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.     

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.     

Seasonal variation of dominant free-floating and attached Ulva species in Rudong coastal area,China

In this paper, species compositions and seasonal variations of attached Ulva species on Porphyra aquaculture rafts and free floating Ulva species at Rudong coastal area, Jiangsu Province of China were investigated during 2010–2011. Based on the sequences analysis of nuclear-encoded ITS (including 5.8S rDNA regions) and 5S rDNA spacer regions, dominant species of both attached and free-floating Ulva samples were identified as Ulva compressa, Ulva linza, Ulva prolifera and Ulva flexuosa. Phylogenetic tree based on sequences of ITS and 5S rDNA spacer regions for attached and free-floating Ulva species was constructed, respectively. Species compositions of the Ulva population attached on aquaculture rafts varied with seasons, and U. prolifera was only found on aquaculture rafts in March 2011 during the 2010–2011 Porphyra yezoensis cultivation season, which had the same sequences of ITS and 5S rDNA spacer regions as that of the dominant species bloomed in the Yellow Sea of China in 2008. Dominant species of the free-floating Ulva population at the early stage of the green tide were U. compressa, U. flexuosa, and U. linza. Free-floating U. prolifera appeared in the middle of May, 2011. ITS sequence similarity rates of U. compressa and U. flexuosa between the attached and free-floating species were 100%. And ITS and 5S rDNA spacer sequences of the attached and the free-floating U. prolifera population also showed no differences. Further study showed that there were two types of free-floating U. prolifera population (Type 5S-A and Type 5S-B) based on 5S rDNA spacer sequences. The present study would provide some useful information for clarifying the outbreak mechanism of green tides occurred in the Yellow Sea, China.     

Examination of prezygotic and postzygotic isolating barriers in tropical Ulva (Ulvophyceae,Chlorophyta): evidence for ongoing speciation

Phylogenetic clades based on DNA sequences such as the chloroplast rbcL gene and the nuclear ITS region are frequently used to delimit algal species. However, these molecular markers cannot accurately delimit boundaries among some Ulva species. Although Ulva reticulata and Ulva ohnoi occasionally bloom in tropical to warm‐temperate regions and are clearly distinguishable by their reticulate or plain blade morphology, they have few or no sequence divergences in these molecular markers and form a monophyletic clade. In this study, to clarify the speciation and species delimitation in the U. reticulata‐ohnoi complex clade, reproductive relationships among several sexual strains from the Philippines and Japan including offspring that originated from the type specimen of U. ohnoi were examined by culturing and hybridization in addition to the ITS‐based analysis. As a result, both prezygotic and postzygotic reproductive isolation were revealed to occur between genetically perforated U. reticulata and imperforate U. ohnoi. They were also separated on the basis of sequence analysis of the ITS region. That strongly supports that the two taxa are independent biological species. Although no prezygotic barrier among the Philippine and Japanese strains of U. reticulata was observed, unexpectedly zoospores produced by hybrid sporophytes in some of their combinations mostly failed to develop, indicating partial formation of a postzygotic barrier despite a 0.2% divergence in the ITS sequence. These findings suggest speciation is still ongoing in U. reticulata.     

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.     

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.     

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.     

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.     

A new green-tide-forming alga, Ulva ohnoi Hiraoka et Shimada sp. nov. (Ulvales, Ulvophyceae) from Japan

Ulva ohnoi Hiraoka et Shimada sp. nov. (Ulvales, Ulvophyceae) is described from southern and western Japan and is characterized by the following combination of features: (i) the large, fragile, easily torn thalli, which are 30–55 μm thick in the upper and middle regions and often have microscopic marginal teeth; (ii) the production of zoids in the upper marginal region; (iii) a regular alternation of dioecious gametophytes and a sporophyte; (iv) the production of free‐floating thalli from torn‐off attached thalli, which reproduce vegetatively by fragmentation and form green tides in summer to autumn; (v) disorderly arranged cells that are polygonal or quadrangular in the upper and middle regions; and (vi) the chloroplast covering the outer face of cell, with 1–3 pyrenoids. Ulva ohnoi differs from U. armohcana Dion et al., U. fasciata Delile, U. reticulata Forsskal, U. scandinavica Eliding and U. spiulosaOkamura et Segawa, which all possess microscopic marginal serrations, in thallus shape, cell shape or life history pattern. It is also distinguished from morphologically similar species by sequences of the nuclear encoded internal transcribed spacers and the 5.8S ribosomal RNA gene and the plastid encoded large subunit of ribulose‐l,5‐bisphosphate carboxylase/oxgenase gene. Furthermore, crossing tests demonstrate that there is a reproductive boundary between U. ohnoi and the most closely related species, U. fasciata and U. reticulata.     

Ulva and Enteromorpha as indicators of heavy metal pollution

We studied the use of two genera of green macroalgae, Ulva and Enteromorpha, as indicators of heavy metal contamination on the northwest coast of Spain. We evaluated the extent of external contamination by fine particles adhering to the algal thallus and found that although not a problem in Ulva, it may be in Enteromorpha samples. The mean levels of metals in both seaweeds were in accordance with previously reported levels in different species of the genera in clean areas. A large number of significant correlations between levels of metals in macroalgae and in sediment (for both the total and labile fractions) were found. However, almost all of the coefficients of correlation decreased after geochemical normalization of sediment metal concentrations.     

