Universal Plastid Amplicon Research Articles

Seasonality of tropical airborne algae: a 16-month study based on high-throughput sequencing in the Hawaiian Islands

The airborne environment is a critical juncture linking terrestrial and aquatic habitats, yet the algae of this environment are not well-characterised and understood. The goal of this study was to examine seasonality patterns of airborne algae in a tropical environment. Airborne algae were collected monthly in spore traps on a building rooftop on the island of Oʻahu, Hawaiʻi, U.S.A. from December 2015 to March 2017. Metabarcoding of Universal Plastid Amplicon (UPA) and 16S ribosomal RNA (rRNA) markers was used to characterise the algal diversity in the samples relative to meteorological data. Modelled back-trajectories of air masses arriving at the site demonstrated that months with the highest rainfall corresponded to those with the strongest tradewind signal. Thirty airborne algal Exact Sequence Variant (ESV)s were identified using the UPA marker including Cyanobacteria (20), Chlorophyta (4), Streptophyta (3), Chrysophyceae (1), Synurophyceae (1), and unknown Stramenopila (1). Forty-four ESVs were identified using the 16S marker and consisted of Cyanobacteria (43) and plastids of Streptophyta (1). The number and diversity of sequence reads obtained varied strongly both among samples for a given time period and from month to month. Highest rainfall and air temperatures corresponded to months with the largest cyanobacterial relative abundance according to the UPA marker. Although cyanobacteria were the most common group identified using the 16S marker, the dominant cyanobacterial taxon varied by month. Diversity or abundance patterns were not explained by meteorological conditions, which may be due to the relatively small annual amplitude in conditions in the Hawaiian Islands.

A metabarcoding comparison of windward and leeward airborne algal diversity across the Ko'olau mountain range on the island of O'ahu, Hawai'i1.

Airborne algae from sites on the windward (n=3) and leeward (n=3) sides of the Ko'olau Mountain range of O'ahu, Hawai'i, were sampled for a 16 d period during January and February 2015 using passive collection devices and were characterized using Illumina MiSeq sequencing of the universal plastid amplicon marker. Amplicons were assigned to 3,023 operational taxonomic units (OTUs), which included 1,189 cyanobacteria, 1,009 heterotrophic bacteria, and 304 Eukaryota (of which 284 were algae and land plants). Analyses demonstrated substantially more OTUs at windward than leeward O'ahu sites during the sampling period. Removal of nonalgal OTUs revealed a greater number of algal reads recovered from windward (839,853) than leeward sites (355,387), with the majority of these being cyanobacteria. The 1,234 total algal OTUs included cyanobacteria, diatoms, cryptophytes, brown algae, chlorophyte green algae, and charophyte green algae. A total of 208 algal OTUs were identified from leeward side samplers (including OTUs in common among samplers) and 1,995 algal OTUs were identified from windward samplers. Barcoding analyses of the most abundant algal OTUs indicated that very few were shared between the windward and leeward sides of the Ko'olau Mountains, highlighting the localized scale at which these airborne algae communities differ. Back trajectories of air masses arriving on O'ahu during the sampling period were calculated using the NOAA HY-SPLIT model and suggested that the sampling period was composed of three large-scale meteorological events, indicating a diversity of potential sources of airborne algae outside of the Hawaiian Islands.

A New Species of Rhodymeniaceae (Rhodophyta, Rhodymeniales) from the Northern Brazilian Coast: Botryocladia franciscana sp. nov.

A new species of marine macroalgae, Botryocladia franciscana sp. nov. (Rhodophyta, Rhodymeniaceae) is described for the south-west Atlantic Ocean. B. franciscana was collected from protected rocky crevices at Coqueiro Beach, Piaui, Brazil. Molecular characterization was based on two plastidial genes, the V domain of the 23S rRNA gene (UPA, universal plastid amplicon), and the large subunit of ribulose-1, 5-bisphosphate carboxylase oxygenase gene (rbcL), and the mitochondrial cytochrome oxidase I gene (cox1). Phylogenetic trees from rbcL sequences clearly set B. franciscana apart from morphologically similar B. bermudana and B. caraibica. Morphological description was performed using anatomical and morphological features, including vesicle medulla and wall thickness, shape and size of the mucous-filled vesicles, pattern of vesicles’ outer cortication, number of secretory cells and type of cell bearing the secretory cells. The observed morphological characters do not correspond to any previously described Botryocladia species and DNA sequence diverge values indicate it is distinct from B. franciscana and B. bermudana; therefore, we present B. franciscana as a new species from the Brazilian coast, with illustrations and morpho-anatomical characteristics.

