Morgan, Raymond P.

Ulanowicz, Nancy I.

English

Aquatic Commons

C o n t r .  N o .  632 UMCEES REF-NO.  76-105 
The Frequency of Muscle Protein Polymorphism 
in Menidia meniclia (Atherinidae) along the 
Atlantic Coast 
Reprinted from COPEIA, 1976, No. 2, May 17 
pp. 356-360 
Made in United States of America 
UMCEES Ref. NO. 76-105 CBL 
The Frequency of h4uscle Protein Polymorphism 
in Menidia menidia ( Atherinidae ) along the 
Atlantic Coast 
Electrophoretic separation of the soluble muscle proteins from the 
Atlautic silverside (Menidia menidia L.) revealed three phenotypes (Mu 
AA, AB and BB) postulated to be genetically controlled by two codominant 
alleles, independent of the sex and size of the fish. The  frequency, MU*, 
of the fastest migrating phenotype difEered among samples collected 
from 11 localities along the Atlantic coast. Phenotypes of the muscle 
proteins are related to body depth of the Menidia. 
T HE Atlantic silverside, hlenid ia  naenidia (Atherinidae), ranges from Florida to 
Nova Scotia (Kendall, 1902). It is an important 
forage fish throughout its range for striped bass 
(Merriman, 1941). 
M .  menidia  menidia  and M .  m ~ n i d i a  notata 
are the two recognized subspecies (Kendall, 
1902). Subspecies menidia  is the southern form, 
whereas nojato is the northern form. Inter- 
gradation between the two is at a maximum iri 
the Chesapeake region but ir~tergrades are found 
almost throughout the entire species range (Ken- 
dall, 1902). 
P~evious work by the senior author disclosed 
two muscle-protein phenotypes in hi. naenidia. 
We present the results of a study on the fre- 
cpency of the muscle protein polymorphism in 
populations along the Atlantic coast and the 
relation of the polymorphism to the subspecies. 
Juvenile and adult specimens were obtained 
by beach seining over a three-month period (in 
1972) from 11 locations along the .4tlantic (Fig. 
1 ) .  Identification as /\/I. menidia  was based on 
meristic characteristics and scale morphology 
(Bayliff, 1950). The  fish were stored on ice 
and frozen until assayed. For each fish, seh 
arid fork length were recorded. ,\fenldlc~ rol- 
lected during 1973 and 1974 were also checked 
for sex. In addition to measuring fork lengths, 
two other rrieasurements were made. One was 
the distance from the stlout to tlle first dorsal 
fin and the other was the depth o l  the body 
posterior to the head and anterior to the first 
dorsal fin at the greatest body depth (no ripe 
females were used for these rneasuremcnts). 
Tliese measurements were used to obtain ratios 
of first dorsal position and body depth relative 
to the overall length of a lish. 
The  epaxial muscle nlass from gill slit to first 
dorsal fin was removed, macerated in an equal 
volume of distilled water, and sonified (Bronwill 
Biosonik IV) for 1 min to further liberate any 
~olul)le muscle protei~ls. After low speed cen- 
trifugation, 25 ,pliters of the supernatent was 
MORGAN I1 AND ULANOWICZ-MENZDIA PROTEIN POLYhlORPHlSM 357 
Fig. 1. Station locations for Menidin ~nenid ia  collections along the Atlantic coast. The numbers corre- 
spond to those in Table 1. 
used for electrophoresis. hluscle protein applied 
to each column, as determined by an A / O  T S  
refractometer, was maintained between 200-400 
grams. 
Soluble muscle proteins were separated elec- 
trophoretically on 8% acrylamide gels (Davis, 
1964) with a Canalco Model 1200 bath and a 
Beckman Duostat power supply. T h e  acryl- 
amide gels were formed in glass tubes (7 mm 
O.D., 5 mm I.D., 67 mm long). T h e  25 pliter 
sample was separated at room temperature (20- 
23 C) with a tris (0.005 M)-glycine (0.039 M) 
buffer, pH 8.3. T h e  buffer was discarded after 
each run. A few drops of bromophenol blue 
(0.005% w/v) solution were added to the buffer 
prior to electrophoresis. Electrophoresis was ter- 
minated when the dye band had migrated to 
within 5 mm of the gel end. A constant d-c cur- 
rent of 4.2 rnA/gel was applied to the system; 
voltage varied from 300-410 V initially to 220- 
3 4 W  at completion of the electrophoresis. 
