Molecular assessment of the species of Gracilariaceae (Gracilariales, Rhodophyta) from the Yucatan Peninsula, Mexico, including two new records for the Mexican Atlantic

Background. The Gracilariaceae is one of the most diverse and abundant families of marine red algae. Most species in this family exhibit high morphological variability and overlapping of characters. In the Yucatan Peninsula 30 species have been historically recorded, but the phylogenetic identity for many of them is still unknown. Questions: Is the current diversity of the family in the area overestimated? Studied species: Crassiphycus caudatus, C. corneus, C. usneoides, Gracilaria flabelliformis, G. hayi, G. cf isabellana, G. microcarpa, G. occidentalis, G. suzanneae, Gracilariopsis tenuifrons. Study site and dates: Campeche: km 33 carretera Champotón, Bahía Tortuga, Puente Xen Kan III, Punta Xen, Sabancuy, 2017, 2018; Yucatan: Puerto Sisal, Puerto Progreso, Puerto Chicxulub, Puerto Telchac, 2018; Quintana Roo: Playa 88, Xcalacoco, Punta Esmeralda, 2019. Methods. Phylogenetic analysis (with COI-5P and rbcL sequences), supported by DNA species delimitation methods, genetic distances and morphological comparisons, allowed us to molecularly identify the specimens collected. Results. The phylogenetic identity of 10 species of Gracilariaceae was confirmed, two of which are new records for the Mexican Atlantic: G. hayi and G. suzanneae. Conclusion. The study demonstrated that the molecular assessment has proved to be very useful for the diversity evaluation, thus, the future phylogenetic identifies for the rest of morphospecies recorded in the area will allow a better approximation of Gracilariaceae diversity species.

DNA barcoding-based assessment of the invasive and native non-crustose Codium species in the central Cantabrian Sea, southern Bay of Biscay

In this work, we identified non-crustose invasive (Codium fragile subsp. fragile) and native Codium spp. (Codium tomentosum and Codium vermilara) in the central Cantabrian Sea using DNA barcoding (tufA and rbcL genes). We designed a new FCOtufA genetic marker for identifying Codium spp. in fresh and herbarium material. The tufA and rbcL sequences revealed three different single haplotypes for each of the species and a lack of intraspecific genetic diversity. The FCOtufA genetic marker revealed one new haplotype of C. fragile within the native region (South Korea), suggesting the possibility of higher genetic diversity in the donor region of this invasive species.

Discovery of Wilsonosiphonia fujiae (Rhodomelaceae, Rhodophyta) from the Maldives Islands and transfer of Polysiphonia rhizoidea from Hawaiʻi to Wilsonosiphonia

Wilsonosiphonia is a newly described marine algal genus with three species. The genus was previously confused with the widely distributed taxon Polysiphonia, and because of this, the taxonomy and distribution of Wilsonosiphonia is likely underestimated. We report the discovery of a minute filamentous red alga, Wilsonosiphonia fujiae (Rhodophyta) in the Maldives Islands, a taxon which was considered endemic to Brazil. Specimens were collected at Fulhadoo, Goidhoo Atoll and Dhidhdhoo Islands, South Ari Atoll, which are ca. 160 km apart, during January and July, 2019, respectively. Thalli formed dense patches on the upper intertidal in regions of shaded rock. Both mitochondrial cox1 and plastid rbcL sequencing confirmed the occurrence of W. fujiae in the Maldives, which is also in agreement with morphological observations. Based on the low pairwise divergences of COI-5P and rbcL sequences between the Maldives and Brazil specimens, we suggest that W. fujiae is a recently introduced yet cryptogenic species in the Maldives. Additionally, as a result of our phylogenetic analyses, it became evident that sequences from the Hawaiian Islands, which were listed as members of the genus Polysiphonia, represent species-level diversity within the genus Wilsonosiphonia. Based on both morphological characteristics and the COI-5P phylogeny, we here reinstate Polysiphonia rhizoidea from Hawaiʻi and transfer it to Wilsonosiphonia, as W. rhizoidea comb. nov.

