Dr. Rob Dillon, Coordinator

Thursday, June 9, 2022

Cytoplasmic Male Sterility in Physa!

Three cheers for our good friend Dr. Patrice David of the CNRS-CEFE, who together with a team from the Universities of Montpellier and Lyon, has opened a new window into the mysteries of sex allocation in pulmonate snails, casting fresh light on mitochondrial evolution across the entire animal kingdom in the process.  Our colleagues from France have discovered the first case of cytoplasmic male sterility ever documented outside the vascular plants [1].  And they have done so in the most underappreciated experimental model of our time, the freshwater gastropod Physa acuta.

Male-fertile & sterile [3]
Genetic phenomena now understood to result from cytoplasmic male sterility were reported in plants as early as 1884 [2]. Many wild populations of the thistle Cirsium oleraceum, for example, are composed of mixtures of (normal) monoecious plants producing monoecious offspring and (male-sterile) female plants producing only female offspring, although always pollinated from monoecious plants. In a series of papers published between 1916 and 1937, the German geneticist Carl Correns demonstrated that the “female C. oleraceum plants contained in their cytoplasms some properties which inhibited the development of male floral parts.”  And in 1939, the obscure Argentinian geneticist E. Gini reported controlled crosses conclusively demonstrating cytoplasmic male sterility in certain varieties of maize, which might, however, be modified by a nuclear gene or genes segregating in the male parent.

Thistles are ordinary dicots, in the sense that their flowers bear both stigma and anther, both pollen and ova.  Maize is a monocot, and among the just 5% of all angiosperms with separate male and female inflorescences; the pollen being produced by the tassel on top, the ovum in the cob below.  What unites maize and thistles is that at least sometimes both demonstrate a mating system called “gynodioecy,”  where both hermaphroditic and female individuals co-exist in a single population.

Between 1940 and 1970, cytoplasmic male sterility was discovered in hundreds of species of (at least occasionally) gynodioecious plants, including many important as crops.  Nuclear genes that moderated the effects of CMS were discovered as well.  In the hands of plant breeders, CMS became a powerful tool to promote hybridization in normally self-pollinating varieties of maize, wheat, rice, carrots, onions, and beets.  And as the beet fields blossomed forth, so too did funding for research into CMS.

By the mid-1980s it was clear that CMS was caused by mitochondrial gene alteration.  By the mid-1990s sterility had been pegged to a variety of genes variably-inserted into otherwise normal plant mitochondria, encoding proteins that interfered with pollen formation [3].  And by the mid-2000s, the sequence data were rolling in.  It materialized that the plant mitochondrial genome is strikingly different from animal mtDNA in its evolutionary dynamics: variably sized, highly repetitive, and characterized by frequent gain, loss, duplication and rearrangement of genes [4].

Now, back to France.  You have heard me sing the praises of Dr. Philippe Jarne, his (1993-96) student Dr. Patrice David, and a bouquet of Montpellier friends and colleagues in many previous essays posted on this blog.  Most recently we featured their collaboration with a host of researchers from across the Americas to work out the evolution of the lymnaeid subgenus Galba, the crappy little amphibious snails serving as intermediate hosts for the sheep liver fluke Fasciola worldwide [5].  Philippe and Patrice also have research interests in Physa, primarily as a model for the study of sex allocation, extending all the way back to 1995.  See note [6] below for a selection of my favorite Physa papers published by Philippe, Patrice, and their colleagues over the years.
Graphical abstract from David et al. [1]
That said.  A phenomenon as startling as cytoplasmic male sterility in little brown trash snails is not something one can design an experiment to discover.  Patrice tells me that the breakthrough was initially made by Dr. Emilien Luquet of the University of Lyon, who has adopted Physa acuta as a model for the study of phenotypic plasticity.  Routinely sequencing the CO1 gene from a sample of Physa collected in a backwater of the Rhone River east of Lyon, Dr. Luquet obtained a couple sequences that were, to use the precise, technical terminology especially developed in Lyon to describe the phenomenon, “weird” [7].

Dr. Luquet, together with a team of four colleagues from the University of Lyon, (Plénet, Lefébure, Konecny and Deglétagne) then developed and characterized 34 isofemale lines of Physa from the Rhone River backwaters, 29 with normal mitochondrial genotypes and 5 weird.  The weird lines bore mitochondria with a median CO1 sequence divergence more than 20% different from the other members of their population, or indeed, any of the hundreds of Physa sequences in GenBank.  Sequences of the mitochondrial 16S gene returned the same remarkable result.

At this point Dr. Luquet sent samples to our mutual friend Patrice, saying “I apparently have Physa that came from outer space, look at them and tell me if this is really Physa acuta.”  Patrice, aware that here in North America we have several species of Physa that are indeed rather cryptic, began a series of experimental crosses with an albino line of P. acuta developed in the CNRS-CEFE laboratory.

When mounted by albino controls mating in the male role, the weird lines of Physa outcrossed readily, and actually showed some evidence of fecundity improved over the normal lines from Lyon.  But it materialized that the weird lines were male-sterile.  They showed much-reduced behavioral tendency to mount partners in the male role, their seminal vesicles apparently shrunken, containing very little sperm.  They could not self-fertilize.  This is the first demonstration of cytoplasmic male sterility in the animal kingdom

… but Amy Wethington and I had a shot at it 20 years ago.

Amy and I started working with Physa when she was an undergraduate student at the College of Charleston in the late 1980s.  And our first samples came from a pond in the state park just around the corner from my house, Charles Towne Landing.  That population of Physa became a standard for 20 papers published over the 20 years that followed [8].  We called it population C.

Amy at Charles Towne Landing

Almost immediately, Amy and I became interested in the evolutionary relationships between our population C, which we initially identified as “Physa heterostropha pomilia,” and all the other populations of trash Physa worldwide, identified with a wide variety of other names.   And my readership with admirably long memory may recall that it was from Philippe Jarne back in 2000 that Amy and I received the batch of French Physa acuta we used in our experiments demonstrating no reproductive isolation between population C and five other populations of Physa that had been known by three different names: Physa acuta, Physa heterostropha, and Physa integra [9].

So given that all six of these populations of Physa from four states and two European countries were conspecific, Amy and I were next curious to estimate the amount of genetic divergence among them.  A CofC undergraduate named Matt Rhett and I ran the allozyme gels [10], and Amy (now in the PhD program at the University of Alabama) sequenced two mitochondrial genes (CO1 and 16S) for approximately 20 snails from each of those six populations: C = Charleston, P = Philadelphia, D = Douglas Lake, Michigan, N = New Harmony, Indiana, F = France, I = Ireland.  The gigantic 16s+CO1 neighbor-joining gene tree that resulted is shown below.

Let’s look at that tree from the bottom up.  At the bottom you see two other good biological species of Physa: three individual Physa gyrina, and three individual Physa pomilia, which we were calling Physa hendersoni at that time [11].  The gigantic middle branch of the tree shows all six of our Physa acuta populations all mixed up: C, P, D, N, F, and I, with a little bit of geographic structure but not much.  Now what the heck is that branch sticking way out at the top?

That top branch, labelled C10, C13, C15 and C18, shows four snails from Charles Town Landing bearing mitochondria with sequences approximately 30% different from all other Physa acuta from four states and two European countries.  Amy called those sequences “whacky.”

So in 2004 Amy drafted a manuscript by Wethington, Rhett, and Dillon to report all our allozyme data and all our sequence data across all six Physa populations.  Meanwhile, I recruited another undergraduate student, Nick DiNitto, and together Nick and I went back to the pond at Charles Town Landing, collected 26 adult Physa, and started isofemale lines.  Once the original mothers had laid eggs, our plan was to enlist the aid of my colleague Bob Frankis to sequence their CO1 genes, hoping to found pure whacky [12] cultures for breeding experiments.

From FWGNA Circular #5 [pdf]

But alas.  Working with Bob, Nick was able to sequence just six of the original mothers before the semester came to a close and the project foundered.  None of those six bore whacky mitotypes [12].  Nick’s poster for the 2004 meeting of the American Malacological Society in Sanibel, Florida, reporting just N=6 unremarkable CO1 sequences, is available for download from note [13] below.

Meanwhile Amy was having no success whatsoever interesting journal editors in publishing the Wethington, Rhett & Dillon manuscript.  In the eyes of reviewers, the whacky result depicted at the top of our Figure 1 invalidated the entire paper.  This must be some sort of lab accident!  Or cryptic speciation?  Either Amy’s technique was hopelessly sloppy, or our premise was hopelessly wrong.  Amy ultimately gave up trying to publish the separate paper, cut the four whacky sequences, and folded the rest of the data into her 2004 dissertation [14].  Her amazing discovery was forgotten.

