THE SYSTEMATIC STATUS OF FUNDULUS KANSAE
AND FUNDULUS ZEBRINUS by
CHARLES THOMAS EVERETT, B.S. in Ed.
A THESIS IN ZOOLOGY Submitted to the Graduate Faculty of Texas Tech University in Partial Fulfillment of the Requirements for the Degree of
MASTER OF SCIENCE
Approved
August, 1972 8QJ> f^tf^/oi"^
m Jin ACKNOWLEDGEMENTS
I am deeply grateful to my major advisor, Dr. John S.
Mecham, for his direction of this thesis. I am thankful to Drs. Francis L. Rose and Robert W. Mitchell for the time and equipment they provided. I sincerely thank Mr. Mike
Bishop, Mr. Scott Simpson, Mr. Greg Mengden, Mr. Tony
Mollhagen, and especially my wife, Donna, for their assis tance in collecting the fish of this study. I am grateful
to Dr. Joe R. Goodin and, again. Dr. Francis L. Rose for
their constructive criticism of this manuscript. This research was supported in part by National Science Founda
tion Grant {GB-13791) under the direction of Dr. John S.
Mecham.
11 CONTENTS
ACKNOWLEDGEMENTS ii
LIST OF TABLES iv
LIST OF FIGURES v
I. INTRODUCTION 1
II. METHODS AND MATERIALS 3
III. RESULTS 10
Morphological Analysis 10
Electrophoretic Analysis 12
IV. DISCUSSION 32
V. SUriMARY 3 9
LITERATURE CITED 41
111 LIST OF TABLES
Table Page 1. Analysis of Variance, Single Classification Males v.s. Females 25 2. Regression of Morphological Characters on Distance from Southernmost Locality 26 3. Descriptive Statistics for Pectoral Rays, Dorsal Rays and Vertical Bar Number-Males 28 4. Descriptive Statistics for Anal Rays and Principal Caudal Rays 30 5. Degree of Correlation between Scale Number and Vertebral Number 31
IV LIST OF FIGURES
Figure Page 1. Sample Localities for Fundulus zebrinus 16
2. Typical Male and Female F. zebrinus 18
3. Descriptive Representation of Lateral Scale Number with Respect to Locality 20 4. Variation in Eye Diameter with Respect to Locality 22 5. Electrophoretic Pattern Differences between F. zebrinus and Cyprinodon rubrofluviatills 24
V CHAPTER I INTRODUCTION
According to the current literature there are two spe cies of the genus Fundulus in the saline, alkaline waters associated with the upper regions of the Central Plains drainage systems. These two fish, F. zebrinus Jordan and
Gilbert and F. kansae Garman, exhibit morphological charac ters not observed elsewhere in the genus. These include a lengthy convoluted gut and weak, slender pharyngial teeth.
Two characters have been utilized in differentiating the two fish. The first character, lateral scale number, is found in many recognized keys (Blair, et al., 1968; Eddy,
1969; Knapp, 1953). In F. kansae the lateral series number is given as 52 to 64 scales while in F. zebrinus the scale count varies from 41 to 49. The other character, eye diam eter, has been only infrequently mentioned (C. L. Hubbs,
1926; Koster, 1957). Hubbs simply reported that F^. zebrinus had a larger eye. Koster compared the eye diameter to the width of the preorbital bone. In F^. kansae the width of the preorbital was reported as being two-thirds or more of the diameter of the eye while in F_. zebrinus the bone width is no greater than one-half to two-thirds the diameter of the eye. According to Koster, the preorbital size is essen tially the same size in both species. There has been some disagreement as to the ranges of the two fish, but the concensus of most contemporary workers is that F. kansae is found from South Dakota to northern
Texas and eastern New Mexico, or more generally the Arkansas
River basin east to Missouri; F^. zebrinus is found in the
Brazos, Colorado, and Pecos River drainages of Texas and New
Mexico.
