Genetic barcoding of dark‐spored myxomycetes (Amoebozoa)—Identification, evaluation and application of a sequence similarity threshold for species differentiation in NGS studies

Abstract

Unicellular, eukaryotic organisms (protists) play a key role in soil food webs as major predators of microorganisms. However, due to the polyphyletic nature of protists, no single universal barcode can be established for this group, and the structure of many protistean communities remains unresolved. Plasmodial slime moulds (Myxogastria or Myxomycetes) stand out among protists by their formation of fruit bodies, which allow for a morphological species concept. By Sanger sequencing of a large collection of morphospecies, this study presents the largest database to date of dark-spored myxomycetes and evaluate a partial 18S SSU gene marker for species annotation. We identify and discuss the use of an intraspecific sequence similarity threshold of 99.1% for species differentiation (OTU picking) in environmental PCR studies (ePCR) and estimate a hidden diversity of putative species, exceeding those of described morphospecies by 99%. When applying the identified threshold to an ePCR data set (including sequences from both NGS and cloning), we find 64 OTUs of which 21.9% had a direct match (>99.1% similarity) to the database and the remaining had on average 90.2 ± 0.8% similarity to their best match, thus thought to represent undiscovered diversity of dark-spored myxomycetes.

A four year survey reveals a coherent pattern between occurrence of fruit bodies and soil amoebae populations for nivicolous myxomycetes

“…Three specimens belonged to the bright-spored genus Trichia ( T. alpina and T. sordida ) and were thus excluded from the comparison with soil OTUs (Operational Taxonomic Unites), as the primers used to target myxamoebae do not amplify these. A total of 537 specimens (75.3% of all determinable records) were sequenced to allow a direct comparison of ribotypes used as species barcodes 20 between fruit bodies (collections) and amoebae (recovered by ePCR of soil). All but four sequences clustered within the clade forming the respective morphospecies – at this level virtually no contradictions were found between the morphospecies determination and barcoding results (Supplementary S2 ).…”

“… Nivicolous morphospecies 25 28 (+2) b 24 n.a. Expected (Chao richness estimator c ) OTUs/Ribotype clusters 208.8 55.1 Morphospecies 34.0 30.3 a Morphospecies for soil samples were assigned from a data base of sequences from fruit bodies 20 , for collections according to morphological determination. b Two morphospecies belong to the bright-spored clade of myxomycetes and cannot be compared with results from ePCR.…”

“… a Morphospecies for soil samples were assigned from a data base of sequences from fruit bodies 20 , for collections according to morphological determination. b Two morphospecies belong to the bright-spored clade of myxomycetes and cannot be compared with results from ePCR.…”

“…a Morphospecies for soil samples were assigned from a data base of sequences from fruit bodies 20 , for collections according to morphological determination.…”

“…Schematic drawings indicate fruit body shapes drawn to relative size, color the degree of peridial calcification, from white = heavily calcified to black = not calcified. Soil OTUs with no match in our database were aligned against uncultured sequences available from 1) a study of soil from three lowland grasslands in Germany 36 (denoted ‘Grass DE’) and 2) a study of soil from the northern Caucasus mountains, Russia 20 (denoted ‘CAU RU’). Monophyletic branches of the same morphospecies are collapsed for improved graphic presentation.…”