Molecular Phylogenetic Tree Research Articles

Molecular phylogeny of Boninia (Platyhelminthes: Polycladida), with description of a new species from the Pacific coasts of Panama.

Mesopsammic polyclad members in the family Boniniidae have attracted attention in terms of their evolutionary shifts of microhabitat and their unique morphology such as a pair of pointed tentacles extending from the anterolateral margins and prostatoid organs harbouring stylets. Here, we establish a new species of this family as Boninia panamensis sp. nov. from the Pacific coasts of Panama, based on its morphological characteristics of (i) four cerebral and 61-80 marginal eyespots, (ii) two prostatoid organs located anterior and posterior to the penis papilla, and (iii) two uterine canals departing from the anterior part of the Lang's vesicle. We also report Boninia cf. uru from Hawai'i, USA, based on its morphological identity with B. uru from Okinawa, Japan, along with their genetic distances for the partial cytochrome c oxidase subunit I (COI ) sequences, which were beyond the range of intraspecific differences observed in congeners in this study. Boninia oaxaquensis is also reported from Panama as a new locality for the species. Involving the above-mentioned three species sequenced herein, we reconstructed molecular phylogenetic trees of Boninia based on the four gene markers (18S rDNA, 28S rDNA, 16S rDNA and COI ). Our phylogenetic trees indicated the synapomorphy within the genus Boninia of the small numbers of stylets (2-4) and the connection route of the uterine canals to the Lang's vesicle. The results also showed a characteristic distribution pattern in which pairs of species in distinct lineages occurred sympatrically with different microhabitats, as observed in Boninia uru and Boninia yambarensis in Okinawa and B. panamensis sp. nov. and B. oaxaquensis in Panama. In addition, we discuss possible speciation pathways in this genus based on the tree topology. ZooBank: urn:lsid:zoobank.org:pub:D414BACD-C14A-4B34-8AF9-7ACBA28F46D0.

New records of three Basidiomycetous species from Iraq using phenotypic and phylogenetic analyses

Wild macrofungi have potential functions in ecosystems and sustainable developments through their roles in waste management, human health and societal upliftment. However, there is limited knowledge of wild macrofungi in Iraq. Three interesting collections within Chlorophyllum, Mycenastrum and Ossicaulis genera related to basidiomycetes were discovered in central and northeastern parts of Iraq in 2022 during a field investigation on wild macrofungi. Molecular identification and morphological characterization were carried out for these collections. The molecular phylogenetic and phenotypic analyses confirmed that isolates are related to three species within two families )Agaricaceae and Lyophyllaceae) belonging to the order Agaricales and class Agaricomycetes. Based on the current publications, these species were the first records from Iraq. Chlorophyllum agaricoides, Mycenastrum corium and Ossicaulis salomii sequences were submitted as the first sequences from Iraq into the international biological database GenBank. The molecular phylogenetic tree formed three independent lineages in each genus with high support. The introduced species can easily be separated from closely related species in an evolutionary framework. The study's results might aid in further investigations into the macrofungi in Iraq.

Viral coexistence and insertional mutations in the ORF8 region of SARS-CoV-2: A possible mechanism of nucleotide insertion

The virus obtained from a swab sample ID: S66 in Hiroshima was reported to have a single T-base insertion in the ORF8 coding region. However, no T insertion was observed when we determined the genomic sequence using another method. We then extracted RNA from the S66 swab sample and sequenced the insertion site using the Sanger method. The resulting waveform was disrupted beyond the insertion site, suggesting the presence of a mixed population of viruses with different sequences. Through plasmid cloning of RT-PCR amplification fragments and virus cloning by limiting dilution, along with TIDE analysis to determine the ratio of components from the Sanger sequencing waveform, it was confirmed that the sample contained a mixture of viruses with varying numbers of T-base insertions. The virus with one T insertion (T1+) was predominant in 70–75 % of the genomes, and genomes with T0, T2+, T3+, T4+, and T5+ were also detected. No T-base insertion mutations were observed in the ORF8 region in three other SARS-CoV-2 samples. In the S66 sample, a C27911T point mutation near the insertion site in the ORF8 region resulted in a sequence of seven or more consecutive T bases, which was the cause of the T-base insertion. When the cloned S66 virus (T1+) was passaged in cultured cells, there was a tendency for viruses with more insertion bases to become dominant with successive generations, suggesting that the T-base insertion was due to polymerase stuttering. The insertion of T bases resulted in synthesis of deletion mutants of the ORF8 protein, but no significant change was observed in the proliferation of the viruses in cultured cells. A search of the GenBank database using NCBI BLAST for viruses similar to S66 with T-base insertion mutations revealed hundreds of viruses widely distributed on the molecular phylogenetic tree. These base insertion viruses were thought to have occasionally arisen during the virus infection process. This study suggests one mechanism of insertion mutations in SARS-CoV-2, and it is important to consider the emergence of future mutant strains.