A New Tropical Species of Gracilariaceae (Rhodophyta, Gracilariales): Gracilaria silviae sp. nov.

A new flat species of Gracilaria, named Gracilaria silviae sp. nov., from the western Atlantic Ocean is described based on comparative morphological and molecular analyses of three molecular markers: the gene encoding cytochrome oxidase I (cox1), the Universal Plastid Amplicon (UPA) and the gene encoding the large subunit of ribulose-1, 5-bisphosphate carboxylase oxygenase (rbcL). Gracilaria silviae has a wide geographic distribution from the Indian River, Florida, USA, to Santa Catarina, southern Brazil, and a wide range of habit phenotypes. Across this geographic range Gracilaria silviae occupies a broad range of habitats, from warm temperate protected estuarine sublittoral areas to exposed tropical intertidal rocky shores and coastal reefs. The most morphologically similar species to G. silviae are not only the phylogenetically closest taxa to this species but also sympatric along its geographic range, i.e. G. tikvahiae and G. isabellana (formerly known as G. lacinulata in the northwestern Atlantic and G. tepocensis in southern Brazil).

DNA barcoding reveals high diversity in the Gelidiales of the Brazilian southeast coast

Abstract Morphological identification of species in the order Gelidiales can be difficult and controversial because of phenotypic plasticity, the low numbers of reproductive specimens and poorly defined taxonomic boundaries. A DNA barcoding survey of Brazilian specimens of Gelidiales, employing neighbor-joining and Automatic Barcode Gap Discovery analyses, indicated the presence of 23 statistically robust primary species hypotheses (PSH). In addition to the cytochrome oxidase I gene (COI-5P), the chloroplast universal plastid amplicon (UPA) marker was also sequenced and submitted to the same analyses. Representatives of each COI-5P/UPA PSH were selected for rbcL sequence analysis to further corroborate the occurrence of 23 species and to infer their phylogenetic relationships. These analyses confirmed the identity of six species previously cited for Brazil: Gelidiella acerosa, G. ligulata, Gelidium crinale, G. floridanum, Pterocladiella bartlettii and P. capillacea. Three new reports for Brazil were also detected: Gelidium microdonticum, Pterocladiella beachiae and P. australafricanensis. Fourteen species remain unidentified and require detailed morphological evaluation.

Phylogeny of Gracilariaceae (Rhodophyta): evidence from plastid and mitochondrial nucleotide sequences.

Gracilariaceae are mostly pantropical red algae and include ~230 species in seven genera. Infrafamilial classification of the group has long been based on reproductive characters, but previous phylogenies have shown that traditionally circumscribed groups are not monophyletic. We performed phylogenetic analyses using two plastid (universal plastid amplicon and rbcL) and one mitochondrial (cox1) loci from a greatly expanded number of taxa to better assess generic relationships and understand patterns of character distributions. Our analyses produce the most well-supported phylogeny of the family to date, and indicate that key characteristics of spermatangia and cystocarp type do not delineate genera as commonly suggested. Our results further indicate that Hydropuntia is not monophyletic. Given their morphological overlap with closely related members of Gracilaria, we propose that Hydropuntia be synonymized with the former. Our results additionally expand the known ranges of several Gracilariaceae species to include Brazil. Lastly, we demonstrate that the recently described Gracilaria yoneshigueana should be synonymized as G.domingensis based on morphological and molecular characters. These results demonstrate the utility of DNA barcoding for understanding poorly known and fragmentary materials of cryptic red algae.

