STATUS AND GENETIC STRUCTURE OF THE CHANNEL CATFISH COMPLEX (GENUS ICTALURUS) IN NEW MEXICO AND TEXAS SHERRI ANN MCCLURE-BAKER

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1 STATUS AND GENETIC STRUCTURE OF THE CHANNEL CATFISH COMPLEX (GENUS ICTALURUS) IN NEW MEXICO AND TEXAS By SHERRI ANN MCCLURE-BAKER Bachelor of Science in Zoology Oklahoma State University Stillwater, Oklahoma 2002 Submitted to the Faculty of the Graduate College of the Oklahoma State University in partial fulfillment of the requirements for the Degree of MASTER OF SCIENCE December, 2005

2 STATUS AND GENETIC STRUCTURE OF THE CHANNEL CATFISH COMPLEX (GENUS ICTALURUS) IN NEW MEXICO AND TEXAS Thesis Approved: Anthony A. Echelle Thesis Adviser Ronald Van Den Bussche Margaret Ewing A. Gordon Emslie Dean of the Graduate College ii

3 ACKNOWLEDGMENTS I wish to express my sincere gratitude to my mentor and advisor Dr. Anthony Echelle. His support began when I was an undergraduate and continues even now. His contributions to this research and to my personal and professional growth are greatly appreciated. I wish to extend that same appreciation to the additional members of my committee: Dr. Ronald Van Den Bussche and Dr. Margaret Ewing. In addition I would like to thank Alice Echelle, Bryan Bevins, Nathan Allan, Gary Garrett, John Karges, Tim Bonner, David Propst, Bob Larson, Jim Brooks, Chris Hoagstrom, Jim Miller (and class) and Bruce Moring for assistance with collection efforts. I am also grateful to Chris Hughey at the Oklahoma State University Veterinarian Medical Center for all of his assistance with radiographing, Sarah Weyandt and all other personnel in Dr. Van Den Bussche s genetics laboratory, and Dean Hendrickson for his continuing advice. I must also recognize the faculty and staff of the Department of Zoology and the Oklahoma Cooperative Fish and Wildlife Research Unit for their time and guidance. And last but certainly not least, without the constant support and understanding of my daughter LeslieHope McClure and my husband Brian K. Baker, I would not be where I am today. iii

4 TABLE OF CONTENTS Chapter Page I. INTRODUCTION...1 II. REVIEW OF LITERATURE Distribution and Habitat...3 Morphologic Differences...4 Allozyme and Geographic Hybrid Variation...6 III. METHODLOGY Fish Collections...8 Morphology...9 Mitochondrial DNA...10 IV. FINDINGS Morphology...12 Mitochondrial DNA...17 Evidence of Introgressive Hybridization...19 V. CONCLUSION...22 REFERENCES...27 APPENDIX...29 iv

5 LIST OF TABLES Table Page I. Anal-fin ray counts for Ictalurus spp. in New Mexico and Texas...14 II. Vertebral counts for Ictalurus spp. in New Mexico and Texas...15 III. Mitochondrial DNA haplotype occurrences in Ictalurus spp. from Texas, New Mexico, and Reference localities outside of the study area...18 v

6 LIST OF FIGURES Figure Page 1. Collection sites in the study area Vertebral counts versus score for a canonical discriminant function (Kelsch 1995) designed to separate I. lupus and I. punctatus Relationships among mtdna haplotypes...17 vi

7 CHAPTER I INTRODUCTION The purposes of this study were to assess the status and geographic pattern in genetic structure of the headwater catfish (Ictalurus lupus) in New Mexico and Texas. The species has been reported from the Pecos River Basin in New Mexico and west Texas, other tributaries of the Rio Grande in Texas, several Gulf Coast streams flowing eastward off the Edwards Plateau of Texas, and Gulf Coast streams in Tamaulipas, Mexico (Kelsch and Hendricks 1990). Historical records are sketchy but I. lupus had declined throughout its range by the 1980s and it apparently had been extirpated from Gulf Coast tributaries north of the Rio Grande (Kelsch and Hendricks 1986). Possible reasons for the declining status are competition and hybridization with the morphologically similar Channel Catfish, I. punctatus, primarily as a result of anthropogenic habitat changes favoring the latter species (Yates et al. 1984, Kelsch and Hendricks 1986, Sublette et al. 1990). The present status of I. lupus is poorly understood, in part because its external morphology can be confused with that of the more common I. punctatus (Kelsch 1995). They differ in anal-fin ray count and body shape, but there is overlap in the former and body shape is confounded by geographic variation and allometric changes within species (Humphries and Miller 1982; Kelsch 1995). In addition, there is the added complication of confusing I. lupus with the so-called Chihuahua catfish (I. sp.), an undescribed and 1

8 poorly understood species also present in the Rio Grande system (Sublette et al. 1990, J. M. Humphries and R. R. Miller, unpubl. manuscript). In this thesis, I refer to I. lupus, I. punctatus, and the Chihuahua catfish as the channel catfish complex. Previous genetic studies of I. lupus in Texas and New Mexico were surveys of allozyme variation in the Pecos River of New Mexico (Yates et al. 1984) and Devils River of Texas (Kelsch and Hendricks 1986). The Pecos River results showed fixed differences at multiple allozyme loci and low levels of hybridization with no evidence of genetic introgression. The only hybrid was an apparent backcross product in a sample that otherwise included five I. lupus and 14 I. punctatus. In contrast, one of the eight specimens examined from Devils River was considered an F 1 hybrid and two were considered products of backcrossing between a hybrid and I. lupus. The remaining five had genotypes expected of I. lupus, based on a sample of that species from the Rio Soto La Marina basin in Mexico. My objective was to assess the status of I. lupus by using a combination of mtdna and various morphological characters, including a canonical discriminant function described by Kelsch (1995) was used. 2

