Dr. Rob Dillon, Coordinator

Thursday, June 2, 2016

The Shape-shifting Pleurocera of North Alabama

The Tennessee River south of Chattanooga reminds me of Danica Patrick on three tires.  Swerving wildly out of the fertile Valley & Ridge Province of East Tennessee, she slices through the Cumberland Plateau at Walden Ridge, bounces off the NW Georgia guard rail, and plunges into the Interior Plains of North Alabama.  The character of the freshwater gastropod fauna changes slightly but perceptibly – not so much in the main river as in the tributaries.  The little black Pleurocera simplex populations, so common in springs and small streams of East Tennessee, disappear west of Chattanooga, to be replaced by populations of the larger and more impressively-shelled P. laqueata.

The most conspicuous element of the Tennessee River macrobenthos through this entire region is Pleurocera canaliculata (Say, 1821) bearing shells of the typical, robust form.  The smaller tributaries are inhabited by a more slender and lightly-shelled ecophenotypic variant, originally described by T. A. Conrad in 1834 as “Melania pyrenellum.”  In 2013 Stephen Jacquemin, Mark Pyron and I presented evidence that the distinction between Conrad’s pyrenellum in small tributaries and Say’s canaliculata in the main Tennessee River is “cryptic phenotypic plasticity,” intrapopulation morphological variance so extreme as to prompt an (erroneous) hypothesis of speciation [1]

Fig 1. Typical Pleurocera canaliculata from the Tennessee R at Site #1.
The sample of typical P. canaliculata canaliculata depicted in Figure 1 above was collected from the Tennessee River at Decatur, Alabama – marked as site #1 on the map below [2].  Stephen, Mark, and I showed that this population is conspecific with the population of P.canaliculata pyrenellum depicted in Figure 2 below, collected from Limestone Creek at Site #2.  I reviewed the evolutionary biology of P. canaliculata throughout eastern North America generally in a pair of related essays posted on this blog back in June of 2013 [3].

Fig 2.  Pleurocera canaliculata pyrenellum from Limestone Creek at Site #2.
The situation is similar in the case of P. clavaeformis, another notorious shape-shifter.  Pleurocera clavaeformis is the most common freshwater gastropod in East Tennessee, ranging from small creeks into large rivers, although not inhabiting the main Tennessee River itself.  Populations of clavaeformis bearing more robust shells in the larger rivers were for many years identified (erroneously) as the distinct species Pleurocera unciale and P. curtum [4].  I first documented cryptic phenotypic plasticity in East Tennessee populations of P. clavaeformis in February of 2007, returning to the subject again in October 2009 and March 2011 [5].

Fig 3.  The Tennessee River west of Knoxville.
Populations of P. clavaeformis are common in several of the Tennessee tributaries of North Alabama, including the Elk, Flint, and Paint Rock Rivers.  These populations generally seem to bear robust shells, however, and have historically been identified as “Pleurocera curtum.”  Goodrich, in fact, stated that Pleurocera (Goniobasis) clavaeformis “does not make the westward turn around Walden Ridge” because he was unaware of any North Alabama populations bearing shells of the lighter, more typical form inhabiting streams of the Cumberland Plateau or Interior Plains [6].

I myself cannot boast of extensive field experience in the state of Alabama.  But I collected the mixed sample of P. clavaeformis and P. canaliculata shown in Figure 4 below from the Paint Rock River SE of Huntsville in March of 1988 (marked as Site #4 above).  The shell variation was rather dramatic in the P. clavaeformis population (top row), ranging from medium-typical to robust-curtum.  The P. canaliculata population (bottom row) was more uniformly heavy-shelled, ranging only from skinnyish-typical to plain-vanilla-typical.

Fig 4.  Pleurocera clavaeformis and P. canaliculata from the Paint Rock at Site 4.
Most significantly, notice that my 1988 sample of Pleurocera from the Paint Rock River did not include any shells demonstrating the pyrenellum morphology, as depicted in Figure 2.  Phenotypic plasticity manifests itself most cryptically where small streams like Limestone Creek empty directly into large rivers, like the Tennessee.  In such situations, the population bearing the more gracile shell morphology will differ strikingly from the population bearing the robust shell, sometimes even mixing unconformably at the mouth of the creek, looking like reproductively-isolated biological species.  But in rivers that grow larger gradually, such as the Paint Rock, the shell morphology of the pleurocerid population changes gradually, and taxonomists are usually not fooled.

