W. L Minckley a Revision of the Gambusia Nobilis Species Group, with Descriptions of Three New Species, and Notes on Their Variation, Ecology, and Evolution

13 REPRINTED FROM THE TEXAS JOURNAL OF SCIENCE, VOL. IX, NO. 3, SEPTEMBER, 1957, ." 1 W. L Minckley A Revision of the Gambusia nobilis Species Group, With Descriptions of Three New Species, and Notes On Their Variation, Ecology, and Evolution

by CLARK HUBBS and VICTOR G. SPRINGER The University of Texas

In the last review of the Gambusia nobilis species group, Carl L. Hubbs (1929) recognized four species: G. nobilis (Baird and Girard), G. senilis Girard, G. gaigei Hubbs, and G. affinis Baird and Girard. The species with which the name affinis was associated then only, is here- in accorded a new name, G. geiseri. True G. affinis does not belong to this species group. Two other previously unreported forms, G. hurtadoi and G. alvarezi, are also described. The variation, ecology, and evolution in the several species are discussed. Carl L. Hubbs (1926) included three genera ( Heterophallus Regan, Belonesox Kner, and Gambusia Poey) in the tribe Gambusiini. He divided the genus Gambusia into four subgenera ( Heterophallina Hubbs, Gambusia Poey, Arthrophallus Hubbs, and Schizophallus Hubbs). The primary dichotomy within the subgenus Gambusia in his key divided the forms from southern Mexico and the Antilles from those inhabiting northeastern Mexico and southwestern United States. Later (1929) he called the latter fishes the nobilis group. He distinguished these fishes from the other species of Gambusia by: "The distal spines of ray 3 (of the modified male anal fin) are very long, projecting far beyond the hook at tip of ray 4; the largest segment is about equal to the combined basal length of all the spine-bearing segments. Other common characters more or less diagnostic of the nobilis group are the development of a diffuse lateral band extending from eye to caudal base, and of dusky or black markings on the anal fin of the female. The dorsal rays are usually 8, sometimes 7 or 9." We concur with this description with the following modifications: the largest segment of the spines of ray 3 may even exceed the combined basal length of all spine-bearing segments, and the anal rays are usually 7 to 9, sometimes 10. 279 280 THE TEXAS JOURNAL OF SCIENCE KrumHoltz (1948) stated that Hubbs and Walker (unpublished MS) consider that the two nominal species (affinis and holbrookii) comprising the nominal subgenera Arthrophallus and Schizoph.allus respectively, regularly integrade in nature and therefore are to be assigned to the same species. Haskins and Rosen (unpublished MS) also have demonstrated integradation between the two nominal species. On the basis of gonopodial types, Rosen and Gordon (1951) divided the tribe Gambusiini into five "species groups." Despite their statement of the need for a new revision of the Gambusiini, their five gonopodial types represent the three subgenera of Gambusia and the two other gambusfine genera currently recognized.

ACKNOWLEDGEMENTS We are indebted to Drs. Reeve M. Bailey and Robert R. Miller of the Museum of Zoology, University of Michigan, as well as Mr. Loren P. Woods of The Chicago Natural History Museum for the loan of specimens. Dr. Ernest A. Lochner provided information on specimens in the U.S. National Museum. Mr. Oscar f. Wiegand assisted in several collections and served as a translator during a collecting trip in December, 1954, and January, 1955. Dr. Kirk Stravvn, Dr. William F. Pyburn, Mr. Chesley Woods, Mr. William H. Brown, Dr. David Pettus, Mr. Jerome Dorf, and Mr. Elgin M. C. Dietz, assisted in Collecting specimens in Texas. Mr. Pablo Guzman-Rivas collected specimens of fishes from three localities in the Rio Sota la Marina drainage. Dr. Jose Alvarez del Villar and Sr. Jorge Carranza of the Direccion General de Pesca aided in obtaining a permit for collecting in Mexico. Sr. Ing. Leopoldo Hurtado Olin of the Department° de Economia of Chihuahua assisted the June field trip and informed us of the location of El Ojo de la Hacinda Dolores. Dr. Ross Maxwell, Mr. Lemuel A. Garrison, both former Superintendents of the Big Bend National Park, Mr. George W. Miller, presently Superintendent of the park, and Mr. John Palmer, formerly District Ranger at Bo- quillas Ranger Station, gave information on warm springs in the park. Sr. Eduardo Rojas of Valle de Allende gave much information on the post-1900 changes of water connections resulting from irrigation. He also informed us of the location of El Ojo de San Gregorio. Sr. Jesus Pallares of La Escuela Normal de Salicias gave information on the present flow of irrigation water. Sr. Raul Miramontes of El Sauz informed us on the disappearance of the Rio Sauz. Mr. William E. Humphrey of DeGolyer and MacNaughton, Inc., gave information on the geology of the Estaci6n Troya region. Dr. William J. Koster provided locality data for G. nobilis in New Mexico. Drs. Carl L. Hubbs A REVISION OF THE GAMBUSIA NOBILIS SPECIES GROUP 281 and Royal D. Suttkus have read and criticized this manuscript. Dr. Hubbs has also examined specimens in the U.S. National Museum, University of Michigan Museum of Zoology, and the Museum of Com- parative Zoology, and provided color notes on living Gambusia nobilis. The figures and descriptions of the gonopodial suspensorium in these species are abstracted from an unpublished manuscript by Luis Howell Rivero and Carl L. Hubbs. The other drawings were mare by Mrs. Grace H. Groce and Miss Nancy Walker. Mr. George G. Hender- son, Jr., made the photographs. Dr. Kirk Strawn and Mr. Walter E. Fosberg assisted by feeding the living stocks. Miss Gail Kaufman has conducted the mate preference experiments. The project is supported by two grants from the National Science Foundation.

METHODS The measurements and counts used in this work are those defined by Hubbs and Lagler (1947). The rays of the gonopodium are num- bered 3 to 5 and the anterior and posterior branches are indicated by the symbols 4a, 4p, and 5a. All dorsal and anal rays are counted, including the two small anterior anal rays, and the last two elements are counted as one. The special designations for gonopodial structures are labelled on figure 1. Many of the specimens are catalogued at several museums. The following abbreviations for these museums are used; CNHM—Chicago Natural History Museum; ENCB—Escuela Nacional de Ciencias Bio- logicas of Mexico; MCZ—Museum of Comparative Zoology (Harvard University) ; SU—Stanford Natural History Museum; TNHC—Texas Natural History Collection; UMMZ—University of Michigan Mu- seum of Zoology; and USNM—United States National Museum. We have virtually omitted proportional measurements from the species descriptions. Our laboratory experiments with five of the species in this group have shown that these characters may vary con- siderably due to environmental influence. For instance, the caudal peduncle depth of wild G. hurtadoi is contained 1.1 to 1.3 times in the caudal length and 0.8 to 0.9 times in laboratory-raised fish (kept under very favorable nutritional conditions). In general, laboratory-raised stocks have deeper bodies and shorter fin rays (see figure 13 and 14) than do wild fish. However, such morphometric differences between the species are undoubtedly in part of genetic origin. Wild or laboratory-raised G. gaigei are more deep-bodied than their G. hurtadoi counterparts, although laboratory-raised G. hurtadoi are more deep- bodied than wild G. gaigei. We have not noticed any variation of gonopodial structure between laboratory stocks and wild specimens 3 S

Sa Sp ::

SERRAE

ELBOW

SPINE TERMINAL HOOKS

Fig. 1. Anal fin rays of laboratory raised Gambusia hurtadoi male (TNHC 45861 showing special designations of gonopodial structures. A REVISION OF THE GAMBUSIA NOBILIS SPECIES GROUP 283 of the same form. Likewise, although laboratory fish are paler than wild ones, their basic colors and color patterns are similar. All color markings used (except in G. nobilis, which has not been kept in our laboratory) are evident in laboratory-raised individuals and are thus considered genetic. Variations in fin-ray numbers attributable to the environment are less than the differences between species. In tables 2 through 5 all fish with no trace of gonopodium on 10 to 45 X magnification are tallied as females. The dorsal rays of some females were not counted. Immature males are those whose gonopodium is more or less elongated but not fully developed. Key to Wild Adults of the Gambusia nobilis Species Group la. First and/or second proximal enlarged spines on ray 3 of gonopodium with recurved hook (Fig. 2, A and B) ; terminal hooks of rays 4p and 5a angular at tip; dorsal rays usually 7 (occasionally 8) ; postanal streak prominent (darker than markings on scale pockets); a median row of spots on caudal fin and some at its base; a median row of spots on dorsal; in females distance from front of dorsal to caudal equal to distance from front of dorsal to first predorsal body scale; caudal peduncle depth in females contained 1.5 to 1.8 times in caudal fin length ...... G. geiseri lb. None of the enlarged spines on ray 3 of gonopodium with recurved hooks; terminal hooks of rays 4p and 5a rounded at tip; dorsal rays S or 9 (occasionally 7 or 10) ; postanal streak weaker than markings on scale pockets; no prominent spots on caudal; either no spots or a subbasal row on dorsal; in females distance from front of dorsal to caudal equal to distance from front of dorsal to second or third predorsal body scale; caudal peduncle depth contained 0.8 to 1.4 times in caudal length ...... 2 2a. Ray 4a of gonopodium extends to or beyond tip of 4b (Fig. 2, C and D) ; lateral stripe thin and threadlike; caudal with dark margin; dark margins on scale pockets the most prominent color; markings on side rounded - specks; predorsal streak strong ...... G. nobilis 2b. Tip of ray 4a does not extend to tip of 4b but approximately to base of terminal hook of 4b; lateral stripe broad (of variable intensity) ; caudal without prominent dark margin; dark markings on scale pockets either weak or obscured by other color markings; markings on sides crescentic or absent; predorsal streak weak to strong ...... 3

284 THE TEXAS JOURNAL OF SCIENCE

I 111 LT1:,,S\ ollanimpip4 4au e

Fig. 2. Distal tips of gonopodia of males of three species of Gambusia. A + B. G. geiseri from TNHC 5. C + D. G. nobilis from UMMZ 137101. E-H. G. senilis from TNHC 4209. A REVISION OF THE GAMBUSIA NOBILIS SPECIES GROUP 285 3a. Elbow on ray 4a of two to four segments (Fig. 2, E—G) ; dark markings on edges of scales below midline prominent, more so than any above midline; no chin bar; no black spot around anus ...... G. senilis 3b. Elbow on ray 4a of one or rarely two segments (Fig. 3) ; dark markings more frequent above midline, never on abdomen; a weak to strong chin bar; a dark spot around anus, at least in females ...... 4 4a. Dorsal rays 8 (rarely 7 or 9) ; suborbital bar weak; chin bar faint (scarcely darker than adjacent color) ; anal spot of females restricted to anus; few crescentic lateral marks; predorsal streak weak to absent ...... G. gaigei 4b. Dorsal rays 9 (rarely 8 or 10) ; suborbital bar prominent; chin bar usually well developed (markedly darker than adjacent color) ; anal spot of females seldom restricted to anus; many crescentic lateral marks; predorsal streak mod- erate ...... 5 5a. Crescentic marks in a regular pattern; black margin of dorsal lighter than subbasal row of spots; ventral surface iridescent blue ...... G. hurtadoi 5b. Crescentic marks irregular; black margin of dorsal darker than subbasal row of spots; ventral surface 'yellowish ....G. alvarezi

Gambusia geiseri HUBBS AND HUBBS, NEW SPECIES' Gambusia patruelis, Jordan and Gilbert; 1886: 22, 24 (in part; Rio San Marcos and Rio Cornal, Texas) ; Evermann, 1892: 88 (San Marcos and Coma! Creek specimens, in part). Gambusia affinis, Evermann and Kendall, 1894: 107 (literature in part) ; Carl L. Hubbs, 1926: 33 (synonomy in part; description; San Marcos) ; 1929: 1 (comparison with G. gaigei), Clark Hubbs, Kuehne, and Ball, 1953: 232 (one misidentified record). Gambusia senilis, Geiser, 1923: 197, figs. 15-18. Gambusia sp. Clark Hubbs, Kuehne, and Ball, 1953: 237, fig. 4 (range; including some specimens of G. affinis speciosa) ; Jurgens and Hubbs, 1953: 14; Clark Hubbs, 1957b: 8. This description has been prepared by Carl L. Hubbs and Clark Hubbs.

