A New Look At F. Similis And F. Majalis DNA

I've been on a new burst of activity analyzing cytochrome b DNA sequences using the Geneious software package. I've realized that Bayesian inference, a somewhat quirky statistical method, is the best approach to comparing sequences of DNA and building a tree so as to easily visualize patterns of relatedness and divergence. The image above is a cleaned-up, simplified analysis of what I have to date in my quest for looking at the relatedness between two very similar coastal killifishes, Fundulus majalis and F. similis. The two fish at the top of the cladogram are related species present to serve as outgroups, for comparison purposes to "root" this analysis. The numbers at the nodes of the branches are consensus numbers, basically a measure of how strong the decision to put a branch at that spot on the tree is from running lots of replicates of the tree building algorithm.

The two species separate out cleanly in this tree, but there's not very much distance between them; the scale bar showing 0.03 (substitutions per base, really) is about how different they are, and that's not a big difference between species. The other interesting feature of this tree is that the Key West, Florida, F. similis is relatively removed from others of that species. There has been discussion over the years about whether the Key West population is really a separate species. I would say that it doesn't appear to be, but it does rate consideration as a distinct population about halfway to being a distinct species, with the other Florida group being close to it (I'm not sure where in Florida that's from, I downloaded that sequence from GenBank and it was only labeled Florida). I have a thought that because Florida has so much coastline that has been suitable habitat for a long period of time (even during the last glaciation) that Florida populations are more ancient and have had time to diversify. Conversely, F. majalis is only found on the Atlantic coast and has probably been affected by glacial cycles that greatly reduce available habitat, leading to lower genetic diversity as a result of long periods of reduced population. That's my working theory, anyway. I hope to have more DNA sequences in the not-so-distant future (thanks, Tony!) and see how this plays out with more localities.

Is This Really A Minnow Trap Sculpture?!?

This sculpture appeared with no warning at the main entrance to the Shelby Center at UAH last week. I approve of public installation art, so that's cool. But what made me laugh are the cones sticking out from the piece. My first thought was, "My god, it's a minnow trap sculpture!" They're not exactly like minnow traps, of course, but close enough. How did they know? I'm honored.

Estill Fork For All The Darters We Could Catch

We went out to Estill Fork at the Baptist Church site today to collect darters for Robert's gill parasite project. It was a cool, clear day, with a good solid water flow as you can see in the picture above. That view is pretty much the area we worked. We were interested in tennessee snubnose, rainbow, fantail, stripetail and redline darters, and kept a banded just as a voucher of sorts. To get the fantails and stripetails, we set the seine and kick and disturb rocks just upstream of it, since those two species more than others are hunkering down around rocks. And it worked well for us.
This spot used to be a ford across Estill Fork before the bridge was built, since it has exposed bedrock not always too deep. Above are Robert and Brian on the road leading into the ford from the east, with the baptist church behind them. Needless to say, this is an easy access point once you find it.
At the end of our trip Robert waded out into the pool at the ford to net a few more large stripetails, which was successful. Fantails and stripetails are pretty similar until you pick up the distinguishing traits, like stripetails have a dark submarginal band on the first dorsal that fantails don't, and the stripetails have some vertical banding on their sides while fantails typically have lateral lines of dots. We're interested in them because they seem to carry more parasites than the tennessee snubs in particular, which are the most common darter in this system.

The Flint River At Winchester, Now THAT'S A Riffle

Five of us made it out to the Flint today to do our habitat survey and darter catch & release. The weather was almost mild at 50 deg. F, but with a steady north wind. The river was down from our last visit but water was still flowing fast. We caught a good number of fishes, especially banded, black and redline darters with a few rainbows and a single greenside. There was a very sharp distinction between where the bandeds and the blacks were found; if deeper, fast water over rock there were bandeds and some redlines, along the shore in less turbulent water were blacks and some redlines.

Here's our first transect line today, Jeremy and Robert on the left, Brian paying attention to the flow meter rod and Alex on the far distant shore. The river was about 38 m wide here.

Here are Robert, Jeremy and Brian carefully crossing the river at our fourth transect point, where the water is fast as it narrows down at the top of this riffle system. This was the most well-defined niche of all, with only bandeds and redlines in the area where everyone's walking, and blacks in the flooded grassy bank about 10 m behind them.
Another shot of the fourth transect, with the depth and flow measurements being made.
And, here's a view upstream from the first transect line to the bridge, with the fourth transect just this side of the bridge. If you look closely you can see whitecaps on the water surface showing breaking water in this riffle.