Several authors (Miller, 1955; Metcalf, 1966; Pfliger,
1971) have suggested the possibility of conspecificity be tween F. kansae and F. zebrinus but have lacked sufficient evidence to support this contention. This study was under taken with the objective of determining the systematic relationships of the two animals. CHAPTER II
METHODS AND MATERIALS
Specimens for analysis were collected at the 20 local ities indicated in Figure 1. These localities were dis tributed from the lower portion of the Pecos drainage in southwestern Texas northward to the Smokie Hill River in the west-central portion of Kansas (least distance approxi mately 573 miles). Eight distinct drainages were sampled, these being from north to south; Smokie Hill River, Arkansas
River, Cimarron River, Canadian River, Red River, Brazos
River, Colorado River, and Pecos River. Samples from numer ous localities within the Canadian, Red and Brazos River drainages were studied in order that the supposed range limits of the fish could be closely inspected.
All fish were collected with a fine mesh, 20 foot seine.
The fish were most generally found in the stream bed proper where the water was shallow with a moderate rate of flow, or in the side pools adjoining the main body of water. The fish definitely seemed to prefer a substrate of sand or fine gravel.
The fish were kept in styrofoam containers for transpor tation to the laboratory. Upon arrival in the laboratory a random sample of the fish were preserved in 10% formalin; the remaining fish were kept in aquaria, styrofoam coolers, and large plastic containers. All fish were kept in an 3 environmental room with the water temperature stabilized at
20.0 - 1.0°C. The fish were maintained on a diet of shrimp
flakes and commercial tropical fish food.
Freshly preserved fish were allowed to set for several days before measurements and counts were taken. This was done to allow completion of preservation, as these fish would be grouped with aged museum specimens. Setting also affected erosion of the mucous layer covering the scales.
This layer seriously hinders the counting of scales.
Four measurements were recorded (to the nearest 0.1 mm) for each fish. These were: standard body length, predor- sal length, eye diameter, and body depth immediately poste rior to the operculum. A binocular, widefield scope was used as an aid in taking all measurements. Counts were made of six meristic characters for each fish. These in cluded numbers of pectoral rays, dorsal rays, anal rays, principal caudal rays, vertical bars, and lateral scales
(from the first scale in contact with the operculum to the last large scale in the region of the hypural plate).
Scales were counted on both sides and averaged if there was a difference. Any mean value ending in 0.5 was raised to the next whole number.
The scales in these fish are normally quite difficult to count, and a technique was devised which greatly simpli fied this problem. After all other measurements and counts 5 were taken, the fishes were placed in close proximity (7-10 cm) to an incandescent bulb and allowed to dry slightly.
They were then placed in a solution of methylene blue in distilled water (1:200) for a period of approximately three minutes. They were again dried for three to five minutes under the lamp. With the aid of a dissecting scope the scales could then be easily and accurately counted. Upon return to the formalin the fish gradually lost their blue color and were in no way harmed. The ray counts generally proceeded without difficulty, but on occasion drying or backlighting was necessary.
The fish were also sexed at the time of measurement.
This is a relatively simple procedure. The males have much bolder barring than do the females (Fig. 2). The male has a rounded anal fin while in the female it is angular. The female also has a low sheath around the anterior portion of the anal fin. Another sexual difference, previously un recorded, is a black spot located above the pectoral fin just posterior to the operculum. This spot is consistently present in the males and absent in the females.
When possible, thirty animals, including fifteen males and fifteen females, were analyzed from each locality. A total of thirteen localities afforded this number of animals.
Where only a lesser number could be obtained, the animals were measured and counts taken in the same manner but the 6 sample was not included in the analyses of variance or re gression analyses as unequal sample size greatly burdened the analysis. All samples were analyzed in terms of descriptive statistics.
Each character was first analyzed for the presence of sexual dimorphism. This was accomplished by single class ification, analysis of variance. If sex influenced the phenotypic expression of a character the males and females were compared separately; if sex did not create a bimodal distribution they were combined for study.
Data for certain characters suggested clinal variation.
