We Have To PCR The PCR Product, And Back To Cleburne County

Travis and I tested the concentration of the stippled studfish PCR-amplified DNA that was purified from gel bands. Eight samples ranged from 1.5 - 10 nanograms of DNA per microliter, and we need about 50 ng/ul for sequencing. So, we can PCR the purified PCR products, a pain in the ass but not too bad; we only have 37 of them. I knew this might be the outcome of cutting out the bands; the product is exactly what we want, but relatively little of it. To make this easier I just ordered a powerful handheld ultraviolet lamp so that we can more easily cut out bands from gels. I have to give Travis credit for doing a good job so far, since we've had to visualize the bands on a large UV box, make marking cuts, and then punch out the gels away from the UV so that no one gets their eyes burned by UV.

And speaking of stippled studfish, I think we're going out next Tuesday to check the Little Tallapoosa River and its tributaries for stippled studfish. This river flows from GA into AL to the south of where we went two weeks ago. There are no records of stippleds from this river, to my knowledge, but I find it hard to believe that the species wasn't there originally. If we don't find any stippleds in this area, we can pretty definitively say that the species doesn't exist in the Tallapoosa drainage upstream from Cornhouse Creek to the south in Randolph County. Maybe they are somewhere, but they would be pretty scarce.

Right now I'm giving and grading final exams, everyone concerned looks pretty punchdrunk.

Telescope Shiners Article Introduction

Since I've been having dreams about writing a journal article on our research project with telescope shiners, I finally sat down several times over the last several days and hammered out an Introduction for such an article. This is the hardest part of a journal article, since you have to concisely state the context and reason for your work preferably in no more than three paragraphs. One of my colleagues says that for him, the introduction takes 50% of his effort on a paper. I might go with more like 35% for me, but that's still significant. Banging out the materials & methods, and results, is almost easy, and then there's another big effort necessary to wrap it all up in a discussion.

So here it is, a work in progress. I'll almost certainly tweak it before submission but I want to put it into play. If nothing else, I'll see and read it again and maybe realize that I could state it better. I hope to submit this to the journal American Midland Naturalist.

Begin:

Cyprinid stream fishes of temperate North America typically spawn in the spring to allow larvae to grow enough to survive their first winter. The details of each species’ reproductive schedule and effort vary as an important part of its ecological niche. With the high species diversity of cyprinid stream fishes in the southeastern United States there are many variations on the theme of vernal spawning and thus in the ecological niches of these species.

One such cyprinid is Notropis telescopus Cope (telescope shiner), found in the Cumberland and Tennessee river drainages east of the Mississippi River and the White and Arkansas drainages in the Ozarks west of the Mississippi. N. telescopus is a common inhabitant of runs and flowing pools over sand and gravel in creeks and small rivers with clear water. Etnier and Starnes (1993) report observations of the species spawning in Tennessee from mid-April through mid-June.

The purpose of this study is to determine the schedule of reproduction (sensu Heins and Rabito 1986) of N. telescopus by examining gonadal condition over the course of a possible breeding season, February through August. No previous study has addressed this issue. Both males and females in the genus Notropis and closely related genera are known to be multiple spawners, with females producing more eggs than are found in the ovary at any one time (Dahle 2001, Heins and Clemmer 1976, Stallsmith et al. 2007).

DNA Extractions In Gear

Travis has been working on extracting PCR-amplified stippled studfish DNA from gel cut-outs. Fourteen of 37 samples have been done now. This basically prepares the DNA for sequencing, which we can hopefully send out in the next several weeks at the most. We still have to test the DNA concentration in the samples but I have faith it will be good (and we'll know after we do it!).

My slow-motion experiment with the young scarlet shiners is still on track. There are currently 7 fish that have been exposed to a higher dose of 11-KT, 10 fish that have been exposed to a lower dose of 11-KT and 5 fish that are controls with no exposure to 11-KT. These aren't extremely strong numbers statistically but as at least a pilot project I hope this shows some pattern of influence on brain development.