New Seine Is Ready To Go

I received a new seine from Memphis Net & Twine last week. It's 12 feet long, with a 1/16" mesh on it. That's fine enough so that we'll net surprisingly small fish, which is the desired effect. It will also be harder to pull up in fast water; we'll see how bad that is. And that might be on Wednesday - we'll got out to the Flint River and do our flow/depth/darter habitat survey. It hasn't rained for a few days, and shouldn't too much between now and then. Hopefully it will almost be easy...

Black Darter Discussion, From Unpublished Manuscript

This is a little odd, but I want to "publish" the Discussion from my manuscript on observing black darters, Etheostoma duryi, from two creeks near Huntsville, AL, several years ago. This is the manuscript that was accepted by Southeastern Naturalist but I had to withdraw it when the co-author disappeared with all of the original data so that I couldn't make modifications on our Results asked for by the editor. We collected and examined both male and female darters from two populations, preparing stained slides of cross-sections through their gonads to estimate size and describe reproductive status. Rachel, the other author, also counted eggs and described their developmental stage from female fish. Town Creek is in Athens, AL, and is an urban, somewhat eutrophic creek. Limestone Creek is in the NW corner of Madison County, AL, in a rural area with lower nutrient inputs. Nanostoma is the subgenus to which black darters belong. I'll omit listing References at the end of this blurb...

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Two populations of Black Darter from nearby creeks did not significantly differ in reproductive investment or in reproductive output. There were, however, significant differences in the total oocyte complement between the sites, best attributable to significant body size differences. Town Creek females were larger, and had attained this larger size going into the breeding season. The month of capture had a significant effect, the site of capture had a highly significant effect and the sex of the fish had a highly significant effect on the mean standard length. Town Creek fish had a greater mean standard length than Limestone Creek fish, and males were longer than females. In darter species the males grow faster and larger than females, so this result was expected (Page 1983).

The observed increase in standard length of Limestone Creek females (but not of Town Creek females) may indicate that any significant growth at Town Creek had occurred prior to the beginning of the study and resulted in larger females. One key difference for Town Creek females was that as standard length increased, the GSI decreased. This indicates that larger females at Town Creek expend less energy toward reproductive investment and more toward physical growth than do smaller females. Fuller (1998) demonstrated that E. caeruleum females in Michigan have a similar relationship between body growth and reproductive development.

Heins et al. (1992) found significant differences between the mature (MA) oocyte weight and the ripening (MR) oocyte weight. If females from Limestone Creek were slow in maturing, they could just be beginning MR development, during which oocytes can have varying sizes. If most of the MR oocytes from Limestone Creek are in the early ripening stage and most of the MR oocytes from Town Creek are in the later ripening stage, the result could be a significant difference in total body weight. Furthermore, if more Town Creek females have ripe, hydrated eggs than do Limestone Creek females, then the water from hydration could increase the overall body weight.

As could have been predicted by the results regarding standard length and weight, there was a highly significant trend for a positive increase in the ratio of weight/standard length in females from Limestone Creek, but no such trend existed for Town Creek females. This suggests that some Town Creek females may have spawned some ripe eggs prior to capture, thereby conferring less of an increase in the ratio than seen in Limestone Creek females. For males, there were no significant trends in the mean ratio of weight/standard length over the three months studied. Males appear to be at their optimum condition in January, prior to any significant female development.

The two populations showed similar patterns in the development of reproductive phenotype, with Town Creek individuals tending to develop earlier. At Town Creek, females were gravid in the February capture. Male breeding coloration began to stand out during the same time period. Limestone Creek females were beginning to become gravid in February, and males began exhibiting the orange breeding colors typical of Black Darters. In March, the abdomens of Town Creek females were greatly extended, and thus gravid, and male breeding colors brightened, with blue-green additions around the opercular/cheek area and at the caudal peduncle, and along the nape. The fin rays were a brick red, and had multiple striations. Similarly, in early April, Limestone Creek females were gravid, and male colors brightened.