It is perhaps for this reason that Goodrich [6] did not report Pleurocera of the pyrenellum form from the Paint Rock River drainage, or indeed from any of the larger tributaries of the Tennessee in North Alabama, such as the Flint or the Elk.  Goodrich gave the distribution of pyrenellum as “tributaries of the Tennessee River in Morgan and Limestone Counties” downstream from the Paint Rock, “and Walker County, Georgia” upstream from the Paint Rock, but not in Madison or Jackson Counties, through which the Paint Rock River runs.  The canaliculata population of the Paint Rock subdrainage just looks like typical canaliculata.  It doesn’t fool anybody.

So for the last three months, we’ve been studying (in excruciating detail) the recent paper by Nathan Whelan and Ellen Strong on mtDNA sequence divergence among the pleurocerid populations of North Alabama [7].  Previously our attention has focused on populations of Leptoxis [8].  But Whelan & Strong also sampled five populations they identified as “Pleurocera prasinata” from tributaries of the Cahaba River draining south through Alabama into the Mobile Basin, and one population they identified as “Pleurocera pyrenellum” from a tributary of the Paint Rock River in Jackson County [9].  That Paint Rock tributary site is marked as Site A on the map above.

Whelan & Strong's Fig 7.  Row C shows putative "Pleurocera pyrenella."  
Whelan & Strong’s Figure 7 is reproduced above, showing example shells from their sample of “Pleurocera pyrenellum” on Row C, at the bottom.  These specimens do not look like the pyrenellum form of P. canaliculata to me.  The shells borne by pyrenellum are flat-sided, almost trapezoidal, essentially demonstrating no suture or indeed any whorl, as shown in Figure 2.  North Alabama pyrenellum populations also typically bear shells marked with spiral cords, especially around the aperture.  But the shells depicted in Row C of Whelan & Strong’s figure seem to lack spiral cords, and demonstrate the slightly rounded whorls and impressed sutures typical of Pleurocera clavaeformis

I hasten to stipulate that I have no personal observations from the Paint Rock subdrainage as far upstream as the Whelan & Strong sample.  And of course, the entire theme of the present essay has been one of caution regarding the use of shell morphology to distinguish species of pleurocerids.  So what might the mtDNA sequence data tell us about the genetic relationships between Whelan & Strong’s Pleurocera populations (all six of them) and Pleurocera populations sampled from elsewhere in the drainage of the Tennessee?  Tune in next time…


[1] Dillon, R. T., S. J. Jacquemin & M. Pyron (2013)  Cryptic phenotypic plasticity in populations of the freshwater prosobranch snail, Pleurocera canaliculata.  Hydrobiologia 709: 117-127.  [PDF]

[2] Here are the complete locality data for all four sites mentioned in this month’s blog:
  • Site #1 – Tennessee River at Decatur, Morgan County, AL.  (34.6279N; -86.9564W).  This was site “CT” of Dillon et al. (2013).
  • Site #2 – Limestone Creek at Nick Davis Road, 2 km N of Capshaw, Limestone County, AL. 
  • (34.8027N; -86.8163W).  This was site “PT” of Dillon et al. (2013).
  • Site #4 - Paint Rock River at US 431 bridge, 25 km SE of Huntsville, AL.  (34.4992, -86.3905).
  • Site A – Estill Fork at Co. 140 bridge, Jackson Co, AL.  (34.9653, -86.1537).
[3]  Cryptic phenotypic plasticity in P. canaliculata:
  • Pleurocera acuta is Pleurocera canaliculata [3June13]
  • Pleurocera canaliculata and the process of scientific discovery [18June13]
[4] Dillon, R. T. (2011)  Robust shell phenotype is a local response to stream size in the genus Pleurocera (Rafinesque 1818). Malacologia 53: 265-277. [PDF]

[5] Cryptic phenotypic plasticity in P. clavaeformis:
  • Goodrichian Taxon Shift [20Feb07]
  • Mobile Basin III: Pleurocera Puzzles [12Oct09]
  • Goodbye Goniobasis, Farewell Elimia [23Mar11]
[6] Goodrich, C. (1940) The Pleuroceridae of the Ohio River system. Occas. Pprs. Mus. Zool. Univ. Mich., 417, 1-21.

