1. Observed Heterozygosity Expected Heterozygosity
Microsatellite Analysis of several Darter species as part of Marcellus Shale Bio-monitoring in the Tenmile Creek system, Pennsylvania
Ranajit Das, Elizabeth Dakin and Brady Porter , Department of Biological Sciences
Introduction
Significantly low genetic diversity and observed heterozygosity (HO) in Darters from Bates Fork as compared to the Fonner Run (control stream).
High FST values between Johnny and Fantail darters from Fonner and Bates Fork.
The Darters from the disturbed areas will not be in Hardy Weinberg Equilibrium (HWE).
Predictions
[1 = fef, 2 = fen, 3 = bef, 4 = ben, 5 = tec]
STRUCTURE determined the optimal number of groups to be three.
Each vertical bar represents an individual’s proportional assignment to three populations:
Red = Fantail, Green = Johnny, and Blue = Rainbow
Results
- Genetic verification
Marcellus Shale is marine sedimentary rock found in Eastern USA throughout Appalachian Basin. The Shale contains a large reserve of untapped natural gas. To find out the impact of Marcellus Shale drilling on organisms, a multidisciplinary bio-monitoring program has been developed in collaboration between the University of Pittsburgh, Carnegie Mellon University, Carnegie Museum of Natural History, and Duquesne University. As a part of the bio-monitoring program, Dr. Brady Porter’s lab is focusing on the impact of Marcellus shale drilling on stream fish populations.
Darters are chosen as indicator species for bio-monitoring because of their sensitivity to manmade disturbances including chloride and total dissolved solids
1. Heterozygosity in each population
Populations
Polymorphic Loci
Observed Heterozygosity
Expected Heterozygosity
fef
(N = 5)
Eca46
0.6
0.93
Eca11
Not Polymorphic
Eca48
1.0
0.88
fen
0.25
0.82
0.8
0.77
bef
(N = 6)
0.95
0.7 (P <0.05)
0.84
ben
(N = 4)
0.5
0.86
0.89
tec
(N = 20)
0.9
0.93 (P <0.05)
Method
DNA was extracted from either fin clips or the muscle tissues from the Darters using standard Phenol-Chloroform method.
Three loci (Eca11, Eca46 and Eca48 ) previously developed for rainbow darter ( Tonnis, 2006) were PCR amplified and genotyped for all the Darters.
Presence of distinct population clusters were inferred
by STRUCTURE v
Heterozygosity, FST values and tests for Linkage Disequilibrium and HWE were analyzed by Arlequin 3.5.
Hypothesis
Habitat destruction due to the Marcellus Shale drilling will decrease the population size of Darter species, reducing genetic diversity and heterozygosity.
2. Population structure
Experiment Design
Darter samples were collected from three streams, which are geographically close to each other except that one of them is downstream of the Marcellus drilling while the others are not:
Bates Fork is the disturbed site as Marcellus drilling is taking place upstream of this site
Fonner Run is the control stream where no drilling is taking place
Tenmile Creek is mainstem site downstream of Bates Fork and Fonner Run
The following Darter samples were collected and used in this study:
Horizontal axis = Base pairs; Vertical axis = Intensity of the flurophore signals
Eca11 = Yellow = Black
Eca46 = Red
Eca48 = Green
Size Standard (500 Liz) = Orange
3. Pair-wise FST among five populations
fef
fen
bef
ben
0.37 (P <0.01)
0.14 (P <0.01)
0.20 (P <0.01)
0.35 (P <0.01)
 0.0 (P = 0.85)
0.18 (P <0.01)
tec
0.23 (P <0.01)
0.11 (P <0.01)
0.09 (P <0.01)
0.07 (P <0.01)
Johnny Darter:
Etheostoma nigrum
Collected from Fonner Run and Bates Fork
Future direction
The sample size of this current study is limited, especially in case of Bates Fork and Fonner Run Darters. To conclude the effect of Marcellus drilling on Darter populations confidently, we need a bigger sample size (20 for each). Also, more loci (10 or so) need to be incorporated and genotyped to substantiate the result.
The highlighted comparisons are between the same species from Fonner Run and
Bates Fork
Fantail Darter:
Etheostoma flabellare
Collected from Fonner Run and Bates Fork
Conclusions
Three distinct population clusters were found. The populations from the same species look similar.
Fantail Darters from Bates Fork are significantly differentiated from those of Fonner Run. The same is not true for the Johnny Darters.
Due to extremely small sample size for Fonner Run and Bates Fork Darters, this prediction cannot be validated with confidence.
The observed and expected heterozygosity are similar in all populations and for all loci. Populations are putatively in HWE.
Fantail Darters in Bates Fork may be genetically more diverse, but more samples are needed to confirm this.
Rainbow Darter:
Etheostoma caeruleum
Collected from Tenmile Creek
Acknowledgement
We thank Mr. Anthony Honick for collecting the Darter samples and DNA extraction. I also thank Ms. Maria Wheeler for her help during genotyping as well as running Peak Scanner. Funding for this study is provided by the Heinz Foundation.
Reference
Tonnis B. D Microsatellite DNA markers for the rainbow darter, Etheostoma caeruleum (Percidae), and their potential utility for other darter species. Molecular Ecology Notes 6:
Five populations were designated: Johnny at Fonner Run (fen), Fantail at Fonner Run (fef), Johnny at Bates Fork (ben), Fantail at Bates Fork (bef) and Rainbow at Tenmile Creek (tec)