PHYLOGENETIC SYSTEMATICS OF THE ULVACEAE (ULVALES,ULVOPHYCEAE) USING CHLOROPLAST AND NUCLEAR DNA SEQUENCES1

Systematic hypotheses for the Ulvaceae were tested using phylogenetic analysis of sequences for the gene encoding the large subunit of RUBISCO, small subunit rDNA and a combined data matrix. Representatives of eight putative ulvaceous genera and twelve additional taxa from the Ulvophyceae and Trebouxiophyceae were included in analyses using maximum parsimony and maximum likelihood criteria. Molecular data supported hypotheses for the Ulvaceae that are based on the early development of vegetative thalli and motile cell ultrastructure. Ulvaceae sensu Floyd and O'Kelly, including Percursaria Bory de Saint‐Vincent, Ulvaria Ruprecht and a complex of closely related species of Chloropelta Tanner, Enteromorpha Link and Ulva L. was supported; however, monophyly of Enteromorpha and Ulva was not supported. The Ulvales and Ulotrichales sensu Floyd and O'Kelly were monophyletic. Blidingia Kylin and Kornmannia Bliding were allied with the former and Capsosiphon Gobi with the latter, although relationships among these and other taxa in these orders remain uncertain. The Ulvales are characterized by an isomorphic life history pattern, gametangia and sporangia that are identical in structure and development, motile cells with bilobed terminal caps and proximal sheaths consisting of two equal subunits. Method of motile cell release and the gross morphology of vegetative thalli are not systematically reliable characters.     

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.     

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.     

Identification of species in the angiosperm family Apiaceae using DNA barcodes

Apiaceae (Umbelliferae) is a large angiosperm family that includes many medicinally important species. The ability to identify these species and their adulterants is important, yet difficult to do so because of their subtle fruit morphological differences and often lack of diagnostic features in preserved specimens. Moreover, dried roots are often the official medical organs, making visual identification to species almost impossible. DNA barcoding has been proposed as a powerful taxonomic tool for species identification. The Consortium for the Barcode of Life (CBOL) Plant Working Group has recommended the combination of rbcL+matK as the core plant barcode. Recently, the China Plant BOL Group proposed that the nuclear ribosomal DNA internal transcribed spacer (ITS), as well as a subset of this marker (ITS2), be incorporated alongside rbcL+matK into the core barcode for seed plants, particularly angiosperms. In this study, we assess the effectiveness of these four markers plus psbA‐trnH as Apiaceae barcodes. A total of 6032 sequences representing 1957 species in 385 diverse genera were sampled, of which 211 sequences from 50 individuals (representing seven species) were newly obtained. Of these five markers, ITS and ITS2 showed superior results in intra‐ and interspecific divergence and DNA barcoding gap assessments. For the matched data set (173 samples representing 45 species in five genera), the ITS locus had the highest identification efficiency (73.3%), yet ITS2 also performed relatively well with 66.7% identification efficiency. The identification efficiency increased to 82.2% when using an ITS+psbA‐trnH marker combination (ITS2+psbA‐trnH was 80%), which was significantly higher than that of rbcL+matK (40%). For the full sample data set (3052 ITS sequences, 3732 ITS2 sequences, 1011 psbA‐trnH sequences, 567 matK sequences and 566 rbcL sequences), ITS, ITS2, psbA‐trnH, matK and rbcL had 70.0%, 64.3%, 49.5%, 38.6% and 32.1% discrimination abilities, respectively. These results confirm that ITS or its subset ITS2 be incorporated into the core barcode for Apiaceae and that the combination of ITS/ITS2+psbA‐trnH has much potential value as a powerful, standard DNA barcode for Apiaceae identification.     

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.     

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.     

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.     

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.     

Discrimination of the common macroalgae (Ulva and Blidingia) in coastal waters of Yellow Sea,northern China,based on restriction fragment-length polymorphism (RFLP) analysis

Since 2007, reoccurring large-scale green algae blooms have caused deleterious effects to the estuarine ecosystem of Yellow Sea, northern China and subsequent economical losses. Previous surveys indicated the green tides were initiated in the coastal water of southern Jiangsu province where Porphyra farming was intensively conducted; however, the main ‘seed source’ of floating green algae is still under debate. Ulva prolifera was confirmed to be the major causative species of green tides. The multiple sympatric ulvoid species in the natural environment has complicated species identification in both field surveys and laboratory studies due to their morphological plasticity. Thus, we developed a genetic identification key based on restriction fragment length polymorphism (RFLP) analysis of the ITS nuclear marker to discriminate the common Ulva and Blidingia species in the Yellow Sea. Ten genetic lineages (1 in Blidingia, 9 in Ulva) were detected along the coast of China through phylogenetic analysis of ITS sequences. They can be separated by virtual restriction digestion using the four selected restriction enzymes (BspT107 I, EcoO109 I, Hin1 I and VpaK11B I). With additional PCR amplification of the 5S spacer region, we were able to discriminate U. prolifera from Ulva linza. Using this genetic key, we screened macroalgal samples collected from the coast of the Yellow Sea, and the results indicated 6 common lineages (U. prolifera, U. linza, Ulva compressa, Ulva pertusa, Clade 6 and Blidingia sp.) in this region, which could be explicitly distinguished by a single enzyme (BspT107 I) coupled with 5S spacer polymorphism. U. prolifera was confirmed to be present on the Porphyra aquaculture rafts with seasonal variation in the species composition. This genetic key will facilitate our long-term field surveys to investigate the origin of the floating U. prolifera and furthermore to explore its bloom dynamics along the coast of the Yellow Sea. It also provided a framework for the future inclusion of more Ulva species, which will expand the usage of this key.     

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.