Dataset: Menidia menidia - Genotyped Parents

Name: Genotyped parents used in a study of survival of Menidia menidia larvae under high CO2 conditions; conducted at Southampton Marine Station from 2011-2015
Published: 2015-02-24
Keywords: biota, oceans

Cite This Dataset

Spatial Extent: N:40.969 E:-73.088 S:40.969 W:-73.088

Project:

Will rising pCO2 levels in the ocean affect growth and survival of marine fish early life stages? (OA Fish)

Principal Investigator:

Hannes Baumann (University of Connecticut, UConn)

Co-Principal Investigator:

Christopher Gobler (Stony Brook University - SoMAS, SUNY-SB SoMAS)

Contact:

Hannes Baumann (University of Connecticut, UConn)

BCO-DMO Data Manager:

Shannon Rauch (Woods Hole Oceanographic Institution, WHOI BCO-DMO)

Version:

24 Feb 2015

Version Date:

2015-02-24

Validated:

Yes

Current State:

Final no updates expected

Genotyped parents used in a study of survival of Menidia menidia larvae under high CO2 conditions; conducted at Southampton Marine Station from 2011-2015

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Description

The investigators reared offspring, selected from a wild coastal fish population (Atlantic silverside, Menidia menidia), at high CO2 conditions (~2300 uatm) from fertilization to 15 days posthatch, which significantly reduced survival compared to controls. Perished and surviving offspring were quantitatively sampled and genotyped along with their parents, using eight polymorphic microsatellite loci, to reconstruct a parent–offspring pedigree and estimate variance components.

Related Dataset: Menidia menidia - Genotyped Offspring

This dataset provides the source data to:
Alex J Malvezzi, Christopher S Murray, Kevin A Feldheim, Joseph D DiBattista, Dany Garant, Christopher J Gobler, Demian D Chapman, Hannes Baumann. 2015. Offspring sensitivity to ocean acidification changes seasonally in a coastal marine fish. Evolutionary Applications. doi: 10.1111/eva.12248

Note: This dataset has also been contributed to Dryad and can be found at http://dx.doi.org/10.5061/dryad.bd6vs

Methods & Sampling

For more information on the dataset acquistion and processing methods, see Malvezzi et al. (2015) (doi: 10.1111/eva.12248). A summary follows:

Species origin:
We collected ripe, adult M. menidia at the beginning of the spawning season (25 April 2013) from a tidal salt marsh on the North shore of Long Island (Poquot, 40°58.12'N, 73°5.28'W). Males and females were caught with a 30 × 2 m beach seine, then transported to our laboratory facility (Flax Pond Marine Laboratory) and held overnight in separate temperature-controlled baths (200 L, 21 degrees C). The next morning, 29 females were strip-spawned by gently squeezing their hydrated eggs into a large shallow container containing seawater-activated sperm of 42 males and a large sheet of window screen (1 mm mesh).

Estimation of 'Days survived at high CO2' (DS):
To quantify DS for each hatched larva, the bottom of every high CO2 rearing container was gently siphoned twice daily, and all dead individuals were removed, recorded, and individually transferred to a tissue lysis solution (100 uL tissue lysis buffer + 12 uL proteinase K) for subsequent genomic DNA extraction. Frequent siphoning was critical, because fish larvae decompose beyond recognition within hours after death (at 24 degrees C). The pattern of daily posthatch mortality was typical for early larval fish, with mortality peaking soon after hatch (1–4 dph), but then declining exponentially over the remaining days of the experiment. After two consecutive days without any mortality (days 14 and 15 posthatch), the experiment was terminated and all surviving larvae (i.e., DS = 15) were individually transferred to tissue lysis solution for DNA extraction. To specifically evaluate the resistance of the population to elevated CO2, only hatched larvae of the 10 high CO2 replicates were sampled for genetic analyses.

DNA extraction and amplification:
Adult spawner DNA was extracted from tail clips (15–35 mg) following the animal tissue protocol of Qiagen DNeasy kits. Larval DNA (DS ranging from 1 to 15 dph) was extracted using a more cost-effective ‘salting out’ protocol that yielded useable DNA from larval silversides. Ten polymorphic microsatellite loci for M. menidia were amplified for all parents and offspring in a 10 uL reaction containing 1× PCR buffer, 10× bovine serum albumin, 1.5–3.5 mm MgCl2, 0.12 mm dNTPs, 0.16 um of the reverse primer and fluorescently labeled M13 primer, 0.04 um of the species-specific forward primer and 1 unit Taq polymerase, and 10–40 ng of genomic DNA. Thermal cycling consisted of 5 min at 94 degrees C followed by 35 cycles of 94 degrees C for 30 s, primer specific annealing temperature (Ta) for 30 s and 72 degrees C for 60 s with a final extension at 72 degrees C for 10 min. Fluorescently labeled PCR products were electrophoresed on an ABI 3730 DNA analyzer along with an internal fluorescent ladder (LIZ-500; Applied Biosystems). Alleles were scored by a single analyst (AJM) using the software Peakscanner 1.0 (Applied Biosystems, Life Technologies, Grand Island, NY, USA). A subset of approximately 10% of genotypes was verified by a second analyst (KAF) using GENEMAPPER v4.0 (Applied Biosystems). One locus (Mm09) did not amplify and another one did not yield easily scored peaks (Mm119), hence only 8 of 10 loci were eventually used for parentage assignment and genetic analyses.

