Molecular phylogenetics of Malesian Diospyros (Ebenaceae) based trnL-F spacer sequences

Wanda IF, Djuita NR, Chikmawati T. 2021. Molecular phylogenetics of Malesian Diospyros (Ebenaceae) based trnL-F spacer sequences. Biodiversitas 22: 4106-4114. Diospyros is a genus composed of potential species as an economic commodity with high diversity. However, there is limited information on the phylogenetic relationship of this genus in the Malesian region. This study aimed to provide information on the species diversity through a DNA barcoding approach and revealing the phylogenetic information of Diospyros spp. in the Malesian region. This study used 20 Diospyros accessions from Bogor Botanical Garden collections, 40 Diospyros accessions, and four outgroup accessions obtained from the NCBI database. The DNA barcoding primer utilized comes from plastids, trnL-F intergenic spacer. The phylogenetic trees were constructed using the Maximum-Parsimony method. A total of 20 accessions of Diospyros were validated with sequence data on the genebank. The result showed that all accessions had relationships with 44 other Diospyros species globally. Here, we reported 10 new trnL-F intergenic spacer sequences of Malesian Diospyros species. A phylogenetic tree grouped 64 monophyletic Diospyros species into seven clades. The phylogenetic results supports the biogeographic hypothesis: the Malesian region, the Malesian-Caledonian Region, and Cosmopolite species in almost all bioregions.

Morphological and Molecular Characterization of Paragonimus skrjabini Complex from Yunnan, China: A Brief Report

Purpose To perform environmental sampling and molecular identification of Paragonimus in endemic regions, which may help in minimizing transmission among humans. Methods Mountain crabs from the genus Potamiscus were collected and the encysted metacercariae were extracted and subjected to morphological identification, followed by animal inoculation in Sprague–Dawley (SD) rats. After 112 days of infection, animals were killed and adult worms were extracted from lungs and muscles. The morphology of adult worms was characterized by microscopy and molecular identification was done by polymerase chain reaction, followed by sequencing of cox1 and ITS2 genes. Phylogenetic analysis was done by maximum parsimony method. Results A total of 447 crabs were captured from the streams of Tongchang Town, Jinping County, Yunnan Province, China. The infection rate was found to be 41% (186 out of 447 crabs). The metacercariae of Paragonimus skrjabini was identified by the characteristics round or spherical encysted form measuring 410 to 460 × 400 to 460 µm. After animal infection in SD rats, adults were presumptively confirmed to be P. skrjabini, which was also confirmed by gene amplification and sequence analysis of cox1 and ITS2 regions. Paragonimus skrjabini clustered with previously reported P. skrjabini from Yunnan and Vietnam. The confidence values of their branches were > 95%. Phylogenetic analysis of the ITS2 region revealed two distinct clusters with distinct geographical grouping. Phylogenetic analysis with the combined data sets reiterated the geographical grouping with P. skrjabini from Yunnan clustering with strains from Vietnam. Conclusion Metacercariae of P. skrjabini was discovered in freshwater crabs in Yunnan province, China, and the strains were phylogenetically related to P. skrjabini from Vietnam.

Isolation and identification of mycorrhizal fungus from an epiphytic orchid (Rhynchostylis retusa L. Bl.)

Isolation and identification of mycorrhizal fungi from the roots of Rhynchostylis retusa indicated that the cultural and microscopic features, namely colony appearance, colony colour, diameter of vegetative hyphae, presence of monilioid cells, right angle branching pattern of the fungal endophyte corroborated the identity of the fungus Rhizoctonia like anamorphs of Ceratobasidium species. Fungal identity was further confirmed through sequencing and analysis of internal transcribed spacer (ITS) sequences of the nuclear ribosomal DNA (nrDNA). ITS sequence of the isolate RhY10A6 (accession no. MN120903.1) showed > 99% similarity with several isolates of the teleomorphic fungus Ceratobasidium sp. in NCBI mega blast search. The phylogenetic analysis based on maximum parsimony method, this orchid mycorrhizal fungus clustered with several isolates of Ceratobasidium sp. or Rhizoctonia like fungi. It showed near distant relation with Ceratobasidium ramicola (GeneBank accession no. NR_138368.1) which is an orchid mycorrhizal fungus. Therefore, molecular characterization validated the morphological data. The techniques established in this orchid will facilitate to isolate and accurate identification of mycorrhizal fungus.

