We are using mitochondrial and nuclear genetic markers and modern molecular techniques to assess degrees of DNA sequence variation at the levels of populations, species and genera within subfamily Ictiobinae.  The mitochondrial genome is one of the best studied of all types of DNA (Kocher and Carleton 1997).  Mitochondrial genes have been used in studies ranging from phylogeny reconstruction and gene evolution to intraspecific phylogeography and gene flow (Avise 1994, 2000, Kocher and Stepien 1997).  Its rapid rate of evolution, clonal inheritance, and lack of recombination have made it a valuable tool for evolutionary studies (Brown 1985, Avise 1994, 2000).  Researchers have used mtDNA to assess degrees of intraspecific genetic diversity and to delineate species boundaries (Frost et al. 1998, Eitner et al. 1999, Tanka-Ueno et al. 1999). Cyt b is perhaps the best studied of all mitochondrial genes, particularly for fishes (Lydeard and Roe 1997).  The gene has both conserved and variable regions, and has proven to be useful for investigating relationships of both closely and distantly related species.  Studies of cyt b sequence variation have shown this region to be well adapted for studying evolutionary relationships in Actinopterygian fishes (Lydeard and Roe 1997).

            The Growth Hormone gene (GH) is proving to be a useful nuclear genetic marker for fishes and other organisms (Chen et al. 1994).  The gene comprises five exons (coding regions), and four  (non-coding) introns in cypriniformes.  The exons are evolutionarily conserved, but the introns evolve at a rate suitable for interspecific and intergeneric level studies.  GH introns have recently been used in subfamily-level phylogenetic studies in salmonid fishes (Oakley and Phillips 1999).  Like salmonids, catostomids are tetraploids (Uyeno and Smith 1972) and thus should have duplicate (paralogous) copies of the GH gene.  Sequencing paralogous copies of GH exons and introns has the potential to reveal interesting facts about ictiobine evolution, as it has for salmonids (Oakley and Philips 1999). 

           Single-stranded conformation polymorphism (SSCP) offers a simple and inexpensive, yet sensitive (capable of detecting differences in DNA sequence as small as 1-2 bp), method for screening large numbers of individuals, and thus represents an efficient means of assessing population-level variability without sequencing (Sunnucks 2000, Sunnucks et al. 2000).  We propose to use SSCP to assess variability in cyt b and GH intron for multiple individuals for each ictiobine study population. As illustrated below, the method is also useful for identifying interspecific hybrids and for determining which individuals need to be cloned and/or sequenced. 

Methods

            The following methods were used in the preliminary SSCP, cyt b and GH intron sequencing work described below, and are similar to the methods that will be used for the proposed molecular variation work. 

Cyt b sequencing: Total genomic DNA was extracted from frozen or ethanol preserved tissues using the DNeasy Tissue Kit  (Qiagen, Inc.).  The 1140 bp Cy -b gene was isolated by PCR.  We used the oligonucleotides GLU (5'-TAA CCG AGA CCA ATG ACT TG) and THR (5'-- ATC TTC GGA TTA CAA GAC CG) (Brady Porter unpublished) to amplify the gene.  Six additional internal sequencing primers were designed with OLIGO primer analysis software (Molecular Biology Insights, Inc.).  Reactions were cycled according to the following temperature profile: 94�C for 1 min., 57�C for 1 min., and 72�C for 1:15 min., for 32 cycles.  PCR products were isolated with the QIAquick PCR Purification Kit  (Qiagen, Inc) and used in cycle sequencing reactions (Applied Biosystems Inc.) according to the manufacturer's recommendations.  Excess dye terminators, primers, and nucleotides were removed by gel filtration (Edge Biosystems) prior to sequencing. Sequences in both directions were determined with an ABI 373A Automated Sequencer.  Reactions were electrophoresed on 6% polyacrylamide gels in 7 M urea (Sooner Scientific).  Raw sequence chromatograms of approximately 400 bp length were assembled into contigs and edited to resolve ambiguities using Sequencher 4.1 (Gene Codes, Inc.). Sequences were aligned to Myxocyprinus asiaticus, an Asian Cycleptine (GenBank AF036176). Amino acid sequences were determined and analyzed with MacVector 4.0 (Oxford Molecular Ltd.).

