Research Projects

Cohort 1 project – taxonomic resilience in a nutrient loaded microbial community

Fine scale functional differences belie taxonomic resilience in a nutrient loaded microbial community

Microbial communities are comprised of a diverse assemblage of taxonomic groups and metabolic functions that carry out important ecosystem processes such as nutrient cycling. The environmental and ecological processes that shape microbial community structure have been of long-standing interest to microbial ecologists.

One context in which the function of microbial communities has important environmental implications is in estuaries, where microbial communities act as an intermediate between human nutrient and pollution runoff and marine systems  This nutrient runoff can have profound implications for fisheries, water quality, and human health (Belkin & Colwell 2006). Thus, the way in which microbial communities respond to nutrient loading has important consequences for better understanding human impacts on ecosystem function and nutrient cycling.

We chose two creeks located in Ipswich, MA, USA for this study, Greenwood and Egypt creek, hereafter referred to as the polluted and reference creeks. Both creeks drain into Plum Island sound and experience tidal fluxes in salinity. The two creeks were located within 5 km of one another, were surrounded by similar salt marsh plant communities and had similar salinity gradients .The Ipswich creek received input from a sewage effluent located near the head of the creek and the reference creek received input water from a local reservoir. We collected metagenomic data from multiple sites along the two creeks and compared our results with studies of microbial community structures based on 16S rDNA amplicon sequencing, in which a single taxonomically informative locus is used to identify the taxonomic composition of a community (Sogin et al. 2006).

Our study identified a clear increase in the abundance of genes involved in the nitrogen cycle in response to nutrient addition from a sewage effluent. In contrast to neutral models, which predict random community assemblage, these results provide clear support for a role of the functional niche that microbes occupy in determining their abundance in sediments. Further, while our work and that of previous studies (Bowen et al. 2011) indicate that taxonomic composition is relatively unchanged under nutrient addition in salt marsh communities, our metagenomic analyses identified a clear difference in the abundance of functional genes related to nutrient metabolism. This discrepancy appears to be due to the fact that similar metabolic functions are often spread among broad taxonomic ranges.

Comparisons of microbial communities among similar environmental samples using 16S amplicon sequencing and functional metagenomics have been met with mixed results, lending support for both neutral and functional responses in communities. Our comparison of microbial communities in salt marsh sediments along a polluted and unpolluted creek reveal a clear role for both processes, with strong functional differences in the abundance of metabolic genes related to nutrient cycling that are spread randomly across a range of functionally redundant taxa.

Cohort 2 – Spartina salt marsh grass and growth form in microhabitats vs. latitudinal gradients

Spartina salt marsh grass and growth form in microhabitats vs. latitudinal gradients

Salt marshes fringe the land-water interface across the Atlantic and Gulf coasts of North America, supporting biologically productive, estuarine ecosystems. The perennial cordgrass Spartina alterniflora, dominates the intertidal of these marshes, creating both habitat and marsh sediment structure. However, human development and global climate change threaten the vitality and productivity of these critical coastal ecosystems.

S. alterniflora exhibits two distinct growth forms across its latitudinal range; a short form (10-40 cm) and a tall form (1-3 m). There is much debate and uncertainty surrounding the environmental and or genetic underpinnings of the two observed growth forms (Smart 1986, Gallagher et. al 1988, Freshwater 1988, Proffitt et. al 2003). Are the two phenotypes of S. alterniflora a plastic response to environmental variables or are the observed differences genetic? Furthermore, if there are genetic differences between the two morphotypes, are they consistent across S. alterniflora’s geographic range?

In this study we aim to resolve the morphotype variation in S. alterniflora alongits latitudinal cline, from Georgia to Massachusetts. Using a double digest RAD-tag sequencing approach (Peterson et. al 2012), we plan to genotype many individuals (tall- and short-form at each of the 7 sites) producing a range of markers (SNPs) across homologous sites. This approach allows us to adequately and efficiently characterize underlying genomic differences among populations. These differences can help inform our knowledge of how populations will respond to new conditions imminent in a changing climate.

Leaf tissue samples (20cm) of 20 individuals of both tall and short Spartina alterniflora were collected from seven sites along the US east coast for later genetic analysis. Sites were selected along the latitudinal gradient to encompass topographic, climatic and marsh structural diversity. We will look at differences in SNPs across a genome, using double digest Restriction Associated DNA sequencing (ddRADseq), a protocol that sequences only regions generated by double digests, we can reduce the fraction of each individual genome sequenced, but increase the number of individuals sampled, thus making the problem tractable (Peterson et. al 2012)

Cohort 3 – Parasite load in salt marsh amphipods

Parasite load in salt marsh amphipods

Located on the northeastern coast of Massachusetts, the Plum Island Ecosystem (PIE) – Long Term Ecological Research Network (LTER) serves as a model system for studying long-term ecosystem level responses of high salt marsh communities to large-scale nutrient additions across scales of space and time.

