Phylogenetic Maximum Likelihood Research Articles

A Piece of the Piper Puzzle: Systematics of Piper section Enckea, a Neotropical Section in a Giant Genus

Abstract— Piper (Piperaceae) is a species-rich pantropical genus (∼2000 species) with distinctive morphological characteristics such as swollen nodes, lack of a perianth, and one-seeded berry fruits. Sub-generic lineages within Piper are often associated with morphological traits and are well supported with molecular data, but sampling in previous studies was incomplete and conflict exists between morphological data and molecular inference. In this paper, Piper section Enckea is evaluated for monophyly by pairing DNA, primarily from herbarium material, with the Angiosperms353 bait set, high throughput sequencing, recently developed bioinformatic techniques, and two methods of phylogenetic reconstruction, maximum likelihood and multi-species coalescence. The results suggest continued support for recognition of Piper sect. Enckea as a monophyletic section and contribute to a better understanding of relationships among species within the section. However, they also raise questions about the morphological features used to define P. sect. Enckea and delimit species within the section.

Ecological distribution and phylogenetic diversity of measles virus genotypes in West Africa, 2001 to 2020

IntroductionMeasles remains a significant threat to public health in developing countries, particularly among children under the age of 5. A pivotal aspect of the measles eradication initiative involves the genetic characterization of wild-type viruses to better understand transmission patterns and inform vaccination strategies. ObjectiveThis study aims to investigate the ecology and genotype diversity of the measles virus in West Africa from 2001 to 2020, utilizing available sequence data from the GenBank. MethodologyWe conducted a comprehensive analysis using maximum-likelihood phylogenetics, focusing on the N450 fragment from measles virus isolates found in West Africa between 2001 and 2020. Additionally, pairwise sequence comparison analysis was carried out to determine the evolutionary divergence of various genotypes in West Africa and their genetic distance from vaccine strains. ResultsOur findings indicate that over the past 2 decades, B3, D3, and D8 isolates have been circulating in various West African countries. Notably, B3 isolates have been identified as the primary contributors to endemic transmission, as evidenced by the concurrent presence of the same isolate in different countries within the subregion. Furthermore, our analysis reveals a significant shift in the circulation of D3 and D8 isolates, which were originally reported exclusively in New Guinea over 15 years ago but are now dominated by the B3 genotype. ConclusionOver the past 2 decades, B3, D3, and D8 measles virus genotypes have circulated in 10 West African countries. Particularly, B3.1 isolates currently dominate, especially in Nigeria, indicating endemic transmission. However, despite the informative value of N450, complete genome sequencing data is highly needed to accurately understand the evolutionary dynamics of the measles virus in West Africa.

First Report of Nigrospora hainanensis Causing Leaf Blight on Brachiaria Griseb in China.

Brachiaria Griseb is an important gramineous forage grown in tropical regions, and also a main grass species uses to restore grasslands in tropical and subtropical regions of China. In August 2022, symptoms of leaf blight were observed on nearly 30% of the Brachiaria forage grass in the base of the Chinese Academy of Tropical Agricultural Sciences, Hainan, China. Symptomatic leaves initially exhibited small, reddish-brown, round or oval spots on their tips, subsequently expanding in size along the leaf margin, and gradually becoming wilted and dry. Twenty leaves showing typical symptoms were randomly collected and pieces (5×5 mm) from the junction of diseased and healthy region were cut, sterilized with 75% alcohol for 30 s, followed by 5% sodium hypochlorite for 30 s. Rinsed three times with sterile water and dried with sterile filter paper. Leaf pieces were placed on potato dextrose agar (PDA) and incubated at 28℃. The colonies were white on the surface and gray on the reverse side. The conidiogenous cells were monoblastic, hyaline, globose or ampulliform, and 6 to 8.7(13.1) ×5 to 7.2 (9) m (n=200). Conidia is single celled, smooth, black, spherical, or ellipsoidal, and (11)13 to 16.5 × (8.2) 10.3 to16.1 m (n=100). Setae were not observed. The morphological characteristics of the isolates were consistent with Nigrospora species. A representative isolate (LNH-5) was selected for genomic DNA extraction. Sequences of the transcribed spacer region of rDNA (ITS), partial translation elongation factor (TEF1), and beta-tubulin fragment (TUB) were amplified using primer pairs ITS1/ITS4(White et al. 1990), EF-728F and EF-986R (Carbone et al. 1999) and Bt2a and Bt2b (Glass et al. 1995), respectively. The sequences of ITS (OQ473493), TEF1 (OQ506059) and TUB gene (OQ506055) were submitted to GenBank. They were 99 to 100% identical to the Nigrospora hainanensis ITS(OM283581.1)(538 out of 519 bp),TEF1(YK019415.1)(274 out of 276 bp),and TUB (OK086377.1)(405 out of 405 bp) sequences. The phylogenetic maximum likelihood analysis using the combined ITS, TEF1 and TUB sequences indicated that the isolate was part of the N. hainanensis clade (100% bootstrap value) that also contained the type isolate LC6979 of this species. Pathogenicity was tested on 15 healthy Brachiaria plants. Fungal conidia were harvested by flooding two-week-old single conidial cultures with sterile water, centrifuging, and adjusting the concentration to 107 spores/mL. Then 10 μL of conidial suspension was dropped onto the surfaces of leaves wounded with a sterile needle. Sterile distilled water was used for control treatment. The test was repeated three times. After inoculation, the plants were kept at 90~100% relative humidity at 25 to 28°C in a greenhouse for two weeks, and monitored daily for lesion development. Seven days post inoculation, all the inoculated leaves presented symptoms similar to those observed under natural conditions, while the control leaves showed no symptoms. The fungus was re-isolated from the diseased tissues by the single spore isolation method (Choi et al. 1999) to complete Koch's postulates. This pathogen has been reported on sugarcane in China (Raza et al., 2019; Zheng et al., 2022). To our knowledge, this is the first report of N. hainanensis causing leaf blight on Brachiaria plants in China.