Until today.  Today I am making the original 2004 manuscript of A. R. Wethington, J. M. Rhett and R. T. Dillon, entitled “Allozyme, 16S, and CO1 sequence divergence among populations of the cosmopolitan freshwater snail, Physa acuta” available for download as FWGNA Circular #5, here: 

-  Wethington, Rhett & Dillon (2022)  -

And perhaps I should have written “almost forgotten” two paragraphs above.  For in footnote (3) of my [15Mar16] essay on mitochondrial superheterogeneity, I made passing reference to “a striking case of mtSH (both COI and 16S) in a Charleston population of Physa acuta.”  Which leads me to offer two final hypotheses, ask two final questions, and make an appeal.

First hypothesis.  It seems quite likely to me that mtSH = CMS in pulmonate snails broadly.  The first report of the phenomenon I subsequently dubbed “mitochondrial superheterogeneity” came in the 1996 paper of Thomaz and colleagues, working with the land snail Cepaea nemoralis [15].  Cases of mtSH have been reported at least three additional times in the land snails that I know of, and one other time in a freshwater pulmonate, the limpet Laevapex fuscus.  See footnotes (2) and (3) of my [15Mar16] essay.

Second hypothesis. It seems possible that CMS -> DUI in bivalves.  Doubly-uniparental inheritance of mitochondria was first reported in the marine mussel Mytilus in the early-1990s and has subsequently been documented in a wide variety of bivalves, including the unionid mussels of American freshwaters.  As the name implies, researchers have found that mitochondria can be passed by sperm as well as by egg in these bivalve groups.  And most intriguingly, the genomes of the male mitochondria and the female mitochondria are always found to be strikingly divergent.  Sophie Breton of the University of Quebec and a team of bivalve researchers including our friend Randy Hoeh have just very recently published a nice paper making this argument in compelling fashion [16].

First question.  Could CMS -> MtSH in pleurocerids and other prosobranch snails?  That is a stretch I am not intellectually limber enough to make.  Sexes are separate in the pleurocerids, as in almost all other prosobranch groups, mechanism of sex determination unknown.  If you wade out into the creek, fish up a nice sample of adult pleurocerids and crack them open, you will almost always find sex ratios significantly biased toward the female [17].  This is a secondary sex ratio, of course.  I don’t think anybody knows the primary sex ratio for any prosobranch population.

I would love to hypothesize that the mitochondrial superheterogeneity so often demonstrated by freshwater prosobranch populations – ten cases listed in footnotes (4), (5), (6), and (7) in my essay of [15Mar16] alone – might be related to such sex ratio biases.  But Whelan & Strong [18] did not find any relationship between sex and mt haplotype in their superheterogeneous populations of Leptoxis from Alabama, and I don’t know where else to look for evidence.

And second question.  What is the origin of CMS in the Physa population of the Rhone River?  Patrice and colleagues ultimately sequenced the weird mitochondrial genome in its entirety, documenting extreme divergence everywhere they looked, summing to an eye-popping 44.4% median deviation from the normal mitotype.  Yet no divergence in the nuclear genome was apparent.  Sequencing the (nuclear) 28S gene returned no significant difference across their entire sample of 34 lines, nor were any differences in microsatellite gene frequencies apparent.  The Mendelian genetics confirm that the Rhone River at Lyon is inhabited by a single randomly interbreeding population of Physa acuta.

Which brings us to my appeal.  To all of our colleagues who persist in defining species with mtDNA gene trees, please stop.  You’re embarrassing us.

Patrice went on to offer several compelling lines of evidence suggesting that the weird mitotype results from an acceleration of mutation rate, perhaps due to some defect in the mitochondrial DNA repair mechanism, rather than selection on mitochondrial functions more broadly.  He further observed that high mutation rates might both produce male-sterility genes, and help them persist over evolutionary time, counter to the evolution of nuclear genes restoring male fertility.  There are all sorts of selfish-DNA-type directions one could go from here.

And so, to conclude.  None of the thoughts, questions, hypotheses and speculations outlined above could have flickered through my brain before the marvelous paper by Patrice David and his colleagues appeared last month.  Job well done, all of you!  La reussite scientifique de la France est intimement lie au bonheur de l’Amerique [19].


[1] David, Patrice, Cyril Degletagne, Nathanaëlle Saclier, Aurel Jennan, Philippe Jarne, Sandrine Plénet, Lara Konecny, Clémentine François, Laurent Guéguen, Noéline Garcia, Tristan Lefébure, Emilien Luquet (2022) Extreme mitochondrial DNA divergence underlies genetic conflict over sex determination.  Current Biology 32: 2325-2333.  https://doi.org/10.1016/j.cub.2022.04.014.

[2] The historical review in this paragraph is extracted from:

  • Edwardson, J.R. (1956) Cytoplasmic male-sterility.  Botanical Review 22: 696-738.
  • Edwardson, J.R. (1970) Cytoplasmic male-sterility.  Botanical Review 36: 341 – 420.

[3] Schnable, P.S., and R.P. Wise (1998)  The molecular basis of cytoplasmic male sterility and fertility restoration.  Trends in Plant Science 3: 175 – 180.

[4] Galtier, N. (2011) The intriguing evolutionary dynamics of plant mitochondrial DNA. BMC Biol. 9, 61. http://www.biomedcentral.com/1741-7007/9/64

[5] Alda, Pilar, M. Lounnas, A.Vázquez, R. Ayaqui, M. Calvopiña, M. Celi-Erazo, R.T. Dillon Jr., L. González Ramírez,  E. Loker, J. Muzzio-Aroca, A. Nárvaez, O. Noya, A. Pereira, L. Robles, R. Rodríguez-Hidalgo, N. Uribe, P. David, P. Jarne, J-P. Pointier, & S. Hurtrez-Boussès (2021) Systematics and geographical distribution of Galba species, a group of cryptic and world-wide freshwater snails.  Molecular Phylogenetics and Evolution 157: 107035. [pdf] [html].  For a review, see:

  • The American Galba and the French Connection [7June21]
  • The American Galba: Sex, Wrecks, and Multiplex [22June21]
  • Exactly 3ish American Galba [6July21]
  • What Lymnaea (Galba) schirazensis is not, might be, and most certainly is [3Aug21]

[6] Here are a few of my favorite Physa papers from Philippe and colleagues:

  • Jarne, P., M-A Perdieu, A-F Pernot, B. Delay, and P. David (2000)  The influence of self-fertilization and grouping on fitness attributes in the freshwater snail Physa acuta: population and individual inbreeding depression. J. Evol. Biol. 13:645-655.
  • Bousset, L., P-Y. Henry, P. Sourrouille, & P. Jarne (2004)  Population biology of the invasive freshwater snail Physa acuta approached through genetic markers, ecological characterization and demography. Molec. Ecol., 13: 2023-2036.
  • Tsitrone A, Jarne P, David P: Delayed selfing and resource reallocations in relation to mate availability in the freshwater snail Physa acuta. Amer Natur 2003, 162:474-488.
  • Henry PY, Bousset L, Sourrouille P, Jarne P: Partial selfing, ecological disturbance and reproductive assurance in an invasive freshwater snail. Heredity 2005, 95:428-436
  • Noel, E., Chemtob, Y., Janicke, T., Sarda, V., Pelissie, B., Jarne, P., and David, P. (2016). Reduced mate availability leads to evolution of self-fertilization and purging of inbreeding depression in a hermaphrodite. Evolution 70, 625–640.

[7] Quoting Patrice, “Initially we called them W for weird; we later opted for D as in Divergent which sounds more serious and doesn't induce confusion with W chromosomes.”

[8] I have reviewed the many years of fruitful collaboration that Amy and I enjoyed in quite a few essays previously posted on this blog.  Here are my favorites:

  • To identify a Physa, 1989 [3Oct18]
  • Albinism and sex allocation in Physa [5Nov18]
  • To identify a Physa, 2000 [6Dec18]
  • TRUE CONFESSIONS: I described a new species [7Apr10]
  • What is a Species Tree? [12July11]

[9] Dillon, R. T., A. R. Wethington, J. M. Rhett and T. P. Smith (2002) Populations of the European freshwater pulmonate Physa acuta are not reproductively isolated from American Physa heterostropha or Physa integra.  Invertebrate Biology 121: 226-234.  [pdf]

[10]  I know that an allozyme survey sounds hopelessly outdated.  A postcard from 1982.  But it was allozyme markers that allowed us to do all our breeding studies.  We could not have verified outcrosses without baseline allozyme surveys such as my students and I labored over for years.  There are a lot of research groups who wish they had that hopelessly-outdated 1982 technology today.

