tufA Gene Research Articles

Halimeda incrassata (Bryopsidales, Chlorophyta) in Rhodes, Greece, Eastern Mediterranean

The first record of the tropical green seaweed Halimeda incrassata (Bryopsidales, Chlorophyta) in the Eastern Mediterranean Sea is presented, based on several thalli found in the stomach of a silver-cheeked toadfish (Lagocephalus sceleratus), collected off Plimmiri beach, Rhodes, Greece. Species identification was based on morphological and molecular identification using the tufA gene as a molecular marker. The finding comes 10 years after a report on the species in Mallorca (Western Mediterranean Sea), where H. incrassata has spread rapidly. The pathway of its introduction in the Eastern basin is unknown, although shipping or the aquarium trade could be involved in this new introduction. Further studies are necessary for visual documentation of the infested areas around Rhodes and assessment of its possible spread in the following years.

Investigating seed bank potential of crustose coralline algae using DNA metabarcoding.

To examine the potential for the autogenic ecosystem engineers, crustose coralline algae (CCA), to serve as seed banks or refugia for life stages of other species, it is critical to develop sampling protocols that reflect the diversity of life present. In this pilot study on two shallow water species of CCA collected from Raoul Island (Kermadec Islands; Rangitāhua) New Zealand, we investigated two preservation methods (ethanol vs. silica gel), sampled inner and outer regions of the crusts, and used DNA metabarcoding and seven genes/gene regions (16S rRNA, 18S rRNA, 23S rRNA, cox1, rbcL, and tufA genes and the ITS rRNA region) to develop a protocol for taxa identification. The results revealed immense diversity, with typically more taxa identified within the inner layers than the outer layers. As highlighted in other metabarcoding studies and in earlier work on rhodoliths (nodose coralline algae), reference databases are incomplete, and to some extent, the use of multiple markers mitigates this issue. Specifically, the 23S rRNA and rbcL genes are currently more suitable for identifying algae, while the cox1 gene fares better at capturing the diversity present inclusive of algae. Further investigation of these autogenic ecosystem engineers that likely act as marine seed banks is needed.

Molecular identification of the ubiquitous green algae Ulva reveals high biodiversity, crypticity, and invasive species in the Atlantic-Baltic Sea region

Correct species identification is fundamental for assessment and understanding of biodiversity. Erroneous species identification may impede conservation management and may delay detection of invasive species. The ubiquitous green algal genus Ulva is known for its wide environmental tolerance, plastic morphology, occurrence of cryptic species and ambiguous species concepts that hinder clear identification. We used molecular monitoring to assess species diversity and distribution of Ulva along the full Atlantic-Baltic Sea salinity gradient (> 10,000 km). Ulva specimens were collected from Denmark, Finland, Germany, Norway, and Sweden. DNA barcoding analysis of the tufA gene revealed 20 genetic entities in total, of which 11 could be identified to species level (U. californica, U. flexuosa, U. torta, U. linza, U. prolifera, U. fenestrata, U. australis, U. intestinalis, U. compressa, U. gigantea, U. lacinulata). Nine entities (Ulva sp. 1–9; [Ulva capillata]) yielded novel sequence reads that belonged to either unidentified species, species complexes, or singletons. At least 3 of the discovered species (U. australis, U. californica, U. gigantea) are considered non-native and potentially invasive. Furthermore, considerable differences between the observed and the historically estimated species distributions were found. The highest diversity was recorded in the Atlantic and Skagerrak region whereas only two entities of taxonomically accepted species where found north-east the Blekinge coast. Our study shows that the species diversity of Ulva in the study area is diverging from previous reports, and that molecular methods are imperative for species identification in this morphologically plastic genus. Furthermore, the presence of non-native species indicates a necessity for further fine-scale monitoring in specific areas to e.g. mitigate formation of green tides.

