Molecular Phylogenetic Tree Research Articles

Eutetrarhynchus pacificus n. sp. (Cestoda: Trypanorhyncha) from Raja inornata Jordan & Gilbert (Batoidea: Rajiformes) off the coast of California with comments on congeners.

Eutetrarhynchus pacificus n. sp. is described from the spiral valve of Raja inornata Jordan & Gilbert off the coast of California, USA. The new species is distinguished from E. ruficollis (Eysenhardt, 1829) and E. leucomelanus (Shipley & Hornell, 1906) in having acraspedote rather than craspedote segments and a saccate rather than a branched uterus. It is distinguished from E. platycephali Palm, 2004 in lacking an enlarged hook in the eighth row of the basal armature and from E. beveridgei Schaeffner, 2013, which has a basal swelling and a distinctive basal armature. A partial redescription of E. ruficollis, the type-species of the genus, is provided based on available museum specimens, highlighting the need for a comprehensive redescription of this species to better define the characteristics of the genus. The presence of an undescribed species in museum collections is also noted. Based on the 28S ribosomal gene, the new species clustered with Dollfusiella in a molecular phylogenetic tree. The delimitation of Eutetrarhynchus and its relationship with Dollfusiella is discussed.

Taxonomy and phylogenetics ofAlliumsectionDecipientia(Amaryllidaceae): morphological characters do not reflect the evolutionary history revealed by molecular markers

AbstractPhylogenetic and phylogeographic patterns in Allium section Decipientia (subgenus Melanocrommyum) were analysed, including most species of this section and representative species of most other sections of the monophyletic Allium subgenus Melanocrommyum. We constructed a molecular phylogenetic tree based on predominantly non-coding sequences from nuclear ribosomal RNA and four plastid regions. In Allium section Decipientia, 2n = 16 and 2n = 20 chromosomes were confirmed and, in part, newly reported. All plants with 2n = 20 form a clade that is sister to the European A. decipiens and A. quercetorum with 2n = 16 in the ITS analysis and are genetically distant from the Eurasian steppe species with 16 chromosomes, A. tulipifolium and A. robustum. The plastid analysis does not reveal a clear relationship between these three monophyletic groups. The karyotype of A. tulipifolium is similar to those of other members of subgenus Melanocrommyum, but only preliminary chromosomal dimensions could be presented for the species with 2n = 20 chromosomes. The latter group comprises A. grande, A. subscabrum, A. viridulum and the newly described taxa A. koksuense and A. lepsicum. Allium tulipifolium and A. robustum have been placed in a new section Tulipifolia, and all species with 2n = 20 have been placed in a new subsection, Viceniprason, of section Decipientia. A nomenclatorial conspectus and a key for determination of the members of Allium sections Decipientia and Tulipifolia are also provided. According to the time divergence estimation, subgenus Melanocrommyum diversified c. 25 Mya in the late Oligocene. The split between most species of the section Decipientia and other taxa in this subgenus was estimated as early Miocene, which furthermore diversified into two sister clades in mid Miocene. Allium decipiens split off from the ancestors of the 2n = 20 species also in the mid Miocene. Further branching in this species group is of rather young age, and occurred within the last 6 Myr (late Miocene to late Pliocene and Pleistocene).

Aymoreana(Nelsonioideae, Acanthaceae), a New Genus Endemic to Brazil

Abstract—A species previously treated inStaurogyne(S. nitida) is elevated to the category of a new genus of Acanthaceae, subfamily Nelsonioideae, based on morphological and molecular data. The sole species,Aymoreana nitida, occurs in the Atlantic Forest of eastern Brazil, from southern Bahia to northern Espírito Santo.Aymoreanadiffers from other genera of Nelsonioideae by the combination of the calyx with subequal segments, the slightly zygomorphic corolla, the four didynamous stamens, and the asymmetric gynoecium. Morphological information is accompanied by a molecular phylogenetic tree, ecological data, a preliminary conservation assessment, and illustrations.