Maybe Another Cleburne County Trip

Maybe I'm a glutton for punishment, but I'm thinking about another trip to Cleburne County in search of stippled studfish. This trip would visit the easternmost edge of the county, along the Georgia line where the Tallapoosa River enters Alabama. I have no reason to expect to find the fish in this area to the NE of where we went 10 days ago, but it would enable me to say with greater confidence that the species is not to be found in this area, if it ever was, reinforcing the idea that the species apparently is limited to six creek systems. I might even try to put together a trip next Tuesday. At least I'm not going today, seeing how the temperature in Huntsville is about 100 degrees F. right now(!).

I've been having dreams about writing a paper on our telescope shiner findings, so I should really do it. The paper will have three figures and maybe one table, so all I have to do is come up with text connecting this graphic information. Soon, I hope!

Scouring Cleburne County, Alabama, For Fundulus bifax

Last Friday four of us spent the day driving around the southern tier of Cleburne County, Alabama, SE of Anniston, AL. The Tallapoosa River enters the state from Georgia here, flowing south into Randolph County and other counties where we've already found bifax. To my knowledge no one has ever collected the species in Cleburne County, which represents a gap between the known populations to the south and populations that used to exist upstream in Georgia. So I thought it was worth trying to connect these two areas by somewhat randomly visiting sites that looked good on the map and see what we found. In short, we found no bifax, but I also now realize that we found no good habitat; in our experience the species is found in clean, clear water running over algae-free sand and we didn't find any such sites in Cleburne County.

Our first stop was the Tallapoosa River itself, where Highway 46 crosses it. GoogleEarth images looked good, showing a boat ramp leading into the river and sandy/gravelly habitat upstream. And the river did look good at first, as shown in the first photo. But, the substrate had lots of hair-like green algae, and other macroalgaes growing off of rocks. We found lots of fish, but too many centrarchids like redbreast sunfish and Coosa bass. The Tallapoosa is reported to be eutrophic coming out of nearby Georgia, and that's what we found. And that's not good for bifax.


Our next site was Dynne Creek, a branching stream coming off of Turkey Heaven Mountain (no, really!). The county road was unpaved leading up to it, which is usually a good sign since it implies little traffic. Below is a picture of Charlie peering into the creek before we sampled.


It was a good-looking creek, with rock cobble, some gravel, and some puffy sediments for substrate (see photo below). We caught lots of Tallapoosa shiners here, including beautiful breeding males. We also caught a single streamline chub, a rare species in Alabama, which I didn't ID until I had it back in the lab. And, we found a subadult bluehead chub, a species I usually don't encounter. But, once again, we found no bifax. My impression is that the species was never in this creek, since again it was a wrong microenvironment rather than significantly polluted or altered (although we encountered a cattle excluding fence across the creek as we worked upstream).

And the last creek we sampled that day was Lockhelooge Creek, in some ways similar to Dynne Creek, but with more exposed bedrock forming a series of descending pools, runs and small falls with little sand. It was a striking creek, and the picture below doesn't really do it justice. The interesting species we found here was the bandfin shiner, Luxilus zonistius. They look a lot like their relative the striped shiner at first, but with a golden brown body color and more color in their dorsal fin (hence the name). The bandfins were apparently the most common shiner in this creek, along with a few pretty and Tallapoosa shiners. But, no bifax were caught or seen, and like Dynne Creek I don't think the species was ever here; it's the wrong microhabitat with almost no sand, much less exposed sand bars alongside slow moving pools.

Several other creeks we visited near Highway 431, the main road leading to Anniston, were badly degraded by typical stupid land uses; one entire creek branch was gone, with the water impounded for a pond next to a church and the streambed now a covered drainage culvert. We stopped at one creek that looked OK at first until we noticed several big fat carp swimming through the channel under the bridge, and the heavily drifted-in garbage along the banks. A guy driving by stopped and engaged us in an incoherent chat about fishing for carp, I'm still not clear if he liked it or thought the carp were too tough to eat or something. More than anything else he probably wondered who the hell we were, four guys staring intently into a dirty creek from a bridge with no guard rails.

So Cleburne County is apparently a big gap in the historic distribution of bifax, and the species may not have been present in the smaller creeks in the area (or at least is long gone from the damaged creeks). Maybe the Tallapoosa was historically a connector between various scattered tributary creeks, and maybe parts of the river were suitable breeding habitat for bifax. The Tallapoosa site we saw was definitely not a good breeding site, with all of the heavy algae growth that would smother any eggs deposited in sand as bifax do when breeding. So with the species apparently extirpated in Georgia, and not present just downstream in Cleburne County, the six creek populations we've identified might be the only remaining populations now separated by alterations in the Tallapoosa. It's a stark thought, but the evidence supports it at the moment.