Structural Development of Marine Green Alga (Ulva rigida C. Agardh, 1823) during Cultivation

Ulva rigida C. Agardh (sea lettuce) is a marine green alga. It grows naturally near shorelines in areas with freshwater influence. It can also be cultivated easily on farms and can be developed into many healthy food products, which bring in revenue for Thailand. It is important to develop an effective way to cultivate the alga. In this research, the molecular biology of sea lettuce using the tufA gene for the identification of Ulva genus and morphological characters at different culturing periods of U. rigida thallus were investigated using a light microscope (LM), a scanning electron microscope (SEM) and a transmission electron microscope (TEM). The results revealed that the U. rigida alga consisted of various cell shapes including elliptic, irregular triangle, irregular quadrilateral, pentagonal and polygonal. The cell size increased with increasing periods of culture. However, when the alga was cultured for 28 days, the cell sizes were smaller than the original cells due to cell division during aging, resulting in an increase in cell numbers. U. rigida had grown rapidly in 14 days which was a relatively short period of cultivation. The results of this study indicate that U. rigida has a potential to be scaled up in pilot and commercial scales. HIGHLIGHTS The molecular analysis of sea lettuce from Phetchaburi Coastal Aquaculture Research and Development Center, Sub District: Bang Kaeo, District: Ban Laem at Phetchaburi Province, Thailand was found to be rigida The morphological and anatomical characters of rigida thalli were studied with light microscope, transmission electron microscope and scanning electron microscope techniques The cell sizes of rigida thalli changed with culturing days. They were the largest at 14 days in the surface view, and at 14 and 21 days in the transverse section GRAPHICAL ABSTRACT

Species diversity and distribution of the calcareous green macroalgae Halimeda in Taiwan, Spratly Island, and Dongsha Atoll, with the proposal of Halimeda taiwanensis sp. nov.

The calcified green algal genus Halimeda is one of the most ecologically important but morphologically diverse seaweeds in sub-tropical and tropical waters. Because of its high morphological plasticity, the identification of Halimeda species based on morphological characters is challenging without the assistance of molecular analysis. To date, the species diversity of Halimeda in Taiwan and its overseas territories has not been investigated with the assistance of DNA sequencing, and this taxonomic knowledge gap should be filled. The present study initiates a systematic examination of the species diversity and distribution of Halimeda in Taiwan, Spratly Island, and Dongsha Atoll in the South China Sea, using DNA sequence data (plastid tufA gene and rbcL) and morphological data. Our DNA analyses revealed the presence of 10 Halimeda species (Halimeda borneensis, Halimeda cylindracea, Halimeda discoidea, Halimeda distorta, Halimeda macroloba, Halimeda minima, Halimeda opuntia, Halimeda renschii, Halimeda taiwanensis sp. nov., and Halimeda velasquezii) in the waters around Taiwan, Spratly Island, and Dongsha Atoll. The majority of the species could be readily distinguished by their morphological and anatomical characters. The proposed new species, H. taiwanensis, was differentiated not only by our algorithmic species delimitation analyses (statistical parsimony network analysis and automated barcode gap discovery), but also by its morphological features. The proposed new species differs from two externally resembled species, H. cuneata and H. discoidea, in having an undulated segment margin, the complete fusion of medullary siphons at the node, the lack of segment stalk, and the presence of a large primary utricle. Here, we present the up-to-date taxonomic account, molecular diversity, and geographical distribution of Halimeda spp. in Taiwan and associated areas of the South China Sea. Environmental factors that might drive the occurrence and latitudinal distribution of the species are also discussed.

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

SUMMARYSpecies 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.

DNA Barcoding and ITS-tufA multi-local molecular phylogeny of nitrophilic alga Prasiola crispa growing on penguin guano at Larsemann Hills, Eastern Antarctica

Antarctica is the coldest and driest continent globally and has always been an exciting habitat to study extremophiles. The study reveals a monostromatic nitrophilic green alga Prasiola crispa (Trebouxiophyceae) growing on Adelie penguin guano at a penguin rockery, Larsemann Hills, Eastern Antarctica. This study is the first report of the barcode of this algal genus from Eastern Antarctica in general and the Larsemann Hills in particular. There are 35 species currently accepted in this genus, while four were reported from Antarctica. The present study relied on morphological diagnoses as well as the phylogenetic inference based on nuclear-encoded ITS gene and plastid-encoded tufA gene for species identification. The study generated phylogenetic reconstruction at the two selected loci for the first time for this species from Antarctica.

Molecular genetic diversity of seaweeds morphologically related to Ulva rigida at three sites along the French Atlantic coast.

