Benchmarking assembly free nanopore read mappers to classify complex millipede gut microbiota via Oxford Nanopore Sequencing Technology.

Millipedes are key players in recycling leaf litter into soil in tropical ecosystems. To elucidate their gut microbiota, we collected millipedes from different municipalities of Puerto Rico. Here we aim to benchmark which method is best for metagenomic skimming of this highly complex millipede microbiome. We sequenced the gut DNA with Oxford Nanopore Technologies' (ONT) MinION sequencer, then analyzed the data using MEGAN-LR, Kraken2 protein mode, Kraken2 nucleotide mode, GraphMap, and Minimap2 to classify these long ONT reads. From our two samples, we obtained a total of 87,110 and 99,749 ONT reads, respectively. Kraken2 nucleotide mode classified the most reads compared to all other methods at the phylum and class taxonomic level, classifying 75% of the reads in the two samples, the other methods failed to assign enough reads to either phylum or class to yield asymptotes in the taxa rarefaction curves indicating that they required more sequencing depth to fully classify this community. The community is hyper diverse with all methods classifying 20-50 phyla in the two samples. There was significant overlap in the reads used and phyla classified between the five methods benchmarked. Our results suggest that Kraken2 nucleotide mode is the most appropriate tool for the application of metagenomic skimming of this highly complex community.

Resolving relationships at the animal-fungal divergence: A molecular phylogenetic study of the protist trichomycetes (Ichthyosporea, Eccrinida).

Trichomycetes is a group of microorganisms that was considered a class of fungi comprising four orders of commensal, gut-dwelling endosymbionts obligately associated with arthropods. Since molecular phylogenies revealed two of those orders (Amoebidiales and Eccrinales="protist trichos") to be closely related to members of the protist class Ichthyosporea (=Mesomycetozoea), trichomycetes have been considered an ecological association of both early-diverging fungi and protists. Understanding of the taxonomy, evolution, and diversity of the protist trichos is lacking largely due to the difficulties inherent in species collection that have contributed to undersampling and understudy. The most recent classification divides the protist trichos between two families, Amoebidiidae and Eccrinidae (suborder Trichomycina, order Eccrinida). However, there is no comprehensive molecular phylogeny available for this group and major questions about the systematics of protist trichos remain unanswered. Therefore, we generated 18S and 28S rDNA sequences for 106 protist tricho samples and combined them with publicly available Eccrinida sequences for phylogenetic analyses. We also sequenced a conserved protein-coding gene (heat-shock 70 protein) to obtain a multigene data set. We conducted ancestral state reconstruction (ASR) and Bayesian tip-association significance test (BaTS) analyses by mapping six morphological and ecological characters onto the resulting phylogenetic trees. Our results demonstrate: (1) several ecological and morphological character states (habitat, host type, host stage at time of infestation, location within host, spore production, and growth form) are significantly correlated with the phylogeny, and (2) two additional protist tricho families should be incorporated into the taxonomy to reflect phylogenetic relationships. Our data suggest that an integrated strategy that combines morphological, ecological, and molecular characters is needed to further resolve and clarify the systematics of the Eccrinida.

Paratrechina longicornis ants in a tropical dry forest harbor specific Actinobacteria diversity.

The diversity of Actinobacteria associated with Paratrechina longicornis, an ant species that prefers a high protein diet, in a subtropical dry forest (Guánica, Puerto Rico) was determined by culture methods and by 16S rDNA clone libraries. The results of both methodologies were integrated to obtain a broader view of the diversity. Streptomyces, Actinomadura, Nocardia, Ornithinimicrobium, Tsukamurella, Brevibacterium, Saccharopolyspora, Nocardioides, Microbacterium, Leifsonia, Pseudonocardia, Corynebacterium, Geodermatophilus, Amycolatopsis, and Nonomuraea were found associated with the ants. The genera Streptomyces and Actinomadura were the most abundant. Also, the diversity of Actinobacteria associated with the soil surrounding the nest was determined using 16S rDNA clone libraries. In total, 27 genera of Actinobacteria were associated with the nest soils. A dominant genus was not observed in any of the soil samples. We compared statistically the Actinobacteria communities among P. longicornis nests and each nest with its surrounding soil using the clone libraries data. We established that the communities associated with the ants were consistent and significantly different from those found in the soil in which the ants live.

Seasonality and prevalence of the protistan trichomycete Enterobryus halophilus (Ichthyosporea: Eccrinales) in the mole crab Emerita portoricensis.