Myxobolus coriumicus n.sp. (Cnidaria: Myxozoa) Causing Scale Disease Associated with Skin Lesions in Indian Cultured Carp, Cirrhinus mrigala in Punjab.

The motive of the present study was to investigate incidences of myxozoan parasite infection in commercially important fishes and their characterization using morphological attributes and molecular approach. The specimens of cultured Cirrhinus mrigala were sampled and various organs were examined. The plasmodia were detected on the scales. The identification of species was based on the myxospore morphology and 18S rDNA sequence analyses. For phylogenetic analysis, maximum likelihood (ML) and Bayesian inference (BI) methods were employed. The SEM and histological studies were performed to assess the damage to the scales. The infected fishes had white patches on the scales and red haemorrhagic lesions on the skin. The plasmodia of Myxobolus coriumicus n. sp. were located on the dorsal surface of the scale towards its exposed part. SEM study indicated that lepidonts were damaged in the infected scales. The myxospores were spherical in shape, 9.0-10.0 × 8.0-9.0[Formula: see text]m in size, with two ovoid equal polar capsules 3.13-4.0 × 2.03-2.33[Formula: see text]m in size, having 4-5 sutural edge markings at the posterior-lateral margins of the shell valves and binucleated sporoplasm. BLAST search based on 18S rDNA revealed 93.87% sequence similarity with M. rewensis, (MZ230381). The prevalence of infection was 6.3% and Scale Plasmodium Index (SPI) was 3 indicating heavy infection. Morpho-molecular data generated during this study enables us to conclude that the present species, M. coriumicus n. sp. infecting scales of C. mrigala is new to the science causing significant damage to the scales.

What do molecular data and vibrational communication tell us about the taxonomy of the genus Tyrrhenoleuctra Consiglio, 1957 (Plecoptera: Leuctridae) in Spain?

In this paper we present the results of a study of different populations of the genus Tyrrhenoleuctra, which includes morphologically cryptic species, in the Iberian Peninsula. A total of six populations were analysed from a molecular point of view and a behavioural study was conducted on three of them. Two phylogenetic trees, maximum likelihood (ML) and neighbour joining (NJ), were constructed using both new COI sequences and COI sequences already available in GenBank. Furthermore, intra- and interspecific p-distances were calculated within and between sequences of the different populations. As regards behavioural analyses, the male call of individuals from three of the studied populations was recorded, analysed and described. Both phylogenetic approaches used, ML and NJ, were congruent in discriminating five well-defined clusters, representing the four known Iberian Tyrrhenoleuctra species and a previously unknown taxon. The sequences from the six studied populations produced in this study fell within two clades: T. lusohispanica (four populations) and the new taxon (two populations). These results were supported also by the behavioral study, in which the drumming calls of individuals belonging to the two clades differed in some important parameters such as the number of sequences, intersequence interval and interbeat interval. The obtained results expand the previously known distribution of T. lusohispanica and show the existence of a new species (Tyrrhenoleuctra hynesi sp. n.) within this genus from a more restricted area, that will be described in this paper.

Evolutionary history of Euteliidae (Lepidoptera, Noctuoidea)

