Jones, F. A. | bioRxiv Science

bioRxiv2021

A new oomycete metabarcoding method using the rps10 gene

Oomycetes are a group of eukaryotes related to brown algae and diatoms, many of which cause diseases in plants and animals. Improved methods are needed for rapid and accurate characterization of oomycete communities using DNA metabarcoding. We have identified the mitochondrial 40S ribosomal protein S10 gene (rps10) as a locus useful for oomycete metabarcoding and provide primers predicted to amplify all oomycetes based on available reference sequences from a wide range of taxa. We evaluated its utility relative to a popular barcode, the internal transcribed spacer 1 (ITS1), by sequencing environmental samples and a mock community using Illumina MiSeq. Amplified sequence variants (ASVs) and operational taxonomic units (OTUs) were identified per community. Both the sequence and predicted taxonomy of ASVs and OTUs were compared to the known composition of the mock community. Both rps10 and ITS yielded ASVs with sequences matching 21 of the 24 species in the mock community and matching all 24 when allowing for a 1 bp difference. Taxonomic classifications of ASVs included 23 members of the mock community for rps10 and 17 for ITS1. Sequencing results for the environmental samples suggest the proposed rps10 locus results in substantially less amplification of non-target organisms than the ITS1 method. The amplified rps10 region also has higher taxonomic resolution than ITS1, allowing for greater discrimination of closely related species. We present a new website with a searchable rps10 reference database for species identification and all protocols needed for oomycete metabarcoding. The rps10 barcode and methods described herein provide an effective tool for metabarcoding oomycetes using short-read sequencing. Interpretive summaryOomycetes are a group of eukaryotes related to brown algae and diatoms, many of which cause diseases in plants and animals. Improved methods are needed to rapidly characterize the diversity of oomycete species found in environmental samples. We have identified the mitochondrial 40S ribosomal protein S10 gene (rps10) as being useful for oomycete community sequencing. We evaluated its utility relative to a popular barcode, the internal transcribed spacer 1 (ITS1), by sequencing environmental samples and a community we synthesized in the laboratory. The amplified rps10 region is predicted to have a higher taxonomic resolution than ITS1, allowing for greater discrimination of closely related species. We present a new website with a searchable rps10 reference database for species identification and all protocols needed for oomycete community sequencing. The rps10 barcode and methods described herein provide an effective tool for characterizing oomycetes using environmental DNA sequencing.

microbiology↗

bioRxiv2021

Testing for pollinator recognition in multiple species of Heliconia

Many plants have evolved floral traits that, in effect, filter pollinator communities and promote pollination by efficient pollinators. Most documented pollinator filter traits act to deter or reduce visitation rates by a subsect of the community of floral visitors. However, a recently described pollinator filter termed pollinator recognition (PR) acts at a stage after a pollinator visit. PR was first documented experimentally in Heliconia tortuosa whereby pollen tube germination - a proxy for reproduction - was enhanced following visits from morphologically specialized pollinators, but not generalists. This is thought to promote outcrossing among plants by preferentially investing in reproduction following visits by long-ranging hummingbirds with specialized bill shapes. To date, this plant behavior has only been described in H. tortuosa, but, if widespread, could have important ecological implications; given declines in abundances of specialist pollinators, visits by generalists would not buffer the loss of pollination services to plants with PR. We therefore tested for PR in four taxa spread widely across the Heliconiaceae. We corroborated previous results that visits by long-billed, but not short-billed hummingbirds increased pollen tubes in H. tortuosa with aviary experiments that standardized pollen quality and minimized variation in pollen quantity. Across species, we found great variation in pollen tube responses to experimental treatments. For one species (H. rostrata), we found increased numbers of pollen tubes in those visited by hummingbirds compared to hand pollination alone, regardless of the visiting birds bill morphology, indicating recognition of hummingbirds in general. In other cases, hummingbird visits decreased pollen tube counts compared to hand pollinations alone. Furthermore, our results could not substantiate any specific mechanism for pollinator recognition and highlight the need for further work on the complexities of and variability in reproductive strategies across plant taxa.

ecology↗