Environmental DNA sequencing primers for eutardigrades and bdelloid rotifers

Abstract

Background: The time it takes to isolate individuals from environmental samples and then extract DNA from each individual is one of the problems with generating molecular data from meiofauna such as eutardigrades and bdelloid rotifers. The lack of consistent morphological information and the extreme abundance of these classes makes morphological identification of rare, or even common cryptic taxa a large and unwieldy task. This limits the ability to perform large-scale surveys of the diversity of these organisms.Here we demonstrate a culture-independent molecular survey approach that enables the generation of large amounts of eutardigrade and bdelloid rotifer sequence data directly from soil. Our PCR primers, specific to the 18s small-subunit rRNA gene, were developed for both eutardigrades and bdelloid rotifers.

Results: The developed primers successfully amplified DNA of their target organism from various soil DNA extracts. This was confirmed by both the BLAST similarity searches and phylogenetic analyses. Tardigrades showed much better phylogenetic resolution than bdelloids. Both groups of organisms exhibited varying levels of endemism.

Conclusion: The development of clade-specific primers for characterizing eutardigrades and bdelloid rotifers from environmental samples should greatly increase our ability to characterize the composition of these taxa in environmental samples. Environmental sequencing as shown here differs from other molecular survey methods in that there is no need to pre-isolate the organisms of interest from soil in order to amplify their DNA. The DNA sequences obtained from methods that do not require culturing can be identified post-hoc and placed phylogenetically as additional closely related sequences are obtained from morphologically identified conspecifics. Our non-cultured environmental sequence based approach will be able to provide a rapid and large-scale screening of the presence, absence and diversity of Bdelloidea and Eutardigrada in a variety of soils.

Figure 1

Gel image of PCR results for eutardigrade specific 18s rDNA primers. First four lanes are from replicate individuals from a single population eutardigrades. Lanes five through 7 are from heterotardigrades. Lanes eight through ten are nematodes.

Figure 2

Gel image of PCR results for bdelloid specific 18s rDNA primers. Lane 1 is the Hyper ladder 1 from Bioline USA Inc. MA, Brachionus plicatilis is the Monogonont in lane 2. Lanes three through six are from individual representatives of the following bdelloid rotifers: Philodina, Adineta, Macrotrachela, and Habrotrocha. The final two lanes are from unidentified bdelloids taken from a pond.

Figure 3

Cladogram representations of phylogenetic trees obtained from TNT [44] and MrBayes [45] on tardigrades. Bootstrap values below 50 and posterior probability values below 70 are not represented. All environmental sequences fall within the Eutardigrada.

Figure 4

Cladogram representations of phylogenetic trees obtained from TNT [44] and MrBayes [45] on bdelloid rotifers. Bootstrap values below 50 and posterior probability values below 70 are not represented. All environmental sequences fall within the bdelloidea.