Mitochondrial DNA Variation in Oryzomys palustris Populations in the Everglades and Oryzomys argentatus populations in the Florida Keys

 Beatriz Blanco

University of Miami, Department of Biology

P.O. Box 249118, Coral Gables, Florida 33124, U.S.A.

Abstract

Nucleotide variation in the D-Loop of Mitochondrial DNA (mtDNA) was examined within and between two Oryzomys palustris population in the East and Central Everglades and an O. argentatus population in the Florida Keys, in order to determine the effects of habitat fragmentation on these species. We focused our attention on the O. argentatus population because its taxonomic status has not been established yet. MtDNA was isolated from approximately 1 cm of tail tip, amplified via PCR, purified, and sequenced using the DNA Silver Sequence Sequencing System. A total of 34 sites out of 291 bp were polymorphic (P = 0.12). The average nucleotide diversity within populations was 0.015. The Florida Keys population had no detectable variation and had a unique sequence that was not found in the Everglades population. The average nucleotide diversity between the Florida Keys population and both Everglades populations was not significantly greater than the diversity between the two Everglades populations. Taken together these data suggest that the isolated O. argentatus population has not had enough evolutionary time to diverge genetically, and should be given a subspecies title. This research was supported by grant number GM 50083 from NIGMS and grant number 71195-54102 from HHMI.

Figure 1. Oryzomys palustris

Introduction

For many years now, house mice and its related species have been studied and used in many areas of research. Now with the recent development of new molecular techniques such as protein electrophoresis, DNA Restriction Fragment Length Polymorphism (RFLP), single-copy nuclear DNA (scnDNA) hybridization (She et al. 1990) and DNA sequencing, evolutionary changes are being studied at the molecular level (Ferris et al. 1983). One of the most accurate ways of determining the exact genetic variation of a group of individuals is by sequencing their DNA. This technique is being used to determine the effects of habitat fragmentation on genetic variability of the two Oryzomys species. One of the two species, O. argentatus, was listed as endangered by the U.S. Fish and Wildlife Service in 1991(Gaines et al. 1997) due to the continuous destruction of the freshwater mash habitat (Spitzer and Lazell 1978). For many years now the taxonomic status of the O. argentatus has been debated by those who believe it should be a species and those who believe it should be a subspecies (Goodyear 1991; Humphrey and Setzer 1989).

Figure 2. Beatriz Blanco with her mentor, Dr. Michael Gaines.

O. palustris (Figure 1) were trapped on two different sites. In the Central Everglades, Everglades National Park (Figure 3), we trapped animals from 17 different hammock islands, and in the East Everglades, Chekika, we trapped on 6 different islands. Islands were separated by range of 20 m to 360 m, and their area ranged from 0.0005 ha to 0.45 ha. The O. argentatus were trapped in Raccoon Key, Monroe County in the lower Florida Keys.

Figure 3. Aerial view of Hammock Islands in Everglades National Park

To determine the effects of habitat fragmentation on the two species we looked at nucleotide sequences in the hypervariable region 1 (HV1) of the D-Loop in mtDNA. MtDNA has become very useful in evolutionary studies because of its small size, its high abundance in the cell, its maternal inheritance, and its evolutionary rate.

Materials and Methods

Field procedures: O. palustris were live trapped on a monthly basis over a period of one year in the Central Everglades (Everglades National Park) and East Everglades (Chekika). O. argentatus were live trapped on a daily basis for five days at Raccoon Key, Monroe County in the Florida Keys. Sherman live traps were used in all captures.

Approximately 2c m of tail tip was cut from each individual and placed in liquid nitrogen or ice until they could be taken to the laboratory and stored in a -80°C freezer.

Laboratory procedures: DNA was isolated from approximately 1 cm of tail in accordance with Invitrogen’s Easy DNA Kit Protocol #8-Mouse Tails (1996) (Figure 2). Following isolation, the HV1 region of the D-Loop was amplified via PCR using chemicals and a Thermal Cycler from Perkin Elmer, and using primers L15997 and H16401, forward and backward primers respectively. The amplified fragment was checked in a 2% agarose gel against 100 bp ladder. The band was then excised from the gel and purified using the QIAEX II Clean Kit from Qiagen, and eluted in TE buffer. The purified DNA was then used in the sequence reaction of the Silver Sequencing System, run in a 6% polyacrylamide gel, and stained with silver nitrate in accordance with the Silver Sequencing System (Promega Co.)