AbstractWe performed a molecular phylogenetic analysis on the family Euteliidae to clarify deep divergences and elucidate evolutionary relationships at the level of the subfamily, tribe, and genus. Our dataset consists of 6.3 kbp of one mitochondrial and seven nuclear DNA loci and was analysed using model‐based phylogenetic methods, that is, maximum likelihood and Bayesian inference. Based on the recovered topology, we recognize two subfamilies, Euteliinae and Stictopterinae, and the tribes Stictopterini and Odontini. We identify apomorphic morphological character states for Euteliidae and its component subfamilies and tribes. Several genera (e.g., Targalla, Paectes, Marathyssa, Eutelia) were found polyphyletic and require taxonomic revision. Two new genera (Niklastelia Zahiri & Holloway gen.nov. and Pellinentelia Holloway & Zahiri gen.nov.) are described and a number of taxonomic changes (new combinations and new synonymies) are established. The Neotropical genus Thyriodes, currently included in Euteliidae, is found to be associated with Erebinae (Erebidae). The divergence time estimate for the split between the Euteliidae and Noctuidae is at 53 Ma, and the Euteliidae subfamilies Euteliinae and Stictopterinae are estimated to have diverged at 42 Ma. In Stictopterinae, the tribes Stictopterini and Odontodini split at 31 Ma, while Euteliinae began diversifying at 34 Ma. Malpighiales are inferred to have been the ancestral larval hostplant order for Euteliidae. The ancestors of Stictopterinae also appear to have been Malpighiales feeders, but then diverged to Malvales specialists (Odontodini) and Malpighiales specialists (Stictopterini) hostplants. Larvae of Stictopterini appear to be restricted primarily to Clusiaceae, apart from a few records from Dipterocarpaceae. In Euteliinae, Anacardiaceae are predominant as larval hosts. Thus, all hosts in the family are lactiferous, possibly providing some degree of pre‐adaptation for exploiting Dipterocarpaceae.

Middle East respiratory syndrome coronavirus—a 10-year (2012-2022) global analysis of human and camel infections, genomic sequences, lineages, and geographical origins

ObjectivesThe World Health Organization priority zoonotic pathogen Middle East respiratory syndrome (MERS) coronavirus (CoV) has a high case fatality rate in humans and circulates in camels worldwide. MethodsWe performed a global analysis of human and camel MERS-CoV infections, epidemiology, genomic sequences, clades, lineages, and geographical origins for the period January 1, 2012 to August 3, 2022. MERS-CoV Surface gene sequences (4061 bp) were extracted from GenBank, and a phylogenetic maximum likelihood tree was constructed. ResultsAs of August 2022, 2591 human MERS cases from 26 countries were reported to the World Health Organization (Saudi Arabia, 2184 cases, including 813 deaths [case fatality rate: 37.2%]) Although declining in numbers, MERS cases continue to be reported from the Middle East. A total of 728 MERS-CoV genomes were identified (the largest numbers were from Saudi Arabia [222: human = 146, camels = 76] and the United Arab Emirates [176: human = 21, camels = 155]). A total of 501 ‘S’-gene sequences were used for phylogenetic tree construction (camels [n = 264], humans [n = 226], bats [n = 8], other [n=3]). Three MERS-CoV clades were identified: clade B, which is the largest, followed by clade A and clade C. Of the 462 clade B lineages, lineage 5 was predominant (n = 177). ConclusionMERS-CoV remains a threat to global health security. MERS-CoV variants continue circulating in humans and camels. The recombination rates indicate co-infections with different MERS-CoV lineages. Proactive surveillance of MERS-CoV infections and variants of concern in camels and humans worldwide, and development of a MERS vaccine, are essential for epidemic preparedness.

Burkholderia semiarida sp. nov. and Burkholderia sola sp. nov., two novel B. cepacia complex species causing onion sour skin

Two putative novel Burkholderia cenocepacia lineages found in the semi-arid region of north-east Brazil causing onion sour skin were studied using genomic approaches to determine their taxonomic position. Four strains belonging to one novel lineage (CCRMBC16, CCRMBC33, CCRMBC74, and CCRMBC171) and one strain (CCRMBC51) belonging to another novel lineage had their whole genome sequenced to carry out taxogenomic analyses. The phylogenomic tree built using the type (strain) genome server (TYGS) clustered the strains CCRMBC16, CCRMBC33, CCRMBC74, and CCRMBC171 into the same clade, while grouped the strain CCRMBC51 separately. Average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) analysis showed values above 99.21 % and 93.2 %, respectively, among the strains CCRMBC16, CCRMBC33, CCRMBC74, and CCRMBC171, while ANI and dDDH values between these strains and the strain CCRMBC51 were below 94.49 % and 56.6 %, respectively. All these strains showed ANI and dDDH values below 94.78 % and 58.8 % concerning type strains of the B. cepacia complex (Bcc) species. The phylogenetic maximum likelihood tree constructed based on the multilocus sequence analysis of core genes (cMLSA) clustered the strains CCRMBC16, CCRMBC33, CCRMBC74, and CCRMBC171 and the strain CCRMBC51 in two exclusive clades, which did not cluster with any known species of the Bcc. Therefore, combined data from TYGS, ANI, dDDH, and cMLSA demonstrated that the strains represent two novel species of the Bcc, which we classified as Burkholderia semiarida sp. nov. and Burkholderia sola sp. nov., and proposed the strains CCRMBC74T (=IBSBF 3371 T = CBAS 905 T) and CCRMBC51T (=IBSBF3370T = CBAS 904 T) as type strains, respectively.

First Report of Anthracnose of Sanhua Plum Caused by Colletotrichum aeschynomenes in Guangxi, China.