After the gel was removed from the glass tube, 
the front edge of the dye band was cut. T h e  cut 
end of the gel allowed measurements of relative 
mobility. T h e  protein stain used was 0.1% 
Buffalo Black NBR in 7% acetic acid. Gels were 
stained for a minimum of 1 h r  and the stain 
was discarded. Destainirig of gels was done in 
a Canalco Quick Gel Destainer with 7% acetic 
acid. Gels were stored in 7% acetic acid in 
10 x 75 mm culture tubes following destaining. 
A chi-square test was used to test the relation- 
ship between observed and expected Hardy- 
Weinberg pllenotypic distributions in hf. 
558 COPEIA, 1976, NO. 2 
Fig. 2. Acrylamide gels showing the three ~iluscle 
protein phenotypes in Menidia menidia. Frorn left 
to right, phenotype Mu BB, BB, AB,  A B ,  A A  and 
A A .  The fastest moving dark band is conlmon to 
all of the polymorphic muscle protein patterns. 
menidia .  Other statistical procedures followed 
Sokal and Rohlf (1969). 
Electrophoretic separation of soluble muscle 
proteins from M. menidia  reveals three pheno- 
types (Fig. 2). These phenotypes are Mu AA, 
the fast migrating homozygote; Mu dB, the het- 
erozygote; and Mu BB, the slow migrating homo- 
zygote (Fig. 2). Phenotypic expression of the 
muscle protein polymorphism is based on two 
codominant autosomal alleles since only three 
phenotypes are observed and the phenotypic 
distributions did not deviate significantly from 
the expected distribution. T h e  observed pheno- 
typic distribution for the eleven localities cor- 
responds closely to the expected distribution as 
calculated assuming a Hardy-Weinberg equi- 
librium (Table 1). Gene frequencies (of MuA) 
range from 0.31 at Great Peconic Bay, N. Y., 
the northernmost station, to 0.98 at Murrells 
Inlet, S. C., the southernmost station (Table 1 
and Fig. 1). 
There is no significant relationship of the 
observed to sex-male, female or 
immature (G = 7.85, 4 df ,  ns). Also, there is 
no significant relation (G = 1.37, 2 df, ns) be- 
tween phenotype and length (less than 75 mm 
or  greater than 75 mm) of the Menidia .  In  the 
case of Menid in ,  75 mm is used as a break-off 
between young-of-the-year and one-year or  older 
fish (Baylilf, 1950). 
T h e  relationship of phenotype to body mea- 
surements of Menidia  is tested by one-way 
analyses of variance wit11 unequal sample silt 
(Sokal and Rohlf, 1969). First, there is no rela I 
tion of phenotype to the ratio obtained wher 1 
the distance from the snout to the first dorsal f i r  
is divided by the total length. F value of 0.45 
(2. 63 df) is obtained for this ratio with pheno. 
type MLI AA having a mean value of 0.473, Ali 
of 0.481 and BB of 0.478. Second, there is 3 
highly significant relationship of the musclr 
protein phenotype to the ratio of the bod! 
depth divided by the total length (F = 60.4; 2, 
6.3 df). Testing of the means through use of 
the Student-Newman-Keuls test indicates thar 
each phenotype is significantly (at p < 0.05) 
different from each other. Table 2 summarizer 
the body depth ratio measurements. I n  addi- 
tion, analyses of variance for the relationship 
of body measurement ratios to sex by phenotype 
are all insignificant at p > 0.05. 
Menidia  from Murrells Inlet, S. C. (Table 
1 and Fig. 1) are different in body measure- 
ments than the Chesapeake sample. T h e  dorsal 
fin ratio is greater (5 = 0.507, SD = 0.0118. 
CV = 2.34%, n = 12) and the body depth ratio 
is larger (2 = 0.165, SE = 0.00617, CV = 3.74%. 
n = 12). All of the fish are phenotype Mu AA. 
Men id in  collected from near Duxbury, Massa- 
chusetts are also different from the Chesapeake 
sample. T h e  dorsal fin ratio is smaller (% = 
0.470, SD = 0.0107, CV = 2.28%, n = 16) and 
the body depth ratio is slightly smaller (k = 
0.142, SD = 0.00336, CV = 2.37%, n = 16). 
All of the Menid ia  in this sample are phenotype 
Mu BB. 