9 CHAPTER II REVIEW OF LITERATURE Distribution and Habitat The Rio Grande Basin supports three species in the channel catfish species complex. The complex includes the Channel Catfish (I. punctatus), a widespread species in eastern North America, and two species restricted to the Rio Grande Basin or closely associated basins in south Texas and northern Mexico: the Headwater Catfish (I. lupus) and an undescribed species referred to as the Chihuahua Catfish (Ictalurus sp.). Ictalurus lupus occupies clear waters generally with a moderate gradient. It has persisted in headwater streams or in fluctuating tailwaters of dams in the Pecos River. Some recent reports of I. punctatus from the Pecos drainage have included this species (Cowley and Sublette 1987). Ictalurus lupus is native to the Pecos drainage downstream of Sumner Reservoir. Historical records of I. lupus from New Mexico and Texas are sketchy, but populations appear to be declining in the Pecos River drainage (Sublette et al. 1990). 3

10 Morphologic Differences A previous study by Humphries and Miller (1982) used multivariate techniques to distinguish between I. lupus and I. punctatus. Characters included gill rakers, anal-fin ray, vertebral counts and a variety of morphometric variables. In a principle components analysis (PCA) of the morphometric variables, the second axis (PCII) contributed most to the discrimination, primarily because of interspecific differences in axis length of pectoral spine and distance from pectoral-spine base to orbit. Axis I from PCA of meristic variables primarily reflected vertebral number and anal-fin ray count and provided nearly complete separation of the two species. This axis together with PCII from the morphometric analysis, produced almost complete discrimination (Humphries and Miller 1982). For most drainages the vertebral counts for I. lupus and I. punctatus were and 49-50, respectively, (Humphries and Miller 1982). Variables identified as most useful in separating I. lupus from I. punctatus indicate that I. lupus is more robust and has a lower anal ray count than I. punctatus (Kelsch and Hendricks 1986). Overall, I. lupus tends to be broader with shorter pectoral spines, and it has a shorter anal-fin base than I. punctatus (Kelsch 1995). Kelsch and Hendricks (1986) used PCA in another attempt to explain variation in morphological characteristics. Principal component II explained 14% of the sample variance and was primarily influenced by vertebral count and anal ray count with loadings of and 0.648, respectively. The next most influential variable was length of the longest caudal ray with a loading of This component clearly separated I. lupus from I. punctatus (Kelsch and Hendricks 1986). 4

11 The two species differ in a number of osteological characters, including vertebral count, as previously mentioned, and presence or absence of contact between the supraoccipital process and the supraneural spine. Another distinguishing character is the supraneural spine. In I. punctatus, the spine extends forward from the base of the dorsal spine and articulates in a v-shaped notch at the posterior extent of the supraoccipital spine. Ictalurus lupus falls short of making any connection (Lundberg 1982). Finally the supraethmoid of I. lupus superficially resembles that of I. punctatus but is flatter, much narrower and more extended (Calovich and Branson 1964). Anal ray count is the character most often used to distinguish the two species. However, many individuals have counts that fall into a range of overlap (25-26), reducing the value of this character. Kelsch (1995) found that a canonical discriminate analysis (CDA) of four variables correctly identified 91% of test individuals. He originally measured 14 characters to determine which were most useful in distinguishing taxa (Kelsch and Hendricks 1986). These variables were standard length, length of longest caudal ray, depth of caudal peduncle, length of anal fin base, head length, head width, snout width (just posterior to insertion of maxillary barbels), diameter of orbit, interorbital width, distance between anterior nostrils, maximum mouth width (when closed), distance from tip of snout to origin of dorsal fin, anal ray count, and vertebral count. The CDA retained five of the 14 characters, namely anal ray count, pectoral spine length, caudal peduncle depth, and mouth width. Analysis of covariance revealed significant differences between the means of I. lupus and I. punctatus for 11 of the 12 morphometric variables analyzed. Only predorsal length was not significantly different (Kelsch 1995). He noted the possibility that some I. 5

12 lupus from New Mexico, particularly three individuals with anal ray counts of 21, may be an undescribed species (Sublette et al. 1990) that could have been in the system from which these individuals were collected (Kelsch 1995). Allozyme and Geographic Hybrid Variation Ictalurus lupus and I. punctatus are potentially sympatric throughout much of the range of I. lupus and are known to hybridize in some areas (Yates et al. 1984, Kelsch and Hendricks 1986, Kelsch 1995). In all previous studies, the two species were considered sympatric in Devils River. Humphries and Miller (1982) found specimens from Devils River to be morphometrically more similar to I. punctatus than to I. lupus, whereas Kelsch and Hendricks (1986) protein electrophoretic survey indicated that three of eight individuals from Devils River were I. lupus x I. punctatus hybrids. One individual was potentially an F 1 hybrid, being heterozygous at each diagnostic locus. The remaining hybrids appeared to be products of backcrossing between hybrids and I. lupus (Kelsch and Hendricks 1986). The absence of pervasive introgression, in spite of species contact, suggests restricted gene flow, supporting the species status of I. lupus (Kelsch and Hendricks 1986). Allozyme analyses indicate substantial genetic divergence between I. punctatus and I. lupus (Yates et al. 1984, Kelsch and Hendricks 1986). The two species exhibited fixed differences in New Mexico (Yates et al. 1984). Of 20 individuals examined from the Pecos River, five contained alleles characteristic of I. lupus, 14 contained alleles found in I. punctatus, and only one individual appeared to be a hybrid (Yates et al. 1984). Sympatry of these two forms in the Pecos River, along with the apparent low level of 6