In Puerto Rico the protist Enterobryus halophilus (Ichthyosporea: Eccrinales) has been reported associated with a new host, Emerita portoricensis (Malacostraca: Hippidae). A study on prevalence and abundance in populations of E. halophilus was conducted during 18 mo to determine whether sex-dependent infection exists in Em. portoricensis, as suggested (1958) for the original description of E. halophilus in Em. talpoida. Individuals (1440) of Em. portoricensis were collected and their guts dissected on microscope slides. The thalli of E. halophilus were counted under a microscope with random grid squares. A factorial ANOVA with interaction revealed that the prevalence and the abundance of E. halophilus do not depend on the sex of its host. Furthermore the data indicate that E. halophilus populations undergo seasonal variations in infestation following the population dynamics of the host.

Specificity in the symbiotic association between fungus-growing ants and protective Pseudonocardia bacteria.

Fungus-growing ants (tribe Attini) engage in a mutualism with a fungus that serves as the ants' primary food source, but successful fungus cultivation is threatened by microfungal parasites (genus Escovopsis). Actinobacteria (genus Pseudonocardia) associate with most of the phylogenetic diversity of fungus-growing ants; are typically maintained on the cuticle of workers; and infection experiments, bioassay challenges and chemical analyses support a role of Pseudonocardia in defence against Escovopsis through antibiotic production. Here we generate a two-gene phylogeny for Pseudonocardia associated with 124 fungus-growing ant colonies, evaluate patterns of ant-Pseudonocardia specificity and test Pseudonocardia antibiotic activity towards Escovopsis. We show that Pseudonocardia associated with fungus-growing ants are not monophyletic: the ants have acquired free-living strains over the evolutionary history of the association. Nevertheless, our analysis reveals a significant pattern of specificity between clades of Pseudonocardia and groups of related fungus-growing ants. Furthermore, antibiotic assays suggest that despite Escovopsis being generally susceptible to inhibition by diverse Actinobacteria, the ant-derived Pseudonocardia inhibit Escovopsis more strongly than they inhibit other fungi, and are better at inhibiting this pathogen than most environmental Pseudonocardia strains tested. Our findings support a model that many fungus-growing ants maintain specialized Pseudonocardia symbionts that help with garden defence.

Dipteran-associated Harpellales from lowland and submontane tropical rain forests of Veracruz (Mexico).

We report on the species of Harpellales found in dipteran hosts during two surveys (32 field d) in the state of Veracruz, Mexico. One new morphospecies, Genistellospora dorsicaudata, is described with particular attention to the position of the terminal cell associated with fully developed fertile thalli bearing sexual spores. We emend the description of G. guanacastensis to include morphometrics on the zygospores, based on discovery of the sexual spores for that species in our collections. Thirteen other previously described species, which are new for Mexico, include G. homothallica, Pennella montana, Simuliomyces microsporus, Smittium aciculare, S. brasiliense (in a new host type), S. culisetae, S. dipterorum, S. microsporum, S. simulii and the unbranched species Harpella melusinae, H. tica, Stachylina grandispora and S. paucispora. Some species have been described but not named, specifically one each of Harpella, Pennella and Smittium. All taxa are identified morphologically, illustrated and additional details on their ecology are provided.

First report of harpellales from the Dominican Republic (Hispaniola) and the insular effect on gut fungi.

We present the first report of Harpellales (Kickxellomycotina) from Hispaniola. The Dominican Republic was surveyed, and members of the ecological group trichomycetes were encountered in the gut of their arthropod hosts. Among Harpellales we describe a new species, Smittium caribense, and report the presence of seven previously known species that are new for Hispaniola: Genistellospora homothallica, Harpella amazonica, Harpella tica, Legerio-m myces rarus, Smittium culisetae, Smittium dipterorum and Stachylina grandispora. The dominance of cosmopolitan, dipteran-associated species is the most outstanding effect resulting from the insularity on gut fungi.

Variation in Pseudonocardia antibiotic defence helps govern parasite-induced morbidity in Acromyrmex leaf-cutting ants.

