Lactate Dehydrogenase Isozymes of Darters 
and the Inclusiveness of the Genus Percina 
Tue parters (Etheostomatini) , with over 110 recog- 
nized species, comprise the second largest group of North 
American freshwater fishes. At present, the most widely 
accepted classification of darters is a proposal of Bailey 
progressively formulated in Bailey (1951), Bailey et al. 
(1954), and Bailey & Gosline (1959) 
The latter paper lists three darter genera (Percina, 
Ammocrypta, Etheostoma) and 22 subgenera and ar- 
ranges them according to evolutionary advancement. 
This classification is based on years of study by Bailey 
and reflects to a high degree the true relationships among 
the darters. However, little documentation was present- 
ed in the 1955 paper, and little has been presented sub- 
sequently, for the classification. Thus some of the 
groupings and arrangements deserve closer scrutiny. 
Collette (1965) compared breeding tubercle patterns 
among all darters and used the same genera and _ sub- 
genera as had Bailey & Gosline except for the recognition 
of Villora as an additional subgenus of Etheostoma. 
More recently Doration and Vaillantia have also been 
recognized as valid subgenera of Etheostoma (Cole 1967, 
Howell 1968). 
Systematic studies of darters have been mainly 
descriptions of species. Few attempts have been made 
to elucidate relationships among species. Although 
recent descriptions of new species include remarks on 
similarities between the species novum and the assumed 
closest relatives, they seldom contain analyses involving 
more than two or three species. 
A controversial aspect of Bailey’s classification has 
been the acceptance of the genus Percina. Some ichthy- 
ologists believe Percina caprodes and its closest relatives 
P. rex, P. burtoni and P. macrolepida deserve separate 
generic status and assign the remaining species to 
Hadropterus (Hubbs & Lagler 1958, Hubbs 1967, Minck- 
ley 1963, Stevenson 1971, Winn 1958) or to Hadropterus 
and Cottogaster (Curd 1967). 
The decision to combine’ the species of Percina and 
Hadropterus in the same genus was based primarily on 
the ubiquitous presence of modified (enlarged and strong- 
ly toothed) scales on the breasts of males of all species in 
both groups (Bailey et al. 1954). Darters in other 
genera do not have the modified scales. 
Data are herein presented on another characteristic 
that is seemingly ubiquitous among, and nearly unique to, 
the species of Percina. The characteristic is the electro- 
phoretic mobility of a readily distinguishable homopoly- 
meric lactate dehydrogenase (LDH) isozyme. The pres- 
This paper is published by authority of the State of Illinois, IRS Ch. 
127, Par. 58.12. It is a contribution from the Section of Faunistic Surveys 
and Insect Identification of the Illinois Natural History Survey. Dr. 
Lawrence M. Page is an Assistant Taxonomist at the Survey. Dr. Gregory 
S. Whitt is an Associate Professor of Zoology at the University of Illinois. 
2 
Lawrence M. Page and Gregory S. Whitt 
ence of a unique morphological characteristic and a 
nearly unique enzymatic characteristic among species 
of Percina is considered strong evidence of a monophy- 
letic origin, attests to the genetic continuity of the group, 
and strongly supports relegation of Hadropterus to the 
synonymy of Percina. 
We appreciate the assistance of Jerry F. Howell, Hart- 
ley F. Hutchins, John C. Marlin, Michael P. Marlin, 
Douglas W. Schemske, Philip W. Smith, Richard L. 
Smith, Lewis J. Stannard, John A. Tranquilli, and Don- 
ald W. Webb in collecting specimens for this study. 
Dr. Smith also reviewed and offered helpful comment 
on the manuscript. 
Dr. William J. Richards, National Marine Fisherie 
Service, and Dr. James B. Shaklee, Yale University 
served as guest reviewers. 
We also acknowledge the assistance of the Illinoi 
Natural History Survey editorial and graphics staff 
the photographs of the gels were taken by Wilmer Zeht 
Fig. 5 and the cover design were executed by Lloyd Le 
Mere, and final editing was done by O. F. Glissendor' 
This research was supported in part by NSF researc 
erant GB-16425 to G. S. Whitt and in part by NS 
doctoral dissertation grant GB-28471 to Philip W. Smit! 
METHODS AND MATERIALS 
The head of each darter was homogenized by har 
in two volumes of tris-HCl buffer (0.1 M, pH 7.0) : 
4°C, The homogenates were centrifuged for 30 minut 
in a Sorvall RC2-B centrifuge at 4°C at 48,200 g. Tl 
supernatant was recentrifuged under the same conditior 
The final supernatant fraction was immediately subject 
to starch gel electrophoresis. The immunochemic 
procedure was described in Whitt (1970). 
Vertical starch gel electrophoresis (Biichler insti 
ments) was accomplished in a 14 percent gel at 8V/c 
for 16-20 hours at 4°C. Two different buffer syste! 
were employed. The tris-citrate pH 6.8 stock bufl 
contained 0.75 M tris and 0.25 M citric acid (mor 
hydrate). Dilution of the stock buffer for both electro 
chambers was 1:7 and for the gel it was 1:20. T 
EBT stock buffer contained EDTA (tetrasodiu 
1 x 10M), boric acid (2.5 x 10°M) and tris (4.5 
102M) and had a pH of 8.7. Dilutions of the stock buf 
for gel, cathode chamber, and anode chamber were 1: 
1:5, and 1:7, respectively. The staining procedure 
LDH isozymes has been described in Shaw & Pra: 
(1970). The control for nonspecific reductants or “no 
ing dehydrogenases” was accomplished by incubat 
one-half of the gel in all the staining components exc 
for the substrate, L-lactate. | 
