of 2008 and 2009, massive algal blooms repeatedly broke out in the Yellow Sea of China. These were undoubtedly caused by the accumulations of one or more species in the macroalgal genus Ulva. In previous reports, morphological observation indicated that the species involved in this phenomenon is Ulva prolifera but molecular analyses indicated that the species belongs to an Ulva linza–procera–prolifera (LPP) clade. Correct identification of the bloom species is required to understand and manage the blooms, but the taxonomic status of the bloom species remains unclear. In the current study, the taxonomic status of 22 selected specimens from the Yellow Sea was assessed by using both morphological and molecular (ITS and rbcL sequences) data. In addition, 5S rDNA analyses were performed for those samples clustering in the LPP clade, and phylogenetic tree and ribotype analyses were constructed for determining the possible origin of the bloom. Three free-floating and two attached Ulva species were distinguished and described: Ulva compressa Linnaeus and Ulva pertusa Kjellman were found in free-floating samples; U. linza Linnaeus was found on rocks; and U. prolifera O.F. Müller was found in both habitats. Diversity in free-floating Ulva of the Yellow Sea appears to be greater than previously thought. The dominant free-floating Ulva species, U. prolifera, was not closely related to local populations attached to rocks but was closely related to populations from Japan.     

Comparing the low‐salinity tolerance of Ulva species distributed in different environments

The green macroalgal genus Ulva (Ulvales, Ulvophyceae, Chlorophyta) is distributed worldwide from marine to freshwater environments. Comparative analyses of hyposalinity tolerance among marine, brackish, and freshwater Ulva species were performed by fluorescein diacetate viability counts. The subtidal marine species Ulva sp., collected from a depth of 30 m, showed the poorest tolerance to low salinity. This species died in 5 practical salinity units (PSU) artificial seawater or freshwater within 1 day. Its closely related species U. linza L. (an intertidal species) and U. prolifera Müller (a brackish species) showed varying tolerances to low salinity. After 7 days of freshwater exposure, the viability of U. linza L. decreased to approximately 20%, while U. prolifera Müller showed nearly 100% viability. The freshwater species U. limnetica Ichihara et Shimada, not yet found in coastal areas, was highly viable in seawater.     

Molecular phylogenetic analyses of the Japanese Ulva and Enteromorpha (Ulvales, Ulvophyceae), with special reference to the free-floating Ulva

In order to elucidate the species composition of free‐floating Ulva that cause green tide in several bays in Japan, and to clarify the generic status of Ulva and Enteromorpha (Ulvales, Ulvophyceae), the nuclear encoded internal transcribed spacer (ITS) region including the 5.8S gene and the plastid encoded large subunit of ribulose‐1, 5‐bisphosphate carboxylase/ oxgenase (rbcL) gene sequences for 15 species were determined. Both ITS and rbcL analyses indicate that free‐floating Ulva samples are divided into four different lineages that correspond to Ulva lactuca Linnaeus, U. pertusa Kjellman, U. armoricana Dion etal. and U. fasciata Delile. These four species are distinguished by cell morphology including the arrangement of cells, the shape and size of cells and the position of chloroplasts. Molecular data also indicated that Ulva and Enteromorpha are not separated as respective monophyletic groups within a large monophyletic clade and congeneric as shown by previous molecular studies using the ITS sequences alone. This strongly suggests that these genera are congeneric and Enteromorpha should be reduced to the synonym of Ulva.     

Phylogeography of the genus <Emphasis Type="Italic">Ulva</Emphasis> (Ulvophyceae,Chlorophyta), with special reference to the Japanese freshwater and brackish taxa

The nuclear-encoded ITS and associated 5.8S rDNA regions were sequenced for 72 specimens of Ulva collected from 44 rivers across Japan, including U. prolifera Müller from the Shimanto River, Kochi Prefecture, as well as 26 samples originally identified as U. linza L. from 20 coastal marine areas. Sequence data revealed that the samples fall into six distinct clades: the U. flexuosa Wulfen clade (2 samples), the Ulva linza-procera-prolifera (LPP) complex clade (75 samples), Ulva sp. 1 clade (3 samples), Ulva sp. 2 clade (7 samples), Ulva sp. 3 clade (4 samples) and Ulva sp. 4 clade (7 samples). The LPP complex contained a mixture of 26 samples collected from seashores and 49 samples obtained from rivers, including U. prolifera from the Shimanto River, and GenBank data for U. linza and U. procera Ahlner. The samples of the LPP complex differed by only 0–7 substitutions (0–1.149%). Subsequent phylogeographic analyses of the LPP complex based on the 5S rDNA spacer region revealed the presence of two further groupings: a group including 22 strictly marine littoral U. linza samples and a U. prolifera group composed of a mixture of 4 marine samples and all 49 river samples. The monophyly of all river samples indicates that adaptation to low salinity might have occurred only once in the evolutionary history of the LPP complex.     

