Mycoparasites, Gut Dwellers, and Saprotrophs: Phylogenomic Reconstructions and Comparative Analyses of Kickxellomycotina Fungi.

Improved sequencing technologies have profoundly altered global views of fungal diversity and evolution. High-throughput sequencing methods are critical for studying fungi due to the cryptic, symbiotic nature of many species, particularly those that are difficult to culture. However, the low coverage genome sequencing (LCGS) approach to phylogenomic inference has not been widely applied to fungi. Here we analyzed 171 Kickxellomycotina fungi using LCGS methods to obtain hundreds of marker genes for robust phylogenomic reconstruction. Additionally, we mined our LCGS data for a set of nine rDNA and protein coding genes to enable analyses across species for which no LCGS data were obtained. The main goals of this study were to: 1) evaluate the quality and utility of LCGS data for both phylogenetic reconstruction and functional annotation, 2) test relationships among clades of Kickxellomycotina, and 3) perform comparative functional analyses between clades to gain insight into putative trophic modes. In opposition to previous studies, our nine-gene analyses support two clades of arthropod gut dwelling species and suggest a possible single evolutionary event leading to this symbiotic lifestyle. Furthermore, we resolve the mycoparasitic Dimargaritales as the earliest diverging clade in the subphylum and find four major clades of Coemansia species. Finally, functional analyses illustrate clear variation in predicted carbohydrate active enzymes and secondary metabolites (SM) based on ecology, that is biotroph versus saprotroph. Saprotrophic Kickxellales broadly lack many known pectinase families compared with saprotrophic Mucoromycota and are depauperate for SM but have similar numbers of predicted chitinases as mycoparasitic.

Phylogenetic and morphological analyses of the mycoparasitic genus Piptocephalis.

The Piptocephalidaceae (Zoopagales, Zoopagomycota) contains three genera of mycoparasitic, haustoria-forming fungi: Kuzuhaea, Piptocephalis, and Syncephalis. Although the species in this family are diverse and ubiquitous in soil and dung, they are among the least studied fungi. Co-cultures of Piptocephalis and their hosts are relatively easy to isolate from soil and dung samples across the globe, making them a good model taxon for the order Zoopagales. This study focuses on the systematics of the genus Piptocephalis. Despite the fact that there are approximately 40 described Piptocephalis species, there are no modern taxonomic or molecular phylogenetic treatments of this group. Minimal sequence data are available, and relatively little is known about the true diversity or biogeography of the genus. Our study addresses two aspects: Piptocephalis systematics and analyses of the length and inter- and infraspecific variation of the nuc rDNA internal transcribed spacer (ITS1-5.8S-ITS2 = ITS) region. First, we generated a large subunit (28S) nuc rDNA phylogeny and evaluated several morphological characters by testing their correlation with the phylogeny using Bayesian Tip-association Significance testing (BaTS). We found monophyly of Piptocephalis species identified based on morphological traits, but morphological character states were not conserved across clades, suggesting that there have been multiple gains and losses of morphological characters. We also found that Kuzhuaea is nested within Piptocephalis. Second, we amplified the ITS from many Piptocephalis isolates, created a sequence alignment, and measured the lengths using the software ITSx. Piptocephalis species had ITS regions that were longer than the average for most Dikarya but were similar in length to those of the related genus Syncephalis.

Phylogenetic systematics of Syncephalis (Zoopagales, Zoopagomycotina), a genus of ubiquitous mycoparasites.

We examined phylogenetic relationships among species of the mycoparasite genus Syncephalis using sequences from three nuclear ribsosomal DNA genes (18S, 5.8S, and 28S nuc rDNA) and a gene encoding the largest subunit of RNA polymerase II (RPB1). Our data set included 88 Syncephalis isolates comprising 23 named species and several unnamed taxa. We also revived a culturing technique using beef liver and cellophane to grow several Syncephalis isolates without their host fungi to obtain pure parasite DNA. Most isolates, however, were grown in co-cultures with their host fungi, so we designed Syncephalis-specific primers to obtain sequence data. Individual and combined data sets were analyzed by maximum likelihood (ML) and Bayesian methods. We recovered 20 well-supported lineages and 38 operational taxonomic units (OTUs). Most major clades contained isolates from distant localities on multiple continents. There were taxonomic and nomenclature issues within several clades, probably due to high phenotypic plasticity or species dimorphism. We also conducted an analysis of Syncephalis nuc rDNA internal transcribed spacer (ITS) sequences for 31 phylogenetically diverse isolates, and we determined that most Syncephalis species have long ITS sequences relative to other fungi. Although commonly employed eukaryotic and fungal primers are compatible with diverse Syncephalis species, the ITS sequences of Syncepahlis are nonetheless rarely recovered in environmental molecular diversity surveys.