Results

Our data shows no variability within the O. argentatus population in the Florida Keys; furthermore, the pattern found within the animals of this population was unique to that population and was not found in any animal in the Everglades area. Because of its unique pattern, we used this population as a standard and compared all other populations to it. Out of the 291 bp we were able to sequence, 34 polymorphic sites were found (P = 0.12) (Figure 5). We calculated nucleotide diversity within and between populations using the equations developed by Lynch and Crease (1990). The average nucleotide diversity within and between populations are summarized in Tables 1 and 2. The average nucleotide diversity within populations was v(w)=0.0151, showing no significant variation within the three populations. To our surprise, the average nucleotide diversity between populations was v(b)=0.023. Nucleotide diversity between the two Everglades populations was 0.0258, and nucleotide diversity between the populations in the Everglades (Central and East) and the Florida Keys was 0.0211 and 0.0221, respectively. These numbers suggest that the variability between the two Everglades populations is higher than the variability between the Florida Keys and both Everglades populations, contrary to our prediction.

A phylogenetic tree (Figure 4) was constructed with the neighbor-joining method (Saitou and Nei 1987). Mus spratus was used as the root of the tree. The tree shows that rice rats form Central and East Everglades are not genetically different. Also the Florida Keys population is not very different from the Everglades populations based on the length of the branches.

Table 1. Mitochondrial DNA variation within Oryzomys populations.

Table 2. Mitochondrial DNA divergence between Oryzomys populations.

Figure 4. Neighbor-joining tree for O. palustris and O. argentatus in the central Everglades, east Everglades, and the Lower Florida Keys.

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Figure 5. DNA sequence showing variation in Oryzomys among localities.

Discussion

The taxonomic status of the silver rice rat, O. argentatus, has been in question for some time now. Opposing points of view on this matter have been presented by Goodyear, and Humphrey and Setzer. Goodyear (1987) suggests that O. argentatus could have colonized the Florida Keys towards the end of the Sangamon Interglacial period, or during the Wisconsin Glacial Period. This suggests that this population of O. argenetatus has been isolated of a very long time. Goodyear (1991) also asserts that there is enough morphological evidence to support the view that O. argentatus is a valid species. Humphrey and Setzer (1989), on the other hand, suggest that this population of silver rice rat has been isolated in the lower Florida Keys for only a short amount of time; less than two thousand years. Also, they were unable to find any species-level differences when they conducted their research.

All samples sequenced from the Florida Keys showed the same nucleotide sequence, which supports the view of Humphrey and Setzer. According to Ferris et al. (1983) "…the average rate of point mutational divergence in mtDNA [for rats and other mammals] is 2-4% per million years". The identical nucleotide sequences in this population suggests that it has been isolated for a short period of evolutionary time, and it has not had sufficient time to diverge genetically. Based on our analysis of between population diversity, the population of O. argentatus in the Florida keys is not very different form both populations of O. palustris in the Everglades, consequently we have to agree with Humphrey and Setzer that the status of O. argentatus should be that of a subspecies.

Our data also shows that the diversity between both populations in the Everglades is slightly higher than the diversity between Everglades populations and the Florida Keys. We were surprised to see this because we had expected the diversity between both Everglades populations to be less than the diversity between Everglades populations and the Florida Keys. Even though there is a slight difference, in no case is it statistically significant. Furthermore out data suggest that the habitat fragmentation observed in the Everglades, does not restrict the movement of O. palustris between neighboring islands, thus explaining the low genetic diversity.

Acknowledgments

I am very grateful to Dr. Gaines for letting me become a part of this research. I also want to thank Dr. Xiaoyuan Kong for teaching me all the laboratory techniques necessary for this research, and NIGMS grant number GM 50083 and HHMI grant number 71195-54102 for funding this project. Robb Wright and Mark Chiappone of The Nature Conservancy provided technical assistance in poster development.

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