Aphanta asiatica sp. nov. (Orthogonacladiaceae, Rhodophyta), a new species from the Asia-Pacific region with the first description of reproductive structures in this genus

We analyzed mitochondrial COI-5P and plastid rbcL sequences from specimens of Aphanta collected from China and Japan and accessed morpho-anatomical data in detail. The results revealed the presence of a novel species of this genus, Aphanta asiatica, described here. Aphanta asiatica was characterized by a turf-forming habit of thalli with anastomosing branches, and a thick, wide, and flattened axis with subpinnate to pinnate branches, and characterized by its robust and branched prostrate system bearing complex peg-like haptera which were either typical or non-typical. Rhizoidal filaments were abundant in the inner cortex and distal ends of the axis, and interspersed in the medulla. Tetrasporangial and spermatangial sori were produced on the terminal ends of the branchlets and axes. Tetrasporangia were irregularly arranged and cruciately divided, and spermatangia were cut off from the outermost cortical cells. In phylogenies of the COI-5P and rbcL sequences, A. asiatica was closely related to Aphanta pachyrrizha. In conclusion, the present study is the first to document reproductive structures in, and contribute further to our understanding of, the genus Aphanta.

Botanical, genetic characteristics and preliminary screening of the phytochemical constituents of Hydnophytum formicarum Jack. in Phu Quoc forest, Vietnam

In Vietnamese folk medicine, Bi ky nam (H. formicarum) tuber has been widely used to treat rheumatism, liver and intestinal diseases. This work aimed to study botanical, genetic characteristics and screening of the phytochemical constituents of wild H. formicarum of Phu Quoc Island, Vietnam. Anatomical characteristics of the plant material were described. Fresh leaves were used to analyze DNA barcodes based on rbcL region amplified by PCR. Sequences of DNA products were identified by Sanger method and BioEdit 7.0.5 software, then compared to the control rbcL sequences published in GenBank by BLAST. The tuber powder was studied for pharmacognostic parameters, preliminary phytochemical screening and total phenolic contents by Folin-Ciocalteu method. Results showed that the similarity between the rbcL sequences of H. formicarum leaves collected in Phu Quoc and the control one published in Genbank was 99%. Moisture content, total ash value and acid insoluble ash value of dried tuber powder were 11.06%, 9.60% and 0.70%, respectively. Raw material contained carotenoids, triterpenoids, flavonoids, phenolics, tannins, saponins, reducing substances and amino acids. Total phenolics content was about 58.847 mg pyrrogallol equivalent/g dried powder weight. Our results provided information about botanical, genetic and preliminary phytochemical characteristics of H. formicarum growing on Phu Quoc Island. This could be useful for the authentication of H. formicarum as a medicinal material.

A new synonym of Rohdea annulata (Asparagaceae) and the first report of its wild distribution in China

Tupistra annulata H.Li & J.L.Huang in Huang & Li (1990: 51) was described based on the materials from cultivated plants in Kunming Botanical Garden, and its precise wild collecting locality was unknown. Its perianth with a ringed, fleshy, smooth, cushion like appendage in throat makes it distinctly different from other species in the genus. Tamura et al. (2000) separated Campylandra Baker (1875: 582) from Tupistra Ker Gawler (1814: 1655) and transferred this species into Campylandra as C. annulata (H.Li & J.L.Huang) Tamura et al. (2000: 159). Later, Tanaka (2003) transferred some species of Campylandra, Tupistra and Gonioscypha Baker (1875: 581) into Rohdea Roth (1821: 196) based on morphological analysis but not including Campylandra annulata. Supported by the evidence from parsimony analysis of trnK and rbcL sequences and karyotype data, Yamashita & Tamura (2004) merged the whole genus Campylandra in Rohdea. Therefore this species was combined as R. annulata (H.Li & J.L.Huang) Yamashita & Tamura (2004: 369).

