The Bifax DNA, She Is Being Sequenced

At least I hope that's the case. I drove to Tuscaloosa yesterday and handed over our purified, amplified DNA for sequencing to the Molecular Systematics Lab at Bryant Hall. I hadn't seen Phil Harris since the St. Louis ASIH meeting, so it was nice to chat. I also talked with Bernie Kuhajda and Michael Sandel. Bernie thanked me for telling them last year that Tuscumbia darters can be found in lower Limestone Creek. They collected 30(!) in the area I pointed them to. One of the doctoral students there is doing his dissertation on Tuscumbias, a likely candidate for Federal protection in the near future. Michael will be at the ASB meeting later this week in Birmingham, making a presentation in the same session I will on Friday morning. He will, of course, talk about Elassoma pygmy sunfish.

It was refreshing to visit this extended lab group. They have most of a floor in an academic building, with the same broad interests I have. Not feeling like a freak or oddball was enjoyable.

My big plan in this busy week: keep plugging away at boiling down Enrique's thesis into journal format. I still have to read the editor's Authors instructions before I attack the Results section.

A Tuscaloosa Trip Tomorrow

I'm driving to Tuscaloosa tomorrow morning after my morning class to hand-deliver our purified DNA amplifications from the stippled studfish project. The U. Alabama Biology Department's DNA lab has agreed to do the sequencing, thanks to Phil Harris, so away we go. I no longer trust the "overnight" services after a few bad experiences, and with all of the work we've gone through in collecting the fish, and doing the lab work, I feel it's worth it to extend myself a little more.

An hour ago I uploaded my PowerPoint talk for the Association of Southeastern Biologists meeting later this week in Birmingham. My talk, "Reproductive Biology of the Telescope Shiner, Notropis telescopus, in Alabama" is at 9:15 Friday morning, part of the (single) Herpetology & Ichthyology session. Ruth and I are driving down to B'ham Thursday afternoon to stay overnight at the Birmingham Sheraton, so I'll be in easy position for the meeting talk. My Friday morning intro biology course is canceled, needless to say. It should be fun, I hope!

Scarlet Shiner Brain Abstract

I've made some serious headway boiling down Enrique's thesis for a journal article in the 'zine NeuroReport. Below is the current version of the Abstract, at 300 words. I think that's a decent length for this journal, but in truth I still have to read the author's instructions which I just printed. The title will be something close to, "Sexual Dimorphism In A Teleost Fish's Central Nervous System: Are Dominant Males Smarter?", with authors Stallsmith, Sosa, Eguchi, Suessman.

ABSTRACT
The teleost fish, Lythrurus fasciolaris (scarlet shiner), is known to be both a seasonal breeder and sexually dimorphic. Wild scarlet shiners were collected at intervals throughout the breeding season. Morphological and molecular changes that may underlie sexual dimorphism were examined in three key regions of the brain: the cerebellum, optic tectum and the telencephalon. Allometric analysis of whole brain mass relative to body mass showed that males were significantly more variable (R2 = 0.29) during the breeding season than females (R2 = 0.84). Males were also found to have larger average volumes than females in all three brain regions relative to total brain mass (p<0.01). The quantity and location of NMDA receptors in relation to sex and reproductive status was examined. Four groups were examined: dominant males, non-dominant males, reproductive females and non-reproductive females. At the beginning of the breeding season, dominant male shiners have at least a 3.5-fold higher expression of NMDARs than non-dominant males in all three brain regions (p<0.01). Non-reproductive females had 4-fold higher NMDAR in the cerebellum then reproductive females. At the peak of the breeding season, the telencephalon region of non-reproductive female fish exhibits 3.5-fold higher expression of NMDAR than non-dominant males. Dominant males and reproductive females both exhibit about a 2-fold higher expression than non-dominant males. In the cerebellum, both female groups exhibited a 2-2.5-fold higher expression level of NMDAR than either male group. In the optic tectum, the non-reproductive females showed the highest difference with nearly a 25-fold higher concentration of NMDAR than non-dominant males. Reproductive females exhibited a 10-fold higher level and dominant males an 8-fold higher level. This study suggests that NMDARs undergo both temporal and sex-specific regulation. These findings show quantifiable morphological and molecular changes in sexually dimorphic teleost fish that may correlate to changes in breeding and dominance requirements, with implications for all vertebrates.