To evaluate a possible clinal change in phenotypic expres sion the various mean values for each character were re gressed against locality on a south-north axis, the southernmost locality being assigned a value of zero. All other values were expressed in miles from this locality.
The locality was considered the independent variable and the character, the dependent variable.
Twenty animals, five from each of four localities, were examined for possible correlation between vertebral number and lateral scale number. The scale counts were made as previously described except that the scales were not stained,
The vertebral counts were made after clearing and staining the fish in a modified method of Taylor (1967) as described by Mitchell (1971). Vertebral counts were taken from the 7 first unfused neural spine to the last neural spine on the hypural plate.
Logrithms were utilized where ratios were involved in the evaluation of a character. Instead of dividing one character by another, the log of the smaller value was sub tracted from the log of the larger. This technique mini mizes problems of normality often associated with ratios.
This procedure was used in evaluating eye diameter (log standard length - log eye diameter) and predorsal length
(log standard length - log predorsal length).
Attempts were made to analyze four protein systems, these systems being, hemoglobins, aromatic esterases, gen eral proteins, and lactic dehydrogenase. Horizontal starch gel electrophoresis was the method applied in studying these systems. Animals for electrophoretic study were collected from ten different localities, ranging from the Smokie Hill
River in west-central Kansas to the Pecos River in south western Texas (Fig. 1; sites listed alphabetically). They were maintained in the laboratory as previously described.
At least five fish from each locality were analyzed for each system.
Each animal was prepared for study in the following manner. The fish was held in one hand, and with a pair of sissors in the other, the body was transversely cut into two pieces, the cut being made just posterior to the anal 8 opening. The anterior portion of the fish was immediately dropped into a graduated centrifuge tube (15 ml) containing two drops of Sigma 14-5 anti-coagulant solution and six ml amphibian Ringer's solution. The tube was gently shaken as the fish was bled to prevent clotting of the blood. The fish was removed after bleeding had stopped and the tube was placed in ice. The liver of the fish was then removed and placed in a centrifuge tube surrounded by ice.
The tube containing the blood was then centrifuged for five minutes at 300 rpms, the supernate removed, six ml
Ringer's added, the red blood cells suspended by shaking and again centrifuged. This procedure was repeated again and the supernate discarded. Three drops of distilled water were added to the "RBCs" as a hemolyzing agent. The cells were further hemolyzed by freezing and thawing. Again the tube was centrifuged to free the hemoglobin of cellular debris. The hemoglobin was then placed in a freezer for subsequent study.
Three drops of distilled water were added to the tube containing the liver. It then was reduced to a homogenate with a glass rod. The homogenate was centrifuged and frozen for later use.
Separation of blood hemoglobins was accomplished by utilizing the pH 8.6 buffer of Smithies (1959). The gels were formed in a 12 x 20 x 0.8 cm mold using 12% 9
Electrostarch and stock buffer diluted one volume buffer to four volumes de-ionized, distilled water; the electrode buffer was not diluted. Electrophoresis was accomplished using both the "comb plate" method and the paper tab method.
Both proved similarly successful. DC voltage was supplied by a GELMAN Model # 38201 power supply. The gels were run under refrigerated conditions for five hours at 200V. Stain ing was effected with Amido-schwartz lOB (0.2 gm in 250 ml methanol); acetic acid: distilled water (10:2:10). De- staining and hardening of the gel was carried out in the
just mentioned solution minus the Amido-schwartz black.
The methods applied in the esterase study were essen tially the same as that previously described, the exception being that liver homogenate was used rather than blood and a different staining technique was utilized. The reagent for esterase staining contained one ml alpha-napthyl acetate
(1 gm/100 ml acetone), 20 mg Fast Blue RR and 50 ml Tris- malate buffer at a pH of 7.0. The sliced gel was placed in this solution for a period of five to thirty minutes.
The buffer systems used in the analysis of LDH were the same as previously described. Staining was accomplished by the tetrazolium method (Fine and Costella, 1963).