Immunoinformatic investigation of three structural protein genes in Indonesian SARS-CoV-2 isolates

Introduction: SARS-CoV-2 has crossed the species barrier to infect human. It is a rapidly spreading virus that has poses a significant public threat and is a considerable burden on the global economy and human health. Objective: We characterized the nucleocapsid phosphoprotein (N), membrane protein (M), and envelope protein (E) genes of Indonesian isolates to investigate genetic composition, predict B-cell epitopes, and construct a molecular phylogenetic tree. Methods: In the present work, we retrieved the sequences of 16 Indonesian isolates from the GISAID EpiCoV and the Wuhan-Hu-1 isolate (reference sequence) from GenBank, NCBI. We used MEGA X to identify mutations in the three structural protein genes and to construct a molecular phylogenetic tree. The IEDB was employed to reveal the linear B-cell epitopes and other parameters. Allergenicity prediction was evaluated using AllerTOP and ToxinPred was performed to analyze non-toxic antigen prediction. Results: In this study, we report the genetic composition of three structural protein genes in Indonesian SARS-CoV-2 isolates. Furthermore, we identified the peptide RGPEQTQGNFGDQELIRQGTDYK from nucleocapsid phosphoprotein to generate a peptide-based vaccine contrary to SARS-CoV-2. Conclusion: In summary, we propose a candidate for a peptide-based vaccine contrary to SARS-CoV-2. However, advance trials such as in vitro and in vivo testing are involved for validation.

The Destructive Tree Pathogen Phytophthora ramorum Originates from the Laurosilva Forests of East Asia.

As global plant trade expands, tree disease epidemics caused by pathogen introductions are increasing. Since ca 2000, the introduced oomycete Phytophthora ramorum has caused devastating epidemics in Europe and North America, spreading as four ancient clonal lineages, each of a single mating type, suggesting different geographical origins. We surveyed laurosilva forests for P. ramorum around Fansipan mountain on the Vietnam-China border and on Shikoku and Kyushu islands, southwest Japan. The surveys yielded 71 P. ramorum isolates which we assigned to eight new lineages, IC1 to IC5 from Vietnam and NP1 to NP3 from Japan, based on differences in colony characteristics, gene x environment responses and multigene phylogeny. Molecular phylogenetic trees and networks revealed the eight Asian lineages were dispersed across the topology of the introduced European and North American lineages. The deepest node within P. ramorum, the divergence of lineages NP1 and NP2, was estimated at 0.5 to 1.6 Myr. The Asian lineages were each of a single mating type, and at some locations, lineages of “opposite” mating type were present, suggesting opportunities for inter-lineage recombination. Based on the high level of phenotypic and phylogenetic diversity in the sample populations, the coalescence results and the absence of overt host symptoms, we conclude that P. ramorum comprises many anciently divergent lineages native to the laurosilva forests between eastern Indochina and Japan.

Role of lipid metabolism and systemic inflammation in the development of atherosclerosis in animal models

Systemic inflammation makes a significant contribution to the pathogenesis of atherosclerosis and has been the subject of numerous studies. Works aiming to analyze the mechanisms of atherosclerosis development often include experiments on animals. A primary task of such research is the characterization, justification, and selection of an adequate model.
 Aim. To evaluate the peculiarities of lipid metabolism and systemic inflammation in chronic obstructive pulmonary disease (COPD) in the development of atherosclerosis in animal models.
 Materials and Methods. Analyses of cross-links between species-specific peculiarities of lipid metabolism and the immune response, as well as a bioinformatic analysis of differences in Toll-like receptor 4 (TLR4) in mice, rats, and rabbits in comparison with its human homolog, were carried out. A search for and analysis of the amino acid sequences of human, mouse, rat, and rabbit TLR4 was performed in the International database GenBank of National Center of Biotechnical Information and in The Universal Protein Resource (UniProt) database. Multiple alignments of the TLR4 amino acid sequences were implemented in the Clustal Omega program, version 1.2.4. Reconstruction and visualization of molecular phylogenetic trees were performed using the MEGA7 program according to the Neighbor-Joining and Maximum Parsimony methods.
 Results. Species-specific differences of the peculiarities of lipid metabolism and the innate immune response in humans, mice, and rabbits were shown that must be taken into account in analyses of study results.
 Conclusion.Disorders in lipid metabolism and systemic inflammation mediated by the innate immune system participating in the pathogenesis of atherosclerosis in COPD possess species-specific differences that should be taken into account in analyses of study results.

