44 Ray E. Ashton, Jr., Alvin L. Braswell, Sheldon I. Guttman 
spotted N. punctatus were collected from Naked Creek, PeeDee River 
drainage, in the Sandhills region of Robeson County, North Carolina. 
Animals were killed in the laboratory and an organ homogenate pre- 
pared from the heart, liver, rinsed stomach and upper part of intestine, 
and kidney and tongue of each. The specimen remains are housed in the 
North Carolina State Museum collection. The tissues of individual 
animals were then homogenized in an equal volume of 2% 2- 
phenoxyethanol and centrifuged at 25,000 g at 4° C for 45 minutes. The 
supernatant of soluble proteins was then decanted and stored at -70° C 
until used a maximum of 48-hours following preparation. 
The 17 loci coding for proteins consistently resolved are as follows: 
malate dehydrogenase (NAD-dependent) (Mdh-1); indophenol oxidase 
(Ipo-1); ^-glycerophosphate dehydrogenase (a-Gpdh-l); isocitrate 
dehydrogenase (NADP-dependent) (Idh-1); phosphoglucomutases, three 
loci (Pgm-1, Pgm-2, Pgm-3); glutamate oxalate transaminases, two loci 
(Got-1, Got-2); glutamate dehydrogenase (Gdh-1); phosphoglu- 
coisomerase (Pgi-1); malic enzyme, two loci (Me-1, Me-2); 6 
phosphogluconate dehydrogenase (6-Pgdh-l); sorbitol dehydrogenase 
(Sdh-1), glyceraldehyde-3-phosphate dehydrogenase (G-3-pdh-l); and 
lactate dehydrogenase (Ldh-2). 
Techniques of horizontal starch gel electrophoresis and protein 
staining were similar to those described by Selander et al. (1971), with the 
following modifications: Idh, Pgm, Mdh, Gdh, and Me were examined 
with their continuous tris-citrate buffer (pH 8.00); 6-Pgdh, Got, Sdh and 
G-3-pdh were demonstrated with the tris-borate-EDTA buffer of Ayala 
et al. (1973). Staining methods for Gdh, G-3-pdh and Sdh were as 
described by Brewer (1970). All gels were 12.5% starch (Electrostarch Lot 
mi). 
Genetic inferences from electrophoretic results are based on the pat- 
terns being consistent with known molecular configurations for the pro- 
teins analysed, i.e. two-banded patterns are observed for the 
heterozygotes for a protein that is a monomer and three-banded patterns 
are observed for a dimeric heterozygote. The genes coding for each en- 
zyme are represented by italicized abbreviations. 
If several forms of the same enzyme are present and each is con- 
trolled by a separate gene locus, the hyphenated numeral serves to dif- 
ferentiate the loci. The enzyme with the greatest anodal migration is 
designated one, the next two, and so on. When allelic variation occurs, 
the allele with the greatest anodal migration is called a, the next b, and so 
on. 
RESULTS 
The two N. maculosus samples were essentially identical genetically 
(genetic distance, D = 0.000; Nei 1972). One heterozygote was found at 
each of the two loci (Pgm-2, Idh-1) in the Massachusetts sample, the only 
heterozygotes found. 