Host-parasite associations are potentially shaped by evolutionary reciprocal selection dynamics, in which parasites evolve to overcome host defences and hosts are selected to counteract these through the evolution of new defences. This is expected to result in variation in parasite-defence interactions, and the evolution of resistant parasites causing increased virulence. Fungus-growing ants maintain antibiotic-producing Pseudonocardia (Actinobacteria) that aid in protection against specialized parasites of the ants' fungal gardens, and current evidence indicates that both symbionts have been associated with the ants for millions of years. Here we examine the extent of variation in the defensive capabilities of the ant-actinobacterial association against Escovopsis (parasite-defence interactions), and evaluate how variation impacts colonies of fungus-growing ants. We focus on five species of Acromyrmex leaf-cutting ants, crossing 12 strains of Pseudonocardia with 12 strains of Escovopsis in a Petri plate bioassay experiment, and subsequently conduct subcolony infection experiments using resistant and non-resistant parasite strains. Diversity in parasite-defence interactions, including pairings where the parasites are resistant, suggests that chemical variation in the antibiotics produced by different actinobacterial strains are responsible for the observed variation in parasite susceptibility. By evaluating the role this variation plays during infection, we show that infection of ant subcolonies with resistant parasite strains results in significantly higher parasite-induced morbidity with respect to garden biomass loss. Our findings thus further establish the role of Pseudonocardia-derived antibiotics in helping defend the ants' fungus garden from the parasite Escovopsis, and provide evidence that small molecules can play important roles as antibiotics in a natural system.

First survey of arthropod gut fungi and associates from Vieques, Puerto Rico.

This is the first survey of trichomycetes for Vieques, Puerto Rico. Several types of arthropods, including millipedes, crabs, mosquitoes, bloodworms, amphipods and isopods, were collected from marine, freshwater and terrestrial habitats and their guts examined for the presence of trichomycetes. A new species of Enterobryus, which was found attached to the gut of the millipede Anadenobolus arboreus (Diplopoda), is described as Enterobryus viequensis sp. nov. We report the presence of Smittium culisetae in mosquito larvae and also provide information on two eccrinids associated with the fiddler crabs Uca rapax and U. burgersi and an amphipod.

First report of zygospores in Asellariales and new species from the Caribbean.

We report the inedited occurrence of zygospores in symbiotic Asellariales (Zygomycota) from Puerto Rico and Dominican Republic. Two new species of Asellaria are described, Asellaria dactylopus and A. jatibonicua, both found in the hindgut of terrestrial isopods. The presence of spherical zygospores in the latter species represents a novelty for the order. The related gut-fungi Harpellales were known to produce sexual spores, characteristically conical or biconical. Order Asellariales is emended to include the description of zygospores. An undetermined species of Orchesellaria also is reported from the Caribbean. Comments are provided for each of the species as well as drawings and photographs to illustrate each taxon.

Harpellales in the digestive tracts of Ephemeroptera and Plecoptera nymphs from Veracruz, Mexico.

This is the first report of Harpellales (Zygomycota) from Mexico, including herein only the endosymbiotic species of gut fungi in the digestive tracts or shed exuviae of Plecopteran and Ephemeropteran nymphs. Four new species are described: Allantomyces zopilotei, Bojamyces olmecensis, Gauthier-omyces viviparus and Graminella ophiuroidea. Among previously known Harpellales, Lancisporomyces nemouridarum and Zygopolaris ephemeridarum are southern range extremes and new records for Mexico. All species are illustrated and discussed relating to biogeographic implications of the new reports from Mexico, as well as the particular environmental circumstances of the Harpellales in the tropics.

Low host-pathogen specificity in the leaf-cutting ant-microbe symbiosis.

Host-parasite associations are shaped by coevolutionary dynamics. One example is the complex fungus-growing ant-microbe symbiosis, which includes ancient host-parasite coevolution. Fungus-growing ants and the fungi they cultivate for food have an antagonistic symbiosis with Escovopsis, a specialized microfungus that infects the ants' fungus gardens. The evolutionary histories of the ant, cultivar and Escovopsis are highly congruent at the deepest phylogenetic levels, with specific parasite lineages exclusively associating with corresponding groups of ants and cultivar. Here, we examine host-parasite specificity at finer phylogenetic levels, within the most derived clade of fungus-growing ants, the leaf-cutters (Atta spp. and Acromyrmex spp.). Our molecular phylogeny of Escovopsis isolates from the leaf-cutter ant-microbe symbiosis confirms specificity at the broad phylogenetic level, but reveals frequent host-switching events between species and genera of leaf-cutter ants. Escovopsis strains isolated from Acromyrmex and Atta gardens occur together in the same clades, and very closely related strains can even infect the gardens of both ant genera. Experimental evidence supports low host-parasite specificity, with phylogenetically diverse strains of Escovopsis being capable of overgrowing all leaf-cutter cultivars examined. Thus, our findings indicate that this host-pathogen association is shaped by the farming ants having to protect their cultivated fungus from phylogenetically diverse Escovopsis garden pathogens.