The fourth system, general proteins, was run with the same technique as described for hemoglobins except that liver homogenate was used in the slots. CHAPTER III
RESULTS
Morphological Analysis
Thirteen of the 20 localities provided a sample of 15 males and 15 females. Studies of sexual dimorphism and clinal variation were made on these samples.
Of the seven characters analyzed for sexual dimorphism only one revealed sexual variation. The position of ante rior insertion of the dorsal fin is located more posteriorly in females than in males. For the samples studied, the dif ference was found to be highly significant (0.001 level).
No other characters, other than those mentioned in the previous section, showed any evidence of sexual dimorphism
(Table 1, A-G). Although no statistical testing was con ducted on the smaller samples with regard to sex it was assumed that these samples do not differ markedly from the larger samples in this respect. For this reason the males and females were combined for analysis of the descriptive statistics for all characters except predorsal length and barring (limited to males). This was done in order to provide a larger sample size.
The regression of scale number upon location on a north- south axis proved to be statistically highly significant
(0.001 level). In other words, as distance from the
10 11 southernmost locality increased, there was a corresponding increase in scale number (Fig. 3). As may be seen, the correlation is not perfect but a definite trend is indi cated. The several noticeable reversals in scale number seem to be random in nature as the variation in one partic ular stream is in some cases more pronounced than that observed in adjacent drainages.
As eye diameter rather than preorbital width appears to vary geographically, eye diameter was analyzed in terms of standard body length, the margin of error being less critical. As mentioned previously, log values were used to alleviate problems of normality. The resultant log value was regressed on locality and again the results proved to be highly significant (0.001 level). Some component of location affects eye diameter in a statistically significant manner. The results of this regression is given in Table 2F.
As log values were used in analyzing eye diameter, confi dence interval and standard deviation are not presented.
The difference in terms of log values for each locality is graphed in Figure 4 so that some concept of the geographic variation in eye diameter may be observed. In this case the larger logrithmic values denote a smaller comparative eye diameter.
The characters, pectoral fin ray number, dorsal fin ray number, anal fin ray number, and principal caudal ray number 12 were evaluated in the manner described for the previous two characters. Tables 3 and 4 give the mean values, one stan dard deviation and 95% confidence intervals for each local ity. These data are not presented graphically as there are no consistent geographic trends. Significant variation is observed in comparing certain localities but this variation does not appear to be correlated with north-south locus or drainage system.
Body depth was recorded for each fish but it was later decided to disregard this character. The body design of the fish is such that nutritional state and stomach contents affect body depth.
Vertical bar number was recorded for all animals but only analyzed in the males, as barring in the females was sometimes faint or absent. Here too, minor variation of number could not be attributed to either stream system or north-south location (Table 3C).
Both methods, pooled and unpooled, of correlating ver tebral number and scale number (Table 5, A-B) gave the same results. Scale number is significantly correlated with number of vertebrae (0.05 level).
Electrophoretic Analysis
Of the four systems studied, only two, the hemoglobins and esterases, gave results adequate for critical analysis.
Neither system revealed any differences among localities 13 and there was no indication of isozyme polymorphism at any locality. The two systems are shown in Figure 2. To facil itate presentation of the different electrophoretic patterns the typical complete banding is drawn for each system.
This method of presentation is used for two reasons: one, no difference was observed among the various localities studied, and two, no single gel was representative of all localities. One or two locations always seemed to run
"lopsided" or streaked. Each system was run repeatedly, so in the case where a sample ran poorly on one gel it could be checked for consistency on another gel.
The most highly resolved system proved to be the hemo globins. Four anodal and four cathodal bands were expressed
(Fig. 5). The leading anodal band appeared wide but clearly and consistently defined. The two intermediate bands were separated only slightly with the faster being the better defined band. The slowest of the four was well defined but showed little movement. The cathodal bands appeared as two distinct pairs, one slow, the other intermediate in speed.
Another member of the family Cyprinodontidae (Cyprinodon rubrofluviatilis) was run with the Fundulus for comparison.