SSCP: A 300 bp fragment of GH Intron B fragment was isolated by PCR using oligonucleotide primers specific to copy I of the gene.   The 50 �l PCR reactions contained 100 ng total genomic DNA, 1.5mM MgCL2, 1X platinum taq reaction buffer (Invitrogen), 2mM each dNTP, 40 pmol each primer, and 1U platinum taq DNA polymerase (Invitrogen). The PCR thermal profile consisted of an initial 94�C hot start for 2 min., then 30 cycles of 94�C for 30 sec, 56�C for 30 sec, and 72�C for 30 sec.  Five �l of the PCR reaction was electrophoresed in a 1% agarose gel for 1 hr at 100V. The gel was stained with ethidium bromide for 10 min and products were visualized with a UV transilluminator to check the quality of the PCR reactions.  3-5 ul of the reaction was diluted with 5 �l of SSCP loading buffer (95% formamide, 20mM EDTA, 0.05% bromophenol blue, 0.05% xylene cyanol). The mixture was then heated at 95�C for 3 min and immediately chilled on ice for 5 min. The samples were loaded onto a 19cm (W) X 16 cm (L), 10% polyacrylamide gel (37.5:1 acrylamide: bisacrylamide) and electrophoresed at 5W for 15 hrs. The gel apparatus was kept cool during the run by placing it in a 4�C refrigerator.  At the end of the run, the glass plates were disassembled and the gel was silver stained.  SSCP gels were dried at room temperature overnight, then visualized with a light box and photographed with a digital camera.

GH sequencing: Fresh pituitaries were harvested from adult I. bubalus and immediately frozen at -70 C until use. Total RNA was extracted from 100 mg of pituitary tissue using a modified acid guanidinium thiocyanate-phenol-chloroform protocol (Ultraspec, Biotecx) and the amount and purity of RNA was determined by spectrophotometry. First strand cDNA was reverse-transcribed from total RNA using the Super script RT-PCR kit (Invitrogen) and 2 �l of the reaction served as template for subsequent PCR amplifications. Approximately 550bp of the coding sequence of the GH gene was amplified using Oligo 6.6 designed (MBI) degenerate primers from alignments of cypriniform GH sequences deposited on GenBank. PCR reactions contained 2 �l of first strand reaction, 1.5mM MgCl₂, 1X PCR buffer, 0.2mM each dNTP, 20 pmols of primers GHF and GHR (sequences available on request), and 0.5 U platinum taq DNA polymerase (Invitrogen). The thermal profile consisted of an initial "hot start" of 94 C for 2 min to activate enzyme. Then 30 cycles of 94 C for 30 sec, 52 C for 30 sec, and 72 C for 1 min. PCR products were electrophoresed in 1% agarose at 100V for 1hour and stained with ethidium bromide. PCR products were visualized with a 312nm variable intensity transilluminator.  

            PCR products were inserted and ligated to pCR4-TOPO vectors, and transformed into TOP 10 competent cells (TOPO TA Cloning Kit, Invitrogen). Plasmid DNA was extracted from bacterial colonies with Qiagen QIaprep kit and insert size was checked by agarose gel electrophoresis after digestion with EcoR1.  We sequenced 20 clones to discriminate PCR or Taq errors. Multiple clones were sequenced using dye terminator cycle sequencing reactions (Applied Biosystems) and sequences from both directions were determined with an ABI 373A Automated Sequencer. Additionally, several internal sense and antisense sequencing primers were designed and used in sequencing reactions to assure complete coverage of clones with high quality sequence data. Raw sequence chromatograms of 400 bp length were assembled into contigs and edited using Sequencher 4.1 (Gene Codes). Intron boundaries were determined by aligning cDNA sequences to other cypriniform GH sequences. Intron spanning primers were designed to include approximately 50bp of flanking exon so these areas could be aligned with known GH sequences to confirm that the correct intron sequence was isolated.

