Mentor: Dr. George Clokey Simon Schmidt Kara Kamps Serenity Mutchler R ESOURCE P ARTITIONING :

♦A strategy developed to reduce competition for resources between two species with similar ecologic niches that occupy the same area.

♦Previous studies suggest that resource partitioning does occur between

Peromyscus maniculatus

(deer mouse), and

Peromyscus leucopus

(white-footed mouse).

• Based on tail morphology which can be problematic due to the two species being almost identical (see images below).

Overlapping Range: mice show resource partitioning with P. maniculatus occupying the trees and P. leucopus occupying the ground Range of

P. maniculatus:

mouse occupies both habitats Range of

P. leucopus:

mouse occupies both habitats

R E EXAMINING THE QUESTION :

♦Our lab decided to re-examine the question using new identification techniques based on PCR amplified mtDNA rather morphology.

♦Both species are supposedly sympatric in Southeastern Wisconsin.

♦Preliminary data showed only the presence of

P. maniculatus

in both arboreal and ground-layer habitats.

• A number of samples yielded no DNA fragments when amplified by PCR.

♦Several possibilities could explain the lack of

P. leucopus

DNA: • We do not have a sufficient number of samples.

• The PCR protocol we are using is not amplifying the DNA • •

P. leucopus

is not found in our area.

♦Solutions to these issues: Determine whether or not the PCR protocol was working by using know tissue samples of both species.

• Optimize [Mg ++ ].

♦Tail tissue samples of

P. maniculatus

and

P. leucopus

were obtained from the Peromyscus Genetic Stock Center.

♦Approximately 3 mm of tissue were clipped from each tail.

DNA P REP :

♦Samples were ground on ice in 0.5 ml of normal saline using a micro-pestle in a 1.5 ml microfuge tube ♦0.5 ml 10% Chelex was added ♦Samples were incubated in a boiling water bath for 10 minutes ♦Tissue debris and Chelex were pelleted by a 30 second spin down in an Eppendorf microcentrifuge at 14,000 rpm ♦The supernatant (ca 0.5 ml) was saved and the pellet discarded.

M ULTIPLEX PCR P ROTOCOL :

♦Reactions were run using puReTaq Ready-to-Go PCR Beads yielding a final concentration of: • 2.5 U Taq DNA polymerase • 0.2 mM dNTP • 10 mM Tris HCL • 50mM KCl • 1.5 mM MgCl 2 ([Mg ++ ] was adjusted as noted in Table 1) ♦Primers (P.mani-F-9197, P.leuco-F-9263, and H9375) were used at 0.5 mM.

PCR C YCLES

♦94 o C for 120 seconds ♦30 cycles of: • 94 o C for 60 seconds (Denature) • 56 o C for 90 seconds (Annealing) • 72 o C for 90 seconds (Polymerization) ♦72 o C for 10 minutes ♦4 o C Soak

DNA A

•

NALYZED

♦Run at 100V

:

♦2% Agarose gel in TAE Sample lanes run with Xyline Cyanol as loading dye only • Lanker lanes run with Bromophenol Blue as lead dye) Table 1:

[Mg

++

] for the PCR reactions are as noted.

numbers to Lane the agarose gel shown in Figure 2

.

[Mg ++ ] 3.5 mm 3.0 mm 2.5 mm 2.0 mm 1.5 mm

Figure 1: DNA Analysis.

PCR amplified DNA from

P. leucopus

and

P. maniculatus

tissue were run as described.

Lanes 1 & 6, Loading dye with Xylene Cyanol and Bromophenol blue; lanes 2 & 5, pGEM DNA markers (Promega, Madison, WI); lane 3,

P. leucopus

PCR amplified DNA and lane 4,

P.

maniculatus

DNA.

PCR amplified Well Set 1 Well Set 2 Lane 1 Lane 1 Lane 2 Lane 3 Lane 4 Lane 5 Lane 2 Lane 3 Lane 4 Lane 5 Figure 2: Varying [Mg ++ ].

PCR amplified DNA from

P. maniculatus

in Well Set 1 and

P. leucopus

in Well Set 2.

See table 1 for [Mg ++ ] and lane number.

Lane 6 for both Well Sets contains loading dye with Xylene Cyanol.

♦We now know that our PCR protocol is working.

♦From here, we will work on obtaining a larger sample size from local woodlots.

♦If indeed we find only

P. maniculatus

in local woodlots, it will bring into question the sympatry of the two species in Wisconsin.

Special Thanks to:

♦Dr. George Clokey for his time and patience ♦The

Peromyscus

Genetic Stock Center of the University of South Carolina ♦Dr. Nathalie Tessier of the University of Montreal, Department of Biological Science ♦The Undergraduate Research Program of UW-Whitewater