The ubiquitous saltmarsh cordgrass (Spartina alterniflora) dominates the high marsh vegetation community across nitrogen enriched and control creeks. Accordingly, studies at the PIE LTER have shown that the density and biomass of Spartina alterniflora and macroinvertebrate communities in the Spartina habitat were significantly greater in the creeks enriched with nitrogen fertilizer, than in reference creeks (Johnson 2011). Previous studies also suggest invertebrate density and biomass increased with nitrogen enrichment as well (Bertness et al. 2008, Johnson and Fleeger 2009, Wimp et al. 2010).

Focusing on the addition of nitrogen to a northeast American salt marsh, we aim to dissect the levels of diversity that exist among a parasitic trematode (Levinseniella byrdii), its second intermediate host (Orchestia grillus), and among L. byrdii within a single O. grillus. Using a double-digest restriction associated DNA (ddRAD) sequencing, we aim to: 1) Pioneer a partial draft de novo genome for each previously unsequenced species, 2) Characterize genetic diversity of the amphipod O. grillus and its associated trematode L. byrdii, 3) Compare genetic diversity within and between two naturally-occurring O. grillus populations exposed to different levels of nitrogen, 4) Characterize variation in trematode load and intraspecific diversity within each trematode population, 5) Evaluate genomic differences between infected and uninfected amphipods.

Previous sequencing work on trematodes infecting amphipods has taken the form of targeted sequencing to address a specific hypothesis (Keeney et al, 2007.) Less work has been done to apply the full power of next generation sequencing to host/parasite interactions in a habitat. To this end, we will sequence whole amphipod samples–including trematode parasites. We hope to extract information about: parasitic load and diversity within each amphipod and amphipod and trematode population structure of as measured by SNPs.

We hypothesize that increased nitrogen fertilization will be a driving force for genetic differentiation between the amphipods at each site. This study will be the first to characterize diversity across the genome of Orchestia grillus and the first to assess genome-wide diversity in any Levinseniella species.

Cohort 4 – Microbial diversity through time in sediment cores of Sider’s Pond

Microbial diversity through time in sediment cores of Sider’s Pond

Sediment cores have provided us with a great deal of information about the past. By taking cores and analyzing the chemical and biological components of the sediments therein, we have been able to glimpse what life on Earth was like thousands of years ago and how it evolved in response to changing climate, ecological and geological conditions (Hays et al., 1976; Smol et al., 2005; Tierney et al., 2010; Zachos et al., 2001). Recently, with the development of next generation sequencing technologies, we have become better able to utilize a new component of these sediments in our reconstructions: DNA. (Giguet Covex et al., 2014; Redou et al., 2014).

While next generation sequencing techniques provide an advantage for analyzing the more degraded DNA found within sediment cores, the depositional environment controls how much DNA degradation occurs. For this reason, we have chosen a study site that exhibits many DNA preserving qualities. Our site, Sider’s Pond, is a small, meromictic kettle pond located in the town of Falmouth, MA just 550 meters from the open ocean. The pond’s meromixis is maintained by the balance of fresh groundwater inputs and salt water inflows during exceptionally high tides. These conditions combined with the pond’s location in a cold temperate environment, lead us to believe that its sediments likely provide a high quality archive of DNA. Here we propose to obtain this DNA by piston coring Sider’s Pond. Through next generation sequencing and our bioinformatics pipeline, we propose to use these DNA analyses to understand how disturbances, both natural and anthropogenic, affected the biological diversity and composition of Sider’s Pond over the last 1,000 years.

Given that the pond has experienced increased nutrient loading in a developed watershed, we expect commensurate change in species composition and genetic diversity. As the pond became increasingly eutrophied, we predict that species diversity decreased as fewer and fewer species could cope with this new, more extreme environment. We also predict that metagenomic diversity would decrease and become more specialized to cope with the environment.

We will use piston cores to sample the sediment in Sider’s pond.  Core chronologies will be inferred using 210Pb analyses, and these data will be correlated with microbial 16S rRNA sequence data from Illumina libraries of core sections that sample multiple time slices through the core.  Our goal is to test the hypothesis that microbial diversity shows a shift in composition associated with increased evidence for human impact as European settlers and suburbanization altered the nutrient input to the pond over the past few centuries.

Cohort 5 – From Tides to Nucleotides: Ecological Genomics of Semibalanus Balanoides Inhabiting Tidal Microhabitats with Different Degrees of Environmental Stress

The cohort IGERT V project seeks to characterize genomic mechanisms involved in adaptation to environmental heterogeneity focusing particularly on evolutionary responses and host-microbiome interactions of the northern acorn barnacle (Semibalanus balanoides). Briefly, the traineed will compare and analyze genome wide differences in allele frequencies observed in barnacles inhabiting the upper and lower end of the rocky intertidal, two microhabitats with different degrees of environmental stress.

The project has three fundamental goals: (1) Assemble a high quality reference genome for S. balanoides, (2)  assemble a transcriptome from barnacles living in microhabitats at the high and low tidal extremes of the intertidal, and (3) use the two aforementioned tools to characterize allele frequencies and microbiome composition differences across the upper and lower extremes of the intertidal zone. The trainees will tackle these goals through the use of second generation (Illumina’s HiSeq platform) and third generation (PacBio’s SMRT platform) sequencing tools.