Morphophylogenetic evidence reveals four new fungal species within Tetraplosphaeriaceae (Pleosporales, Ascomycota) from tropical and subtropical forest in China.

Tetraplosphaeriaceae (Pleosporales, Ascomycota) is a family with many saprobes recorded from various hosts, especially bamboo and grasses. During a taxonomic investigation of microfungi in tropical and subtropical forest regions of Guizhou, Hainan and Yunnan provinces, China, several plant samples were collected and examined for fungi. Four newly discovered species are described based on morphology and evolutionary relationships with their allies inferred from phylogenetic analyses derived from a combined dataset of LSU, ITS, SSU, and tub2 DNA sequence data. Detailed illustrations, descriptions and taxonomic notes are provided for each species. The four new species of Tetraplosphaeriaceae reported herein are Polyplosphaeriaguizhouensis, Polyplosphaeriahainanensis, Pseudotetraploayunnanensis, and Tetraploahainanensis. A checklist of Tetraplosphaeriaceae species with available details on their ecology is also provided.

Appressoria-Producing Sordariomycetes Taxa Associated with Jasminum Species.

Appressoria are specialized structures formed by certain phytopathogenic fungi during the early stages of the infection process. Over the years, significant advancements have been made in understanding the formation, types, and functions of appressoria. Besides being formed primarily by fungal pathogens, many studies have reported their occurrence in other life modes such as endophytes, epiphytes, and saprobes. In this study, we observed the formation of appressoria in fungal genera that have been found associated with leaf spots and, interestingly, by a saprobic species. We used morphological descriptions and illustrations, molecular phylogeny, coalescent-based Poisson tree processes (PTP) model, inter- and intra-species genetic distances based on their respective DNA markers, and Genealogical Concordance Phylogenetic Species Recognition Analysis (GCPSR) to establish a new species (Pseudoplagiostoma jasmini), a Ciliochorella sp., and a new host record (Coniella malaysiana). The Ciliochorella sp. is reported as a saprobe, while Pseudoplagiostoma jasmini and Coniella malaysiana were found to be associated with leaf spots of Jasminum species. All three taxa produce appressoria, and this is the first study that reports the formation of appressoria by a Ciliochorella sp. and a Pseudoplagiostoma sp.

Fungicolous Mucor on mushrooms: One novel species and six host records from southwest China and northern Thailand.

Mucor species are a group of common soil-borne fungi, known to cause infections on humans and animals, interfere in food production, and act as useful agents in biotechnological applications. This study reports one new Mucor species, M. yunnanensis, which was found to be fungicolous on an Armillaria sp. from southwest China. Further, M. circinelloides on Phlebopus sp., M. hiemalis on Ramaria sp. and Boletus sp., M. irregularis on Pleurotus sp., M. nederlandicus on Russula sp., and M. yunnanensis on Boletus sp. are reported as new host records. Mucor yunnanensis and M. hiemalis have been collected from Yunnan Province in China, whereas M. circinelloides, M. irregularis, and M. nederlandicus have been collected from Chiang Mai and Chiang Rai Provinces in Thailand. All the Mucor taxa reported herein were identified based on both morphology and phylogenetic analyses of a combined nuc rDNA internal transcribed spacer region ITS1-5.8S-ITS2 (ITS) and partial nuc 28S rDNA (28S) sequence matrix. Comprehensive descriptions, illustrations, and a phylogenetic tree are provided for all the taxa reported in the study to show the placements of taxa, and the new taxon is compared with its sister taxa.

Two new fungal genera (Diaporthales) found on Dipterocarpaceae in Thailand.

Diaporthales is a species-rich order of fungi that includes endophytes, saprobes, and pathogens associated with forest plants and crops. They may also occur as parasites or secondary invaders of plant tissues injured or infected by other organisms or inhabit living animal and human tissues, as well as soil. Meanwhile, some severe pathogens wipe out large-scale cultivations of profitable crops, timber monocultures, and forests. Based on morphological and phylogenetic analyses of combined ITS, LSU, tef1-α, and rpb2 sequence data, generated using maximum likelihood (ML), maximum parsimony (MP), and MrBayes (BI), we introduce two new genera of Diaporthales found in Dipterocarpaceae in Thailand, namely Pulvinaticonidioma and Subellipsoidispora. Pulvinaticonidioma is characterized by solitary, subglobose, pycnidial, unilocular conidiomata with the internal layers convex and pulvinate at the base; hyaline, unbranched, septate conidiophores; hyaline, phialidic, cylindrical to ampulliform, determinate conidiogenous cells and hyaline, cylindrical, straight, unicellular, and aseptate conidia with obtuse ends. Subellipsoidispora has clavate to broadly fusoid, short pedicellate asci with an indistinct J- apical ring; biturbinate to subellipsoidal, hyaline to pale brown, smooth, guttulate ascospores that are 1-septate and slightly constricted at the septa. Detailed morphological and phylogenetic comparisons of these two new genera are provided in this study.

