University of Tennessee, Knoxville TRACE: Tennessee Research and Creative Exchange
Supervised Undergraduate Student Research Chancellor’s Honors Program Projects and Creative Work
Spring 5-2000
Population Genetics of Eastern Red Bats, Lasiurus borealis
David Michael Wills University of Tennessee - Knoxville
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Recommended Citation Wills, David Michael, "Population Genetics of Eastern Red Bats, Lasiurus borealis" (2000). Chancellor’s Honors Program Projects. https://trace.tennessee.edu/utk_chanhonoproj/442
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Signed: __~ 27 Population Genetics of Eastern Red Bats, Lasiurus borealis By: David Wills Mentor: Dr. Gary McCracken Abstract variation in DNA microsatellites was examined in eastern red bats (Lasiurus borealis) in 82 individuals from Michigan, Pennsylvania, Ohio, Indiana, West Virginia, Kentucky, Virginia, North Carolina, Tennessee, and South Carolina. 3mm wing biopsy punches were taken from eastern red bats caught in mist nets. DNA was extracted from the samples and analyzed using two different microsatellite loci. The first loci, MM5 was highly variable with 33 alleles present in my sample. The second loci, NN8 showed low variability with only 3 alleles present in my sample. I divided my 82 samples into nine populations based on region, and used Genetic Data Analysis software to perform cluster analysis on the populations using both alleles. The largest genetic distance was an insignificant 0.013, showing no evidence for genetic structuring of eastern red bat populations. Introduction The eastern red bat, Lasiurus borealis, is a common insectivorous bat found from southern Canada, through the eastern United States and into northern Mexico. Eastern red bats typically roost singly in trees, hanging by one foot so as to appear as a dead leaf. Mating is thought to occur in flight, with the sperm stored through winter hibernation. Conception occurs in early spring with one to five offspring born in June. Eastern red bats are thought to be migratory, particularly in the northern extent of their range. (Shump and Shump, 1982). Previous genetic studies with migratory bat species have shown strikingly different results. In lesser long-nosed bats, Leptonycteris curasoae, Wilkinson and Fleming found evidence for two distinct subpopulations based limited gene flow between migration corridors (1996). Brazilian free-tailed bats, Tadarida brasiliensis, are believed to have both a migrating and nonmigrating subspecies. In a pair of studies, no evidence for genetic structuring was observed in Brazilian free-tailed bats between these behaviorally distinct subspecies (McCracken and Gassel, 1997). Past genetic studies with eastern red bats have looked exclusively at phylogenetic relationships (Baker et aI, 1988; Morales and Bickham, 1995). In this study, I am examining genetic structuring ln eastern red bat populations through a large portion of their range. Using DNA microsatellites, I have compared eastern red bat populations extending from southwest North Carolina to the lower Michigan peninsula. My results allow me to look at heterozygosity as a measure of genetic diversity and to measure genetic distance among local populations to assess possible genetic structuring. Materi als and Methods Sampling and DNA Extraction Eighty-two eastern red bats were captured and sampled in ten states (Figure 1) covering the center of their range. Figure 1: Map of Eastern red bat's range with a closer view of the sampling area. Size of ovals denotes relative area and the number of individuals is listed next to each loca Eastern Red Bat Range Through U.S. and Canada. They are common throughout most of this range. The majority of samples were harvested from eastern red bats caught by field researchers in the summers of 1998 and 1999 (no sites were sampled consecutively from 1998 to 1999). A 3-mm biopsy punch was used to excise a tissue sample from