Taxonomic reassessment of Gracilaria cearensis (Rhodophyta, Gracilariales), a poorly defined yet common flattened species based on morphological and molecular analysis including topotype collections

The genus Gracilaria is the major source of agarose in the world today and is one of the most species-rich genera in the Rhodophyta. Flat Gracilaria species are among the most taxonomically challenging taxa due to their widespread phenotypic plasticity among all species. Gracilaria cearensis is a flat species described in 1965 from Brazil. Its original description is not conducive to accurate taxonomic identifications and the delineation of this taxon remains elusive. New samples of G. cearensis were collected across a coastal length of 500 km including its type locality. Universal Plastid Amplicon (UPA) and the rbcL gene were used to confirm the phylogenetic and taxonomic status of this poorly known species. Results showed that this is a distinct species despite morphological similarities to G. hayi, G. cuneata, G. curtissiae, G. brasiliensis and G. galetensis. G. cearensis is sister to G. hayi yet the genetic divergence between these two species was 2% for rbcL and 1.08% for UPA, enough to consider them distinct taxa. However, newly generated molecular data placed G. smithsoniensis, another morphologically similar species, as conspecific with G. cearensis with only 0.07% rbcL sequence divergence between them.

Comparison of potential diatom 'barcode' genes (the 18S rRNA gene and ITS, COI, rbcL) and their effectiveness in discriminating and determining species taxonomy in the Bacillariophyta.

Diatoms form an enormous group of photoautotrophic micro-eukaryotes and play a crucial role in marine ecology. In this study, we evaluated typical genes to determine whether they were effective at different levels of diatom clustering analysis to assess the potential of these regions for barcoding taxa. Our test genes included nuclear rRNA genes (the nuclear small-subunit rRNA gene and the 5.8S rRNA gene+ITS-2), a mitochondrial gene (cytochrome c-oxidase subunit 1, COI), a chloroplast gene [ribulose-1,5-biphosphate carboxylase/oxygenase large subunit (rbcL)] and the universal plastid amplicon (UPA). Calculated genetic divergence was highest for the internal transcribed spacer (ITS; 5.8S+ITS-2) (p-distance of 1.569, 85.84% parsimony-informative sites) and COI (6.084, 82.14%), followed by the 18S rRNA gene (0.139, 57.69%), rbcL (0.120, 42.01%) and UPA (0.050, 14.97%), which indicated that ITS and COI were highly divergent compared with the other tested genes, and that their nucleotide compositions were variable within the whole group of diatoms. Bayesian inference (BI) analysis showed that the phylogenetic trees generated from each gene clustered diatoms at different phylogenetic levels. The 18S rRNA gene was better than the other genes in clustering higher diatom taxa, and both the 18S rRNA gene and rbcL performed well in clustering some lower taxa. The COI region was able to barcode species of some genera within the Bacillariophyceae. ITS was a potential marker for DNA based-taxonomy and DNA barcoding of Thalassiosirales, while species of Cyclotella, Skeletonema and Stephanodiscus gathered in separate clades, and were paraphyletic with those of Thalassiosira. Finally, UPA was too conserved to serve as a diatom barcode.

DNA barcode assessment of Ceramiales (Rhodophyta) in the intertidal zone of the northwestern Yellow Sea

A total of 142 specimens of Ceramiales (Rhodophyta) were collected each month from October 2011 to November 2012 in the intertidal zone of the northwestern Yellow Sea. These specimens covered 21 species, 14 genera, and four families. Cluster analyses show that the specimens had a high diversity for the three DNA markers, namely, partial large subunit rRNA gene (LSU), universal plastid amplicon (UPA), and partial mitochondrial cytochrome c oxidase subunit I gene (COI). No intraspecific divergence was found in our collection for these markers, except for a 1–3 bp divergence in the COI of Ceramium kondoi, Symphyocladia latiuscula, and Neosiphonia japonica. Because short DNA markers were used, the phylogenetic relationships of higher taxonomic levels were hard to evaluate with poor branch support. More than half species of our collection failed to find their matched sequences owing to shortage information of DNA barcodes for macroalgae in GenBank or BOLD (Barcode of Life Data) Systems. Three specimens were presumed as Heterosiphonia crispella by cluster analyses on DNA barcodes assisted by morphological identification, which was the first record in the investigated area, implying that it might be a cryptic or invasive species in the coastal area of northwestern Yellow Sea. In the neighbor-joining trees of all three DNA markers, Heterosiphonia japonica converged with Dasya spp. and was distant from the other Heterosiphonia spp., implying that H. japonica had affinities to the genus Dasya. The LSU and UPA markers amplified and sequenced easier than the COI marker across the Ceramiales species, but the COI had a higher ability to discriminate between species.

