V.   References:

Bernardi, G., and D. A. Powers.  1995.  Phylogenetic relationships among nine species of the genus Fundulus (Cyprodontiformes, Fundulidae) inferred from sequences of the cytochrome b gene.  Copeia:  469-471.

Bernatchez, L., H. Glemet, C. C. Wilson, and R. G. Danzmann.  1995.  Introgression and fixation of Arctic char (Salvelinus alpinus) mitochondrial genome in an allopatric population of brook trout (Salvelinus fontinalis).  Can.  J. Fish. Aquat. Sci. 52 (1):  179-185.

Duvernell, D. D., and N. Aspinwall.  1994.  Introgression of Luxilus cornutus mtDNA into allopatric populations of Luxilus chrysocephalus (Teleostei:  Cyprinidae) in Missouri and Arkansas.  Molecular Ecology, 4:  173-181.

Chen, T. R.  1970.  A Comparative Chromosome Study of Twenty Killifish Species of the Genus Fundulus (Teleostei:  Cyprinodontidae).  Chromosoma, 32:  436-453.

Ghedotti, Michael J. and Michael J. Grose.  1997.  Phylogenetic Relationships of the Fundulus nottii Species Group (Fundulidae, Cyprodontiformes) as Inferred from the Cytochrome b Gene.  Copeia, 4:  858-862.

Mayr, Ernst.  Systematics and the Origin of Species from the Viewpoint of a Zoologist.  New York:  Columbia University Press, pg. 102-129.

Pflieger, William L.  1997.  The Fishes of Missouri.  Jefferson City:  Missouri Department of Conservation, pg. 235-244.

Setzer, Paulette Y. 1970.  An Analysis of a Natural Hybrid Swarm by Means of Chromosome Morphology.  Trans. Amer. Fish. Soc., 1:  139-146.

Thomerson, Jaime E.  1966.  A Comparative Biosystematic Study of Fundulus notatus and Fundulus olivaceus (Pices:  Cyprodontidae).  Tulane Stud. Zool. 13 (13):  29-47.

Thomerson, Jaime E.  1967.  Hybrids Between the Cyprodontid Fishes, Fundulus Notatus and Fundulus Olivaceus in Southern Illinois.  Transactions Illinois Academy of Science.  375-379.

Thomerson, Jaime E., and David P. Wooldridge.  1970.  Food Habits of Allopatric and Syntopic Populations of the Topminnows Fundulus olivaceus and Fundulus notatus.  American Midland Naturalist, 84:  573-576.

VI.   Budget Justification

                The molecular techniques used in the study will require commodities as follows.

Item	Quantity	Supplier	Price
Bio-Rad AquaPure genomic DNA Isolation Kit	200 extractions	Bio-Rad	.10
Ex Taq DNA Polymerase	250 units	Fisher	.00
Sigma Genosys custom oligonucleotides	2 oligos	Fisher	.92
Agarose	100 grams	Fisher	.92
Tris-Acetate-EDTA buffer, 50X Solution	500 mL	Fisher	.10
PCR Tubes, 0.5 mL	1000	Fisher	.00
Restriction Enzyme:  HaeIII	3000 units	NEB	.00
Restriction Enzyme:  HhaI	2000 units	NEB	.00
Restriction Enzyme:  NsiI	1000 units	NEB	.00
Shipping and Handling			.06
		Total Cost:	.00

The Bio-Rad Kit will be used to extract the DNA samples from the muscle tissue of the fish.  Taq DNA polymerase is a heat resistant enzyme that aids in the PCR process.  The oligonucleotides are the primers that flank the targeted regions on the mtDNA cytochrome b gene, used during PCR amplification.  The agarose and the Tris-Acetate-EDTA buffer are both used in the electrophoresis process.  The PCR tubes are thin walled tubes specifically designed for use in the PCR machine.  Restriction enzymes were chosen to assay specific nucleotide fixed differences that are diagnostic for F. olivaceus and F. notatus mtDNA sequences.  Many of the items required for this study are available from the labs in the SIUE Science Department or will be covered by the experimenters, due to financial limitations.  Items such as: pipette tips (various sizes), reaction tubes (various sizes), seines, traps, thermal cycler, centrifuge, heating blocks, power supply, electrophoresis apparatus, gas, mileage, travel (to Harrisburg, IL, from Edwardsville, IL:  ~150 miles).

