Latest News | Puritz Lab of Marine Evolutionary Ecology

Assessing Atlantic Horseshoe Crab (Limulus polyphemus) Population Structure within Southern New England

Wed, 27 Apr 2016 00:00:00 +0000

The Atlantic horseshoe crab, Limulus polyphemus, is a commercially, ecologically, and economically important species. Current management practices of this species may not be well informed enough to avoid jeopardizing the future health of these animals. This thesis argues that population differences, as defined by morphometrics, behavior, and genomics, may be visible over smaller geographic scales than current fisheries management observes. Specifically, this work focuses on three states in southern New England: Rhode Island, Massachusetts, and Connecticut. Ten sampling locations were observed over two years, 2020-2021, from which over 500 crabs were sampled. Width and weight data were collected to assess whether size differs by location using non-parametric approaches. Tagging data from the US Fish and Wildlife Service was analyzed to assess whether small, localized movement patterns or broad range geographic movement was more prevalent throughout the range. Tissue samples were processed to extract genetic information (single nucleotide polymorphisms) to inform upon adaptive and migratory traits across the range. Morphometric data identified that 36% of pairwise comparisons were significant. Tagging data showed 69% of recaptured crabs were caught in the same water body they were originally released. Genomics tools suggested that outlier loci, more so than neutral loci, were driving the population structuring observed. Cumulatively, these results suggested that population differences can be observed over a smaller scale than currently employed for fisheries management.

Coastal Stressors

Wed, 27 Apr 2016 00:00:00 +0000

Marine species face a complex suite of stressors that span multiple temporal and spatial scales from long-term global ocean change to localized episodes of coastal acidification. The cumulative and concurrent impacts of multiple stressors remain relatively unknown and requires investigating their synergistic impacts across all life stages. Two common stressors in coastal environments are hypoxia, or low dissolved oxygen (LDO), and coastal acidification (CA). Hypoxia and coastal acidification are linked to daily cycles of respiration and photosynthesis, even in pristine bays and estuaries.

The Puritz lab has two concurrent projects that look at the interaction of CA and LDO with other coastal stressors. One, funded by the National Science Foundation looks at the interaction of LDO, CA, and low salinity pulses. Coastal waters are also affected by pulses of natural and artificial freshwater runoff driven by rainfall and storm events. Pulses of freshwater can cause short-term, low salinity conditions, another stressor, that are expected to worsen with climate change.

Our second project, funded by RI Sea Grant, looks at the CA, LDO, and sewage effluent, a common source of eutrophication in many urbanized estuaries.

For many marine species, larval stages are the only means of migration and genetic exchange, and larvae are likely encountering hypoxia, coastal acidification, and low salinity stressors while they are in shallow coastal waters. Additionally, early juveniles may encounter extended periods of all three stressors. The interaction of early life-history stages with repeated and combinations of coastal stressors has the potential to result in an increase of larval/juvenile mortality or the removal of less tolerant larvae. The consequences of this differential mortality are being investigated in the eastern oyster using laboratory multi-stressor exposure experiments and in the field through genomic surveys of natural populations. Patterns of genetic selection are being analyzed by combining genomic and environmental data to elucidate how multiple stressors are shaping marine populations.

Both projects take a two phase framework:

Phase 1 will determine how larval and juvenile genotypes and phenotypes respond to multiple stressors across different developmental time points. This will be done with two larval short-term exposures to factorial combinations of DO/CA and LS and a long-term juvenile exposure to factorial combinations of DO/CA diurnal cycling and LS. A cost-effective exome capture method will be used to sequence the coding sequences of genes expressed during exposures. Phase 1 will (i) characterize how multiple stressors affect larval growth, respiration, and survival, (ii) how allele frequencies in larval and juvenile pools respond to multiple stressors across three different life-history stages and (iii) if specific alleles convey higher survival.