There is nearly 5,800 ha of Sanhua plum (Prunus salicina Linn) planted in Babu district in Hezhou, Guangxi, with over 67,000 tons of annual output. In August 2021, anthracnose symptoms were observed on Sanhua plum leaves in three different cultivated towns in Babu district in Hezhou, Guangxi (N23°49' - 24°48', E111°12' - 112°03'). The plant disease incidence was over 50% with approximately 20 to 30% of leaves on a plant being symptomatic. The disease outbreak occurred in the warm and damp climate (June to August) in Hezhou. Initially, small chlorotic spots developed on the leaves which gradually enlarged to larger irregular dark brown sunken lesions with yellowish halos, necrotic lesions abscised and formed holes at a later stage. In severe cases, the whole leaf withered and defoliated. Three symptomatic leaf samples were collected from three different cultivated towns in Hezhou. Margins of infected tissues were cut into 3×3 mm pieces, surface disinfected with 75% alcohol for 10 s, 2% NaOCl for 2 min followed by three washes in sterile distilled water and transferred to potato dextrose agar (PDA) plates. In total, forty-one isolates were obtained after 4 days of incubation at 25℃ on PDA, and thirty-one of them were Colletotrichum (average isolation frequency 76%). Three representative isolates (HZ18-1, HZ22-3, and HZ46-3) were selected for further study. After 7 days on PDA at 25℃, isolates had white to light grey cottony aerial mycelium on the obverse and revealed dark grey on the reverse. Conidia were hyaline, cylindroid, tapering slightly near both ends, measuring 16.3 ± 1.2 μm × 5.6 ± 0.4 μm, 16.1 ± 1.4 μm × 6.4 ± 0.7 μm, 16.2 ± 1.1 μm × 6.0 ± 0.4 μm (n=90) for HZ18-1, HZ22-3, and HZ46-3, respectively. Appressoria were brown, elliptic or fusoid, deeply lobed, measuring 10.2 ± 1.6 μm × 6.8 ± 1.0 μm, 10.7 ± 1.3 μm × 6.6 ± 0.8 μm, 9.3± 1.3 μm × 6.9 ± 0.9 μm (n=90) for HZ18-1, HZ22-3, and HZ46-3, respectively. These characteristics were consistent with the descriptions of Colletotrichum aeschynomenes B. Weir & P. R. Johnst (Weir et al. 2012). The internal transcribed spacer (ITS) region and the intergenic region and flanking regions of Apn2 and MAT1-2-1 (ApMAT) were amplified using ITS1/ITS4 and AM-F/AM-R primers, respectively (White et al. 1990; Silva et al. 2012). BLASTn analysis of the sequences showed over 99% identity with the corresponding loci from the culture collection C. aeschynomenes ICMP 17673 (ex-type). Sequences from the three isolates were deposited in GenBank (Accession Nos.: ITS, OM838335, OM838339, OM838370; ApMAT, OM816771, OM816775, OM816806). Phylogenetic maximum likelihood analysis with RAxML version 8.2.10 based on the concatenated sequences of ITS and ApMAT showed that the three isolates clustered with the ex-type specimen of C. aeschynomenes ICMP 17673. Pathogenicity was confirmed on leaves with and without wounds of 24 two-year-old Sanhua plum plants in a greenhouse. The wound was made with a sterilized toothpick. Wounded and unwounded leaves were inoculated with 20 μL of conidial suspension (106 conidia/mL) of the three isolates and control plants were inoculated with sterile distilled water (20 leaves/plant, 3 plants/treatment). All plants were covered with plastic bags to maintain high humidity. After 8 days of incubation at 25℃ with constant light, necrotic lesions were observed on inoculated leaves, whereas control plants showed no symptoms. To fulfill Koch's postulates, all fungi were successfully reisolated from symptomatic leaves. This species has been reported on Aeschynomene virginica in the United States (Weir et al. 2012), Manihot esculenta in Thailand (Sangpueak et al. 2018), Theobroma cacao (Nascimento et al. 2019) and Myrciaria dubia (Matos et al. 2020) in Brazil. To our knowledge, this is the first report of C. aeschynomenes causing Sanhua plum leaf anthracnose in China. The results will provide valuable information for management of anthracnose associated with Sanhua plum.

Mitochondrial phylogenomics of Acanthocephala: nucleotide alignments produce long-branch attraction artefacts