Why Is Brier Fork Of The Flint River Running So Muddy?

Allison from the Flint River Conservation Alliance (Madison County, Alabama) sent me these pictures that she took yesterday. She noticed that Brier Fork of the Flint River is running really muddy, while the main stem of the river is relatively clear. The first and third pictures below show the contrast where Brier Fork runs into the Flint at Winchester Road. The contrast is strong. She also drove up Brier Fork as far as Highway 431 and the stream was muddy all the way up. No one knows exactly why the fork has such a high sediment load. My own guess is that several sources are contributing to this as Brier Fork's drainage is still subject to lots of construction and road tweaking, and many contractors around here are sloppy about sediment fences.

This is unfortunate since it's Brier Fork that contains a disproportionate share of the vulnerable aquatic species in the greater Flint system, species such as slackwater darters and (scattered) flame chubs. Given that this is Alabama, state authorities aren't exactly all over the situation.

We Found No Stippled Studfish In Cleburne County, AL

We had a good trip today driving around the southern tier of Cleburne County in the Tallapoosa River drainage, southeast of Anniston, AL. We even seined in the Tallapoosa for the first time, where Highway 46 crosses it. But we found no stippled studfish. The short reason seems to be that the correct habitat doesn't exist in the area: clearwater streams over sand with no nutrient overload fueling rich algae growth. The Tallapoosa itself has both obvious nutrient enrichment and extensive bank erosional processes. We caught lots of sunfishes, bassses, shiners and even blackspotted killifish, but the right microhabitat for the stippleds wasn't there; it took us a while to clean out all of the thick, green, hair algae from the net afterward.

I'll post a more complete posting later.

Ideas For Bifax

Travis PCR'd our 4 Sofkahatchee stippled studfish DNA extractions today. I guess we'll run the gels next week. And, we're preparing for our trip to Cleburne County, AL, on Friday. My plan now is to survey random good-looking streams starting at Hwy 431 in the SW corner of the county and move east on county roads along the southern edge of the Tallapoosa River. There are some reasonably large mountains in this area, so we'll be moving on their northern edge looking at streams running off of them. Like I said before, I have no idea what we'll find; this area is an historic gap in studfish distribution from what I can tell, but the fish should be there in theory.

I've been reading the book Conservation and the Genetics of Populations by Allendorf and Luikart as a broad review/refresher on the subject. The one idea for future work I've gotten from this through the first five chapters is to examine chromosome structure of this species and relatives, i.e. trying to make a good ol' fashioned karyotype. Maybe someone has done this work for all Fundulus species and I just haven't found it yet (I know it's been done for olivaceous and notatus, anyway). But I have no idea how many chromosomes the various studfish species have. And that would be a useful datum for examining the functional genomics of these species. But, we'll see.

DNA Extraction From Sofkahatchee Stippled Studfish

This entry's title is more of a mouthful than I intended. But, yesterday Travis and I extracted DNA from four of the bifax from Sofkahatchee Creek that Joe Scanlan caught and gave to me. It was Travis' first extraction, using the old-fashioned PCI/CI phase separation method with white muscle tissue. Everything seemed to go well. We didn't get big pellets at the end, which is usually a good sign since pellets contain more protein and salts than DNA, and in truth I could see just indistinct smears along the tubes' walls. Leland graciously agreed to help us used the Nano Drop spec in the Podila lab, and we measured DNA concentration values of 30-90 nanograms/microliter with 260/280 ratios of 1.6-1.8. I was a little put off by these values at first, since our first round of extractions had concentrations more in the range of 100-300 ng/ul and ratios of 1.8-2.1. But Leland wasn't phased, saying that was their typical values for PCR work in their lab. Maybe we just did a better, cleaner job of extracting DNA with few contaminants? I guess so; or the Nano Drop wasn't as clean before, which Leland said was also the case? We'll know once we run PCRs on these samples. We haven't been able to assay concentrations for a while, and probably our most recent extractions have the same values as yesterday's and produced good bands on gels after PCR. I guess the real proof will be looking at sequences, hopefully sooner than later.