Molecular Detection and Phylogenetic Tree of Rotavirus from Human and Sheep, Iraq

Group A rotaviruses (RVA) have been linked to acute gastroenteritis in children, as well as young domestic and wild animals. In Iraq few molecular studies involved the genotype profile of rota virus and this is the first study target VP1 gene. Data were collected from January to February 2018 and subsequently examined utilizing a PCR and because of low viral concentration we utilized Nested PCR. Sequenced PCR result at 327 bp fragment in 4 A rota virus-positive samples deposited in GenBank under accession number (MH11809.1, MH118097.1) for human and (MH118095.1, MH118094.1) to sheep. Children samples were identical to the other human A rota virus at (99-100%), with Japan feline-like human G6P and G6P[14] isolated from cattle as well as, animal rota virus (99-100%) similarity with cross relation with human Wa-Like and porcine, moreover Morocco characterized caprine, bovine rotavirus and Slovenian Roe deer ARV.

Molecular detection and phylogenetic tree assay of Pseudomonus aeruginosa isolated from otitis cases of cats and humans, Iraq

The aim of this study was toward isolation and identification of P. aeruginosa from cats with otitis externa in Basra city Using culture media, Biochemical tests and Polymerase chain reaction. Pseudomonas aeruginosa was isolated from 46 isolates that involved 32 cases out of 75 of feline otitis externa (21.3%) and in 14 (9.3%) from 75 human samples .the clinical signs of otitis externa were head shaking, scratching, excessive ear wax, malodor, pain during palpation, alopecia and pus and/or blood. Culture was done on Pseudomonas chrome agar revealed positive samples with production of greenish-blue pigmentation by the bacteria .polymerase chain reaction revealed that isolates were Pseudomonas aeruginosa using specific primers for P. aeruginosa identification 16S rRNA and the result was positive for the 15 isolates. a phylogenetic tree analysis of P. aeruginosa was performed using the 16S rRNA gene sequence analysis which illustrated the phylogenetic relationship between P. aeruginosa strains isolated from cats and other related genera. In conclusion, this study confirmed the higher prevalence of P. aeruginosa in otitis externa infection of cats when compared to human. The using of molecular assay and 16S rRNA gene contributed effectively in detection of P. aeruginosa. Therefore, this study suggests the application of molecular techniques in identification of different microorganisms. Additionally, moreover studies are necessary to detect the prevalence of P. aeruginosa in different infections in animals as well as human.

The Cretaceous Diatom Database: A tool for investigating early diatom evolution.

The Cretaceous period is the time of the first appearance of the diatoms in the fossil record. These fossils give us direct evidence of the age and early evolution of the diatom lineage. The fossil record, however, is incomplete and therefore often extrapolated through time-calibrated phylogenies. These two approaches offer different perspectives on the early evolution of diatoms, which is still poorly understood. We compiled the first comprehensive Cretaceous Diatom Database, a tool to investigate the taxonomy, diversity, and occurrence of the earliest known diatom lineages. To further aid the integration and use of the oldest diatom fossils in molecular clock analyses, we present a set of well-documented Cretaceous fossils that can be placed onto molecular phylogenetic trees of extant and extinct species, making them ideal candidates for the calibration of molecular clocks. The analysis of the fossil record and the Cretaceous Diatom Database revealed Cretaceous diversity is substantially greater than previously thought, yet considerable taxonomic work is still needed. The Cretaceous Diatom Database and the list of Cretaceous fossils for calibrating molecular clocks represent valuable resources for future evolutionary and taxonomic studies of modern and fossil diatoms.