CDNA cloning, expression and bioinformatical analysis of Tssk genes in tree shrews

Tree shrews are more closely related to primate animals than rodents in many aspects.In addition, they also possess several advantageous characteristics including small body size, high brain-to-body mass ratio, low cost of feeding and maintenance, short reproductive cycle and life span, which make them promising novel laboratory animals to replace more precious larger primate animals. Testis-specific serine/threonine kinase (Tssk) plays important roles in spermatogenesis and/or the regulation of sperm function. However, studies on Tssk in tree shrews have not been reported yet. In the present study, the full-length sequences of five members of the Tssk family in tree shrews were cloned and their CDS region sequences were analyzed by basic bioinformatics. The phylogenetic tree and prokaryotic protein expression system of Tssk gene of tree shrews were constructed. The mRNA expressions of Tssk genes in 11 tissues/organs from tree shrews were studied. The results showed that: 1. the length of the CDS region of tree shrew Tssk gene for Tssk1B, Tssk2, Tssk3 (variant X1 / X2), Tssk4 (variant X1 / X2) and Tssk6 is 1080bp, 1077bp, 867 / 807bp, 1014 / 984bp, 822bp, respectively, encoding 359, 358, 288/268, 337/327 and 273 amino acids, respectively; the cloned sequences of Tssk genes have been submitted to GenBank with the following accession numbers: KX091161(Tssk1B), KX091162(Tssk2), KX091163(Tssk3 variant X1)/KX091164(Tssk3 variant X2), KX091165(Tssk4variant X1)/KX091166(Tssk4variant X2), KX091160(Tssk6). 2. All tree shrew Tssk proteins distribute in cytoplasm, indicating that they are hydrophilic and non-secretory proteins, with multiple phosphorylation sites of serine and/or threonine. In addition, they are all mixed proteins with similar tertiary structures sharing a highly conserved functional domain of S_TKc (Serine/Threonine protein kinases, catalytic domain). 3.The molecular phylogenetic tree of five Tssk genes in tree shrews indicates that they are neither rodent nor primate animal, but are closely related to primate animals. 4. Five members of the Tssk recombinant proteins in tree shrews were successfully obtained using the constructed prokaryotic protein expression system. 5. Five Tssk genes are specifically expressed in the testis and/or sperm of tree shrews. Additionally, small amount of Tssk1B was expressed in several tissues other than testis and sperm. Limited mRNA levels of Tssk2 and Tssk4 were expressed in the brain, while mRNA of Tssk3 or Tssk6 could only be detected in the testis and sperm.This study will provide fundamental data on reproductive biology of tree shrews, which paves a way for further studying Tssk’s biological function in this novel model animal.

Investigation on antibiotic resistance property of bacteria enumerated from human pus samples

This study was focused on evaluating the presence of commercial antibiotic resistant pathogenic bacteria present in the pus samples of patients from secondary care hospital Namakkal district of Tamil Nadu. About 494 pus samples from a total of 729 samples contained antibiotic resistant pathogenic bacteria. Among several bacterial species, the strains of Pseudomonas, Staphylococcus, Klebsiella, Escherchia, Enterococcus, and Micrococcus were found as the most predominant bacteria from pus samples. About 4 isolates showed resistance to more commercial antibiotics. These 4 strains were identified as P. aeroginosa CM1 to CM4. These strains showed resistance up to 98% for more number of commercial antibiotics than other strains. These strains were identified through molecular characterization (16S rRNA) and phylogenetic tree analysis. Fortunately, these 4 numbers of P. aeruginosa strains were susceptible to meropenem and amikacin antibiotics. These results declared that the pathogenic bacteria present in the soft tissue of wounds and others could create resistance to the commercial antibiotic, which is frequently administered to the patients.