Endophytic Colletotrichum (Sordariomycetes, Glomerellaceae) species associated with Citrusgrandis cv. "Tomentosa" in China.

Colletotrichum species are well-known plant pathogens, saprobes, endophytes, human pathogens and entomopathogens. However, little is known about Colletotrichum as endophytes of plants and cultivars including Citrusgrandis cv. "Tomentosa". In the present study, 12 endophytic Colletotrichum isolates were obtained from this host in Huazhou, Guangdong Province (China) in 2019. Based on morphology and combined multigene phylogeny [nuclear ribosomal internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (gapdh), chitin synthase 1 (chs-1), histone H3 (his3) actin (act), beta-tubulin (ß-tubulin) and glutamine synthetase (gs)], six Colletotrichum species were identified, including two new species, namely Colletotrichumguangdongense and C.tomentosae. Colletotrichumasianum, C.plurivorum, C.siamense and C.tainanense are identified as being the first reports on C.grandis cv. "Tomentosa" worldwide. This study is the first comprehensive study on endophytic Colletotrichum species on C.grandis cv. "Tomentosa" in China.

Neogrisphenol A, a Potential Ovarian Cancer Inhibitor from a New Record Fungus Neohelicosporium griseum.

From the rice fermentation product of a new record fungus, Neohelicosporium griseum, two new polyketides, neogrisphenol A (1) and neogrisphenol B (2), one new isochroman-1-one, (S)-6-hydroxy-7-methoxy-3,5-dimethylisochroman-1-one (3), and four known compounds (4-7) were isolated. Their structures were determined using 1D- and 2D-NMR, mass spectrometry, and chemical calculations. The C-3~C-2' polymerization mode between the two α-naphthalenone derivative moieties is uncommon in compounds 1 and 2. Meanwhile, compounds 1-2 and 5 exhibited antibacterial activity against Bacillus subtilis, Clostridium perfringens, Staphylococcus aureus, and Staphylococcus aureus, with MIC values ranging between 16 and 31 µg/mL. In addition, compound 5 showed antifungal activity against Sclerotinia sclerotiorum and Phytophthora nicotianae var. nicotianae, with respective IC50 values of 88.14 ± 2.21 µg/mL and 52.36 ± 1.38 µg/mL. Compound 1 showed significant cytotoxicity against A2780, PC-3, and MBA-MD-231 cell lines with respective IC50 values of 3.20, 10.68, and 16.30 µM, and the cytotoxicity against A2780 cells was even higher than that of cisplatin (CDDP). With an IC50 value of 10.13 µM, compound 2 also exhibited cytotoxicity against A2780. The in vitro results showed that compound 1 inhibited A2780 cell proliferation, induced apoptosis, and arrested the cell cycle at the S-phase in a concentration-dependent manner.

Morphology and Phylogeny Reveal Three Montagnula Species from China and Thailand.

Four stains were isolated from two fresh twigs of Helwingia himalaica and two dead woods during investigations of micro-fungi in China and Thailand. Phylogenetic analyses of four gene regions LSU, ITS, SSU and tef1-α revealed the placement of these species in Montagnula. Based on the morphological examination and molecular data, two new species, M. aquatica and M. guiyangensis, and a known species M. donacina are described. Descriptions and illustrations of the new collections and a key to the Montagnula species are provided. Montagnula chromolaenicola, M. puerensis, M. saikhuensis, and M. thailandica are discussed and synonymized under M. donacina.

Four novel Pleurocordyceps (Polycephalomycetaceae) species from China.