How Phenograms and Cladograms Became Molecular Phylogenetic Trees

Tree diagrams are the prevailing form of visualization in biological classification and phylogenetics. Already during the time of the so-called Systematist Wars from the mid-1960s until the 1980s most journal articles and textbooks published by systematists contained tree diagrams. Although this episode of systematics is well studied by historians and philosophers of biology, most analyses prioritize scientific theories over practices and tend to emphasize conflicting theoretical assumptions. In this article, I offer an alternative perspective by viewing the conflict through the lens of representational practices with a case study on tree diagrams that were used by numerical taxonomists (phenograms) and cladists (cladograms). I argue that the current state of molecular phylogenetics should not be interpreted as the result of a competition of views within systematics. Instead, molecular phylogenetics arose independently of systematics and elements of cladistics and phenetics were integrated into the framework of molecular phylogenetics, facilitated by the compatibility of phenetic and cladistic practices with the quantitative approach of molecular phylogenetics. My study suggests that this episode of scientific change is more complex than common narratives of battles and winners or conflicts and compromises. Today, cladograms are still used and interpreted as specific types of molecular phylogenetic trees. While phenograms and cladograms represented different forms of knowledge during the time of the Systematist Wars, today they are both used to represent evolutionary relationships. This indicates that diagrams are versatile elements of scientific practice that can change their meaning, depending on the context of use within theoretical frameworks.

A Phenomenon: What Are the Minuscule Grey Moths Abundant in the Dry Season in the Tropical Dry Forests of the Pacific Coast of Honduras?

Our investigation centered on the tropical dry forests along the Pacific coast of Honduras, aiming to elucidate the presence and abundance of minuscule grey moths during the dry season. Through specimen dissections and the taxonomic identification of the collected material, we have described three new species: Acalyptris podenasi sp. nov., A. palpiformis sp. nov., and A. tortoris sp. nov. Additionally, we documented two species previously known from neighboring countries, A. lascuevella Puplesis & Robinson and A. basicornis Remeikis & Stonis. The females of A. lascuevella were previously unknown and are documented here for the first time. Morphological examinations were complemented by DNA barcoding, particularly highlighting variation in A. lascuevella. The paper's primary significance lies not only in the description of new species but also in uncovering their taxonomic, morphological, and molecular importance. We found that these species are unique and indicative of the previously unstudied dry forests as a distinct ecosystem. Our findings revealed several novel atypical morphological traits within the studied Nepticulidae, including unusually large signum cells in the female genitalia, a dorso-ventrally divided uncus, and asymmetrical valvae in the male genitalia. These discoveries underscore the morphological diversity of Acalyptris Meyrick and their significance in evolutionary biology. Consequently, the paper addresses a previously unknown phenomenon of the occurrence and astonishing abundance of minuscule plant-mining micromoths in dry deciduous forests during the peak of the dry season. We hope that this paper will encourage Lepidoptera taxonomists to explore micromoths in other tropical dry forests, which, while limited in distribution, hold global importance. The paper is extensively illustrated with photographs of Acalyptris adults and their genitalia, along with maps, habitats, and molecular phylogenetic trees.

Molecular phylogeny and taxonomic review of the family Pomacentridae from Korea

A total of eight genera and 21 species of the family Pomacentridae have been reported in Korea, but the classification is uncertain. Therefore, this study aims to clarify molecular phylogeny and classification of eight genera and 17 species of the family Pomacentridae from Korea based on mtDNA sequences (16S rRNA 473 bp), nDNA sequences (RAG1 812 bp) and morphological traits. Molecular phylogenetic trees (ML, and BI) showed that four groups (Chrominae, Glyphisodontinae, Pomacentrinae, and Microspathodontinae) were largely formed, and monophyletic relationship was observed four groups. Each subfamily is divided by the number of spiniform procurrent caudal rays, the shape and rows of the teeth, and the margin of suborbital and preopercle. Each genus is divided by the number of dorsal fin spines and anal fin soft rays, the shape and rows of the teeth, and body coloration. Through an extensive examination of pomacentrids specimens loaned from museum or universities, it is believed that Chromis analis, Neopomacentrus violascens and Pomacentrus chrysurus are not distributed in Korea in spite of their previous records. Finally, we provide key to the species of the family Pomacentridae from Korea.

