Gracilaria parva sp. nov. (Gracilariales, Rhodophyta) a Diminutive Species from the Tropical Eastern Pacific

DNA sequencing of the plastid encoded rbcL gene supported by morpho-anatomical features reveals Gracilaria parva sp. nov. from Panama and Ecuador in the tropical eastern Pacific Ocean. In the rbcL phylogram, G. parva occurs in a clade sister to the western Atlantic species G. galatensis. Morphologically and anatomically, G. parva is distinguished from two similar, described tropical eastern Pacific species, G. brevis and G. veleroae by its small size, to 2.5 cm tall with branch widths mostly <2 mm occasionally to 4 mm, and by its two to three cell layered cortex. Gracilaria brevis and G. veleroae are taller, have wider branches, and a one cell layered cortex. DNA sequencing is needed to resolve the many diminutive species in the tropical eastern Pacific, particularly those occurring in turf communities. DNA sequencing of historical type specimens from the 19th and 20th centuries is also needed to correctly apply names in this region.

Genetic Diversity of Orobanche cumana Populations in Serbia

In this study, we report genetic characterization of Orobanche cumana, the causal agent of sunflower wilting in Serbia. The genetic diversity of this parasitic plant in Serbia was not studied before. Random amplified polymorphic DNA (RAPD) markers and partial rbcL gene sequences analysis were used to characterize the O. cumana populations at the molecular level. While phylogenetic analyses of RAPD-PCR amplicons were performed using unweighted pair-group Method analyses, rbcL gene sequences were analyzed using neigbor joining method and minimum spanning tree. Molecular analyses of RAPD-PCR analysis revealed high genetic diversity of O. cumana populations which indicated high adaptive potential of this parasitic weed in Serbia. Further analyses of rbcL gene using minimum spanning tree revealed clear differences among diverse sections of Orobanche genus. Although this molecular marker lacked the resolution to display intrapopulation diversity it could be a useful tool for understanding the evolution of this parasitic plant. Our results suggested that O. cumana has great genetic potential which can lead to differentiation of more virulent races which is important for determining crop breeding strategies for their control.

Improved direct lysis PCR amplification method of microalgal culture for sequencing and species identification

Molecular approach plays important role in species identification for microalgae which involves sequencing of specific DNA barcode present in the genome. This approach involved preparation of template DNA for polymerase chain reaction (PCR) which is time consuming and requires large amounts of algal cells. Microalgal direct PCR have been used frequently for species identification, which simplified the DNA isolation procedure. However, the recent attempts to amplify the rbcL gene of microalga using the previously reported protocol led to poor repeatability. In this study, Nannochloropsis gaditana NIES-2587 was cultured in f/2 liquid medium. The culture growth was estimated on optical density value and the lysis process was improved using gradual temperature procedure during the PCR process. The same culture was extracted using manual DNA extraction method for comparison. The DNA obtained from both methods were amplified using RbclN primer pair to amplify 1486 bp partial sequence of Nannochloropsis rbcL gene, followed by the sequencing of the PCR product. Molecular identification based on the sequence result and BLAST analysis indicated that direct PCR and manual DNA extraction methods successfully produced high sequences result and confirmed the identity of microalgae species into N. gaditana strain CCMP527 with a genetic similarity of >99%.

Morphological and Molecular Characterization of Some Egyptian Six-Rowed Barley (Hordeum vulgare L.)

Barley production is essential in Egypt. In the present study, 15 different six-rowed Egyptian barley cultivars were studied. To differentiate between the different cultivars under study in terms of morphological characteristics and ISSR, molecular characterization reactions were carried out. Moreover, four cultivars (Giza 123, Giza 126, Giza 136, and Giza 138) were selected for further studies using scanning electron microscopy (SEM). Computational analysis of the DNA barcoding sequences of the two plastid markers rbcL and matK was executed, and the results were deposited in the NCBI database. The morphological traits showed low statistical significance among the different cultivars under study via the data collected from two seasons, suggesting that the mean field performance of these Egyptian cultivars may be equal under these conditions. The results showed that the phylogenetic tree was divided into four groups, one of which contained the most closely related genotypes in the genetic distance, including Giza 124, Giza 130, Giza 138, Giza 136, and Giza 137, which converge in the indicative uses of farmers. The seed coat of the studied cultivars was “rugose”. The elevation folding of the rugose pattern ranged from 11 ± 1.73 µm (Giza 126) to 14.67 ± 2.43 µm (Giza 123), suggesting variation in seed quality and its uses in feed and the food industry. According to the similarity matrix of ISSR analysis, the highest similarity value (93%) was recorded between Giza 133 and Giza 132, as well as between Giza 2000 and Giza 126. On the other hand, the lowest similarity value (80%) was recorded between Giza 130 and (Giza 133 and Giza 132), indicating that these cultivars were distantly related. Polymorphism information content (PIC) ranged from 0.26 for the primer ISSR UBC 835 to 0.37 for the primers ISSR UBC 814 and ISSR UBC 840. The current study showed that the matK gene is more mutable than the rbcL gene among the tested cultivars.