T l ~ e  muscle protein polymorphism observed 
in M. menidia  is similar to that found in 
Catostomus catostomus (Tsuyuki et al., 1967), 
Anop lopoma  fimbria (Tsuyuki and Roberts, 
1969), Sebastodes elongatus (Tsuyuki et al., 
1968), and Stizostedion v i t r eum v i t r eum (Uthe 
and Ryder, 1970). Polymorphisms in soluble 
muscle proteins of fish are rare (Uthe and 
Ryder, 1970). U p  to 1971, the above four spe- 
cies are the only examples of a muscle protein 
polymorphism in fish even though a large num- 
ber of species had been examined (primarily 
northern species, and primarily by Canadian 
investigators). So far, all muscle polymorphisms 
appear to be controlled by only two codominant 
a;;osomal alleles at one locus. 
T h e  muscle protein polymorphism in M. 
men id ia  may also be important regarding the 
two subspecies. Distinguishing cl~aracters for 
M .  m .  menid ia ,  the southern subspecies, are the 
MORGAN I1 AND ULANOWICZ-IMENIDIA P R O T E I N  POLYMORPHISRl  359 
TABLE 1. THE DISTRIBUTION F PHENOTYPES AND A ALLELE (MuA) FREQUENCY I K  ~Menidia menidia MUSCLE 
nv LOCALITY. The  chi-square values were not significant a t  the 5% level \vith one degree of freedom. ( 0  = 
observed, E = expected). T h e  numbers in parentheses following the location refer to Fig. 1. 
-- - - 
Phenotype 
I<o. of 
Location Samples A A AB BB MuA xa 
Great Peconic (I) 50 0 6 22 22 0.34 0.019 
Bay, N. Y. E 5.78 22.44 21.78 
Patuxent River, (2) 150 0 17 68 65 0.34 0.035 
41tl. E 17.34 67.32 65.34 
Nanticoke River, (3) 50 0 6 24 20 0.36 0.14 
hfd. E 6.48 23.04 20.48 
Isle of IVight, (4) 50 0 7 23 20 0.37 0.045 
Md. E 6.48 23.31 19.84 
Choptank River, (5) 45 0 6 23 16 0.39 0.24 
Md. E 6.85 21.41 16.75 
Potornac River, (6) 30 0 7 13 10 0.45 0.31 
Md. E 6.07 14.85 9.08 
Lerves, Del. (7) 50 0 13 24 13 0.50 0.080 
E 12.50 25.00 12.50 
Sinepuxent Bay, (8) 50 0 17 18 15 0.52 0.84 
Md. E 13.52 24.96 11.52 
York River, (9) 37 0 10 19 8 0.53 0.089 
Va. E 10.39 18.43 8.17 
IVachapreague, (10) 100 0 43 42 15 0.64 0.29 
Va. E 40.96 46.08 12.96 
blurrells lnlet: (11) 50 0 48 2 0 0.98 0.021 
S. C. E 48.02 1.96 0.02 
--- 
1Testing of this population violates the assumptions of the chi-square distribution due to one cell being zero. 
more posterior position of the first dorsal fin, 
fewer longitudinal and  cross series scales, and  
deeper body (Kendall, 1902). Kendall (1902) 
notes that intergrades of the two subspecies ap- 
pear over a wide area from Woods Hole,  Mass. 
to North Carolina. I n  Chesapeake Bay, the 
distinctness of these characters breaks down and  
intergrades are most common. 
Phenotypic and  morphological characteristics 
of Menidia menid ia  from the Atlantic Coast 
appear to follow a bimodal pattern.  T h e  south- 
ern subspecies (or geographical population) M. 
m. menidia may be characterized as having a 
more posterior position of the first dorsal fin, 
a deeper body and  a predominance of M u a  
alleles in the population. I n  contrast, the north- 
ern subspecies M. m .  notata is described a s  
having a more anterior position of the first dor- 
sal fin, a thinner body, a n d  a predominance 
of hluB alleles i n  the population. Kcndall  
(1902) states that the tzotata form is most perfect 
above Cape Cod, the metzidia form most per- 
fect below South Carolina. Intergrades of these 
two subspecies are a t  their  greatest i n  the Chesa- 
peake. O u r  observations o n  the frequency a n d  
pattern of the muscle protein polymorphism i n  
M. menidia  agree with Kendall's work. T h e  
morphological intergradation of the two forms 
reaches i ts  greatest point i n  the Chesapeake Bay 
TABLE 2. BODY DEPTH (ACTUALLY THE RATIO OF 
B O D Y ~ E P T H  DIVIDED BY TOTAL LENGTH) OF iMenidia 
BY PHENOTYPE. T h e  66 fish are all fro111 the 
Patuxent River (MuA = 0.394). 
-- 
Muscle phenotype 
Statistic A A AB BB 
360 COPEIA, 1976, NO. 2 
area (Kendall, 19021, the area where we ob- 
served a great deal of variation in MuA. 