[11] Dillon, R. T., J. D. Robinson, and A. R. Wethington (2007)  Empirical estimates of reproductive isolation among the freshwater pulmonates Physa acuta, P. pomilia, and P. hendersoni.  Malacologia 49: 283 - 292.  [pdf]

[12] Actually, Nick preferred to call the weird sequences “zany” rather than “whacky.”  So that’s what we called them on his poster…

[13] DeNitto, N. W., R. C. Frankis and R. T. Dillon (2004) Extensive mitochondrial CO1 sequence diversity in a population of the freshwater snail, Physa: Admixture or cryptic speciation?  American Malacological Society, Sanibel Island, FL. [Poster, pdf]

[14] Amy’s dissertation was ultimately published as: Wethington, A.R., & C. Lydeard (2007) A molecular phylogeny of Physidae (Gastropoda: Basommatophora) based on mitochondrial DNA sequences. Journal of Molluscan Studies 73: 241 - 257. [pdf]  For a review, see:

  • The classification of the Physidae [12Oct07]

[15] Thomaz, D., Guiller, A. & Clarke, B. (1996) Extreme divergence of mitochondrial DNA within species of pulmonate land snails. Proc. of the Royal Society of London B: Biological Sciences, 263, 363–368.

[16] Breton, Sophie, Donald T. Stewart, Julie Brémaud, Justin C. Havird, Chase H. Smith, and Walter R. Hoeh (2022)  Did doubly uniparental inheritance (DUI) of mtDNA originate as a cytoplasmic male sterility (CMS) system?  Bioessays 44: 2100283. https://doi.org/10.1002/bies.202100283

[17] Cipiaris, S., W. F. Henley and J.R. Voshell (2012)  Population sex ratios of pleurocerid snails (Leptoxis spp.): Variability and relationships with environmental contaminants and conditions.   Amer. Malac. Bull. 30: 287 - 298.

[18] Whelan, N.V. & E. E. Strong (2016)  Morphology, molecules and taxonomy: extreme incongruence in pleurocerids (Gastropoda, Cerithiodea, Pleuroceridae). Zoologica Scripta 45: 62 – 87.  For a review, see:

  • Mitochondrial superheterogeneity: What we know [15Mar16]
  • Mitochondrial superheterogeneity: What it means [6Apr16]
  • Mitochondrial superheterogeneity and speciation [3May16]

[19] The scientific success of France is closely tied to the good fortune of America.  I offer this benediction as an echo of the Marquis de Lafayette quote with which I ended my essay of [7June21].

Monday, May 16, 2022

Freshwater Gastropods of the Tennessee/Cumberland

Today we are pleased to announce the expansion of our FWGTN coverage from its East Tennessee origins though the entirety of the Tennessee and Cumberland River drainage basins, increasing our sampling area from approximately 22,000 square miles to over 58,000.  We document 54 species of freshwater gastropods with 16 additional subspecies in this malacologically rich region, offering ecological and systematic notes for each, as well as detailed distribution maps, a dichotomous key and a photo gallery.  This expanded web resource, coauthored by R.T. Dillon, M. Kohl and R. Winters, is available here:

The Tennessee/Cumberland

The previous version of our FWGTN website, brought online in 2011 by Dillon & Kohl, covered only the Tennessee River drainage system from SW Virginia and western North Carolina through East Tennessee to skim the top of North Georgia and stop at the Alabama border.  Our 2011 database included 1,674 records from approximately 767 discrete sites, documenting 39 species and 2 subspecies.  The expanded database we release today includes 4,003 records from approximately 1,700 discrete sites, ranging though North Alabama and Middle Tennessee to clip the corner of NE Mississippi, plus a big slice across southern Kentucky as well.

Click for larger

Among many interesting findings, we report here that three pleurocerid species previously thought restricted to East Tennessee range significantly further west: Pleurocera simplex (with its subspecies ebenum), Pleurocera troostiana (with subspecies perstriata, edgariana, and lyonii) and Pleurocera clavaeformis (subspecies unciale).  We have also discovered that Pleurocera semicarinata, previously unknown further south than Kentucky, ranges through Cumberland drainages well into Tennessee.  The distributions of several hydrobioid species are also clarified and expanded – more about this in coming months.

Our complete FWGNA database, covering the drainages of The Ohio as well as Atlantic drainages from Georgia to the New York line, now contains 22,044 records documenting 107 species of freshwater gastropods, with 21 subspecies.  We have updated our overall website with a new continent-scale biogeographic analysis, dividing records into North Atlantic, South Atlantic, Ohio, and Tennessee/Cumberland subsets.  Our analysis suggests that natural selection has been more important in the evolution of freshwater pulmonates than gene flow restriction, but that gene flow restriction has been more important in the evolution of freshwater prosobranchs than natural selection.

We also announce today the publication of an updated “Synthesis v3.1,” ordering our 107 species by their incidence in our continental database and assigning fresh FWGNA incidence ranks to all.

So, visit the FWGNA web resource again, for the first time!

Tuesday, April 5, 2022

The ham, the cheese, and Lithasia jayana

Editor’s Note – This is the fifth and final installment of my series on the population genetics and systematic relationships of the Duck River Lithasia.  If you are interested in the science, you probably ought to review my posts of 7Dec21, 4Jan22, 3Mar22, and 28Mar22 before proceeding.  Or you could simply enjoy this essay for what it is, a true tale from the wild west days of American malacology.

“Well, Ahlstedt, is this jayana or is it not?”  That was the punchline of a story told me by my major advisor, Dr. George Davis, shortly after I arrived at the Philadelphia Academy of Natural Sciences in the summer of 1977.  Why was that question so weighty?  And why have I remembered the saga from which it springs over 40 years now, to pass it along to future generations of malacologists?

Previously, on the FWGNA blog!  In December and January, we obsessed at great length over Lithasia geniculata, with its three subspecies, extending down the 275 mile length of the Duck River of Middle Tennessee, bearing smooth, oblong shells in the headwaters, developing robust, bumpy shoulders in the lower reaches. In his seminal (1940) monograph [1], Calvin Goodrich also identified a second species of Lithasia in the Duck, bearing a more acute apex and angled (sometimes even tuberculate) shell, extending only from about river mile 186 to the mouth.  These he identified as Lithasia duttoniana.

This month’s episode begins in Washington, walking the hallowed halls of Congress.  In 1969, just before the dawn of the environmental movement, federal funding was secured for the construction of two new dams on the Duck River: the smallish Normandy Dam at RM 248, and a larger and more expensive Columbia Dam, downstream around RM 145.  The Normandy Dam was completed in 1976.

Normandy Dam (TVA)

But the National Environmental Policy Act went into effect January 1, 1970, the Clean Water Act in 1972, the and the Endangered Species Act in 1973.  The TVA found itself required to file an environmental impact statement for the Columbia Dam, even as construction proceeded apace.  And Dr. George M. Davis, fresh out of the Army and sitting in the Pilsbry Chair of Malacology at the ANSP, was awarded a contract to survey the Duck for potentially-endangered pleurocerid snails.

George Davis’ pleurocerid taxonomy was idiosyncratic.  He began by noticing that in almost all aspects of their biology, including body size, life habit, and shell morphology, pleurocerid populations of the Haldeman's 1840 genus Lithasia are not strikingly different from Lea's monotypic genus Io of 1831. Davis therefore synonymized Lithasia under Io and recognized five taxa in the Duck River: Io geniculata geniculata, Io geniculata pinguis, Io salebrosa, Io armigera duttoniana and Io armigera jayana.  The identities of Davis’ Io geniculata geniculata, Io geniculata pinguis, and Io armigera duttoniana will by now be obvious to my readership.  “Io salebrosa” was Davis’ name for what Goodrich would have called Lithasia geniculata fuliginosa.  What was “Io armigera jayana?”

The idea that a pleurocerid population matching Isaac Lea’s nomen “jayana” in the Duck River was especially controversial when Davis filed his report in 1974 [2].   Isaac Lea [3] published a brief, Latinate description of “Melaniajayana in July [4] of 1841: “Hab. Cany Fork, DeKalb Co., Tenn. – Dr. Troost [5].”  In his more complete English description of 1846, Lea emphasized “It very closely resembles the M. armigera (Say), in most of its characters, but may at once be distinguished by the double row of tubercles, the armigera never possessing distinctly more than one row [6].”