Phylogenomics of the pantropical Connaraceae: revised infrafamilial classification and the evolution of heterostyly

Connaraceae is a pantropical family of about 200 species containing lianas and small trees with remarkably diverse floral polymorphisms, including distyly, tristyly, homostyly, and dioecy. To date, relationships within the family have not been investigated using a targeted molecular phylogenetic treatment, severely limiting systematic understanding and reconstruction of trait evolution. Accordingly, their last infrafamilial classification was based only on morphological data. Here, we used phylogenomic data obtained using the Angiosperms353 nuclear target sequence capture probes, sampling all tribes and almost all genera, entirely from herbarium specimens, to revise infrafamilial classification and investigate the evolution of heterostyly. The backbone of the resulting molecular phylogenetic tree is almost entirely resolved. Connaraceae consists of two clades, one containing only the African genus Manotes (4 or 5 species), which we newly recognize at the subfamily level. Vegetative and reproductive synapomorphies are proposed for Manotoideae. Within Connaroideae, Connareae is expanded to include the former Jollydoreae. The backbone of Cnestideae, which contains more than half of the Connaraceae species, remains incompletely resolved. Reconstructions of reproductive system evolution are presented that tentatively support tristyly as the ancestral state for the family, with multiple parallel losses, in agreement with previous hypotheses, plus possible re-gains. However, the great diversity of stylar polymorphisms and their phylogenetic lability preclude a definitive answer. Overall, this study reinforces the usefulness of herbarium phylogenomics, and unlocks the reproductive diversity of Connaraceae as a model system for the evolution of complex biological phenomena.

Characterization of corazonin signaling in a molluscan model species, Lymnaea stagnalis

In recent years, new concepts have emerged regarding the nomenclature, functions, and relationships of different peptide families of the gonadotropin-releasing hormone (GnRH) superfamily. One of the main driving forces for this originated from the emerging evidence that neuropeptides previously called molluscan GnRH are multifunctional and should be classified as corazonin (CRZ). However, research articles still appear that use incorrect nomenclature and attribute the same function to molluscan CRZs as vertebrate GnRHs. The aim of the present study was to further support the recent interpretation of the origin and function of the GnRH superfamily. Towards this goal, we report the characterization of CRZ signaling system in the molluscan model species, the great pond snail (Lymnaea stagnalis). We detected a CRZ-receptor-like sequence (Lym-CRZR) by homology-searching in the Lymnaea transcriptomes and the deduced amino acid sequence showed high sequence similarity to GnRH receptors and CRZ receptors. Molecular phylogenetic tree analysis demonstrated that Lym-CRZR is included in the cluster of molluscan CRZRs. Lym-CRZR transiently transfected into HEK293 cells was found to be localized at the plasma membrane, confirming that it functions as a membrane receptor, like other G protein-coupled receptors. The signaling assays revealed that the previously identified Lym-CRZ neuropeptide stimulated intracellular Ca2+ mobilization in a dose-dependent manner, but not cyclic AMP production, in HEK293 cells transfected with Lym-CRZR. Finally, we demonstrated a wide tissue distribution of Lym-CRZR. These results suggest that Lym-CRZ is a multifunctional peptide and provide further insights into the evolution of the GnRH neuropeptide superfamily. The present study also supports the notion that previously termed molluscan “GnRH” should be classified as “CRZ”.

Apis cerana Fabricius, 1793 in Sumatra: Haplotype Variations of Mitochondrial DNA and the Molecular Relationship with the Asian Honey Bees (Hymenoptera: Apidae)

Honey bee Apis cerana is widely distributed in Asia and the Indonesian archipelago, including Sumatra. We studied the molecular variations of A. cerana using cytochrome c oxidase subunits 1 and 2 genes (cox1 and cox2) and the cox1/cox2 intergenic spacers (igs) in several altitudes in the six provinces of Sumatra. We explored the haplotype distributions of those three mtDNA markers for A. cerana in the low-, mid-, and highlands of Sumatra. We also analyzed their relationship with A. cerana in Sundaland and Asia using those markers. Our study revealed 12 new haplotypes of A. cerana cox1 in Sumatra, while nine and eight new haplotypes for cox2 and igs, respectively. Apis cerana in North Sumatra, Lampung, and South Sumatra had the three highest haplotype variations. Most of the specific haplotypes of inter-colony A. cerana from Sumatra were found in the lowlands, while most were in the highlands for intra-colony variations. We found low gene flow among populations of A. cerana in Sumatra. One haplotype, Sumatra4 cox2 from North Sumatra, was the same as Java3 haplotype, presumably due to anthropogenic impact. The molecular phylogenetic tree of A. cerana in the Sundaland revealed that A. cerana from Sumatra has a close relationship to those of Borneo compared to Java.