Glossary

Allopatric - occurring in separate, nonoverlapping, geographic areas

Assay - an analysis or examination

Backcrossing - mating of a hybrid with an organism of the parental species

Chromosomal rearrangement - the duplication, deletion, inversion, or movement of a nucleotide or group of nucleotides found on the chromosome

Dichotomous key - method of classification by a series of one or the other choices, until a final conclusion is made

Disturbed [habitat] - a habitat that has been altered (polluted, fluctuating dryness)

DNA digestion - breaking down of DNA into smaller, more manageable segments

Eukaryotic - a single-celled or multicellular organism whose cells contain a distinct membrane-bound nucleus

Gene pool - the genetic composition of an entire population

Genetic variation - the difference between each individual organism within a population

Hybrid - the offspring of genetically dissimilar parents, such as different species of parentals

Interbreed - mating of an organism with another organism of the same species 

Interfertile - one species has the ability to fertilize an egg of another species

Introgression - the infiltration of the genes of one species into the gene pool of another, through repeated backcrossing

Mitochondrial DNA - genetic material found within the mitochondria organelle in eukaryotic organisms

Morphology - the form and structure of an organism or one of its parts

MS-222 - an anesthetic for aquatic animals, chemically known as Tricaine Methanesulphonate

Nucleotide - one of the four nitrogen-based molecules, Adenine, Thiamine, Guanine, and Cytosine, joined together to code for different proteins; can be used to identify genes and genetic differences

Phylogenetic - the lineage history of a type of organism based on evolutionary development or history

Rare hybridization - creation of a hybrid individual that occurs only scarcely in nature

Reproductive barriers - physical or instinctual mechanism that prevents organisms of one species mating with organisms of another

Restriction enzyme - organic substance that cuts strands of DNA nucleotide locations specific to the enzyme

Sympatric - occupying the same or overlapping geographic areas without interfering

Viability - the ability to produce fertile offspring

2003-2004 Proposal "Spatial Analysis of a Hybrid Zone Between Two Species of Fundulus"

I.   Introduction and Significance:

                Fundulus olivaceus and Fundulus notatus are two of the most widely distributed species of the North American cyprinodontid genus Fundulus (10).  They are known respectively as the Black-spotted topminnow and the Black-striped topminnow.  Their wide geographic distribution in the central and southwestern United States includes a zone of contact in southern Illinois where the two species can be found sympatrically.  While these species have overlapping broad-scale geographic ranges, they are rarely found occupying the same stream or pool.  A previous study outlined testing populations samples from areas of sympatry for evidence of past hybridization events, by testing for genetic introgression.  My recent studies have demonstrated pronounced introgression in certain sympatric populations of Fundulus.  These results can be found in Appendix A of this proposal.

                Our research seeks to survey a single stream in its entirety in an attempt to discern a distribution pattern of the two species.  We will be testing two types of hybrid zone structure, clinal and mosaic.  A clinal hybrid zone results if there is a smooth transition from one species to another along a linear transection (e.g. a single stream).  The hybrid zone would be restricted to a defined region where the species overlap.  In contrast, the mosaic pattern of hybridization would result if the two species exhibit a patchy distribution along the stream in response to local environmental conditions.  Important environmental factors, such as substrate type, stream depth, and current, may lead to environmental selection which could maintain clinal or mosaic hybrid zone patterns.  We will be concentrating on a fine-scale spatial context, looking at a single stream.  Analyzing the spatial distribution patterns will prove both interesting and important in determining character distribution and possible past hybridization events.

II.   Literature Review:

                These two species of Fundulus are of particular interest because much has been previously researched regarding extensive phylogenetic relationship and hybrid characterization.  Despite their overlapping ranges, these two species are rarely found together.  Artificial crosses have demonstrated that F. olivaceus and F. notatus remain interfertile (11) despite marked chromosomal differences stemming from several chromosomal rearrangements (4).  One notable exception was reported in which extensive hybridization was discovered (9).  This hybridization was attributed to ecological disturbance at the local level that may have resulted in the breakdown of behavioral isolating mechanisms between the two species, rendering individuals less discriminate in mate choice.  The mtDNA (mitochondrial DNA) is a good candidate to search for genetic introgression as it has been shown to readily introgress between fish species that have undergone hybridization (3, 2).  Several mitochondrial genes have been sequenced in each of these species revealing a number of distinctive nucleotide differenced between the two species that may be readily assayed using either DNA sequencing or restriction enzyme digestion techniques. 