286 THE TEXAS JOURNAL OF SCIENCE

A B

C D

E F

Fig. 3. Distal tips of gonopodia of males of three species of Gambusia. A. G. gaigei from TNHC 4213. B. Laboratory-raised G. gaigei from TNHC 4626. C. G. hurtadoi from TNHC 4205. D ± E. Laboratory-raised G. hurtadoi from TNHC 4203. F. Wild G. hurtadoi from TNHC 4205. G ± H. G. alvarezi from TNHC 4207. A REVISION OF THE GAMBUSIA NOBILIS SPECIES GROUP 287 Description. Dorsal rays 7 (rarely 8) ; anal rays 9 or 10; scale rows 28 to 32; scale rows around caudal peduncle 16. This species has the most slender body and most anterior dorsal of any member of the species group. The tip of the gonopodium is unique in that it is the only member of the G. nobilis species group that has recurved hooks on the proximal spines of ray 3 and in which the terminal hooks on rays 4p and 5a are more or less angular at the tip (this condition occurs in other Gambusia species that are not in the G. nobilis group) . As in G. nobilis, ray 4a extends to near the tip of the gonopodium. Gambusia geiseri resembles G. gaigei, G. hurtadoi, and G. alvarezi in that the gonopodial elbows of a large percentage of the specimens are composed of only one segment, but it differs from those species in that more than half of the males have a two-segmented elbow. The most distal serra of 4p is one segment proximal to the elbow. The gonopodial suspensorium of Gambusia geiseri is illustrated in figure 5, taken from a manuscript by Luis Howell Rivero and Carl L. Hubbs, who have indicated that this structural complex is of prime value in poeciliid taxonomy (Howell Rivero and Hubbs, 1936, and Howell Rivero, 1946). The following description is based on a paratype from San Marcos. There are three gonapophyses, but the third is scarcely developed (it is well developed in some species) . The first is

Fig. 4. A. 20 mm. Gambusia geiseri male from TNHC 2651. B. 34 mm. G. geiseri female from TNHC 2651.

288 THE TEXAS JOURNAL OF SCIENCE

BRAE GAUD 4

SU SPENSORY LIGAMENT AIR B LADDER

AIR BLADDER

Fig. 5. Gonopodial suspensorium of a male Gambusia geiseri collected from San Marcos, Texas. weakly curved downward, with a very small rib-bearing parapophysis. The second gonapophysis, which lacks a parapophysis and rib, is broadly triangular with a straight axis and a long, slender uncinatoid process. The weak third gonapophysis is only about half as long as the following haemal spine and ends in a slight knob with a small median backward process. The air bladder extends under the first two caudal vertebrae. In the compound interhaemal, the lateral edges of the second interhaemal flare to form an anterior groove which be- comes funnel-like distally when the lateral edges unite. The color pattern of this species is rather consistent and distinctive. The ground color is silvery with yellowish and iridescent blue overtones. The pronounced dark margins of the scale pockets on the body produce a cross-hatched appearance. Dark scale pocket markings are not evident in the area anterior to the anal base and below the eye. They are obscured by the darker postanal streak, the middorsal streak, and the dark coloration over the neurocranium. There is a fine axial black streak which may be supplemented by a wider but fainter dark band. The body is covered by specks at the posterior end of the scales (most frequently in smaller fish). There is no black spot around the anus in the females. The dorsal and caudal have rows of dark spots A REVISION OF THE GAMBUSIA NOBILIS SPECIES GROUP 289 one-third to two-thirds of the way out on the rays (often absent in small individuals). Spots often occur at the caudal base. The anal in females often has a faint median row of spots and a grayish margin; in males it is grayish. Both lips are dark, especially in adults, but the darkness of the upper lip is slightly obscured by the darkened top of the head. There is no darkening of the lower parts of the head posterior to the middle of the eye, i.e., the suborbital bar is absent. Range. Gambusia geiseri is known from nine localities (Fig. 6), all in Texas: San Marcos Springs and the adjacent section of the San Marcos River; Comal Springs and adjacent section of Comal Creek; in the headwaters of Bear Creek, Comal County (misidentified as G. affinis by Clark Hubbs, Kuehne, and Ball, 1953) ; the head springs of the South Fork of the Guadalupe River, 8 miles southwest of Hunt, Kerr County; the head springs of the South Concho River, 4 miles

_ • 0 gemeri II G. nobilis • G. goiget • G. sanilis U G. hurtodoi X G. olvoresi

— 30

— 25

23 109 96

Fig. 6. Recorded collection localities of the six species of the Gambusia nobilis species group. Doubtful published localities are followed by a question mark.

290 THE TEXAS JOURNAL OF SCIENCE south of Christoval, Tom Green County; the head springs of the Dev- il's River, Lat. 30° 03' N., Long. 101° 00' W.), Val Verde County; an irrigation ditch, 1 mile east of Toyahvale, Reeves County; Comanche Springs at Fort Stockton and vicinity; and Tunis Springs, 20 miles east of Fort Stockton. No other fish species has so disjunct a range in Texas. Specimens were collected from the two localities along the Balcones Escarpment during the nineteenth century. Seven specimens of Gambusia nobilis were the only mosquitofish taken in the Trans- Pecos springs during the boundary survey from 1851 to 1853. The first record of G. geiseri in these springs was in 1937, when C. Basil Jordan collected two at Fort Stockton. The next year Carl L. Hubbs and party collected many more along with a series of G. nobilis. On October 8, 1956, Clark Hubbs and Jerome Dorf collected over 600 specimens but no G. nobilis. In 1940, Leo T. Murray and party collected some from Tunis (Escondido) Springs 20 miles east of Fort Stockton. On October 13, 1956, Clark Hubbs and Jerome Dorf collected forty-four specimens of G. geiseri and only five of G. nobilis from an irrigation ditch near Toyahvale. In this area many collections that do not contain G. geiseri specimens have been made. As the first collections at the remaining localities were made since 1950 no data are available on historical faunal changes there. All of these collections were made after the fish hatchery in San Marcos had stocked game fishes in the area. Moreover, as Dr. C. P. Coogle of the Texas State Board of Health informed Carl L. Hubbs in 1938, mosquito fish from San Marcos had been distributed for mosquito control to many sections of the state. These circumstances, along with the unique distribution and the late discovery of the species in localities other than along the Balcones Escarpment support our belief that these stocks were brought there from San Marcos, directly or indirectly. It must be remembered, however, that few fish collections were made in Texas between 1853 and 1937, and that only seven mosquitofish were collected from the suspected localities in the nineteenth century. The record from the head springs of the South Fork of the Guada- lupe River strongly indicates that introduced G. geiseri from San Mar- cos might survive in spring-fed waters. This region was extensively collected during 1951 and no specimens were obtained (Clark Hubbs, Kuehne, and Ball, 1953). Since that time more than 100 additional collections have been made in that region, 15 of them at that locality, without any G. geiseri having been collected. On January 14, 1956, we released between 100 and 150 individuals collected at San Marcos. As a cold wave was prevalent over Central Texas at that time, the A REVISION OF THE GAMBUSIA NOBILIS SPECIES GROUP 291 environment was not highly favorable to the survival of introduced spring-inhabiting fishes. However, on July 17, 1956, Drs. Kirk Strawn and Clark Hubbs collected several young and adult G. geiseri. Some of the Tunis Springs males are larger than any from San Marcos. However, we suspect that different conditioning during ontogeny may cause the differences, as most of our laboratory-raised males of this species group are larger than any wild males. Both G. geiseri and nobilis have been collected together in Comanche Springs and in irrigation ditches near Toyahvale, indicating definitely that they are distinct species. In both San Marcos and Comal Springs G. geiseri lives with populations of G. affinis. Literature.—Geiser (1923), who was the first to recognize this fish as distinct from G. affinis of Baird and Girard, identified it as G. senilis. Carl L. Hubbs (1926) determined that this form was distinct from G. senilis. He knew that Baird and Girard (1854) had proposed two names, affinis and patruelis, for mosquitofishes in southcentral Texas. On the basis of Girard's descriptiohs and figures, he assumed that the form here described was widely distributed in that area and attempted to match specimens with the incomplete descriptions. Since the original description of G. affinis fits the specimens of the present species rather better than it does that of G. patruelis, and since no type specimens of affinis had been located, he called this fish G. affinis. Later, on finding in the Academy of Natural Sciences, Philadelphia, two cotypes of Heterandria affinis (from "Rio Medina and Rio Salado") he real- ized that the name affinis had properly been applied to the wide- ranging species of the Gulf of Mexico drainage basin, and assigned the species from San Marcos the name geiseri, but did not publish a description of it. On his advice the name affinis was properly applied by several authors, including D'Ancona (1939) who definitely resynony- mized patruelis with affinis affinis, and Krumholz (1948) who briefly recounted the confusion. Clark Hubbs, Kuehne, and Ball (1953) wrongly included specimens of G. affinis in their report on this species. The misidentified specimens differ from typical G. affinis and are referable to the nominal subspecies G. a. speciosa (Girard). They incorrectly postulated the extinction of that form in Comal Springs. Material. The 17 mm. male holotype (U1VIMZ 168974) was collected by Kirk Strawn and William F. Pyburn from the San Marcos River just downstream from the Rio Vista Dam in San Marcos, Texas. Four hundred thirty-five paratypes (TNHC 5, 19, 60, and 2651, UMMZ 72565, 72570, 72571, 108090, 111005, 120247, 166018, and 166043) were collected from various localities on the San Marcos 292 THE TEXAS JOURNAL OF SCIENCE River between Spring Lake (the head spring) and the Cotton Gin, one and three-quarters miles south of the head spring at Brown School. The other three thousand six hundred and twenty-seven specimens are not designated as paratypes as considerable morphologic variation has been noted between specimens from different localities: one hundred and seven (TNHC 73 and UMMZ 120262) from the San Marcos River at Martindale, six and one-half miles southeast of San Marcos, Texas; one hundred and fifty (TNHC 2328, 2344, and 2351 and UMMZ 111012) from Comal Creek at New Braunfels, Texas; eight (TNHC 2991) from Bear Creek nine and one-half miles northwest of New Braunfels, Carnal County, Texas; one (TNHC 4569) from the South Fork of the Guadalupe River eight miles southwest of Hunt, Kerr County, Texas; one thousand two hundred and five (TNHC 3115 and 4559) from the South Concho River four miles south of Christo- val, Tom Green County, Texas; seventy-three (TNHC 4231) from the head springs of the Devil's River, nine miles southwest of Juno, Val Verde County, Texas; forty-four (TNHC 4863) from an irrigation ditch one mile east of Toyahvale, Reeves County, Texas; one thousand seven hundred and sixty-six (TNHC 4866 and UMMZ 103550, 120357, and 120358) from Comanche Springs and vicinity at Fort Stockton, Texas; and 273 from Tunis Springs 20 miles east of Fort Stockton. Named geiseri for Dr. Samuel W. Geiser of Southern Methodist Uni- versity, in recognition of his studies on this and other species of the genus Gambusia.