This fish is commonly found in much the same habitat as the plains killifish. While no differences were evident among the killifish the pupfish was obviously different. Its pat tern is shown in Figure 5B. It separated into three anodal 14 bands, the lead band migrating only as far as that of the
second Fundulus band. There were five distinct cathodal
bands for Cyprinodon as opposed to four in Fundulus.
The study of aromatic esterases in Fundulus gave the
same results as did the hemoglobins: no variation was
noted. A wide leading anodal band (Fig. 5C) followed by a
weakly defined, narrow band was observed for Fundulus, while
Cyprinodon expressed a weak leading band and a narrow, well
defined second band. No cathodal bands were observed for
either of the two fish. 15 16
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(ti ci)oco+Jcocn5H^cnO(ti(ti^i O ^d O cn o (ti O >irH G X O O dCU -O •5H05H(ti(ti-H5Hg Ho CN OO in r^ cTi iH CN 00 in r- 00 oooor^ooorHooooooooinoooo OOOO OOiHOOrHfOrHOOOOOOCMOOrHOOOOOOOO CO >H u '=) CO + 1 rHOOOOOOOOOOOOOOOOOOO U H EH CO r--0(TiOincNrHinr~r^rHvor-vo'*cMrHHfNjin H r^c^lOcT^a^cNvoooof^J^~-tN^lnoovDr-c^l0^r^ Ln'^oovDvo'=i'vDinLninLnvoinvD'=i'ooinvoinvo < cn EH OOOOOOOOOOOOOOOOOOOO CO +1 p:i > H EH i^i^oooooor^'^oooor^i^cyioooooooooo 04 VDVDOO'«*OOVOVDOOrOVOVOOOOOOOOfO(^ H CO Pi >H l>H vocrivorHocNvofooinininvDvo =H= HCNoo^invor-oocTiOHOMoo rj" in ijD r^ 00 cTi o rH rH H H rH rH CM 31 TABLE 5 DEGREE OF CORRELATION BETWEEN SCALE NUMBER AND VERTEBRAL NUMBER Locality Correlation Little Silver Creek, Tex. Method One—Pooled values for scale # vertebrae # each locality-equal weight LS 1 42 28 n=4 d.f.=2 LS 2 45 30 LS 3 46 29 r^2 = 0.959* LS 4 46 30 LS 5 48 30 Y 45.4 29 ^.05 (2)= 0.950 t.01(2)= °-950 Dbl. Mtn. Fork, Brazos, Post, Tex. scale # vertebrae # p 1 45 Method Two--Pooled P 2 46 29 localities P 3 50 28 n=18 d.f.=16 P 4 41 29 P 5 43 29 ^12 = 0.611** Y 45. 0 28. 75 '^.05(16)= 0.468 .01(16)= 0-590 Pease Riv., Roaring Sp., Tex. scale # vertebrae # RS 1 46 30 RS 2 47 29 RS 3 51 30 RS 4 50 RS 5 49 29 Y 48.6 29.5 Smokie Hill River, Kansas scale # vertebrae # SH 1 57 30 SH 2 58 30 SH 3 57 30 SH 4 58 32 SH 5 54 32 Y 56.8 30.8 CHAPTER IV DISCUSSION Most of the characters analyzed in this study exhibited remarkably little geographic variation. Variation in pec toral ray number, dorsal ray number, anal ray number, prin cipal caudal ray number, and vertical bar number (males) appeared to be for the most part of a random nature. For each character there are localities with mean values that appear to be significantly different from those of certain other localities, but no consistent geographic pattern is indicated by these differences. The localities showing the greatest divergence for one character may exhibit almost no difference for another. The two characters, eye diameter and lateral scale num ber, exhibit geographic variation in terms of a north-south cline. Eye diameter decreases with increasing north lati tude (Fig. 5) while scale number increases with increasing north latitude (Fig. 3). The expression of these characters is related to their location at a statistically, highly significant level. While the cause of these clines may well be of a ge netic origin there is persuasive evidence that one or both characters may be under environmental influence. Salinity and water temperature, as well as other environmental 32 33 factors, can modify morphological appearance in some ecto- thermal vertebrates and invertebrates. Previous workers have found that temperature in particular can affect pheno typic expression in fishes. One of the earliest workers to discuss the effects of temperature was Garman (1895). In the very paper that he decrees F. kansae a species distinct from F. zebrinus he implies that the characters that dis tinguish them may not be valid. The following paragraph is a direct quote from his paper: By recent discussion attention has been directed to a decrease in the number of vertebrae, of fish in general, in and toward the torrid zone, and sev eral theories have been propounded to account