A Molecular Survey of Ulva (Chlorophyta) in Temperate Australia Reveals Enhanced Levels of Cosmopolitanism

The green algal genus Ulva includes a speciose group of marine macroalgae inhabiting shallow seas worldwide. Although algal blooms in Asia highlight the opportunistic nature of several “nuisance” species, recent research clearly reveals important positive benefits of Ulva. Applied research requires accurate, reliable, and rapid identification, however, identification of Ulva spp. has met with con‐siderable difficulty. Consequently, many have turned to molecular markers to aid in taxonomy. Previous studies of plants and algae have relied heavily on ITS and rbcL. Recently, tufA has been presented as a suitable barcoding gene to facilitate species‐level identification of green macroalgae and it is used here to explore the diversity of Ulva spp. in temperate Australia. Ninety Ulva specimens collected from 38 sites across five states were sequenced for this gene region with exemplars from each genetic group also sequenced for rbcL to test for congruence. Collections of Australian Ulva spp. were compared to samples from Asia and North America and exhibited trends consistent with recent studies in terms of species relationships. Results support an overwhelmingly cosmopolitan flora in temperate Australia that contrasts with other Australasian surveys of Ulva that report a greater number of endemics and new species. Four new records, as well as numerous range extensions for taxa already known from the country, are documented. Evidence for three nonindigenous Ulva species in temperate Australia is discussed.     

Exhaustive reanalysis of barcode sequences from public repositories highlights ongoing misidentifications and impacts taxa diversity and distribution

Accurate species identification often relies on public repositories to compare the barcode sequences of the investigated individual(s) with taxonomically assigned sequences. However, the accuracy of identifications in public repositories is often questionable, and the names originally given are rarely updated. For instance, species of the Sea Lettuce (Ulva spp.; Ulvophyceae, Ulvales, Ulvaceae) are frequently misidentified in public repositories, including herbaria and gene banks, making species identification based on traditional barcoding unreliable. We DNA barcoded 295 individual distromatic foliose strains of Ulva from the North-East Atlantic for three loci (rbcL, tufA, ITS1). Seven distinct species were found, and we compared our results with all worldwide Ulva spp. sequences present in the NCBI database for the three barcodes rbcL, tufA and the ITS1. Our results demonstrate a large degree of species misidentification, where we estimate that 24%–32% of the entries pertaining to foliose species are misannotated and provide an exhaustive list of NCBI sequences reannotations. An analysis of the global distribution of registered samples from foliose species also indicates possible geographical isolation for some species, and the absence of U. lactuca from Northern Europe. We extended our analytical framework to three other genera, Fucus, Porphyra and Pyropia and also identified erroneously labelled accessions and possibly new synonymies, albeit less than for Ulva spp. Altogether, exhaustive taxonomic clarification by aggregation of a library of barcode sequences highlights misannotations and delivers an improved representation of species diversity and distribution.     

TRUE IDENTITY OF THE EUROPEAN FRESHWATER ULVA (CHLOROPHYTA,ULVOPHYCEAE) REVEALED BY A COMBINED MOLECULAR AND MORPHOLOGICAL APPROACH1

A set of 18 freshwater and morphologically similar marine samples of Ulva were collected from inland and coastal waters throughout Europe to assess their taxonomic identity and invasive potential. An additional 11 specimens were obtained from herbaria. The material was studied using a combination of classical morphological methods and molecular techniques; the latter included sequencing of the nuclear internal transcribed spacer (ITS) region (ITS1‐5.8S‐ITS2) and the chloroplast RUBISCO LSU (rbcL) gene and comparison of the ITS2 secondary structure predictions. Based on classical methods, all the specimens could be determined as U. flexuosa Wulfen and could be further divided into three groups matching three infraspecific taxa. This pattern was generally well supported by molecular phylogenetic analyses. All sequenced samples formed a monophyletic lineage within Ulva, showing a putative synapomorphy in the ITS2 secondary structure. The individual subspecies corresponded to phylogenetic clusters within this lineage. In freshwater habitats, the dominant taxon was U. flexuosa subsp. pilifera, but subsp. paradoxa was also occasionally recorded. In marine habitats, only U. flexuosa subsp. flexuosa and subsp. paradoxa were located. These findings support the view that U. flexuosa subsp. pilifera is primarily a freshwater alga that probably dominates in Europe. As confirmed by the study of herbarium specimens, U. flexuosa should be regarded as indigenous, although it has a tendency to form blooms under certain conditions. Besides clarifying the identity of prevailing European freshwater Ulva, the study provides novel data concerning the distribution and morphological plasticity within the U. flexuosa complex.