Gambusia nobilis (BAIRD AND GIRARD) Heterandria nobilis Baird and Girard, 1853: 390 (described from Co- manche and "Leona" Springs). Gambusia nobilis, Girard, 1859: 71, pl. 39, figs. 8-11 (description, Leon's Spring, Comanche Spring, "Zoquito," Texas) ; 1859b: 120 (Comanche Spring) ; Gunther, 1866: 33; Jordan and Gilbert, 1883: 346 (description, "Texas") ; Carl L. Hubbs, 1926; 32 (synonomy, Sulphur Spring, south of Sulphur Lake, not located by us; North Spring River near the northwest limits of Roswell; and from north of Hondo, all in New Mexico) ; 1929: 1 (description, comparison with G. gaigei, Zimmerman Reservoir near Imperial, and 1 and 1.5 miles northwest of Toyahvale, all in Texas) ; Jordan, Evermann, and Clark, 1930: 185 (range) ; Miller, 1951: 104 (range, Pecos River drainage of New Mexico and western Texas) ; Rosen and Gordon, 1953: fig. 31a (drawing of gonopodial tip) ; Jurgens and Hubbs, 1953: 14; Clark Hubbs, 1957b: 8. A REVISION OF THE GAMBUSIA NOBILIS SPECIES GROUP 293 Gambusia nobilis nobilis, Scrimshaw, 1945: 234 (development) ; 1946: 21 (egg size) ; Knapp, 1953:94 (range in part) . Gambusia affinis, Evermann and Kendall, 1894: 107 (literature and range in part). Gambusia gracilis, Garman, 1895: 85 (synonorny in part) . Description. Dorsal rays 8 (rarely 9) ; anal rays 9; scale rows 29 to 31; scale rows around caudal peduncle 16. Gonopodial differences separate all of the adult males examined from those of any of the other members of the species group. There are no recurved hooks on the elongate spines of ray 3. Ray 4a reaches to or beyond the tip of 4p. The elbow is formed of 3 or 4 segments. The distal serra on ray 4p reaches to the distal end of the elbow. The terminal hooks on 4p and 5a are rounded. The gonopodial suspensorium of G. nobilis, described and drawn (Fig. 7) by Luis Howell Rivero from an adult male collected in Phan- tom Lake, Toyahvale, Texas, is similar in pattern to that of G. geiseri, but differs in several details. There are three gonapophyses, but the third is scarcely developed. The first is straight and slightly thickened medially. Its medium-sized parapophysis bears a rib. The second gonapophysis lacks a parapophysis and rib. Its body grades evenly into the thick and straight subhorizontal axis. Its uncinatoid process is long and thin. The weak third gonapophysis is about three-fourths

Fig. 7. Gonopodial suspensorium of a male Gambusia nobilis collected in Phantom Lake, Toyahvale, Texas. 294 THE TEXAS JOURNAL OF SCIENCE as long as the following haemal spine, thick basally, laterally compressed distally, and weakly curved backward medially and distally. The air bladder extends beneath two caudal segments. The compound interhaemal does not exhibit the prominent anterior groove described for G. geiseri. The color pattern is close to that of G. geiseri. The ground color is silvery with wax-yellow and iridescent blue overtones. Individuals are usually lighter and yellower than those of G. geiseri. The pronounced dark margins of the scale pockets give the fish a cross-hatched appearance, although less markedly than in G. geiseri. Dark scale-pocket markings are lacking in the area anterior to the anal base and below the eye, and elsewhere they are obscured by the darker middorsal streak and by the dark coloration over the neurocranium. The postanal streak does not obscure the scale-pocket markings. The fine axial black streak may be supplemented by a wider but fainter dark band. The body has many specks at the posterior end of the scales. The black spot around the anus in females is not restricted to the anus. The dorsal 1 has a subbasal row of spots less than /4 of the way out. The caudal has spots in a faint median row or no spots. The dorsal and caudal have a darkened margin. The anal of females has a darkened margin; that of males is grayish. Both lips are dark, especially in adults, but marks on the upper lip are slightly obscured by the darkened top of the head. The lower lip marking is diffuse on the margins and often has a median posterior extension. Except for the suborbital bar, there is no darkening of the lower parts posterior to the eye. Range. G. nobilis has been collected in many localities in the western tributaries of the Pecos River in Texas and New Mexico (Fig. 6). None has been recognized in the many recent collections from the Pecos River in New Mexico (William J. Koster, personal communication, 1955). The recorded locality nearest to the Pecos River is that of canals from Zimmerman Reservoir near Imperial, Texas (Carl L. Hubbs, 1929). The specimens (UMMZ 89484) were first catalogued as "canals in Pecos system, near Fort Stockton." The phrase "near Fort Stockton" is handwritten on the typed card. Presumably the col- lector, W. A. Clark, further restricted the locality in an oral report to Carl L. Hubbs. Zimmerman Reservoir receives its water supply from the now heavily polluted segment of the nearby Pecos River. During the summer of 1954 when Zimmerman Reservoir and nearby canals were dry, we made eight collections from the Pecos River in Texas. Many specimens of Gambusia affinis were collected, but none of G. nobilis. We suggest four possible explanations for the discrep- ancy: (1) the Zimmerman Reservoir record is based on faulty lo- A REVISION OF THE GAMBUSIA NOBILIS SPECIES GROUP 295 cality data; (2) the Zimmerman Reservoir specimens resulted from introduction as bait or from recent high waters that washed them down from the spring areas; (3) the Pecos River has changed between 1927 and the 1940's, when Koster started his collecting, from one inhabit- able by G. nobilis to one that is too saline or otherwise unsuited for them; or, (4) G. affinis has eliminated G. nobilis in these waters. As the locality label has been modified we suggest the first explanation to be most probable. Girard (1859a) listed two specimens of G. nobilis from Zoquito, Texas. In the original report on the other Boundary Survey specimens, Baird and Girard (1853 and 1854) did not include the Zoquito record. Girard also includes a record of Astyanax argentatus = A. fasciatus from Zoquito. The fishes were listed as having been collected in 1851 by John H. Clark, who was with Col. J. D. Graham. We can find no locality in Texas that is spelled Zoquito. Graham (1852) listed Zoquete Creek of which Girard's name, Zoquito, appears to be a spelling variant. At that time variations in the spelling of place names were common. We have attempted to locate Zoquete Creek. Unfortunately the name is not now listed on modern maps for any creek in Texas. Gra- ham (1852) and Emory (1857) both listed a Zoquete Creek as being between Fort Inge (Uvalde, Texas) and the first crossing of the Rio San Pedro (Devil's River). We have looked at several old maps of the per- tinent area and find Zoquete Creek listed on four of them. Three, Richardson (1867 and 1873) and Spaight (1882), located the name on the stream now known as Pinto Creek and one Roessler (1874), placed the name on the stream now called the East Fork of Sycamore Creek. Apparently the name Zoquete Creek has been applied to both streams. Emory (1857) listed four creeks between the Rio Nueces and Rio San Pedro. They are: Las Moras, Piedras Pintas, Zoquete, and San Felipe. The first and the last still bear those names. Pinto and Sycamore creeks are the two largest streams between Las Moras and San Felipe creeks. Apparently Emory called the present Sycamore Creek by the name Zoquete. However, we believe that Graham's Zo- quete Creek is the modern Pinto Creek. On page 238 Graham listed Zoquete as being 44.9 miles from Fort Inge and 32.1 miles from the lower crossing of the Rio San Pedro. As the course of the wagon road used by Graham approximated that of U. S. Highway 90 in this area, it is possible to get estimates of distances between Uvalde (Fort Inge) and the Devil's Diver (Rio San Pedro) bridge to the pertenent streams. The mileages for Sycamore Creek (55.2 and 21.5) are far less satis- factory than those for Pinto Creek (44.9 and 31.8). Graham's chart of 296 THE TEXAS JOURNAL OF SCIENCE barometric profiles shows eight indentations (stream beds) between Fort Inge and the Rio San Pedro. Zoquete is identified as the fifth. We have calculated the proportional distance of each indentation (stream bed) from Fort Inge and find that they approximate the locations of the following streams respectively: Nueces River, Turkey Creek, Elm Creek, Las Moras Creek, Pinto Creek, Sycamore Creek, Sacatosa Creek, and San Felipe Creek. Pinto Creek has the same relative locality as Zoquete Creek. It is possible that Perdido Creek (a smaii tributary of Pinto Creek) can be Zoquete. However, the stream bed of Perdido Creek is not pronounced and has been dry during all of our visits except one during a heavy rainstarm. Pinto Creek has a notice- able stream bed at the highway crossing and has water in all but the most extreme drought conditions. We feel that Graham's Zoquete Creek is not the same as Emory's and is the present Pinto Creek. A collection of fishes is available from Pinto Creek at the U. S. Highway 90 bridge, virtually the same locality as Clark presumably made his collection. Dr. Ernest A. Lachner of the U. S. National Museum (personal communication, 1955) reports that the Zoquito specimens can not be found on the shelves. As Girard's identifications are not always trust- worthy and the morphological description of G. nobilis was obviously based on other specimens, we consider the identity of the missing fish questionable. They must belong to one of three species. (1) Gambusta nobilis. As the nearest known locality of this species is 180 miles dis- tant and on the other side of the salty Pecos River, we consider this possibility at best very remote. (2) Gambusia geiseri. This possibility is deemed unlikely as this species is now restricted to the spring heads of much larger streams. (3) Gambusia affinis. We suggest this identification as the geographic and ecologic conditions of Pinto Creek are approximately the same as those now occupied by this species. Moreover, the collection from Pinto Creek contains G. affinis. Various authors, including Woolman (1894), Jordan and Ever- mann ( 1896 ) , Alvarez (1950 ) , and Knapp (1953 ) , incorrectly included parts of Mexico in the range of G. nobilis. As Miller (1951) clearly pointed out, the fish does not occur outside of the Pecos River drainage of New Mexico and western Texas. On the basis of unpublished work by Carl L. Hubbs, Scrimshaw (1945 and 1946) and Knapp used a trinomial but did not list any other subspecies. On the basis of imperfect material, the stocks of G. nobilis inhabiting various springs in western Texas seem to be more or less differen- tiated from the Comanche Spring population, but further studies will be needed to determine the degree of differentiation. A REVISION OF THE GAMBUSIA NOBILIS SPECIES GROUP 297 Material. Only four of the seven types can be found. All of them are females from Leon Spring, Pecos County, Texas (MCZ 1298 and 1455). The one specimen in MCZ 1455 is of the same length as the drawing in Girard (1859, pl. 39, fig. 8). For this reason we designated MCZ 1455 as lectoholotype and MCZ 1298 as lectoparatypes. Ten males (UMMZ 137101) from Phantom Lake, and five specimens (TNHC 4862) from an irrigation ditch one mile east of Toyah- vale, Texas, were examined. In addition to the locality records published by Carl L. Hubbs (1926 and 1929), Dr.0 William J. Koster has collected specimens from a spring (Lat. 33 30' N., Long. 104° 25' W.) on the Bitter Lakes Refuge northeast of Roswell and Blue Spring (Lat. 32° 12' N., Long. 104° 20' W.) near Black River Village, both in New Mexico. Many other specimens have been collected at Phantom Lake, Reeves County, Texas, and Comanche Springs, Pecos County, Texas.