Random Amplified Polymorphic DNA (RAPD) Markers Protocol of Bacterial Isolates from Two selected General Hospitals Wastewater (HWW)

In this research work, Random amplified polymorphic DNA (RAPD) markers Protocol was used to characterize bacterial isolates from two selected General Hospitals Waste Water (HWW). The hospital environment and its wastes accumulate diseases from both inward and outward patients. It is pertinent to investigate the wastewater and study its microbial community. Wastewater samples were collected from two major General Hospitals in Akoko area, namely, Ikare and Akungba-Akoko General Hospitals. Samples were microbiologically examined for the presence of bacterial colonies. Isolated bacterial species were preliminarily identified through conventional biochemical tests and were further characterized using 16s rRNA sequencing protocol. The bacterial gene was amplified using selected primers. DNA was isolated, purified, and amplified using 16s rRNA gene sequence protocol, and the results were analyzed and assessed with the standard bacteria sequence in the NCBI database. Molecular evolutionary analyses and Phylogenetic trees were mapped out from the results obtained. it was observed that the Bacterial counts in Cfu/ml range between 10.0 x 103Cfu/ml to 24.0 x 103Cfu/ml. The highest bacterial count was observed from the test sample from Ikare followed by Akungba Akoko General Hospitals with the Cfu/ml value of 24.0 x 103 and 18.0 x103 Cfu/ml respectively. Molecular data sequence, when compared with NCBI database using BLAST showed 99.60 –99.87% bactaria phylogenic identity and E-value equal to 0 for all closely related taxa. The following bacteria were characterized using Random amplified polymorphic DNA (RAPD) markers protocol namely: Streptococcus pneumoniae, Staphylococcus aureus, Bacillus cereus, and Bacillus subtilis. this study has revealed the presence of different bacterial species in hospital waste water which pose threat to public health. Hence, proper monitoring and sewage treatment should be encouraged in different hospital settings before the disposal of these wastewaters to the public water runways

First Report of Penicillium oxalicum Causing Leaf Blight on 'Hongyang' Kiwifruit in China.

In September 2022, leaf blight symptoms (Fig. 1) were detected on six-year-old kiwi trees (Actinidia chinensis cv. 'Hongyang') in Xuzhou municipality (117.29º E, 34.23º N), Jiangsu Province. Early-stage disease symptoms included light brown necrotic lesions of irregular shape ranging in length from 0.2 to 2.4 cm, which turned into leaf blight after approximately 2 weeks. Those symptoms were similar to those previously reported during a Pestalotiopsis sp. infection on kiwi trees in Turkey (Karakaya 2001). Approximately 20% of the leaves from 300 trees examined in one kiwi orchard, 3000 m2 in size, showed the disease symptoms. Ten leading edges of symptomatic leaves were sterilized with 2% sodium hypochlorite for 1 min, rinsed twice with sterile ddH2O and cultured at 26ºC for 3 days on PDA medium containing 50 µg/ml chloramphenicol. The fungal colonies were collected, and the single spore isolation method was used to obtain four isolates. The obtained isolates showed white aerial mycelia that turned greyish after 2 days of cultivation on PDA medium at 26ºC. ITS (OR054113, OR054153-OR054155), TUB2 (OR060951-OR060953, OR249978), and CMD (OR255947-OR255950) genes were amplified using the ITS1/ITS4, BT2a/BT2b and CMD5/CMD6 primers, respectively (Visagie et al. 2014a). The obtained ITS, TUB2, and CMD sequences shared 99.81%-100%, 96.72%-96.96%, and 90.17%-92.58% homology compared to the ex-type strain P. oxalicum CBS 219.30 (MH855125, KF296462, and KF296367), while the obtained ITS and TUB2 sequences showed 99.62%-99.81%, and 96.46%-96.72% identity compared to the representative strain P. oxalicum DTO 179B9 (KJ775647 and KJ775140) (Visagie et al. 2014b). The sequences obtained also showed high homology compared to P. oxalicum HP7-1 (ITS: 99.81%-100% homology; TUB2: 98.98%-99.38% homology; CMD: 94.71%-95.10% homology) (Li et al. 2022). A molecular phylogenetic tree was constructed using MEGA X with representative Penicillium strains retrieved from GenBank (Fig. 2). Microscope observations revealed the presence of curved septate hyphae. Conidia were colorless, unicellular, and ellipsoidal (5-8 μm in length; > 2000 observations), whereas conidiophores were mainly monoverticillate (approximately 20% of the conidiophores were biverticillate) (50-70 μm in length; 43 observations) and contained cylindrical phialides (13-15 μm in length). These findings are consistent with P. oxalicum morphology (Wu et al. 2022; Zheng et al. 2023). The pathogenicity of the four isolates was screened using healthy non-detached 'Hongyang' kiwi leaves. Fifteen leaves from five different two-month-old trees were used for each isolate, with three repetitions. For inoculation, a 10 mL solution containing 1 × 106 spores/mL was sprayed on the leaves. Sterilized water was used in the control experiment, which was carried out using fifteen leaves from five different two-month-old trees, with three repetitions. Inoculated trees were stored at 26ºC and 60% relative humidity for 2 days. All the infected leaves had necrotic lesions and leaf blight symptoms comparable to those found in the field, but the control leaves had no lesions. The pathogen was recovered, and its identity was confirmed by ITS sequencing and morphology analysis, fulfilling Koch's postulates. P. oxalicum is a common cause of blue mould in postharvest fruits (Tang et al. 2020). P. oxalicum has been recently reported as the causal agent of leaf spot in pineapple (Wu et al. 2022; Zheng et al. 2023), and leaf blight on maize (Han et al. 2023). Although Alternaria sp., Glomerella cingulate, Pestalotiopsis sp., Phomopsis sp., and Phoma sp. were previously isolated from kiwi leaves with blight symptoms (Kim et al. 2017), this is the first report of P. oxalicum causing leaf blight on kiwi trees worldwide. P. oxalicum is a well-known source of mycotoxins, such as secalonic acid (Otero et al. 2020), indicating that its presence in kiwifruit orchards may pose a significant risk to human health. The discovery of this hazardous pathogen in kiwi trees must drive the development of management strategies. Kiwifruit is an important dietary source of vitamins, fiber, folate, and potassium, and China is the major producer of kiwifruit, with more than 1.2 million metric tons harvested in 2021. This report will help to generate a better understanding of the pathogens affecting kiwifruit orchards in China.