BackgroundClassification of the Acanthocephala, a clade of obligate endoparasites, remains unresolved because of insufficiently strong resolution of morphological characters and scarcity of molecular data with a sufficient resolution. Mitochondrial genomes may be a suitable candidate, but they are available for a small number of species and their suitability for the task has not been tested thoroughly.MethodsHerein, we sequenced the first mitogenome for the large family Rhadinorhynchidae: Micracanthorhynchina dakusuiensis. These are also the first molecular data generated for this entire genus. We conducted a series of phylogenetic analyses using concatenated nucleotides (NUC) and amino acids (AAs) of all 12 protein-coding genes, three different algorithms, and the entire available acanthocephalan mitogenomic dataset.ResultsWe found evidence for strong compositional heterogeneity in the dataset, and Micracanthorhynchina dakusuiensis exhibited a disproportionately long branch in all analyses. This caused a long-branch attraction artefact (LBA) of M. dakusuiensis resolved at the base of the Echinorhynchida clade when the NUC dataset was used in combination with standard phylogenetic algorithms, maximum likelihood (ML) and Bayesian inference (BI). Both the use of the AA dataset (BI-AAs and ML-AAs) and the CAT-GTR model designed for suppression of LBA (CAT-GTR-AAs and CAT-GTR-NUC) at least partially attenuated this LBA artefact. The results support Illiosentidae as the basal radiation of Echinorhynchida and Rhadinorhynchidae forming a clade with Echinorhynchidae and Pomporhynchidae. The questions of the monophyly of Rhadinorhynchidae and its sister lineage remain unresolved. The order Echinorhynchida was paraphyletic in all of our analyses.ConclusionsFuture studies should take care to attenuate compositional heterogeneity-driven LBA artefacts when applying mitogenomic data to resolve the phylogeny of Acanthocephala.Graphical

Genetics and Pathogenicity of Natural Reassortant of Infectious Bursal Disease Virus Emerging in Latvia

Infectious bursal disease virus is an immunosuppressive pathogen that, despite applied vaccination, is affecting the poultry industry worldwide. This report presents the genetic and pathotypic characterization of a natural reassortant emerging in Europe (Latvia). Genetic characterization showed that strain 25/11/Latvia/2011 represents genotype A3B1, whose segment A is derived from very virulent strains, while segment B is from the classic-like genogroup. Phylogenetic maximum likelihood inference of the B-segment sequence clustered the reassortant strain together with the US antigenic variant E strain. However, the obtained full-length sequence of 25/11/Latvia/2011 revealed that not only reassortment but also dozens of mutations shaped the unique genetic makeup. Phenotypic characterization showed no mortality and no clinical signs of disease but a severe bursa of Fabricius atrophy and splenomegaly in the convalescent birds at 10 days post infection. The results obtained indicate that the acquired genetic constellation contributed to a decrease in virulence; nevertheless, the infection causes severe damage to lymphoid organs, which can lead to impaired immune responses.

Molecular revision of Rhagiini sensu lato (Coleoptera, Cerambycidae): Paraphyly, intricate evolution and novel taxonomy

Due to classical taxonomy, the subfamily Lepturinae is divided into two large tribes Rhagiini s.l. and Lepturini s.l. However, this division is clearly artificial and does not correspond to the evolutionary relationships between the groups of genera with different morphologies that are part of these two tribes. However, there is a consensus among researchers supporting the monophyly of Lepturini s.l. while there is no such consensus for Rhagiini s.l. Over the past three decades, there have been several attempts to revise the tribe Rhagiini s.l. and divide it into smaller tribes. These attempts were somewhat successful based on larval and adult morphology. In general, eight tribes are distinguished within Rhagiini s.l. These include Teledapini, Xylosteini, Encyclopini, Oxymirini, Enoploderini, Rhamnusiini, Rhagiini and Sachalinobiini. However, the new system of Rhagiini s.l. is not always unambiguously accepted by different researchers, which causes discussions among experts. First of all, this is due to the fact that this system is only partially natural and far from fully reflects the phylogeny not only of Rhagiini s.l., but also of entire subfamily Lepturinae. In addition to the classical morphologic methods of studies, the use of the modern molecular phylogenetic methods opens up wide prospects for solving this puzzle. However, there have been very few such studies. Moreover, all of them were based on the use of only one gene. In this study, I used a general time-reversible (GTR) model of sequence evolution based on three mitochondrial (12S rRNA, 16S rRNA, COI) and two nuclear (18S rRNA, 28S rRNA) genes. My analysis yielded a well-resolved phylogenetic maximum likelihood tree, which clearly demonstrates the paraphyly of Rhagiini s.l. It consists of at least three clades representing different and distantly related evolutionary branches: 1) PaRh, 2) OSaXyR-SEP, 3) GAC. The extant Rhagiini s.l. are mostly heterogeneous relict groups with an intricate evolutionary and phylogeographic history. Most of these groups are represented by one or very few species, often isolated geographically on different continents. In particular, numerous cases of parallel and convergent evolution and homoplasy, a chimeric combination of plesiomorphic and apomorphic morphological characters, were found in all clades. Therefore, the evolutionary "tree" of Rhagiini s.l. is more like a "bush" with numerous relict branches. Finally, I proposed a new taxonomic model of Rhagiini s.l., which is the most consistent with their natural phylogeny, with new developments in nomenclature.

First report of Anthracnose on 'Purple Dream' Solanum melongena in Malaysia Caused by Colletotrichum siamense.