Preliminary phylogeny of Coemansia (Kickxellales), with descriptions of four new species from Taiwan.

Four new species of Coemansia from Taiwan are described. Three produce spirally twisted sporangiophores, and these new taxa increase the number of species in the Coemansia spiralis complex from three to six. Each new taxon is morphologically unique. Coemansia biformis, sp. nov., has two different asexual reproductive types on the same thallus; one is straight and the other has a spiral fertile region. Coemansia helicoidea, sp. nov., has stoloniferous sporangiophores with a helicoid fertile region. Coemansia pennisetoides, sp. nov., has a sporangiophore with a fertile region that resembles the inflorescence of the plant genus Pennisetum. Coemansia umbellata, sp. nov., has an umbellate sporangiophore branching pattern and a spirally twisted fertile region on the lowest branches. A dichotomous key was provided to identify the 23 accepted Coemansia species. Phylogenetic analysis based on a combined data set of D1-D2 domains of nuc 28S ribosomal RNA (rDNA) and partial nuc 18S rDNA identifies several independent evolutionary lineages within Coemansia and suggests that Spirodactylon aureum and Kickxella alabastrina may be nested within the genus Coemansia. Sequences of nuc rDNA ITS1-5.8S-ITS2 (internal transcribed spacer [ITS] barcode) are also used to support the description of these new species of Coemansia.

Five new species of the obligate mycoparasite Syncephalis (Zoopagales, Zoopagomycotina) from soil.

More than 520 soil samples were surveyed for species of the mycoparasitic zygomycete genus Syncephalis using a culture-based approach. These fungi are relatively common in soil using the optimal conditions for growing both the host and parasite. Five species obtained in dual culture are unknown to science and are described here: (i) S. digitata with sporangiophores short, merosporangia separate at the apices, simple, 3-5 spored; (ii) S. floridana, which forms galls in the host and has sporangiophores up to 170 µm long with unbranched merosporangia that contain 2-4 spores; (iii) S. pseudoplumigaleta, with an abrupt apical bend in the sporophore; (iv) S. pyriformis with fertile vesicles that are long-pyriform; and (v) S. unispora with unispored merosporangia. To facilitate future molecular comparisons between species of Syncephalis and to allow identification of these fungi from environmental sampling datasets, we used Syncephalis-specific PCR primers to generate internal transcribed spacer (ITS) sequences for all five new species.

Two new Ramicandelaber species from Taiwan.

Two new species of Ramicandelaber isolated from soil in Taiwan are described. Ramicandelaber fabisporus sp. nov. is characterized by having bean-shaped spores that are the smallest in the genus and also by the absence of lateral branches on the sporangiophores. Ramicandelaber taiwanensis sp. nov. is characterized by small, fusiform spores and it produces lateral branches that arise from the sporangiophores. Morphological characters and molecular phylogenetic analysis (D1/D2 domains of the LSU rRNA genes and ITS region) justify these new species of Ramicandelaber. A key is provided to the recognized species of Ramicandelaber.

A population-based follow-up study on gallstone disease among type 2 diabetics in Kinmen, Taiwan.

AIM: To assess the incidence of and risk factors for gallstone disease (GSD) among type 2 diabetics in Kinmen, Taiwan. METHODS: A screening program for GSD was performed by two specialists who employed real-time abdominal ultrasound to examine the abdominal region after patients had fasted for at least eight hours. Screening, which was conducted in 2001, involved 848 patients diagnosed with type 2 diabetes. After exclusion of 63 subjects with prevalent GSD, 377 participants without GSD were invited in 2002 for a second round of screening. A total of 281 (74.5%) subjects were re-examined. RESULTS: Among the 281 type 2 diabetics who had no GSD at the first screening, 10 had developed GSD by 2002. The incidence was 3.56% per year (95% CI: 1.78% per year-6.24% per year). Using a Cox regression model, age (RR = 1.07, 95% CI: 1.00-1.14), waist circumference (RR = 1.12, 95% CI: 1.01-1.29), and ALT (RR = 1.13, 95%CI: 1.01-1.26) appeared to be significantly correlated with development of GSD. CONCLUSION: Older age is a known risk factor for the development of GSD. Our study shows that greater waist circumference and elevated ALT levels are also associated with the development of GSD among type 2 diabetics in Kinmen.