Lea’s selection of Mr. Say’s Melania armigera as a point of comparison is especially significant.  Described by Thomas Say in 1821 from “The Ohio River” [7], the range of populations today identified as Lithasia armigera extends through much of the lower Cumberland and Tennessee Rivers as well [1], including the Tennessee at the mouth of the Duck.  And the resemblance between Mr. Lea’s jayana and Mr. Say’s armigera is indeed close, as shown in the montage of Tryon’s [8] figures below.

Two L. armigera left, two L. jayana right [8]
Goodrich recognized neither armigera nor jayana in the Duck River.  Goodrich identified all the Lithasia bearing acutely-spired shells with tubercles or spines inhabiting the Duck River as L. duttoniana, a species Lea described in brief Latinate form about five months prior [4] to his description of M. jayana: “Hab. Waters of Tennessee, Dr. Troost.  Duck River, Maury Co. Tenn., Mr. Dutton [9].”  Lea never compared his M. duttoniana to any other species of what we would identify today as a Lithasia: armigera, jayana or anything else.

The only population of L. jayana that Goodrich recognized in his 1940 monograph was the Caney Fork type population, and that population seems to have been extincted by the closing of the Center Hill Lake dam and associated development in 1948.  So if Davis’ Duck River record of “Io armigera jayana” was to be believed, the last remaining population of a genuinely endangered species would be smack in the middle of the Duck River where the Columbia Dam was even at that time being constructed.

The scene now shifts to the sleepy little East Tennessee company town of Norris, were in 1974 the TVA hired a promising young biologist named Steven A. Ahlstedt to work in its Department of Forestry, Fisheries, and Wildlife Development [10].  Steve is an excellent scientist, with superb field skills and a great eye for freshwater mollusks.  He had a reprint of Tryon’s monograph on his shelf, and all of Goodrich’s papers in the top drawer of his filing cabinet, but very little of Goodrich’s work is illustrated, and to match Goodrich with Tryon is a bitch.  And this was six years before the EPA published The Gospel According To Jack Burch [11].  So the TVA sent Steve to the University of Michigan Museum of Zoology to learn freshwater malacology hands-on.

And the stage is now set.  In the fall of 1975 the telephone rang on George Davis’ desk at the Academy of Natural Sciences in Philadelphia.  It was the receptionist downstairs.  She reported that a team of TVA biologists had presented themselves at the front door and were requesting admission to the Malacology Department to examine his Duck River pleurocerid collections.  They had no appointment.

Two L. duttoniana [8]

The members of the TVA team were John Bates, Billy Isom, Steve Ahlstedt and Donelly Hill.  Bates was, at that time, a professor at Eastern Michigan University and Research Associate at the UMMZ [12].  Isom worked for the TVA at their Muscle Shoals office [13].  Hill was a supervisor, with a fisheries background.

Like my Momma used to say, “You could have called.”  George Davis was still livid about the surprise nature of this audit when he told me the story two years later.  The TVA team asserted that they had a right to examine the Duck River collections, since the agency they represented had paid for them.  Davis countered that he would need reimbursement for the time required by his curatorial staff to pull the lots.  The TVA team went away threatening to get a court order but returned the next day with a purchase order instead.

Davis would not admit either Bates or Isom into the collection.  They were notorious guns-for-hire in the wild west era of American freshwater malacology, and Davis did not trust them.  Neither did Steve Ahlstedt, for that matter.  So, in the end, Donelly Hill sent the most junior member of the team, alone, up the elevator into the ANSP malacology collection to face the Wrath of George.  Steve tells me that he felt like he was “caught between the ham and the cheese.”

At high noon the fateful shot rang out over Benjamin Franklin Parkway, “Well, Ahlstedt, is this jayana or is it not?”  And Steve’s reply was … (dramatic pause)… affirmative.

The lower reaches of the Duck River, from about RM 60 to the mouth, are indeed inhabited by a population of Lithasia bearing shells indistinguishable from type collections made of L. jayana in the Caney Fork.  And since Isaac Lea and his contemporaries defined gastropod species by shell morphology alone, and since the Duck shells in George Davis’ right hand matched the Caney Fork types, they were, by definition, Lithasia jayana.

So, Davis’ pleurocerid identifications stood.  And in 1984, after a ten-year period of comment, revision and study, his taxa were included in a gigantic laundry-list of candidate invertebrates offered for review by the USFWS as Lithasia jayana, L. duttoniana, L. geniculata, L. pinguis, and L. salebrosa [15]. Nothing ever came of that proposal, however, and Lithasia jayana receded back into sincere obscurity, as opposed to the mere obscurity it had enjoyed in the 1970s.

The abandoned Columbia Dam, 1986 [14]

But it did not matter.  By that point, Steve Ahlstedt and his colleagues had documented populations of genuinely-endangered unionids in the Duck – species that had already been entered into the Federal list, as early as 1977.  Construction was halted on the 90% complete Columbia Dam in 1983, never to resume.  According to Steve, “They left their coffee cups on their desks.” The $83M project was demolished in 1999, and the 12,800 acres it was planned to inundate turned over to the state wildlife resources agency.

But although the name of Lithasia jayana disappeared from the public eye, it remained printed in the pages of dusty tomes, and scrawled on the labels of moldering museum collections.   And in 2003 our colleagues Russ Minton & Chuck Lydeard pulled it back from sincere obscurity into mere obscurity once again in the paper we reviewed early last month [16].  Russ and Chuck reported no mtDNA sequence divergence between the robust, doubly-spined populations that Davis identified as jayana and more lightly shelled populations, tuberculate at best, that Davis (and everybody else prior to 2003) identified as Lithasia duttoniana.

Nor is there any allozyme difference between jayana and duttoniana.  Late last month I posted an unusually technical report on this blog, which I did not advertise at the time, so most of you probably missed it.  If you’re serious about the science, you might want to open this link in a new window [28Mar22].  Or you could just scroll down directly below the present post, skim that essay and scroll back.  Or you could just believe the paragraph that follows.

When Paul Johnson sent me all those Lithasia samples from the Duck River back in the summer of 2002, there were three bags from Site E and three bags from Site F – one labelled L. geniculata geniculata (which we talked about in December, January, and early March), one labelled L. duttoniana (which we talked about in January, early March, and late March) and one labelled L. jayana.  Using gene frequencies at three allozyme-encoding loci, I was indeed able to distinguish L. geniculata from L. duttoniana.  But there is no genetic distinction whatsoever between L. duttoniana and L. jayana.  They seem to be simple shell forms of the same biological species.

So now the time has come to sum up, over all five essays in this extended series.  The Duck River is inhabited by two biological species of large-bodied pleurocerid snails referable to the genus Lithasia.  One of those species bears smooth, high-spired, shells in the headwaters, becoming more robust, oblong and bumpy downstream and out into the main Tennessee River.  The modern era of American malacology was born in 1934, when Calvin Goodrich realized that populations bearing all those smooth-to-bumpy shells are conspecific, lowering the specific nomina assigned to two upstream forms, pinguis and fuliginosa, to subspecific status under the downstream nomen Lithasia geniculata.

Gastropod magafauna of Caney Fork, 500 A.D. [17]

Living together with Lithasia geniculata in the lower reaches of the Duck River is a second species of Lithasia, genetically quite similar to L. geniculata, but morphologically distinct and reproductively isolated.  These snails also demonstrate a cline in shell morphology, from a single row of light tubercules upstream, becoming more robust and heavily (even doubly) spined downstream, out into the main Tennessee River.  Then by analogy with L. geniculata, let us lower the specific nomina assigned to the two upstream forms, duttoniana and jayana, to subspecific status under the downstream nomen Lithasia armigera.

This hypothesis is supported by the genetic data, both by the duttoniana/jayana allozyme results offered late last month [28Mar22], and by the L. armigera CO1 sequences published by Minton & Lydeard, reviewed early last month [3Mar22].  It was originally suggested on the basis of shell morphology by Calvin Goodrich himself, way back in 1921 [18], although by 1940, he had apparently changed his mind [1].  It was also advocated by Davis in 1974 [2].

So this morning I have uploaded three fresh pages to the FWGNA website: Lithasia armigera duttoniana, L. armigera jayana, and L.armigera armigera.  And I remind my readership that the FWGNA defines the word “subspecies” to mean “populations of the same species in different geographic locations, with one or more distinguishing traits.”  This is the definition of the term as developed by the architects of the Modern Synthesis, and means precisely what it says, neither more nor less [19].