Data Processing Description

Genetic analyses:
We used CERVUS 3.0 (www.fieldgenetics.com) to calculate summary statistics (e.g., number of occurrences of each allele at each locus) and assess the suitability of loci for parentage analysis. CERVUS was then used to calculate the following four measures for each of the 10 high CO2 replicates: relative allelic richness (i.e., NAlleles, replicate/NAlleles, total), observed heterozygosity, number of dams, and number of sires. Linear regression was used to relate the four proxies to survival. Subsequently, CERVUS was used to assign individual offspring to candidate mothers and fathers. For each offspring tested, candidate parents were assigned with at least 95% confidence using exclusion probability first and the maximum likelihood score second. Comparing each offspring to all parent genotypes, exclusion of candidate parents followed if mismatches occurred at more than one locus. In total, 704 of 772 individuals (91%) were successfully assigned to parents, a success rate comparable to other studies on fish (75–95%).Unassigned offspring, resulting from poor amplification at some loci, were excluded from further analyses. All females, but only 35 of 42 males, were assigned to offspring, likely because the sperm from seven males was not activated or the fertilized eggs were not selected for in the experiment.

Last, we ran a hierarchical set of univariate ‘animal models’ for the trait DS, using the restricted maximum likelihood (REML) software ASReml v3.0.5 (VSN International Ltd, Wood Lane, Hemel Hempstead, UK). An ‘animal model’ is a mixed model (i.e., a form of linear regression with ‘fixed’ and ‘random’ effects as explanatory variables) that has been found advantageous for estimating phenotypic variance components in wild populations. For fixed effects, we only considered replicate ID (1–10), which corresponds to the experimental vessel, given that age (all individuals had the same day of fertilization) and sex (individuals sampled in their entirety) were not relevant factors. Replicate ID significantly influenced DS (df = 9, P < 0.001, univariate general linear model SPSS; IBM) therefore all subsequent analyses included replicate ID as a fixed effect to remove its effect prior to the estimation of genetic parameters. We then estimated the heritability of DS by testing three distinct models: (i) the base model including only the fixed effect, (ii) a model including the fixed effect and an additive genetic random effect (αi with variance VA), and (iii) a model including the fixed effect, additive genetic random effect, and dam identity (mi with variance VM) as a random effect. The components of total phenotypic variance in DS (VDS = VA + VM + VR) were then used to calculate narrow sense heritability h2 of DS as the ratio of additive genetic variance to total phenotypic variance (h2 = VA/VDS) and maternal effects m2 of DS as the ratio of maternal variance to total phenotypic variance (m2 = VM/VDS). Statistical significance was assessed with likelihood-ratio tests [LRT = −2 * (likelihood base model–likelihood model with random effects)] tested against the chi-square distribution (df = 1).

BCO-DMO Processing:
- Replaced spaces with underscores in species column.
- Replaced blanks with 'nd' to indicate 'no data'.
- Modified parameter names to conform with BCO-DMO naming conventions.
- Changed positive degrees west (long) to negative degrees east.

Data Files

File
genotyped_parents.csv (Comma Separated Values (.csv), 37.22 KB) MD5:95b3809d743fbf7d16053a277ae2cd8d Primary data file for dataset ID 551857

More Information about this dataset

Funding Sources

Funding Source	Award Number
NSF Division of Ocean Sciences	OCE-1129622

Deployments

Deployment	Synonyms	Start Date	Platform	Investigator
lab_Baumann_Gobler_FP		2011-09-01	Flax Pond Marine Lab	Hannes Baumann (Principal Investigator) Christopher Gobler (Co-Principal Investigator)	data info

Instruments

Thermal Cycler

Supplied Description:

Instrument Type

Generic Name: Thermal Cycler

Generic Description:

A thermal cycler or "thermocycler" is a general term for a type of laboratory apparatus, commonly used for performing polymerase chain reaction (PCR), that is capable of repeatedly altering and maintaining specific temperatures for defined periods of time. The device has a thermal block with holes where tubes with the PCR reaction mixtures can be inserted. The cycler then raises and lowers the temperature of the block in discrete, pre-programmed steps. They can also be used to facilitate other temperature-sensitive reactions, including restriction enzyme digestion or rapid diagnostics.

(adapted from http://serc.carleton.edu/microbelife/research_methods/genomics/pcr.html)

Parameters

Date and time formats are described in python strftime() syntax.

Supplied Name	Supplied description	Supplied Units	Standard Name
species	Name of the species studied. This study was done on the Atlantic Silverside (Menidia menidia).	unitless	species
date_sampled	Date of sampling in mm/dd/yyyy format. All parents used in this study were caught on 4/25/2013 .	unitless	date
lat	Latitude of sample site (Poquot, Long Island, NY).	decimal degrees	lat
lon	Longitude of sample site (Poquot, Long Island, NY).	decimal degrees	lon
sample_id	Sample ID number (F1-29 and M1-41). F and M stand for female and male, respectively. This study used a total of 29 females and 41 males to produce offspring.	unitless	sample
locus	This study used 9 polymorphic loci (Mm02, 108, 119, 202, 204, 240, 248, 251, and 272).	unitless	no_bcodmo_term
allele_1	Microsatellite allele 1.	base pairs	no_bcodmo_term
allele_2	Microsatellite allele 2.	base pairs	no_bcodmo_term

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Database

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Dataset: Menidia menidia - Genotyped Parents