Foliose species of the genus Ulva are notoriously difficult to identify due to their variable morphological characteristics and high phenotypic plasticity. We reassessed the taxonomic status of several distromatic foliose Ulva spp., morphologically related to Ulva rigida, using DNA barcoding with the chloroplastic tufA and rbcL (for a subset of taxa) genes for 339 selected attached Ulva specimens collected from three intertidal rocky sites. Two of the collection sites were in Brittany and one site was in Vendée, along the Atlantic coast of France. Molecular analyses included several museum specimens and the holotype of Ulva armoricana Dion, Reviers & Coat. We identified five different tufA haplotypes using a combination of phylogenetic analysis, with the support of several recently sequenced holotypes and lectotypes, and a species delimitation method based on hierarchical clustering. Four haplotypes were supported by validly named species: Ulva australis Areschoug, Ulva fenestrata Postels & Ruprecht, Ulva lacinulata (Kützing) Wittrock and U. rigida C. Agardh. The later was additionally investigated using rbcL. The fifth haplotype represented exact sequence matches to an unnamed species from European Atlantic coasts. Our results support: (1) the synonymy of both U. rigida sensu Bliding non C. Agardh and U. armoricana with U. lacinulata. This finding is based on current genetic analysis of tufA from the U. armoricana holotype and recent molecular characterization of the lectotype of U. laetevirens, which is synonymous to U. australis, (2) the presence of U. australis as a misidentified introduced species in Brittany, and (3) the presence of U. fenestrata and U. rigida in southern Brittany. The taxonomic history of each species is discussed, highlighting issues within distromatic foliose taxa of the genus Ulva and the need to genetically characterize all its available type specimens.

Molecular Characterization of Caulerpa racemosa (Caulerpales, Chlorophyta) from Indonesia Based on the Plastid tufA Gene

The green seaweed Caulerpa racemosa is a seaweed of high prospect that is being given emphasis by the Indonesian government. However, C. racemosa in Indonesia may include multiple species level-entity exhibiting morphological overlap and require molecular analysis for species identification. Molecular documentation of species richness of indigenous populations of C. racemosa is essential to underpin cultivar development and conservation of the species to avoid overharvesting. The present study aimed to determine the genetic diversity of C. racemosa and document the haplotype network of the specimens from four different locations (Bintan Island, Jepara, Takalar and Osi Island) using the chloroplast tufA gene. Twenty individuals from four areas were collected and amplified with the chloroplast-encoded gene tufA for species identification against publicly available data. The identification of C. racemosa based on the tufA gene showed that the species found in four locations were C. cylindracea (previously C. racemosa var. cylindracea), C. macra (previously C. racemosa var. macra), C. racemosa, and C. oligophylla (previously C. racemosa var. lamourouxii). This study records the existence of C. cylindracea in Takalar and Jepara, Indonesia for the first time. The most diverse C. racemosa species was in Osi Island, where the exploitation of this seaweed is very low. In contrast, the lowest number of C. racemosa varieties were found in Takalar, where exploitation is very high. There were only minor light variations of Caulerpa species in the tufA gene in four different sites with only four haplotypes found, and each haplotype corresponded to another species.

Molecular Identification, Nutritional Profile and Heavy Metals Content of Edible Caulerpa from Binuangeun Coast, Banten

Two species of Caulerpa, locally known as “Pedesan” and “Latuh” have been traditionally consumed by coastal communities at Binuangeun, Banten. This study aimed to identify “Pedesan” and “Latuh” using the DNA barcoding method and to evaluate their nutrient and heavy metal contents. Fatty acids were determined by Gas Chromatography Flame Ionization Detector (GC FID), amino acids using Ultra Performance Liquid Chromatography (UPLC), and minerals using Inductively Coupled Plasma Optical Emission Spectrometry (ICP OES). Based on the tufA gene sequences, the “Pedesan” was identified as Caulerpa racemosa var. macrophysa and “Latuh” as Caulerpa chemnitzia. Thirteen fatty acids were detected in C. racemosa var. macrophysa and twelve fatty acids in C. chemnitzia. Of the total fatty acid content, C. racemosa var. macrophysa contained 41.0% unsaturated fatty acids, dominated by linolenic acid and eicosapentaenoic acid. Meanwhile, C. chemnitzia contained 47.5%, dominated by oleic acid. Both seaweeds contained fatty acids with the w6/w3 ratio lower than 10, which could prevent heart disease risk based on World Health Organization (WHO) recommendation. The primary amino acids content in C. racemosa var. macrophysa were glutamic acid, alanine, serine and aspartic acid, while those in C. chemnitzia were glutamic acid, serine, aspartic acid, and glycine. The high content of glutamic acid in both samples indicated their potential use as food flavor enhancer. The Na/K ratio of C. racemosa var. macrophysa (40.31) and C. chemnitzia (27.48) were higher than those recommended by WHO. Heavy metals were not detected in either “Pedesan” nor “Latuh”, indicating that they are safe for consumption.