13 hybridization, suggests co-existence without significant genetic introgression (Yates et al. 1984). Kelsch (1995) assayed 191 individuals from the channel catfish complex from New Mexico, Texas, Mexico, and North Dakota. In accordance with Yates et al. (1984) and Kelsch and Hendricks (1986), he found fixed allozyme differences between I. lupus and I. punctatus at several gene loci. 7

14 CHAPTER III METHODOLOGY Fish Collections A total of 141 specimens of the channel catfish complex were collected. 78 additional specimens were obtained from other sources (see Acknowledgments). All specimens were collected from 32 locations in New Mexico and Texas (Fig. 1) in 2002 and Collections were made with baited hoop nets, trotlines and juglines, seines and backpack electroshocking. Sample sizes at each site ranged from 1 to 21. For those collected in our presence, individuals less than about 80 mm TL were immediately preserved directly in 100% ethanol. Larger fish were individually labeled and preserved in 10% formalin, following removal of tissues (muscle or adipose fin and a maxillary barbel), which were stored in individually labeled tubes containing 100% ethanol. Specimens obtained from other sources were either handled as just described or they were placed on ice in the field and subsequently frozen until transferred to us, and then treated as described. All specimens preserved in formalin were later transferred to 45% isopropyl alcohol. Eventually, all specimens will be deposited in the Oklahoma State University Collection of Vertebrates. 8

15 Figure 1. Collection sites in the study area. All sites are in the Rio Grande Basin except See Appendix A for detailed locality information Morphology For 125 specimens, x-ray radiographs were used to make vertebral and anal-fin ray counts and to assess presence or absence of contact between the dorsal-fin supraneural bone and the supraoccipital process. The vertebral counts included five representing the Weberian apparatus. For anal-fin rays, there normally is one ray per proximal pterygiophore. Exceptions include the most posterior ray, which is not associated with a pterygiophore and two or three short anterior rays associated with a single pterygiophore. Following Kelsch and Hendricks (1986), our counts include the posterior-most ray, and the 1-3 rays associated with the first pterygiophore are counted as one. For each of these 125 specimens, standard length (SL) pectoral spine length (PSL), 9

16 caudal peduncle depth (CPD), and mouth width (MW) were also recorded. These were measured with dial calipers and recorded to the nearest 0.1 mm, except that SL in large specimens was measured to the nearest mm with a metered rule. Each of the 125 specimens was subjected to the following formula to obtain a score for a canonical discriminant function (Kelsch 1995) designed to separate I. lupus and I. punctatus on the basis of external characters (AR = anal-fin ray count): S = S 1 + S 2 + S 3 + S 4 ; S 1 = (AR 25.2)/1.66; S 2 = 0.2[log e PSL ( *log e SL) ]/0.144; S 3 = 0.276[log e CPD ( *log e SL) 0.057]/0.087; S 4 = [log e MW ( *log e SL] 0.096]/ Mitochondrial DNA Genomic DNA was isolated from 217 specimens within the study area by using either a phenol extraction method (Longmire et al. 1997) or the Qiagen Dneasy protocol for animal tissue. Three primers described by Kocher et al. (1989), H16249, L15058, and H15149, were used to sequence the first 399 base pairs of the cytochrome b (cytb) gene. Primer L15058 was used in combination with either H15149 or H16249 to amplify, with the polymerase chain reaction (PCR), respectively, the first 399-bp segment of cytb or the entire gene. PCR conditions were as follows: 94 C for 3 min; 35 cycles of 94 C for 1 min, 50 C for 1 min, and 72 C for 2 min; 72 C for 30 min. PCR products were purified using the Wizard PCR Preps DNA Purification System (Promega). 10

17 Sequencing reactions were conducted with separate reactions for primers L15058 and H15149 and sequences were resolved with the ABI 377 automated sequencer. Two sequences from I. lupus were obtained from GenBank, one from Dark Canyon, Eddy County, New Mexico (GenBank accession number, AY327267), and one from San Felipe Creek, Val Verde County, Texas (AY458885). As reference material for mtdna of I. punctatus from outside the study area, six additional sequences were obtained from GenBank: AY from Mud River, Kentucky; AY from San Marcos State Fish Hatchery (broodstock from Hays Co., Texas), AY from Lamoine River, Illinois, AF and AB from introduced populations in China and Japan, and AB from Lake Kasumiguara in Japan. As additional reference material, cytb was sequenced from three I. punctatus collected in the Arkansas River Basin, two from the Arkansas River in Kansas and one from the Illinois River in Oklahoma. For outgroups in the phylogenetic analysis, the following GenBank sequences were used: AF from Blue Catfish (Ictalurus furcatus) and AY from Flathead Catfish (Pylodictis olivaris). Sequences were aligned with Clustal X (Thompson et al. 1994), and, using MEGA2 (vers. 2.1; Kumar et al. 2001), the sequences were subjected to a neighborjoining tree based on uncorrected pairwise p-values. Bootstrapping with 1000 replicates was used to assess support for nodes. For Independence Creek populations, the exact probability test in ARLEQUIN ( was used to test for haplotype frequency differences. 11