Phase 2 will determine the role of natural and anthropogenic forces shaping the evolution of oyster populations by testing (i) if selective regimes differ and interact across life-history stages and (ii) if the frequencies of both neutral and resistant genotypes correlate to environmental conditions and if adaptive loci partitioned across life stage. Environmental data will be integrated into a seascape genomics framework using panel of genomic markers, including potential loci under selection during early-life history to survey the genome of adult populations across 24 localities from four urbanized estuaries.

Coastal Stressors across urbanized estuaries

Wed, 27 Apr 2016 00:00:00 +0000

Our second project, funded by RI Sea Grant, looks at the CA, LDO, and sewage effluent, a common source of eutrophication in many urbanized estuaries.

Both projects take a two phase framework:

dDocent

Wed, 27 Apr 2016 00:00:00 +0000

dDocent is simple bash wrapper to QC, assemble, map, and call SNPs from almost any kind of RAD sequencing. If you have a reference already, dDocent can be used to call SNPs from almost any type of NGS data set. It is designed to run on Linux based machines with large memory capacity and multiple processing cores, and it can be modified for use on HPC.

Why use dDocent?

Accuracy

de novo assembly

dDocent employs a series of data reduction techniques, aligment based clustering (using CD-hit), and, for PE assembly, a specialized RAD assembly software (rainbow. This combination allows for accurate and effecient de novo assembly.

Bayesian, haplotype based, population-aware, genotyping from FreeBayes

FreeBayes is a Bayesian genetic variant detector designed to detect SNPs, INDels (insertions and deletions), and complex events (composite insertion and substitution events) smaller than the length of a short-read sequencing alignment. FreeBayes is haplotype-based, in the sense that it calls variants based on the literal sequences of reads aligned to a particular target, not their precise alignment, and for any number of individuals from a population and a to determine the most-likely combination of genotypes for the population at each position in the reference.

Eastern oyster population genomics

Wed, 27 Apr 2016 00:00:00 +0000

The eastern oyster, Crassostrea virginica, is a valuable fishery and aquaculture species that provides critical services as an ecosystem engineer. Oysters have a life-history that promotes high genetic diversity and gene flow while also occupying a wide range of habitats in variable coastal environments from the southern Gulf of Mexico to the southern waters of Atlantic Canada. To understand the interplay of genetic diversity, gene flow, and intense environmental selection, we used whole genome re-sequencing data from 90 individuals across the eastern United States and Gulf of Mexico, plus 5 selectively bred lines. Our data confirmed a large phylogeographic break between oyster populations in the Gulf of Mexico and the Atlantic coast of the USA. We also demonstrated that domestication has artificially admixed genetic material between the two ocean basins, and selected lines with admixed ancestry continue to maintain heterozygosity at these sites through several generations post admixture, possibly indicating relevance to desirable aquaculture traits. We found that genetic and structural variation are high in both wild and selected populations, but we also demonstrated that, when controlling for domestication admixture across ocean basins, wild populations do have significantly higher levels of nucleotide diversity and copy number variation than selected lines. Within the Atlantic coast, we detected subtle but distinct population structure, introgression of selected lines within wild individuals, an interaction between structural variation and putatively adaptive population structure, and evidence of candidate genes responding to selection from salinity. Our study highlights the potential for applying whole genome sequencing to highly polymorphic species and provides a road map for future work examining the genome variation of eastern oyster populations.

EecSeq Bioinformatics

Wed, 27 Apr 2016 00:00:00 +0000

To develop EecSeq into an exome capture method for any organism, the accompanying bioinformatics pipeline needs to be capable of de novo assembly of exon loci directly from captured genomic reads. Assembling exon loci is a new bioinformatic challenge as traditional exome capture relies on designed probes from genomic and/or transcriptomic data. Accurate de novo assembly is critical to population level inference derived from reduced representation data sets, and errors, artifacts, and biases in assembly are still problematic in both RADseq and RNAseq. Leveraging the chromosome-level assembly of the eastern oyster genome, we are developing two complementing de novo assembly methods for EecSeq: one utilizing only captured genomic reads and a second hybrid method that will utilize sequences from the cDNA probes (when sequenced) and captured genomic reads. The final output will be an open source bioinformatics pipeline for EecSeq.