I'm Writing A Scarlet Shiner Brain Article, And Kris Has Increasingly Cool DNA Data

Well, I'm not really writing that article but rather I've started to boil down Enrique's thesis on scarlet shiner brain size as it relates to sexual dimorphism. He seems to have gone missing, so I'm seizing the moment and doing it myself. We're going to submit it to the journal Neuroreport (I'm pretty sure) and they don't like long Introductions on articles. The starting point for the Introduction was 29 pages; I'm aiming for three pages once I do another round of cutting tomorrow from where I have it at five pages.

Kris came by and showed me some refinements on his analysis of the mummichog DNA data. All of the DNA differences he's pinpointed between the various populations are single nucleotide polymorphisms (SNPs), and they're all apparently neutral -- they don't change the amino acid sequence. Most are third position SNPs, but a surprising number are neutral first position changes. We don't have a firm transversion/transition count yet. He's working on a map to show the different collecting sites, and to try to construct a cladogram or dendrogram that would match the map. We just have to be clear in our mind where Wiscassett, Maine, is since that's a sequence we download from the NCBI data bank. As I remember it's on the central Maine coast...

A Nice, Long, Sequence For The Mummichog DNA

I met with Kris yesterday afternoon for an update on his mummichog population genetics project. He now has phylogenetic trees based on a sequence of 775 base pairs of the cyt-b mtDNA gene, a good stretch of the total 1160 base pairs. Analysis of this longer stretch of gene confirms and clarifies what we'd seen with shorter stretches: that the New England populations have very little genetic difference between them, and that the New England populations are measurably different from populations from the Delmarva peninsula south to Georgia. The latter point is hardly new in of itself, but our data strongly suggest that these New England fish are descended from a very small founding population that colonized the New England coast after the glaciers retreated ~13,000 years ago. One way to quantify this separation is by percent differentiation: the New England and southern populations are about 1.4% divergent. By way of comparison to another Fundulus species, the longnose killifish, F. similis, which we're using as an outgroup is about 7.5% divergent from the mummichog populations.

I just realized we're due for another trip to Limestone Creek to collect scarlet shiners. The pair I have in a lab aquarium are beginning to peak in terms of breeding coloration and apparent egg production. Those pituitary hormones are powerful chemicals.

On To Flagler County, Florida, In May

I realized that my first choice for a site to collect longnose killifish in Texas, the Port Bolivar peninsula on the east side of Galveston Bay, was ground zero for Hurricane Ike last summer. So rather than go there this May, Ruth and I will head for Flagler County, Florida. This is the boundary between what's usually recognized as a northern species, Fundulus majalis (striped killifish), and the southern species, Fundulus similis (longnose killifish). It's also the ecotone (boundary) between Spartina salt marshes typical of the northern Atlantic coast and the mangrove swamps typical of the southern Atlantic coast. So, I'd like to collect fish from the northern edge of Flagler County and the southern edge. I finally read the 1995 paper by Charles Duggins et al. in the journal Heredity entitled, "Analysis of a hybrid zone in Fundulus majalis in a northeastern Florida ecotone." They used protein electrophoresis to examine genetic differentiation between northern and southern populations of these species, from South Carolina down around to the Florida panhandle. Their conclusion was that there's really one species present, with two distinct populations with easy gene flow at the ecotone in Flagler County. That's been my working hypothesis that these two species are really a long cline of distinct populations along the Atlantic and Gulf coasts from New Hampshire down to Tampico, Mexico. My expectation is that once I get all of my mitochondrial DNA sequences done from sites within this range, the data would reflect what Duggins et al. found using protein electrophoresis. Other work has shown statistically significant morphological differences between the two currently recognized species (and I feel stupid because I can't remember the citation!).

On a different note, we made a terrible mistake in our garden this week. We got a truckload of composted horse poop from a farm in Tennessee and put most of it on our garden. Now we have a scary ant infestation, likely fire ants. So tomorrow we'll spend the day digging out the top 6-12 inches that we just put in to physically remove the ants and hopefully avoid using heavy chemicals. The poop was free, and worth every penny apparently.

It's Spring Break!