While it appears that physical growth and development may have occurred more slowly at Limestone Creek than at Town Creek, it may be the case that fish at Limestone Creek just grow to a different size than fish at Town Creek. Both sites had females with ripe eggs in January, although the numbers were very small. Although the Limestone Creek sample in January had a smaller proportion of ripe (RE) females than the Town Creek samples, both sites had at least 50% RE females in February. Town Creek females were exhibiting the occasional atretic follicle as early as January and throughout the study. No atretic follicles were found in females from Limestone Creek, but this could have been due to chance of sampling.

During all months of capture, male samples from Town Creek were at an earlier stage of development than samples at Limestone Creek. However, sampling error could have influenced these results. Male darters tend to be ahead of females in terms of growth, and, thus, maturation (Page 1983). It is possible that errors occurred in differentiating Stage IV from Stage V in males. Stage V in described by Leino et al. (2005) as having numerous spermatids in the lumen of the testes. If some males were captured just after releasing their milt they may have been classified erroneously as being in Stage IV development.

There could be several different causes for the size difference between the two populations. One cause could have been the differences in water temperatures between the two sites. While not extremely different, Town Creek had higher water temperatures during the first two months of the study. In the third month of the study, Limestone Creek had a higher water temperature than Town Creek. However, this should not have affected the size of the fish, since most should have been at their optimum size by their March/April capture.

Another possibility for the size difference between the sites could be the food availability for larval development in each habitat. Limestone Creek had a greater depth and swifter current with little vegetation. Town Creek, however, was of shallow depth and a low current speed with heavy vegetation in the capture areas. Heavy vegetation would offer protection for the offspring of many forage species, including insect larvae and crustaceans, a staple in the diet of congeners such as E. simoterum (Page and Mayden 1981).

The total dissolved solids (TDS) could have affected the overall size of the fish in this study. Fallo and Warren (1982) studied another Nanostoma species, E. atripinne, in Kentucky, finding that they were in better condition and more successful in streams with high levels of TDS. Town Creek consistently had higher TDS readings than Limestone Creek.

Many studies have tried to elucidate the spawning season for various darters of the subgenus Nanostoma (e.g., E. coosae, O'Neil (1981); E. baileyi, Clayton (unpublished 1984); E. zonale, Hubbs (1985); E. rafinesque, Weddle and Burr (1991)). Considering the trends in the current study in mean oocyte and mean RE egg size and mass and trends in GSI, the spawning season for Black Darters likely begins in early-to-mid March, with the peak possibly occurring in April and, perhaps, into May. We urge that further research into the reproductive biology of other Etheostoma species be done with histological methods similar to ours to better clarify the seasonal patterns of gonadal growth and function in this speciose and ecologically important North American genus.

Fish & Wildlife Designates Critical Habitat For The Vermilion Darter

The U.S. Fish & Wildlife Service released its report in the Federal Register last Tuesday designating 21 km of critical habitat for the federally Endangered Vermilion Darter, Etheostoma chermocki. This species is only found in the Turkey Creek system in the northeastern suburbs of Birmingham, Alabama. This habitat designation was in response to a lawsuit brought by the Center for Biological Diversity in 2007. This designation doesn't prevent land owners from doing what they want with their property, but it makes it much more difficult for federal money to be used in the area in ways that might affect the creek and the darter, for instance by highway or sewer construction. So it does have teeth, but not what most people would expect.

I happened to be in that area of Pinson, AL, yesterday on other business so I took some photos of an upstream tributary to Dry Creek, which in turn is a tributary to Turkey Creek. This area has gone through rapid development as a suburb in the last 20 years, creating threats to the creek's biotic integrity including increased sediment run off, altered stream flow, and other physical factors making the creek warmer and more eutrophic in general. This area is at the southern terminus of a long, high ridge called Sand Mountain that runs northeast into Georgia.

So, here are two pictures I took of this area yesterday, about 3 km upstream of the critical habitat designation. The water is clear and flowing reasonably, not always the case at points downstream. The area is overrun with exotic privet and Japanese honeysuckle, symptomatic of profound land use changes.

Manuscript Submission To Southeastern Naturalist

I just submitted the gill parasites/telescope shiner manuscript to Southeastern Naturalist. It was easier than I anticipated to reformat it for SENA. The title is now, "Impact of a Gill Parasite Upon the Minnow, Notropis telescopus". As always, I hope they like it. But really, who wouldn't like a research article about any shiner species? Let's get real!