ВЫЯВЛЕНИЕ ПРИНАДЛЕЖНОСТИ ВИДОВ ГИДРОБИОНТОВ ОЗЕРА ВЕРХНИЙ КАБАН Г.КАЗАНИ К ЗОНАМ САПРОБНОСТИ ПО МАРКЕРНОМУ ГЕНУ 18S рРНК

The paper presents the results of a study of the hypothetical evolution of unicellular hydrobionts from Verhniy Kaban Lake of Kazan by 18S rRNA marker gene with aim of identifying species saprobity based on molecular phylogenetic analysis. Verhniy Kaban Lake is an isolated freshwater reservoir. Its coastline is relatively smooth except for the northern end, which forms swampy and overgrown bay. To the West of lake an urban settlement is situated and to the East are highway and garden plots. Lake length is about 1 km, average and maximum widths are 245 m and 370 m respectively. Deepest point is 15 m; average depth is 8 m; lake area – 25 hectares. Quality of lake water is classified as polluted. One of methods used for ecological assessment of water reservoirs is the bioindication method based on ability of indicator species to survive in water of certain pollution degree. At present, freshwater hydrobiont species from V.Sladechek’s list (1973) are used as indicators. Indicator species lists expand very slowly due to the long process of experimental observations. At the same time, price drop of the next-generation sequencing technologies allows to identify more organisms and run molecular phylogenetic analysis with aim of identifying species saprobity based on study of hypothetic evolution of hydrobionts by marker genes including 18S rRNA. The advantage of using 18S rRNA/16S rRNA ribosomal genes is that they are present in all organisms. Ribosomal genes are ones of the most conservative genes. Therefore, the systematic position of the organism and the time of divergence with closely related species can be determined via analysis of similarities and differences in rRNA sequences. Molecular phylogenetic trees were constructed by the neighbour joining (NJ) and maximal parsimony (MP) methods for Verhniy Kaban Lake hydrobionts identified with next-generation sequencing method (Illumina) by 18S rRNA marker gene (SRR7510986, SRR7465374, SRR7516513). Phylogenetic analysis of hydrobionts of Verhniy Kaban Lake by 18S rRNA marker gene revealed 12 clusters with a high bootstrap value (68–100 %), containing 12 existing indicator species of hydrobionts and 13 unicellular algae and ciliates – indicator species of different saprobity that did not previously have the status of bioindicators – algae: Diplosphaera mucosa (o-b-saprobity), Mucidosphaerium pulchellum (p-a-saprobity), Dinobryon pediforme (b-mesosaprobity), Gomphoneis minuta (b-mesosaprobity), Nitzschia amphibia (o-b-saprobity), Rhabdonema adriaticum (o-b-saprobity), Synura mammillosa (b-o-saprobity), Cryptomonas ozolini (b-mesosaprobity); ciliates: Astylozoon enriquesi (b-mesosaprobity), Blepharocorys curvigula (p-saprobity), Enchelys gasterosteus (p-saprobity), Monocoronella carnea (a-b-mesosaprobity), Plagiopogon loricatus (b-a-mesosaprobity). Most identified indicator species group near b-mesosaprobity. This indicates ecological state of Verhniy Kaban Lake as polluted by ecological assessments.

A new aphelench nematode, Basilaphelenchus brevistylus n. sp. (Aphelenchoididae: Tylaphelenchinae) from Pinus massoniana in China.

Basilaphelenchus brevistylus n. sp. was isolated from masson pine (Pinus massoniana) in Guangdong province, China. The new species is characterized by an offset lip region, short stylet (female stylet 4.5-5.5 μm and male stylet 4-5 μm long) with three elongate posteriorly directed knobs, posteriorly located metacorpal valve and lateral field composed of three lines. The female has an elongate postuterine sac and a short conical tail, uniformly narrowing toward a sharp tip, or tapering to a slightly offset mucronate tip in a few individuals. The male has a conical tail with a sharp terminal mucro, three pairs of caudal papillae, and small arcuate spicules with a bluntly rounded condylus and small pointed rostrum. B. brevistylus n. sp. can be distinguished from all described Basilaphelenchus nematodes by numerous morphological and morphometrical traits, especially the tail morphology of both sexes and stylet length. In addition, molecular phylogenetic trees inferred from rRNA small subunit and D2-D3 expansion domains of large subunit revealed that this nematode belongs to the Basilaphelenchus, and is clearly different from all the other Basilaphelsenchus species.

