Paralichthys Spp.) from Baja California, Mexico

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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

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

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).

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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

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.

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.

ABSTRACT

THE MORPHOLOGICAL AND GENETIC SIMILARITY AMONG THREE SPECIES OF HALIBUT (PARALICHTHYS SPP.) FROM BAJA CALIFORNIA, MEXICO

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.

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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

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

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,

Haaker 1971 & 1975). The level of infestation is likely to vary with location, but can be considerable. Haaker (1971 & 1975) estimated that almost half of the population in

Anaheim Bay was infested with nematodes (48% nematodes, 34% trematodes, and 2% cestodes).

Species of Paralichthys are exploited by a variety of predators. Although predation of halibut has not been thoroughly studied or quantified, it is likely that natural predators differ with a change in halibut body size, from various fish to marine birds and mammals (Fitch and Lavenberg 1971). Even larger halibut have been found to prey on smaller conspecifics (Feder et al. 1974). But the greatest predators of halibut are undoubtedly humans. Aside from the fact that humans destroy and otherwise encroach on halibut nursery habitat, these fish have long been important to the recreational and commercial fishing industries of many countries, and consequently halibut populations have suffered from over-fishing and other anthropomorphic factors (Allen MJ and

Herbinson 1990). The impact of commercial halibut fishing in the eastern Pacific Ocean has been observed as far back as the 1920s. Thompson (1950) first observed that an increase in fishing effort did not relate proportionately to a larger catch, and since then the annual halibut catch has been highly variable, with an overall decline (Frey 1971,

Allen LG 1988, Maunder 2011). The most current estimate of abundance is 3.9 million halibut in southern California, and in central California it is 700,000 halibut (Wertz

2001).

California halibut are caught most easily using nets (e.g., trawls, trammel nets), but hook-and-line fishing from a boat or kayak also works, as long as the depth is greater than about six meters (Frey 1971). Commercially they are very important and are fished

throughout their ranges. The halibut caught in Mexico are usually exported to Southern

California, where restrictions on net size are more stringent and fish must be longer than

22-inches. Many fishing villages in Baja owe their livelihoods to the commercial importance of halibut, particularly the village of La Bocana in Baja California Sur. The co-op factory in the village (owned by fishermen) processes halibut and transports them for sale to restaurants and grocery stores in southern California, with a daily average of

600 halibut when in season (pers. observ.).

Although halibut can be caught year-round, the peak halibut season in California is early spring and summer, but in Mexico it is different for each location (pers. observ.).

In the Gulf of California, halibut are generally caught in all months except from July through September, as seen in San Felipe and Bahia de Los Angeles; and on the Pacific coast, the commercial season can be as short as a few months, from May through July, as is typical in Magdalena Bay. The reasons for this seasonality are probably latitudinal effects (such as water temperature) and that halibut move to inshore waters to spawn, returning to deeper waters where they cannot be reached with conventional fishing techniques after spawning (Clark 1930a, Clark 1930b, Kramer and Sunada 1992).

Halibut eggs and larvae are planktonic, residing near shore throughout the water column (Clark 1930a, 1930b, Allen LG 1988). The larvae spend about a month in nearshore surface waters before they are transported onshore and mature in nursery habitats (Moser and Pommeranz 1999). Juveniles have a growth rate of 0.79 cm per month, according to the growth equation Y = 8.98 + 9.51X (Hammann and Ramirez-

Gonzalez 1990). After about a year, these halibut move into deeper water and are considered “subadults” (Kramer 1991). The habitats these fish use for nurseries are

mostly embayments and protected areas of the open coast, and to a lesser extent lagoons and estuaries. After about two or three more years they are considered adults (Allen LG

1988, Allen MJ and Herbinson 1990, Fodrie and Levin 2008). Males and females grow at the same rate until sometime after two years, and then when the fish reach three years of age the males are significantly smaller than females of the same age (Hammann and

Ramirez-Gonzalez 1990). An important question is whether the subadults and adults live near or far from each other (Gillanders 2002). Through elemental fingerprinting techniques (Campana 1999, Brown 2006) it was found that halibut migrate less than 10- km from their nursery origins (Fodrie and Levin 2008), and adults do not travel far throughout their lifetime (Frey 1971). The level of connectivity among populations has only recently been investigated, and it appears that a substantial amount of gene flow occurs for the California halibut over its entire range that has resulted in low nucleotide diversity among populations (π = 0.003), with a high number of haplotypes (Craig et al.

2011).

Considering the cryptic diversity of these three halibut species, a genetic analysis is one of the best approaches to estimate their evolutionary relationships. Genetic analyses have been used for decades, and comparisons of nuclear sequences and mitochondrial DNA (mtDNA) sequences have become a common method for making comparisons between species, especially in fish (Graves et al. 1984, Graves and Dizon

1989, Graybeal 1993, Scoles and Graves 1993, Shulman and Bermingham 1995, Stepien

1995, Sedberry et al. 1996, Miya and Nishida 1997, Waters and Burridge 1999, Salomon

2005). Therefore, a genetic analysis was judged to be ideal for this study.