[7] Whelan, N.V. & E. E. Strong (2016)  Morphology, molecules and taxonomy: extreme incongruence in pleurocerids (Gastropoda, Cerithiodea, Pleuroceridae). Zoologica Scripta 45: 62 – 87.  Open Access: [html]

[8]  I have featured the Leptoxis mtDNA data set of Whelan & Strong in three previous essays:
  • Mitochondrial Superheterogeneity: What we know [15Mar16]
  • Mitochondrial Superheterogeneity: What it means [6Apr16]
  • Mitochondrial Superheterogeneity and speciation [3May16]
[9] Our good friends Nathan Whelan and Ellen Strong seemed unaware that Conrad’s pyrenellum is an ecophenotypic variant of Say’s canaliculata, and indeed entirely neglected to cite the (2013) paper by Jacquemin, Pyron, and myself.  I’m sure this was just a simple oversight.

Tuesday, May 3, 2016

Mitochondrial Superheterogeneity and Speciation

Editor’s Note – This is the third essay in my series on the phenomenon of double digit intrapopulation mtDNA sequence variation, which I have begun calling “mitochondrial superheterogeneity.”  The points I plan to advance here will depend upon my crazy “wildebison” model of 6Apr16, which (in turn) depended on the field observations I reviewed in my essay of 15Mar16.

Understood in their proper context, typical mtDNA sequence data sets yield weak, null models of population relationship, not directly relevant to the question of speciation [1], but certainly correlative.  A mitochondrial gene sequence is a perfectly fine single character.  Given a decent sample size per population, typical mtDNA sequence data can reasonably be used to estimate interpopulation genetic divergence, which (especially in combination with data from other sources) is generally correlated with the likelihood of speciation [2].

So in the last couple months we have focused on the mtDNA sequence data collected by Whelan & Strong from five populations of Leptoxisampla” inhabiting tributaries of the Cahaba River, draining south through Alabama into the Mobile Bay [3].  Whelan & Strong also sampled n = 20 Leptoxis praerosa from a tributary of the Tennessee River, draining west across the top of Alabama toward the Mississippi River.  Their data set included many striking examples of mitochondrial superheterogeneity.  So while intriguing as an evolutionary phenomenon, doesn’t the occurrence of up to 20% sequence divergence within conspecific populations of “ampla” compromise the utility of mtDNA as a tool for distinguishing “ampla” from praerosa?

No, perhaps just the opposite.  But before I develop that surprising point, let’s back up a few years and review what we actually know about the evolutionary biology of Leptoxis populations in Alabama.

The Leptoxis fauna of the Mobile Basin was last comprehensively monographed by Calvin Goodrich in 1922 [4], prior to his 1934 - 1941 awakening to the Modern Synthesis [5].  The entire pleurocerid fauna of Alabama was then decimated by a massive program of impoundment and channelization, leaving behind what may be the single biggest taxonomic mess anywhere in North American malacology [6].

In 1998, Chuck Lydeard and I published a survey of genetic variation at 9 allozyme-encoding loci in eight populations representing all four nominal species of Leptoxis known (at that time) to persist in the Mobile Basin of Alabama [7], calibrated with three populations of the widespread and well-studied Leptoxis praerosa of Tennessee.  Our Alabama populations included three identified as Leptoxis ampla from the Cahaba drainage [8], two populations identified as L. taeniata from tributaries of the Coosa River, and one population of Leptoxis picta from the main Alabama River way downstream in Monroe County [9].  The allozyme variation among those six populations was comparable to that observed among our three control populations of Leptoxis praerosa, and negligible compared to the genetic divergence between praerosa and the ampla+taeniata+picta group combined.  Thus our (9 gene, 11 population, 333 individual) results suggested that Leptoxis ampla (Anthony 1855) is a junior synonym of L. picta (Conrad 1834) [10].

So returning to the mtDNA sequence data of Whelan & Strong.  It is reassuring to note that, despite the high incidence of superheterogeneity W&S observed scattered among the five Cahaba drainage pleurocerid populations of (what should better be referred to as) Leptoxis picta, their 16S+CO1 gene tree nevertheless depicted the single population of Leptoxis praerosa they sampled from that Tennessee tributary as genetically distinct.