To amplify and compare GH intron sequences across several ictiobine taxa, PCR reactions were performed with 100ng total genomic DNA using copy specific primers that anneal to exons 2 and 3 of the representative Ictiobine GH gene. PCR protocol was similar to that described earlier except annealing temperature was set to 55 C. Small aliquots of the PCR reactions were run on agarose gels to check the quality and size of PCR products. PCR products were purified using Qiagen QIAquick PCR extraction kits. PCR products were directly sequenced as described above.

Phylogenetic analysis: Pairwise sequence divergence and transition-transversion ratios were calculated to examine patterns of variation in cyt b and GH. Phylogenetic trees were generated with PAUP*4.0 (Swofford 1998) under Maximum Parsimony (MP) and Maximum Likelihood (ML) models. Robustness of inferred nodes from parsimony analysis was assessed with bootstrap analysis.  The best fit model of nucleotide substitution for ML analysis was determined through likelihood ratio tests using the program MODELTEST 3.04 (Posada and Crandall 1998).   Posterior probabilities (i.e. confidence) of nodes in ML trees were estimated using MRBAYES (Huelsenbeck and Rondquiust, in press)

Preliminary Results 

Cyt b sequencing: To date, complete cyt b sequence data have been obtained for 20 individuals representing all of the currently recognized extant species in subfamily Ictiobinae (and multiple populations for most of the species) and five catostomid outgroup taxa.  Sequences for an additional 5 outgroups (an Asian sucker, 2 minnows and 2 cobitids) were obtained from Genbank. These data were used to infer relationships within subfamily Ictiobinae (Bart et al. in review).  Space limits will only allow us to give general results of this study.  Reviewers who wish to view the complete manuscript may visit http://www.tubri.org/museum/ictiobin/MPE2002.htm/.

At the level of ictiobines, cyt b sequences contain 172 variable sites, the vast majority of which (94%) are at the 3rd codon position. The Ti/Tv ratio is 7:1. Within genus Ictiobus 74 sites are variable (89% 3^rd position substitutions) and transitions outnumber transversions 20:1. There are 28 variable sites within genus Carpiodes, all but one of which is at the 3rd position, and the Ti/Tv ratio is 47:1. Sequences show evidence of saturation only for 3^rd position transitions and the distinct anti-G bias at the 2^nd and 3^rd codon positions typical of mitochondrial genes.  None of the base substitutions in genus Carpiodes alter the amino acid sequence of the cyt b protein.  Ictiobus sequences show four amino acid differences (2 conservative, 2 nonconservative).

Sequence divergences among Carpiodes species are low, ranging from 0-1.3% (mean = 0.85%). Cyt b sequences for the two C. velifer specimens are identical. Sequences for three Miss. River Basin C. cyprinus specimens and two of four Miss River Basin C. carpio specimens differ at only two positions, suggesting that the two C. carpio specimens are hybrids with C. cyprinus cyt b haplotypes. Sequences for two other Miss. R. C. carpio specimens are 0.96 to 1.16% divergent from the typical Miss R. C. cyprinus haplotype, 0.43 to 0.61% divergent from the Miss. R. C. velifer haplotype, and 0.35% divergent from each other.  We regard these latter C. carpio sequences as more typical of the true C. carpio haplotype.