Genetic Diversity of Natural Morchella spp. from the Southern Gansu Province Based on rDNA-ITS, China

Background: China is a mainland country rich in natural Morchella spp. resources. At present, about half of the natural Morchella spp. in the world has been recorded in China. The current study was aimed at the classification and cultivation of Morchella spp. This study provided a more accurate molecular trait for the systematic classification of Morchella spp. in southern Gansu Province, China.Methods: From April to May 2019. Based on the molecular biological analysis of 16 natural Morchella spp. strains collected from the southern Gansu province, China. the ITS fragments between transcripts of the rDNA gene were amplified by PCR and sequenced.Result: The results of BLAST comparison according to GenBank showed that the 16 tested strains of Morchella spp. were classified into 5 species, which were M.angusticeps, M.esculenta, M.elata, M.crapssipes and M.conica. According to the molecular evolutionary trees constructed by the Maximum-Parsimony method (MP) and the Neighbour-Joining method (NJ), the topological structures of the two molecular evolutionary trees were similar and the Bootstrap verification had a high support rate, indicating that the phylogenetic relationship had high credibility.

Molecular record for the first authentication of Isaria cicadae from Vietnam

The entomopathogenic fungus T011, parasitizing on nymph of Cicada, collected in the coffee garden in Dak Lak Province, Vietnam, was preliminarily morphologically identified as Isaria cicadae, belonged to order Hypocreales and family Clavicipitaceae. To ensure the authenticity of T011, phylogenetic analysis of the concatenated set of multiple genes including ITS, nrLSU, nrSSU, Rpb1, and Tef1 was applied to support the identification. Genomic DNA was isolated from dried sample T011. The PCR assay sequencing was applied to amplify ITS, nrLSU, nrSSU, Rpb1, and Tef1 gene. For phylogenetic analysis, the concatenated data of both target gens were constructed with MEGAX with a 1,000 replicate bootstrap based on the neighbor-joining, maximum likelihood, maximum parsimony method. As the result, the concatenated data containing 62 sequences belonged to order Hypocreales, families Clavicipitaceae, and 2 outgroup sequences belonged to order Hypocreales, genus Verticillium. The phylogenetic analysis results indicated that T011 was accepted at subclade Cordyceps and significantly formed the monophyletic group with referent Cordyceps cicadae (Telemorph of Isaria cicadae) with high bootstrap value. The phylogenetically analyzed result was strongly supported by our morphological analysis described as the Isaria cicadae. In summary, phylogenetic analyses based on the concatenated dataset were successfully applied to strengthen the identification of T011 as Isaria cicadae.

Taxonomic revision and morphological cladistics analysis of the diatom genus Anorthoneis (Cocconeidaceae), with description of Anorthoneis arthus-bertrandii sp. nov.

We present here an ultrastructure-based revision of the diatom genus Anorthoneis using both light (LM) and electron microscopy (SEM). The original slides and type material of all known Anorthoneis species were analysed and new, previously unseen, morphological features of the frustules, such as rota-like vela and ligulate structures around the valve margin, were revealed. The phylogenetic relationships among Anorthoneis taxa were investigated based on morphological characters using a maximum parsimony method. This analysis indicated a large, well-supported clade that included taxa that exhibited similarities in external and internal details of the central areas in both raphe-sternum and sternum valves. Revealed synapomorphies within the genus include externally and internally transversely dilated central area on both sides of the raphe-sternum valve, externally transversely dilated central area on both sides of the sternum valve, sternum valve striae parallel at the center and radiate at the apices, and areolae with hymenes in both valves. During a survey a new species Anorthoneis arthus-bertrandii sp. nov., was found on seagrass leaves collected from Siladen Island (Indonesia). This taxon is sister to A. hyalina, but can be easily differentiated from the latter by smaller valve dimensions, central area size and shape, and rota-like vela occluding sternum valve areolae - a feature that has never been observed in other Anorthoneis taxa.

Phylogenetic analysis of eyeworm (Oxyspirura petrowi) in northern bobwhite (Colinus virginianus) based on the nuclear 18S rDNA and mitochondrial cytochrome oxidase 1 gene (COX1)

Oxyspirura petrowi is a heteroxenous nematode found in northern bobwhite (Colinus virginianus) of the Rolling Plains ecoregion of Texas. Despite its impact on this popular gamebird, genetic level studies on O. petrowi remain relatively unexplored. To accomplish this, we chose the previously studied nuclear rDNA 18S region as well as the mitochondrial COX1 gene region of O. petrowi to investigate phylogenetic relations between O. petrowi and other nematode species. In this study, we generate primers using multiple alignment and universal nematode primers to obtain a near-complete 18S and partial COX1 sequence of O. petrowi, respectively. Phylogenetic trees for O. petrowi’s 18S and COX1 gene regions were constructed using the Maximum Likelihood and Maximum Parsimony method. A comparative analysis was done based on the nuclear and mitochondrial region similarities between O. petrowi and other nematode species that infect both humans and animals. Results revealed a close relation to the zoonotic eyeworm Thelazia callipaeda as well as a close relation with filarial super family (Filarioidea) such as the human eyeworm Loa loa and Dirofilaria repens eyeworm of dog and carnivores.

