Bermuda Land Snails make up a genus called Poecilozonites that is endemic to Bermuda and is extensively present in its fossil record. These snails were also integral to the creation of the theory of punctuated equilibrium. The DNA of mollusks is difficult to sequence because of a class of proteins called mucopolysaccharides that are present in high concentrations in mollusk tissue, and are not removed with standard DNA extraction methods. They inhibit Polymerase Chain Reactions (PCRs) and interfere with Next Generation Sequencing methods. This paper will discuss the DNA extraction methods that were designed to remove the inhibitory proteins that were tested on another gastropod species (Pomacea canaliculata). These were chosen because they are invasive and while they are not pulmonates, they are similar enough to Bermuda Land Snails to reliably test extraction methods. The methods that were tested included two commercially available kits: the Qiagen Blood and Tissue Kit and the Omega Biotek Mollusc Extraction Kit, and one Hexadecyltrimethylammonium Bromide (CTAB) Extraction method that was modified for use on mollusk tissue. The Blood and Tissue kit produced some DNA, the mollusk kit produced almost none, and the CTAB Extraction Method produced the highest concentrations on average, and may prove to be the most viable option for future extractions. PCRs attempted with the extracted DNA have all failed, though it is likely due to an issue with reagents. Further spectrographic analysis of the DNA from the test extractions has shown that they were successful at removing mucopolysaccharides. When the protocol is optimized, it will be used to extract DNA from the tissue from six individuals from each of the two extant species of Bermuda Land Snails. This DNA will be used in several experiments involving Next Generation Sequencing, with the goal of assembling a variety of genome data. These data will then be used to a construct reference genome for Bermuda Land Snails. The genomes generated by this project will be used in population genetic analyses between individuals of the same species, and between individuals of different species. These analyses will then be used to aid in conservation efforts for the species.
Improvements in sequencing technology now allow easy acquisition of large datasets; however, analyzing these data for phylogenetics can be challenging. We have developed a novel method to rapidly obtain homologous genomic data for phylogenetics directly from next-generation sequencing reads without the use of a reference genome. This software, called SISRS, avoids the time consuming steps of de novo whole genome assembly, multiple genome alignment, and annotation.
Results
For simulations SISRS is able to identify large numbers of loci containing variable sites with phylogenetic signal. For genomic data from apes, SISRS identified thousands of variable sites, from which we produced an accurate phylogeny. Finally, we used SISRS to identify phylogenetic markers that we used to estimate the phylogeny of placental mammals. We recovered eight phylogenies that resolved the basal relationships among mammals using datasets with different levels of missing data. The three alternate resolutions of the basal relationships are consistent with the major hypotheses for the relationships among mammals, all of which have been supported previously by different molecular datasets.
Conclusions
SISRS has the potential to transform phylogenetic research. This method eliminates the need for expensive marker development in many studies by using whole genome shotgun sequence data directly. SISRS is open source and freely available at https://github.com/rachelss/SISRS/releases.
Nutrient recycling by fish can be an important part of nutrient cycles in both freshwater and marine ecosystems. As a result, understanding the mechanisms that influence excretion elemental ratios of fish is of great importance to a complete understanding of aquatic nutrient cycles. As fish consume a wide range of diets that differ in elemental composition, stoichiometric theory can inform predictions about dietary effects on excretion ratios.
We conducted a meta-analysis to test the effects of diet elemental composition on consumption and nutrient excretion by fish. We examined the relationship between consumption rate and diet N : P across all laboratory studies and calculated effect sizes for each excretion metric to test for significant effects.
Consumption rate of N, but not P, was significantly negatively affected by diet N : P. Effect sizes of diet elemental composition on consumption-specific excretion N, P and N : P in laboratory studies were all significantly different from 0, but effect size for raw excretion N : P was not significantly different from zero in laboratory or field surveys.
Our results highlight the importance of having a mechanistic understanding of the drivers of consumer excretion rates and ratios. We suggest that more research is needed on how consumption and assimilation efficiency vary with N : P and in natural ecosystems in order to further understand mechanistic processes in consumer-driven nutrient recycling.