First Report of Fusarium luffae Causing Leaf Blight on Loquat in Eastern China.

During May-June 2021 and 2022, leaf blight symptoms were observed on loquat leaves (Eriobotrya japonica cv. 'Mogi') in Jiangsu Province (Xuzhou municipality, 117.17° E, 34.13° N) in China. Approximately 10% of the leaves on the two hundred trees studied in a six-year-old loquat orchard exhibited round lesions that changed from light yellow to reddish-brown in 8-10 days. Approximately 3% of the infected leaves exhibited numerous lesions that coalesced, leading to expansive blighted areas. Twenty-five samples of symptomatic tissue, approximately 0.2 cm2 in size, were collected in May 2022 from five different trees (five samples per tree), sterilized in 2% NaOCl for 1 min, washed twice with sterilized ddH2O, and incubated at 26°C for 5 days on PDA medium containing 50 µg/mL chloramphenicol. Six isolates were obtained via single spore isolation. ITS (OQ954852-OQ954857), TUB2 (OQ968488-OQ968493), EF1-α (OQ971890-OQ971895), RPB1 (OQ971896-OQ971901), and RPB2 (OR037266-OR037271) genes were amplified using the ITS1/ITS4, T1/T22, EF1-728F/EF1-986R, RPB1-R8/RPB1-F5, and fRPB2-7CF/fRPB2-11aR primers, respectively (O'Donnell et al. 2010). The species was identified using the Fusarioid ID database (Crous et al. 2021), revealing that all obtained isolates showed high homology to representative F. luffae strains. Upon combining the ITS, TUB2, EF1-α, RPB1, and RPB2 sequences, the isolates showed 99.42%-97.85% and 99.59%-98.10% identity to F. luffae CGMCC 3.19497 (ex-type strain) and NRRL 32522, respectively. A molecular phylogenetic tree was constructed using MEGA X, with a selection of representative Fusarium strains. Microscope observations showed septate mycelium, microconidia (6.86 ± 0.91 µm length, 1.67 ± 0.24 µm width, containing 1 septum; number of observations = 21), fusiform macroconidia (15.88 ± 1.43 µm length, 1.66 ± 0.24 µm width, containing 1 septum; number of observations = 45), and linear chlamydospores (79.36 ± 28.36 µm length, 12.03 ± 3.37 µm width; number of observations = 152). These observations are consistent with the morphology of F. luffae (Wang et al. 2019). All isolates exhibited identical morphological characteristics. All isolates were evaluated for pathogenicity in vivo using healthy non-detached loquat leaves. A total of 15 leaves from 5 different three-month-old 'Mogi' loquat trees were used for each isolate. Experiments were performed three times. A suspension of 1 × 106 spores/mL obtained from a seven-day-old colony (10 mL per 15 leaves), was sprayed on non-wounded leaves for inoculation. Sterilized ddH2O was used in the control experiment. Inoculated trees were stored at 26°C and 70% relative humidity for four days. Leaf blight symptoms were observed in all inoculated leaves, and the symptoms were observed in all repeated trials. The pathogen was recovered, and its identity was confirmed by ITS sequencing and morphological analysis, fulfilling Koch's postulates. In recent years, F. luffae has been reported to cause fruit rot on muskmelon, flower rot on kiwifruit, soybean pod rot, and leaf spot on cherry in China (Yu et al. 2022; Zhang et al. 2022; Zhao et al. 2022; Zhou et al. 2022), demonstrating the host promiscuity of this pathogen. Although F. solani has been identified as the causal agent of root rot and fruit rot on loquat (Abbas et al. 2017; Wu et al. 2021), this is the first report of F. luffae causing leaf blight on loquat worldwide. This report will help to understand the pathogens affecting loquat orchards in China.