for the phenomenon. The species of this family (Cyprinodontidae) and others have been somewhat carefully studied,-first to determine the facts, and second, to test the theories . . . It is true a decrease obtains (vertebral number), with few exceptions, in the direction of warmer waters, but warmth of water is attended by both increase in the amount of food and decrease in the need of it, thus lessening the comparative activity of the species. Some would ascribe the differences directly to natural selection. This hypothesis of course cannot be proved; it begs the entire question. It is also found that with the decrease in number of vertebrae, there is in some cases a decrease in the number of fin rays and scales. A considerable amount of experimental work has been done by various workers which tend to support and explain some of the basic ideas of Garman. Gabriel (1944) found that in F. heteroclitus vertebral number was higher in fish hatched at lower temperatures and, conversely, was lower at higher temperatures. Svardson (1952) in working with the 34 coregonids found that by transplanting these fish he was able to demonstrate the environmental plasticity of certain characters. He indicated that scale number could be af fected by temperature and nutritional state of the female parent. Taning (1952) delved deeper into the problem and was able to relate the effect of temperature on the pheno typic expression of a certain character to a particular time frame in the embryological life of the fish. Salinity may also have an effect upon expression of scale number. While Taning found that vertebral number is set before the vitelline membrane becomes permeable to salts Motley (19 34) , in working with Salmo kamloops, found that scale number was set later in ontogeny. The vitelline mem brane, may at this time, be permeable to various ions. It is apparent that environmental factors may affect the development of certain morphological characters in fishes, although these factors may not have the same effect in all species. Only future work will provide the exact cause of clinal variation in lateral scale number and eye diameter in Fundulus. For the present it can only be said that although genetic differences may be involved, there is reason to suspect that the variation is the result of the direct effect of some component of the environment. Only two of the electrophoretic systems studied gave sufficient resolution to be considered reliable. These' two 35 systems, hemoglobins and esterases, were run repeatedly and all results were the same--no difference could be observed at the molecular level. In comparing the electrophoretic bands with another member of the same family, Cyprinodon rubrofluviatilis, obvious differences were noted. On the basis of the evidence of this study, there seems no justification for maintaining any taxonomic distinction between the two fish. They quite obviously do not merit separation at the specific level and there does not appear to be sufficient justification for separation at the sub- specific level. Lateral scale number and eye diameter are the only characters studied which show appreciable varia tion and this is of a clinal nature. This geographic varia tion could be an expression of environmentally influenced phenotypic plasticity rather than an expression of genetic differences. Various workers, with other fish, have shown that scale number can be influenced by various components of the environment. Though the effects of various environ mental components upon eye diameter have not been investi gated it may well be that the same forces are operating in this case. In accepting the fact that the two fish are conspecific the problem of what they should properly be called then arises. After a thorough review of the literature it was concluded that Fundulus zebrinus Jordan and Gilbert stands 36 as the first available name for the fish in question. The following sijmmary of the literature is offered in support of that conclusion. It also provides a taxonomic history of the fish. Fundulus kansae=zebrinus