The characterization of enzymatically amplified eukaryotic 16S-like rRNA-coding regions

Medlin, Linda K.; Elwood, Hille J.; Stickel, Shawn K.; Sogin, Mitchell L.

doi:10.1016/0378-1119(88)90066-2

Cited by 2,677 publications

(1,642 citation statements)

References 22 publications

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“…82 ) and Euk-B (ref. 83 ). PCR products were cleaned via a MinElute PCR Purification Kit from Qiagen and ligated to a pGEM®-T vector (Promega) and cloned.…”

Section: Statistical Analysesmentioning

confidence: 99%

Parasites dominate hyperdiverse soil protist communities in Neotropical rainforests

et al. 2017

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High animal and plant richness in tropical rainforest communities has long intrigued naturalists. It is unknown if similar hyperdiversity patterns are reflected at the microbial scale with unicellular eukaryotes (protists). Here we show, using environmental metabarcoding of soil samples and a phylogeny-aware cleaning step, that protist communities in Neotropical rainforests are hyperdiverse and dominated by the parasitic Apicomplexa, which infect arthropods and other animals. These host-specific parasites potentially contribute to the high animal diversity in the forests by reducing population growth in a density-dependent manner. By contrast, too few operational taxonomic units (OTUs) of Oomycota were found to broadly drive high tropical tree diversity in a host-specific manner under the Janzen-Connell model. Extremely high OTU diversity and high heterogeneity between samples within the same forests suggest that protists, not arthropods, are the most diverse eukaryotes in tropical rainforests. Our data show that protists play a large role in tropical terrestrial ecosystems long viewed as being dominated by macroorganisms.S ince the works of early naturalists such as von Humboldt and Bonpland 1 , we have known that animal and plant communities in tropical rainforests are exceedingly species rich. For example, one hectare can contain more than 400 tree species 2 and one tree can harbour more than 40 ant species 3 . This hyperdiversity of trees has been partially explained by the Janzen-Connell model 4,5 , which hypothesizes that host-specific predators and parasites reduce plant population growth in a density-dependent manner 6,7 . Sampling up in the tree canopies and below on the ground has further led to the view that arthropods are the most diverse eukaryotes in tropical rainforests 8,9 .The focus on eukaryotic macroorganisms in these studies is primarily because they are familiar and readily observable to us. We do not know whether the less familiar and less readily observable protists-microbial eukaryotes that are not animals, plants or fungi 10 -inhabiting these same ecosystems exhibit similar diversity patterns. To evaluate if macroorganismic diversity patterns are reflected at the microbial scale with protists, we conducted an environmental DNA metabarcoding study by sampling soils in 279 locations in a variety of lowland Neotropical forest types in La Selva Biological Station, Costa Rica, Barro Colorado Island, Panama and Tiputini Biodiversity Station, Ecuador. This metabarcoding approach has the power to uncover known and new taxa on a massive scale 11 . By amplifying DNA extracted from the soils with broadly targeted primers for the V4 region of 18S rRNA and sequencing it using the Illumina MiSeq platform, we were able to detect most eukaryotic lineages, and assess the diversity and relative dominance of free-living and parasitic lineages.

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“…82 ) and Euk-B (ref. 83 ). PCR products were cleaned via a MinElute PCR Purification Kit from Qiagen and ligated to a pGEM®-T vector (Promega) and cloned.…”

Section: Statistical Analysesmentioning

confidence: 99%

Parasites dominate hyperdiverse soil protist communities in Neotropical rainforests

et al. 2017

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“…Partial sequences of the rRNA genes LSU and SSU and complete 5.8s/ITS genes were amplified using previously published primers: SS3, SS5 (Medlin et al, 1988), D1R, D3b (Scholin et al, 1994), and ITSfor, ITSrev (Murray et al, 2012a,b). For the amplification of sxtA genes, sxtA1 and sxtA4 domains were amplified using the primers sxt001, sxt002 (A1) and sxt007 and sxt008 (A4) .…”

Section: Culture Maintenancementioning

confidence: 99%

Gene duplication, loss and selection in the evolution of saxitoxin biosynthesis in alveolates

Murray

Diwan

Orr

et al. 2015

Molecular Phylogenetics and Evolution

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a b s t r a c tA group of marine dinoflagellates (Alveolata, Eukaryota), consisting of $10 species of the genus Alexandrium, Gymnodinium catenatum and Pyrodinium bahamense, produce the toxin saxitoxin and its analogues (STX), which can accumulate in shellfish, leading to ecosystem and human health impacts. The genes, sxt, putatively involved in STX biosynthesis, have recently been identified, however, the evolution of these genes within dinoflagellates is not clear. There are two reasons for this: uncertainty over the phylogeny of dinoflagellates; and that the sxt genes of many species of Alexandrium and other dinoflagellate genera are not known. Here, we determined the phylogeny of STX-producing and other dinoflagellates based on a concatenated eight-gene alignment. We determined the presence, diversity and phylogeny of sxtA, domains A1 and A4 and sxtG in 52 strains of Alexandrium, and a further 43 species of dinoflagellates and thirteen other alveolates. We confirmed the presence and high sequence conservation of sxtA, domain A4, in 40 strains (35 Alexandrium, 1 Pyrodinium, 4 Gymnodinium) of 8 species of STX-producing dinoflagellates, and absence from non-producing species. We found three paralogs of sxtA, domain A1, and a widespread distribution of sxtA1 in non-STX producing dinoflagellates, indicating duplication events in the evolution of this gene. One paralog, clade 2, of sxtA1 may be particularly related to STX biosynthesis. Similarly, sxtG appears to be generally restricted to STX-producing species, while three amidinotransferase gene paralogs were found in dinoflagellates. We investigated the role of positive (diversifying) selection following duplication in sxtA1 and sxtG, and found negative selection in clades of sxtG and sxtA1, clade 2, suggesting they were functionally constrained. Significant episodic diversifying selection was found in some strains in clade 3 of sxtA1, a clade that may not be involved in STX biosynthesis, indicating pressure for diversification of function.

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“…DNA was extracted and purified using cetyltrimethylammonium bromide (CTAB) follOWing the protocol of Wright et al (1997). Primer A and Primer B above (Medlin et al 1988) were used in a PCR amplification using a PTC-100 thermal cycler (MJ Research Inc., Watertown, MA). The PCR product was excised under longwave ultraviolet light « 60 s exposure) and purified using the GeneClean kit (Qbiogen).…”

Section: Dna Extraction and Amplification: (A)mentioning

confidence: 99%

Molecular Phylogeny of Litostome Ciliates (Ciliophora, Litostomatea) with Emphasis on Free-Living Haptorian Genera

Strüder‐Kypke

Wright

Foissner

et al. 2006

Protist

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The characterization of enzymatically amplified eukaryotic 16S-like rRNA-coding regions

Cited by 2,677 publications

References 22 publications

Parasites dominate hyperdiverse soil protist communities in Neotropical rainforests

Parasites dominate hyperdiverse soil protist communities in Neotropical rainforests

Gene duplication, loss and selection in the evolution of saxitoxin biosynthesis in alveolates

Molecular Phylogeny of Litostome Ciliates (Ciliophora, Litostomatea) with Emphasis on Free-Living Haptorian Genera

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