Preliminary placement and new records of an overlooked Amazonian tree, Christiana mennegae (Malvaceae)

Christiana mennegae is a phylogenetically enigmatic taxon and represents a case in point of a species whose presence escaped the radar of the Amazon lists and the Brazilian Flora project. Here we expand its distribution by adding new records from Peru and overlooked ones from Brazil. To investigate its phylogenetic placement in the Brownlowioideae, part of the rbcL gene of the plastid and the intergenic ITS2 region were sequenced. Macro- and micro-morphological investigation of features of C. mennegae using SEM of foliar, flower, fruit and seed structures are presented. A lectotype for the name is designated here. The morphology of trichomes revealed five types of trichomes ranging from glandular to branched and unbranched and we also report stomata on the seed surface for the first time in Brownlowioideae. Christiana mennegae and C. africana were recovered as sister species in the phylogenetic analysis, albeit with low to moderate support, and more species of this and closely related genera must be sampled and analyzed in order to obtain a clearer picture of the group’s affinities and relationships. We provide an update of its conservation status from Vulnerable to Least Concern. We also highlight the need for investment in the digitization of biological collections, botanical capacity building at the local level and the importance of the availability of online literature to speed the study of Amazonian plant diversity.

Chloroplast Genome Draft of Dryobalanops aromatica Generated Using Oxford Nanopore Technology and Its Potential Application for Phylogenetic Study

Kapur (Dryobalanops aromatica) is an important dipterocarp species currently classified as vulnerable by the IUCN Red List Threatened Species. Science-based conservation and restoration efforts are needed, which can be supported by new genomic data generated from new technologies, including MinION Oxford Nanopore Technology (ONT). ONT allows affordable long-read DNA sequencing, but this technology is still rarely applied to native Indonesian forest trees. Therefore, this study aimed to generate whole genome datasets through ONT and use part of these data to construct the draft of the chloroplast genome and analyze the universal DNA barcode-based genetic relationships for D. aromatica. The method included DNA isolation, library preparation, sequencing, bioinformatics analysis, and phylogenetic tree construction. Results showed that the DNA sequencing of D. aromatica resulted in 1.55 Gb of long-read DNA sequences from which a partial chloroplast genome (148,856 bp) was successfully constructed. The genetic relationship was analyzed using two selected DNA barcodes (rbcL and matK), and its combination showed that species of the genus Dryobalanops had a close relationship as indicated by adjacent branches between species. The phylogenetic tree of matK and the combination of the matK and rbcL genes showed that D. aromatica was closely related to Dryobalanops rappa, whereas the rbcL gene showed group separation between D. aromatica and D. rappa. Therefore, a combination of the matK and rbcL genes is recommended for future use in the phylogenetic or phylogenomic analysis of D. aromatica.

The perspectives for DNA barcoding of Rhaponticum carthamoides (Willd.) Iljin using rbcL gene sequence

The methods of biological species identification using nucleotide sequences of short genome regions (DNA barcoding) are actively developed. The universal DNA barcode for plants remains to be discovered, and one of the leading candidates is the plastid gene of the large subunit of ribulose-bisphosphate carboxylase gene (rbcL). In our study, we estimated the part of rbcL gene as a possible marker for molecular identification of Rhaponticum carthamoides (Willd.) Iljin. Due to its officinal properties, the species is susceptible to uncontrolled and illegal harvesting from natural populations. Today, the species needs to be protected and therefore is included into the Red Data Books of the Russian Federation and certain regions. The study was carried out using plants from the natural populations sampled from the Khamar-Daban Ridge (South Siberia) and considering now as Rh. carthamoides var. chamarense (Peschkova) O S Zhirova. It was shown that rbcL gene can be used to identify Rh. carthamoides at least from the populations of the Khamar-Daban Ridge using a fragment of the maximum length or its 3’ region. Apparently, the 5’ region of the gene (rbcLa) most often used as DNA barcode for plants may be of lesser importance for Rh. carthamoides. The rbcL gene sequences can be also used for the development of approaches for Rh. carthamoides identification in the medicinal preparations and products containing dried tissues to prevent their falsification and illegal harvesting of this species. The combination of rbcL gene with additional markers seems to be highly desirable to create effective DNA barcodes for Rhaponticum species.