Sequence divergence among Ictiobus species average 1.51%.  However, much of this is due to the high divergences between I. labiosus and other Ictiobus species (average of 6%).  Divergences among I. bubalus, I. cyprinellus and I. niger average 0.46%.  Divergence within these latter three species - even among widely separated populations of I. bubalus (inclusive of I. meridionalis) - are much lower.  Cyt b sequences for Ictiobus bubalus specimens from the R�o San Fernando in northeastern Mexico and the Amite River (Lake Pontchartrain Basin), just east of the lower Mississippi River, are identical. Sequences for the I. meridionalis specimen from R�o Usumacinta (extreme southeastern Mexico) and I. bubalus from the Upper Mississippi River differ by only one base.  Mean sequence divergence within I. bubalus (inclusive of I. meridionalis) is 0.22%.  Two base substitutions separate the sequences of Ohio and Upper Mississippi R. specimens of I. niger, and only one base substitution separates the sequences of Ohio and Upper Mississippi R. specimens of I. cyprinellus.   The cyt b sequence for the Amite River specimen of I. cyprinellus is unusual in that it is 0.57% divergent on average from other I. cyprinellus sequences, but only 0.09% and 0.25% divergent, respectively, from upper Mississippi and Ohio R. I. niger cyt b sequences, suggesting that the Amite River I. cyprinellus specimen is expressing an I. niger cyt b DNA haplotype.

Sequence divergences among Carpiodes and Ictiobus species average 10.5% overall.  Sequence divergences between ictiobines and the two Cycleptines (Myxocyprinus asiaticus and Cycleptus elongatus) average 14.8%. As a group, Ictiobines are as divergent from Moxostomini as from minnows (17.2-17.4%).  Ictiobines are most divergent from loaches among non-catostomid outgroups (20-20.4%).

Of the 478 variable sites at the level of all taxa, 416 are parsimony informative.  At the level of ictiobines, 131 of the172 variable sites are parsimony informative.  However, phylogenetic signal falls off considerably within ictiobine genera. Within genus Carpiodes, 14 of the 28 variable sites are parsimony informative. In genus Ictiobus, only 8 of 74 variable sites are parsimony informative.  The likelihood ratio test in Modeltest identified GTR+I+G as the model of base substitution that best fit the data.  ML analysis and MP analysis excluding 3^rd position transitions (which show evidence of saturation) produced trees with nearly identical topologies.  Only the ML tree is shown (Fig 5).  Both trees resolve Family Catostomidae, Subfamily Ictiobinae, and genera Carpiodes and Ictiobus as monophyletic with high bootstrap support.  However, the trees fail to resolve a number of the species complexes as monophyletic (namely C. carpio, C. cyprinus, I. cyprinellus, and I. niger).  We attribute the low sequence divergence and low phylogenetic resolution observed at the species level for cyt b to intrageneric, interspecific hybridization, particularly within the Mississippi River Basin. 

The hypothesis that hybridization is influencing patterns of cyt b sequence variation in ictiobines is entirely plausible.  Hybrids among I. bubalus, I. cyprinellus, and I. niger have been observed in nature (Robison and Buchanan 1989, Etnier and Starnes 1993) and produced in experimental ponds (Stevenson 1964).  Moreover, all three of the species are known to hybridize introgressively in reservoirs (Johnson and Minckley 1969).  Four morphologically divergent catostomine sucker species in the Klamath River Basin (presently assigned to three genera) similarly show very low levels of mitochondrial DNA sequence divergence, which has also been attributed to interspecific hybridization (see reports under "Genetics" at http://www.mp.usbr.gov/kbao/esa/). 

SSCP: Figure 6 shows preliminary SSCP results for 5 individuals each of I. bubalus, I. cyprinellus and I. niger from the Wisconsin River (Upper Mississippi River Basin).  Three different conformational patterns are evident in I. bubalus and I. cyprinellus, and four patterns are evident in I. niger.  Similarities are evident when  patterns are compared  across species (e.g., lanes 2, 7, and 13), suggesting that some of individuals are interspecific hybrids. Nevertheless, species-specific SSCP patterns can be identified for specimens of I. bubalus (lanes 3 and 5), I. cyprinellus (lanes 6,9 and 10), and I. niger (12 and 14).  By sequencing GH I Intron B for specimens with these patterns, together with specimens from other populations of these species, I. cyprinellus (3 populations) formed a monophyletic group related to I. niger, with these species sister in turn to I. bubalus. Thus, SSCP enabled us to assess genetic variability within populations, identify interspecific hybrids, and identify individuals for sequencing that allowed us to better resolve species of Ictiobus as monophyletic. We expect similar results using SSCP for cyt b.