There need be no additively-genetic basis for the “distinguishing traits.”  Indeed, in the genus Pleurocera, a very closely analogous trend from gracile shells in the headwaters to robust shells in the big rivers has been convincingly attributed to cryptic phenotypic plasticity [20]. The heritable component of the shell morphological variance among populations identifiable as jayana, duttoniana, and armigera (s.s.) remains an open question.


[1] Goodrich, C. 1940. The Pleuroceridae of the Ohio River drainage system. Occasional Papers of the Museum of Zoology, University of Michigan 417:1 - 21.

[2] Davis, G.M. 1974.  Report on the rare and endangered status of a selected number of freshwater Gastropoda from southeastern U.S.A. U.S. Fish & Wildlife Service. Washington, DC. 51 p.

[3] For more about “The Nestor of American Naturalists” see:

  • Isaac Lea Drives Me Nuts [5Nov19]

[4] Lea, I (1841) Proceedings of the American Philosophical Society 2 (19): 83.  There has historically been much controversy regarding the exact publication dates for Lea’s descriptions.  The date on the cover of PAPS 2(19) is “July – October 1841,” a four month window.  Lea’s description of jayana appears in the “Stated Meeting of July 16,” prefaced by a statement that the paper itself was “read on the 18th of June last.”  That’s as far down the rabbit hole as I care to go.

[5] For a brief biography of Gerard Troost, see:

  • On the Trail of Professor Troost [6Dec19]

[6] Lea, I (1846) Continuation of Mr. Lea’s paper on fresh water and land shells.  Transactions of the American Philosophical Society 9(1) 1 – 31.

[7] Say, T. (1821) Journal of the Academy of Natural Sciences of Philadelphia (First Series) 2: 178.

[8] Tryon, G. W. (1873)  Land and Freshwater shells of North America Part IV, Strepomatidae.  Smithsonian Miscellaneous Collections 253: 1 - 435.

[9] Lea, I. (1841) Proceedings of the American Philosophical Society Volume 2(16) 15.

[10] I met Steve in 1975, the summer after my sophomore year at Virginia Tech, when I was blessed to be offered an hourly job with the TVA in Norris.  Malacological posterity will owe him a debt of gratitude for sharing his recollections of the events recorded here.

[11] This is a difficult work to cite.  J. B. Burch's North American Freshwater Snails was published in three different ways.  It was initially commissioned as an identification manual by the US EPA and published by the agency in 1982.  It was also serially published in the journal Walkerana (1980, 1982, 1988) and finally as stand-alone volume in 1989 (Malacological Publications, Hamburg, MI).

[12] John Morton Bates (b. 1932) began his career at the ANSP 1956 – 1966, then accepted a tenured position at Eastern Michigan University, with a research associate appointment at the UMMZ.  He left Michigan to found “Ecological Consultants” of Shawsville, VA, with Sally Dennis.  I’m not sure what became of him after that.

[13] Billy G. Isom (b. 1932) was an ecologist for the Tennessee Stream Pollution Control Board 1957 – 1963 before joining the TVA as a supervisor in the Limnology Section at Muscle Shoals, AL.  I think he is still alive and living in Killen, AL at the age of 89.

[14] Tennessee Valley Authority (1999) Use of Lands Acquired for the Columbia Dam Component of the Duck River Project. Final Environmental Impact Statement.

[15] Federal Register FR-1984-05-22. Endangered and threatened wildlife and plants; Review of invertebrate wildlife for listing as endangered or threatened species.  49(100) 21664 – 21675.

[16] Minton, R. L. and C. Lydeard. 2003. Phylogeny, taxonomy, genetics, and global heritage ranks of an imperiled, freshwater snail genus Lithasia (Pleuroceridae). Molecular Ecology 12:75-87.  For my review, see:

  • The third-most amazing research results ever published for the genetics of a freshwater gastropod population. And the fourth-most amazing, too. [3Mar22]

[17] This necklace was discovered by Cousin Bob Winters in a rock shelter on the north shore of Center Hill Reservoir, DeKalb County, TN.

[18] Goodrich, C. (1921)  Something about Angitrema.  The Nautilus 35: 58 – 59.

[19] If the clean, clear definition of the word “subspecies” confuses you, read:

  • What is a subspecies? [4Feb14]
  • What subspecies are not [5Mar14]

[20] Cryptic phenotypic plasticity is defined as “intrapopulation morphological variation so extreme as to prompt an (erroneous) hypothesis of speciation.”  For examples, see:

  • Goodrichian Taxon Shift [20Feb07]
  • Goodbye Goniobasis, Farewell Elimia [23Mar11]
  • Pleurocera acuta is Pleurocera canaliculata [3June13]
  • Elimia livescens and Lithasia obovata are Pleurocera semicarinata [11July14]

Monday, March 28, 2022

No reproductive isolation between Lithasia populations of the duttoniana and jayana forms in the Duck River, Tennessee

Editor’s Note.  We have always tried to avoid excessively technical posts on the FWGNA blog.  I typically publish formal research results elsewhere first and subsequently refer to those results here in a more casual tone.  Two of the three essays I have posted in recent months about the Lithasia of the Duck River [7Dec21] and [4Jan22] were, for example, preceded by brief, technical notes published in Ellipsaria (the newsletter of the Freshwater Mollusk Conservation Society) in 2020.

The post that follows, however, is technical.  Its publication was suppressed by FMCS Newsletter editor Dr. John Jenkinson because one of our mutual colleagues on the FMCS Board [1] called to Dr. Jenkinson’s attention that Ellipsaria content is being indexed by Google Scholar, and hence that the hypotheses I have proposed below [2] might fall upon naïve and uncritical eyes.  Why is the research that follows so dangerous?  You be the judge!

The taxonomic history of the pleurocerid genus Lithasia (Haldeman 1840) is a long and complicated one.  Goodrich (1940) recognized 16 taxa of Lithasia with smooth shells, 3 with tuberculate shells, and 5 taxa bearing shells with spines or acute protuberances.  This last category comprised Lithasia duttoniana, described by Lea (1841) from the Duck River, L. jayana, also described by Lea (1841) from Caney Fork (of the Cumberland), and three subspecies of L. armigera, described by Say (1821) from the Ohio River.

Goodrich considered both Lithasia jayana, a heavily-shelled species bearing two rows of spines, and Lithasia duttoniana, a more lightly-shelled species typically bearing (at most) a single row of small protuberances, endemic to the rivers from which they were described.  Lithasia armigera, as understood by Goodrich, ranged from the lower Ohio and Wabash Rivers through most of the Cumberland River and much of the Tennessee River as well.

In an unpublished report to the U.S. Fish and Wildlife Service, Davis (1974) suggested that Lithasia be subsumed under the genus Io of Isaac Lea (1831).  Davis then went on to recognize three smooth-shelled taxa in the Duck River, which do not concern us here, and two spiny taxa, which he identified as Io armigera duttoniana and Io armigera jayana.  After a period of comment and revision, Davis’ spiny taxa were offered for review under the Endangered Species Act in the Federal Register (May 22, 1984) as Lithasia duttoniana and Lithasia jayana.

Minton & Lydeard (2003) surveyed mitochondrial CO1 sequence variation in Lithasia populations from the Duck River and many other river systems of the American southeast. The 4 unique sequences they obtained from 19 Duck River snails (1 jayana, 4 duttoniana, and 14 of smooth-shelled taxa, in 9 Genebank submissions) did not resolve into consistent clades.  Thus Minton & Lydeard synonymized all Duck River populations, bearing both smooth and spiny shells, under a single smooth-shelled nomen, L. geniculata (Haldeman 1840).

Dillon (2020 b, c) has recently reported, however, a survey of allozyme variation in Duck River Lithasia confirming Goodrich’s (1940) hypothesis that populations of the spiny duttoniana form are reproductively isolated from the more smooth-shelled form, which Dillon followed Goodrich in identifying as Lithasia geniculata.

The large and diverse samples of Lithasia analyzed by Dillon (2020 b, c) were collected incidentally during a survey of the Duck River mussel fauna conducted by Ahlstedt et al. (2017).  In addition to populations bearing shells of the (smooth-shelled) geniculata form and the lighter, single-spined duttoniana form, the collections made by Ahlstedt and colleagues at two of their most downstream sites also contained Lithasia bearing shells of the heavy, doubly-spined jayana form.  Here I compare those doubly-spined jayana samples to sympatric samples of the lightly-shelled duttoniana form using gene frequencies three allozyme-encoding loci.

