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

Editor’s Notes - 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.  This essay was ultimately published as: Dillon, R.T., Jr. (2023b)  No reproductive isolation between Lithasia populations of the duttoniana and jayana forms in the Duck River, Tennessee.  Pp 175 – 182 in The Freshwater Gastropods of North America Volume 6, Yankees at The Gap, and Other Essays.  FWGNA Project, Charleston, SC 

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In an unpublished report to the U.S. Fish and Wildlife Service, Davis (1974) suggested that Lithasia be subsumed under the genus Io of Isaac Lea (1831).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.

References

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.

Notes

[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.

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 Notes - 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. This essay was subsequently published as: Dillon, R.T., Jr. (2023b)  The third-most amazing research results ever published for the genetics of a freshwater gastropod population, and the fourth-most amazing, too.  Pp 163 – 173 in The Freshwater Gastropods of North America Volume 6, Yankees at The Gap, and Other Essays.  FWGNA Project, Charleston, SC.

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!

Notes

[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.