Molecular presence of Felis catus gammaherpesvirus -1 in cats with ocular disorders in Turkey

Felis catus gammaherpesvirus-1 (FcaGHV-1) is a newly discovered feline virus, and its effects on the health of cats are not certainly known. Known feline viruses act a huge role in ocular disorders in domestic and wild cats. Also, emerging viruses may be responsible for ophthalmic diseases of cats. We aimed to investigate the presence of FcaGHV-1, Feline calicivirus (FCV), Feline herpesvirus -1 (FHV-1), Feline immunodeficiency virus (FIV), Feline infectious peritonitis virus (FIPV), Feline leukemia virus (FeLV), Feline panleukopenia virus (FPV) in cats in Turkey by molecular methods, and to examine the relationship between the virus and ocular disorders by obtained data. Swab, blood and biopsy samples were collected from 45 owned cats with ocular disorders. Molecular techniques including viral genome amplification and sequencing were used in this study. Both blood-swabs and biopsy samples were in two out of 45 cats (2/45; 4.4%) found to be FcaGHV-1 and FIV positive. Our two sequences and a reference sequence from Japan (LC437925) have constructed a separated subgroup in the molecular phylogenetic tree. In this study, we have not found a tight relationship between ocular disorders and FcaGHV-1. This is the first study exhibiting the presence of FcaGHV-1 and its molecular status in Turkey. Acquired knowledge suggests performing further studies on FcaGHV-1.

First Report of Botryosphaeria dothidea Causing Stem Canker on Soybean in China.

In September 2020, widespread stem canker on soybean (Glycine max) was detected in southeastern Jiangsu (Nantong municipality; 120.76° E, 32.23° N) in China. Mature plants, 14 weeks of cultivation, exhibited brown necrotic lesions and dried-up stem. The symptoms were observed in eleven soybean fields, 1.6 ha in total, and approximately 80% of the plants were symptomatic. The symptoms were consistent with those previously reported for stem canker on soybean caused by Diaporthe aspalathi, D. caulivora and D. sojae (Ghimire et al. 2019; Mena et al. 2020). Small pieces, approximately 0.4 cm2 in size, of symptomatic tissue were surface sterilized in 1.5% NaOCl for 1 min, and washed twice with sterile ddH2O. The pathogen was isolated and cultured on potato dextrose agar (PDA), containing chloramphenicol (50 µg/mL), under darkness at 28 ºC for 7 days. Amplification of internal transcribed spacer (ITS), elongation factor 1-α (EF1-α) and β-tubulin (TUB2) genes was performed using ITS1/ITS4, EF1-728F/EF1-986R and Bt2a/Bt2b primers, respectively (Jia et al. 2019). Sequences were submitted to GenBank under accession numbers MW130133 (ITS), MW147481 (EF1-α) and MW147482 (TUB2). Blast search revealed that the amplified sequences had 99.65% (ITS; B. dothidea JZB310202, MN945381), 100% (EF1-α; B. dothidea ZB-77, MH726166) and 99.75% (TUB2; B. dothidea ZB-1, MN642587) matches to multiple B. dothidea strains, whereas all reported Diaporthe strains showed no nucleotide identity to the amplified sequences. Molecular phylogenetic tree was constructed using MEGA7 to confirm the identity of the pathogen. ITS, EF1-α and TUB2 sequences were blasted separately in Muscle (https://www.ebi.ac.uk/Tools/msa/muscle/) and then combined together to make the phylogenetic tree. The evolutionary history was inferred by using the Maximum Likelihood method based on the Tamura 3-parameter model, and the tree with the highest likelihood (-4291.3981) is shown in Figure 1. Diaporthe strains found causing stem canker on soybean, some Phytophthora sojae strains (which also cause dried-up stem on soybean) (Yang et al. 2019), and B. dothidea strains found in China in other hosts were included in the phylogenetic tree. To confirm pathogenicity, a sterilized spatula was used to make wounds (3 mm diameter, 1 mm depth) on the stem of 2-week old soybean plants. Mycelial plugs from a 7 day-old culture on PDA were placed on the wounds and covered with Parafilm. Sterilized PDA plugs were used as control. Inoculated plants were maintained in a growth chamber at 28 °C and 60% relative humidity. Typical stem canker symptoms were observed 5 days after inoculation (Figure 2). Microscope observations showed the presence of septate mycelium, fusiform conidia and round conidiomata, and agreed with those previously reported for the morphology of B. dothidea strains (Phillips et al. 2013). During recent months, B. dothidea was reported to cause stem canker and leave wilt on a number of plant species in China (Huang et al. 2020; Ju et al. 2020; Wang et al. 2020a, 2020b, 2020c), confirming the expansion and host promiscuity of this pathogen. Stem canker on soybean has been thoroughly associated to Diaporthe strains; however, this is the first report of B. dothidea causing this disease. We believe that our results will help to better understand the pathogens affecting soybean production in China.