The bottom half [11] of Whelan & Strong’s Figure 2 (reproduced above) shows negligible sequence divergence within their 20-individual sample of L. praerosa, but approximately 13% divergence between modal ("clade 9") L. praerosa and modal (“clade 15”) L. picta [12].  Granted, the sequence variance within populations of L. picta (“ampla”) does indeed range well above 13%.  But a mode-to-mode difference of such magnitude is certainly consistent with an hypothesis of speciation.

And we have thus far given short shrift to the histone H3 sequence data adduced by Whelan & Strong, which is especially interesting because the gene is nuclear, and tends to evolve quite slowly.  Figure 3 of W&S (reproduced below) shows essentially zero sequence divergence within or among any of the five L. picta (“ampla”) populations, but 2.0% divergence between picta and praerosa [13].  

Levels of genetic divergence in allopatry, no matter how striking, do not directly address the question reproductive isolation, and hence can only constitute indirect evidence of speciation.  But the nine allozyme-encoding genes of Dillon & Lydeard, plus the 13% mode-to-mode sequence divergence W&S report for the CO1 gene, plus the 2.0% sequence difference at histone H3, taken together with whatever morphological evidence may have accumulated [14], certainly combine to suggest that the specific distinction between L. picta (aka “ampla”) and L. praerosa is indeed a valid one.

Now begging your indulgence, let me return to Figure 4 of Whelan & Strong – the third time I have reproduced the figure below in three months.  And let’s focus once again on the situation at red-star-14 in the lower left corner, showing that the Shades Creek Leptoxis sample included 16 copies of the modal haplotype and 1 copy of a 12% divergent haplotype, modal in the Cahaba River at US52.  (The red-star numbers in Fig 4 below correspond to the clade numbers in Fig 2 above.)  Last month I suggested that cases of mitochondrial superheterogeneity such as this are the signatures of very rare dispersal events, as though a wildebeest were airlifted into a herd of bison, and successfully interbred.  Seen in this light, the results depicted at red-star-14 constitute direct, positive evidence that the Leptoxis population of Shades Creek is conspecific with the Leptoxis population of Cahaba-US52.

And similarly at red-star-12.  Here we see that the Leptoxis populations of Cahaba-Bibb/Shelby and Cahaba-US82 share a unique, rare CO1 haplotype that Whelan & Strong didn’t discover modally anywhere.  This would seem to constitute direct, positive evidence that the Bibb/Shelby and the US82 Leptoxis populations are conspecific both with some third (unsampled) population where that particular haplotype is much more common, and with each other, as well.

So more broadly, I would suggest that rare, superdivergent mtDNA haplotypes be understood as markers of genetic compatibility with second populations some distance removed.  And at red stars 10, 11, and 13, we see three additional markers of genetic compatibility with three other populations of Leptoxis that Whelan & Strong did not sample.  All of these populations must be conspecific – the populations of Leptoxis picta in which W&S found the markers, and the four other populations elsewhere.  In an ideal world, given complete sampling, it seems possible to me that the phenomenon of mitochondrial superheterogeneity might afford a direct, positive tool to reconstruct specific relationships among extensive sets of highly allopatric populations in their entirety.

Well, perhaps you’d like to join us back here in the real world, Dillon?  The pleurocerid fauna of real Alabama is just a ghost of its former self.  And the Leptoxis populations where the sequences shown at red stars 10, 11, 12 and 13 are modal may well have been extinguished 80 years ago.  Or longer?  And those mtDNA haplotypes might be genetic fossils, of populations long gone to glory?  Interesting to think about.

But turning the page.  As I am sure most of my readership will have by now noticed, the mtDNA survey of Whelan & Strong extended to cover six populations of Pleurocera, sampled alongside the six populations of Leptoxis upon which we have focused these last three months.  Their Pleurocera results offer some striking contrasts with their Leptoxis results, one of which is illustrated in the copy of their Figure 3 I have reproduced above.  Why doesn’t the population W&S identified as “Pleurocera pyrenella” from the Tennessee drainage appear genetically distinct from the five populations they identified as “Pleurocera prasinata” from the Mobile Basin?  Tune in next time!