Two New Species of Freshwater Planarian (Platyhelminthes, Tricladida, Dugesiidae, Dugesia) from Southern China Exhibit Unusual Karyotypes, with a Discussion on Reproduction in Aneuploid Species

Two new species of the genus Dugesia from Southern China are described by applying an integrative approach, including morphological, karyological, histological, and molecular information. In the molecular phylogenetic tree, the two new species, Dugesia pendula Chen & Dong, sp. nov. and Dugesia musculosa Chen & Dong, sp. nov., fall into an Eastern Palearctic/Oriental clade and an Oriental/Australasian clade, respectively, while sharing only a rather distant relationship. The separate specific status of the two new species is supported also by their genetic distances. Dugesia pendula is characterized by the following features: symmetrical openings of the oviducts into the bursal canal, a duct between seminal vesicle and diaphragm, small diaphragm, dorsally located seminal vesicle, a penis papilla suspended from the dorsal wall of the male atrium, and mixoploid karyotype with diploid complements of 2n=2x=14+0−1 B-chromosome and triploid complements of 2n=3x=21+0−1 B-chromosome, with all chromosomes being metacentric. Dugesia musculosa is characterized by the following features: asymmetrical openings of the oviducts into the bursal canal; a ventrally displaced ejaculatory duct with a terminal opening; two diaphragms; a bursal canal provided with a strong, thick layer of circular muscle, which extends from the copulatory bursa to the common atrium and gonoduct; the left vas deferens opening at the midlateral wall of the seminal vesicle, while the right sperm duct opens at the dorsolateral wall of the seminal vesicle; and karyotype consisting of complicated diploid and aneuploid mosaicism, with diploid complements of 2n=2x=16 and 2n=2x=16−17th-18th, with all chromosomes being metacentric. The uncommon karyotypes, combined with the asexual reproduction of aneuploid animals, are evaluated in the context of the relationship between ploidy levels and reproductive modalities in the genus Dugesia.

Integrative description of a new species of Dugesia (Platyhelminthes, Tricladida, Dugesiidae) from southern China, with its complete mitogenome and a biogeographic evaluation

A new species of freshwater flatworm of the genus Dugesia from Guangdong Province in China is described through an integrative approach, including molecular and morphological data, as well as mitochondrial genome analysis. The new species, Dugesia ancoraria Zhu & Wang, sp. nov., is characterised by: (a) a highly asymmetrical penis papilla, provided with a hunchback-like dorsal bump; (b) a short duct between seminal vesicle and ejaculatory duct; and (c) a postero-ventral course of the ejaculatory duct, which opens to the exterior at the subterminal, ventral part of the penis papilla. The molecular phylogenetic tree obtained from the concatenated dataset of four DNA markers (18S rDNA, ITS-1, 28S rDNA, COI) facilitated determination of the phylogenetic position of the new species, which shares a sister-group relationship with a small clade, comprising D. notogaea Sluys & Kawakatsu, 1998 from Australia and D. bengalensis Kawakatsu, 1983 from India. The circular mitogenome of the new species is 17,705 bp in length, including 12 protein coding genes, two ribosomal genes, and 22 transfer RNAs. Via analysis of gene order of mitochondrial genomes, the presently available pattern of mitochondrial gene rearrangement in the suborder Continenticola is discussed.