Variasi Cendana (Santalum album Linn.) Berdasarkan Morfologi Daun dan Bunga di Desa Petir, Rongkop, Gunungkidul

Cendana (Santalum album Linn.) merupakan salah satu spesies yang digunakan sebagai tanaman rehabilitasi, dikenal mampu tumbuh di lahan dengan keterbatasan hara, dan memiliki nilai ekonomi tinggi dari kandungan minyak santalol yang wangi. Cendana merupakan spesies asli di Provinsi Nusa Tenggara Timur (NTT) namun dijumpai permudaan alami di beberapa kawasan Kabupaten Gunungkidul, Daerah Istimewa Yogyakarta (DIY), salah satunya di Desa Petir, Kecamatan Rongkop, Kabupaten Gunungkidul. Permudaan alami tersebut memiliki variasi morfologi yang relatif besar pada populasinya. Penelitian ini bertujuan mendeskripsikan variasi cendana dan mengklasifikasikan cendana yang ditemui di lokasi penelitian berdasarkan karakter morfologi. Bahan yang digunakan dalam penelitian ini berupa (1) cendana (meliputi organ generatif dan vegetatif) yang diambil secara sengaja (purposive) dan (2) Ximenia americana sebagai spesies pembanding. Data dideskripsi secara morfologi dan dilakukan analisis filogeni menggunakan metode maksimum parsimoni dengan penanda morfologi. Karakter morfologi cendana bervariasi pada ukuran dan bentuk daun, warna tenda bunga (perigonium), bunga, bentuk cuping, bentuk appendiks, warna batang, bentuk dan ukuran buah. Ditemui dua karakter cendana yang belum dijelaskan pada sumber sebelumnya di Indonesia yaitu bentuk daun lanset dan bulat telur serta karakter warna tenda bunga (perigonium), bunga oranye. Hasil analisis filogeni menunjukkan karakter daun kecil cendana lebih dekat dengan yang dimiliki oleh spesies pembanding. Karakter diagnostik secara tegas mengelompokan ukuran daun kecil dan ukuran daun besar. Ukuran daun kecil pada kisaran 2,8-7,7 cm sampai dengan 1,1-2,2 cm dan 7,9-10 cm sampai dengan 3,1-4 cm untuk cendana berdaun besar. Variasi bentuk daun serta warna bunga ditemukan pada masing-masing kelompok ukuran daun.Variation of Sandalwood (Santalum album Linn.) Based on Morphology of Leaves and Flowers at Petir Village, Rongkop, GunungkidulAbstractSandalwood (Santalum album Linn.) is one of the flora that are commonly used as rehabilitation species, known as a plant that can grow in a lack of nutrient soil, and has a high economic value of santalol oil. This plant is indigenous in East Nusa Tenggara but it has a natural regenaration in some areas of Gunungkidul Yogyakarta, including in Petir Village, Rongkop, Gunungkidul. Morphological variation of Sandalwood in this area was relatively high. This research aimed to describe and classify sandalwood variations that exist in the research site based on the morphological characters. The materials used in this study were (1) sandalwood as the target species (including generative and vegetative organs) with purposive sampling and (2) Ximenia americana as the outgroup species. Data were analyzed using description and phylogeny analysis using maximum parsimony method with morphological markers. Morphological characters of Sandalwoods were varied in size and leave shapes, flowers perigonium, lobes shapes, appendices shapes, stem color, shapes and fruit size. There were two characters that have not been described in the previous sources in Indonesia, form of leaves (oblong and oval) and orange color of perigonium. Phylogeny results showed that a small leaf sandalwood character was closer to the outgroup character. Diagnostic characters explicitly classified small leaf size and leaf size large. Size of small leaf was from 2.8 -7.7 cm to 1.1-2.2 cm and from 7.9-10 cm to 3.1-4 cm for large leaf. Leaf form and colour of flower variation was found in each group of leaf size.