each wing. The tissues were stored in 20% DMSO in 2 mL microfuge tubes. Upon arrival in the lab, the samples were digested with proteinase K, 20mg/mL at 50 degrees C until the tissue was dissolved. The DNA was then purified using phenol/chloroform extraction (protocol from Hillis et al.,1996) Remaining samples were collected from eastern red bat corpses which tested negative for rabies at the University of Tennessee Veterinary Hospital. Larger wing tissue samples were extracted in the same manner from these specimens. Location and Analysis of Microsatellite Loci For my study, two microsatellite loci, MM5 and NN8, were analyzed for each individual (Table 1). Table 1: Array description and PCR primer sequence for the two loci examined in this study. These loci were initially identified by Petri et aI., 1997. Species Locus Array Primers MM5 GT/CA11 TIC ATC CAG TIC TGG Myotis myotis GIT GAT ITA ACA TGC NN8 GT/CA 11 ITG TGT TIT AAA GAA AA T CC Nyctalus noctula AAC ACA AAA TIT CIT ITA GG These loci and the flanking PCR primers were obtained from a study by Petri et al. and were originally identified in Myotis myotis and Nyctalus noctula, respectively (1997). Microsatellite loci were then analyzed using 6% denaturing manual sequencing gels as in Hoelzel, (1998). Each individual was genotyped for each loci. Statistical Analysis Population for Genetic Structuring Each individual was assigned to one of nine separate subpopulations as shown in Figure 1. Subpopulation assignments are not based on behavioral observations, but on groupings designed to divide the sample into as small a geographic area as possible. Small geographic groupings were selected to avoid obscuring any genetic structuring. Subpopulations were compared using genetic identity and distance as calculated by Genetic Data Analysis (Weir, 1996). A UPGMA phenogram was the constructed using those values. Results Descriptive Statistics for the Entire Sample Locus MM5 amplified successfully in 80 of 82 individuals and showed a total of 33 different alleles. Locus NN8 amplified in 56 of 82 individuals and showed only 3 different alleles. On average, the population is polymorphic at 100% of its loci (See Table 2) The observed heterozygosity was 0.55 Table 2:Descriptive statistics for the entire eastern red bat sample. Listed below is average sample size (n), proportion of loci polymorphic (P), average number of alleles per locus (A) and polymorphic locus (Ap), expected proportion of heterozygotes (He), observed proportion ofheterozygotes (Ho), and inbreeding coefficient (t). fis estimated using the Method of Moments (Weir, 1996) n P A Ap He Ho f Total Sample 69.5 1 18 18 0.653 0.551 0.158 Comparison of Subpopulations When the sample is divided into subpopulations, a maximum genetic distance was calculated to 0.013 (Table 3). T a bl e 3 DIstance' matnx WIt' h genetic. I'd entity a b ove th e d'Iagona an d genetic. d'Istance b e IOW t h e dIagona.' ETN& EKY& WTN WNC SC IN WVA MI OH WV PA WTN ---- 0.052 -0.043 -0.051 0.006 0.039 -0.010 0.006 -0.019 ETN& WNC 0.053 ---- -0.056 -0.041 0.029 -0.042 0.024 0.016 -0.004 SC -0.043 -0.054 ---- 0.067 -0.013 0.057 -0.013 -0.010 -0.019 IN -0.049 -0.040 -0.065 ------0.034 -0.072 -0.046 -0.042 -0.063 EKY& WVA 0.006 0.029 -0.013 -0.033 ---- 0.053 -0.010 -0.008 -0.040 MI 0.040 -0.042 0.058 -0.007 0.054 ---- 0.028 0.028 0.031 OR -0.010 0.025 -0.0125 -0.045 -0.010 0.028 ---- -0.008 -0.033 WV 0.006 0.016 -0.010 -0.041 -0.010 0.029 -0.008 ---- -0.010 PA -0.019 -0.005 -0.018 -0.061 -0.039 0.0311 -0.032 -0.009 ---- The UPGMA phenogram demonstrates the slight distance between the east Tennessee/west North Carolina and Michigan subpopulations from the remaining subpopulations (Figure 2) . Figure 2: UPGMA phenogram calculated using genetic distance with all negative numbers set to zero. UPGMA phenogram + POPWT +0 +lPOPSC I +2POPIN I +l3POPOH II +______16 POPPA I I+_POPKV 17 +15 I + POPWV I I +POPET + ______14 +POPMI 0 . 0l3 0.010 0.006 0 . 003 0 . 