Gambusia senilis (GIRARD) Gambusia senilis Girard, 1859a: 122 (description from Chihuahua River = Rio Chivuscar) ; Jordan and Gilbert, 1883: 894 (description, Chihuahua River) ; Regan, 1913: 989, fig. 168E; Carl L. Hubbs, 1926: 32 (synonomy, range) ; 1929: 1 (comparison with G. gaigei); Jordan, Evermann, and Clark, 1930: 183 (range) ; Al- - varez, 1950: 84 (range). Gambusia nobilis, Woolman, 1894: 60 (Rio de los Conchos at Chihua- hua); Jordan and Evermann, 1896: 682 (exclusive of most of synonorny and geographic range) ; Alvarez, 1950: 84 (range following above). Gambusia nobilis nobilis, Knapp, 1953: 94 (range in part) . Gambusia affinis, Evermann and Kendall, 1894: 107 (literature in part) ; Meek, 1902: 99; 1904: 130 (description in part, only specimens from Rio Conchos drainage of Chihuahua and those probably erroneously attributed to the Rio Sota la Marina drainage of Ta- maulipas at La Cruz and Garza Valdez). Description. Dorsal rays 8 (rarely 7 or 9) ; anal rays 10; scale rows 29 to 32; scale rows around caudal peduncle 16. Gonopodial characters distinguish all adult males from all those of the other members of the species group. There are no recurved hooks on the elongate spines of ray 3. Ray 4a reaches only to the terminal hook of 5a. The elbow is formed of two to four segments. The distal serra on 4p reach only to the middle of the elbow. The terminal hooks on 4p and 5a are rounded. The color pattern is distinctive. The body ground color is silver with 298 THE TEXAS JOURNAL OF SCIENCE

Fig. 8. A. 22 mm. Gambusia senilis male from TNHC 4209. B. 34 mm. G. senilis female from TNHC 4209. C. 20 mm. G. senilis male of spotted race from TNHC 4210. D. 34 mm. G. senilis female of spotted race from TNHC 4210. A REVISION OF THE GAMBUSIA NOBILIS SPECIES GROUP 299 little yellow or iridescent blue. The clear areas on the median fins are yellowish. The dark margins of the scale pockets are obscured by other color markings. The scale-pocket markings are obscured by the diffuse coloring around the middorsal streak, by the underlying wide lateral band, and by the darkness of the neurocranium. They are absent on the ventral edge of the caudal peduncle and on the region anterior to the anal and ventral to the eye. The very prominent dark crescents on the side behind the pectoral fin are the dominant color markings. These dark markings extend farther ventrally than do the abdominal markings of other members of the species group. In large individuals they extend onto the caudal peduncle as specks or crescents. These abdominal marks are faint or absent in males and small females. The 1 dorsal has a subbasal row of spots less than /4 of the way out. There are no markings on the caudal fin. There is a darkened margin on the dorsal of each sex and on the anal of the female. In mature females the anal has black subbasal dashes on the rays. The gonopodium is grayish. There are no dark markings on the jaws. There is usually a well-developed suborbital bar and a darkened opercle. Range. All our specimens come from the Rio Conchos and its tributaries in Chihuahua and Durango (Fig. 6). In addition to Rio Conchos records, Meek (1904) collected it from the Rio Sauz, an isolated stream in Chihuahua with faunal affinities to the Rio Conchos. We have checked on his collection locality. When we asked for the locality we were informed that our car was parked in the river bed. It had dried up in 1947, and according to Sr. Raul Miramontes of El Suaz, there is now no permanent surface water in the valley. Carl L. Hubbs (1926) included Meek's (1904) records from La Cruz and Garza Valdez in the Rio Sota la Marina system in the synonymy of G. senilis. He, as well as we, have determined that Meeks' specimens from La Cruz and Garza Valdez agree well with Rio Conchos material, but no specimens of senilis are included in any of the numerous, more recent collections from the Sota la Marina system, which has a fauna quite different from that of the Conchos. A mistake in labelling is suspected. Carl L. Hubbs also referred Garman's 1881 record of G. patruelis and 7R his 1895 record of G. gracilis from Monclova, Coahuila, to the syn- , onomy of G. senilis (on circumstantial evidence, he tells us). He has examined the material and considers it to represent an undescribed species belonging to the subgenus Heterophallina. Our collections indicate that G. senilis is widely distributed in the Rio Conchos. Meek's collection from San Andres shows that it occurs in the mountains. Our collection from Julimes is the farthest down- , stream. Collections from near the mouth of the Rio Conchos in the

300 THE TEXAS JOURNAL OF SCIENCE vicinity of Ojinaga contain G. affinis but not G. senilis. Future collections from between Ojinaga and Julimes may include both species. Spotted specimens. Specimens from the five collections have ir- regularly scattered black spots (Fig. 8, C and D) . They are rare in smaller individuals, more abundant at intermediate sizes, and always present in the largest size groups. None of the other specimens of G. senilis have these spots. The spotting does not have the distinct swelling and discrete margin of pathogenic concentrations of melanophores. There is no indication of any associated parasite. In nature the spotting is correlated with poor condition of the fish. Those with such spots have on the average a displacement volume 10 per cent less than do those of equal length taken in the same collection, without the spots. It seems unlikely that the spotting is caused by physical factors in the environment, as Mr. William E. Humphrey lists no geologic features in the region (personal communication, 1955) not occurring within the range of non-spotted G. senilis. Moreover, we have raised two stocks of spotted fish and a stock of unspotted G. senilis from the Rio San Pedro at Meoqui and the laboratory raised individuals of neither the spotted nor the nonspotted stocks have the other pheno- typic condition. Because the spotted and nonspotted stocks breed true in the laboratory we believe it probable that the spotting is controlled by genetic factors. As all the large individuals of spotted stocks have *The spots, the factor is thought to be homozygous. The only variable is probably that of the time of expression. The distribution of the spotted type, as represented by the collections is disjunct (Fig. 9). Three of the series are from the Rio Valle de Al- lende, a tributary to the Rio Florido. The other two are from an irrigation ditch carrying water from a spring, El Ojo Almoloya, near Es- taciOn Troya. This latter area is part of an isolated basin on the divide between the Rio Florido and the Rio Parral. We do not believe, however, that the two stocks are of different origin. During 1954 (extreme drought), irrigation water from the Rio Valle de Allende reached Porvenir, near a reservoir which stored water for EstaciOn Troya. Mr. Oscar f. Wiegand and Clark Hubbs traced irrigation ditches that passed over the divide. Sr. Eduardo Rojas reports that irrigation water has flowed from the Rio Valle de Allende to EstaciOn Troya via irrigation ditches. We feel that the fish fauna of El Ojo Almoloya may have originated from this source. The similarity of the spotting supports this hypothesis. Although more than 50 years have passed since the Rio Valle de Allende has carried water (other than floods) into the Rio Florido, the streams had permanent contact during earlier wet periods. A REVISION OF THE GAMBUSIA NOBILIS SPECIES GROUP 301 Material. Five hundred and twenty-six specimens of the nonspotted race were collected from Chihuahua, Mexico; twenty-three (TNHC 4208) from the Rio Conchos at Julimes; twenty-three (TNHC 4483) from the Rio Conchos at La Cruz; forty-four (TNHC 4209) from the Rio Sacramento, one mile north of Chihuahua City; one hundred and thirty-seven (TNHC 4212) from an irrigation ditch one

men' 105. • Camara°

•-A I : Boquillas N.

•

. ° 27 30' •

s Typical senilii b Black spotted senilis Is hurtadoi a. alvarezi 0 1 4.71 • Estacion Troya JIminez —13 Sierra PorVenir Hacienda Almoloya Dolores...

. ° San 27 00' yi,Gregorio galiclas dr•-•1

isParral „Valle de Al lende

._; Talamantes •"'"" Dam

Rio...... -• DRAINAGE CONDITIONS IN 1954 —-Permanent Water / • - Intermittant Water * 10 miles 414+04 Irrigation Ditch busol =1 - .. ... Stream Channel . No information • Spring

Fig. 9. Recorded collection localities of three species of the Gambusia nobilis species group in southern Chihuahua. 302 THE TEXAS JOURNAL OF SCIENCE mile southeast of Bachimba; two hundred and fifteen (TNHC 4351, 4515, and 4560) from the Rio San Pedro between its junction with the Rio Conchos and one mile southwest of Meoqui; twenty-four (TNHC 4368 and 4602) from tributaries to the Rio Conchos, twelve and fifteen miles southwest of Camargo; fifty (CNHM 3553) from the Rio Florido at Jiminez, and ten (TNHC 4344) from a tributary to the Rio Florido, 15.4 miles SSE Parral. Four specimens (TNHC 3711 and 4079) were collected from the Rio Florido in Durango at Villa Ocampo and Esperito Santo. Eleven hundred and ninety specimens of the spotted race were examined. Eight hundred and twelve (TNHC 3696 and 4211) were from El Ojo Almoloya and irrigation ditches one or two miles west of Estaciem Troya, Chihuahua, and three hundred and seventy-eight (TNHC 3702, 4210, and 4561) were from the Rio Valle de Allende, between Valle de Allende and Miramontes Dam. Meek's specimens (CNHM 4447 and 4452) allegedly from Garza Valdez and La Cruz, Tamaulipas, were also examined. Dr. R. R. Miller has sent us three records for this species collected in Chihuahua by an expe- dition from the University of California at Berkeley: one from Rio San Juan (no precise localifiy), the second from an irrigation ditch five miles north of Meoqui, and the last from Rio San Pedro at Meoqui.