[1]  Gene trees and species trees are not the same thing:
[2] Examples of the correlative relationship between mtDNA sequence divergence and speciation:
  • Rhodacmea Ridotto [8Aug11]
  • The Lymnaeidae 2012: Stagnalis yardstick [4June12]
[3] Whelan, N.V. & E. E. Strong (2016)  Morphology, molecules and taxonomy: extreme incongruence in pleurocerids (Gastropoda, Cerithiodea, Pleuroceridae). Zoologica Scripta 45: 62 – 87.  Open Access: [html]

[4] Goodrich, C. (1922) The Anculosae of the Alabama River Drainage. Misc. Publ. Univ. Mich Mus. Zool. 7: 1-57.

[5] Goodrich’s (1934 – 1941) “Studies of the Gastropod Family Pleuroceridae” series may represent the earliest application of the modern evolutionary synthesis to malacology – I don’t know of any earlier.  For more, see my essays:
  • The Legacy of Calvin Goodrich [23Jan07]
  • Mobile Basin II: Leptoxis lessons [15Sept09]
[6]  But we can’t blame the entire mess on Alabama Power Company and the Corps of Engineers.  As was true throughout The South, the state of Alabama was blanketed with cotton and other row crop agriculture through the nineteenth century and well into the twentieth.  Bank-to-bank farming dumped tremendous loads of silt into the smaller rivers and streams, which still chokes most of the pleurocerid habitat, even to this day.

[7] 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]

[8] One of the three Leptoxis ampla sites of Dillon & Lydeard matched one of the five Whelan & Strong sites precisely – the Cahaba River at HWY 52 bridge.

[9] We also included two populations of Leptoxis plicata from the Black Warrior drainage, not relevant to this particular essay.  We did not include the subsequently rediscovered Leptoxis “foremani/downiei” or Leptoxis “compacta.”

[10]  Our good friends Nathan Whelan and Ellen Strong neglected to cite Dillon & Lydeard (1998) in their more recent work on the Alabama Leptoxis under review here.  I’m sure this was just a simple oversight.

[11] The upper half of Whelan & Strong’s Figure 2 depicted the 16S+CO1 genetic relationships among their six populations of Pleurocera.  We’ll return to those data next month.

[12] For this 13% estimate I blasted randomly-chosen L. praerosa CO1 sequence KT164014 against randomly-chosen clade 15 L. picta sequence KT163938.

[13] To obtain this 2.0% estimate, I blasted randomly-chosen L. praerosa H3 sequence KT164460 against randomly-chosen L. picta (“ampla”) H3 sequence KT164387, just as in note [12] above.

[14]  Whelan & Strong did not offer any morphological observations on L. praerosa.  But it is my subjective impression that there may be subtle differences in the whorl shape or spire height between praerosa and picta (“ampla”) – perhaps noticeable only in juveniles, or in adults inhabiting calmer, more protected waters [15].

[15] And it is a crying shame that the captive-rearing study published by Whelan and colleagues (2015) in J. Moll. Stud. 81:85-95 was not controlled for current speed.

Wednesday, April 6, 2016

Mitochondrial Superheterogeneity: What it means

Last month [1] we reviewed, or at least touched upon, 15 separate reports of double-digit mtDNA heterogeneity within gastropod populations published over the last 20 years, the paper by Whelan & Strong [2] among the best and most recent.  Three additional reports from pulmonate snail populations have subsequently come to my attention [3].  The discussion sections of almost all of these works have featured lists of possible explanations for the phenomenon, including population fragmentation, great antiquity, introgression from other species and cryptic speciation, many of which do not seem to fit the data as it has now accumulated, none of which is complete. 

So Rob Dillon’s model to explain mitochondrial superheterogeneity begins with an observation (not an assumption, in the case of pleurocerid snails) of large numbers of super-isolated populations.  Then I add two more super-assumptions: super-long time and super-rare dispersal events.  And let me begin with an extended analogy.