Phylogeny of the Zygomycota based on nuclear ribosomal sequence data.

The Zygomycota is an ecologically heterogenous assemblage of nonzoosporic fungi comprising two classes, Zygomycetes and Trichomycetes. Phylogenetic analyses have suggested that the phylum is polyphyletic; two of four orders of Trichomycetes are related to the Mesomycetozoa (protists) that diverged near the fungal/animal split. Current circumscription of the Zygomycota includes only orders with representatives that produce zygospores. We present a molecular-based phylogeny including recognized representatives of the Zygomycetes and Trichomycetes with a combined dataset for nuclear rRNA 18S (SSU), 5.8S and 28S (LSU) genes. Tree reconstruction by Bayesian analyses suggests the Zygomycota is paraphyletic. Although 12 clades were identified only some of these correspond to the nine orders of Zygomycota currently recognized. A large superordinal clade, comprising the Dimargaritales, Harpellales, Kickxellales and Zoopagales, grouping together many symbiotic fungi, also is identified in part by a unique septal structure. Although Harpellales and Kickxellales are not monophyletic, these lineages are distinct from the Mucorales, Endogonales and Mortierellales, which appear more closely related to the Ascomycota + Basidiomycota + Glomeromycota. The final major group, the insect-associated Entomophthorales, appears to be polyphyletic. In the present analyses Basidiobolus and Neozygites group within Zygomycota but not with the Entomophthorales. Clades are discussed with special reference to traditional classifications, mapping morphological characters and ecology, where possible, as a snapshot of our current phylogenetic perspective of the Zygomycota.

Phylogenetic analysis of mutualistic filamentous bacteria associated with fungus-growing ants.

The attine ant-microbe system is a quadripartite symbiosis, involving a complex set of mutualistic and parasitic associations. The symbiosis includes the fungus-growing ants (tribe Attini), the basidiomycetous fungi the ants cultivate for food, specialized microfungal parasites (in the genus Escovopsis) of the cultivar, and ant-associated mu tualistic filamentous bacteria that secrete antibiotics specifically targeted to suppress the growth of Escovopsis. In this study, we conduct the first phylogenetic analysis of the filamentous mutualistic bacteria (actinomycetes) associated with fungus-growing ants. The filamentous bacteria present on 3 genera of fungus-growing ants (Acromyrmex, Trachy myrmex, and Apterostigma) were isolated from 126 colonies. The isolated actinomycetes were grouped into 3 distinct morphological types. Each morphological type was specific to the ant genus from which it was isolated, suggesting some degree of host specificity. The phylogenetic position of the 3 morphotypes was estimated using 16S rDNA for representative strains. The 8 isolates of actinomycetes sequenced are in the family Pseudonocardiaceae (Actino mycetales) and belong to the genus Pseudonocardia. Transmission electron microscopy examination of the actino mycete associated with the cuticle of Acromyrmex sp. revealed bacterial cells with an outer electron-dense membrane, consistent with actinomycetes in the genus Pseudonocardia. Ant-associated Pseudonocardia isolates did not form a monophyletic group, suggesting multiple acquisitions of actinomycetes by fungus-growing ants over their evolutionary history.

Eccrinales (Trichomycetes) are not fungi, but a clade of protists at the early divergence of animals and fungi.

The morphologically diverse orders Eccrinales and Amoebidiales have been considered members of the fungal class Trichomycetes (Zygomycota) for the last 50 years. These organisms either inhabit the gut or are ectocommensals on the exoskeleton of a wide range of arthropods--Crustacea, Insecta, and Diplopoda--in varied habitats. The taxonomy of both orders is based on a few micromorphological characters. One species, Amoebidium parasiticum, has been axenically cultured and this has permitted several biochemical and phylogenetic analyses. As a consequence, the order Amoebidiales has been removed from the Trichomycetes and placed in the class Mesomycetozoea. An affinity between Eccrinales and Amoebidiales was first suggested when the class Trichomycetes was erected by Duboscq et al. [Arch. Zool. Exp. Gen. 86 (1948) 29]. Subsequently, molecular markers have been developed to study the relationship of these orders to other groups. Ribosomal gene (18S and 28S) sequence analyses generated by this study do not support a close association of these orders to the Trichomycetes or to other fungi. Rather, Eccrinales share a common ancestry with the Amoebidiales and belong to the protist class Mesomycetozoea, placed at the animal-fungi boundary.