Geographic boundaries like mountain chains and canyons can affect the ecology of organisms in myriad ways, including evolutionary relationships (Pielou 1979, Cox and

Moore 1993). Moreover, boundaries exist not only in the terrestrial realm but in the

Ocean as well, and can cause speciation events and affect population connectivity

(Eckman 1953, Gee and Warwick 1996, Blanchette et al. 2008). For example, the closure of the Panamanian isthmus about 3.1-3.5 million years ago (Keigwin 1978, Coates et al.

1992) is the ultimate cause of modern amphi-American fishes (Donaldson and Wilson

1999, Craig et al. 2008). In Baja California, boundaries in the marine realm have been created by factors such as coastal currents, islands, headlands, and upwelling (Moser et al. 1993, Longhurst 2007, Blanchette et al. 2008). Often, these factors influence the gene flow between populations and affect the distribution of species, usually limiting their potential to fill available habitat (Scott et al. 2002). Phylogeography is the study of relationships between phylogenies and geography, where the genetic structure between geographically separate populations is compared intra- and interspecifically with geographic landmarks or patterns (Avise et al. 1987, Avise 1992). Although phylogeography is generally used in terrestrial studies, it is also an old and accepted approach in the study of both freshwater and marine fishes (Tranah 1996, Waters and

Burridge 1999, Salomon 2005).

This study sought to assess multiple hypotheses about halibut relationships. I investigated whether the species Paralichthys californicus and P. aestuarius are morphologically or genetically different from each other, and assessed their relationships to P. woolmani. I also investigated phylogeographic differences among populations. And

finally, I hoped to resolve the evolutionary history of these three species in relation to other taxa.

Materials & Methods

2.1 Collections

Paralichthys were collected in Baja California during June and July of 2009, as well as July of 2010. Due to restrictions on the use of fishing nets, it was necessary for fish to be purchased in an uncut, fresh condition directly from Mexican fishermen. Fish were collected in Mexico from four locations, and tissue samples from locations in the southern California bight were collected by Mara Morgan (1997). Along the Gulf of

California, specimens were collected from San Felipe and Bahia de Los Angeles (Figure

1). Along the Pacific coast, fish were collected from La Bocana and Magdalena Bay.

These sites offered a wide sampling of distributions, spanning both sides of the Baja peninsula and at least two recognized biogeographic provinces (Hall 1960 &1964,

Valentine 1966). More importantly, these were the only locations where fishermen could be found who owned the correct types of nets.

Specimens were immediately placed on a flat plywood board with a ruler in direct mid-day sunlight and photographed using a Nikon D80 camera from directly overhead.

Fish were sealed in labeled plastic bags and packed in crushed ice. After transportation, the specimens were stored in laboratory freezers (-20ºC). Muscle tissue samples were taken from each specimen using a scalpel and tweezers sterilized with ethanol, and samples were stored in 15-ml polypropylene conical tubes with 5X NET buffer

(Appendix). Muscle tissue vouchers were also taken and stored in 1-ml cryogenic tubes and frozen at -40ºC at the Natural History Museum of Los Angeles (NHM), and additional fin clip vouchers were likewise stored at the Southern California Marine

Institute (SCMI). Voucher specimens and tissue samples were numbered and labeled according to location and date of capture. Fish preservation was carried out according to

Nagorsen and Peterson (1980). Specimens were then measured and donated to the NHM fish collection as vouchers.

2.2 Measurement Techniques and Analysis

Nineteen morphometric variables were recorded for each specimen.

Morphological measurements were rounded to the nearest tenth of a millimeter using

Mitutoyo digital calipers on preserved halibut specimens according to Miller and Lea

(1972) and Cailliet et al. (1986), using the standard “landmarks” of body geometry that meet the criteria outlined by Zelditch et al. (2004). The variables recorded are as follows: standard length, caudal peduncle depth, caudal peduncle length, predorsal length, length of dorsal fin base, length of anal fin base, height of dorsal fin, height of anal fin, length of pectoral fin, length of pelvic fin, head length, head width, snout length, suborbital width, gape width, orbit to preopercle length, eye diameter, upper-jaw length, and body depth

(Figures 2 and 3).

In addition, the sides of the body with eyes (left or right) were recorded for each specimen, and body weight was recorded to the nearest 0.01-g using a digital scale. Gill rakers were also counted on each specimen using the second gill arch on the ocular side according to Miller and Lea (1972).

Specimens were also examined for the presence of physical anomalies that have been observed in the Paralichthyidae family (Norman 1934, Dawson 1964, 1966).

Specifically, fish were examined for the presence of a fleshy hook over the eye, partial or full hypermelanosis of the non-ocular side (ambicoloration), or anomalous scales on the

non-ocular side, such as ctenoid instead of cycloid (Haaker 1971, Venizelos and Benetti

1999). Fish were also checked for external parasites before they were preserved (Bane and Bane 1971, Haaker 1971, 1975). Fish from the NHM collection were not checked for parasites because the preservation process may have dislodged or otherwise disguised parasites from view.