Example Project

Wed, 27 Apr 2016 00:00:00 +0000

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Expressed Exome Capture Sequencing

Wed, 27 Apr 2016 00:00:00 +0000

Understanding the interaction between genotype, phenotype, and the environment is one of the greatest challenges in biology. Researchers face a two-fold challenge in experimental design: 1) sampling enough individuals to accurately characterize populations and 2) sequencing the most informative part of the genome, the base pairs that cause phenotypic change. Even with major technological advances in DNA sequencing, it is still too expensive, for most organisms, to sequence the entire genome of multiple individuals. Using probes to target specific locations in the genome has the advantage of focusing sequencing on the functional areas of the genome. However, current methods require preexisting genomic resources for probe design and substantial financial resources for probe synthesis, limiting use to already well-studied organisms and long-term projects. This risky project will develop a method for capturing specific areas of the genome for sequencing with no need for existing genomic resources, removing the time and cost of probe development. This new method will enable the rapid and cost-effective sequencing of the portions of the genome involved in adaptation and will provide unprecedented capacity to detect selection in captive and wild populations. If successful, this new method will enable the assessment of rapid adaptation to short-term ecological disasters and long-term climate change, directly assisting with successful mitigation, conservation, and restoration efforts.

The over-arching goal of the proposed research is to develop Expressed Exome Capture Sequencing (EecSeq) into a cost and time efficient method of exome capture for any organism. To do this, the project takes a three phased approach, (1) focusing on laboratory protocol optimization and improvement, (2) validation with whole genome sequencing (WGS), traditional exome capture, and RNAseq, and (3) the development of an open source, reproducible bioinformatics pipeline, including de novo assembly. Phase one will optimize three key elements in the EecSeq protocol: probe and insert length, probe and capture pool diversity, and the hybridization process. Optimizing all three parameters will maximize the number of sequenced exomic basepairs and on-target reads, increasing the number of individuals that can be sequenced simultaneously while greatly reducing costs. The second phase of the project will use two independent approaches to validate EecSeq genotypes, including a comparison of EecSeq to traditional exome capture, a set of reference individuals with WGS data. The final phase will leverage the chromosome-level assembly of the eastern oyster genome along with results from both previous phases to develop two complementing de novo assembly methods for EecSeq: one utilizing captured genomic reads and a hybrid method that will utilize sequences from the cDNA probes (when sequenced) and captured genomic reads. All experiments, data analysis, and presentation will take place in a completely open and reproducible science pipeline, which should lead to an efficient step-by-step laboratory protocol and a de novo bioinformatic pipeline for EecSeq that incorporates locus assembly and annotation for any organism.

External Project

Wed, 27 Apr 2016 00:00:00 +0000

Fiddler Crabs

Wed, 27 Apr 2016 00:00:00 +0000

Coastal urban areas, home to over 50% of the population in the USA and over 60% of the population of the world, are a major source of marine pollution in coastal environments. This pollution enters the marine environment via either sewage effluent (wastewater) from wastewater treatment facilities, or runoff (stormwater) from rivers and municipal drainage systems. Stormwater and wastewater actively transport a variety of chemicals and substances that are known to be harmful to marine organisms. However, few studies have focused on the biphasic life cycle of marine organisms, that consists of a benthic, low migrating adult stage with low dispersal potential and a pelagic larval stage that spends highly variable amounts of time in the water column. Species with this life cycle are dependent on the early life stages for long-distance dispersal to increase gene flow across populations. One population connectivity study found that stormwater and wastewater are effective barriers to larval dispersal and significantly reduce gene flow between populations in a sea star species in coastal California (Puritz & Toonen 2011).