The even-toed ungulates first evolved in the Eocene Epoch, about 50 mybp.  So today, the mtCOI sequence divergence between American bison (JF443195) and the wildebeest of the Serengeti (JX436977) is approximately 15%.  I suggest that when we stand on the bank of the Green River in Polk County, NC, and look down at the population of Pleurocera proxima grazing over the rocks, it is as though we were Lewis and Clark, looking over a vast herd of American bison [4].  And should we drive 80 km west to Buncombe Co, NC, and look down on the Pleurocera proxima population inhabiting Bent Creek, it is as though we were Frederick Russell Burnham standing before the wildebeests. 

Now suppose that every million generations or so, a wading bird with sticky feet transports one snail from Buncombe County to Polk County, as though a modern zookeeper were to airlift a wildebeest to Wyoming.   I don’t know, but it seems likely to me that our jet lagged wildebeest might find itself perfectly well-adapted to graze on the plains of Wyoming in all respects, morphologically and physiologically. 

But it does not seem likely to me that a single wildebeest could reproduce in a herd of American bison.  I imagine a variety of prezygotic reproductive isolating mechanisms have evolved between wildebeest and bison – appearances, smells, behaviors and so forth.  And even if a mating should occur, I feel certain all sorts of postzygotic incompatibilities have evolved - the chromosomes of the two species probably don’t match, a million screw ups occur in development, and nothing comes of it.

So (I admit) the most implausible element of my model is that, in the case of pleurocerid snails, a super-rare immigrant arriving via sticky-bird express must successfully mate within a host population from which it has been isolated for millions of generations.  There are several factors that make this phenomenon marginally less-implausible for pleurocerids, specifically.  Pleurocerid snails are aphallic, and no obvious mating behavior has been reported.  Apparently the male simply crawls over the female and deposits a spermatophore in her gonoduct, so unceremoniously that human observers typically don’t even notice it.  There’s very little light down where the snails live, so appearances don’t matter.  And pleurocerid populations typically inhabit waters with significant flow rates, so it seems unlikely that chemical mating cues could evolve.

Now why postzygotic reproductive incompatibilities have not evolved over millions of generations among such isolated gastropod populations, I do not know.  North American pleurocerids seem to demonstrate negligible anatomical diversity [5], and negligible chromosomal diversity to match [6].  I was tempted to add “super-stable environment” to my list of assumptions in paragraph two at the top of this essay, because it seems possible to me that stabilizing selection may have worked to prevent reproductive isolating mechanisms from evolving.

But I should hasten to add that very little of the reproductive biology I have reviewed directly above applies to pulmonate land snails, and that it was in land snail populations mitochondrial superheterogeneity was first discovered [7].  The bottom line seems to be that, over a wide range of mating systems, populations as isolated as bison and wildebeest have not speciated in gastropod world.  The plains of snail-America and snail-Africa are both grazed by reproductively compatible “wildebison.” 

Rob Dillon’s patented “wildebison model” fits all six of the points I reviewed last month regarding mitochondrial superheterogeneity as a general phenomenon [1].  So let us return to the Leptoxis population inhabiting Shades Creek in northern Alabama, marked in dark blue above.  Whelan & Strong reported 16 copies of the modal CO1 sequence (the “bison” sequence, at red-star-15) and one copy of a 12% divergent sequence, which we’ll call “wildebeest.”  The wildebeest sequence almost exactly matched the modal sequence in the Cahaba River at the US52 bridge, shown in black above (at red-star-14).  And not only did the rare CO1 sequence of Shades Creek match the modal CO1 sequence of the Cahaba River, the rare 16S sequence of Shades Creek matched the modal 16S sequence of the Cahaba River.  The conclusion seems almost inescapable that at some point in the (probably distant) past, a Cahaba wildebeest has been airlifted into the Shades Creek bison population.

The first-most amazing thing about this data set is that an individual Cahaba wildebeest was apparently able to mate into the Shades Creek bison herd, despite millions of generations of isolation.  And the second-most amazing thing is that Whelan & Strong happened to sample both Shades Creek and the Cahaba River, to show us what happened.  In most data sets of this sort, we simply find no clue. 

So for example, in addition to the 17 copies of the modal CO1 sequence Whelan & Strong reported from the Cahaba River at US52, they also reported two copies of a 20% divergent sequence that matches nothing else in their database, way off at red-star-11 [8, 9].  Did that rare mitochondrial genome come from a third population of “wildecamels,” currently grazing on some other continent that W&S did not sample?  Or might that genome have originated in an extinct population of “wilde-Irish-elk,” now gone?   A fascinating question.