Characterization of Hawaiian freshwater and terrestrial cyanobacteria reveals high diversity and numerous putative endemics

SummaryFifty samples of cyanobacteria were collected and characterized as part of the Hawaiian Freshwater Algal Biodiversity Survey. Fifty‐two percent of the samples originated from Oahu, while the remainder were collected from Kauai (14%), Maui (20%) and Hawaii (14%). A diversity of habitats (e.g. streams, wet walls, taro fields, terrestrial areas and ditches) was represented by the collection sites, which is reflective of the abundance of suitable non‐marine algal substrata in these isolated, humid, subtropical islands. Most samples were isolated and cultured for observation of morphological features, and all were sequenced for both the Universal Plastid Amplicon (UPA) marker (partial 23S rRNA) and 16S rRNA gene. Alignments of both markers (separately and concatenated) with additional GenBank sequences for phylogenetic representation were analyzed using Maximum Likelihood, Bayesian and Maximum Parsimony approaches. Nodal support was highest for the concatenated UPA+16S rRNA gene alignment, and phylogenetic analyses indicated a monophyletic Nostochophycidae, and monophyly, although with low support, for the Oscillatoriophycidae and Synechococcophycidae. A conservative estimate is that 11 of the taxa are putative endemics to the Hawaiian Islands, further highlighting the uniqueness of this isolated and understudied flora, and the potential for discovery of novel taxa through taxon‐focused biodiversity surveys.

Taxonomy of Grateloupia (Halymeniales, Rhodophyta) by DNA barcode marker analysis and a description of Pachymeniopsis volvita sp. nov.

We applied DNA barcoding analysis to the red alga Grateloupia because it is one of the most complicated taxa in the family Halymeniaceae. We used two DNA barcoding markers, the 5′ end of the cytochrome c oxidase I gene and the universal plastid amplicon of the 23S rRNA gene, to define levels of genetic diversity and species relationships within rbcL phylogeny. We detected nine species of Grateloupia, four species of Pachymeniopsis, and one Kintokiocolax in Korea: G. angusta, G. asiatica, G. catenata, G. divaricata, G. imbricata, G. jejuensis, G. kurogii, G. subpectinata, G. turuturu, P. elliptica, P. gargiuli, P. lanceolata, a new species, and K. aggregato-cerantha. The COI-5P barcode successfully differentiated species, with interspecific divergence values ranging between 3.7 and 14 %. Barcoding data provided a rapid and accurate methodology for species-level identification and for taxonomic explorations when used in combination with morphological data. We describe Pachymeniopsis volvita sp. nov. based on DNA barcoding analysis. Fourteen species detected in four different subclades with strong bootstrap support within rbcL phylogenetic tree.

Resolving species diversity in the red algal genus Callophyllis (Kallymeniaceae, Gigartinales) in Canada using molecular assisted alpha taxonomy