18 CHAPTER IV FINDINGS Morphology The canonical discriminant function scores indicated no overlap in distributions for two groups of catfish collections (Fig. 2) that were used in this project as operational reference groups for morphological comparisons. One group clustered as I. punctatus (i.e., score > 0.00; Kelsch 1995), with scores ranging from to 4.736, and comprised all collections from the Nueces River and the Pecos and Rio Grande mainstems. Individuals in the second group were from the only locations (Rocky Arroyo, New Mexico and San Solomon Spring, Texas) isolated by dry land from the mainstem rivers. All except one fish from these two locations had scores < 0.00 (range = to 0.083). The two reference groups had non-overlapping anal-fin ray counts, with 23 to 25 in I. lupus and 27 to 29 in I. punctatus (Table I), and they were almost non-overlapping in vertebral counts respectively, 44 to 47 and 47 to 51 (Table II). Finally, the reference groups differed in presence/absence of contact between the dorsal-fin supraneural and the supraoccipital process (in contact = I. punctatus; not in contact = I. lupus). This character was difficult to evaluate from the x-rays, and will not be mentioned elsewhere in this study. 12

19 Figure 2. Vertebral counts versus score for a canonical discriminant function (Kelsch 1995) designed to separate I. lupus and I. punctatus. Ellipses in the two graphs are in the same position. 13

20 TABLE I ANAL-FIN RAY COUNTS FOR ICTALURUS SPP. IN NEW MEXICO AND TEXAS. SITE NUMBERS AS IN FIGURE 1. Anal-fin ray count Locality and site number New Mexico 1. Pecos R., Ft. Sumner State Park 1 2. Pecos R., Pecos R. at Old Ft. Sumner 3 3. Pecos R. at Bosque Draw 1 4. Pecos R. at Highway Pecos R. near Hagerman 1 7. Pecos R. at Ten-Mile Reservoir 3 9. Rocky Arroyo 2 2 Texas Rio Grande, Big Bend National Park San Solomon Springs & 13. Independence Creek a 15. Devils River upstream of Dolan Creek Dolan Creek Devils River below Dolan Falls b. Mud Creek Pinto Creek 5 20, Nueces River Totals a This was the only specimen from Independence Creek identified as I. punctatus in the field. 14

21 TABLE II VERTEBRAL COUNTS FOR ICTALURUS SPP. IN NEW MEXICO AND TEXAS. SITE NUMBERS AS IN FIGURE 1. Number of Vertebrae Locality and site number New Mexico 1. Pecos R., Ft. Sumner State Park 1 2. Pecos R., Pecos R. at Old Ft. Sumner Pecos R. at Bosque Draw 1 4. Pecos R. at Highway Pecos R. near Hagerman 1 7. Pecos R. at Ten-Mile Reservoir 3 9. Rocky Arroyo RA 3 1 Texas Rio Grande, Big Bend National Park San Solomon Springs & 13. Independence Creek a 15. Devils River upstream of Dolan Creek Dolan Creek Devils River below Dolan Falls b. Mud Creek Pinto Creek , Nueces River Totals a This is the only specimen from Independence Creek identified as I. punctatus in the field (2 specimens from an impoundment at Oasis Ranch were identified as I. punctatus, but they were not preserved for detailed morphological analysis). 15

22 It is unlikely that the collections included the undescribed Chihuahua catfish (I. sp.), although the possibility cannot be completely discounted. An unpublished species description indicates that, for the morphological characters used, Ictalurus sp. and I. lupus overlap broadly, with the former being modally somewhat more divergent from I. punctatus (J. M. Humphries and R. R. Miller unpubl.). The identification problem is compounded by the occurrence of putative hybrids between I. sp. and I. lupus in a morphological survey of museum specimens (Humphries and Miller op. cit.). Based on the description presented by Humphries and Miller (op. cit.), the four specimens from Rocky Arroyo are the only potential representatives of I. sp. in the collections. However, the pectoral spine serration is more similar to that of I. lupus (fine, recurved, sharp) than to that of I. sp. (few, non-recurved, blunt). The canonical discriminant scores for the two larger collections from tributaries connected with the Pecos River and the Rio Grande showed wide ranges of variation, respectively, to for fish from Independence Creek and to for those from the Devils River area. For the fish from both areas, the distributions of discriminant function scores and counts of anal-fin rays and vertebrae were skewed toward I. lupus, but one individual from each of the two areas fell within the range of variation for the reference collections of I. punctatus. In Devils River, the specimens from outside of Dolan Creek, three from upstream of Dolan falls and three from just downstream of the falls, were closer to the reference I. punctatus cluster than were any of the 17 specimens from Dolan Creek (Fig. 2). The four fish from Pinto Creek fell well within the range of variation for I. punctatus. 16