v;as first recorded by Dr. C. M. Girard (1859). The Fish were collected by a Lieuten ant J. C. Ives, the locality being listed as "between Fort Defiance and Fort Union, New Mexico." Girard proposed the taxon, Hydrargyra zebra, "in allusion to the numerous lat eral bars." The generic allocation, however was incorrect. Giinther (1866) assigned the name, Fundulus zebra, to the fish and gave its distribution as the "upper affluents of the Rio Grande del Norte." Jordan and Gilbert (1882) again listed the fish of Girard, calling it F_. zebra. In the same publication, however, F. zebra was also listed as a synonym of F. adinia. In the addendum to their work of 1882 (p. 891), Jordan and Gilbert noted that Fundulus zebra was pre occupied in the genus and proposed the replacement name, Fundulus zebrinus. Gilbert (1884) gave a full description of F. zebrinus based on three specimens from Ellis, Kansas. He stated that they were the first specimens seen since the original discovery of the species in 1859. The name, Fundulus kansae, was proposed in 1895 by Garman who placed it in a separate subgenus (Plancterus). He indicated that this fish was not the one to which F. zebrinus was first 37 applied. The type locality was given as Kansas. Jordan and Evermann (1896), however, stated that: . . . There is no doubt that the original F zebra is the species called zebrinus by us and ka^nsae by Garman. It came from some point between 'Fort Union and Fort Defiance.' In other words, it came from the headwaters of the Canadian River or the Rio Grande. No species of this type has been re corded from the upper Rio Grande, but the species called zebrinus and kansae is in all the upper waters of the Arkansas basin, to which the Canadian River belongs, and doubtless in the streams above Fort Union. In the same paper Jordan and Evermann suggestec3 that the subgenus (Plancterus), possibly merited generic status. Carl Hubbs (1926) did elevate Plancterus to generic rank and included two species, P. zebra and P_. kansae, the former with 41 to 49 scale rows, a more robust body and larger eyes--the latter with 52 to 64 scale rows. The name, Plancterus, was subsequently used by Koster (1948) in a description of the spawning activities of the killifish. In Knapp's work of 1953, however, the generic epithet, Fundulus, was used. He listed F. zebrinus with a distribution that included the Rio Grande and tributaries from Brownsville to New Mexico, and F. kansae with a distribution that included the upper Arkansas River basin and upper parts of the Red, Colorado, and Brazos Rivers. Miller (1955) reviewed the genus, Fundulus, and denounced Hubbs' recognition of Plancterus. He gave the distribution of F. zebrinus as the upper por tions of the Colorado, Brazos, and Pecos River drainage, 38 and that of F. kansae as from South Dakota to Texas (Red River) and New Mexico (Arkansas River). Subsequent authors have followed Miller. It seems clear that the name Fundulus zebrinus, pro posed by Jordan and Gilbert in 1882 as a replacement name for the Fundulus (Hydrargyra) zebra of Girard, is the earliest available name, predating the name, Fundulus kansae, by some thirteen years. The name of the Plains killifish, therefore, should be Fundulus zebrinus Jordan and Gilbert. CHAPTER V SUMMARY According to the current literature, there are two species of the genus Fundulus in the saline alkaline waters of the Central Plains region of the United States. These fish are F. zebrinus and F. kansae. These forms have been distinguished on the basis of lateral scale number and eye diameter. Recently, several authors have suggested the possibility of conspecificity between the two fish. An analysis was made of variation in a number of char acters in order to determine the systematic status of the two forms. Emphasis was placed on morphological analysis. Ten characters were analyzed, either singly, or in combina tion if size was a factor (i.e., eye diameter v.s. standard body length). Only two characters yielded variation of in terest. Both lateral scale number and eye diameter vary in a clinal