DNA metabarcoding reveals fine scale geographical differences of consumed algae in the Galápagos marine iguanas (Amblyrhynchus cristatus)

Galápagos marine iguanas are primarily associated with the marine environment and show special nutritional adaptations. They are the only lizards worldwide that forage on marine macroalgae. Until now, consumed algae have been identified by direct observations during their feeding activities and microscopic identification in faeces samples. In this study, we use a novel DNA metabarcoding approach to identify consumed algal species from the faeces of marine iguanas. We developed primers for the ribulose-bisphosphate carboxylase (rbcL) gene and applied a metabarcoding approach to 25 individual faeces samples collected in four representative sites of two subspecies (Amblyrhynchus cristatus mertensi and A. c. godzilla), found in the San Cristóbal Island. We detected 18 consistently occurring macroalgal operational taxonomic units (OTUs). Most of the OTUs were assigned to Rhodophyta (red algae) and only one OTU to Chlorophyta (green algae). Despite the number of consumed algal species did not differ between two subspecies (OTU richness; P = 0.383), diet overlap level between A. c. mertensi and A. c. godzilla was low (Schoener index = 0.345), suggesting that both subspecies consumed different algal species in their natural environment. Further studies are needed to understand whether the difference of consumed algae reflects disparities in the abundance of algal species between sites, or whether iguanas of the two genetically differentiated subspecies prefer distinct algal species.

Codon Usage of Chloroplast Gene rbcL in Laurencia sensu lato (Rhodophyta) species

Recent advances in molecular biology make it possible to sequence not only genes or genomes, but also to understand codon dynamics. For the organelle genes of these organisms, a small set of preferred codons are used for encoding proteins. For the first time, this paper treats the divergence of synonymous codon usage and its bias in the rbcL gene within the Laurencia complex of red algae. We observed that the synonymous codon preference biases in rbcL are large and differ among species. A clear distinction in codon usage between genera is evident: the genera Dasya and Delesseria use a set that fluctuated between 53 and 58 codons. Whereas, in the genera Ceramium, Chondrophycus, Chlamydomonas, Chlorella, Laurencia s.s, Osmundea, and Palisada codon usage indicates a higher restriction fluctuating between 40 to 51 codons. Laurencia complex genera and other representative algae showed a defined composition pattern, with lower percentage values of NNC/G (7-24.9%). Dasya and Delesseria showed a selective pattern tendency because of high percentage values of NNC/G (54-55%). The estimated codon bias parameters were tested to infer systematic relationships and match suitable codons with the NNC / G codon percentages. Cluster analysis based on Codon Usage supports phylogenetic relationships between Chondrophycus, Palisada, Laurencia, Osmundea, and Yuzurua species.

Endoplura jejuensis sp. nov. and Endoplura koreana sp. nov. (Ralfsiales, Phaeophyceae) from Korea based on molecular and morphological analyses

The crustose brown algal genus Endoplura has been known as a monotypic genus characterized by its intercalary plurangial reproductive structures composed of 2–4 separate parallel filaments terminated by 2–5 sterile cells and by containing several to many chloroplasts per cell. In this study, Endoplura jejuensis sp. nov. and E. koreana sp. nov. from Korea are newly described based on molecular and morphological analyses. Our phylogenetic analyses of the rbcL gene reveal that E. jejuensis sp. nov. and E. koreana sp. nov. are placed in the same clade with “E. aurea” from Japan with a strong bootstrap supporting value. E. jejuensis is characterized by small and light to dark brown crustose thalli of less than 1 cm diameter, tufts of hairs arising from the basal disc, plurangia composed mostly of two separate parallel reproductive filaments terminated by 2–4 sterile cells, and sessile unangia each with a single paraphysis. E. koreana is distinguished by olive or yellowish-brown crustose thalli of up to 3 cm diameter, tufts of hairs arising from the basal disc, and apical parts of erect filaments, plurangia with 2–5 separate reproductive filaments terminated by 2–8 sterile cells, and sessile unangia with 1–2 paraphyses. Our studies also show that “E. aurea” specimens from Japan may be recognized to be a different species from other Endoplura species.