I will close with two final notes.  First, I should emphasize that I have not evoked selection at any point during the essay above.  I very nearly used the S-word when I was waving my hands about the apparent absence of reproductive isolation among highly isolated gastropod populations with a surprising range of evolutionary backgrounds, but (ultimately) held off.  The wildebison model is grudgingly neutral.

And second, my good friend John Robinson and I are currently simulating the evolution of mitochondrial superheterogeneity using the “ms” program of R. R. Hudson [10]. Our preliminary results have not yielded the phenomenon in single-population models given any reasonable values of mutation rate (u) and population size (Ne).  Our two-population models do, however, yield mitochondrial superheterogeneity within reasonable values of u and (perhaps-surprisingly low values of) Ne, given (super-rare) migration rates around Nem = 0.0001 genomes per generation.

I find all this quite interesting as a study of evolutionary science, pure as the driven snow, and (I feel sure) a large fraction of my readership does as well.  But in situations such as the pleurocerid fauna of Alabama, research efforts have been at least partly motivated by conservation concerns regarding the distribution of potentially endangered species.  What might mtDNA sequence data sets such as developed by Whelan & Strong, riddled (as they are) with superheterogeneity, tell us about the species relationships of the populations sampled?  Tune in next time!


[1] Mitochondrial Superheterogeneity: What we know [15Mar16]

[2] Whelan, N.V. & E. E. Strong (2016)  Morphology, molecules and taxonomy: extreme incongruence in pleurocerids (Gastropoda, Cerithiodea, Pleuroceridae). Zoologica Scripta 45: 62 – 87.  Open Access: [html]

[3] Pinceel, J, K. Jordaens & T. Backeljau (2005)  Extreme mtDNA divergences in a terrestrial slug (Gastropoda, Pulmonata, Arionidae): Accelerated evolution, allopatric divergence and secondary contact.  J. Evol. Biol. 18: 1264 – 1280.  Parmakelis, A., P. Kotsakoizi & D. Rand (2013)  Animal mitochondria, positive selection and cyto-nuclear coevolution: Insights from Pulmonates.  PLOS ONE 8(4): e61970.  Nolan, J. R., U. Bergthorsson & C. M. Adema (2014)  Physella acuta: atypical mitochondrial gene order among panpulmonates (Gastropoda).  J. Moll. Stud. 80: 388 – 399. 

[4] The populations of Pleurocera I’m using as examples here were sampled in 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].  I reviewed the Dillon & Robinson results in my blog post of March, 2009.  See:
  • The Snails The Dinosaurs Saw [16Mar09]
[5] Dazo B (1965) The morphology and natural history of Pleurocera acuta and Goniobasis livescens (Gastropoda: Cerithiacea: Pleuroceridae). Malacologia 3:1–80.   Strong, E., 2005. A morphological reanalysis of Pleurocera acuta Rafinesque, 1831, and Elimia livescens (Menke, 1830) (Gastropoda: Cerithioidea: Pleuroceridae). Nautilus 119: 119–132

[6] Dillon, R.T. (1989) Karyotypic evolution in pleurocerid snails. I. Genomic DNA estimated by flow cytometry. Malacologia 31: 197-203. [PDF]   Dillon, R.T. (1991) Karyotypic evolution in pleurocerid snails. II. Pleurocera, Goniobasis, and Juga. Malacologia 33: 339-344.  [PDF

[7]  I almost cut the two paragraphs about the absence reproductive isolating mechanisms in pleurocerid snails entirely out of this essay.  But then I figured, hell, if the land snail people don’t like it, they can start their own blog.

[8]  For this calculation I used KT164003 as an example as the modal CO1 sequence from the Cahaba River at US52, and KT164004 as an example of the rare, divergent sequence, and compared the two using blastn.

[9] And somewhat amazingly, Whelan & Strong also found one mitochondrial haplotype in the Cahaba River at US52 that matches the Shades Creek mode.  So their Figure 4 (reproduced above) should have shown one little black circle near their big blue circle, as well as that single little blue circle near their big black.  I have added one little black circle at red-star-15 above.  More about this next month.