manner. Relative eye diameter decreases in a north ward direction on a north-south axis while scale number in creases in the same direction. This variation seems to be in no way associated with the various drainage systems or with the presumed distributional limits between the two fishes. Direct environmental effects rather than genetic differences may be the cause of this variation. Electrophoretic analysis of two protein systems, hemo globins and esterases, failed to reveal any differences in 39 40 the fish. There was no indication of molecular variation among the various localities studied, nor was there evidence of isozyme polymorphism at any particular locality. It was concluded that there is no evidence to support separation of £. kansae and F. zebrinus as distinct species. A review of the taxonomic literature indicates that the name Fundulus zebrinus Jordan and Gilbert should be applied to the populations formerly included in F. zebrinus and F. kansae. LITERATURE CITED Blair, W. F., A. P. Blair, P. Brodkorb, F. R. Cagle, and G. A. Moore. 1968. Vertebrates of the United States. McGraw-Hill Book Company, New York, 616 pp. Eddy, Samuel. 196 9. How to know the fresh water fishes. Wm. C. Brown Company Publishers, Dubuque, Iowa, 286 pp. Fine, I. H., and L. A. Costello. 1963. The use of starch electrophoresis in dehydrogenase studies. In Colowick, S. P., and N. O. Kaplan (eds.), Methods in enzymology, Vol. 6, Academic Pre»s, New York. Gabriel, M. L. 1944. Factors effecting the number and form of vertebrae in Fundulus heteroclitus. J. Exp. Zool., 95:105-147. Garman, Samuel. 189 5. The Cyprinodonts. Mem. Mus. Comp. Zool., Harvard Coll., 19:1-179. Gilbert, C. H. 1884. Notes on the fishes of Kansas. Bull. Washburn Lab. Nat. Hist., 1:15. Girard, Charles M. 1859. Ichthyological notices. Proc. Acad. Nat. Sci. Phil., pp. 60-61. Giinther, A. 1866. Catalog of the fishes in the British Museum, Vol. 6, 8 8 pp. Hubbs, Carl L. 1926. Studies of the fishes of the Order Cyprinodontes. VI. Material for a revision of the American genera and species. Misc. Publ. Mus. Zool. Univ. Mich., 16:1-86. Jordan, D. S., and B. W. Evermann. 1896. The fishes of North and Middle America. Bull. U.S. Nat. Mus., 47, 4. vols., 3,313 pp., 392 pis. Jordan, D. S., and C. H. Gilbert. 1882. Synopsis of the fishes of North America. Bull. U.S. Nat. Mus., 16, 8 vols., 1,074 pp. Knapp, F. T. 1953. Fishes found in the fresh watery of Texas. Ragland Studio and Litho Printing Company, Brunswick, Georgia, 166 pp. Koster, W. J. 1948. Notes on the spawning activities and the young stages of Plancterus kansae (Garman). Copeia, 1948(1) :25-33. 41 42 1957. Guide to the fishes of New Mexico. Univ. New Mexico Press, Albuquerque, New Mexico, 116 pp. Metcalf, A. L. 1966. Fishes of the Kansas River system in relation to zoogeography of the Great Plains. Mus. Nat. Hist., Univ. Kans. Publ., 17 (3) : 23-189. Miller, Rcpbert R. 1955. An annotated list of the American Cyprinodontid fishes of the genus Fundulus, with the description of Fundulus persimilis from Yucatan. Occ. Papers Mus. Zool., Univ. Mich., 568:1-25. Mitchell, R. W., and R. E. Smith. 1971. Some aspects of the osteology and evolution of the neotenic spring and cave salamanders (Eurycea, Plethodontidae) of central Texas. Tex. J. Sci., 23:343-362. Mottley, C. McC. 1934. The effect of temperature during development on the number of scales in Kamloops trout, Salmo kamloops Jordan. Contr. Canad. Biol., 8:254-263. Pflieger, William L. 1971. A distributional study of Missouri fishes. Mus. Nat. Hist., Univ. Kans. Publ., 20 (3) :225-570. Smithes, O. 1959. Zone-electrophoresis in starch gels and its application to studies of serum proteins. Advan. Protein Chem., 14:65. Svardson, G. 1952. The coregonid problem. IV. The sig nificance of scales and gill rakers. Inst. Freshwater Research, Drottningholm, No. 33:204-232. Tuning, A. Vedel. 1952. Experimental study of meristic characters in fishes. Biol. Rev., 27:169-193. Taylor, W. R. 196 7. An enzyme method of clearing and staining small vertebrates. Proc. U.S. Nat. Mus., 132(3596):1-17.