For all that really matters. I was supposed to meet with Kris today but he isn't feeling well, so we'll meet on Thursday. My last two whitetail shiners were dead when I came in today; I'm not sure if it's me, or if they're harder to keep in aquaria than I'd thought. My super alpha scarlet shiners are all about ready to spawn, which would basically just feed the darters in that tank. I'm not sure what I can do about that.

I'm thinking about doing a Texas trip the first week of May, after the Sea Lab meeting. I want to collect some Fundulus similis from hopefully two sites on the Texas coast. Ruth and I haven't ever really been to that coast, so hopefully it would be fun to see someplace different. I don't think that any decent sandy beaches exist west of Pass Christian, Mississippi, until you get well into Texas since Louisiana is all bayou which makes coastal collecting tough. But, maybe I'm wrong. This is all part of my other long-term project to compare the mtDNA of F. similis and F. majalis from Cape Cod, MA, down to wherever I can get in Tejas. Getting the new, improved version of Geneious for analyzing sequences might be the last missing piece of the puzzle for me. At least I hope so!

A New, Improved, Dendrogram For Mummichog mtDNA

For all you gene jockeys out there, I generated a dendrogram through the Geneious program's Tree package using HKY Neighbor Joining with 10 bootstraps and a minimum of 50% consensus support for nodes. I used a stretch of 669 bases in the gene with the available sequences we have. And it shows a somewhat different picture from only looking at 272 bases, not surprisingly; the New England fish resolve as a clade with Nantucket being slightly different, the Charleston, SC, fish resolve into a double-lobed clade with Sapelo Island, GA, off to the side, and Chincoteague, VA, and Virginia Beach, VA, resolve as a surprisingly distinct and distant clade. The outgroups, Fundulus majalis and a Rivulus species, aren't shown in this figure and are down to the lower right. Here it is: (you can expand the image by clicking on it):

This reinforces my working theory that the New England fish are the result of a founders effect, and are very different from even nearby more southerly fish. I'll show you the whole story later.

We Now Have A Bigger DNA Dataset For The Mummichog Project

Kris acquired the new, improved version of Geneious, the software package we've been using to align and interpret DNA data. With this software our usable stretch of cyt-b DNA has increased from 272 to 719 bases because it can read what had seemed to be garbled sequences to the old software. So that's a big step forward, given that the whole gene is ~1160 bases long (and I'd thought that our PCR/sequencing work was better than 272 bases, anyway). We might also be able to recover a sequence from a Boston Harbor fish, one of the first ones I captured back in 1995 in front of UMass/Boston. That makes me about as happy as you can get about DNA data....

Another Successful Trip To Estill Fork, With Good Weather

I went to Estill Fork with Andrew and Brittany yesterday. We netted 37 telescope shiners, and 2 scarlet shiners. Water was a little bit higher than on Jan. 31 but not enough to be a problem. We installed my custom driftnet for about 45 minutes, and the steel-core tomato stakes I bought at Lowe's held in a reasonably strong water chute. We kept the take, and when we examined it at the lab found some material we wouldn't have found otherwise such as juvenile springtail instars. So I'm psyched about that, Ruth is willing to make more bags if she can see this one again to remind her what she did.

Here are a few photos from the trip. The first one is a downstream view of the riffle system we collected the telescopes in. The driftnet was placed in a flowing chute just to the left of where this photo was taken.

Here are Andrew and Brittany at the site where we installed the driftnet, although the chute isn't really apparent.

And here's some real science in action, Andrew doing the simple water chemistry tests we routinely carry out--pH, total dissolved solids, temperature.

I think we'll go back in mid-April.

Back To Estill Fork Saturday

We'll go out to Estill Fork Saturday to collect telescope and scarlet shiners. Once again we seem to be lucky with the weather, the prediction is for high in the 70's F and sunny. There hasn't been rain since last weekend so water level should be manageable.

Brittany has been doing more statistical analysis of data from the 11-KT scarlet shiner experiment. Females exposed to 11-KT compared to wild-caught females had significantly larger brain weight/body weight ratios in a U-test, with p<0.001. Males exposed to 11-KT compared to wild-caught males also had a significantly larger ratio, but only with p=0.05. This makes sense, because the wild-caught males have been influenced by their endogenous 11-KT, while wild-caught females would have almost no endogenous 11-KT. This seems to be a good story, and the results encourage me to go on to a more ambitious experimental manipulation later this year.