Gambusia gaigei CARL L. HUBBS Gambusia gaigei Carl L. Hubbs, 1929: 1 (description, comparison with G. nobilis, G. senilis and G. affinis ( =G. geiseri); 1940: 11 (range) ; Jurgens and Hubbs, 1953: 14; Knapp, 1953: 94 (range) ; Clark Hubbs, 1957b: 8. Description. Dorsal rays 8 (rarely 7 or 9) ; anal rays 9 or 10; scale rows 28 to 31; scale rows around caudal peduncle 16. This species has the deepest body of any in the species group. Carl L. Hubbs (1929) remarked on the small size of this species when compared with its relatives. His largest female was only 25 mm. in standard length. The largest of our 223 wild females is only 28 mm.; however, two wild females held in the laboratory for one year reached 45 mm. The tip of the gonopodium is essentially identical with that of G. hurtadoi and G. alvarezi and is quite distinct from that of the other species. There are no recurved hooks on the elongate spines of ray 3. Ray 4a reaches to the middle of the terminal hook on 5a. The elbow of all males examined is of one or two segments. The distal serra on ray 4p reaches to the elbow. The terminal hooks on rays 4p and 5a are rounded. The gonopodial suspensorium (Fig. 11) as described and drawn

A REVISION OF THE GAMBUSIA NOBILIS SPECIES GROUP 303

Fig. 10. A. 18 mm. Gambusia gaigei male from TNHC 4213. B. 25 mm. G. gaigei female from TNHC 4213. from a paratype by Luis Howell Rivero, is similar to that of G. geiseri and G. nobilis, but is different in some details. There are three gonapophyses, but the third is only moderately specialized. The first is long, thin, and slightly curved. It has a rib-bearing parapophysis. The second gonapophysis is long, thin, and straight. Its thin uncinatoid process is relatively short because basally it grades into the body of the gonapophysis. The third gonapophysis, unlike that of geiseri and nobilis, has a forward-bent projection, but the forward bend is weak and the projection is only about half as long as the rest of the gonapophysis. The process is laterally compressed and slightly curved. At the angle there is a slight backward swelling that does not form a definite uncinatoid process. The air-bladder extends to the third caudal segment. The color pattern is weaker than that of the other members of the G. nobilis species group. The ground color is silvery with an iridescent blue overtone. There is considerable yellowish-orange on the clear areas of the unpaired fins. The markings on the margins of the scale

304 THE TEXAS JOURNAL OF SCIENCE

Fig. 11. Gonopodial suspensorium of a male Gambusia gaigei collected from Boquillas, Texas. pockets are faint. There are none anterior to the anus or below the eye. The middorsal streak and dark coloration of the neurocranium cover and obscure the scale-pocket markings, but the postanal streak does not obscure these markings. The faint, broad lateral band often obscures the scale-pocket markings on one scale row. There are a few dark crescents on the scale rows surrounding the lateral band. The anal spot in females is restricted to the anus. The dorsal has a subbasal row of black spots and a dark margin. The caudal has no dark markings. The anal of females has a dark margin; that of males is grayish. There is only a trace of a dark chin bar. Except for the suborbital bar there is no darkening of the lower parts posterior to the eye. Racial variation. Specimens of G. gaigei have been obtained from two localities, Boquillas Spring and Graham Ranch Warm Spring. The individuals differ from each other by a number of morphologic characters. The Boquillas Spring specimens have a weaker axial streak, more numerous and more widely scattered crescent shaped marks on the sides, and a lighter lateral band. The Graham Ranch specimens have greater sexual dimorphism of the dorsal insertion, i.e., the dorsal to caudal distance of Graham Ranch specimens is contained 1.65 to 1.7 in predorsal length of females and 1.35 to 1.5 in males, and, in the A REVISION OF THE GAMBUSIA NOBILIS SPECIES GROUP 305 Boquilas Spring collection, 1.5 to 1.65 in females and 1.4 to 1.6 in males. All of the fish from Graham Ranch have nine anal rays whereas two-thirds of those from Boquillas Spring have ten anal rays and one- third have nine. All of the Graham Ranch males have only one segment in the elbow of ray 4a and five of seven from Boquillas have two segments there. These differences are sufficient for subspecific sepa- ration following the criteria of Carl L. Hubbs and Hubbs (1953). We do not propose subspecific names for the populations for two reasons. First, the collections are not contemporaneous; i.e., the Boquillas specimens were taken in 1928 and the Graham Ranch specimens were collected in 1954 and 1956 when the Boquillas population was extinct. Second, there has been considerable controversy concerning the valid- ity of formalizing infraspecific differentiation in zoological nomenclature (see Wilson and Brown, 1953; Hubbell, 1954; Mayr, 1954; and others) . Local populations of many fish species are often morpho- logically separable from all other local populations of that species. Therefore we do not believe that racial populations of fishes need to be formalized with scientific names until the problems of racial variation have been investigated further. Range. G. gaigei has been collected from the Graham Ranch Warm Spring and Boquillas Spring near Boquillas Canyon in Brewster County, Texas (Fig. 12) . The Boquillas Spring from which Dr. Fred- erick M. Gaige collected the types undoubtedly is not the Graham Ranch Warm Spring. The Boquillas Spring is a different one at which a bathhouse was built (Frederick M. Gaige, personal communication, 1956) . Dr. Ross Maxwell reports seeing "little fish" there while he was superintendent of Big Bend National Park. District Park Ranger John Palmer, who has occupied the Boquillas Ranger Station for three years (1954-56), has informed us that the spring ceased flowing a few years ago. The spring has been examined and found devoid of fish life. We have examined all other known springs near Boquillas and found no mosquitofish. Undoubtedly the population from which the types were obtained is extinct. During June, 1954, the Rio Grande was on a rise so our collecting from the river was not extensive. We did obtain G. affinis from the Rio Grande, Tornillo Creek, and Graham Ranch Warm Spring. In April, 1956, the Rio Grande was clear. We obtained G. affinis in all collections, but G. gaigei only at Graham Ranch. The clear warm-water springs are so different from the peri- odically muddy, temperature-fluctuating river that we do not believe that G. gaigei lives in the river except, possibly, as strays. The outlet from the Graham Ranch Spring, a ditch 1 to 10 feet wide and 2 feet deep, has been dredged about 40 feet from the spring to form a pool 306 THE TEXAS JOURNAL OF SCIENCE

Boguillas Canyon

Tornillo C reek

Fig. 12. Collection localities for Gambusia gaigei in Big Bend National Park. about 20 feet wide, 40 feet long, and 4 feet deep. G. gaigei and G. affinis live in the pool, inlet ditch, and rock-lined spring pool. Cattails choke the inlet ditch and are on the sides of the pool. The drainage from the pool is confined to surface seepage which falls over a 15' clay bank into the Rio Grande. No fish were seen between the pool and river. There is little imminent danger of the Rio Grande eroding away and obliterat- ing the current geographic range of G. gaigei. Dr. Ross A. Maxwell does not believe that flood waters would reach the springs. Mr. Lemuel A. Garrison reported that "catfish" (Ictalurus punctatus) and "perch" (Lepomis cyarzellus) had been placed in the pool. Perhaps G. affinis as well as Notropis lutrensis and N. braytoni, which we also collected in the spring, were introduced with the game species. Carl L. Hubbs (1940) stressed the importance of avoiding the introduction of game fish into the waters of the Big Bend Park area that contain endemic fishes. During the 1954 visit to Graham Ranch nearly 95% of the mosquitofish collected were G. gaigei. At the time of the 1956 visit, approximately 95% were G. a ffinis. Although habitat changes may be responsible for the replacement of G. gaigei by already existing stocks of G. affinis, it is more likely that the more successful G. affinis were introduced. Therefore, the senior author has recommended the elimi- A REVISION OF THE GAMBUSIA NOBILIS SPECIES GROUP 307 nation of the G. affinis population that apparently has been introduced into the Graham Ranch Warm Spring, and urged every possible effort to maintain the remaining population of G. gaigei. As a part of this effort stocks of G. gaigei have been introduced into Glenn Springs without success and into a spring near the Boquillas ford in Big Bend National Park where they have reproduced. Material. Two hundred and fifty-six specimens (TNHC 4213, 4573, and 4575) collected from Graham Ranch Warm Spring were examined. Fifty-two specimens (TNHC 4571, 4572, and 4626) reared from Graham Ranch stocks in the laboratory were also examined. Dr. Carl L. Hubbs compared Graham Ranch specimens with the type and 63 paratypes (UMMZ 84527 and 84528) from Boquillas Spring.

Gambusia hurtadoi, NEW SPECIES Description. Dorsal rays 9 (rarely 8 or 10) ; anal rays 10 (rarely 9 or 11) ; scale rows 28 to 31; scale rows around caudal peduncle 16 (rarely 14 or 15). The tip of the gonopodium is apparently identical with that of G. gaigei and G. alvarezi and is quite distinct from that of the other species. There are no recurved hooks on the elongate spines of ray 3. Ray 4a reaches only to near the base of the terminal hook on 4p. The elbow is formed from one (334), two (27) or three (1) segments. The distal serra on 4p reaches the elbow. The terminal hooks on 4p and 5a are rounded. The color pattern is darker and has more iridescent blue than that of other members of the G. nobilis species group. The ground color is iridescent bluish-silver. There is less orange on the body than in G. alvarezi. The clear areas on the median fins are yellowish orange. The markings on the margins of the scales are dark but diffuse, and are often obscured by other more prominent markings. There are no marks anterior to the anus and none below the preopercle. The lateral diffusion of the dark middorsal streak often reaches the lateral band. The thin postanal streak does not obscure the scale-pocket markings. The lateral band is broad and dark. The dark crescents on the side are concentrated along the lateral band and often cover and obscure it. They follow the scale-pocket margins. The anal spot is not restricted to the anus. The dorsal has a subbasal row of spots, which are darker than the darkened margin. The caudal has no dark markings. The anal of females has a dark margin; that of males is grayish. The prominent dark chin bar is often interrupted medially. The suborbital bar is dark. Three color variants have been noted. These may be designated Spotted, Gray, and Golden. All three lack the dorsal streak, the post- 308 THE TEXAS JOURNAL OF SCIENCE anal streak, and the lateral band. Spotted also has no dark markings on the scale-pockets and no suborbital bar; the color pattern on the body consists chiefly of the crescents which are often grouped and not concentrated along the location of the lateral band. Gray lacks the crescents typical of Spotted. Its coloration somewhat resembles that of G. geiseri except that the scale-pocket markings are more diffuse. Golden has no dark markings except the dark margin of the dorsal, three small patches of scale-pocket margins in front of the dorsal and above the pectoral bases, the spotting of the peritoneum, and the dark eye. In life it is an attractive golden yellow. There is no iridescent blue. All three color phases were present in a collection made by Clark Hubbs and Oscar f. Wiegand at El Ojo de la Hacienda Dolores on De- cember 31, 1954. The preserved collection contained a random sample of 1,342 individuals. Later a single Golden individual 16 mm. long was collected. Approximately 500 more specimens were collected in the subsequent work. Sixteen Spotted and thirteen Grays have been noted in the preserved sample. Although 502 of the specimens are longer than 20 mm., none of the color variants are. It is possible that they have a reduced survival rate. This hypothesis is supported by our failer to bring the Golden individual back to Austin alive. Range. G. hurtadoi is known only from El Ojo de la Hacienda Do- lores, seven miles south of Jiminez, Chihuahua, Mexico, and from irrigation ditches carrying water from it. As the Rio Florido was dry and the water did not reach the channel, we do not know if G. hurtadoi will breed with G. senilis. However, Meek's collection from the Rio Florido at Jiminez contained typical G. senilis. All 181 specimens collected from an irrigation ditch 5 miles north of the spring are typical of G. hurtadoi. The species has had opportunity to breed with G. senilis in the past. Sr. Eduardo Rojas reports that until about 1900, at Haci- enda Dolores (on the banks of the Rio Florido), there was a mill that got its power from a water wheel run by water from the spring. Material. The male holotype (UMMZ 168975) was collected by Clark Hubbs and Oscar f. Wiegand from El Ojo de la Hacienda Do- lores, seven miles south of Jiminez, Chihuahua, Mexico, on December 31, 1954. Thirteen hundred and forty-one paratypes (CNHM 61604, ENCB P 350, SU 48133, TNHC 4206, UMMZ 168976, and USNM 164252) were collected with the holotype. Five hundred and forty-six paratypes (TNHC 4205 and UMMZ 168977) were collected from the

