Immunological Comparisons of Four Subgenera of Ground Squirrels

Immunological precipitin tests were used to determine relative serological propinquity of four subgenera of Spermophilus (Otospermophilus, Callospermophilus, Spermophilus, and Ictidomys). Serum from another sciurid genus, Sciurus, was cross-reacted with antiserum against each of the subgenera of Spermophilus. Interest in the relationships of North American sciurids, especially ground squirrels (Spermophilus), has increased recently as a result of cytogenetic investigations by Nadler ( 1962, 1966, and elsewhere) and by Nadler and Sutton (1962). The genus Spermophilus was revised by Howell (1938), and extensively studied later by Bryant (1945) and Black (1963). By use of immunological precipitin tests (see Boyden, 1926, 1942), we have attempted to further elucidate the interrelationships of four of the subgenera of Spermophilus (Otospermophilus, Callospermophilus, Spermophilus, and Ictidomys) and have compared these to another sciurid genus, Scirus. MATERIALS AND METHODS Sciurids from which serum was collected are as follows: Spermophilus (Otospermophilus) variegatus, from, 1 mi. NW Boulder, Boulder Co., Colorado (19, 1 &); Spermophi'lus (Callospermophilus) lateralis, from Boulder Creek Canyon, 8 mi. WSW Boulder, Boulder Co., Colorado (29, 2 &); Spermophilus (Spermophilus) richardsonii, from Magnolia Road, 6 mi. W Boulder, Boulder Co., Colorado ( 1 & ); Spermophilus (Ictidomys) tridecemlineatus, from Fort Leavenworth, Leavenworth Co., Kansas (39, 3&); Sciurus niger, from Lawrence, Douglas Co., Kansas (1 &). All specimens except the Sciurus niger are preserved as skins and skulls in The University of Kansas Museum of Natural History. Animals were bled by cardiac puncture; sera were separated by centrifugation and preserved by freezing at -150C. Levine and Moody (1939) demonstrated that storage of sterile antigen does not affect the precipitin reaction. Nadler and Hughes (1966) observed intraspecific electrophoretic variations in protein fractions from the sera of two species of ground squirrels. Disc-electrophoresis and starch-gel electrophoresis of the serum of individuals of Spermophilus tridecemlineatus and S. lateralis in this study also showed intraspecific protein polymorphisms. To mitigate the effect of such variation, sera from each species, if represented from two or more individuals, were pooled. Antisera against whole sera were prepared in rabbits. A 1 ml mixture of 0.5 ml of serum, 1 gm per cent protein-nitrogen (PN), and 0.5 ml of complete Freund's adjuvant, was injected subcutaneously as a sensitizing dose. An immunizing dose (prepared as above) was given 30 days later then repeated every other day for 6 days. Rabbits were partially bled 8 days later. After 9 days, another series of three injections was given; 9 days later the rabbits were bled a second time. All tests were conducted using antisera from the second bleeding. The method of precipitin testing used in this study followed that outlined by Leone and Wiens (1956). A constant amount of antiserum, 0.2 ml, was added to each of 12 tubes of a doubling dilution series of serum. The first tube of each dilution series, except that of S. variegatus, contained 400 IgPN/ ml in 1 ml of phosphate buffered saline, pH 7.0. That for S. variegatus contained 200 ygPN/ml of saline. A control tube consisting of 0.2 ml of antiserum in buffered saline was made for each series. The tubes were