23 Mitochondrial DNA 14 cytb haplotypes were detected (designated A to N) in the 219 fish assayed for mtdna variation, and two additional haplotypes (O and P) were among the six sequences obtained from GenBank. The phylogenetic analysis resolved two primary clades (Fig. 3), a punctatus clade of 14 haplotypes (C-P) and a lupus clade comprising haplotypes A and B. Paired haplotypes between the two clades differed by transition substitutions (0 transversion substitutions), giving uncorrected p values ranging from to (= 4.8% to 6.3% divergence for the 399 base pairs assessed). Figure 3. Relationships among mtdna haplotypes. Neighbor-joining tree based on uncorrected pairwise p-values. Abbreviations for haplotypes are as in Table III. Rectangle encloses a haplotype reported by Hardman (2004) for a specimen identified as I. lupus from Dark Canyon, New Mexico. Haplotypes in circles were detected in I. punctatus from outside the study area; those in thick circles also occurred in the study area. Numbers below nodes = bootstrap support (1000 replications). 17

24 TABLE III MITOCHONDRIAL DNA HAPLOTYPE OCCURRENCES IN ICTALURUS SPP. FROM TEXAS, NEW MEXICO, AND REFERENCE LOCALITIES OUTSIDE OF THE STUDY AREA. SFH=STATE FISH HATCHERY; BBNP=BIG BEND NATIONAL PARK. SITE NUMBERS AS IN FIGURE 1. Haplotype lupus punctatus Locality and site number A B C D E F G H I J K L M N O P I. punctatus outside of study area Kansas (Arkansas River) 2 Illinois (LaMoine River) a 1 Kentucky (Mud River) 1 Oklahoma (Illinois River) 1 Texas (San Marcos SFH) b,c 1 Unknown localities (China & Japan) d 1 1 New Mexico 1. Pecos R., Ft. Sumner State Park Pecos R., Pecos R. at Old Ft. Sumner Pecos R. at Bosque Draw 1 4. Pecos R. at Highway Pecos R. near Hagerman 1 6. Pecos R. below Brantley Dam 1 7. Pecos R. at Ten-Mile Reservoir Pecos R. at Harroun Crossing a. Rocky Arroyo 4 9b. Dark Canyon a,e 1 Texas 32. Rio Grande near Presidio Rio Grande, BBNP San Solomon Springs & 13. Independence Creek 4 1 f Impoundment at Oasis Ranch Pecos River at Pandale Crossing Devils River above Dolan Creek Dolan Creek Devils River below Dolan Falls 3 18a. San Felipe Creek b 1 18b. Mud Creek Pinto Creek Nueces River basin a From Hardman (2004). From Wilcox et al. (2004). From broodstock taken in Hays Co., Texas. From Matsuo et al. (2001). e Identified as I. lupus and catalogued as such at Museum of Southwestern Biology (MSB45448). f This is the one specimen from Independence Creek identified as I. punctatus in the field. 18

25 With one exception, haplotype P from Illinois, the five haplotypes used as reference mtdna samples for I. punctatus were identical to one or another of the three common haplotypes of the punctatus mtdna clade (C, F, and J) detected in the study area. Haplotype O, which was not detected in the samples, was in the punctatus mtdna clade although it occurred in a specimen from Dark Canyon, New Mexico that was morphologically identified as I. lupus in a previous study (Hardman 2004). The haplotypes of the punctatus mtdna clade differed from one another by 1 to 5 transition substitutions (no transversion substitutions), giving divergences of 0.8% to 1.3%. The lupus mtdna clade included two haplotypes (A and B) with non-overlapping geographic distributions. Haplotype A was restricted to the Pecos River basin, occurring in all fish in the reference collections of I. lupus from Rocky Arroyo (n = 4) and San Solomon Spring (n = 19), and in 4 of 26 fish from Independence Creek proper (not counting two I. punctatus from a nearby springfed lake on Oasis Ranch). Haplotype B of the lupus mtdna clade was found in three tributaries of the Rio Grande downstream from the mouth of the Pecos River. It occurred in all fish from the Devils River watershed (n = 23), 1 of 5 from Pinto Creek, and the one GenBank sample from San Felipe Creek. Sequence divergence between haplotypes A and B was 1.0% (4 transition substitutions, 0 transversion substitutions). Evidence of Introgressive Hybridization The small collection (n = 5) from Pinto Creek included 1 fish with haplotype B of the lupus mtdna clade, 3 fish with haplotype C; and 1 with haplotype K, all of the punctatus clade. Morphologically, these fish rather homogeneously resembled I. 19

26 punctatus, and, as previously mentioned, they fell well within the range of that species in counts and measurements. Thus, this collection appears to represent I. punctatus, but with some individuals carrying the mtdna of I. lupus. In Independence Creek, 4 of the 25 fish field-identified as I. lupus had haplotype A of the lupus mtdna clade, whereas 21 (84%) carried haplotype D of the punctatus clade. These two haplotype groups showed no significant difference in anal-fin ray or vertebral counts, or in canonical discriminant score (t-tests; P = ). Their morphology was strongly skewed toward I. lupus (Fig. 2), and some fell well within the range of variation for the two reference collections of that species (San Solomon Springs and Rocky Arroyo). However, t-tests comparing them with the reference collections showed a significant shift toward I. punctatus in anal-fin ray count (P < 1*10-5 ) and canonical discriminant score (P < 1*10-5 ), but not in vertebral count (P > 0.28). This difference held when both of the reference collections, San Solomon Springs and Rocky Arroyo were included in the comparison and when the latter was excluded. All 23 fish from the Devils River watershed were carrying mtdna from the lupus clade. Morphologically, however, the six from the mainstem Devils River clustered closer to I. punctatus than did any of the17 fish from Dolan Creek, a large, springfed tributary of the river, and one of the six fell within the range of variation for the reference collections of I. punctatus (Fig. 2). The fish from Dolan Creek and the mainstem of Devils River differed significantly in anal-fin ray count (t-test; P < 1*10-6 ) and canonical discriminant score (P < 1*10-5 ), but not in vertebral count (P = 0.59), indicating that the latter have an ancestry in hybridization. 20