000 Discussion Eastern red bats are a widespread and common bat throughout North America. They have a typical home range of 500 hectares, which is shared with other eastern red bats. With their large numbers and high vagility, gene flow can occur across local natural barriers. The results support this idea. The observed heterozygosity was 55% for the two observed loci. Typically, 30% is considered a high heterozygosity value. Eastern red bats are reported as highly migratory (Shump and Shump, 1982). If breeding is restricted to migration corridors, then it would be expected to observe genetic structuring within this high genetic diversity. However, no such structuring was observed. For the two loci analyzed, only a negligible maximum genetic distance of 0.13 was observed. It would appear that no gene flow restrictions are imposed by migration. The observed findings should only be viewed as moderate support at this point. There was a great disparity in allele numbers between the MM5 and NN8 loci. This disparity is likely skewing the data in one direction or the other. My sample is largely from the center of the range. Any reproductive isolating mechanism acting on the edges of the range was not observed in my sample. Additional tissue samples from west of the Mississippi would be most likely to demonstrate genetic structuring. Samples from Canada and Mexico of the north and southern extreme might also reveal structuring not present in my sample. Acknowledgments First, I must thank the researchers across the nation who gathered samples for my study: Lynda Andrews, Dr. Mike Gannon, Dr. Michael Harvey, Dr. James Kiser, Robert Kiser, Dr. Al Kurta, Alex Menzel, Dr. David New, Dixie Pearson, Katrina Schultes, Jeff Schwierjohann, and Joe Settles. Otherwise, I would be mist netting myself for the next decade. Thanks to the staff, graduate students, and undergraduates in Dr. McCracken's lab for the support and expertise given to me as I slowly progressed up the learning curve. Thanks go to the Threshold Program at the University of Tennessee which funded my research through a grant from the Howard Hughes Medical Foundation. I am most grateful for additional funding for this research being generously given by Mr. and Mrs. Leland La Roche. Finally, thanks to Dr. Gary McCracken for this opportunity to do a research project that was both biochemical and ecological. Works Cited Baker, Robert J., John C. Patton, Hugh H. Genoways, and John W. Bickham. 1988. Genic Studies of Lasiurus (Chiroptera: Vespertilionidae). Occasional Papers: The Museum of Texas Tech University. 117:1-12. Hillis, D., and C. Moritz. 1990. Molecular systematics. Sinauer Associates, Sunderland, MA. 588. Hoelzel, A.R. 1998. Molecular genetic analysis of populations: a practical approach. In The practical approach series. IRL Press at Oxford University Press, Oxford. 445. Koopman Karl, F., and Gary F. McCracken. 1998. The taxonomic status of Lasiurus (Chiroptera: Vespertilionidae) in the Galapagos Islands. American Museum Novitates. Nov.:1 6. McCracken, G.F., and M.F. Gassel. 1997. Genetic structure in migratory and nonmigratory populations of Brazilian free tailed bats. Journal of Mammalogy. 78:348 57. Morales Juan, C., and W. Bickham John. 1995. Molecular systematics of the genus Lasiurus (Chiroptera: Vespertilionidae) based on restriction_site maps of the mitochondrial ribosomal genes. Journal of Mammalogy. 76:730 749. Petri, B., S. Paeaebo, A. Von Haeseler, and D. Tautz. 1997. Paternity assessment and population subdivision in a natural population of the larger mouse eared bat Myotis myotis. Molecular Ecology. 6:235 242. Shump, K.A., Jr., and A.U. Shump. 1982. Lasiurus borealis. Mammalian Species. 183:1 6. Weir, B. S. 1996. Genetic Data Analysis II. Sinauer Associates, Sunderland, MA. 588. Wilkinson, G.8., and T.H. Fleming. 1996. Migration and evolution of lesser long_nosed bats Leptonycteris curasoae, inferred from mitochondrial DNA. Molecular Ecology. 5:329 339.