Fig. 13. A. 19 mm. Gambusia hurtadoi male from TNHC 4205. B. 26 mm. G. hurtadoi female from TNHC 4205. C. 26 mm. laboratory-raised G. hurtadoi male from TNHC 4203. D. 31 mm. laboratory-raised G. hurtadoi female from TNHC 4203. -

310 THE TEXAS JOURNAL OF SCIENCE same locality on June 30, 1954. Two hundred and seventy-seven paratypes (TNHC 4204) were collected from an irrigation ditch two miles south of Jiminez. Thirty-one paratypes (TNHC 4377) were collected from an irrigation ditch six miles south of Jiminez. A large number of laboratory raised individuals including (TNHC 4203 and 4586, and UMMZ 168978) were also examined. These are not designated paratypes. Named hurtadoi for Sr. Ing. Leopoldo Hurtado Olin, in appreciation for his aid during our collecting in the summer of 1951.

Gambusia alvarezi NEW SPECIES Description. Dorsal rays 9 (rarely 8 or 10) ; anal rays 10 (rarely 9); scale rows 29 to 31; scale rows around caudal peduncle 16. There are no recurved hooks on the elongate spines of ray 3 of the gonopodium. Ray 4a reaches to the middle of the terminal hook on 5a. The elbow is formed from one (62) or two (24) segments. The distal serra on ray 4p reaches to the elbow. The terminal hooks on 4p and 5a are rounded. The color is more yellow and orange than that of any other members of the G. nobilis species group. The ground color is yellow-orange. There is little iridescent blue on the body. All clear areas on the fins are yellow-orange. The diffuse dark markings on the margins of the scales are often obscured by more prominent markings. There are no marks anterior to the anus or below the eye. The middorsal streak is dark, but its lateral diffusion does not reach ' o the lateral band. The dim postanal streak often obscures the scale-pocket markings. The lateral band is broad and dark. The dark crescentic marks on the side are concentrated along the lateral band. They are not numerous and more than three are seldom interconnected. The lateral band can easily be traced through the areas between the scattered groups of crescents. The black spot around the anus is large. The dorsal has a subbasal row of spots and a darker margin. The caudal has no dark markings. The anal of females has a dark margin; that of males is uni- formly grayish. The prominent dark chin bar is often interrupted medially. The suborbital bar is dark. Range. G. alvarezi is known only from El Ojo de San Gregorio, Chihuahua, Mexico, and the adjacent creek. As the Rio Parral was reported to be dry we did not investigate the entry of the spring water

Fig. 14. A. 20 mm. Gambusia alvarezi male from TNHC 4207. B. 20 mm. G. alvarezi female from TNHC 4207. C. 24 mm. laboratory-raised G. alvarezi male from TNHC 4570. D. 25 mm. laboratory-raised G. alvarezi female from TNHC 4570. A REVISION OF THE GAMBUSIA NOBILIS SPECIES GROUP 311

A 312 THE TEXAS JOURNAL OF SCIENCE into its bed. Although the one collection we have made in the Rio Parral two miles west of Parral did not contain any G. senilis, it is likely that G. senilis occurs in that stream. At times of higher water G. senilis undoubtedly has lived in the Rio Pan-al at its junction with the spring run from El Ojo San Gregorio. As the Rio Parral bed in Parral is heavily polluted with mining wastes, it is questionable whether fish would now live downstream from Parral even if water were there. Under the present conditions it is not likely that the two forms will come into contact. Material. The male holotype (UMMZ 168979) was collected by Clark Hubbs and Oscar f. Wiegand from El Ojo de San Gregorio, Chi- huahua, Mexico, on December 31, 1954. Six hundred and fifty paratypes (CNHM 61605, ENCB P 349, SU 48134, TNHC 4207, UM.MZ 168980, and USNM 164253) collected with the holotype were examined. Many laboratory-raised individuals (including TNHC 4570) were also examined. Named alvarezi for Dr. Jose Alvarez del Villar for his assistance to us and for his work on Mexican fishes.

ECOLOGY The members of the G. nobilis species group appear to be most suited to spring life. Gambusia geiseri inhabits the waters of the two large springs along the Balcones Escarpment of Texas. The flow of the springs is rather constant and large. Between 1928 and 1946 the discharge from Comal Springs ranged between 235 and 420 second feet, with an average of 324, and San Marcos Springs flowed between 51 and 286 second feet, with an average of 153 (George, 1947). These springs have an average temperature of 22° C. at San Marcos Springs (Kirk Strawn, personal communication, 1955) and 23.3° at Comal Springs (George, 1947). G. geiseri inhabits only those waters close to or in the springs them- selves. The annual temperature range to which they are exposed probably does not exceed 3° on either side of the spring temperatures. These clear-water springs are rich in calcium carbonate and are heavily vegetated. The mosquitofish are usually found on the stream edges. This species is also known from other Texas springs; we suspect that they were introduced to these locations (seep. 290). Gambusia nobilis also typically inhabits spring waters. Except for the specimens once reported from Zimmerman Reservoir, all known examples are from clear spring-fed waters. The best available work on the physical factors of the waters in which G. nobilis occurs is the treatise of White, Gale, and Nye (1941) on the water resources of the A REVISION OF THE GAMBUSIA NOBILIS SPECIES GROUP 313 Balmorhea area (near Toyahvale). They reported that the flow of the five major springs there varied from 53 to 104 second feet. They also reported that the discharge of Leon's Springs varied between 16 and 18 second feet and that the flow of Comanche Springs ranged from 40 to 50 second feet. These springs definitely vary more than these figures indicate, as Comanche Springs has ceased flowing several times in the last few years. Phantom Lake Springs near Toyahvale had surface 0 water temperatures varying between 21.7 and 25.6° C. It is likely that the temperature of waters inhabited by G. nobilis does not vary far from those figures. In all regions the water is clear, high in calcium carbonate, and heavily vegetated. Gambusia senilis has been collected in varied habitats. It is exposed to a great variation of temperatures, chemical conditions, and water flow. It occurs in nearly all of the irrigation ditches and semi-permanent roadside ditches in the upper Rio Conchos valley. We have collected it at temperatures ranging between 12° and 30° C. Oddly, both

extremes were in the Rio San Pedro near Meoqui. The low tempera-0 ture occurred on December 30, 1954, with an air temperature of 7 , and the high was on June 24, 1954, with an air temperature of 33°. Obviously G. senilis can tolerate considerable temperature variations. The spotted race likewise can tolerate considerable temperature range, from 14.5° C. to 29° in the Rio Valle de Allende near Valle de Allende. Like the Rio San Pedro temperatures, the high recording was in the summer, June 27, 1954, and the low in the winter, December 30, 1954. More extreme temperatures are to be expected. Gambusia senilis usually inhabits the quiet weed-choked surface waters. It has been collected in other habitats, especially when other fishes usually inhabiting those habitats are scarce or absent. G. senilis has been collected in clear spring water and in very turbid water. At the majority of the collection localities that we visited, the water was so clear that the bot- tom was or would have been visible at more than ten feet. It was collected in the Rio Florido near Villa Ocampo, Durango, where the bot- tom was not visible at depths of over one inch. It is likely that the excessive turbidity was temporary as many of the other species in the same collections, including Catostomus conchos, Dion':la episcopa, Campostoma ornatum, and Notropis chihuahua, are typically found in clear waters. We have also collected G. senilis in waters containing extensive organic pollution in both the Rio San Pedro and the Rio Sac- ramento, Chihuahua. Gambusia gaigei has been collected in only two very restricted localities. We have ecologic data on only the Graham Ranch Warm Spring. The spring temperature was 34.5° C. on April 27, 1956, and

314 THE TEXAS JOURNAL OF SCIENCE 0 the pool near the spring varied between 25 and 32.5°. Air tempera- 0 ture varied between 20 and 26.5° C. These springs were cooler than the nearby hot springs (38° to 40°), which contained no fishes. G. gaigei can withstand lower temperatures as they will live in aquaria which have temperatures near 20°. The water of the spring is clear, high in calcium carbonate (and other minerals), and heavily vegetated. G. gaigei were most common in the shallower and more heavily- vegetated areas. Except for G. affinis, the other species collected with G. gaigei were concentrated in the open waters. G. gaigei can stand high organic concentration as our stock tanks occasionally have become "dirty" and the fish have survived. The spring flow is less than 0.3 second feet. Gambusia hurtadoi inhabits another warm spring. We have winter and summer temperature data for the spring. On June 30, 1954, when the air temperature was 25° C., the water temperature varied from 33° in the boil to 32.5° on the other side of the spring hole. On Decem- ber 31, 1954, when the air reading was 9°, the water temperature was 29.5° in the boil and 29° on the other side of the spring hole. We suspect that the spring temperature varies seasonally. G. hurtadoi is not restricted to this temperature range. The main irrigation ditch was checked extensively for the species for five miles north of the spring. It occurred throughout that region. On July 1, 1954, temperatures ranged as low as 31°, and on December 31, 1954, to 21° C. (air temperature 13.3°). G. hurtadoi is most common in heavily vegetated habitats. The waters are crystal-clear and apparently contain much calcium carbonate. G. hurtadoi can stand high organic concentrations as our stock tanks occasionally have become "dirty" and the fish have survived. The spring flow was estimated at 12 second feet on June 30, 1954. Only a species of Cyprinodon (probably new) was collected in the spring. Three cyprinids ( Notropis lutrensis, N. chihuahua, and Pimephales promelas) were taken in the irrigation ditches. Gambusia alvarezi inhabits a spring, which was visited only on December 31, 1955, when air temperature was 3° C. A hard freeze occurred the previous night, so temperatures must have been consider- ably lower. The water temperature was 24° and one-third of a mile downstream 19°. Ice-covered side pools did not contain G. alvarezi, but all locations examined with temperatures between 19° and 24° did. Like the other species, G. alvarezi is most common in heavy vege- tation. It was more common in the running water, perhaps due to the coldness of the edges or to the absence of other fishes. The spring flow was approximately one-third of that in El Ojo de la Hacienda Dolores the same day.