27 The Dolan Creek population of I. lupus was morphologically skewed toward that species. However, t-tests comparing them with the reference collection of that species from San Solomon Springs (and from the combined collections from San Solomon Springs and Rocky Arroyo) showed a shift toward I. punctatus for anal-fin ray count (P < ), vertebral count (P < 0.004), and canonical discriminant score (P < 0.01). Finally, although no catfish were found in Dark Canyon, results from previous reports, together with this analysis, indicate that this population of I. lupus is genetically introgressed by I. punctatus. Based on allozymes, Yates et al. (1984) considered the 21 fish they examined to be pure I. lupus. However, my analysis demonstrates that the cytb sequence reported for one of those specimens (Hardman 2004) was haplotype O of the punctatus mtdna clade. That specimen was deposited as I. lupus in the Museum of Southwestern Biology (Cat. No ). 21

28 CHAPTER V CONCLUSION There is reason to believe that hybridization between I. lupus and I. punctatus is a recent anthropogenic effect, rather than a product of distant evolutionary history. Koster (1957) and Sublette et al. (1990) suggested, with no explanation, that I. punctatus is not native to the Rio Grande basin, including the Pecos River, in New Mexico, and an ongoing investigation of historical records by D. Hendrickson (pers. comm.) preliminarily suggests that the species might not be native to the Rio Grande of Texas. Relatively recent introduction is supported by the low sequence divergence detected within the punctatus mtdna clade (W1.3%), with individuals from streams far removed from our study area carrying cytb sequences identical to the common sequences detected in the Rio Grande basin. Results indicate that the mainstem Pecos River and Rio Grande of New Mexico and Texas primarily support I. punctatus. No evidence of I. lupus was found in such waters, although the possibility remains that it may be present. As recently as the early 1980s, an allozyme (Yates et al. 1984) analysis of 20 fish from the Pecos River just downstream of Sumner Reservoir detected 5 I. lupus, 14 I. punctatus, and 1 backcross product. Collections from Independence Creek, Devils River, and Pinto Creek clearly indicate past or ongoing hybridization. The five fish examined from Pinto Creek rather 22

29 homogeneously resembled I. punctatus, although one of them carried the lupus mtdna clade. Additional collecting in Pinto Creek might reveal the presence of I. lupus. The Independence Creek population of I. lupus, which has not previously been examined genetically, appears introgressed by I. punctatus. The collection field-identified as I. lupus included individuals carrying the mtdna of either I. lupus (n = 4) or I. punctatus (n = 21). In morphology, they were strongly skewed toward I. lupus, but there was a statistically significant shift toward I. punctatus. This might reflect either genetic introgression or geographic variation within I. lupus. But, regardless, morphology and the mtdna distribution together clearly indicate genetic introgression. Despite evidence for genetic introgression of Independence Creek I. lupus, two lines of evidence argue that the local populations of that species and I. punctatus exist without fusion as a single reproductive population. First, three fish were field-identified as I. punctatus, two (not preserved for counts and measurements) from the springfed lake on Oasis Ranch and one from Independence Creek proper. Correspondingly, the lastmentioned fish was the only one of the 26 morphologically analyzed fish that was within the range of variation expected of I. punctatus in anal-fin ray and vertebral counts and canonical discriminant score. This fish was well separated morphologically from the 25 fish identified as I. lupus (Tables I and II; Fig. 2). The distribution of mtdna haplotypes also indicates two distinct populations in Independence Creek. The fish identified as I. lupus were carrying either lupus haplotype A or punctatus haplotype D, neither of which was in the three fish identified as I. punctatus. Those three carried either haplotype C or F of the punctatus clade. The difference in haplotype frequencies between the two groups was statistically significant, 23

30 both when all three fish identified as I. punctatus were included (Exact Probability Test in Arlequin; P = ) and when the two from the lake were removed, leaving only the one I. punctatus taken together with I. lupus (P = 0.04). Previous studies of the Channel Catfish complex in Devils River indicated hybridization between I. lupus and I. punctatus. A morphological analysis identified 3 potential hybrids in a collection that otherwise included 43 I. lupus (Humphries and Miller 1982). A subsequent allozyme analysis identified 5 I. lupus and 3 apparent hybrids, including a putative F 1 hybrid and 2 potential progeny of hybrid backcrossing to I. lupus (Kelsch and Hendricks 1986). In my results, all fish from the Devils River watershed were carrying mtdna from the lupus clade. As a group, however, the six from the mainstem Devils River were morphologically intermediate between those from Dolan Creek and the reference collections of I. punctatus, and one individual fell within the range of variation for that species. Those six specimens apparently reflect past or ongoing hybridization between the two species. The genetic history of the Dolan Creek population of I. lupus is unclear. All 17 fish surveyed were carrying the lupus mtdna clade. Morphologically, they were skewed toward I. lupus, but, relative to the reference samples of that species, there was a significant bias toward I. punctatus. This might reflect either genetic introgression by I. punctatus or geographic variation in I. lupus without genetic introgression. Further clarification of the genetic status of the Dolan Creek population will require analysis of nuclear genetic markers. 24

