Genetic Diversity in Alabama Gopher Frogs

Krista Ruppert

PhD Student at Mississippi State University

November 2023

An adult gopher frog making its way to the breeding pond. Gopher frogs spend most of their lives in upland burrows but make yearly migrations to breeding ponds. – Krista Ruppert

One major issue facing conservation of small populations is the potential for loss of genetic diversity. As genetic diversity is lost, the fitness of populations may also decrease, reducing the abilities of a population to endure disturbance, disease, or other events (Reed and Frankham 2003). This process is known as genetic drift and can be detrimental for small populations. As amphibian populations are often small with limited dispersal abilities, they are especially vulnerable to drift, particularly when habitats are fragmented and connectivity between populations is lost (Allentoft and O’Brien 2010). There are several ways that populations maintain genetic diversity and variation; genetic variation can be introduced by random mutations in a breeding population, where more breeding individuals increases the probabilities of these mutations, or through transfer of genes from individuals originating in other populations. As populations shrink or become more isolated, this variation can be lost.

One species that may be especially vulnerable to this loss of genetic variation is gopher frogs (Rana [Lithobates] capito). While gopher frogs are capable of dispersing relatively long distances (up to 3.5km, Humphries and Sisson 2012), they are also habitat specialists, requiring fire-maintained longleaf pine uplands as well as fishless, ephemeral breeding wetlands. As longleaf pine forests are now estimated to occur at less than 3% of their original extent (Jose et al. 2006), gopher frogs have lost a large amount of historic habitat, subjecting populations to reductions and isolation. Previous studies have assessed the genetic diversity of gopher frogs in Florida (where genetic diversity remains high, Devitt et al. 2023) and North Carolina (where recent genetic divergence between populations is evident, Arbogast et al. 2022).

An adult gopher frog crossing a sandy road to reach the breeding pond. Female frogs will leave the pond shortly after laying eggs, while males may stay up to several months for the chance to breed. – Krista Ruppert

In Alabama, gopher frogs are only known to consistently breed at two ponds and are a species of greatest conservation need. These ponds are approximately 2km apart from each other but are almost 100km away from the next nearest known breeding population, which is separated by rivers and major highways that would be extremely difficult for these frogs to cross. Since these Alabama gopher frogs are so isolated and occur in relatively small numbers, I wanted to investigate their genetics. Could these frogs be experiencing a loss of genetic diversity, and if so, do we need to step in to try to help?

A southern leopard frog considers entering the funnel trap placed along the drift fence. – Krista Ruppert

To look at the genetics of these frogs, we first needed to get DNA samples. We set up a drift fence fully surrounding one of the breeding ponds. On rainy nights during the breeding season, we closed up the gaps in the fence, set funnel traps along the fence to capture frogs, and waited for the frogs to move. Every half hour or so, we would walk around the fence and check the traps. Any gopher frogs we caught leaving the pond were weighed and measured. To take DNA samples, we swabbed the mouths of the frogs and froze the swabs to preserve the DNA. The frogs were released and continued on their merry way, perhaps a little confused by the experience, but no worse for wear.

A buccal swab being taken on a gopher frog in hand. While the frogs do not enjoy the experience, it is a safe and minimally invasive way to obtain DNA from amphibians. – Kolby Altabet

We also conducted egg mass surveys and dip net surveys for gopher frogs. We took a single egg from any gopher frog egg masses we found, and a small tail clip from any gopher frog tadpoles we caught to increase our sample size. Altogether we collected 27 swabs, 23 tail clips, and 7 egg mass samples for our genetic analysis.

A gopher frog tadpole in the hand, identifiable by its globular body and lack of pale facial markings. Tail clips are a safe and minimally invasive method to obtain DNA from tadpoles. – Krista Ruppert

Now that I have my samples, I have plenty of lab work ahead of me! Next, I will extract the DNA from these samples, amplify specific regions (called microsatellite markers), and analyze these regions to determine the genotypes of each individual. I will then calculate genetic diversity indices, relatedness, and recent migration rates, and compare the genetic diversity of the Alabama population to previous studies in Florida and North Carolina.

Microsatellite markers are areas of repeated base pairs. Different alleles will have different numbers of repeats, allowing for genotyping. – Krista Ruppert

While my hope is that we find no evidence of a loss of genetic diversity or drift in the Alabama gopher frog population, there are efforts we can implement to help if things aren’t looking good. One of the best ways to increase and maintain genetic diversity over time is to increase the breeding population size and improve connectivity with other populations. Although there are not any nearby populations to improve connectivity with, the restoration or creation of breeding wetlands near the occupied ponds could increase the breeding population size and function as the creation of new subpopulations in the landscape. This would also help bolster the population against any major issues; currently, if something happens to one of the two gopher frog ponds in Alabama, the result could be severe. With more breeding ponds, the population would be more resilient.

Another option for quickly increasing genetic diversity is by moving individuals from one population to another (translocation). This can immediately inject new alleles into the population but can lead to other genetic problems and does not address the base reasons for the loss of diversity.

That said, I am excited to see what we find as we look further into the genetics of this population! This project is part of a larger project looking into the post-breeding movements, upland microhabitat use, and community breeding ecology of gopher frogs in Alabama, and I am hopeful for the continuation (and expansion!) of this population for many years to come.

A male gopher frog poses for a picture in hand before being released into the breeding pond. – Krista Ruppert

Literature Cited

Allentoft, M., and J. O’Brien. 2010. Global amphibian declines, loss of genetic diversity and fitness: A review. Diversity 2:47–71.

Arbogast, B. S., S. J. Kamel, N. T. Akers, and J. G. Hall. 2022. Kinship and breeding site philopatry drive fine-scale genetic structure in fragmented populations of the gopher frog (Rana capito) in North Carolina. Journal of Herpetology 56:249–257.

Devitt, T. J., K. M. Enge, A. L. Farmer, P. Beerli, S. C. Richter, J. G. Hall, and S. L. Lance. 2023. Population Subdivision in the Gopher Frog (Rana capito) across the Fragmented Longleaf Pine-Wiregrass Savanna of the Southeastern USA. Diversity 15:93.

Humphries, W. J., and M. A. Sisson. 2012. Long distance migrations, landscape use, and vulnerability to prescribed fire of the gopher frog (Lithobates capito). Journal of Herpetology 46:665–670.

Jose, S., E. J. Jokela, and D. L. Miller. 2006. The Longleaf Pine Ecosystem. Pages 3–8 in S. Jose, E. J. Jokela, and D. L. Miller, editors. The Longleaf Pine Ecosystem: Ecology, Silviculture, and Restoration. Springer Series on Environmental Management, Springer, New York, NY.

Reed, D. H., and R. Frankham. 2003. Correlation between fitness and genetic diversity. Conservation Biology 17:230–237.