Miller Research Presentation 1

Effects of forest road fragmentation on genetic differentiation in northern red-backed salamanders William Miller Messi...

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Effects of forest road fragmentation on genetic differentiation in northern red-backed salamanders

William Miller Messiah College, Au Sable Institute Tim Evans Grand Valley State University, Au Sable Institute

Background: Little research done on effects of small roads on genetic differentiation in salamanders

Question: Does habitat fragmentation caused by forest roads cause increased genetic differentiation between populations? Experiment: Tested 4 microsatellite loci for observable polymorphisms between fragmented populations Conclusion: Further research is required

Introduction 







Roads are a significant source of human-mediated habitat fragmentation 1,2,3,4 Roads create physical barriers between isolated populations • Consequences:  Reduced gene flow 5  Increased genetic differentiation 6 The effects of road fragmentation has been extensively studied in urban areas5 However, effects of small, secondary roads remains largely unknown • In Michigan, forest fragmentation caused by forest roads is quite common

Background 



Forest roads have been shown to reduce dispersal success in northern red-backed salamanders by as much as 51% 7 Gene flow between subpopulations fragmented by smaller roads was not compromised and genetic differentiation was minimal 8

Study Species Plethodon cinereus

Photo by: Mark Tegges, www.newtowson.edu/herpetology/amphibians

Study Species Plethodon cinereus 









Small, lungless salamander (Plethodontidae)9 Found in a variety of mature forests in Northeastern North America 9 Found in areas of moist soil under downed course woody debris 10 Does not disperse across dry, hot areas 11 Effective population size of 500 – 9,000 per hectare 9

Background: Little research done on effects of small roads on genetic differentiation in salamanders Question: Does habitat fragmentation caused by forest roads cause increased genetic differentiation between populations? Experiment: Tested 4 microsatellite loci for observable polymorphisms between fragmented populations Conclusion: Further research is required

Hypothesis 



Objective: examine if fragmentation by seasonal roads leads to genetic variation and differentiation in populations of northern red-backed salamanders (Plethodon cinereus) Hypothesis: Fragmentation by forest roads will result in higher genetic differentiation and lower genetic variability between fragmented sub-populations

Study Sites 

 

2 road segments in Kalkaska Co., MI One lane, sand and gravel roads Surrounding habitat is northern hardwood forests

Field Sampling 









Sites visited June 2011 24 redback salamanders sampled from each site • Site split into 2 subpopulations on each side of road (N=12 for each) • Salamanders were sampled at random by flipping course woody debris within a 1 ha segment on each side of road About 1 cm tissue collected from distal end of tail aseptically12 Distance from road margin recorded categorically (>20m, <20m) Tissue put on ice and stored in storage freezer

Molecular Analysis 







Extracted tissue using DNeasy mini tissue extraction protocol 13 Genotyping for 48 samples was run at 2 dinucleotide microsatellite loci 14 2 more dinucleotide microsatellite loci to be done 14 6-fam and VIC primers used to fluorescently tag sequences

Microsatellite Analysis  

Repeating, polymorphic segments of DNA Common tool to assess differentiation in population genetics

Figures from: Noel and Lapointe 2010

Results 





Successful DNA extraction for every sample

Agarose gels showed successful PCR amplifications No heterozygosity or polymorphisms observed

However, no usable results were found after genetic analysis 

Figure 1: No usable data (typical)

Figure 2: Representation of expected results

Background: Little research done on effects of small roads on genetic differentiation in salamanders Question: Does habitat fragmentation caused by forest roads cause increased genetic differentiation between populations? Experiment: Tested 4 microsatellite loci for observable polymorphisms between fragmented populations Conclusion: Further research is required

Discussion 





DNA extraction and PCR amplification are successful However, genetic analysis failed to produce results Possible problems: • User error • Dilution • Computational error

Background: Little research done on effects of small roads on genetic differentiation in salamanders Question: Does habitat fragmentation caused by forest roads cause increased genetic differentiation between populations? Experiment: Tested 4 microsatellite loci for observable polymorphisms between fragmented populations Conclusion: Further research is required

Continued Study 



Redo analysis for primers used Redo PCR reactions with fresh reagents

Literature Cited

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Fahrig, L., Pedlar, J.H., Pope, S.E., Taylor, P.D., and J.F. Wegner. 1995. Effect of road traffic on amphibian density. Biological Conservation 73:177-182. Gibbs, J.B. 1998. Distribution of woodland amphibians along a forest fragmentation gradient. Landscape Ecology 13:263-268. Kolozsary, M.B., and R.K. Swihart. 1999. Habitat fragmentation and the distribution of amphibians: patch and landscape correlates in farmland. Canadian Journal of Zoology 77:1288-1299. Marsh, D.M., and N.G. Beckman. 2004. Effects of forest roads on the abundance and activity of terrestrial salamanders. Ecological Applications 14:1882-1891. Holderegger, S.R., and M. Di Guilio. 2010. The genetic effects of toads: A review of emperical evidence. Basic and Applied Ecology 11:522-531. Noel, S., Ouellet, M., Galois, P., and F. Lapointe. 2006. Impact of urban fragmentation on the genetic structure of the eastern red-backed salamander. Conservation Genetics 2007:599-606. Marsh, D.M., Milam, G.S., Gorham, N.P., and N.G. Beckman. 2005. Forest roads as partial barrier to terrestrial salamander movement. Conservation Biology 19:2004-2009. Marsh, D.M., Page, R.B., Hanlon, T.J., Corritone, R., Little, E.C., Seifert, D.E., and P.R. Cabe. 2008. Effects of roads on patterns of genetic differentiation in red-backed salamanders, Plethodon cinereus. Conservation Genetics 9:603-613. Harding, J.H. 1997. Amphibians and Reptiles of the Great Lakes Region. The University of Michigan Press:91-96. McKenny, H.C., Keeton, W.S., and T.M. Donovan. 2006. Effects of structural complexity enhancement on eastern red-backed salamander (Plethodon cinereus) populations in northern hardwood forests. Forest Ecology and Management 230:186-196. Larson, A., Wake, D.B., and K.P. Yanev. 1984. Measuring gene flow among populations having high levels of genetic fragmentation. Genetics 106:1882-1891. Herpetological Animal Care and Use Committee. 2004. Guidelines for the use of live amphibians and reptiles in field and laboratory research. American Society of Ichthyologists and Herpetologists. QIAGEN. 2006. DNeasy Blood & Tissue Handbook. QIAGEN Group, Austin, Texas, USA. Connors, L.M., and P.R. Cabe. 2003. Isolation of dinucleotide microsatellite loci from redbacked salamanders (Plethodon cinereus). Molecular Ecology Notes 3:131-133. Noel, S. and F. Lapointe. 2010. Urban conservation genetics: Study of a terrestrial salamander in a city. Biological Conservation 133: 2823-2831

Acknowledgements 

    

Dr. Tim Evans Dr. Sam Riffell Dr. Rob Keys Dr. Tom Lee Dr. David Mahan Mr. Dustin Wcisel