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The role of internal transcribed spacer 2 secondary structures in classifying mycoparasitic Ampelomyces.

ABSTRACT: Many fungi require specific growth conditions before they can be identified. Direct environmental DNA sequencing is advantageous, although for some taxa, specific primers need to be used for successful amplification of molecular markers. The internal transcribed spacer region is the preferred DNA barcode for fungi. However, inter- and intra-specific distances in ITS sequences highly vary among some fungal groups; consequently, it is not a solely reliable tool for species delineation. Ampelomyces, mycoparasites of the fungal phytopathogen order Erysiphales, can have ITS genetic differences up to 15%; this may lead to misidentification with other closely related unknown fungi. Indeed, Ampelomyces were initially misidentified as other pycnidial mycoparasites, but subsequent research showed that they differ in pycnidia morphology and culture characteristics. We investigated whether the ITS2 nucleotide content and secondary structure was different between Ampelomyces ITS2 sequences and those unrelated to this genus. To this end, we retrieved all ITS sequences referred to as Ampelomyces from the GenBank database. This analysis revealed that fungal ITS environmental DNA sequences are still being deposited in the database under the name Ampelomyces, but they do not belong to this genus. We also detected variations in the conserved hybridization model of the ITS2 proximal 5.8S and 28S stem from two Ampelomyces strains. Moreover, we suggested for the first time that pseudogenes form in the ITS region of this mycoparasite. A phylogenetic analysis based on ITS2 sequences-structures grouped the environmental sequences of putative Ampelomyces into a different clade from the Ampelomyces-containing clades. Indeed, when conducting ITS2 analysis, resolution of genetic distances between Ampelomyces and those putative Ampelomyces improved. Each clade represented a distinct consensus ITS2 S2, which suggested that different pre-ribosomal RNA (pre-rRNA) processes occur across different lineages. This study recommends the use of ITS2 S2s as an important tool to analyse environmental sequencing and unveiling the underlying evolutionary processes.

SUBMITTER: Prahl RE

PROVIDER: S-EPMC8244850 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundMost phylogenetic studies using current methods have focused on primary DNA sequence information. However, RNA secondary structures are particularly useful in systematics because they include characteristics, not found in the primary sequence, that give "morphological" information. Despite the number of recent molecular studies on octocorals, there is no consensus opinion about a region that carries enough phylogenetic resolution to solve intrageneric or close species relationships. Moreover, intrageneric morphological information by itself does not always produce accurate phylogenies; intra-species comparisons can reveal greater differences than intra-generic ones. The search for new phylogenetic approaches, such as by RNA secondary structure analysis, is therefore a priority in octocoral research.ResultsInitially, twelve predicted RNA secondary structures were reconstructed to provide the basic information for phylogenetic analyses; they accorded with the 6 helicoidal ring model, also present in other groups of corals and eukaryotes. We obtained three similar topologies for nine species of the Caribbean gorgonian genus Eunicea (candelabrum corals) with two sister taxa as outgroups (genera Plexaura and Pseudoplexaura) on the basis of molecular morphometrics of ITS2 RNA secondary structures only, traditional primary sequence analyses and maximum likelihood, and a Bayesian analysis of the combined data. The latter approach allowed us to include both primary sequence and RNA molecular morphometrics; each data partition was allowed to have a different evolution rate. In addition, each helix was partitioned as if it had evolved at a distinct rate. Plexaura flexuosa was found to group within Eunicea; this was best supported by both the molecular morphometrics and combined analyses. We suggest Eunicea flexuosa (Lamouroux, 1821) comb. nov., and we present a new species description including Scanning Electron Microscopy (SEM) images of morphological characteristics (sclerites). Eunicea flexuosa, E. pallida, E. laxispica and E. mammosa formed a separate clade in the molecular phylogenies, and were reciprocally monophyletic with respect to other Eunicea (Euniceopsis subgenus, e.g. E. tourneforti and E. laciniata) in the molecular morphometrics tree, with the exception of E. fusca. Moreover, we suggest a new diagnostic character for Eunicea, also present in E. flexuosa: middle layer sclerites > 1 mm in length.ConclusionITS2 was a reliable sequence for intrageneric studies in gorgonian octocorals because of the amount of phylogenetic signal, and was corroborated against morphological characters separating Eunicea from Plexaura. The ITS2 RNA secondary structure approach to phylogeny presented here did not rely on alignment methods such as INDELS, but provided clearly homologous characters for partition analysis and RNA molecular morphometrics. These approaches support the divergence of Eunicea flexuosa comb. nov. from the outgroup Plexaura, although it has been considered part of this outgroup for nearly two centuries because of morphological resemblance.

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The role of internal transcribed spacer 2 secondary structures in classifying mycoparasitic Ampelomyces.

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