31 A final locality potentially supporting a genetically introgressed population of I. lupus appears to be Dark Canyon in New Mexico. As previously mentioned, allozymes showed no evidence of I. punctatus in this population (Yates et al. 1984), my analysis demonstrates that the cytb sequence reported by Hardman (2004) for a Dark Canyon specimen of I. lupus belongs to the punctatus mtdna clade. Access to the Dark Canyon site was denied, but local residents reported catching channel catfish from an impounded spring at, or near, the locality described by Yates et al. (1984). Two collecting trips to Sitting Bull Canyon Creek, which is part of the Dark Canyon watershed, failed to produce I. lupus, although it was introduced to this area in the 1980s from a population in Delaware River (Sublette et al. M. Hatch, pers. comm.). Results indicate that genetically pure I. lupus in New Mexico and Texas might be limited to areas that, in recent history, rarely have been connected to the mainstem Pecos River or Rio Grande, possibly because of reduced opportunities for contact with I. punctatus. The fish from the relatively isolated San Solomon Spring and Rocky Arroyo were markedly shifted toward the morphology expected of I. lupus, and their mtdna was from the lupus clade. Other such populations of I. lupus might exist in the mountains west and south of the Pecos River in Texas and New Mexico. Our efforts in the summers of 2002 and 2003 usually found dry creek beds except at higher altitudes where we found no catfish. A major exception was Rocky Arroyo, which harbored a previously unreported, apparently small, population of I. lupus. At a lower Delaware River locality in New Mexico, where I. lupus was collected in the early 1980s (Yates et al. 1984), a 100-m stretch of standing water was sampled with only a large number of Yellow Bullhead (Ameiurus natalis) 25

32 found, but no I. lupus or I. punctatus. No attempt was made to collect on private property in an upper reach of Delaware River in Texas where Humphries and Miller (1982) found I. lupus. It should be emphasized that populations of I. lupus with genetic material from I. punctatus remain worthy of attention from the standpoint of conservation. Natural selection can maintain ecologically and behaviorally divergent characters of two species, even in the face of gene flow for selectively neutral or effectively neutral characters like mtdna and other genetic markers typically assayed (Clarke et al. 1998). In Independence Creek, for example, there is a morphotype representing I. punctatus and one identifiable as I. lupus, even though the majority of the latter carry mtdna from the former. In other words, I. lupus is a unique historical entity that persists, even in areas where it carries genetic material from I. punctatus. 26

33 REFERENCES 1. Calovich, F. E., and B. A. Branson The supraethmoid-ethmoid complex in the American catfishes, Ictalurus and Pylodictis. The American Midland Naturalist 72: Clarke, B., M. S. Johnson, and J. Murray How molecular leakage can mislead us about island speciation, p In: Evolution on Islands. P. R. Grant (ed.). Oxford Univ. Press, Oxford. 3. Cowley, D. E., and J. E. Sublette Distribution of fishes in the Black River drainage, Eddy County, New Mexico. The Southwestern Naturalist 32: Hardman, M The phylogenetic relationships among Noturus catfishes (Siluriformes: Ictaluridae) as inferred from mitochondrial gene cytochrome b and nuclear recombination activating gene 2. Molecular Phylogenetics and Evolution. 30: Humphries, J. M. and R. R. Miller Taxonomic status, ecology, and distribution of the headwater catfish, Ictalurus lupus. Final Report, U. S. Fish and Wildlife Service, Project No Kelsch, S. W Patterns of morphometrics variation in the channel and headwater catfishes. Transactions of the American Fisheries Society 124: Kelsch, S. W., and F. S. Hendricks An electrophoretic and multivariate morphometrics comparison of the American catfishes Ictalurus lupus and I. punctatus. Copeia 1986: Kelsch, S. W., and F. S. Hendricks Distribution of the headwater catfish Ictalurus lupus (Pisces: Ictaluridae). Southwestern Naturalist 35: Kocher, T. D., W. K. Thomas, A. Meyer, S. V. Edwards, S. Paabo, F. X. Villablanca, and A. C. Wilson Dynamics of mitochondrial DNA evolution in animals: amplification and sequencing with conserved primers. Proceedings of Natural Academy of Science 86: Koster, W. J Guide to the Fishes of New Mexico. Univ. New Mexico Press, Albuquerque. 27