There is a possibility that the M. meniclia 
polymorphism is due to an introgression of the 
two subspecies. Menidia tend to be non-migra- 
tory (Hoff, 1972, Robbins, 1969) but limited 
genetic exchange in coastal areas could occur 
through larval drift and juvenile nl9)vements. 
Another possible explanation for the present 
M. menidia polymorphisnl is genetic isolation in 
tlie past. During the last glacial period, M. 
rnenidia could have become separated into 
northern and southern populations. Following 
the glacial recession, ranges of the two popu- 
lations met and intermingled. 
One other point needs to be considered in 
discussing M. menidia. Recently, Platz and Platz 
(1973) determined that the hemoglobin pheno- 
types and morphological phenotypes of Rana 
pipiens were well correlated. We observed that 
there are nlorphological characteristics (tlie 
body depth of M. menidia) associated with a 
particular muscle protein phenotype. 
We wish to thank Margaret Flowers, Linda 
Noe, Janet Olmon, Jerry Hardy, David Brown- 
lee, Robert Bertin, Leo Minasian, Rob Kelsey, 
Henry Zion, Wayne Gordon, Michael Castagna 
and Victor Kennedy for their assistance in field 
collections. Frances Younger and Michael Reber 
prepared the figures for the text. Miss Ulano- 
wicz was supported by the National Science 
Foundation Undergraduate Research Participa- 
tion program, grant Gy-10026 to the Chesapeake 
Biological Laboratory, Center for Environmen- 
tal and Estuarine Studies, University of Mary- 
land. We especially thank T e d  S. Y. Koo, di- 
rector of the program, for his assistance during 
the project. This  paper is C,ontribution 632 of 
the Center for Environmental and Estuarinr 
Studies, University of Maryland. 
BAYLIFF, W. M., JR. 1950. The life history of the 
silverside, Menidia menidia (Linnaeus). Md. Dep. 
Res. Educ., Chesapeake Biol. Lab. Contr. 90. 
DAVIS, B. J. 1964. Disc electrophoresis-11. Meth- 
ods and application to human serum protein< 
Ann. N. Y. Acad. Sci. 121:404-427. 
HOFT, J. G. 1972. Movements of adult tidewatri 
silverside, Menidia beryllina (Cope), tagged in 
New England waters. Amer. Midl. Natur. 88: 
499-502. 
KENDALL, W. C. 1902. Notes on the silversides ol 
the genus Menidia of the East Coast of the United / 
States with descriptions of two new subspecies.~ 
Report, U. S. Comm. Fish and Fish. 1901:241-26i.j 
PLATZ, J. E.. AND A. L. PLATZ. 1973. Rana pipienr; 
complex: Hemoglobin phenotypes of sympatrir: 
and allopatric populations in Arizona. Science 
179: 1334-1336. 
ROBRINS, T. W. 1969. A syste~natic study of the 
silversides Membras Ronaparte and Menidia (Lin- 
naeus) (Atherinidae, Teleostei). Unpubl. Ph.D. 
Thesis, Cornell University. 
SOKAL, R. R., AND F. J .  ROHLF. 1969. Biometry. 
Freeman, San Francisco. 
TSUYUKI, H., AND E. ROBERTS. 1969. Muscle protein 
polymorphism of sablefish from the eastern Pa. 
cific Ocean. J .  Fish. Res. Bd. Canada 26:2633- 
2641. 
-, R. H. KERR, J. F. UTHE AND L. W. CLARKE. 
1967. Comparative electropherograms of the 
family Catostomidae. Ibid. 24:299-304. 
-, R. H. LOWES, W. HADAWAY A N D  S. J.  WEST. 
RHEIM. 1968. Contribution of protein electropho- 
resis to rockfish (Scorpaenidae) systematics. Ibid. 
252477-2501. 
UTHE, J. F., AND R. A. RYDER. 1970. Regional 
variation in muscle myogen pol ylnorphisln in 
walleye (Stizostedion vitreutn vitreum) as related 
to morphology. Ibid. 27:923-927. 
CHESAPEAKE BIOLOGICAL LABORATORY, CENTER 
FOR ENVIRONMENTAL N D  ESTL~ARINE STUDIES, 
UNIVERSITY OF MARYLAND, SOLO~IONS, MARY 
LAND 20688. Accepted 3 hlarch 1975. 


The frequency of muscle protein polymorphism in Menidia menidia (Atherinidae) along the Atlantic coast

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