BY ANNELIESE MOLL
For the UAS Whalesong

In 2011 a study involving several groups of researchers from the United States, Canada, Germany, and Mexico who had been working on a study replicating ecological speciation the Atlantic molly (Poecilia Mexicana) and in P. sulphuraria (Poeciliidae) was published in the journal of Molecular Biology and Evolution.

These fish usually live within freshwater or brackish waters, but they have also been colonized several sulfidic springs in southern Mexico. These springs are particularly relevant because they are not just slightly sulfidic. They are extremely hypoxic (lacking oxygen) and have high concentrations of hydrogen sulfide, enough to be lethal to most organisms.

In order to identify speciation within the mollies who are able to inhabit these toxic springs researchers used several different methods of analysis. This meant a phylogenetic analysis to test whether springs were independently colonized, then a phenotypic assessment of body and gill morphology variation to identify convergent patterns of trait differentiation, and then an eco-toxicological experiment to detect differences in sulfide tolerances among ecotypes.

To explain the relationships within the populations within the toxic springs, genomic DNA was extracted from fins which had been preserved in ethanol. From that DNA researchers were specifically looking for the mitochondrial cytochrome b gene. They also had a subset looking at the mitochondrial DNA gene NADH subunit 2.  When it came to data analysis they chose three different approaches: only the cyt b gene, only the ND2 gene, and then cyt b and ND2 together.

The second way that researchers looked the differences in the sulfidic and nonsulfidic populations by quantifying phenotypic by performing a geometric morphometric analysis.  This means that they took lateral radiographs of both populations. Those were then digitized and 13 landmarks points were placed on every image. In addition to the photos, eye diameter was measured and a weight matrix was created. Then to test for variation in body shape between populations they used several statistical tests which consisted of a multivariate analysis of covariance, or MANCOVA, is used in cases where there is more than one dependent variable and control of naturally associated continuous independent variables is required. A mixed-model nested analysis of covariance was also conducted because in the MANCOVA fixed effectes can often inflate error rates if nested terms are significant.

In order to look at the morphology of the gills, they measured the total gill filament length and used that as an index for oxygen uptake. This approach was chosen rather than gill surface area because it’s more likely to reflect spatial restrictions of large gills. To actually quantify total gill filament length they measured the four gill arches. The filament lengths were summed for the 8 hemibranchs and then multiplied by two in order to arrive at an estimated total gill filament length.

Finally, their tolerance to sulfide was tested by subjecting wild-caught fish from the different populations to various concentrations of sulfide. During this part, the wild fish were brought back to a lab and over the course of 24 hours slowly acclimated to water that did not contain any sulfide. Once the fish were ready to slowly be exposed to levels of sulfide, sulfidic water was obtained from one of the springs that is known to have high concentrations. Each fish was placed into a 100 mL container and then 10 mL of the sulfidic water was added at two minute intervals. During this time fish were monitored and the time of their loss of motion control was noted and they were removed so that length and weight could be recorded. They were then placed into a heavily aerated container in order to recover. From this experiment researchers used the model with the highest support retained sulfide, sex, mass, and the interaction between drainage and sulfide in the final analysis.

Now, the moment you’ve been waiting for: the results. From the first portion of the experiment where researchers conducted a phylogenetic analysis. That analysis indicated that their samples of P. mexicana and P. sulphuraria formed a monophyletic group, and within that group there were three highly significant lineages. They also found that their data suggested that P. sulphuraria evolved independently from the P. m. mexicana sulfide spring populations. However, the data did tell much about the relationships between the P. m. mexicana mollies, which includes the    sulfur spring haplotypes. Ultimately, from this part of the experiment they found that their data suggests at least three independent colonizations of sulfidic springs by mollies.

From the the second portion, where morphology was examined, researchers found that body shape different strongly between nonsulfific and sulfidic populations. Between sexes, the position of the anal fin was different. They also found that total gill filament length was strongly correlated with body mass. It was also found that fish from sulfidic springs had larger gills than those from the freshwater streams. Males were also noted to have smaller gills than females.

In their third test regarding tolerance to sulfide was strongly dependent on the body mass. Smaller fish were found to have a higher tolerance to sulfide and that males would generally lose motor function sooner than females.

Ultimately, this study and others similar to it present interesting insights into evolution. In this case the sulfide tolerant fish may be able provide an ideal model system to eventually revealing the underlying adaptions and speciation.

In the references below the paper is cited, and I would definitely recommend taking a look at it. There are many highly informative figures and graphs that could not be included within this article. This study is also being continued by other researchers currently. Just last month a paper was published with a heavier focus on the genetic differences (also an interesting paper).

References

Tobler, M., Palacios, M., Chapman, L. J., Mitrofanov, I., Bierbach, D., Plath, M., … & Mateos, M. (2011). Evolution in extreme environments: replicated phenotypic differentiation in livebearing fish inhabiting sulfidic springs. Evolution, 65(8), 2213-2228.