The COI-5P DNA barcode (the 5′ region of the mitochondrial cytochrome c oxidase I gene) has proven an effective tool for quickly screening large numbers of biological specimens and assigning them to genetic species groups for subsequent examination in an approach known as molecular-assisted alpha taxonomy (MAAT). In the present study, we applied MAAT to Canadian species of the red algal genus Callophyllis in order to resolve confusion surrounding this taxonomically perplexing group. A total of 563 Callophyllis specimens were examined (either molecularly, morphologically or both) – 504 from British Columbia along the Pacific coast of Canada and 59 from the U.S.A., Australia and Chile. In total, 16 genetic species groups were resolved including 12 in Canada although only nine species are reported in the flora. Here we report range extensions of C. beringensis and C. radula from the NW Pacific, C. dissecta from California, U.S.A. and the Chilean species C. variegata (the type species) into the Canadian flora. Our results also uncovered cryptic diversity in the genus necessitating the description of C. schneideri Clarkston & G.W. Saunders sp. nov. Subsequent analyses of a subset of specimens using the internal transcribed spacer of the ribosomal cistron (ITS) resolved the same groups as COI-5P while the universal plastid amplicon (UPA, domain V of theplastid 23S rRNA gene) had poor resolution at the species level and in one case underestimated species diversity. Finally, large-subunit ribosomal DNA (LSU), nuclear elongation factor 2 (EF2), COI-5P and a combined LSU, EF2 and COI-5P alignment were subjected to phylogenetic analyses in order to investigate relationships among Canadian species of Callophyllis. A significant outcome was the resolution of two well-supported lineages for the monocarpogonial versus polycarpogonial species of this genus, as had been posited in the literature.

Applications of three DNA barcodes in assorting intertidal red macroalgal flora in Qingdao, China

This study is part of the endeavor to construct a comprehensive DNA barcoding database for common seaweeds in China. Identifications of red seaweeds, which have simple morphology and anatomy, are sometimes difficult solely depending on morphological characteristics. In recent years, DNA barcode technique has become a more and more effective tool to help solve some of the taxonomic difficulties. Some DNA markers such as COI (cytochrome oxidase subunit I) are proposed as standardized DNA barcodes for all seaweed species. In this study, COI, UPA (universal plastid amplicon, domain V of 23S rRNA), and ITS (nuclear internal transcribed spacer) were employed to analyze common species of intertidal red seaweeds in Qingdao (119.3A degrees-121A degrees E, 35.35A degrees-37.09A degrees N). The applicability of using one or a few combined barcodes to identify red seaweed species was tested. The results indicated that COI is a sensitive marker at species level. However, not all the tested species gave PCR amplification products due to lack of the universal primers. The second barcode UPA had effective universal primers but needed to be tested for the effectiveness of resolving closely related species. More than one ITS sequence types were found in some species in this investigation, which might lead to confusion in further analysis. Therefore ITS sequence is not recommended as a universal barcode for seaweeds identification.

A SURVEY OF BANGIALES (RHODOPHYTA) BASED ON MULTIPLE MOLECULAR MARKERS REVEALS CRYPTIC DIVERSITY(1).

The Bangiales is a diverse order consisting of 28 species in Canada. Morphological simplicity and similarity among species has led to taxonomic confusion and the need for molecular techniques for species identification. This study is the first to employ the standardized DNA barcode marker COI-5P in a broad floristic survey of the Bangiales in Canadian marine waters. A total of 37 species were ultimately sequenced, 29 of which occurred in Canada. Molecular results led to the synonymization of Wildemania cuneiformis with W.amplissima, as well as the description of two new species: Porphyra corallicola sp. nov. and Pyropia peggicovensis sp. nov., and discovery of another five putative new species. Comparison of the performance of COI-5P as a species identification tool relative to rbcL (large subunit of ribulose-1,5-bisphosphate carboxylase oxygenase) and the UPA (universal plastid amplicon) revealed that, although each marker had strengths and weaknesses, the COI-5P showed the highest species-discriminatory power due to its high level of interspecific variation. The rbcL was further used to place the new species into a phylogenetic context, whereas UPA was not recommended for species identification in the Bangiales owing to within-individual heterogeneity between the two copies present in the plastid genomes in some lineages.

The Gracilariaceae Germplasm Bank of the University of São Paulo, Brazil—a DNA barcoding approach

