Editor’s Note. This is the second installment of a three-part series on the discovery of a cryptic pleurocerid species in East Tennessee. You might want to back up and read my essay of 13Sept16 if you haven’t already. The present essay was published in the FMCS Newsletter Ellipsaria 18(2): 16 - 18 [pdf] if you’re looking for something citable.
So when last we left our intrepid malacologist, puzzling at his lab bench in the summer of 2008, he had come around to the realization that the population of Pleurocera simplex inhabiting Pistol Creek at the Courthouse Park in Maryville, Tennessee, was actually two reproductively isolated populations, fats (S6f) and skinnies (S6s). Which population might be the bona fide P. simplex of Thomas Say (1825)? And what might be the identity of the other?
I actually started out with a pretty good hunch. In 2007 John Robinson and I ran allozyme gels on five populations of P. simplex from up in Virginia, including a nice sample from Say’s type locality in Saltville . The Saltville population (S5) was fixed for the Oldh100 allele that turned out to be diagnostic for the Maryville S6f fats. And interestingly enough, the simplex population John and I sampled from Indian Creek at Kesterson Mill way down at the southwest tip of Virginia (S1) was nearly fixed for that Oldh104 allele diagnostic for the Maryville S6s skinnies. I hadn’t noticed any shell morphological differences between the Saltville simplex and the Kesterson Mill population at the time, however.
So in August of 2008  I drove up to Virginia for additional samples from Saltville and from Kesterson Mill, which I returned to Charleston to run alongside a fresh batch of Maryville samples, to make sure all the bands matched up. Sure enough, the Maryville S6f fats were indeed genetically similar to P. simplex collected from its type locality at Saltville S5, not just at the Oldh locus but over all ten of the allozyme loci I have found informative for studies of this sort. And the Maryville S6s skinnies were similar to Kesterson Mill S1.
|Figure 1. See footnote  for methodological details.|
I also took the calipers to a bunch of the shells from Saltville and Kesterson Mill. And sure enough, exactly the same pattern reappeared that we first noticed at Maryville. Dividing the total shell length into body whorl height (B) and apex height (everything else, A), the (N = 37) shells I sampled from Kesterson Mill showed a significantly greater ratio of A to B than the (N = 40) shells from Saltville. Figure 2 shows that the simple regression of apex height on body whorl height for the Saltville type population S5 was A = 0.157B + 2.46 (r = 0.36), very significantly different (ANCOVA t = -9.52, p < 0.0001) from the regression for the Kesterson Mill population S1, A = 0.556B - 0.09 (r = 0.84). Saltville snails are fat, and Kesterson Mill snails are skinny.
I have picked two shells directly off the regression lines to illustrate the difference between the two species. So since the Maryville S6f fats match that lower-sloping S5 sample of N = 40 from Saltville both genetically and morphologically, and since Saltville is the type locality of Pleurocera simplex (Say 1825), clearly the Maryville S6f fats must be the bona fide simplex .
Then what might be the identity of the Maryville S6s skinnies, matching the higher-sloping S1 population from Kesterson Mill both genetically and morphologically? Tune in next time!
 Dillon, R. T., Jr. and J. D. Robinson. 2007. The Goniobasis ("Elimia") of southwest Virginia, I. Population genetic survey. Report to the Virginia Division of Game and Inland Fisheries. 25 pp. [pdf]
 Yes, this was the same trip I featured last month, in which I resampled Maryville. I’ve been sandbagging my story a little bit, to make it unfold in a more linear fashion. To tell you the truth, I got the “hunch” mentioned in this month’s essay pretty much immediately upon reading my first Maryville simplex gel, and last month’s story unfolded pretty much simultaneously with this month’s story.
 Subsamples of 14 individuals from population S5 and 41 from population S1 were analyzed electrophoretically together with the 20f + 51s individuals from Pistol Creek I featured in last month’s blog post. These data were combined with the data sets of N = 34 from population S5 and N = 37 from population S1 previously published by Dillon & Robinson (2007), and with the N = 17f + 13s data I had previously accumulated from Maryville. Then BIOSYS version 1.7 (Swofford & Selander, 1981) was used to calculate matrices of Nei's (1978) unbiased genetic identity (below the diagonal in Fig 1) and Cavalli-Sforza and Edwards (1967) chord distances between all pairs of control populations S1 and S5 and the two cryptic S6 populations co-occurring in Maryville. Chord distances were used as the basis for the neighbor-joining tree shown above the diagonal in Figure 1, as calculated using PHYLIP v3.65 program NEIGHBOR (Felsenstein, 2004).
 Ultimately I only used that sample of 20 + 17 = 37 fats as my S6 Maryville control in Dillon 2011. The existence of a second Pleurocera population at Maryville cryptic under simplex was not mentioned in my 2011 paper at all:
- Dillon, R. T., Jr. 2011. Robust shell phenotype is a local response to stream size in the genus Pleurocera (Rafinesque 1818). Malacologia 53:265-277. [pdf]