Fig. 1. The Duck River, showing sample sites.

My methodology for the resolution of allozyme polymorphism by horizontal starch gel electrophoresis has been previously detailed (Dillon 1982, 1985, 1992).  For the present study, variation interpretable as the product of codominant, Mendelian alleles was resolved at the mannose phosphate isomerase (Mpi) locus using buffers TrisCit6 and TEB8, at the octopine dehydrogenase (Odh) locus using buffers TisCit6 and Poulik, and hexanol dehydrogenase (hexdh) using buffers TEB8 and Poulik.

Sample sites and example shells are shown in Figure 1 above.  (The shell length of dutF is 24.6 mm; the other shells are to scale.)  At their most downstream site, site F, Ahlstedt and colleagues collected 35 Lithasia of the duttoniana form (dutF) and 30 of the jayana form (jayF).  This site, the Watered Hollow Boat launch at Duck River Mile 26.0 (35.9322, -87.7475), was the point at which Minton & Lydeard (2003) collected their sample of L. jayana for mtDNA sequencing.  Upstream at Wright Bend Site E (TNC110, DRM 38.7, 35.8267, -87.6657), Ahlstedt collected 44 Lithasia of the duttoniana form (dutE) and 40 of the jayana form (jayE).

Lithasia bearing shells of the jayana morphology become increasingly rare further upstream and are not effectively collectable above Duck River mile 60.  But populations of the more lightly-shelled duttoniana type extend as far upstream as DRM 186.  Gene frequencies in duttoniana population dutD, collected from the Fountain Creek confluence at DRM 145.5 (TNC 94, 35.5695, -86.9682), are included here for comparison.

Table 1 below shows that no significant allele frequency differences were apparent between samples bearing shells of the duttoniana and jayana forms at either site where they co-occurred.  This was true for the Odh locus (chi-square = 0.688, 2 df at site E, chi-square = 1.62, 3 df at site F), the Mpi locus (Fisher’s p = 0.334 at site F) and the Hexdh locus (Fisher’s p = 0.513 at site E, p = 0.832 at site F).  Judging by Nei (1978) genetic distance, sample jayE was more genetically similar to sample dutE (D = 0.050), and sample jayF was more similar to dutF (D = 0.080) than jayE was to jayF (D = 0.161) or dutE to dutF (D = 0.164).

Gene frequency differences were very significant longitudinally, however, at two of the three loci examined.  Combining the 44 + 40 = 84 samples from site E (DRM 38.7) and comparing to the 35 + 30 = 65 samples from site F (DRM 26.0), chi-square = 26.2 (3 df, p < 0.00001) at the Odh locus and chi-square = 9.91 (1 df, p = 0.002) at the Hexdh locus.  The dutD sample collected upstream at DRM 145.5 also differed significantly at the Odh locus from the combined site E sample (chi-square = 7.84, 2df, p = 0.02).

Tab 1. Gene frequencies at three loci in five samples of Lithasia.

These results reflect no evidence of reproductive isolation between Lithasia bearing the duttoniana shell morphology and those bearing the jayana shell morphology.  The genetic evidence is strong, however, for isolation by distance among the spiny Lithasia populations down this length of river, similar in magnitude to that documented by Whelan et al. (2019) in Alabama Leptoxis, and Dillon (2020a) in North Carolina Pleurocera.

The similarity between these results and those previously published by Dillon (2020b) for the smooth-shelled Lithasia of the Duck River is striking.  Dillon confirmed the hypothesis of Goodrich (1934) that the shells borne by Duck River Lithasia geniculata also become more robust when sampled in a downstream direction, adding bumpy shoulders to the point that 19th-century authorities recognized two additional species, L. fuliginosa and L. pinguis.  Here an identical phenomenon is documented in the spiny Lithasia, populations identified by Goodrich as Lithasia duttoniana developing such robust and heavy shell spines downstream that some authorities have recognized a second species, L. jayana.

Given such levels of shell variability, neither nominal Lithasia duttoniana (Lea 1841) nor Lithasia jayana (Lea 1841) can be distinguished at the specific level from the much more broadly-distributed Lithasia armigera (Say 1821).  The suggestion of Davis (1974) that both of Lea’s 1841 nomina be lowered to subspecific status under Say’s L. armigera would seem to have substantial merit.


Ahlstedt, S. A., J. R. Powell, R. S. Butler, M. T. Fagg, D. W. Hubbs, S. F. Novak, S. R. Palmer and P. D. Johnson. 2017. Historical and current examination of freshwater mussels (Bivalvia: Margaritiferidae: Unionidae) in the Duck River basin Tennessee, USA. Malacological Review 45:1-163.

Davis, G.M. 1974.  Report on the rare and endangered status of a selected number of freshwater Gastropoda from southeastern U.S.A. U.S. Fish & Wildlife Service. Washington, DC. 51 p.

Dillon, R. T., Jr. 1982. The correlates of divergence in isolated populations of the freshwater snail, Goniobasis proxima (Say). Ph.D. Dissertation, The University of Pennsylvania.

Dillon, R. T., Jr. 1985. Correspondence between the buffer systems suitable for electrophoretic resolution of bivalve and gastropod isozymes. Comparative Biochemistry and Physiology 82B: 643-645. [pdf]

Dillon, R. T., Jr. 1992. Electrophoresis IV, nuts and bolts. World Aquaculture 23(2):48-51.

Dillon, R. T., Jr. 2020a. Fine scale genetic variation in a population of freshwater snails. Ellipsaria 22(1): 24-25. [pdf]

Dillon, R. T., Jr. 2020b. Population genetic survey of Lithasia geniculata in the Duck River, Tennessee. Ellipsaria 22(2):19 – 21. [pdf]

Dillon, R. T., Jr. 2020c. Reproductive isolation between Lithasia populations of the geniculata and duttoniana forms in the Duck River, Tennessee. Ellipsaria 22(3): 6 – 8. [pdf]

Goodrich, C. 1934. Studies of the gastropod family Pleuroceridae - I. Occasional Papers of the Museum of Zoology, University of Michigan 286:1-17.

Goodrich, C. 1940. The Pleuroceridae of the Ohio River drainage system. Occasional Papers of the Museum of Zoology, University of Michigan 417:1 - 21.

Minton, R. L. and C. Lydeard. 2003. Phylogeny, taxonomy, genetics, and global heritage ranks of an imperiled, freshwater snail genus Lithasia (Pleuroceridae). Molecular Ecology 12:75-87.

Nei, M. 1978 Estimation of average heterozygosity and genetic distance from a small number of individuals. Genetics 89:583–590.

Whelan, N. V, M. P. Galaska, B. N. Sipley, J. M. Weber, P. D. Johnson, K. M. Halanych and B. S. Helms. 2019. Riverscape genetic variation, migration patterns, and morphological variation of the threatened Round Rocksnail, Leptoxis ampla. Molecular Ecology 28:1593-1610.


[1] Here is the relevant passage from the minutes of the FMCS Board, November 19, 2020:

“Nathan Whelan noticed recently that some Contributed Articles in Ellipsaria are now findable on Google Scholar. This was unexpected for our informal, non-peer-reviewed newsletter.  Nathan also recognized that some recent articles in the newsletter include the analysis of data and/or what could be viewed as proposed taxonomic revisions. In a series of emails, monitored and, occasionally, participated in by the Executive Committee, Nathan and Ellipsaria Editor John Jenkinson agreed that articles including data analysis and/or taxonomic revisions should be peer-reviewed and, therefore, are outside of the intended scope and purpose of our newsletter.”

[2] Science is the construction of testable hypotheses about the natural world.  It is not the handing down of fact.  It appeals to no authority, nor does it merit any.  It is independent of context or culture.  Whether published in a slick international journal or a humble newsletter is irrelevant.  The quality of a work of science is dependent only upon the extent to which the hypothesis proposed matches the natural world, upon rigorous test.  And it stuns me – literally stuns me – to see how few scientists actually understand any of this.

Thursday, March 3, 2022

The third-most amazing research results ever published for the genetics of a freshwater gastropod population [1]. And the fourth-most amazing, too.

Editor's Note:  This is the third installment of what will turn out to be a five-part series on the Lithasia of the Duck River in Middle Tennessee.  It will help to familiarize yourself with my posts of [7Dec21] and [4Jan22] before continuing.