Species diversity and phylogenetic analysis of Heliconia spp. collections of Purwodadi Botanic Garden (East Java, Indonesia) inferred by rbcL gene sequences

Hapsari L, Trimanto, Wahyudi D. 2019. Species diversity and phylogenetic analysis of Heliconia spp. collections of Purwodadi Botanic Garden (East Java, Indonesia) inferred by rbcL gene sequences. Biodiversitas 20: 1266-1283. Heliconia L. is a single genus in the family Heliconiaceae, with approximate consists of 200 species. It has wide morphological variations among and within species which led to problems in species identification. Species diversity and phylogenetic analysis using morphology and rbcL marker subjected to 17 Heliconia spp. living collections of Purwodadi Botanic Garden (PBG) have been conducted. The rbcL gene located in chloroplast genome is one of appropriate proposed marker for plant barcoding assessment. This study aimed to study morphology and genetic variability of the PBG Heliconiaceae collections, to confirms the species name for a more accurate identity record and to reveal the diversity and phylogenetics of the species. Morphological characterization showed high variability among Heliconia species, which included 3 subgenera (Heliconia, Stenochlamys, and Griggsia) and 1 hybrid. Each species possessed unique morphological characteristics. The common morphological characters which distinguished among and within Heliconia species includes leaf form, inflorescence type, and bract characteristics. Key to the Heliconia species examined is presented in this paper. However, molecular confirmation using rbcL sequences showed high conservation level (0.932) and low genetic variability. About 656 nucleotides were monomorphic and 33 positions were polymorphic which comprised 18 singleton variable sites and 15 parsimony informative sites. Twelve haplotypes were produced with haplotype diversity value 0.8952. Pairwise distance analysis shows that they were shared high similarity of rbcL sequences with very low genetic distance (0.022 to 0.000). The topology of phylogenetic tree resulted by Neighbour-Joining algorithm has the best grouping and be able to explain the relationship among species of Heliconia, although supported by low bootstrap (65). It was separated into two clades following its subgen. classification. Clade 1 consists of subgen. Heliconia and Griggsia; while clade 2 consists of subgen. Heliconia and Stenochlamys; also hybrid species. Further, separation of deeper branchings (section) was inconsistent and unclear. Upon this study, rbcL marker was considered too conserved thus less valuable for phylogenetic analysis at lower taxa among and within Heliconia spp. However, rbcL was suggested to distinguish at higher level taxa between closely related genus and above.

Genetic Diversity of Large Japanese Field Mouse Apodemus speciosus Populations and Identification of their Food Plant Resources using DNA Barcoding in an Industrial Green Space

Estimates of the genetic diversity of Large Japanese field mouse Apodemusspeciosus populations and identification of their plant food resources were conducted in an industrial green space, where were constructed on reclaimed land and belonged to the Aichi Refinery of Idemitsu Kosan Co., Ltd., in Aichi Prefecture, Japan. A total of six mitochondrial D-loop haplotypes were identified in 50 mice. Habitat condition with the highest number of captured individuals had abundant broad-leaved trees and understory vegetation. A minimum spanning network, which did not form a ring-shaped network, revealed that the hereditary population structure was weak. The low genetic diversity observed in the study area was thus attributed to isolation from other populations once the population in the study area by sea and road, which is more than 30 m wide. In order to identify which plant food resources were utilized by mice captured inside the industrial green space, partial chloroplast rbcL sequences were amplified by PCR from DNA extracted from 43 feces samples. Calculations of sample completeness curve revealed that 25 of the taxa identified in this study comprised approximately 90% of the food plant resources in the study area. Of the 21 plant families identified from the obtained rbcL sequences, members of the Rosaceae (28.0%), Fagaceae (17.2%), Lauraceae (14.2%) and Oleaceae (7.7%) were dominant. To ensure the continued survival of A. speciosuspopulation in this industrial green space would be to preferentially conserve plant species that are used as food resources by this species.