We are interested in determining population structure within a hybrid zone.  Hybrid zones can be characterized as clinal or mosaic.  In clinal hybrid zones, pure individuals (or populations) meet and hybridize to form a smooth transition (or stepped cline) from one parental type to the other.  Mosaic hybrid zones are characterized by a patchwork of alternating pure parental types (or populations) throughout the zone of hybridization, with abrupt transitions and reversals in the character of individuals and populations (8).  Spatial scale can be an important issue when looking at the structures of hybrid zones because patterns of variation will appear only at specific spatial scales.

III.   Hypothesis:

                We will be testing two hypotheses:  The spatial analysis will show a clinal pattern of species distribution or that of a mosaic distribution.  If the two species occur selectively at opposite ends of the stream, i.e. headwaters versus tail waters, and these topminnows have been observed along the entire stream, there will be a zone of contact at some point along the stream, and if this shows a smooth transition from one species to the other species with a hybrid zone in the middle, then there is a clinal distribution.  Alternatively, if both species are found throughout the entire stream where their relative prevalence is dictated by local ecological conditions (micro-habitats), then this shows a mosaic pattern of distribution.

I V.  Materials, Methods and Timeline:

GENERAL APPROACH

                We will be sampling for both species of Fundulus in a single stream of which both species are known inhabitants.  Population samples will be taken from seven locations along the Little Cache Creek from its origin to its confluence with the Ohio River (Johnson-Massac Counties, IL).  Specimens will be characterized on the basis of morphology and mitochondrial DNA.  Sample sizes of approximately 30 killifish per population (non-species specific) will be seined and anesthetized with a 1% solution of MS-222 (Tricaine Methanesulfonate), a commonly used anesthetic for aquatic animals.  Anesthetized specimens will be preserved immediately in 95% ethanol.  Ecological data will also be collected at each site.  We will be measuring stream velocity, turbidity, stream depth, and stream width.  We will also assay water turbidity and substrate type.  Specimens will then be tested for introgression.

TECHNICAL METHOD

                We will be extracting total DNA from muscle tissue samples using the Bio-Rad AquaPure Genomic DNA Isolation kit (Bio-Rad Laboratories, Hercules, CA).  We will PCR (Polymerase Chain Reaction) amplify portions of the cytochrome b gene using published primers (1).  Double-stranded DNA products will be amplified using the following cycling profile:  denaturing at 94 C for 30 seconds, annealing at 48 C for 30 seconds, and extension at 70 C for 4 minutes.  Extension time will be lengthened 4 seconds each cycle for 40 cycles (5).  Amplified PCR products will be digested with restriction enzymes that cut sites that are diagnostic for the mtDNA sequences of the respective species.  To identify diagnostic restriction endonuclease sites, published sequences for each species (1) were aligned.  Restriction enzymes for F. notatus include NsiI, and HaeIII.  These diagnostic sites will be confirmed in a few individuals from allopatric populations in streams adjacent to the sympatric populations.  Digested DNAs will be electrophoresed in a 1% agarose gel in 1xTAE buffer and visualized by ethidium bromide staining and UV irradiation.  Visualized DNAs will allow us to determine if mtDNA of one species or the other is prevalent.  If the mtDNA of the wrong species is present, then this indicates that hybridization has occurred. 

TIMETABLE

Event	Estimated Time
Collection of specimens:  Collections from 12 locations, with four collections per trip	One three-day trip in July
Extraction of DNA, PCR, Digestion, Analysis of Data (210 specimens)	6 months (August - ~30 specimens)                (September - ~30 specimens)                (October - ~30 specimens)                (November - ~30 specimens)                (December - ~30 specimens)                (January - ~30 specimens)                (February - ~30 specimens)
Analysis of Total Data	1 month (February to March)
Total Time for Project	8 months (July to March)