'Purple Dream' eggplant (Solanum melongena) is widely grown for its edible fruits in Malaysia. In July 2021, anthracnose symptoms were observed on fruit with a disease severity of approximately 80% and an incidence of 10% in a field (14.6 m2) (5°56'50.9"N, 116°04'31.9"E) located in the Penampang district of Sabah province. The symptoms initially appeared as irregular light brown spots. As the disease progressed, the spots enlarged and merged into extensive lesion patches that appeared in concentric circles. The symptomatic fruit tissues (5 x 5 mm) were surface sterilized based on Khoo et al. (2022), and plated onto potato dextrose agar (PDA), and incubated at 25°C in the dark. Colonies with gray-white fluffy mycelia developed after 7 days, and the reverse of the colonies was dark brown. A representative isolate named Penampang was characterized morphologically and molecularly. The conidia were one-celled, cylindrical, blunt at the ends, hyaline, smooth, and measured 13.3 to 16.1 x 3.9 to 6.0 µm (n= 20). Appressoria ranged in size from 7.6 to 9.3 m x 5.5 to 6.6 µm (n= 20) and were spherical to irregular in shape and dark brown in colour. Genomic DNA was extracted from fresh mycelia of isolate Penampang based on Khoo et al. (2021) and Khoo et al. (2022). ITS1/ITS4, CL1C/CL2C, ACT-512F/ACT-783R, CHS-79F/CHS-354R, and GDF1/GDR1 primer pairs were used to amplify the isolate's internal transcribed spacer region (ITS), and partial calmodulin (CAL), actin (ACT), chitin synthase (CHS-1), and glyceraldehyde-3-phosphate dehydrogenase genes (GAPDH) (Weir et al. 2012). PCR products were sequenced by Apical Scientific Sdn. Bhd. (Selangor, Malaysia). Sequences were deposited in GenBank under the accession numbers OL957466 (ITS), OL953035 (CAL), OL953032 (ACT), OL953038 (CHS-1), and OL953041 (GAPDH). They were 99% to 100% identical to the Colletotrichum ti ITS (NR_120143) (515 bp out of 519 bp), and C. siamense CAL (JX009714) (729 bp out of 731 bp), ACT (JX009518) (282 bp out of 282 bp), CHS-1 (JX009865) (299 bp out of 299 bp), and GAPDH (JX009924) (276 bp out of 277 bp) sequences. ITS sequences do not reliably resolve relationships within the C. gloeosporioides complex (Weir et al. 2012). The phylogenetic maximum likelihood analysis using the combined ITS, CAL, ACT, CHS-1, and GAPDH sequences indicated that the isolate was part of the C. siamense clade (100% bootstrap value) that also contained the type isolate ICMP 18578 of this species. Morphological and molecular characterization matched the description of C. siamense (Huang et al. 2021; Ismail et al. 2021). Koch's postulates were performed similarly as described by Chai et al. (2017) but using spray-inoculation (108 spores/ml) of three healthy 'Purple Dream' eggplant fruit with isolate Penampang. Water was sprayed on three additional fruits that served as controls. All the fruits were incubated at 25°C and less than 90% relative humidity. Symptoms similar to those observed in the field developed 5 days after inoculation. No symptoms occurred on controls. The experiment was repeated two more times. The reisolated fungi were identical to the pathogen morphologically and molecularly. To our knowledge, this is the first report of C. siamense causing anthracnose on fruit of 'Purple Dream' S. melongena in Malaysia as well as worldwide. Our findings expand the host range of C. siamense and indicate that the pathogen could potentially limit 'Purple Dream' eggplant production in Malaysia.

Molecular Variation and Phylogeny within Fusarium avenaceum and Related Species

Many recent articles feature research on the Fusarium tricinctum species complex (FTSC), and their authors present different ideas on how the isolates of this species complex can be identified at the species level. In previous studies, our aim was to investigate the phylogeny of FTSC strains, which researchers have morphologically identified as Fusarium avenaceum, Fusarium arthrosporioides, and Fusarium anguioides. In the current study, our phylogenetic maximum parsimony and likelihood analyses of the DNA sequences of the translation elongation factor 1-alpha (TEF1) and combined sequences of TEF1 and beta-tubulin (TUB2) supported the existence of at least four main groups among these strains. Main Group I mainly contains F. avenaceum strains, while Main Group II contains two subgroups, one of which primarily includes F. arthrosporioides strains, and the other mainly includes European F. anguioides strains. Main Group III contains strains from different plants that originated from Asia, including two F. anguioides strains. F. avenaceum strains, which are mostly isolated from different trees, form Main Group IV. A fifth group (Main Group V) was only supported by TEF1 sequences. The main groups previously found by us based on TUB2 sequences could be connected to the new species of the FTSC, which were identified based on TEF1 sequences. In addition, we found strains that significantly differ from Main Groups I-V, and we grouped some of them as single, intermediate, or sister groups. All of the main groups of the present work, and some single and intermediate strains, may represent different species of the FTSC, while the two subgroups of Main Group II constitute intraspecific variation. Regardless of whether they belonged to the main groups, all the analysed strains were able to form different enniatins and 2-amino-14,16-dimethyloctadecan-3-ol, but did not produce beauvericin.