To characterize the evolutionary impacts of sewage effluent on other marine intertidal communities, an experiment was conducted on the mudflat fiddler crab (Uca rapax), a benthic, demersal species that is reliant on pelagic larvae for dispersal. The crabs were sampled near three different wastewater outfalls in the City of Corpus Christi, and at two control sites that are not likely influenced by sewage effluent. This study will determine if:

Fiddler crab populations located near sewage effluent sources will have lower genetic diversity and heterozygosity compared to the control populations. Fiddler crab populations located near sewage effluent sources will have lower genetic connectivity compared to the control populations Candidate genes under selection in response to wastewater will have similar variant frequencies among populations near sewage effluent sources and different variant frequencies among control populations.

Investigating the effects of coastal stressors on the distribution of genomic variation of oyster populations in Narragansett Bay

Wed, 27 Apr 2016 00:00:00 +0000

Investigating how larval response to multiple coastal stressors shapes the distribution of genomic variation of adult oysters

Amy Zyck¹, Rebecca Stevick², Marta Gomez-Chiarri², & Jonathan Puritz¹

University of Rhode Island, Department of Biological Sciences
University of Rhode Island, Department of Fisheries, Animal, and Veterinary Sciences

Background (project description)

During the summer of 2017, exposure trials were conducted using larvae from the eastern oyster (Crassostrea virginica). Wild adult broodstock were collected from Ipswich, MA and Barnstable, MA, and brought into the lab and conditioned for several weeks. Oysters were spawned via thermal induction and eggs from 7 females were fertilized with sperm from 11 males. Larvae were kept in ambient, filtered sea water for 16 hours to ensure all embryos had developed to the trochophore larval stage. After 16 hours, larvae were counted and approximately ~125,000 larvae were transferred to twelve 1 L glass mesocosms and four replicate 100,000 larvae subsamples were flash frozen (T₀). Ambient water was adjusted to 100 mL and larvae were allowed to acclimate for 1 hour. After acclimation, one of four treatments were randomly assigned to each mesocosm (4 per treatment): CON-Control conditions, CA- Coastal Acidification treatment of ~2800 µatm pCO₂ seawater, SE- Sewage Effluent treatment of 5% volume to volume ratio of treated sewage effluent to sea water, CASE- Coastal Acidification and Sewage Effluent treatment of 5% volume to volume ratio of treated sewage effluent to sea water ~2800 µatm pCO₂. At 24 hours post-exposure (T₂₄), larvae were filtered out of their experimental bottles and flash frozen to preserve the DNA and RNA. RNA was extracted from all samples, mRNA libraries were prepared and normalized, and probes were synthesized (adapter removal and biotin labeling).

In 2017, adult oysters were collected from 4 sites in Narragansett Bay (Figure 1), with 10 individuals from each site. In 2020, 10 adult oysters were collected from each of 4 additional sites in Narragansett Bay and the Narrow River site was resampled to serve as a control across sampling years (Figure 2). Rectum, gill, and mantle tissue was dissected from each sample. DNA was then extracted from the tissue following the DNeasy Blood and Tissue extraction protocol and sheared down to 150 base pair fragments. Environmental data was also collected from each sample site by deployed data loggers or fixed site monitoring from other groups. The collected environmental data includes: temperature, salinity, pH, dissolved oxygen, and chlorophyll-a concentrations. We also calculated the potential influence of sewage effluent at each site by identifying wastewater treatment facilities in close proximity to each site (Figure 3) and determining the outflow from each facility.

The probes, generated from the larval oyster mRNA, were then hybridized with the adult oyster DNA to capture expressed exon regions. After washing away non-target DNA regions and eluting the captured DNA off of the beads, the DNA is sequenced, resulting in a direct capture of genomic sequences that correspond to expressed genes related to coastal stressor response in larval oysters (Puritz and Lotterhos 2018). The sequences obtained from this experiment and collected environmental data from Narragansett Bay will allow for the examination of allele frequencies at target (putatively under selection) loci in adult populations that are exposed to coastal stressors in their natural habitat.

