CALIFORNIA STATE UNIVERSITY, NORTHRIDGE THE MORPHOLOGICAL AND GENETIC SIMILARITY AMONG THREE SPECIES OF HALIBUT (PARALICHTHYS SPP.) FROM BAJA CALIFORNIA, MEXICO A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Biology by Nathaniel Bruns May 2012 The thesis of Nathaniel Bruns is approved: ——————————————— ————————— Michael P. Franklin Ph.D. Date ——————————————— ————————— Virginia Oberholzer Vandergon Ph.D. Date ——————————————— ————————— Larry G. Allen Ph.D., Chair Date California State University, Northridge ii ACKNOWLEDGEMENTS I would like to thank my advisors Larry Allen, Michael Franklin, and Virgina Vandergon for their support. Also, my appreciation and thanks to the kind people at the Los Angeles Natural History Museum, specifically Jeff Siegel (retired), Rick Feeney, and Neftalie Ramirez. The help I received from Pavel Lieb (CSUN sequencing facility) was essential to this research. I also owe thanks to Natalie Martinez-Takeshita (USC), and Chris Chabot (UCLA). iii TABLE OF CONTENTS SIGNATURE PAGE ii ACKNOWLEDGEMENTS iii LIST OF FIGURES v LIST OF TABLES vi ABSTRACT v INTRODUCTION 1 MATERIALS AND METHODS 9 RESULTS 17 DISCUSSION 22 REFERENCES 34 APPENDIX A 40 APPENDIX B 41 iv LIST OF FIGURES Figure 1 The locations where fish were collected along the coast of southern California and along the Baja peninsula (highlighted). The numbers represent all flatfish collected at each site that were used in the DNA analyses. Figure 2 A drawing of the ocular side of a right-sided flatfish. Ten morphometric variables are shown Figure 3 A drawing of the anterior half of a flatfish, showing the ocular side of a left-sided head. Eight morphometric variables are shown Figure 4 A canonical scores plot of a stepwise discriminant function analysis. The three species are not significantly different from each other morphologically, but it appears that there is more of a difference between Paralichthys californicus and P. woolmani. Both P. californicus and P. aestuarius appear to be very similar in morphology. Figure 5 Molecular Phylogenetic analysis by the Bayesian and Maximum Likelihood (ML) methods and a visual consensus showing relationships of the three Paralichthys species. v LIST OF TABLES Table 1 Classification results in matrix format of the three nominal species of Paralichthys, based on a stepwise discriminant function analysis. The rows indicate observed classifications. Table 2 The two highly significant morphometric variables in a stepwise discriminant function analysis, based on eighteen morphometric variables, and including weight (g). Data was log-transformed before analysis. N = 24. Table 3 Results of Tajima's relative rate test, which tests the equality of evolutionary rates among taxa. Table 4 Accession numbers for Genbank reference sequences of the RAG-2 gene. Table 5 Estimates of haplotype diversity and nucleotide diversity for each species and population, with the number of haplotypes and sample size. Table 6 Results of an AMOVA with a Kimura 2-parameter distance method. Populations for Paralichthys californicus, P. aestuarius, and P. woolmani were structured into three groups, and then were analyzed with an AMOVA test. Table 7 Pairwise Fst values among nominal species. Numbers in parentheses are P-values ± S.E. vi ABSTRACT THE MORPHOLOGICAL AND GENETIC SIMILARITY AMONG THREE SPECIES OF HALIBUT (PARALICHTHYS SPP.) FROM BAJA CALIFORNIA, MEXICO by Nathaniel Bruns Master of Science in Biology The three nominal species of halibut (Paralichthys californicus, P. woolmani, and P. aestuarius) that occur off Baja California are difficult to distinguish from each other. Whether these are distinctly separate species has come into question in recent years, and the best way to answer this question is through a combination of genetic and morphological techniques. Halibut were collected from locations spanning California and Baja California, including the Gulf of California. Extensive measurements of field specimens as well as museum specimens were taken and analyzed. A portion of the nuclear RAG-2 gene was sequenced for each fish and analyzed. The results of this analysis indicated that P. aestuarius does not deserve separate species status as it is essentially identical to P. californicus, while P. woolmani is a distinct species that appears to be more closely related to the Paralichthys species in the Gulf of Mexico and the southern Atlantic Ocean. vii Introduction The California halibut, Paralichthys californicus, is one of the most popular commercial and recreational fishes in the United States and Mexico (Helvey 1990). In Mexico, halibut are usually the most expensive fillets in fish markets (Barsky 1990). California halibut are present in nearshore waters along western North America, from the Quillayute River in Washington to Magdalena Bay in Baja California (Gilbert and Scofield 1898, Pattie and Baker 1969). These fish are rare north of San Francisco, and it is unknown whether the California halibut is found in the Gulf of California. Two closely related species appear to have