A New Disease for Europe of Ficus microcarpa Caused by Botryosphaeriaceae Species.

The Indian laurel-leaf fig (Ficus microcarpa) is an important ornamental tree widely distributed in the urban areas of Italy. Surveys conducted in 2019 and 2020 on several tree-lined streets, squares, and public parks in Catania and Siracusa provinces (Sicily, southern Italy) revealed the presence of a new disease on mature trees. About 9% of approximately 450 mature plants showed extensive branch cankers and dieback. Isolations from woody tissues obtained from ten symptomatic plants consistently yielded species belonging to the Botryosphaeriaceae family. The identification of the recovered fungal isolates was based on a multi-loci phylogenetic (maximum parsimony and maximum likelihood) approach of the ITS, tef1-α, and tub2 gene regions. The results of the analyses confirmed the presence of three species: Botryosphaeria dothidea, Neofusicoccum mediterraneum, and N. parvum. Pathogenicity tests were conducted on potted, healthy, 4-year-old trees using the mycelial plug technique. The inoculation experiments revealed that all the Botryosphaeriaceae species identified in this study were pathogenic to this host. Previous studies conducted in California showed similar disease caused by Botryosphaeriaceae spp., and the pathogenic role of these fungi was demonstrated. To our knowledge, this is the first report of Botryosphaeriaceae affecting Ficus microcarpa in Europe.

An empirical test of the relationship between the bootstrap and likelihood ratio support in maximum likelihood phylogenetic analysis.

In maximum likelihood (ML), the support for a clade can be calculated directly as the likelihood ratio (LR) or log-likelihood difference (S, LLD) of the best trees with and without the clade of interest. However, bootstrap (BS) clade frequencies are more pervasive in ML phylogenetics and are almost universally interpreted as measuring support. In addition to theoretical arguments against that interpretation, BS has several undesirable attributes for a support measure. For example, it does not vary in proportion to optimality or identify clades that are rejected by the evidence and can be overestimated due to missing data. Nevertheless, if BS is a reliable predictor of S, then it might be an efficient indirect method of measuring support-an attractive possibility, given the speed of many BS implementations. To assess the relationship between S and BS, we analyzed 106 empirical datasets retrieved from TreeBASE. Also, to evaluate the degree to which S and BS are affected by the number of replicates during suboptimal tree searches for S and pseudoreplicates during BS estimation, we randomly selected 5 of the 106 datasets and analyzed them using variable numbers of replicates and pseudoreplicates, respectively. The correlation between S and BS was extremely weak in the datasets we analyzed. Increasing the number of replicates during tree search decreased the estimated values of S for most clades, but the magnitude of change was small. In contrast, although increasing pseudoreplicates affected BS values for only approximately 40% of clades, values both increased and decreased, and they did so at much greater magnitudes. Increasing replicates/pseudoreplicates affected the rank order of clades in each tree for both S and BS. Our findings show decisively that BS is not an efficient indirect method of measuring support and suggest that even quite superficial searches to calculate S provide better estimates of support.

30. SARS-CoV2 Reinfections in a University Teaching Hospital

BackgroundThe consequences of SARS-CoV2 reinfections for patients, healthcare workers and society are unclear. We reviewed the clinical, laboratory, and epidemiological characteristics of patients re-infected with genetically distinct strains of SARS-CoV2 identified by Whole Virus Genome Sequencing (WvGS).MethodsCases were selected based on a positive SARS-CoV-2 Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) test, clinical resolution, a negative interim test and a subsequent positive nasopharyngeal swab. Positive samples were prepared for sequencing by cDNA synthesis, tiled-PCR following the ARTIC protocol and amplicon sequencing using Illumina MiSeq platform. Raw reads were mapped to the reference sequence using bowtie and Samtools was used for variants calling and to generate the consensus sequences. Comparative sequence analysis was conducted by phylogenetic inference maximum likelihood method with RAxML using the multiple sequence aligned by MAFFT. Clades and variants were assigned respectively using Nextstrain and Pangolin COVID-19 lineage assigner (Figure 1). The clinical, radiological and laboratory data were collected from patient medical notes and laboratory information system.ResultsTwo cases of SARS-CoV-2 reinfection were detected by RT-PCR (patient 1 and 2). CT values and strain variants are presented in Table 1. The time between detection of the first and second infection was 67 and 270 days respectively. WvGS confirmed that the second episodes were due to a genetically distinct strain of SARS CoV2. These reflected the dominant contemporaneous variants in circulation.Both patients were immunocompromised from co-morbidities and medications. First and subsequent infections were minimally symptomatic. Both cases were associated with known hospital outbreaks. They passed away within 2 weeks of the second infection of unrelated causes. ConclusionTwo patients in this study were diagnosed with a SARS-CoV-2 reinfection confirmed by WvGS. A common factor in these cases was immunocompromise. Where a previously infected patient test shows a new positive or an unexpected reduction in CT value is observed, we recommend individual risk assessment to determine the timing of discontinuation of isolation and infection control precautions.Disclosures Jérôme Fennell, MB BCh BAO MSc PhD FRCPath FRCPI, Roche Diagnostics (Advisor or Review Panel member)

Identification and characterization of Eimeria spp. in western north American Bison (Bison bison) herds and potential risk of cross-species transmission.