Morphological and Phylogenetic Data Confirm the Identity of Prasiola fluviatilis (Prasiolales, Trebouxiophyceae) from Glacier Streams in the Tianshan Mountains, China

The green alga Prasiola fluviatilis (Sommerfelt) Areschoug ex Lagerstedt occurs in cold lotic environments. The species has a mainly circumarctic distribution, but has also been reported from glacier areas in lower latitude regions in both hemispheres. It was reported from China on a single occasion in the first half of the 20th century, but without description, illustrations or voucher specimens the identity of this record cannot be verified. Here we confirm the presence of P. fluviatilis in the Tianshan Mountains, Xinjiang Province, China based on morphological features, habitat characteristics, and plastid rbcL and tufA gene sequences. The biogeographic distribution of P. fluviatilis and its phylogenetic relationship with other terrestrial and freshwater Prasiola species are discussed.

Multiple genetic marker analysis challenges the introduction history of Ulva australis (Ulvales, Chlorophyta) on French coasts

ABSTRACT The green seaweeds Ulva australis and U. pertusa were described from southern Australia and Japan, respectively. They are conspecific and U. australis, the currently accepted taxon, is native to temperate marine waters in north-eastern Asia, and known to be introduced overseas into Australasia, the Americas and Europe. Although the genetics of U. australis have been investigated elsewhere, along French coasts the origins and history of the introduction of this species need to be clarified. We used mitochondrial, plastid and nuclear markers to differentiate introduced populations of U. australis along the French Atlantic coasts. The plastid tufA gene used as a barcoding marker revealed a well-defined species with a higher haplotype diversity in native vs. introduced areas. The ITS2 region (nuclear) and rbcL (plastid) were used to compare French specimens with the lectotype of U. australis. Putative geographic origins of the genetically determined U. australis were examined using genetic markers with better resolution, the plastid atpI-H combined with the mitochondrial trnA-N. Origin(s) and introduction history of French specimens were inferred from the comparison between their haplotypes and those previously described in native and non-native temperate areas worldwide. Our results indicate that the presence of U. australis along the French Atlantic and Mediterranean coasts is the result of multiple introductions and independent pathways, and suggest that historical oyster transfers from Japan and British Columbia can only partially explain the observed patterns in genetic markers. Alternative hypotheses for the timing and pathways of introductions are proposed in the light of the historic background of maritime transport networks and trade between north-eastern Asia and Europe.

New insights on the distribution and habitat of Ulvella endozoica (Ulvellaceae, Chlorophyta) in the tropical Southwestern Atlantic, based on thallus ontogeny in culture and DNA barcoding

The small size (< 5 mm) of marine microfilamentous green algae makes it difficult to identify based on field collections, remaining largely unnoticed. To catalogue the diversity of these diminutive marine algae, a combination of culture-based taxonomic studies and DNA barcoding has proven to be a successful approach. During subtidal surveys in the northeastern Brazil, microfilamentous green algae were isolated as epiphytes on red algae cultured in laboratory. In this work, we used thallus ontogeny and plastid tufA gene sequences to elucidate the taxonomic identity of these isolates. Molecular and morphological evidence revealed that Brazilian specimens correspond to Ulvella endozoica, a poorly known species thought to be endemic to Florida, its type locality. Since U. endozoica was not found in gorgonian corals, as in the original description, this species does not follow a host specificity pattern, growing in more diverse substrata than previously thought. The origin of U. endozoica in Brazil remains undetermined, and we concluded that it is a cryptogenic species. Limited sampling, troublesome taxonomy, and difficulty in finding Ulvella species make them overlooked in Western Atlantic, resulting in taxonomic and biogeographic gaps. Our results expand southward the known distribution range of U. endozoica, demonstrating that an integrative barcode and laboratory culture-based approach is critical to uncover these inconspicuous algae.

Morphology and molecular phylogeny of Umbraulva spp. (Ulvales, Ulvophyceae), and proposal of Ryuguphycus gen. nov. and R. kuaweuweu comb. nov.