Abstract Of My Smithsonian Research Proposal

I submitted my research proposal to the Smithsonian Tropical Research Institute today for a two week visit in May. Below is my 250 word Abstract for this work:

Parasites are known to be important regulators of energy flow through ecosystems, and also to affect the fitness of individuals. Monogenean trematodes of the genera Dactylogyrus and Gyrodactylus that live in hosts’ gills or on the skin have been shown to be harmful to a variety of fishes under laboratory, aquaculture and natural conditions. In this weakened condition hosts may also have diminished reproductive capacity, measured as smaller functional gonads, lowered gamete production or diminished sexual coloration phenotype. My proposal is to begin a research project on the possible influence of monogenean trematodes on a livebearing Poeciliid fish species, Brachyrhaphis episcopi, endemic to Panama. Previous research on this species conducted at the STRI has shown the species to have life-history responses to the presence or absence of larger piscivorous fishes. Twelve known sites with B. episcopi would be visited and 40 fish collected at each. Fish collected would be examined for how many skin and gill parasites they carry, and would also be examined to determine reproductive condition such as number of eyed embryos carried by adult females as a measure of possible influence of parasite load. This would address two linked hypotheses: one, that higher parasite load is linked to, and possibly causative of, lowered reproductive capacity; and two, that higher parasite loads would be found in host populations that live in environments without predatory fishes such as large characins. Any monogenean parasites found would likely be new species because of the tight co-evolution of these parasites and hosts.

A No From Freshwater Biology, On To Southeastern Naturalist

I heard back from the Editor at Freshwater Biology today. They declined our manuscript on parasite infections of telescope shiners, basically as lacking broad enough interest. Oh well, that's the risk with doing taxon-specific research. So, I think the next move is to reformat it for Southeastern Naturalist and send it to them. Hopefully they'll like it.

Meanwhile, I've almost finished the research proposal I have to submit to the Smithsonian in Panama to be accepted as a visiting scientist this May. It's like writing a short research paper in its own right.

Cold, High Water At The Flint Today

We met at the farmers' market parking lot a little after 1 today to run the driftnets at the Flint, with Brian, Robert, Jeremy, Douglas and myself. It was a sunny, cold day, about 5 deg. C, with a steady north wind. The water was markedly higher and faster than last time. In the photo below you can see fresh debris in the tree branches at the upper left; that was from about 12 cm of rain a week ago that flooded local rivers. The shallow area along the bank in the photo has always been exposed on past trips. Luckily there were five of us today, because the current was so strong we had to hold the nets in place for an hour instead of relying on the zinc rods to anchor them. Water temperature was 7 deg. C, which doesn't sound so bad until you've been standing in it for over an hour in uninsulated waders.
The next photo is an awkward shot I took while holding onto one end of the net. The white poles running to the right are Robert's invention to try to stabilize the net by bracing the skinny zinc rods. They helped today because it's easier to hold on to them than the zinc rods, mostly by bracing one against my leg while standing in place. I don't have the exact figure on current flow, but the raw measurement was 447 propeller rotations in one minute, a pretty good clip. Lots of water went through the nets in the hour we held them in place. Water running around and through the net was very turbulent, as you can see.
Finally, here's a view of one our transects. The two nets were set up at two points on the left in this view. High flow has pretty thoroughly scoured the river of any leaves or other debris. And, you can see in the silhouette than I'm in the Australian hat my father gave me. Luckily a leather hat is pretty good at keeping out wind.

More Stripetails At Estill Fork Than We Thought

Robert spent the afternoon yesterday curating the fishes we collected on Saturday at Estill Fork. Close examination of the stripetails/fantails showed that 7 of 11 are stripetails rather than fantails, reversing our initial assessment. Two traits can be seen in preserved specimens to separate the species: the stripetails have a dark submarginal band on the first dorsal fin, and the striping pattern on the tail is both richer and darker. Also, the stripetails are lighter ventrally and don't have as pointed a head when observed from above. The challenge for us will be to quickly, accurately ID the species when pulling them out of a seine at both the Flint and Estill Fork. We've been kinda OK up to this point with IDs, now I think we can be much more accurate.

We'll be going to the Flint early Monday afternoon to run the driftnets since the new moon is Monday. The high temperature is predicted to be 47 F, so it will be a brisk experience standing around the river. At least the sun should be out.