GH sequencing: Sequencing of clones revealed two unique cDNAs for I. bubalus, which differed in nucleotide sequence by 5.3%.  The validity of this result was suggested two ways.  First, all amino acid changes were conservative and none caused termination of the GH protein.  Second, the paralogs differed at codon 123 by a single nucleotide, agreeing with previously identified paralogs of Carassius auratus GH genes which have a divergent number of total cysteine residues (more specifically either 4 or 5 based on a C->T transition at nucleotide position 369, Law et al. 1996). Moreover, amplification of genomic DNA using primers spanning GH Intron C (i.e., primers binding to Exons 3 and 4) produced two discrete product bands (Fig. 7), indicating that the two paralogous copies of GH intron C are highly divergent.  The two amplified products have been cloned and sequenced, and have characteristics (intron splice donor and acceptor sites) indicating that neither is a psuedogene.  GH I and II are to 93% identical in the short exonic region amplified.  The sequences then diverge rapidly starting at 10 bp past the splicing sites; the variation involves 6 of the first 30 bp, and 16 of the next 30 bp (data not shown).  We were able to use this nucleotide variation in the coding regions to design copy-specific primers.

Intron B, measuring 300 bp in length, was amplified and bi-directionally sequenced for seven Ictiobus specimens (I. bubalus from upper Miss. R., Lake Pontchartrain and R�o San Fernando, I. cyprinellus from upper Miss. R., Ohio R. and Lake Pontchartrain, and I. niger from upper Miss. R.).   Comparisons of the sequences reveals more interspecific divergence (average of 3%) than observed for cyt b sequences of the same taxa (average 0.4%).  Interpopulational divergences average 0.4%.  We also detected taxon-specific indels, which may also prove useful for resolving species and population level questions. Thus, we are confident that sequences from GH introns will give us resolution at the lowest taxonomic levels (species complexes to populations) that we currently lack with cyt b.  Moreover, having sequences from nuclear and cytoplasmic genes will enable us to measure associations between these genomes and test for cytonuclear disequilibria.  Cytonuclear disequilibria provide an array of conceptual tools useful for examining hybrid zones, including levels of gene flow, age of reproductive barriers, directionality of crosses between hybridizing species, levels of assortative mating, and mechanisms of selection on hybrids (Arnold 1993).

Proposed Molecular Work

We request support to isolate total DNA from 1,800 specimens of ictiobines.  In sampling populations of each of the currently recognized species, we will remove fin clips from up to 20 individuals. Half of the specimens will be preserved for detailed morphological study, the rest will be photographed and released. We have identified areas of the distribution of each of the currently recognized species that we will initially target for assessing genetic variability (60 areas in all, Table 1). The areas were selected to represent what we believe to be the major zones of differentiation of ictiobine species complexes based our morphological studies and evidence from other groups of fishes (Wiley and Mayden 1985, chapters in Hocutt and Wiley 1986, Eisenhour 1999). Wide-ranging species are represented by more areas. Areas to be sampled along the Gulf and Atlantic coasts encompass a number of rivers that lack freshwater connections today.  We are targeting at least one area of the distribution of each of the species where other congeners do not occur to get a sense of genetic divergence without the potential for hybridization.

We will run SSCP analysis on cyt b and GH introns for 10 individuals from 3 populations from each of these areas.  SSCP will give us a general sense of genetic diversity within populations and will allow us to identify the variants we need to sequence.  The combination of cyt b and GH intron sequences, and cytonuclear disequilibrium analysis will give us the resolution we need to assess evolutionary relationships among haplotypes and impacts of interspecific hybridization.

            The goal of the molecular portion of this study is to produce independent phylogenies at the levels of Subfamily Ictiobinae, genera Carpiodes and Ictiobus, and currently recognized species complexes.  We will select outgroups for phylogenetic analyses based on existing phlyogenetic hypotheses of basal catostomid relationships (Smith 1992, Harris and Mayden 2001, Bart et al. in review).

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