ABSTRACT The reproductive structures, early development and carotenoid composition of Umbraulva kuaweuweu, which is a deep-water ulvacean species recently reported from Hawaii, are reported for the first time using the specimens collected by dredging at ~30 m depth from Ogasawara Islands and Mageshima in southern Japan. Although Umbraulva kuaweuweu had siphonaxanthin as in U. japonica, a characteristic carotenoid of Umbraulva, U. kuaweuweu lacked α-carotene and β-carotene. U. kuaweuweu differed markedly from U. japonica in thallus morphology and reproductive structures: cell shape, presence/absence of inner rhizoidal filaments in the upper parts of thalli, solid/partly saccate thalli, and developmental processes of zoids. Multigene concatenated DNA sequences of chloroplast rbcL and tufA genes, and nuclear 18S, 5.8S, 28S rDNA and rDNA ITS1 and ITS2 regions indicated separation of U. kuaweuweu from the clade comprised of the generitype U. japonica, U. amamiensis, U. dangeardii and U. kaloakulau. In conclusion, based on the distinctive morphology, life history, carotenoid composition and molecular phylogeny, we propose the establishment of a new genus Ryuguphycus gen. nov. to accommodate U. kuaweuweu, as R. kuaweuweu comb. nov.

Phylogeography of the mediterranean green seaweed Halimeda tuna (Ulvophyceae, Chlorophyta).

Populations of many Mediterranean marine species show a strong phylogeographic structure, but the knowledge available for native seaweeds is limited. We investigated the genetic diversity of the green alga Halimeda tuna based on two plastid markers (tufA gene and a newly developed amplicon spanning five ribosomal protein genes and intergenic spacers, the rpl2-rpl14 region). The tufA sequences showed that Mediterranean H. tuna represents a single, well-defined species. The rpl2-rpl14 results highlighted a genetic separation between western and eastern Mediterranean populations; specimens collected from widely scattered locations in the Adriatic/Ionian region shared a haplotype unique to this region, and formed a group separated from all western Mediterranean regions. Specimens from Sardinia also formed a unique haplotype. Within the western Mediterranean basin, a gradual shift in the frequency of haplotypes was apparent along a West-East gradient. Our results represent the first clear evidence of an East-West genetic cleavage in a native Mediterranean macroalga and offer an interesting perspective for further research into fine-scale seaweed population structure in the NW Mediterranean Sea.

Cryptic species inside the genus Hariotina (Scenedesmaceae, Sphaeropleales), with descriptions of four new species in this genus

ABSTRACT The genus Hariotina was established in 1889 based on the type species Hariotina reticulata. This species was then transferred to Coelastrum by Senn (1899). The genus Hariotina was eventually restored by Hegewald et al. (2002) based on morphological characteristics. Species of Hariotina are ubiquitous in fresh water, especially in some tropical and subtropical areas. The taxonomic position of the genus is now considered to be within the family Scenedesmaceae. However, until now, there was an insufficient understanding of the molecular diversity within this genus, and the related reference sequences in the database were inadequate to resolve the species taxonomy. In this study, 20 strains of Hariotina were identified and successfully cultured in the laboratory. We used a combined approach, joining molecular and morphological analyses, to determine their taxonomic relationships and phylogenetic positions. The molecular analyses based on 18S rDNA+ITS sequences and the tufA gene resolved the analysed strains into at least six species, including the known species H. reticulata and H. polychorda and four new species herein described, i.e. H. hainanensis sp. nov., H. compacta sp. nov., H. guilinensis sp. nov. and H. laxa sp. nov. The genus Hariotina was confirmed in the Scenedesmaceae as a monophyletic clade that was closely related to the genus Coelastrum in tufA gene phylogeny. Strains with H. reticulata morphotype were found to be located on two different branches, and formed sister relationships with the other two morphologically distinct species. In addition, the topology was supported by all three DNA markers. The congruence between the phylogeny and the classical taxonomic system based on morphological criteria for this taxon is discussed.

Four New Members of Foliicolous Green Algae Within the Watanabea Clade (Trebouxiophyceae, Chlorophyta) from China

Members of the Watanabea clade of Trebouxiophyceae are genetically diverse and widely distributed in all kinds of habitats, especially in most terrestrial habitats. Ten new strains of terrestrial algae isolated from the tropical rainforest in China, and four published strains were investigated in this study. Morphological observation and molecular phylogenetic analyses based on the 18S, ITS, rbcL, and tufA genes were used to identify the new strains. Four previously described species were reinvestigated to supplement molecular data and autospores' morphological photographs. The phylogenetic analyses based on 18S only, the concatenated dataset of 18S and ITS, as well as the concatenated dataset of rbcL and tufA, showed the same phylogenetic positions and relationships of these new strains. According to the phylogenetic analysis and morphological comparisons results, we described these 10 strains as four new members within the Watanabea clade, Polulichloris yunnanensis sp. nov., Polulichloris ovale sp. nov., Massjukichlorella orientale sp. nov., and Massjukichlorella minus sp. nov., and two known species, Massjukichlorella epiphytica, and Mysteriochloris nanningensis. Additionally, we provide strong evidence proving that Phyllosiphon, Mysteriochloris, Polulichloris, and Desertella all reproduce through unequal sized autospores.