In 2003, Russ Minton and Chuck Lydeard published a CO1 gene tree for the North American pleurocerid genus Lithasia in Molecular Ecology [2].  Nestled among the branches of that tree was the third-most amazing research result in the history of freshwater gastropod population genetics.  What Minton and Lydeard found was… nothing.

The Minton & Lydeard study was very good by the standards of its day.  Our colleagues did a thorough job collecting U1S2NMT3 individuals [7] from 30 Lithasia populations representing 11 nominal species and subspecies.  We first touched on the M&L results back in [4Sept19], focusing on a single outside branch, labeled “L. geniculata pinguis.”  And here is a quote from that 2019 essay: “To completely unpack the message being telegraphed to us by the enigmatic arboreal specimen (of Minton and Lydeard) would require at least 6 – 8 blog posts of standard length.”  So, what follows is another installment [8].

Minton & Lydeard included in their analysis 19 Lithasia individuals from the main Duck River, identified as follows: 6 geniculata pinguis, 7 geniculata fuliginosa (from three sites), 1 geniculata geniculata, 4 duttoniana (from two sites), and 1 jayana.  And on the basis of mtCO1 sequence, they were unable to distinguish among any of those 19 individuals.  I was agog in 2003 and remain agog 20 years later.

Detail from Minton & Lydeard [2] fig 4, modified

To contextualize.  With no difficulty whatsoever, even at very small sample sizes, workers have easily been able to document 23% CO1 sequence divergence within Pleurocera simplex populations, 21% within Pleurocera catenaria, 19% within Pleurocera proxima, 15% within Leptoxis carinata, and 12% within L. ampla [9].  Then In 2003, in the pages of an international journal, Russ Minton and Chuck Lydeard stunned the world by reporting no more than a couple lousy nucleotides of difference in 19 individual Lithasia sampled down the 200-mile length of the Duck River, bearing five different Latinate nomina.

Well, maybe the M&L result is not terribly surprising for the 14 individual Duck River Lithasia geniculata they included in their survey, of the three subspecies.  In December [7Dec21], we documented evidence of gene flow among subpopulations representing all three of those nomina, attenuated by distance but not much else [11].  The M&L pinguis sample seems to have been sampled from below the falls.

Nor am I terribly shocked by the absence of sequence divergence among the 4 duttoniana sampled by M&L.  In January [4Jan22] we documented similar levels of isolation-by-distance between Duck River subpopulations bearing the DUT shell morphology that we had previously seen in the GEN form [12].

Nor am I even terribly surprised by the absence of sequence divergence between the 4 duttoniana and the singleton snail that M&L identified as “Lithasia jayana.”  This is the first time the specific nomen “jayana” has appeared on the FWGNA blog.  It will not be the last.  We will have much more to say about Lithasia jayana in posts upcoming.  But for now, please accept that there is no significant genetic difference between snails that have been identified as L. jayana on the basis of shell morphology and sympatric Lithasia populations that Goodrich, Minton, and everybody else has always called L. duttoniana.

Rather, the third-most amazing research result ever registered in the annals of freshwater gastropod population genetics is the absence of any detectable sequence divergence between the 14 individual geniculata (all subspecies) and the 5 individual duttoniana + jayana.  Populations historically identified by those two sets of nomina really are morphologically distinct throughout their entire 100+ miles of sympatry in the Duck River, and everywhere else through their combined ranges across the Ohio, Cumberland, and Tennessee.

Can you tell us apart? [13]

Isaac Lea and George Tryon and Calvin Goodrich and all modern workers even unto the present day have all drawn a clear and unambiguous distinction between Lithasia populations bearing a robust, oblong, bumpy shell morphology and those bearing a more acutely-spired shell morphology, with angular whorls often tuberculate or even slightly-spiny.  In our January essay we abbreviated that former morphology “GEN” and that latter morphology “DUT.”  Snails bearing shells of the DUT morphology range only up to around Duck River mile 186 (as opposed to 275 for GEN) and seem more common in the shallows, rather than on rocks in the middle.  No prior worker has ever questioned the distinction between those two groups of taxa.

Setting 200 years of field observation aside, however.  On the basis of their sequence data, Minton and Lydeard synonymized all the Duck River Lithasia taxa: pinguis, fuliginosa, duttoniana, and jayana, under Haldeman’s (1840) geniculata.  Russ Minton then went on, in papers published in 2008 and again in 2018, to perform detailed morphometric analyses on the entire five-taxon GEN/DUT mishmash combined [14].  Bless his heart.

Well, the GEN and DUT populations do differ genetically, but not by much.  When Johnson, Ahlstedt, and their colleagues sent me those Lithasia samples from Fountain Creek (site D), Wright Bend (site E) and Watered Hollow (site F) back in 2002, they divided them (quite naturally and conventionally) into oblong-bumpy subsamples they identified as Lithasia geniculata, and acute-angular subsamples they identified as Lithasia duttoniana.  And in my January essay [4Jan22] I used the allozyme results I obtained from Wright Bend (site E) as an example of stable character phase disequilibrium between GEN and DUT.  Results were the same at Fountain Creek and Watered Hollow.  Hit this link for a pdf of my technical results [Ellipsaria 22(3)].

Lithasia bearing the GEN shell morphology and Lithasia bearing the DUT morphology sympatric in the Duck River do not constitute a single randomly-breeding population.  There is some sort of reproductive isolation between them [15].  They are distinct biological species.

How many species can you see? Click to zoom [16].

But my goodness, the allozyme divergence between GEN and DUT is tiny!  The gene frequencies I published in Table 1 of my paper in Ellipsaria 22(3) were certified in 2020 as the fourth-most amazing research results in the history of freshwater gastropod population genetics, at 88.7 international amazingness units [1].

Again, some context would seem to be in order.  I ran allozyme gels on scores of pleurocerid populations during the 35 years I had access to a biochemical laboratory, 1980 – 2016.  Typically, I would do an initial screening across 15 – 20 allozyme loci (17 in the case of the Duck River Lithasia), and then focus on the polymorphic loci for a detailed analysis.  And very rarely did I ever find a pair of distinct biological species sharing alleles any more than at a couple loci, out of 15 or 20 [17].

Even among populations within pleurocerid species, fixed allozyme differences are not uncommon [18].  Across the 25 populations of P. proxima I surveyed for my 1984 dissertation, for example, it was possible to find conspecific populations sharing no alleles at five loci.  And even within individual pleurocerid populations, sampled from single creeks or rivers, significant allozyme differences among subpopulations are not uncommon, as we witnessed in the P. proxima of Naked Creek in October [12Oct21], and in the L. geniculata of the Duck in December [7Dec21].

So seen in that context, to find no difference between a pair of reproductively-isolated pleurocerid species at 14 of 17 allozyme loci, and merely-statistical differences at the other three, shocked me back in 2002.  And I’m obviously still not over it, any more than I am over the CO1 sequence results published by Minton and Lydeard in 2003.  I was aghast at the time and remain aghast to this day.

Let this be a lesson to any of you high school seniors out there, looking for science fair projects.  There are a lot of online purveyors of simple kits advertising “DNA barcoding” services, promising to identify any sort of unknown bug or slug you might pluck into a tube and mail to Canada.  That’s fun, and I’m sure you’ll learn more from the experience than lying around your bedroom, watching Tik-Tok videos.  But please understand that no serious scientist would ever publish a paper in the peer-reviewed literature relying on “DNA barcoding.”

Do I have time to touch on one additional feature of the 2003 M&L gene tree before you run out of patience with me this month?  Notice this.  Not only is there essentially zero divergence among their 19 Duck River samples of two reproductively-isolated species, we really don’t see much sequence divergence anywhere in the entire top half of the Minton & Lydeard Lithasia tree.

Detail from Minton & Lydeard [2] fig 4, modified.

If you back down one limb below the big Duck River cluster at the top, you’ll see a couple samples labeled, “geniculata fuliginosa” from 23 miles back up a tributary of the lower Duck River called the Buffalo.  M&L did uncover 2.0% sequence divergence between their Duck River N = 19 and their Buffalo River N = 2, upon which basis Russ described a new species, “Lithasia bubala” in 2013 [19].  The allozyme data I reported on [7Dec21] did not support that [11].

Then if you back down two limbs from the M&L Duck River cluster, you find a set of five sequences identified as Lithasia armigera.  These represent 14 individuals collected from five far-flung rivers: the Harpeth River and the Stones River (both tributaries of the Cumberland), the main Tennessee River way down in Alabama, the Wabash River (in Illinois) and the main Ohio River on the IL/KY border.  All 14 of these snails, from five populations, were genetically indistinguishable.  And all differed by just 3.8% from the Duck River group.