Objectives

Use sequence capture probes synthesized from CASE larval mRNA to capture expressed exon regions in adult oyster DNA related to coastal stressor response in larvae
Identify loci putatively under selection across these 4 populations
Analyze associations between environmental data in Narragansett Bay and putatively adaptive loci and neutral loci in adults to examine if selective or neutral proceesses may be shaping population structure in this system
Identify putatively adaptive loci in CASE larvae and examine associations with environmental data in Narragansett Bay to elucidate if selection at the larval stage is shaping the genomic variation of adult oysters
Identify putatively adaptive loci shared by both adult and larval oysters and associations with environmental data to examine shared selective regimes across life-history stages

Samples Sites

Table 1: The names, ID’s, and geographic locations (provided as latitude and longitude) of the 8 sample sites in Narragansett Bay, RI.

Station #	Station name	Station ID	Sample Year	Latitude	Longitude	Add. Location Info
1	Providence	PVD	2017	41.816	-71.391	Bold Point Park
2	Greenwich Bay	GB	2017	41.654	-71.445	Goddard Park Boat Launch
3	Bissel Cove	BIS	2017	41.545	-71.431	Rome Point
4	Narrow River	NAR	2017 & 2020	41.505	-71.453	River Road
5	Barrington	BAR	2020	41.741	-71.305	Barrington Public Library
6	Kickemuit	KIC	2020	41.698	-71.247	Narrows Fishing Area
7	Mary C. Donovan Marsh	MCD	2020	41.547	-71.203	Pond Bridge Road
8	Green Hill Pond	GHP	2020	41.374	-71.620	Beech Road Parking Area

Wastewater Treatment Plants

Table 2: The names, ID’s, and geographic locations (provided as latitude and longitude) of the wastewater treatment plants located in and around Narragansett Bay, RI.

Wastewater Treatment Plant	ID	Latitude	Longitude
Narragansett Bay Commission - Bucklin Point	NBCB	41.8514102	-71.36403
Narragansett Bay Commission - Fields Point	NBCF	41.794747	-71.391006
East Providence Wastewater Treatment Facility	EPW	41.773839	-71.364992
Warren Wastewater Treatment Facility	WAW	41.726278	–71.285239
Somerset Sewer Treatment Plant	SST	41.717475	-71.168569
Brayton Point Station	BPS	41.661614	-71.263894
Bristol Wastewater Treatment Facility	BrW	41.661614	-71.263894
Fall River Wastewater Treatment	FRW	41.676875	-71.191211
East Greenwich Bay Wastewater	EGW	41.658703	-71.448131
Quonset Point Wastewater	QPW	41.588386	-71.408497
Jamestown Wastewater	JW	41.509675	-71.362322
Newport Wastewater Treatment Facility	NWTF	41.512689	-71.318022
South Kingstown Wastewater	SKW	41.425147	-71.475606
Narragansett Wastewater Treatment Facility	NW	41.384167	-71.476736

Sampling Map

Figure 1: Adult oyster individuals were sampled from 4 different sites in Narragansett Bay, RI in 2017.

Figure 2: The 8 adult oysters sites sampled in Narragansett Bay, RI in 2017 and 2020.

Figure 3: Wastewater Treatment Facilities in Rhode Island and Massachusetts that have outflow into Narragansett Bay. Wastewater treatment facilities are blue, original four sample sites are red.

References

Puritz JB & KE Lotterhos (2018) Expressed exome capture sequencing: A method for cost-effective exome sequencing for all organisms. Mol. Ecol. Resour. 18(6):1209-1222. doi: 10.1111/1755-0998.12905.

Next-generation sequencing

Wed, 27 Apr 2016 00:00:00 +0000

The advent of next-generation sequencing (NGS) has rapidly transcended population genetics to population genomics. Current research focuses on adopting next-generation sequencing technology and embracing an ever-adapting genomic toolkit to take advantage of this unprecedented amount of genetic data. Current research focuses on developing novel exome capture methods for non-model organisms.