...111111

A REVISION OF THE GAMBUSIA NOBILIS SPECIES GROUP 315 Effect of environment on growth. Hubbs (1929), distinguished G. GAIGEI from other related species by its small size. Our wild collections also show that species to be much smaller than related species (except G. HURTADOI). Our wild females of G. GAIGEI do not exceed 28 mm. while females of G. NOBILIS reach 40 mm. (Carl L. Hubbs, 1929), G. SENILIS 46 mm., G. ALVAREZI 42 mm., G. GEISERI 37 mm., and G. HURTADOI 36 mm. However, when held in the laboratory, wild- caught individuals of G. GAIGEI and of G. HURTADOI have attained a length of 45 mm. (Table 1). The bulk has increased to an even greater degree, for the laboratory-held fish are much heavier-bodied than those in nature. These fish have grown steadily since being brought into the laboratory. The increased growth may be due to: (1) greater

TABLE 1 Comparative Sizes in Nature and in Laboratory of GAMBUSIA GAIGEI and GAMBUSIA HURTADOI

Sped.- Size Date MENS Range, nun. Mean GAMBUSIA GAIGEI Females Caught in nature VI: 11: '54 2231 Max., 28 Same stock, in laboratory since VI: 16: '54 II:25:'55 10 34-43 37.5 Survivors, in laboratory since VI:16:'54 VI: 15:'55 6 35-45 41.7 Mature males, caught in nature At time of capture VI:11:'54 9 18-21 19.4 Same stock, in laboratory since VI:16:'54 II: 25:'55 3 20-22 21.0 Survivors, in laboratory since VI:16:'54 VI: 15:'55 2 21-21 21.0 F, males, laboratory-raised .. . II:25:'55 4 21-24 22.7 F, males, laboratory-raised. . . VI: 15:'55 23 21-30 24.6 GAMBUSIA HURTADOI Females Caught in nature VI:30:'54 4222 Max., 36 Stock in laboratory since VII:3:'54 II:25:'55 20 33-44 38.7 Survivors in laboratory since VII:3:'54 VI:15:'55 17 38-45 40.5 Mature males, caught in nature At time of capture VI:30:'54 & XII: 31 : '54 320 16-22 18.5 Stock in laboratory since VII:3:'54 II:25:'55 6 19-22 20.7 Survivors in laboratory since VII:3:'54 VI: 15:'55 5 20-22 20.8 F, males, laboratory-raised II:25:'55 39 22-28 24.9 F, males, laboratory-raised.. . VI:15:'55 21 24-31 26.5

1 Including about 10 undifferentiated males. 2 Including about 20 undifferentiated males.

316 THE TEXAS JOURNAL OF SCIENCE age; (2) increased food; (3) lower temperature; or, (4) probably to a combination of such factors. Gambusia males are better suited for length comparisons. Like the males of other poeciliids, they do not grow after becoming mature. Wild males held in the laboratory approximate the length of males captured with them and preserved (Table 1). After a year laboratory- kept G. gaigei males were not larger than the largest preserved wild males. Some females of this species had grown at least 17 nun. in this time. The small sample and the difficulty of precise measurement of living fish may have caused some of the observed variation. In approximately one year in the laboratory males of G. hurtadoi had not exceeded in size the largest preserved wild males. Some females had grown at least 9 mm. in this time. Basing specific distinctions on the size of mature males may lead to incorrect conclusions, for laboratory-raised males of both species far exceed the length of their parents (Table 1). The increased size can- not be due to age as the F„ males must be younger than their fathers on the same date. The difference is probably due to better feeding and/or lower temperatures during ontogeny. Most of the laboratory- raised males developed at temperatures between 22° and 25° C., well below the temperatures recorded from El Ojo de la Hacienda Dolores. Our laboratory stocks undoubtedly get much more food than do wild fish. Populations at El Ojo de la Hacienda Dolores are extremely dense. We have collected over 100 individuals in a two-foot drag of a six-foot Common Sense seine. Food must be at a premium under these conditions. We have raised two G. hurtadoi males that were nearly the same size as the wild males. They were born during the hot summer months at a temperature around 29°, and developed in a tank with Notropis lutrensis, which is possibly a more efficient food gath- erer. Either or both of the two suspected factors may apply to the small laboratory-raised fish. The wild males of G. hurtadoi from the irrigation ditch average slightly longer than those from the spring source. The temperatures in the ditch were cooler during both the June and December collections. Since populations in the ditch are far less dense, the competition for available food is undoubtedly less. The ditch populations are probably thinned from time to time by fluctua- tions in flow, as the water is used in irrigation. Our laboratory F,'s not only exceed parents in length, but also differ from them pronouncedly in other respects (Figs. 13 and 14). They have deeper bodies, shorter fin rays, shorter heads, and lighter colors, and they might easily be mistaken for a different species if they had been collected in nature. A REVISION OF THE GAMBUSIA NOBILIS SPECIES GROUP 317

DORSAL-RAY VARIATION When we first analyzed the June sample of G. hurtadoi from El Ojo de la Hacienda Dolores, we noted that the males averaged more dorsal fin rays than did the females. When the difference was subjected to statistical analysis we attained a p value of 0.004. Partially to check this apparent secondary sexual dimorphism we made the December collection. In that collection no difference was noted between the dorsal-ray counts of the sexes. Our problem seemed to have been resolved when we plotted the June fish according to size. The low ray number in the largest females is the major cause for the lower average number of dorsal rays of the females (Table 2). There is a gradual decrease in the average number of dorsal rays of 0.0396 fin rays per mm. from the small to the large females. The probability that

TABLE 2 Variation in Number of Dorsal Rays Correlated with Size of Individuals in Gambusia hurtadoi Collected at El Ojo de la Hacienda Dolores on June 30, 1954, with Data on Size Frequency of the Sexes

Females Males Standard Dorsal Rays Im- Dorsal Length 8 9 10 Totall mature Mature Ave. 36 2 2 35 3 3 34 ...... 33 1 1 2 32 .. . . 31 .. 1 1 30 1 1 2 •12 9.00 29 1 .. 4 28 I 6 7 27 1 5 8 26 I 12 14 25 4 30 .. 34 24 8 34 2 53 23 11 49 4 67 . . 22 6 25 2 58 ...... 21 3 8 45 .. 20 19 1 }2 9.00 19 13 6 30 18 17 6 22 8.89 17 15 3 9 1 8.80 16 16 35 15 21 . . 14 11 13 5 12 3 11 2

1 Dorsal rays of some females not counted. Total shows size distribution of females. 2 Sex reversal, or delayed differentiation. Regression line: Slope=9.80 — 0.0396 per mm. t=3.4723 318 THE TEXAS JOURNAL OF SCIENCE this is a chance deviation from zero slope is 0.0005. We suspect that the average ray number is negatively correlated with the temperature of the water during development, that the larger fish developed during the previous winter, and that the smaller (younger) the female was, the warmer was the temperature during its developmental period. The small male sample has a statistically insignificant positive slope instead of the negative slope of the females. We suspect that the old males (with low counts) had died previously. Males are more aggressive and subject to predation. They often fight over a female and injure each other. In addition we have had heavier mortality of males transporting them to Austin. Except for extremely gravid females, males have a higher mortality rate in the laboratory. Low viability of males is a well authenticated phenomenon in G. affinis (Hildebrand, 1927). If the variation of dorsal fin rays is correlated with seasonal temperature change at the spring, the females of the December sample should have the dorsal ray number positively correlated with size. This correlation does occur with an average increase of 0.0228 fin rays per mm. and a probability of only 0.0004 that it is by chance (Table 3). The large sample of males also has a positive correlation between the number of dorsal rays and increased size, but the statistical signifi- cance of the difference is uncertain. We have also plotted the dorsal ray counts of FI's against size (Table 4). As our laboratory room is not yet temperature controlled, the large fish, which were born in the summer, were exposed to warmer temperatures than the small fish which were born in Febru- ary. The fish would thus be expected to have a positive correlation of number of dorsal rays with size. This does occur with an average increase of 0.0261 fin rays per mm. and the probability of its being a chance occurrence is less than 0.0001. There is no correlation of the males with regard to size and dorsal ray count; however, they are all from the summer young, as no males were apparent among the winter young. A sample from the irrigation ditch was also plotted. There is no significant correlation of size with number of dorsal rays. In fact, there is a slight and insignificant negative correlation where a positive one would be expected. The temperature of the water in the irrigation ditch probably is more rapidly affected by climatic changes and by diurnal variation. The resulting effect of the irregular temperatures on the number of dorsal rays might obscure the effect of seasonal temperature changes. The collection of G. alvarezi was also plotted according to size. An A REVISION OF THE GAMBUSIA NOBILIS SPECIES GROUP 319

TABLE 3 Variation in Number of Dorsal Rays Correlated with Size of Individuals in Gambusia hurtadoi Collected at El Ojo de la Hacienda Dolores on December 31, 1954, with Data on Size Frequency of the Sexes.