The American bison (Bison bison) is an iconic native wildlife species of the Great Plains of North America. Recently, farmed bison have also gained importance to the livestock industry across the United States and Canada. One of the common diseases in young bison is coccidiosis, a diarrheal disease caused by protozoa that can result in significant morbidity. The goal of the present study was to investigate occurrence and identity of bison coccidia of the genus Eimeria in both farmed and free-ranging bison with focus on potential Eimeria species transmissibility between bison and cattle. Individual bison (up to one year of age) were sampled across Wyoming, Colorado, Nebraska, and South Dakota on six bison ranches (n=137) and in two free-range herds (n=70). Eimeria populations were assessed by fecal analyses. Morphological identification revealed presence of oocysts consistent with Eimeria (E.) bovis, E. zuernii, E. ellipsoidalis, E. cylindrica, E. alabamensis, E. auburnensis, E. canadensis, E. pellita, E. subspherica, and E. bukidnonensis, all of which are described in cattle. Additional Eimeria species specific ITS1 sequencing data along with generated phylogenetic maximum likelihood trees suggest that Eimeria species from cattle, namely E. bovis, E. zuernii, E. alabamensis, E. cylindrica, E. brasiliensis, E. ellipsoidalis, and E. wyomingensis, are genetically consistent with the detected bison coccidia. In conclusion, the study results indicate that bison harbor a variety of Eimeria species and bison Eimeria species appear to be transmissible between different bovine species such as bison and cattle.

New long-horned caddisfly genus and species of Leptoceridae (Insecta: Trichoptera) from Brazil.

Ibyacerina caparao new genus, new species (Trichoptera: Leptoceridae) is described and illustrated from specimens collected at Serra do Capara, Minas Gerais, Brazil. The monotypic genus is characterized by tibial spur formula 0,2,2; preanal appendages originating from a single base with a median process; tergum X broad, heavily sclerotized, saddle-like, wider at apex, and upturned, bearing small stout setae; inferior appendages each 3-branched, setose; and phallic apparatus tubular, curved slightly ventrad, with pair of lateral processes. Phylogenetic Bayesian and maximum likelihood analyses based on concatenated cytochrome oxidase I (COI) and carbamoyl-phosphate synthetase (CAD) partial sequences (1,504 bp) including representatives of 38 leptocerid genera positioned Ibyacerina gen. nov. with good support within Leptocerinae. However, due to low branch support of most relationships among genera within the clade of Leptocerinae, except Mystacidini, Setodini, and Leptocerini, its close affinities and tribal placement are still unknown.

Phylogenetic and Drug-Resistance Analysis of HIV-1 Sequences From an Extensive Paediatric HIV-1 Outbreak in Larkana, Pakistan

IntroductionIn April 2019, an HIV-1 outbreak among children occurred in Larkana, Pakistan, affecting more than a thousand children. It was assumed that the outbreak originated from a single source, namely a doctor at a private health facility. In this study, we performed subtype distribution, phylogenetic and drug-resistance analysis of HIV-1 sequences from 2019 outbreak in Larkana, Pakistan.MethodsA total of 401 blood samples were collected between April–June 2019, from children infected with HIV-1 aged 0–15 years recruited into a case-control study to investigate the risk factors for HIV-1 transmission. Partial HIV-1 pol sequences were generated from 344 blood plasma samples to determine HIV-1 subtype and drug resistance mutations (DRM). Maximum-likelihood phylogenetics based on outbreak and reference sequences was used to identify transmission clusters and assess the relationship between outbreak and key population sequences between and within the determined clusters. Bayesian analysis was employed to identify the time to the most recent common recent ancestor (tMRCA) of the main Pakistani clusters.ResultsThe HIV-1 circulating recombinant form (CRF) 02_AG and subtype A1 were most common among the outbreak sequences. Of the treatment-naïve participants, the two most common mutations were RT: E138A (8%) and RT: K219Q (8%). Four supported clusters within the outbreak were identified, and the median tMRCAs of the Larkana outbreak sequences were estimated to 2016 for both the CRF02_AG and the subtype A1 clusters. Furthermore, outbreak sequences exhibited no phylogenetic mixing with sequences from other high-risk groups of Pakistan.ConclusionThe presence of multiple clusters indicated a multi-source outbreak, rather than a single source outbreak from a single health practitioner as previously suggested. The multiple introductions were likely a consequence of ongoing transmission within the high-risk groups of Larkana, and it is possible that the so-called Larkana strain was introduced into the general population through poor infection prevention control practices in healthcare settings. The study highlights the need to scale up HIV-1 prevention programmes among key population groups and improving infection prevention control in Pakistan.