The University of Sao Paulo Gracilariaceae Germplasm Bank has 50 strains collected mostly in Brazil, but also elsewhere in the world. This bank has been used as a source of material for research developed locally and abroad. With over 200 species, some of which have high economic value, the family Gracilariaceae has been extensively studied. Nonetheless, taxonomic problems still persist by the existence of cryptic species, phenotypic plasticity, and broad geographic distribution. In the case of algae kept in culture for long periods of time, the identification is even more problematic as a consequence of considerable morphological modification. Thus, the use of molecular markers has been shown to be an efficient tool to elucidate taxonomic issues in the group. In this work, we sequenced the 5′-end of the cox1 gene for 41 strains and the universal plastid amplicon (UPA) plastid region for 45 strains, covering all 50 strains in the bank. In addition, the rbcL for representatives of the cox1/UPA clusters was sequenced for 14 strains. The original species identification based on morphology was compared with the molecular data obtained in this work, resulting in the identification of 13 different species. Our analyses indicate that cox1 and UPA are suitable markers for the delineation of species of Gracilariales in the germplasm bank. The addition of DNA barcode tags to the samples in the Gracilariaceae germplasm bank and the molecular identification of the species will make this bank even more useful for future research as the species can be easily traced and confirmed.

MOLECULAR PHYLOGENY OF THE GENUS KUMANOA (BATRACHOSPERMALES, RHODOPHYTA)(1).

Species belonging to the newly established genus Kumanoa were sampled from locations worldwide. DNA sequence data from the rbcL gene, cox1 barcode region, and universal plastid amplicon (UPA) were collected. The new sequence data for the rbcL were combined with the extensive batrachospermalean rbcL data available in GenBank. Single gene rbcL results showed the genus Kumanoa to be a well-supported clade, and there was high statistical support for many of the terminal nodes. However, with this gene alone, there was very little support for any of the internal nodes. Analysis of the concatenated data set (rbcL, cox1, and UPA) provided higher statistical support across the tree. The taxa K.vittata and K.amazonensis formed a basal grade, and both were on relatively long branches. Three new species are proposed, K.holtonii, K.gudjewga, and K.novaecaledonensis; K.procarpa var. americana is raised to species level. In addition, the synonymy of K.capensis and K.breviarticulata is proposed, with K.capensis having precedence. Five new combinations are made, bringing the total number of accepted species in Kumanoa to 31. The phylogenetic analyses did not reveal any interpretable biogeographic patterns within the genus (e.g., K.spermatiophora from the tropical oceanic island Maui, Hawaii, was sister to K.faroensis from temperate midcontinental Ohio in North America). Previously hypothesized relationships among groups of species were not substantiated in the phylogenetic analyses, and no intrageneric classification is recommended based on current knowledge.

DNA barcoding of Canadian Ahnfeltiales (Rhodophyta) reveals a new species – Ahnfeltia borealis sp. nov.

Milstein D. and Saunders G.W. 2012. DNA barcoding of Canadian Ahnfeltiales (Rhodophyta) reveals a new species – Ahnfeltia borealis sp. nov. Phycologia 51: 247–259. DOI: 10.2216/11-40.1The red algal genus Ahnfeltia has only two described species in Canada, Ahnfeltia fastigiata and Ahnfeltia plicata, occurring along the North Pacific and North Atlantic coasts, respectively. However, during an ongoing molecular-based floristic survey of Canadian marine algae using the mitochondrial 5′ end of the cytochrome c oxidase I gene as a screening tool, cryptic diversity was uncovered, indicating the presence of four genetic lineages. These genetic clusters were further characterized using the nuclear ribosomal internal transcribed spacers and the variable domain V of the plastid large subunit (23S) ribosomal gene (universal plastid amplicon), as well as morphological and anatomical observations. Our data indicate the presence of a third species of Ahnfeltia, A. borealis sp. nov., which is typically subarctic in its distribution and intermediate in many key anatomical features between A. fastigiata and A. plicata.

An examination of the red algal genus Pugetia (Kallymeniaceae, Gigartinales), with descriptions of Salishia firma gen. & comb. nov., Pugetia cryptica sp. nov. and Beringia wynnei sp. nov.