Because of the phenomenon of allometric scaling with shape during growth

(Atchley et al. 1976), measurements of specimens could not be directly compared without first limiting the analysis to fish of similar standard lengths. Because male and female halibut have comparable growth-weight relationships that diverge after their second year of life, the analysis was limited to fish of a standard length within 18-30 cm, at which time they are generally considered young adults less than two years of age, and have a diet and behavior like adults.

A logarithmic transformation was applied to the raw morphological data according to Voordouw (2001) so that statistical comparisons could be made (Sokal and

Rohlf 1994). Normality and homoscedasticity was assessed to ensure that parametric assumptions were met, and the data was then analyzed using a multiple discriminant function analysis (Atchley and Bryant 1975). Variables that showed multicollinearity with others were omitted from the analysis, and variables were added in a stepwise fashion to identify the most efficient model (Schinske et al. 2009).

Specimens were distinguished tentatively using the published species descriptions, such as the location of capture and the number of gill rakers as described by

Jordan and Evermann (1896) and Norman (1934). Museum collections (NHM) were included to augment the sample size for morphologic comparisons. Of the museum

specimens, only those that were positively identified to the classification level of

‘species’ were used.

2.2 DNA Extraction, Amplification, DNA Sequencing

Deoxyribonucleic acid (DNA) was isolated from tissue using Quiagen’s DNeasy blood and tissue kit, using their “animal tissue protocol” with muscle samples of approximately 25-mg. The concentration and purity of DNA (ng/μl) dissolved in AE buffer (Appendix) was determined using a nanodrop spectrophotometer and was usually at a concentration of 30-100 ng/μl. If DNA needed to be further concentrated before

PCR, ethanol precipitation was used (Appendix A). Polymerase chain reactions (PCR) were performed using either Qiagen’s HotStarTaq® Plus DNA Polymerase kit, or

Qiagen’s Taq PCR Master Mix kit. Reactions used between 1.5-µl to 4-µl aliquots of

DNA, depending on the DNA concentration of each sample. PCR was performed using a

GeneAmp® PCR System 9700 by Applied Biosystems.

PCR reactions were usually performed with a total volume of 25-µl each, with the following reagents and concentrations in a reaction: 16.4-µl nanopure water (ddH20), 0.5-

µl dNTP (25mM), 1-µl magnesium chloride, 4-µl DNA template, 0.25-µl each of forward and reverse primers (100-ng/µl), 2.5-µl of 10x Taq buffer, and 0.1-µl of Taq Polymerase.

Gel electrophoresis was performed on samples of amplified DNA to separate and identify

DNA fragments by their size using a DNA ladder for comparison (Qiagen).

Electrophoresis was performed using a Thermo Scientific gel box; model Owl

Easycast™ B1 by Owl Separation Systems Inc., Portsmouth, NH USA. The voltage device was made by the E-C Apparatus Corporation, model EC 105. Gels were run at a voltage of either 91 V for 40 min, or 110 V for 30 min, using 1x TAE buffer (Appendix

A), GelRed™ (a safe alternative to Ethidium Bromide), and an agarose concentration between 2-4% w/v. The gels were then examined and photographed under UV light.

Nucleic acids that were successfully amplified were purified before sequencing using the

QIAquick purification kit (Qiagen).

Amplification of double-stranded DNA targeted the Reticulation Activating Gene

2 (RAG-2). This nuclear gene (RAG-2) was chosen because it was of adequate length to design primers (~850 base pairs), and has the property of being highly conserved evolutionarily across multiple taxa (Nei 1987, Pamilo and Nei 1988, Wu 1991, Moore

1995, Springer et al. 2001, Leach and McGuire 2006).

Many sets of primers were designed and tested for RAG-2, but with limited success. Sequences from the following species were aligned using the ClustalW program from the Biology Workbench 3.2 (Subramaniam 1998): Pleuronectes platessa, Solea senegalensis, Platichthys stellatus, and Paralichthys olivaceous. Primers that targeted flanking regions of the Cyt-B gene were then designed using the PRIMER3 program

(UCSD), and then the BLAST program was used to test the specificity of the tentative primer pair (http://www.ncbi.nlm.nih.gov). A literature search was also conducted for

RAG-2 primers. The primer pair RAG2-f2 and RAG2-r3 (Appendix A) designed by

Westneat and Alfaro (2005) was tested and found to be successful in amplifying a portion

(488 bp) of the RAG-2 gene. Primers for amplification were purchased from Operon, Inc.

Amplifications of RAG-2 were performed on a thermocycler with an initial denaturation of template DNA for 3-min at 94ºC after a hot start, and then 30 cycles of the following: denaturation for 30 seconds at 94ºC, annealing of DNA for 35-sec at 54ºC,

and DNA extension for 40-sec at 72ºC. A final extension of DNA was carried out for 10- min at 72ºC, followed by storage of the amplification products at 4ºC.

Forward and reverse halibut DNA sequences were generated at CSUN’s DNA sequencing facility by Pavel Lieb. Sequencing reactions were carried out using an ABI

377 automated DNA sequencer machine with a 96-sample XL upgrade (Applied Bio

Systems, Foster City, CA), with forward and reverse primers at a concentration of 1 ng/µl

(1X).