Trichaleurina javanica from West Java

Trichaleurina is a fleshy mushroom with goblet-shaped within Pezizales. Many genera have a morphology similar to Trichaleurina, such as Bulgaria and Galiella. Some previous reports had been described fungi like Trichaleurina as Sarcosoma. Indonesia has been reported that has Trichaleurina specimen (the new name of Sarcosoma) by Boedijn. This research aimed to obtain, characterize, and determine the Trichaleurina around IPB University. Field exploration for fungal samples was used in the Landscape Arboretum of IPB University. Ascomata of Trichaleurina were collected, observed, and preserved using FAA. The specimen was deposited into Herbarium Bogoriense with collection code BO 24420. The molecular phylogenetic tree using RAxML was used to identify the species of the specimen.
 Morphological data were used to support the species name of the specimen. Specimen BO 24420 was identified as Tricahleurina javanica with 81% bootstrap value. Molecular identification was supported by the morphological data, such as the two oil globules and the size of mature ascospores.

First Report of Colletotrichum brevisporum Causing Soybean Anthracnose in China.

In March 2020, widespread anthracnose was observed on soybean (Glycine max) in southeastern Jiangsu (Nantong municipality; 120.53° E, 31.58° N) in China. Plants exhibited irregular brown necrotic lesions in stem and leaves, and pronounced wilting. The symptoms were detected in one soybean field, 0.42 ha, surrounded by healthy wheat fields. Approximately 65% of the soybean plants showed the disease symptoms, and crop yield was reduced by 28-35% with respect the yield achieved in previous years, when no symptoms were observed. The symptoms were consistent with those previously reported for anthracnose on soybean caused by Colletotrichum chlorophyti, C. cliviae and C. gloeosporioides (Barbieri et al. 2017; Mahmodi et al. 2013; Yang et al. 2012). Diseased, 3-week old plants were collected. Small pieces, approximately 1 cm2 in size, of symptomatic tissue were surface sterilized in 1.5% NaOCl for 1 min, and washed twice with sterile ddH2O. The pathogen was isolated and cultured on potato dextrose agar (Song et al. 2020), containing chloramphenicol (50 µg/mL), under darkness at 28 °C for 3 days. Sequence of internal transcribed spacer (ITS), actin (ACT), β-tubulin (TUB2) and glyceraldehyde 3-phosphate dehydrogenase (GAP/span>DH) genes was performed as reported by Yang et al. (2015). Sequences were submitted to GenBank under accession numbers MT361074 (ITS) and MT415548-MT415550 (ACT, TUB2 and GAPDH). Blast search revealed that the amplified sequences had 100% (ITS; C. brevisporum TCHD, MH883805), 97.66% (ACT; C. brevisporum S38, KY986905), 99.06% (TUB2; C. brevisporum PF-2, KY705061) and 100% (GAPDH; C. brevisporum LJTJ27, KP823797) matches to multiple C. brevisporum strains, whereas all reported C. chlorophyti, C. cliviae and C. gloeosporioides strains showed no similarity to at least 2 of the studied genes. Molecular phylogenetic tree constructed using MEGA7 confirmed the identity of the pathogen. ACT and ITS sequences were blasted separately in Muscle (https://www.ebi.ac.uk/Tools/msa/muscle/) and then combined together to make the phylogenetic tree. The evolutionary history was inferred by using the Maximum Likelihood method based on the Tamura 3-parameter model, and the tree with the highest log likelihood (-1749.2186) is shown in Figure 1. The Colletotrichum strains previously found causing anthracnoseon soybean, and other relevant strains used in taxonomic analyses were included in the phylogenetic tree. Microscope observations showed the presence of 15-µm-long cylindrical conidia and septate mycelium, and agree with those reported for the morphology of C. brevisporum by Damm et al. (2019). To confirm pathogenicity, the mycelia from a 2 day-old culture on PDA was collected and suspended in sterile ddH2O (≈ 106 cells/mL) to prepare the inoculum. The pathogen was sprayed-inoculated on stem and leaves of healthy soybean plants. In control plants, sterile ddH2O was used. Inoculated plants were maintained in growth chamber at 28 °C and 50% relative humidity. Typical anthracnose symptoms were obsered 20 days after inoculation (Figure 2). C. brevisporum was reported to produce anthracnose on pumpkin, papaya, mulberry, coffee, passion fruit and pepper in China (Liu et al. 2017; Liu et al. 2019; Xue et al. 2019). Here, we report for the first time C. brevisporum causing anthracnose on soybean, an economically-relevant crop in China.