Entomopathogenic fungi comprise an ecologically important group of specialized pathogens infecting other fungi, invertebrates, and plants. These fungi are species-rich with high diversity and broad distribution worldwide. The majority of entomopathogenic fungi belong to clavicipitoids, which consist of the hypocrealean families, Clavicipitaceae, Cordycipitaceae, Ophiocordycipitaceae, and Polycephalomycetaceae. The latter is a newly established entomopathogenic family that recently separated from the family Ophiocordycipitaceae to accommodate the genera, Perennicordyceps, Pleurocordyceps, and Polycephalomyces. In recent years, Polycephalomycetaceae has been enriched with parasitic and hyperparasitic fungi. With 16 species spread across China, Ecuador, Japan, and Thailand, Pleurocordyceps is the most speciose genus in the family. In this study, we expand the number of taxa in the genus by introducing four new Pleurocordyceps species from China, namely, P. clavisynnema, P. multisynnema, P. neoagarica, and P. sanduensis. We provide detailed descriptions and illustrations and infer genus-level phylogenies based on a combined 6-loci gene sequence dataset comprising the internal transcribed spacer gene region (ITS), small subunit ribosomal RNA gene region (SSU), large subunit rRNA gene region (LSU), translation elongation factor 1-alpha gene region (TEF-1α), RNA polymerase II largest subunit gene region (RPB1), and RNA polymerase II second largest subunit (RPB2). This study contributes to knowledge with regard to the diversity of Pleurocordyceps specifically and entomopathogenic Hypocreales more broadly.

Description of Four Novel Species in Pleosporales Associated with Coffee in Yunnan, China.

In Yunnan Province, the coffee-growing regions are mainly distributed in Pu'er and Xishuangbanna. During the surveys of microfungi associated with coffee in Yunnan Province, seven taxa were isolated from coffee samples. Based on molecular phylogenetic analyses of combined ITS, LSU, SSU, rpb2, and tef1-α sequence data and morphological characteristics, four new species viz. Deniquelata yunnanensis, Paraconiothyrium yunnanensis, Pseudocoleophoma puerensis, and Pse. yunnanensis, and three new records viz. Austropleospora keteleeriae, Montagnula thailandica, and Xenocamarosporium acaciae in Pleosporales are introduced. In addition, Paracamarosporium fungicola was transferred back to Paraconiothyrium based on taxonomy and DNA sequences. Full descriptions, illustrations, and phylogenetic trees to show the placement of new and known taxa are provided. In addition, the morphological comparisons of new taxa with closely related taxa are given.

Ten decadal advances in fungal biology leading towards human well-being.

Fungi are an understudied resource possessing huge potential for developing products that can greatly improve human well-being. In the current paper, we highlight some important discoveries and developments in applied mycology and interdisciplinary Life Science research. These examples concern recently introduced drugs for the treatment of infections and neurological diseases; application of -OMICS techniques and genetic tools in medical mycology and the regulation of mycotoxin production; as well as some highlights of mushroom cultivaton in Asia. Examples for new diagnostic tools in medical mycology and the exploitation of new candidates for therapeutic drugs, are also given. In addition, two entries illustrating the latest developments in the use of fungi for biodegradation and fungal biomaterial production are provided. Some other areas where there have been and/or will be significant developments are also included. It is our hope that this paper will help realise the importance of fungi as a potential industrial resource and see the next two decades bring forward many new fungal and fungus-derived products.

Lasiodiplodia fici sp. nov., Causing Leaf Spot on Ficus altissima in China.

High temperatures and the seasonality in tropical ecosystems favours plant pathogens, which result in many fungal diseases. Among these, diseases caused by Botryosphaeriaceae species are prominent as dieback, canker and leaf spots. In this research, we isolated one leaf-spot-causing Botryosphaeriaceae species from Ficus altissima leaves, which were collected in Guangzhou, Guangdong Province, China. Isolation and identification of the pathogen were based on morphological and molecular aspects. Based on multigene phylogenetic analysis of combined internal transcribed spacer (ITS), translation elongation factor 1-α gene (tef1) and beta-tubulin gene (tub2), the fungus associated with leaf spots on F. altissima is described as Lasiodiplodia fici, a novel species. Pathogenicity assays were conducted by inoculating the fungus onto detached shoots and plants under controlled environmental conditions. The results revealed that the L. fici isolates can infect the plant tissues under stress conditions by developing disease symptoms on detached shoots within three days. However, when it was inoculated onto the leaves of the host and grown in natural conditions, the progression of the disease was slow. The putative pathogen was re-isolated, and Koch's assumptions were satisfied. This is the first report of Lasiodiplodia species causing disease on Ficus altissima. Results from the present study will provide additional knowledge on fungal pathogens associated with forest and ornamental plant species.