Females Males Standard Dorsal Rays Im- Dorsal Length 8 9 10 Total? mature Mature Ave. 34 1 1 33 . . . . 32 3 3 31 . . 1 2 • 12 9.00 30 . . 4 4 29 1 10 • 2 14 28 1 10 11 27 . . 10 11 26 2 25 33 25 4 27 . . 47 . . . . 24 8 42 2 73 1 2 9.00 23 7 36 1 92 . . . . 22 11 36 2 107 4 t 8.91 21 7 26 91 . . 8j 20 7 26 81 44 8.89 19 8 12 75 12 67 8.89 18 7 15 62 31 87 8.89 17 2 7 70 42 37 8.81 16 . . I 67 18 61 15 1 2 58 2 8.81 14 I 4 47 1 13 18 12 12 11 I 10 1

1 Dorsal rays of some females not counted. Total shows size distribution of females. 2 Sex reversal, or delayed differentiation. Regression line: Slope=8.33 0.0228 per mm. t=3.574 p=0.0004 insignificant negative correlation is found in both sexes, whereas if the dorsal ray number of G. alvarezi and G. hurtadoi are similarly affected by temperature, a positive correlation would occur. A collection of G. senilis from the Rio San Pedro at Meoqui4was also checked for dorsal ray variation with size. All the fish counteci had eight dorsal rays. The sample of G. gaigei collected on June 11, 1954, is plotted according to size (Table 5). There is a positive and significant (p = 0.0035) correlation. The small sample of males is not so correlated. Laboratory-raised G. gaigei did not have any correlation of dorsal rays with size. On superficial examination it appears that the three species studied have different physiological responses to temperature. Gambusia hurtadoi develops more dorsal fin rays with increased temperature, G. 320 THE TEXAS JOURNAL OF SCIENCE

TABLE 4 Variation in Number of Dorsal Rays Correlated with Size of Individuals in Gambusia hurtadoi Laboratory Fi's Preserved in February, 1955, with Data on Size Frequency of the Sexes.

Females Males Standard Dorsal Rays Length 8 9 10 Total Number Dorsal Ave. 32 1 . 1 31 I 1. 2 30 1 2 3 . . . . 29 1 1 . . . . 28 3 3 11 27 . . 5 5 3 I 9.00 26 1 14 15 8J 25 I 16 17 13 8.92 24 1 21 22 7 23 6 24 30 5 1 9.00 22 2 22 25 2J 21 .. 9 9 .. 20 I 2 i 5 19 1 1 18 1 1 17 .. 16 ...... 15 1 . . I 14 1 I 2 13 3 3 6 12 2 5 7 11 10 6 16 Regression line: Slopez----8.26 + 0.626.1 per mm. t=4.4654. p=0.6901 gaigei develops fewer, and G. alvarezi is not affected by temperature change. However, more precise temperature data during development are necessary before reaching a definite conclusion. It is possible that El Ojo de San Gregario has a constant temperature, or that its temperature varies around a point where the temperature has little effect on the dorsal fin rays of G. alvarezi. It is also possible that the temperatures of development of G. gaigei and G. hurtadoi were either higher or hirer than the temperature which produced an extreme dorsal ray count. 'Farling (1952) has demonstrated that median temperatures can produce extremes in meristic characters in Salmo trutta.

SEX RATIO Examination of Tables 2 through 5 can lead one to the conclusion that the sex ratio is not 1:1. All of them show more females than males. However, our laboratory FI stocks, in which all or nearly all of the males are mature, have a 1:1 sex ratio. The data constituting Table 4 are based mostly on immature fishes, and some of the fish recorded as females have not yet developed male characters. The wild A REVISION OF THE GAMBUSIA NOBILIS SPECIES GROUP 321

TABLE 5 Variation in Number of Dorsal Rays Correlated with Size of Individuals in Gambusia gaigei Collected at Graham Ranch Warm Spring on June 11, 1954, with Data on Size Frequency of the Sexes.

Females Males Standard Dorsal Rays Im- Dorsal Length 7 8 9 Total mature Mature Ave. 28 1 1 2 27 3 3 6 26 8 . . 8 25 21 1 22 24 28 15 43 23 30 . . 30 22 .. 16 1 17 . 21 2 20 4 26 2 1 20 2 18 . . 20 1 2 8.10 19 21 1 22 1 3j 18 1 9 10 5 2 t 8.10 17 7 2 16 2 1 3 15 2 1 3 14 1 1 13 2 2 12 • • 11 . . . . 10 . . 1 1 Regression line: Slope=7.55 ± 0.0255 per mm. t=2.•897 •p•=0.0035 • samples contain many more females, whose lengths exceed that of the largest immature male, than they do recognizable males. We believe that this is due to net selection for the larger and fatter females and to the higher survival rate of females.

ABNORMAL GONOPODIAL DEVELOPMENT Three of the males collected from El Ojo de la Hacienda Dolores are believed to be sex-reversed or to have been delayed in development of male secondary sexual characteristics (Tables 2 and 3). They are the three largest males from the spring and exceed the next largest nosnal male by at least 2 mm. One of them (the largest) is irnature. The gonopodia of the two mature individuals are dissimilar from those of any other mature G. hurtadoi males examined. It is not due to size of development of the gonopodium as equally large laboratory- raised F,'s have normal gonopodia. There are 546 females in the two collections that are large enough to be readily recognized if they were to change sex or to differentiate belatedly. Sex reversal or delayed differentiation apparently occurs in about 0.6% of the females. • Comparison of the tips of the gonopodia of the two large transformed males with those of normal males shows many differences, and the

322 THE TEXAS JOURNAL OF SCIENCE differences are most pronounced in the larger individual (Fig. 15). In the smaller individual, the elbow is reduced; it is absent in the larger. In the small individual the terminal hooks are angular; in the larger, the terminal segment of ray 5a is not modified into a definite hook. The spines on the posterior branch of ray 4 are more numerous in both individuals, as is true of gonopodia structures formed by treating females of Gambusia affinis with male hormones (Turner, 1942). The spines on ray 3 are shorter and more numerous, especially in the larger fish. The larger fish has a point on the end of the terminal hook of the posterior branch of ray 4. All these variations approach morphologic structures of the gonopodia of other poeciliids. The first three

OE?C] l 11 b%1 11 1111.111000

%SW

Fig. 15. Gonopodia of Gambusia hurfadoi individuals with sex reversal or delayed transformation. A. 24 mm. individual from TNHC 4205. B. 30 mm. individual from TNHC 4206. are similar to structures found in members of the Heterandriini (a tribe belonging to the same subfamily as Gambusia). The fourth variation is similar to that of the members of the G. nicaraguensis species group, and the subgenus Heterophallina. The fifth variation is similar to structures found in many species of Gambusia including geiseri, lemaitrei Fowler, and a species that has been misidentified as nicaraguensis by Carl L. Hubbs (1926) and Rosen and Gordon (1953). One male G. senilis had an aberrant gonopodium (Fig. 2H). We can offer no explanation for the cause of this anomaly.

EVOLUTION The Gambusia nobilis species group consists of six allopatric species. We believe that the sympatric range of G. nobilis and G. geiseri is due to artificial transfer of the latter species. It is possible that the forms are merely racial variants of one species. We doubt this on four grounds: (1) G. nobilis and G. geiseri now are sympatric and we know of no hybridization; (2) there is no correlation of morphologic A REVISION OP THE GAMBUSIA NOBILIS SPECIES GROUP 323 similarity with geographic nearness of the wide ranging species G. senilis to any other species; (3) laboratory F,'s retain the distinction that their parents are recognized by; and (4) males in the laboratory court females of their own species in preference to those of other species. The formation of these six species is believed to have resulted from isolation. We suggest that the ancestor of nobilis may have been wide- spread. It would have had geographic variation typical of many modern wide-ranging species. This species might be expected to have been adapted to spring life necessary for survival under desert conditions. During an interglacial period the eastern form which was the ancestor of affinis was able to enter the range of the ancestor of nobilis. It first contacted the more eastern populations of that species. The eastern forms of the nobilis type were able to compete successfully with the affinis ancestor only in the large Comal and San Marcos Springs, and perhaps some intervening waters. They were cut off from their relatives by waters inhabited by the affinis type, with which they could not compete elsewhere than in springs. In this isolated locality they evolved into G. geiseri. During the same, or, more probably, a later interglacial period, the streams inhabited by the other populations of the nobilis series were subjected to drought. Some of the fish population were killed. The few that survived did so in springs or mountain headwaters. Each small population became adapted to the conditions of their survival locality. When wet cycles occurred the forms adapted to stream life were able to spread more rapidly and to occupy the previously dry stream courses. They contacted other relic stream populations and fused with them. When they contacted spring populations many of them fused; however, some of the spring forms were so modified that they were ecologically isolated from stream forms. These spring forms might have fused with other spring populations, but were prevented from doing so by the intervening stream populations with which they could not compete in streams. This process may have been repeated several times. At or about the same time, G. affinis entered western Texas and further isolated the forms which were evolving into G. nobilis and G. gaigei. We suspect that other spring endemics of the G. nobilis species group will be found in northern Mexico when the area is more fully explored. The distribution of an endemic species belonging to the Gambusia nicaraguensis species group in Menard County, Texas, also may well result from speciation through isolation resulting from competition with G. affinis (Clark Hubbs, 1957a) .

324 THE TEXAS JOURNAL OF SCIENCE

SUMMARY The members of the Gambusia nobilis species group are defined; three of the species (G. geiseri, G. hurtadoi, and G. alvarezi) are described as new. Five of the six species have been reared in the laboratory and maintain morphologic distinctions used. Laboratory-raised specimens are shown to have great morphometric differences from wild-caught individuals of the same populations. Known ranges are given for all species. Doubt is cast on two published localities each for G. nobilis and G. senilis, and the western records for G. geiseri are considered to result from introduction. A spotted race of G. senilis is recognized and its disjunct distribution is explained by past irrigation diversions. Two populations of G. gaigei are described—one extinct, the other near extinction. An attempt to save the remaining population is recommended. Four color variants of G. hurtadoi are recognized. Three are suspected to have a lower survival than the other. All six species are shown to be abundant in clear spring-fed habitats. Only one, G. senilis, occurs frequently in other habitats. One of the springs is shown to have annual thermal variation. The dorsal ray number correlates with the temperature in two species. Three males are either sex reversed or have been delayed in transformation. These individuals have gonopodia similar to those of other related species and genera. The species are suspected to have evolved while isolated geographically. This isolation is believed to be due to drought and compe- tion with Gambusia affinis.

LITERATURE CITED

ALVAREZ, JOSE, 1950—Claves para la determinacion de especies en los peces de las aquas continentales Mexicanas. Secretaria de Marina, Mexico: 1-144. BAIRD, SPENCER F., AND CHARLES GIRARD, 1853—Descriptions of new species of fishes collected by Mr. John H. Clark, on the U. S. and Mexican Bouiadry Survey, under Lt. Col. Jas. D. Graham. Proc. Acad. Nat. Sci., Phila., 6: 387- 390. 1854—Descriptions of new species of fishes collected in Texas, New Mexico and Sonora, by Mr. John H. Clark, on the U. S. and Mexican Bound- ary Survey, and in Texas by Capt. Stewart Van Vliet, U.S.A. Proc. Acad. Nat. Sci., Phila., 7: 24-29. D'ANCONA, UMBERTO, 1939—A proposito di gambusie. Bull. Zool., Un. Zoo!. Ital., 10: 75-79. EMORY, WILLIAM H., 1857—Report on the United States and Mexican Boundary Survey, made under the direction of the secretary of war. Vol. 1: i-xvi 1-258. EVERMANN, BARTON WARREN, 1892—A report upon investigations made in Texas in 1891. Bull. U. S. Fish Comm., 11: 61-90, 1 map, pls. 29-36.

A REVISION OF THE GAMBUSIA NOBILIS SPECIES GROUP 325

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