Molecular detection and phylogenetic analysis of Vibrio cholerae genotypes in Hillah, Iraq.

Vibrio cholerae is a cause of serious endemic diarrhoea associated with cholera in many regions in the world. A total of 256 stool and rectal swabs were collected from patients suspected to have cholera admitted to three hospitals in Hillah, Babylon Governorate, Iraq, for the period 1 September to 29 December 2017. After the routine culture of samples for isolation and identification of V. cholerae isolates, PCR was performed for molecular detection of V. cholerae isolates based on 16S ribosomal RNA gene. Toxigenicity was detected by RTX toxin genes. PCR technique emphasized molecular detection of V. cholerae for eight isolates. Only two isolates (25%) possessed both the rtxA and rtxC genes, while only three isolates (37.5%) possessed the rtxB gene. DNA sequencing was performed for the eight isolates via analysis and phylogenetic tree. The observed bacterial variants were compared to their neighbour homologous reference sequences using the National Center for Biotechnology Information (NCBI) BLAST server (Basic Local Alignment Search Tool; https://blast.ncbi.nlm.nih.gov/Blast.cgi). The findings indicated that the eight investigated isolates of V. cholerae were positioned in three different phylogenetic positions. Partial sequence dissimilarities were reported between GenBank isolate accession number MK212155.1 and these six clustered GenBank accession numbers of the same species. For the first time in Babylon Governorate, Iraq, the molecular assay, sequencing and phylogenetic tree are reported for V. cholerae and their toxins isolated during the 2017 cholera outbreak.

Molecular and morphological evidence for the identity of the giant mottled eel, Anguilla marmorata in Southeast Asia

Tropical anguillid eels account for two-thirds of all species in the genus Anguilla, however information on the species diversity, biogeography, and life histories is very limited. The anguillid eels found in Southeast Asian waters, i.e. Malaysia, Thailand and Vietnam were identified using molecular genetic analysis after morphological observation. This is the first record of the occurrence of A. marmorata in Thailand and Vietnam that was confirmed by both morphological and molecular genetic analyses. The molecular phylogenetic tree and the haplotype network of A. marmorata in Malaysia, Thailand and Vietnam suggested that the eel might be transported from the western North Pacific spawning area. We also propose possible dispersion and migration of A. marmorata into Southeast Asian waters.

Short Communication: Molecular characteristics and phylogenetic relationships of silurid catfishes (Kryptopterus, Ompok and Phalacronotus) from the Kampar River, Indonesia, based on the cytochrome b gene

Abstract. Elvyra R, Solihin DD, Affandi R, Junior MZ, Suhendra M. 2020. Short Communication: Molecular characteristics and phylogenetic relationships of silurid catfishes (Kryptopterus, Ompok, and Phalacronotus) from the Kampar River, Indonesia, based on the cytochrome b gene. Biodiversitas 21: 3539-3546. The study of molecular characteristics and phylogenetic relationships among silurid catfishes (Kryptopterus, Ompok, and Phalacronotus) is very scarce. Existing data are mostly based on morphological characters. Genetic markers among Kryptopterus, Ompok, and Phalacronotus can be analyzed by exploring the nucleotide and amino acid sequences of the mitochondrial cytochrome b gene region (906 base pairs). This study aims to identify molecular characteristics and phylogenetic relationships of silurid catfishes from the Kampar River based on the cytochrome b gene. This research examined 14 silurids of four species from three genera in the Kampar River of Indonesia. Molecular phylogenetic trees were constructed using the neighbor-joining method. This study revealed that three amino acid sites can be used as specific genetic markers for characterizing Kryptopterus limpok. They are E (glutamic acid) at site 123, V (valine) at site 155, and M (methionine) at site 156. Two amino acid sites can be used as specific genetic markers of Ompok spp., A (alanine) at site 201 for O. hypophthalmus, and S (serine) at site 282 for O. eugeneiatus. Two amino acid sites, I (isoleucine) at site 197 and L (leucine) at site 284, can be used to identify Phalacronotus apogon from the Kampar River, Indonesia. The phylogram results based on genetic distance (p-distance) from nucleotide cytochrome b sequences generally showed that intraspecies of K. limpok, O. eugeneiatus, O. hypophthalmus and P. apogon from the Kampar River formed relationship groups within each species supported by high bootstrap values. Additionally, based on genetic distance, K. limpok data from the Kampar River samples and GenBank formed one relationship group within species of K. limpok with a high bootstrap value. This study proves that cytochrome b gene can be used to identify molecular characteristics and phylogenetic relationships of K. limpok, O. eugeneiatus, O. hypophthalmus, and P. apogon from the Kampar River, Indonesia.