2.3 Sequence Editing, Alignment and Analysis

DNA was sequenced from a total of 115 fish (Figure 1). Specimens of

Paralichthys californicus (83) were positively identified from the following locations:

Morro Bay (8), Southern California (11), La Bocana (32), Magdalena Bay (24), and

Bahia de Los Angeles (8). DNA was also sequenced from 24 specimens of P. aestuarius from San Felipe. Specimens of P. woolmani from Bahia de Los Angeles (6) and from

Magdalena Bay (2) were also sequenced. Additional fish collected during this study were omitted from the DNA analysis due to various time and lab constraints.

Nucleic sequences were edited using either Geneious 4.8.5 (Drummond et al.

2009), or BioEdit (Hall 1999). Raw DNA sequences were aligned with their chromatograms and with their reference sequences (Table 4) to check the reading frame and to detect if insertions or deletions were present (indels). Sequences were edited visually in aligned groups based on their species and by their location of capture. Where nearly overlapping chromatogram peaks were within 95% of each other at a locus, those base pairs were coded as ambiguous according to IUPAC conventions. Sequences were trimmed so as not to include the upstream and downstream primer sites, for a final length

of 400 nucleotides. If sequences were shorter than their reference sequence, the remainders of the upstream and downstream ends were left blank. Forward and reverse sequences were collapsed into a reference sequence for each specimen. Consensus sequences were then generated by population, aligned based on their species and visually assessed. Consensus sequences were then generated for each species, which were deposited in GenBank (http://www.ncbi.nlm.nih.gov) with their voucher and accession information listed in Table 4.

Consensus sequences of RAG-2 for each species were tested using MEGA5 software (Tamura et al. 2011). All consensus sequences were tested for equality of evolutionary rates using Tajima's relative rate test (Tajima 1993). Tajima’s D was also calculated for all sequences. Sequences were then tested for the best nucleotide substitution model in MEGA5 with Modeltest (Posada 1998), using the maximum- likelihood (ML) method and the automatic neighbor-joining tree in the analysis. The

Kimura 2-parameter (K2P) was chosen as the best nucleotide substitution model with default settings (Kimura 1980). Haplotypes for Paralichthys californicus, P. aestuarius, and P. woolmani were analyzed using the DNAsp program (Librado and Rozas 2009).

Phylogenetic trees were constructed using both the Bayesian and ML methods.

The Bayesian analysis (Huelsenbeck and Ronquist 2001) was performed with 3 million generations, and the ML method was run with 5000 bootstrap pseudoreplicates

(Felsenstein 1985). Eleven closely related species were used whose sequences were extracted from GenBank (Table 4). To minimize the possibility of substitution saturation, only similar genera other than Paralichthys were chosen for the analysis, with one outgroup (Kartavtsev 2007). Sequences from the following species were included in the

phylogenetic analysis because they were found to have the highest percentages of pairwise sequence identity: Paralichthys dentatus, P. lethostigma, Atheresthes stomias, A. evermanni, Hippoglossus stenolepis, Clidoderma asperrimum, Verasper moseri, V. variegatus, Pleuronichthys coenosus, P. decurrens, and the outgroup species Chaetodon ulietensis (Table 4). Bayesian and Maximum Likelihood analyses resulted in rooted phylogenies with multifurcating trees. The Bayesian tree was generated using the Mr.

Bayes 2.0.2 plugin for Geneious 4.8.5, and the ML tree was generated in MEGA5.

A molecular analysis of variance (AMOVA) was used to determine relationships among populations and nominal Paralichthys species. The K2P model (Kimura 1980) was used, and standard errors were calculated using 5000 bootstrap pseudoreplicates

(Felsenstein 1985). The overall diversity of genotypes was assessed using the nucleotide diversity (π) and haplotype diversity (h) indices of Nei and Tajima (1981). The extent of differentiation between populations and among Paralichthys species was determined using pairwise fixation indices (Fst) (Wright 1951, 1965, 1978). The degree of genetic variation between populations was compared with geography.

Results

3.1 External Features and Morphology

Of the halibut specimens collected in Baja California (~150), none had physical anomalies. However, a single preserved specimen in the collection at the Los Angeles

Natural History Museum (NHM) was identified as having a fleshy hook over its eyes

(Photo B1, Appendix B). None of the fish that were examined from both the field and the museum exhibited ambicoloration, nor did they have anomalous scales. “Spotting” or

“staining” on the blind side of specimens as described by Norman (1934) was observed twice in the museum collection, but due to the preservation of fish in formalin, it was impossible to determine if these colorations were present at the time of their capture. No such markings were observed on fish collected in the field.

The number of fish collected in the field with visible ectoparasites totaled five specimens. The five parasites were copepods that were identified as members of either

Acanthochondria or Holobomalochus, attached to gills of specimens collected from

Bahia de Los Angeles that were later identified as Paralichthys californicus.