FIRST OCCURENCE OF CAULERPA MACRODISCA (CAULERPACEAE, CHLOROPHYTA) IN MALAYSIA BASED ON THE MOLECULAR AND MORPHOLOGICAL EVIDENCE

The siphonous green macroalgae Caulerpa macrodisca (Decaisne) Weber-van Bosse has been reported to be widely distributed around South-west Asia (Sri Lanka), South China Sea, South-east Asia (Indonesia, Philippines, Singapore and Vietnam) and Pacific Islands (Samoan Archipelago). There was no formal report on the existence of C. macrodisca in Malaysia. This study reported the first occurence of the green algae, C. macrodisca in the coastal area of Menumbok, Sabah, Malaysia. The C. macrodisca was first discovered in March 2016 grew around the sea cage of a local farmer in Sikalong Village, Menumbok. In May and July 2016, C. macrodisca was searched around the coastal area near the sea cage and the existence was discovered at 1.4 to 2.7 m depth. Further scuba search was done in August 2016 and confirmed the occurrence nearby the area at similar depth. Most of the collected green macroalgae were large and reproductive and a few were small and young but precociously reproductive. The samples were collected in May, July and August 2016 for further analysis. The collected specimen was confirmed as C. macrodisca through morphology and genetic analyses using tufA gene as DNA biomarker. In conclusion, the first occurrence of C. macrodisca in Malaysia added a new record for the South China Sea region marine algae checklist.

Morphology and molecular phylogeny of coccoid green algae Coelastrella sensu lato (Scenedesmaceae, Sphaeropeales), including the description of three new species and two new varieties.

The genus Coelastrella was established by Chodat (Bull. Soc. Bot. Geneve, 13 [1922] 66), and was characterized as being unicellular or in few-celled aggregations with many longitudinal ribs on the cell wall. Many species of this genus showed strong ability to accumulate carotenoids and oils, so they have recently attracted much attention from researchers due to its potential applicability in the energy and food industries. In this study, a total of 23 strains of Coelastrella were sampled from China, and three new species and two new varieties were described: C. thermophila sp. nov., C. yingshanensis sp. nov., C. tenuitheca sp. nov., C. thermophila var. globulina var. nov., C. rubescens var. oocystiformis var. nov. Besides 18S rDNA and ITS2 sequences, we have newly sequenced the tufA gene marker for this taxon. Phylogenetic analysis combined with morphological studies revealed four morphotypes within the Coelastrella sensu lato clade, which contained the morphotype of original Coelastrella, original Scotiellopsis, Asterarcys, and morphotype of C. vacuolata and C. tenuitheca sp. nov. The relationships between morphological differences and phylogenic diversity based on different markers were discussed. Our results support that 18S rDNA was too conserved to be used a species-specific or even a genus-specific marker in this clade. The topology of tufA gene-based phylogenetic tree had a better match with the morphological findings.

Homogeneity of Mycoplasma agalctiae vaccine strains in an agalaxy- high-burden environment

Interrogation of the genomic relations between Iranian Mycoplasma agalactiae vaccine strains of Taliqan, Lorestan and Shiraz. Two MLVA (covering VNTR loci of 5, 9, 17 and 19) and MLST (comprising dnaA, gltX, gyrB, C, tufA genes) genotyping systems plus nucleotide structure analysis of P80 gene, was conducted. The shared MLVA pattern represented by the three strains differed to that of the Mag PG2 laboratory strain, only at locus VNTR19 where the PG2 genome hold a 3 bp longer stretch. In MLST analysis, at dnaA, gltX, gyrB, metS and tufA loci, the three strains displayed alleles 1, 21, 2, 2 and 1, respectively. At, gltX locus a new allele (21) was detected where a new sequence type (ST33) was identified. Besides, the trio strains hold an identical nucleotide structure in their ma-mp81 gene. In explanation, lack of efficient disease control measures, has possibly contributed in evolution of a clone or a few clones that gradually overwhelmed the population over the time. Besides, the similarity between the Iranian and the PG2 strains, might be due to homoplasy or farming exercises such as animal importation. Inclusion of further local isolates in next studies will help to assess these assumptions.