And if you back down three limbs from the M&L Duck River cluster, you’ll find a set of three sequences (representing 8 individuals) labelled “geniculata fuliginosa,” two from the Red River (a tributary of the Cumberland about 80 miles north of the Duck) and one sequence from Garrison Fork, an upstream tributary of the Duck River itself.  The sequence divergence between that set of N = 8 and the set of N = 19 from the main Duck was 4.3%.

Let me say that again.  There is less sequence divergence between L. duttoniana of the Duck River and L. armigera of the Wabash River almost 200 miles away, than between L. geniculata fuliginosa of the Duck River and L. geniculata fuliginosa of Garrison Fork, 25 miles upstream.  What in the world does that mean?  Stay tuned!


[1] I apologize for the overly-dramatic title.  For the record, the CO1 sequence homogeneity in the Duck River Lithasia as reported by Minton & Lydeard in 2003 [2] scored 91.5 international amazingness units.  The Bianchi et al. (1994) report of hybridization between P. virginica and P. semicarinata livescens [3] holds first place in the freshwater gastropod population genetics division at 93.2 international amazingness units, with Nathan Whelan’s [4] discovery of a wildebeest sequence in the population of bison he sampled at Shades Creek in second place at 91.9 iau.

For context, in the freshwater gastropod transmission genetics division, Yoichi Yusa’s discovery of multigenic sex determination in Pomacea [5] scored a whopping 98.7 iau in 2007, pushing  Boycott’s (1923) paper on maternal inheritance of chirality in Lymnaea [6] to second all time, at 98.4 iau.

[2] Minton, R. L. and C. Lydeard. 2003. Phylogeny, taxonomy, genetics, and global heritage ranks of an imperiled, freshwater snail genus Lithasia (Pleuroceridae). Molecular Ecology 12:75-87.

[3] Bianchi, T. S., G. M. Davis, and D. Strayer 1994.  An apparent hybrid zone between freshwater gastropod species Elimia livescens and E. virginica (Gastropoda: Pleuroceridae).  Am. Malac. Bull. 11: 73 - 78.

[4] Whelan, N.V. & E. E. Strong (2016)  Morphology, molecules and taxonomy: extreme incongruence in pleurocerids (Gastropoda, Cerithiodea, Pleuroceridae). Zoologica Scripta 45: 62 – 87.  I reviewed Nathan’s findings in a series of posts back in 2016, see note [10] below.

[5] Yusa, Y. 2007. Nuclear sex-determining genes cause large sex-ratio variation in the apple snail Pomacea canaliculata. Genetics 175: 179-184.  For more, see:

  • Ampullariids star at Asilomar [11Aug05]

[6] Boycott, A.E. and C. Diver (1923) On the inheritance of sinistrality in Limnaea peregra.  Proceedings of the Royal Society of London, Series B, Biological Sciences 95: 207 – 213.

[7] Usually 1, Sometimes 2, Never More Than 3.  This has always been the rule-of-thumb in sampling for gene trees.  See:

  • The Lymnaeidae 2012: Stagnalis yardstick [4June12]

[8] Actually, looking back on this post from the bottom, I am afraid I have written an essay of twice what ought to be my standard length.  And this is two installments.  Sorry.

[9] I coined the term “mitochondrial superheterogeneity” on this blog in 2016 to describe double-digit intrapopulation sequence divergence [10].  Here are several prominent examples from the pleurocerids:

  • Dillon, R. T., and R. C. Frankis. (2004)  High levels of DNA sequence divergence in isolated populations of the freshwater snail, Goniobasis.  American Malacological Bulletin 19: 69 - 77.  [PDF]
  • Lee, T., J. J. Kim, H. C. Hong, J. B. Burch, and D. O’Foighil (2006)  Crossing the Continental divide: the Columbia drainages species Juga hemphilli is a cryptic member of the eastern North American genus Elimia.  J. Moll. Stud. 72: 314-317. 
  • Dillon, R T. and J. D. Robinson (2009)  The snails the dinosaurs saw: Are the pleurocerid populations of the Older Appalachians a relict of the Paleozoic Era?  Journal of the North American Benthological Society 28: 1 - 11. [PDF]
  • Dillon, R. T. Jr, and J. D. Robinson (2016)  The hazards of DNA barcoding, as illustrated by the pleurocerid gastropods of East Tennessee.  Ellipsaria 18: 22-24. [PDF]
  • Whelan, N.V. & E. E. Strong (2016)  Morphology, molecules and taxonomy: extreme incongruence in pleurocerids (Gastropoda, Cerithiodea, Pleuroceridae). Zoologica Scripta 45: 62 – 87.

[10] For more about the origin and significance of the phenomenon, see:

  • Mitochondrial superheterogeneity: What we know [15Mar16]
  • Mitochondrial superheterogeneity: What it means [6Apr16]
  • Mitochondrial superheterogeneity and speciation [3May16]

[11] Dillon, R. T. (2020) Population genetic survey of Lithasia geniculata in the Duck River, Tennessee.  Ellipsaria 22(2): 19 - 21. [PDF]

[12] Dillon, R. T. (2020) Reproductive isolation between Lithasia populations of the geniculata and duttoniana forms in the Duck River, Tennessee.  Ellipsaria 22(3): 6 - 8.  [PDF]

[13] From upper left: GEN, GEN, DUT, DUT, GEN, GEN.

[14] Papers in which Russ Minton lumped L. geniculata and L. duttoniana:

  • Minton, R. L., A. P. Norwood & D. M. Hayes (2008) Quantifying phenotypic gradients in freshwater snails: a case study in Lithasia (Gastropoda: Pleuroceridae)  Hydrobiologia 605: 173-182.
  • Minton, R. L., K.C. Hart, R. Fiorillo, & C. Brown (2018) Correlates of snail shell variation along a unidirectional freshwater gradient in Lithasia geniculata (Haldeman 1840) (Caenogastropoda: Pleuroceridae) from the Duck River, Tennessee, USA.  Folia Malacologia 26(2): 95 – 102.

[15] But I’ll bet dollars to donuts that they hybridize.  I think hybridization is widespread in the North American family Pleuroceridae.  See the paper by Bianchi et al from footnote [3] above.

[16] Five pleurocerid species are visible grazing across this rock in the Duck River at the Watered Hollow Boat Launch (RM 26): Pleurocera canaliculata canaliculata, Pleurocera laqueata laqueata, Leptoxis praerosa praerosa, Lithasia geniculata geniculata, and Lithasia armigera jayana.  Notice that no juveniles are apparent whatsoever.  All massively-shelled adults!  I could write an entire essay on that phenomenon alone.

[17] A selection of papers showing typical levels of allozyme divergence between pleurocerid species:

  • Dillon, R.T. and G.M. Davis (1980) The Goniobasis of southern Virginia and northwestern North Carolina: Genetic and shell morphometric relationships. Malacologia 20: 83-98. [PDF]
  • Dillon, R. T., and S. A. Ahlstedt (1997) Verification of the specific status of the endangered Anthony's River Snail, Athearnia anthonyi, using allozyme electrophoresis. The Nautilus 110: 97 - 101. [PDF]
  • Dillon, R. T. and A. J. Reed (2002)  A survey of genetic variation at allozyme loci among Goniobasis populations inhabiting Atlantic drainages of the Carolinas.  Malacologia 44: 23-31. [PDF]

[18] A selection of papers showing typical levels of allozyme divergence among populations within species:

  • Dillon, R.T. (1984) Geographic distance, environmental difference, and divergence between isolated populations. Systematic Zoology 33:69-82.  [PDF]
  • Dillon, R.T. (1988) Evolution from transplants between genetically distinct populations of freshwater snails. Genetica 76: 111-119. [PDF]
  • Dillon, R.T., and C. Lydeard (1998) Divergence among Mobile Basin populations of the pleurocerid snail genus, Leptoxis, estimated by allozyme electrophoresis.  Malacologia. 39: 111-119. [PDF]
  • Dillon, R. T. and J. D. Robinson (2011)  The opposite of speciation: Population genetics of  Pleurocera (Gastropoda: Pleuroceridae) in central Georgia.  American Malacological Bulletin  29: 159-168.  [PDF]

[19] Minton, R. L. 2013. A new species of Lithasia (Gastropoda: Pleuroceridae) from the Buffalo River, Tennessee, USA. The Nautilus 127:119-124.