Clarkston B.E. and Saunders G.W. 2012. An examination of the red algal genus Pugetia (Kallymeniaceae, Gigartinales), with descriptions of Salishia firma gen. & comb. nov., Pugetia cryptica sp. nov. and Beringia wynnei sp. nov. Phycologia 51: 33–61. DOI: 10.2216/11-01.1The red algal family Kallymeniaceae was surveyed along the west coast of Canada using the DNA barcode (COI-5P – 5′ region of the mitochondrial cytochrome c oxidase I gene) as a species identification tool. A total of 253 specimens field identified as Pugetia spp. subsequently resolved as five genetic species groups, although only two are reported in the flora. Additionally, COI-5P data were available for the Chilean P. chilensis, which resolved as a distinct species. Subsequent analysis of the internal transcribed spacer of the ribosomal cistron and the universal plastid amplicon (domain V of the 23S rRNA gene) resolved the same groups as COI-5P. Phylogenetic relationships of the Canadian groups were investigated using large-subunit ribosomal DNA (LSU) and a combined analysis of LSU and COI-5P data. One species was divergent from the Pugetia spp. in all analyses and grouped closely with the kallymeniacean genera Erythrophyllum (Kallymeniaceae, Gigartinales) and Kallymeniopsis (Kallymeniaceae, Gigartinales) − it is here described as Beringia wynnei sp. nov. The other ‘Pugetia’ species fell into two divergent clusters in phylogenetic analyses, differing also in blade thickness, carpogonial branch morphology and the association of the auxiliary cell relative to the carpogonium (procarpic vs nonprocarpic). We retain the genus Pugetia for the type species P. fragilissima and P. cryptica sp. nov. and describe the new genus Salishia for Salishia firma (Kylin) comb. nov., S. sanguinea (Montagne) comb. nov. and S. chilensis (J. Agardh) comb. nov. Finally, we completed morphological and anatomical examinations of other Pugetia species to provide a comprehensive review of the genus.

Molecular diversity of Amansieae (Ceramiales, Rhodophyta) from the Hawaiian Islands: a multi‐marker assessment reveals high diversity within Amansia glomerata

SUMMARYSixty‐one Hawaiian algal specimens corresponding to members of the tribe Amansieae (Amansia and Osmundaria) were compared through DNA sequence analysis. Short DNA barcode‐like sequences of mitochondrial cytochrome c oxidase subunit I (COI) and universal plastid amplicon (UPA) markers were obtained for as many of the specimens as possible, and a subset of specimens was also used for amplification and sequencing of the nuclear small‐subunit rRNA (SSU) gene for phylogenetic inference in a broader taxonomic context. Statistical parsimony analysis of the COI and UPA markers for A. glomerata produced relationships among the samples that were largely congruent with each other, although the UPA marker was more conserved. The COI marker yielded three lineages, and nucleotide divergences for these three lineages were intermediate to those typically reported for intraspecific and interspecific comparisons, suggesting that they represent either incipient species or a complex of closely related species. The COI and UPA sequences demonstrated little to no divergence for Osmundaria obtusiloba and the taxon referred to as Amansia fimbrifolia. In contrast, specimens identified as A. daemelii were identical in sequence to lineage 3 sequences of A. glomerata, and it is recommended that this taxon no longer be included in species lists for the Hawaiian flora. Phylogenetic reconstruction based on the SSU gene was largely unresolved, indicating that this marker may be of limited utility for this purpose in this group of algae, but a small amount of nucleotide variation was found for samples of A. glomerata.

Using DNA Barcodes to Identify Rosaceae

DNA barcoding has recently been proposed as a technique that employs a short, standardized gene region to identify species. DNA barcoding is well established in animals because of a widely appropriate sequence for them, the cytochrome coxidase 1 [1], but there is not any universally accepted barcode for plants till now. Therefore, the primary task for barcoding plants is to find more useful barcodes that can identify as many species as possible. Medicinal plants have been used as traditional Chinese drugs for treating diseases, some of them are similar in morphology, and are often misidentified by chemical fingerprints. Rosaceae includes many medicinal plants with similar morphology and are usually hard to be identified. Here, we chose five potential barcodes, Universal Plastid Amplicon (matK, rpoB, rpoC1, rbcL) and the nuclear ribosomal DNA (rDNA) internal transcribed spacer (ITS), to identify species from different genera in Rosaceae. The results suggest that the nuclear ribosomal DNA (rDNA) internal transcribed spacer (ITS) is a candidate to discriminate all of plant species in Rosaceae. Acknowledgements: We thank all my teachers and classmates in our laboratory very much for their help.