A stepwise discriminant function analysis was run on three groups of fish identified down to species, with eight fish in each group. The morphometric discriminant function analysis computed two functions that accounted for 100% of the variation, based on eighteen external measurements (Fig. 2, 3), and taking into account their weight (g), but not taking into account the number of gill rakers. A canonical scores plot (Fig. 4) illustrated that there is more of a physical difference between Paralichthys californicus and P. woolmani. Overlap occurred for P. aestuarius with both other species, but more

similarities in morphology are apparent with P. californicus based on the ellipsoids. The lack of overlap of the ellipsoids between P. californicus and P. woolmani indicate they are separate species. Also, 12.5% of P. californicus specimens were more correctly grouped with P. aestuarius, and 25% of P. aestuarius specimens were more correctly grouped with P. californicus.

The percent of correctly classified specimens ranged from 75 to 100%, with an overall rate of 88% (Table 1). All of the specimens of P. woolmani were found to be correctly classified. Two of the eighteen morphological variables were found to be highly significant in explaining the variation among species (Table 2). The discriminant factor

#1 on the x-axis explains most of the morphological variation (r2 = 75%) and is mostly a function of body depth, while the y-axis (factor #2) is mostly a function of suborbital width (Fig. 4). P. californicus appears to have a significantly longer suborbital width than

P. woolmani, as well as a thinner body depth than P. woolmani.

The total percent of fish that were examined and were found to be left-eyed was

70.2% out of a total of 84 fish (Table B1). All specimens of Paralichthys woolmani were found to be left-eyed. P. aestuarius were less likely to be left-eyed (63.2%) than the P. californicus specimens (90.5%).

3.2 Analyses of DNA

The average nucleotide composition of the sequenced region of RAG-2 was

22.0% Adenine, 23.6% Thymine (or Uracil), 30.2% Cytosine, and 24.2% Guanine. The average number of nucleotides for sequences of RAG-2 was 387.8 base pairs (bp), with the maximum being 400 bp, and a minimum of 117 bp. The number of polymorphic sites for samples of Paralichthys californicus was 12, for P. aestuarius 2, and 11 for sequences of P. woolmani.

Tests for equality of evolutionary rates were found to be neutral, with equal rates of evolution among all specimens. The result of Tajima’s Neutrality Test on all of the sequences was D = -1.228106 (P > 0.05). Using Tajima's relative rate test (Tajima 1993), the consensus sequences of the species used in the phylogenetic tree were analyzed

(Table 3). The analysis involved three nucleotide sequences: (Paralichthys californicus),

(Atheresthes stomias), with sequence (Chaetodon ulietensis) as the outgroup. All sequence positions containing gaps and missing data were removed, for a total of 400 base pairs in the analysis. The null hypothesis of equal rates of evolution between these lineages is supported, because the P-value is greater than 0.05.

All DNA sequences were checked for the presence of indels (insertions and deletions), and the RAG-2 sequences were checked for heterozygous states. No indels were found for the regions of RAG-2 that were sequenced. A haplotype analysis of the

RAG-2 sequences resulted in 13 haplotypes for Paralichthys californicus, 3 for P. aestuarius specimens, and 8 for P. woolmani (Table 5), based on their unique polymorphic loci (Figure B1).

After DNA editing and alignment, it was possible to identify three unidentified specimens from Bahia de Los Angeles as Paralichthys woolmani based on comparisons of polymorphic sites. The preserved specimen vouchers at NHM that had previously not been examined for these fish were later examined, and using gill raker counts (5+11) it was possible to confirm their identity as P. woolmani. Likewise, two specimens from

Magdalena Bay were also confirmed as P. woolmani.

A phylogenetic tree was constructed for the aforementioned species using both the Maximum Likelihood (ML) estimate in MEGA5, and a Bayesian analysis in

Geneious 4.8.5 (Figure 5). The node for Paralichthys californicus and P. aestuarius had a posterior probability of 1 and a bootstrap value of 99, with no nucleotide differences among their consensus sequences. The analysis resulted in distinct relationships among the different taxa. The Paralichthys genus is clearly a monophyletic group, with a speciation event that occurred which branches the genus into two distinct clades with P. californicus and P. aestuarius in one clade. In a separate clade is P. woolmani and it is more closely related to species from the Gulf of Mexico and the southern Atlantic. The

Astheresthes genus is clearly monophyletic, while another monophyletic group includes the genera Hippoglossus, Clidoderma, Verasper, Pleuronichthys. The outgroup used in the analysis was Chaetodon.

The overall result of the AMOVA test among the three groups was Fst = 0.599, indicating very strong differences among species, with a high level of significance (P =

0.00001 ± 0.00001) (Table 6). Using the qualitative guidelines of Wright (1978) for interpreting pairwise Fst values, there is extremely little genetic variation between P. californicus and P. aestuarius (Table 7), as their Fst values are much less than 0.05 with a

high degree of significance (P << 0.05). Conversely, P. woolmani has great genetic variation when compared with either P. californicus or P. aestuarius, as both of the Fst values are much greater than 0.25, with a high degree of significance (P << 0.05).