On the Occurrence of Metadiaptomus chevreuxi (Calanoida, Diaptomidae, Paradiaptominae) in the Iberian Peninsula, With Notes on the Ecology and Distribution of its European Populations

Temporary ponds are one of the most peculiar ecosystems in the world, being characterized by an extraordinarily rich crustacean fauna, with a high degree of endemism. Among them, diaptomid copepods are among the most biogeographically interesting taxa. However, the present knowledge on diaptomid distribution is still far from being exhaustive, even in the relatively well-studied western European countries. In this study, we report the first record of the diaptomid calanoid copepod Metadiaptomus chevreuxi for the Iberian Peninsula, where it was collected in five temporary ponds in Andalusia (Spain). The characteristics of the new sites are described, the literature dealing with the European localities of the species is reviewed, and a molecular phylogenetic tree has been built, based on new and previously available mitochondrial DNA sequences, thus expanding the knowledge on the ecology and phylogeography of this rare species. The species mainly occupies small isolated temporary ponds in (semi-)arid regions, suggesting adaptations to unpredictable aquatic habitats. The existence of two molecular clades separating the Iberian from the Sicilian and Tunisian populations supports the existence of a longitudinal long-term disjunction, whereas the north-south flow is probably facilitated by migrating birds. Further research on the biota of the small water bodies of the western Mediterranean area may help to expand our knowledge on rare aquatic species, such as M. chevreuxi, and to better interpret their natural history.

Internal transcribed spacer sequence based identification and phylogenic relationship of Herba Dendrobii

Herba Dendrobii, commonly known as "Shi-hu", has been used as a precious traditional Chinese medicine. It is expensive and adulteration are common due to high demand. The method to distinguish the herb from adulterant species is necessary. In the present study, internal transcribed spacers (ITS) region-based analysis was employed to ascertain the phylogenetic relationship among the 11 Dendrobium and two adulterant species Pholidota articulata and Flickingeria comate. Results showed that the length of the ITS regions among the thirteen species ranged from 635 to 641 bp and the GC ratio in ITS (ITS1 + 5.8S + ITS2) regions ranged from 50.55% to 57.25%. Dendrobium species was significantly different from one another by an average of 13.20% and from P. articulata and F. comate by 42.00% and 29.00% respectively. The molecular phylogenetic trees indicated that most of Dendrobium species are closely related and share common clad while both the adulterants outgroup and have separated clad. Therefore, ITS regions can be used as a molecular marker to differentiate medicinal Dendrobium spp. from one another and also from adulterants.

Parvilucifera multicavata sp. nov. (Alveolata, Perkinsozoa), a New Parasitoid Infecting Marine Dinoflagellates Having Abundant Apertures on the Sporangium

The phylum Perkinsozoa is known as an exclusively parasitic group including the parasites of shellfish, fish, dinoflagellates, cryptophytes, and tadpoles and at present comprises seven genera across three families (Parviluciferaceae, Perkinsidae, and Xcellidae), with the genus Parvilucifera having the most abundant species in the family Parviluciferaceae. During intensive sampling along the Korean coast in August and September 2017, a new species of the genus Parvilucifera was discovered and successfully established in cultures. Morphological and ultrastructural observations revealed that the new parasitoid shares almost all known diagnostic characters with other species of Parvilucifera, except that its sporangium has a higher number of apertures although with smaller diameters than those in P. infectans. Molecular phylogenetic trees based on both nuclear small subunit (SSU) and concatenated SSU and large subunit (LSU) ribosomal DNA (rDNA) sequences revealed that the new parasitoid was nested within the family Parviluciferaceae and had a sister relationship with P. infectans. Based on morphological, ultrastructural, and molecular data, we propose to erect a new species, P. multicavata sp. nov., for the new parasitoid found in this study.