Endophytic Fungi Associated with Coffee Leaves in China Exhibited In Vitro Antagonism against Fungal and Bacterial Pathogens.

Coffee endophytes have been studied for almost 74 years, and several studies have demonstrated coffee-endophytic fungi with antibacterial and antifungal potential for human and plant pathogens. In this study, we isolated and identified a total of 235 strains of endophytic fungi from coffee leaf tissues collected in four coffee plantations in Pu'er city, Yunnan province, China. Molecular identification was carried out using maximum likelihood phylogenetic analysis of nuclear ribosomal internal transcribed spacer (ITS1-5.8S rDNA-ITS2) sequences, while the colonization rate and the isolation frequency were also calculated. Two pathogenic fungi (Alternaria alternata and Penicillium digitatum) and two pathogenic bacteria (Pseudomonas syringae and Salmonella enterica subsp. enterica) were used for screening the antagonistic activities of 61 strains of coffee-endophytic fungi by a dual-culture test assay while maximum likelihood phylogenetic analysis confirmed their natural classification. This is the first study of coffee-leaf-endophytic fungal diversity in China, and the results revealed that coffee-endophytic fungi from this study belong to the Ascomycota, distributed among two classes, 10 orders, and 17 families. Concurrently, endophytic fungi isolates distributed in Arthrinium, Biscogniauxia, Daldinia, Diaporthe, and Nigrospora showed strong antagonistic activities against the pathogens. For the pathogens Alternaria alternata and Pseudomonas syringae, Nigrospora XCE-7 showed the best inhibitory effects with inhibition rates of 71.76% and 61.11%, respectively. For the pathogen Penicillium digitatum, Daldinia ME-9 showed the best inhibitory effect with a 74.67% inhibition rate, while Biscogniauxia PTE-7 and Daldinia T5E-1-3 showed the best inhibitory effect with a rate of 60.42% against the pathogen Salmonella enterica subsp. enterica. Overall, our study shows the diversity of coffee endophytes in four coffee-growing areas in Pu'er city, Yunnan province, China, and their potential use as biological control agents against two fungal and two bacterial pathogens.

A Global Overview of Diversity and Phylogeny of the Rust Genus Uromyces.

Uromyces is the second-largest plant pathogenic rust genus, is responsible for numerous diseases, and has major effects on both agricultural and non-agricultural plants. The genus is generally characterized by its unicellular teliospores that help to characterize it and distinguish it from another important rust genus, Puccinia. In this study, a global overview of the diversity and distribution of Uromyces is presented based on both online and offline resources. The information obtained was analyzed for numerical and graphical summaries to provide the diversity and distribution of the genus by country and continent. Besides this, broad taxonomical aspects, a brief life cycle, and other comparative aspects on diversity and distribution were also provided. In addition, a phylogenetic analysis based on the ITS and nLSU DNA sequence data available in GenBank and published literature was performed to examine the intergeneric relationships of Uromyces. The results obtained revealed that the rust genus is found distributed over 150 countries, territories, and occupancies of the world on around 647 plant genera belonging to 95 plant families. Phylogenetic studies based on LSU and ITS sequence data revealed that Uromyces species are polyphyletic and require more DNA-based analyses for a better understanding of their taxonomic placement.

Comprehensive Review of Fungi on Coffee.

Coffee is grown in more than 80 countries as a cash crop and consumed worldwide as a beverage and food additive. It is susceptible to fungal infection during growth, processing and storage. Fungal infections, in particular, can seriously affect the quality of coffee and threaten human health. The data for this comprehensive review were collected from the United States Department of Agriculture, Agricultural Research Service (USDA ARS) website and published papers. This review lists the fungal species reported on coffee based on taxonomy, life mode, host, affected plant part and region. Five major fungal diseases and mycotoxin-producing species (post-harvest diseases of coffee) are also discussed. Furthermore, we address why coffee yield and quality are affected by fungi and propose methods to control fungal infections to increase coffee yield and improve quality. Endophytic fungi and their potential as biological control agents of coffee disease are also discussed.