Discussion

Haaker (1975) found that fish with total ambicoloration (or almost total), also formed the fleshy hook abnormality over their eyes. This hook develops because of the natural migration of the eyes to one side of the body, and the hook forms because the dorsal fin is not yet developed. Once the fin grows forward, it forms the hook. A fish that was previously unrecorded for this anomaly in the Los Angeles Natural History

Museum’s (NHM) collection (Catalogue # W52-163) did not show ambicoloration on its blind side, which appears to be the rarest anomaly for halibut. In Haaker’s study, he found that some level of ambicoloration was usually present, and only one fish out of

1205 in his study had a fleshy hook and no coloration on the blind side. Usually, the fish had total ambicoloration (0.33% of the time). No fish examined in this study in the field or at NHM was identified as having ambicoloration.

Only five fish (out of ~150) were found to have copepod parasites, and all of them came from Bahia de Los Angeles, located in the Gulf of California. In comparison to data collected by Haaker (1971) in Anaheim Bay, which in the 1970’s had a high degree of human disturbance, it is surprising that so few were found to have ectoparasites, since in

Haaker’s study, parasites were fairly common. Parasitism is thought to increase with the age of a fish. Locations with less anthropomorphic disturbance may also have lower rates of infestation. Bahia de Los Angeles is a small town, but is heavily fished by American tourists and locals, so it is no surprise that parasites are present. But out of the two locations where fish were captured in the Sea of Cortez, San Felipe should have had a greater abundance of parasites, as it is a major commercial fishing town. Another baffling

result was that fish from La Bocana showed no signs of ectoparasites, although the area is heavily fished by boat on the Pacific coast. One explanation may involve the ages of fish.

An otolith analysis was not performed, but it is probable that the ages of fish collected in the Sea of Cortez were younger than those from the Pacific coast, as warmer water increases metabolism and speeds maturity, and might explain the extremely low amount of parasites in comparison to Haaker’s study, since older fish tend to have more parasites.

The sites on the Pacific coast were small towns, and consequently may have little pollution, and may explain the complete lack of parasites from La Bocana and Magdalena

Bay.

The standard length of Paralichthys californicus is commonly thought to be longer than the standard length of P. aestuarius, although large specimens may exist. P. californicus is also commonly identified as being heavier. Length and weight are usually attributed to the conditions where these halibut are distributed. The result of the discriminant analysis indicates that physical characters are essentially not that different between these species. Although halibut in general are indeed larger on the Pacific coast, there appears to be no significant difference in the species when compared directly. The large size of fish on the Pacific coast could be due to colder temperatures according to

Bergmann’s rule (1947), and is generally thought to be caused from metabolic benefits associated with delayed maturation. This relationship is seen among the majority of fishes that have distributions spanning the Gulf of California and the Pacific Ocean.

Future studies would be needed to determine if halibut larvae from the Gulf of California would grow into large adults if reared in the Pacific Ocean.

The number of gill rakers among these species of Paralichthyidae appears to be their best feature for use in identification. Aside from Paralichthys californicus and P. aestuarius, which may be the same species and which have 9+20 gill rakers, the next most closely related fish was the Atlantic halibut (P. dentatus), who has 5+15 to 6+18 gill rakers (Jordan and Evermann 1963).

The number of gill rakers that were recorded for specimens of P. woolmani were categorically different (5+11). Every specimen of P. woolmani that was examined was also identified as having this exact number of gill rakers, with no variation. Interestingly, the gill raker counts for P. woolmani are similar to those reported for the southern flounder (P. lethostigma), which has 2+10 rakers (Jordan and Evermann 1963). The southern flounder’s consensus sequence (GenBank Accession No. FJ870491) was also found to be closely related evolutionarily to P. woolmani (Figure 5). Their distributions are separate, however, and the southern flounder has a distribution that ranges from the

South Atlantic to the Gulf coast. Its distribution is separated from P. woolmani by the

Isthmus of Panama. Also, the gulf flounder (P. albigutta) is found between these locations from Mexico to North Carolina (Gilbert and Williams 2002), and it has a gill raker count (3+10) that is roughly intermediate between that of P. woolmani and P. lethostigma. A molecular timeline can be established based on the closure of the

Panamanian isthmus approximately 3.1 million years ago (Coates and Obando 1996,

Bernardi and Lape 2005). If P. woolmani is more closely related to halibut from the Gulf coast than to halibut in the Pacific Ocean, as it appears in the phylogenetic data (Figure

5), then it makes sense that a separate speciation event occurred for P. californicus and P. dentatus, and these relationships agree completely with the pattern found in the numbers

of gill rakers of fish in this genus. Caution must be taken in drawing conclusions based only on gill rakers counts though, as they may indicate differences in feeding habits, not necessarily in relationships.

The number of haplotypes and the variation in haplotypes can indicate relationships between populations. The number of total haplotypes for Paralichthys californicus (13) was greater than those found for P. aestuarius (3) and P. woolmani (8).