Repeated upslope biome shifts in Saxifraga during late-Cenozoic climate cooling.

Mountains are among the most biodiverse places on Earth, and plant lineages that inhabit them have some of the highest speciation rates ever recorded. Plant diversity within the alpine zone - the elevation above which trees cannot grow-contributes significantly to overall diversity within mountain systems, but the origins of alpine plant diversity are poorly understood. Here, we quantify the processes that generate alpine plant diversity and their changing dynamics through time in Saxifraga (Saxifragaceae), an angiosperm genus that occurs predominantly in mountain systems. We present a time-calibrated molecular phylogenetic tree for the genus that is inferred from 329 low-copy nuclear loci and incorporates 73% (407) of known species. We show that upslope biome shifts into the alpine zone are considerably more prevalent than dispersal of alpine specialists between regions, and that the rate of upslope biome shifts increased markedly in the last 5 Myr, a timeframe concordant with a cooling and fluctuating climate that is likely to have increased the extent of the alpine zone. Furthermore, alpine zone specialists have lower speciation rates than generalists that occur inside and outside the alpine zone, and major speciation rate increases within Saxifraga significantly pre-date increased rates of upslope biome shifts. Specialisation to the alpine zone is not therefore associated with speciation rate increases. Taken together, this study presents a quantified and broad scale perspective of processes underpinning alpine plant diversity.

Mating Pheromone (Gamone 1) in Blepharisma: A Glycoprotein Responsible for Species Diversity in Unicellular Organisms (Alveolata, Ciliophora).

The genus Blepharisma (Alveolata, Ciliophora) is a unicellular organism distributed worldwide, even in extreme environments, and comprises numerous species. While usually proliferating through cell division, Blepharisma undergoes sexual reproduction (conjugation) when cells are moderately starved. Conjugation is initiated by mating pheromones (gamone 1 and gamone 2) secreted by complementary mating-type cells. Gamone 1, a glycoprotein, functions in a species-specific manner, while gamone 2, an amino acid derivative, is a common molecule across species. The specific function of gamone 1 suggests the possibility that mutations in gamone 1 might have led to reproductive isolation during the evolutionary process, triggering species diversification. In this study, by comparing the amino acid sequences of gamone 1 homologs from 15 strains (representing five species), we found that mutations resulting in distinct amino acid properties occur across species boundaries and are mainly concentrated at two specific regions within gamone 1. These mutations potentially alter the binding affinity of gamone 1 to its receptors, suggesting their effect in causing reproductive isolation. The interspecies artificial conjugation conducted previously and the molecular phylogenetic tree constructed using the gamone 1 homolog sequences in this study provide insights into the speciation process within the genus Blepharisma.

Nigrohirschioporus violacaeruleum sp. nov. and a new record of Pallidohirschioporus brastagii (Basidiomycota, Hymenochaetales) from India

The present study deals with the descriptions of two Hirschioporaceae taxa from West Bengal, India, using morphological and molecular phylogenetic analyses of nrITS sequence data. Of these two taxa, Nigrohirschioporus violacaeruleum is newly described, while Pallidohirschiorus brastagii is a new record to West Bengal, India. Nigrohirschioporus violacaeruleum is characterized by a dimidiate, applanate, woody basidiocarp; a velutinate to glabrous, azonate upper surface coloured white to greyish violet; circular to angular pores (7–10 per mm) coloured lilac grey; dimorphic cystidia and inamyloid, ellipsoid to elongate basidiospores (3.5–6 × 2.5−3.0 µm). Pallidohirschioporus brastagii possess a sessile imbricate, pileate basidiocarp, an adpressed hispid, brownish orange concentrically zonate upper surface; a poroid to irpicoid hymenophore, apically encrusted, thick-walled cystidia, and inamyloid, cylindrical basidiospores (3‒5.5 × 1.5‒2.5 µm). Full descriptions, field photographs of the fruiting bodies, detailed morphological descriptions, and a molecular sequence-based phylogenetic tree, are provided.