34 11. Kumar, S., K. Tamura, I. B. Jakobsen, and M. Nei MEGA2: Molecular Evolutionary Genetics Analysis software. Bioinformatics 17: Longmire, J. L., M. Maltbie, and R. J. Baker Use of Lysis Buffer in DNA isolation and its implication for museum collections. Occasional Papers of the Museum Texas Tech University 163: Lundberg, J. G The Comparative Anatomy of the Toothless Blindcat, Trogloglanis pattersoni Eigenmann, with a Phylogenetic Analysis of the Ictalurid Catfishes. Miscellaneous Publications of the Museum of Zoology, University of Michigan 163: Matsuo, T., Y. Ogawa, A. Kumamaru, K. Ochi, and Y. Adachi Complete nucleotide sequence of the cytochrome b gene of channel catfish Ictalurus punctatus and comparison of sequence homology among channel catfish and other fishes. Journal of Veterinarian Medical Science 63: Sublette, J. E., M. D. Hatch, and M. Sublette The fishes of New Mexico. University of New Mexico Press, Albuquerque. 16. Thompson, J. D., D. G. Higgins, and T. J. Gibson CLUSTAL W: improving the sensitivity of progressive sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acid Research 22: Wilcox, T. P., F. J. Garcia de Leon, D. A. Hendrickson, and D. M. Hillis Convergence among cave catfishes: long-branch attraction and a Bayesian relative rates test. Molecular Phylogenetics and Evolution 31: Yates, T. L., M. A. Lewis, and M. D. Hatch Biochemical systematics of three species of catfish (Genus Ictalurus) in New Mexico. Copeia 1984:

35 MAP ID APPENDIX Appendix A. Locality descriptions for specimens used in the analysis. Map ID refers to the number associated with each locality in Fig. 1. Site ID is a collection code that is associated with the tissue numbers for each fish analyzed for mtdna. SITE NAME SITE ID COUNTY STATE LAT (DMS) LONG (DMS) NA Arkansas River at 47th Street Bridge in Wichita Ark Sedgwick KS 37 38' " 97 18' " NA Illinois River at Falcon Floats ILL Cherokee OK 34 50' " 94 55' " 1 Ft Sumner below dam C7 DeBaca NM 34 37' " ' " 2 Ft Sumner State Park C6 DeBaca NM 34 29' " ' " 3 Bosque Draw RDL090 Chaves NM 33 46' " ' " 4 Hwy 380 Bridge DLP4887 Chaves NM 33 24' " ' " 5 ACME gage DLP4886 Chaves NM 33 04' " ' " 6 Brantley Reservoir NJL02_01 Chaves NM 32 25' " ' " 7 10 Mile Reservoir C5 Eddy NM 32 19' " ' " 8 Harroun Crossing C9 Malaga NM 32 13' " ' " 9a Rocky Arroyo RA Eddy NM 32 17' " ' " 9b Dark Canyon Dark Canyon Eddy NM Balmorhea State Park BAL Reeves TX 30 55' " ' " 11 Hwy 349 Bridge C1 Terrell TX 30 29' " ' " 12 Oasis Ranch C3 Terrell TX 30 28' " ' " 13 Chandler House C2 Terrell TX 30 26' " ' " 14 Pandale PAN Valverde TX 30 08' " ' " 15 Devils River DR Valverde TX 29 59' " ' " 16 Dolan Creek C4 Valverde TX 29 58' " ' " 17 Below Dolan Falls GPG Valverde TX 29 41' " ' " 18a San Felipe Creek, Del Rio San Felipe Ck Valverde TX b Mud Creek MC Kinney TX 29 22' " ' " 19 Pinto Creek South of Hwy 90 PC Kinney TX 29 16' " ' " 20 Hwy 335 Na Uvalde TX 29 53' " ' " 21 (S) Montell, (N) Hwy 334 Nb Uvalde TX 29 28' " ' " 22 Soldier Camp Springs Nc Uvalde TX 29 18' " ' " 23 Mira Sol Ranch Ng Uvalde TX 29 07' " 99 57' " 24 Hwy 83 Nd Zavala TX 29 03' " 99 54' " 25 Lyles Ranch Nf Zavala TX 29 00' " 99 53' " 26 Peterson Ranch Ne Zavala TX 28 57' " 99 51' " 27 Boquillas - Big Bend BQ Brewster TX 29 11' " ' " 28 Solis - Big Bend SL Brewster TX 29 02' " ' " 29 Talley - Big Bend TLY Brewster TX 28 58' " ' " 30 Gauging Station - Big Bend GS Brewster TX 29 01' " ' " 31 Santa Elena Takeout - Big Bend SE Brewster TX 29 08' " ' " 32 Alamito Creek C8 Presidio TX 29 32' " ' " 29

36 VITA Sherri Ann McClure-Baker Candidate for the Degree of Master of Science Thesis: STATUS AND GENETIC STRUCTURE OF THE CHANNEL CATFISH COMPLEX (GENUS ICTALURUS) IN NEW MEXICO AND TEXAS Major Field: Zoology Biographical: Education: Graduated from Arvada High School, Arvada, Colorado in May Received a Bachelor of Science degree in Wildlife and Fisheries Ecology from Oklahoma State University, Stillwater, Oklahoma in May Completed the requirements for a Master of Science degree with a major in Zoology at Oklahoma State University, Stillwater, Oklahoma in December Experience: 08/2001 to 09/2002. Biologist Aide Internship for the Oklahoma Department of Wildlife Conservation, Aquatics Education Program, 1801 Lincoln, OKC. Job duties included maintaining a database of 400+ volunteers, conducting aquatics education workshops at various schools, maintaining the Trophy Fish Conservationist and Master Anglers Trophies. Graduate Teaching Fellow: NSF funded Graduate Scientist for the Rural Alliance for Improving Science Education (RAISE) in K-12, Department of Geography, Oklahoma State University, July 2004-June 2006 Professional Memberships: Member Golden Key National Honor Society, OSU Student chapter of American Fisheries Society, Oklahoma Academy of Science, Southwestern Association of Naturalists, SEAFWA, Minorities in Natural Resources Committee.