Pleocatenatachiangraiensis gen. et. sp. nov. (Pleosporales, Dothideomycetes) from medicinal plants in northern Thailand.

Pleocatenata, a new genus, is introduced with its type species, Pleocatenatachiangraiensis, which was isolated from withered twigs of two medicinal plants, Clerodendrumquadriloculare (Blanco) Merr (Verbenaceae) and Tarennastellulata (Hook.f.) Ridl (Rubiaceae) in northern Thailand. The genus is characterized by mononematous, septate, brown or dark brown conidiophores, monotretic conidiogenous cells and catenate, obclavate, olivaceous to blackish brown conidia. Phylogenetic analysis of combined LSU, SSU, tef1-α, rpb2 and ITS sequence data showed Pleocatenata forms a distinct phylogenetic lineage in Pleosporales, Dothideomycetes. Therefore, we treat Pleocatenata as Pleosporales genera incertae sedis based on morphology and phylogenetic analyses. Descriptions and illustrations of the new taxa are provided, and it is compared with morphologically similar genera.

Fungal diversity notes 1512-1610: taxonomic and phylogenetic contributions on genera and species of fungal taxa.

This article is the 14th in the Fungal Diversity Notes series, wherein we report 98 taxa distributed in two phyla, seven classes, 26 orders and 50 families which are described and illustrated. Taxa in this study were collected from Australia, Brazil, Burkina Faso, Chile, China, Cyprus, Egypt, France, French Guiana, India, Indonesia, Italy, Laos, Mexico, Russia, Sri Lanka, Thailand, and Vietnam. There are 59 new taxa, 39 new hosts and new geographical distributions with one new combination. The 59 new species comprise Angustimassarina kunmingense, Asterina lopi, Asterina brigadeirensis, Bartalinia bidenticola, Bartalinia caryotae, Buellia pruinocalcarea, Coltricia insularis, Colletotrichum flexuosum, Colletotrichum thasutense, Coniochaeta caraganae, Coniothyrium yuccicola, Dematipyriforma aquatic, Dematipyriforma globispora, Dematipyriforma nilotica, Distoseptispora bambusicola, Fulvifomes jawadhuvensis, Fulvifomes malaiyanurensis, Fulvifomes thiruvannamalaiensis, Fusarium purpurea, Gerronema atrovirens, Gerronema flavum, Gerronema keralense, Gerronema kuruvense, Grammothele taiwanensis, Hongkongmyces changchunensis, Hypoxylon inaequale, Kirschsteiniothelia acutisporum, Kirschsteiniothelia crustaceum, Kirschsteiniothelia extensum, Kirschsteiniothelia septemseptatum, Kirschsteiniothelia spatiosum, Lecanora immersocalcarea, Lepiota subthailandica, Lindgomyces guizhouensis, Marthe asmius pallidoaurantiacus, Marasmius tangerinus, Neovaginatispora mangiferae, Pararamichloridium aquisubtropicum, Pestalotiopsis piraubensis, Phacidium chinaum, Phaeoisaria goiasensis, Phaeoseptum thailandicum, Pleurothecium aquisubtropicum, Pseudocercospora vernoniae, Pyrenophora verruculosa, Rhachomyces cruralis, Rhachomyces hyperommae, Rhachomyces magrinii, Rhachomyces platyprosophi, Rhizomarasmius cunninghamietorum, Skeletocutis cangshanensis, Skeletocutis subchrysella, Sporisorium anadelphiae-leptocomae, Tetraploa dashaoensis, Tomentella exiguelata, Tomentella fuscoaraneosa, Tricholomopsis lechatii, Vaginatispora flavispora and Wetmoreana blastidiocalcarea. The new combination is Torula sundara. The 39 new records on hosts and geographical distribution comprise Apiospora guiyangensis, Aplosporella artocarpi, Ascochyta medicaginicola, Astrocystis bambusicola, Athelia rolfsii, Bambusicola bambusae, Bipolaris luttrellii, Botryosphaeria dothidea, Chlorophyllum squamulosum, Colletotrichum aeschynomenes, Colletotrichum pandanicola, Coprinopsis cinerea, Corylicola italica, Curvularia alcornii, Curvularia senegalensis, Diaporthe foeniculina, Diaporthe longicolla, Diaporthe phaseolorum, Diatrypella quercina, Fusarium brachygibbosum, Helicoma aquaticum, Lepiota metulispora, Lepiota pongduadensis, Lepiota subvenenata, Melanconiella meridionalis, Monotosporella erecta, Nodulosphaeria digitalis, Palmiascoma gregariascomum, Periconia byssoides, Periconia cortaderiae, Pleopunctum ellipsoideum, Psilocybe keralensis, Scedosporium apiospermum, Scedosporium dehoogii, Scedosporium marina, Spegazzinia deightonii, Torula fici, Wiesneriomyces laurinus and Xylaria venosula. All these taxa are supported by morphological and multigene phylogenetic analyses. This article allows the researchers to publish fungal collections which are important for future studies. An updated, accurate and timely report of fungus-host and fungus-geography is important. We also provide an updated list of fungal taxa published in the previous fungal diversity notes. In this list, erroneous taxa and synonyms are marked and corrected accordingly.