The greatest haplotype diversity was found in Magdalena Bay and Bahia de Los Angeles

(Table 5). The high number of haplotypes for P. californicus agrees with data in Craig et al. (2011) who reported 92 haplotypes for P. californicus, although they used

Cytochrome-B mitochondrial sequences. They found that P. californicus is genetically homogeneous with haplotypes evenly dispersed along the Pacific coast, and migration occurring from north to south, with the greatest diversity in the San Diego region. None of the locations in their study had less than two or greater than nine haplotypes. If P. aestuarius is assumed to be the same species, the low number of total haplotypes (4) for

San Felipe may indicate a recent population expansion into the Gulf of California by the more ancestral population along the Pacific coast of the Californias. The low number of haplotypes for P. woolmani is probably due to low sample size.

The phylogenetic analysis resulted in distinct relationships among the different taxa (Figure 5). The Paralichthys genus is clearly a monophyletic group, with two distinct clades. P. woolmani is in a separate clade than P. californicus, and is clearly more related to species from the South Atlantic Ocean and the gulf coast. Astheresthes is also clearly its own monophyletic group, while another monophyletic group includes the

genera Hippoglossus, Clidoderma, Verasper, Pleuronichthys. The outgroup is

Chaetodon.

If the California halibut and the Cortez flounder are the same species, as the

AMOVA results indicate (Table 6), it could change our estimates of fish stocks. It has been estimated that more than half of the flatfish caught in Baja from 1980-86 came from

San Felipe (Escobar-Fernandez 1989). This is where the species P. aestuarius are reportedly found (Norman 1934). If these fish are actually California halibut, as the data from this study seems to indicate, then the abundance of California halibut is much greater than presently believed (Maunder 2011). This is good news for fisherman, although it is mixed news for those concerned with the protection of marine biodiversity, since it would mean one less species of Paralichthys.

Paralichthys aestuarius does not seem to be a subspecies of P. californicus, due to both having a low level of nucleotide diversity (Table 5), which indicates panmixis.

The low number of haplotypes in P. aestuarius is also more typical of a population. As far as the naming of these species, P. californicus is the senior synonym (Ayres 1859), and should replace the name P. aestuarius (Gilbert and Scofield 1898).

Figure 1: The locations where fish were collected along the coast of southern California and along the Baja peninsula. (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. Eleven 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. P. californicus and P. aestuarius appear to be very similar in morphology. Ellipsoids represent 95% confidence limits. Factor 1 describes 75% of the variation among the species and is mostly a function of body depth, while Factor 2 is mostly a function of suborbital width.

Figure 5: Molecular Phylogenetic analysis by the Bayesian and Maximum Likelihood methods. The evolutionary history was inferred by using the Maximum Likelihood method based on the Kimura 2- parameter model of nucleotide substitution (Kimura 1980). The numbers in bold above the nodes are bootstrap proportions inferred from 5000 pseudoreplicates, which indicate the percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (Felsenstein 1985). Numbers below the nodes are posterior probabilities from a Bayesian analysis with 3 million generations. Asterisks denote values indicating 100% support. Initial trees for the heuristic search were obtained by the BIONJ method using a MCL distance matrix. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 14 nucleotide consensus sequences. All positions with less than 95% site coverage were eliminated such that fewer than 5% gaps, missing data, and ambiguous nucleotides were allowed at any position. There were a total of 400 aligned base pairs in the final dataset.

Table 1: Classification results in matrix format of the three nominal species of Paralichthys, based on a stepwise discriminant function analysis (Figure 1). The rows indicate observed classifications. Species Percent correct P. "aestuarius" P. californicus P. woolmani P. "aestuarius" 75 6 1 1 P. californicus 87.5 1 7 0 P. woolmani 100 0 0 8 Total 87.7 7 8 9

Table 2: The two highly significant morphometric variables in a stepwise discriminant function analysis, based on nineteen morphometric variables (Figs 2 and 3), and including weight (g). Data was log-transformed before analysis. N = 24. Variable F Wilks's lambda P Body depth 36.348 0.224 < 0.001 Suborbital width 15.826 0.15 < 0.001

Table 3: Tajima's relative rate test, which tests the equality of evolutionary rates among taxa. The analysis involved 3 nucleotide sequences: A (Paralichthys californicus), B (Atheresthes stomias), with sequence C (Chaetodon ulietensis) used as an outgroup. The χ2 test statistic was 0.05 (P = 0.82726 with 1 degree of freedom). The null hypothesis of equal rates of evolution between these lineages is supported in this case, because the P-value is greater than 0.05. All sequence positions containing gaps and missing data were removed, for a total of 488 base pairs in the analysis. Results from Tajima's test for 3 Sequences Configuration Count Identical sites in all three sequences 397 Divergent sites in all three sequences 5 Unique differences in Sequence A 10 Unique differences in Sequence B 11 Unique differences in Sequence C 65

Table 4: Accession numbers for Genbank reference sequences of the RAG-2 gene.