overlapping distributions with the California halibut: the Cortez flounder (P. aestuarius) and the Mexican flounder (P. woolmani), both of which are also known as “halibut” or “lenguado.” It is unknown how these fish are related to the California halibut, and the characteristics that have been used to tell these species apart remained problematic for many years. The distributions of these three species may overlap in at least one location. The Cortez flounder has a reported distribution that begins at the mouth of the Colorado River in the Gulf of California (Norman 1934) and continues southward, but it is unclear how far around the peninsula it reaches. Specimens from the Los Angeles Natural History Museum (NHM) were collected from various locations south of the Colorado River’s mouth on both shores in the Gulf of California, as far south as La Ventana in the west and Mazatlan in the east, as well as at Magdalena Bay on the Pacific coast of the peninsula (pers. observ.). And a single specimen was also reportedly found on the Pacific side of the peninsula in the Guerrero Negro Lagoon (Martinez et al. 1996). Paralichthys woolmani has a distribution that may slightly overlap that of the Cortez flounder, but this 1 species ranges to the south as far as the Galapagos Islands and Peru (Norman 1934). A parapatric relationship seems to exist in and around Magdalena Bay (Figure 1), where the distributions of all three species seem to overlap (pers. observ.). Prior to the current study only a handful of flatfishes have had some of their genetic material sequenced, so the current phylogeny depends heavily on morphology. The published descriptions for the species P. californicus and P. aestuarius are very similar (Jordan and Evermann 1896, Norman 1934). For example, the approximate body depth that is listed for P. aestuarius falls within the range of body depths listed for P. californicus. Differences in meristic counts of these species are minor and could be explained by within-species variation. The only apparent difference noted was that specimens of P. aestuarius do not grow to be very large (Norman 1934), while specimens of P. californicus can become very large as adults and weigh up to 33-kg (Eschmeyer et al. 1983). However, this may be due to the differences in water temperature between the Pacific Ocean and the Gulf of California. Bergmann’s rule (1847), which states that species within a genus tend to be larger in colder water, may explain this observation (Blackburn et al. 1999, Ashton 2004). Fish that have broad geographic ranges will often show phenotypic plasticity in body size associated with water temperature, and most ectotherms that have been studied grow larger in colder environments (Atkinson 1994, 1995). Differences in morphology are more apparent for P. woolmani. Specifically, the reported number of gill rakers in P. woolmani (5 + 11) is quite different than that reported for both P. californicus and P. aestuarius (9 + 20, respectively), and can be used reliably to identify specimens. The ocular side of P. woolmani is also usually profusely covered 2 with black spots that are rare in the other two forms of Paralichthys studied herein (L.G. Allen, pers. comm.). A contributing factor to the problem of species identification is that many of the differences recorded for these species are not useful in the field. In some cases the differences between species are described in too vague a manner, and in other instances they were recorded for only one species. It seems likely that misidentification occurs in locations where these species have overlapping distributions. In actuality, the distributions may be different than presently known, which has significance for the proper management of these fish stocks. The life history of Paralichthys californicus has been thoroughly described by Haaker (1971), and is widely believed to be similar for P. aestuarius and P. woolmani (Allen MJ 1982, 1990). Halibut are lie-in-wait predators that are capable of blending in with the seafloor. They lack swimbladders and strike upon unsuspecting prey that come within three head-lengths of their mouths (Allen MJ 1982). They feed throughout the diel period, being most active in daylight hours (Allen MJ 1990). Changes occur in their diet as halibut increase in body size. As larvae they tend to eat rotifers or brine shrimp, and as juveniles they eat mostly small crustaceans such as copepods and amphipods (Allen LG 1988). As they mature they start to include small fish in their diet, and when they have grown beyond 230-mm in length they become almost totally piscivorous, preying on larger fish such as Engraulis mordax and Atherinops affinis (Haaker 1971). Like most fish, species of halibut appear to be subject to parasitic infestations. Common endoparasites of the California halibut are cestodes, nematodes, and trematodes, while common ectoparasites are copepods and isopods (Bane and Bane 1971, 3 Haaker 1971 & 1975). The level of infestation is likely to vary with location, but can be considerable.