Three new species of Nigrograna (Dothideomycetes, Pleosporales) associated with Arabica coffee from Yunnan Province, China.

Coffee is one of the most important cash crops in Yunnan Province, China. Yunnan is ranked as the biggest producer of high-quality coffee in China. During surveys of microfungi from coffee plantations in Yunnan, six fungal strains that resemble Nigrogranaceae were collected. Multi-gene analyses of a combined SSU-LSU-ITS-rpb2-tef1-α sequence data matrix were used to infer the phylogenetic position of the new species in Nigrograna while morphological characteristics were used to deduce the taxonomic position of the new species. Six fungal strains isolated from decaying branches of Coffeaarabica represent three new saprobic species in Nigrograna. The three new species, N.asexualis, N.coffeae, and N.puerensis, are described with full (macro and micro characteristics) descriptions, illustrations, and a phylogenetic tree that shows the phylogenetic position of new taxa.

Additions to Neopestalotiopsis (Amphisphaeriales, Sporocadaceae) fungi: two new species and one new host record from China.

Background: In this study, three Neopestalotiopsis taxa were identified, associated with leaves of Zingiberofficinale, Elaeagnuspungens and Salaccazalacca. New information: Based on morphology and multi-gene analyses of the internal transcribed spacer (ITS), beta-tubulin (TUB2) and translation elongation factor 1-alpha (TEF1), the five strains of Neopestalotiopsis represent two novel and one known species. They are introduced with descriptions, illustrations and notes herein.

Campylocarpon fasciculare (Nectriaceae, Sordariomycetes); Novel Emergence of Black-Foot Causing Pathogen on Young Grapevines in China.

The black-foot disease of grapevine is one of the most destructive diseases in viticulture and it is caused by a complex of soil-borne fungi. This study aimed to identify the species associated with black-foot disease in young grapevines in vineyards of China. Fungal isolates were identified as Campylocarpon fasciculare, based on both morphological and multi-locus phylogenetic analysis of ITS, tef1-α and ß-tubulin sequence data. For the first time in China, we report Campylocarpon fasciculare associated with symptomatic young grapevines. Koch's postulates were performed on Vitis vinifera cv. Summer Black (SB) in a greenhouse and to confirm the pathogenicity on grapevines. This work improves the knowledge of black-foot disease in Chinese vineyards and will be helpful to growers in their decisions regarding vinicultural practices, planting and disease management.

Morphology and Phylogeny Reveal Vamsapriyaceae fam. nov. (Xylariales, Sordariomycetes) with Two Novel Vamsapriya Species.

Phylogenetic analyses of combined LSU, rpb2, tub2 and ITS sequence data of representative Xylariales taxa indicated that Diabolocovidia, Didymobotryum and Vamsapriya cluster together and form a distinct clade in Xylariales. Morphological comparison also shows their distinctiveness from other families of Xylariales. Therefore, we introduce it as a novel family, Vamsapriyaceae. Based on morphological characteristics, Podosporium and Tretophragmia, which were previously classified in Ascomycota genera incertae sedis, are now included in the Vamsapriyaceae. In addition, three Vamsapriya species, V. chiangmaiensis sp. nov, V. uniseptata sp. nov, and V. indica are described and illustrated in this paper.