Species Accession number

Paralichthys californicus JX045830 Paralichthys “aestuarius” JX045831 Paralichthys woolmani JX045832 Paralichthys dentatus DQ874762 Paralichthys lethostigma FJ870491 Atheresthes stomias FJ870476 Atheresthes evermanni FJ870469 Hippoglossus stenolepis FJ870499 Clidoderma asperrimum FJ870489 Verasper moseri FJ870493 Verasper variegatus FJ870495 Pleuronichthys coenosus FJ870480 Pleuronichthys decurrens FJ870481 Chaetodon ulietensis EF617092

Table 5: Estimates of haplotype diversity and nucleotide diversity for each species and population, with the number of haplotypes and sample size. Nucleotide diversity is the number of base substitutions per site averaging over all sequence pairs. Standard error estimates are based from 5000 bootstrap pseudoreplicates. Analyses were conducted using the Kimura 2-parameter model (Kimura 1980). The analysis involved 115 individuals. All positions with less than 95% site coverage were eliminated. Fewer than 5% alignment gaps, missing data, and ambiguous bases were allowed at any position. There were a total of 379 nucleotide positions in the final dataset. Sample size No. of Haplotype Nucleotide Species & Locations (n) haplotypes diversity (h) diversity (π) Paralichthys californicus 83 13 0.24872 0.00102 Paralichthys “aestuarius” 24 3 0.5293 0.00112 Paralichthys woolmani 8 8 0.8901 0.01052 Morro Bay (P. californicus) 8 1 0 0.00027 Southern California (P. californicus) 11 2 0.1594 0.0004 La Bocana (P. californicus) 32 2 0.2593 0.00076 Magdalena Bay (P. californicus) 24 4 0.2425 0.00372 Bay of Los Angeles (P. californicus) 8 4 0.5824 0.00291 San Felipe (P. aestuarius) 24 3 0.5293 0.00112 Bay of Los Angeles (P. woolmani) 6 4 0.7778 0.00822 Magdalena Bay (P. woolmani) 2 4 1 0.01042

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 before the analysis. Degrees Percentage Source of Sum of Variance of of Variation Squares Components Freedom Variation Among V = 2 91.51 a 59.94 species 0.46364 Within V = 441 136.641 b 40.06 Species 0.30984 Total 443 228.151 0.77348 100 Fixation 0.59942 P-value < 0.00001 ± 0.00001 Index (Fst)

Table 7: Pairwise Fst values among nominal species. Numbers in parentheses are P-values ± S.E. P. californicus P. “aestuarius” P. woolmani

Paralichthys californicus 0.00000 - - Paralichthys “aestuarius” 0.01279 (0.00001 ± 0.0001) 0.00000 - Paralichthys woolmani 0.79337 (0.00001 ± 0.0001) 0.7571 (0.00001 ± 0.0001) 0.00000

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Appendix A

5X NET buffer (pH 8.0)

2.5 M NaCl 0.25 M EDTA 0.25 M Tris-Cl

AE Buffer

10 mM Tris-Cl 0.5 mM EDTA (pH 9)

TAE Buffer

Tris-acetate buffer (pH 8) EDTA

Ethanol Precipitation Protocol (Sambrook et al. 1989)

1. Add 1/10 volume of Sodium Acetate (3 M, pH 5.2). 2. Add 2.5–3.0 X volume (calculated after addition of sodium acetate) of at least 95% ethanol. 3. Incubate on ice for 15 minutes. In case of small DNA fragments or high dilutions overnight incubation gives best results, incubation below 0 °C does not significantly improve efficiency 4. Centrifuge at > 14,000 x g for 30 minutes at room temperature or 4 °C. 5. Discard supernatant being careful not to throw out DNA pellet, which may or may not be visible. 6. Rinse with 70% Ethanol 7. Centrifuge again for 15 minutes. 8. Discard supernatant and dissolve pellet in desired buffer. Make sure the buffer comes into contact with the whole surface of the tube since a significant portion of DNA may be deposited on the walls instead of in the pellet.

RAG-2 Primers (Westneat and Alfaro 2005)

RAG2-f2: 5’- GAC TGT CCT CCT CAG GTG TTC -3’

RAG2-r3: 5’- GAT GGC CTT CCC TCT GTG GGT -3’

Appendix B

Figure B1: An example of a polymorphic locus for a specimen of Paralichthys woolmani from Bahia de Los Angeles. The alignment shows specimen BLA14’s forward and reverse sequences and their chromatograms, zoomed in on residues 115-122. This specimen was identified as having a heterozygous state at residue 120. Other specimens had a cytosine residue at this locus, consistent with the reference sequence (Genbank accession No. FJ870492). An S ambiguity indicates either a guanine or cytosine nucleotide.

Photo B1: A preserved specimen of Paralichthys californicus from the collection at the Los Angeles Natural History Museum (Cat. No. W52-163). This fish exhibits the physical abnormality known as the “fleshy hook” located above its eyes.

Table B1: Summary statistics below show the number of left- and right-eyed specimens for each species that were measured morphologically. Eye Side Percent Species Frequency Total Right Left Left-eyed californicus 21 28.40% 2 19 90.50% aestuarius 38 51.40% 14 24 63.20% woolmani 14 18.90% 0 14 100% Total 84 100% 24 59 70.20%