Next-generation sequencing

Wed, 27 Apr 2016 00:00:00 +0000

The eastern oyster, a crucial species for US aquaculture, benefits from improved breeding techniques. The Eastern Oyster Breeding Consortium created two SNP arrays to aid in genetic studies. One array, with 566K SNPs, is for screening, while the other, with 66K SNPs, is for breeders’ use. These arrays help identify desirable traits and separate cultivated oysters from wild ones. The arrays also include markers for pathogen detection and mitochondrial identification. Despite challenges like low marker conversion rates due to genetic diversity, the arrays significantly enhance genetic research and breeding efforts for eastern oysters. They offer insights into traits, aid in selecting desirable stocks, and could even help in oyster restoration efforts.

Synergistic effects of Coastal Acidification and Sewage Effluent: A CASE study

Wed, 27 Apr 2016 00:00:00 +0000

During the summer of 2017, preliminary exposure trials were conducted using larvae from the eastern oyster (Crassostrea virginica). Wild adult broodstock were collected from Ipswich, MA and Barnstable, MA, and brought into the lab and conditioned for several weeks. Oysters were spawned via thermal induction, and larvae were kept in ambient, filtered sea water for 16 hours to ensure all embryos had developed to the trochophore larval stage. After 16 hours, larvae were counted and approximately ~125,000 larvae were transferred to sixteen 1 L glass mesocosms and four replicate 100,000 larvae subsamples were flash frozen. After acclimation, one of four treatments were randomly assigned to each mesocosm (4 per treatment): CON-Control conditions, CA- Coastal Acidification treatment of ~2800 $\mu$atm pCO2 sea water, SE- Sewage Effluent treatment of 5% volume to volume ratio of treated sewage effluent to sea water, CASE- Coastal Acidification and Sewage Effluent treatment of 5% volume to volume ratio of treated sewage effluent to sea water ~2800 $\mu$atm pCO2. After 24 hours, three, one mL samples were taken from each mesocosm, stained with neutral red, and preserved in formalin and remaining larvae were filtered out of the treatment water and flash frozen for genetic analysis. Larvae were later counted on a dissecting scope to assess mortality.

Exome capture was performed on all samples, including the four initial replicate samples taken from the overall larval pool before being added to treatment water. Libraries were sequenced on a single lane of Illumina HiSeq, and reads were demultiplexed, quality trimmed, mapped to the oyster genome, and used to call pooled variants with a modified version of the dDocent pipeline (Puritz et al. 2014). After variants were filtered to only bi-allelic SNPs with at least 20X coverage per pool (13,209 SNPs with no missing data), Cochran–Mantel–Haenszel tests (CMH) between final treatment replicates and initial genetic replicates were calculated using the Popoolation2 software package (Kofler et al. 2011). Outliers detected between control and initial replicates were removed to help mitigate experimental artifacts and maternal effects, leaving a final set of 3,263 SNPs. A PCA was performed on the pooled allele frequencies using the pcadapt package (Duforet-Frebourg et al. 2014; Luu et al. 2017).

Our preliminary results show five striking patterns:

Short-term exposure to highly acidified water does not induce larval mortality in oysters
Sewage effluent alone induces significant larval mortality
CA and SE appear to act synergistically producing the highest levels of larval mortality
All three stressor treatments were genetically distinct from both the control and initial samples
Larval pools in CASE treatments were more similar to CA samples than SE samples

The Eastern Oyster Genome

Wed, 27 Apr 2016 00:00:00 +0000

The eastern oyster, Crassostrea virginica, now has its first complete chromosome-level genome assembly. Released and annotated in 2017, this genome is over 97% complete and serves as a vital resource for studying how mollusks adapt to changing environments and for improving selective breeding in aquaculture. To enhance the accuracy of genomic studies, we developed a method to correct assembly errors caused by high levels of genetic variation. This method significantly improves the detection of genetic diversity and population structure, offering a valuable tool for researchers and breeders alike. Our open and reproducible resource ensures better genomic analysis for future studies.