[10] Hudson, R. R. (2002)  Generating samples under a Wright-Fisher neutral model of genetic variation.  Bioinformatics 18: 337-338.  

Tuesday, March 15, 2016

Mitochondrial Superheterogeneity: What we know

Finally some decent sample sizes, thank heaven!  I remember thinking this to myself when the abstract of the recent paper in Zoologica Scripta by Nathan Whelan and Ellen Strong fell upon my eyes in January [1].  Perhaps now, after twenty years of bouncing around on the fringes of evolutionary science, the phenomenon we here define as “mitochondrial superheterogeneity” will finally attract the attention it so richly deserves.

A population can be said to demonstrate “mitochondrial superheterogeneity” when two or more of its members demonstrate 10% sequence divergence or greater for any single-copy mtDNA gene, not sex-linked.  The phenomenon was first documented (as far as I am aware) in 1996 by Thomaz, Guiller and Clarke in European populations of the land snail, Cepaea nemoralis [2].  It has now been reported at least three additional times in land snails [2], twice in populations of freshwater pulmonates [3], once in oriental pomatiopsids [4], three times in Old World cerithiaceans [5] and six times in populations of North American pleurocerids [6, 7].  But the new study by Whelan & Strong is by far the most thorough.

W&S sampled 20 individuals from each of 12 populations of pleurocerid snails collected from North Alabama, sequencing three genes from all 240 individuals [8]: (mitochondrial) COI, (mitochondrial) 16S rRNA, and (nuclear) histone H3.  Six of the populations sampled were of Leptoxis (identified as praerosa in Tennessee drainages or ampla in Alabama drainages) and six of the populations were of Pleurocera (identified as pyrenella in Tennessee drainages or prasinata in Alabama drainages).  The specific identities of these populations are a complicated question, which we will defer to a later essay.  But here are the important findings:
(1) No intermediate sequences.  Given the small numbers of haploid genomes sampled in previous studies, it has seemed possible that individuals bearing mtDNA sequences differing by as much as 10% within populations might be united by (unsampled) individuals bearing intermediate sequences 2%, 4%, 6% and 8% divergent.  But apparently not. 

For example, the COI results obtained by W&S from five of their Leptoxis populations are depicted above.  The 19 snails [8] sequenced from the Shades Creek population (shown in dark blue) had three strikingly different haplotypes: N=16 in the modal category, N=2 about 68 mutational steps (9%) different from the modal, and N=1 about 88 steps (12%) different.  Notice that the red intermediate points in the figure below are mostly quite distant from the branch tips and entirely hypothetical.

(2)  Peculiar patterns of interpopulation haplotype sharing.  Let us focus on that single Shades Creek snail demonstrating a 12% COI sequence divergence from its N = 16 associates, marked with a red star in the figure above.  That CO1 sequence (KT163940) is only 1% different from the sequence demonstrated by N=17 of the 20 snails sampled from the Cahaba River at the US52 bridge, 5 km downstream and then back up the main river about 20 km (e.g., KT164013).

(3)  Tight coupling in the evolution and distribution of separate mitochondrial genes.  W&S confirmed that individuals bearing highly divergent COI sequences also tend to bear highly divergent 16S sequences.  So the starred individual in the COI network above also demonstrated a 16S sequence rather strikingly divergent from the modal Shade Creek 16S sequence.  And that divergent 16S sequence also very nearly matched the modal sequence recovered from the Cahaba River at the US52 bridge [9].

Such results were first reported in 2004 by Dillon & Frankis [5], working with Pleurocera proxima in southern Virginia [10].  Our 2004 mtDNA sample sizes were very small – just three individuals from three P. proxima populations, plus single individuals of P. catenaria dislocata and P. semicarinata.  But we did sequence both the COI and 16S genes, giving us a total of 11 sequences for each gene.  All three of our P. proxima from Naked Creek matched in both sequences, and all three of our P. proxima from Cripple Creek matched in both sequences.  But the individuals we sampled from Nicholas Creek demonstrated superheterogeneity: the CO1 sequence of the (N=2) mode and the (N=1) minority differing by 16.9%.  And that same N=1 minority snail also differed from the (N=2) modal 16S sequence in Nicholas Creek by 14.1%.

(4)  No evidence of cryptic speciation.  Although the sample sizes of mtDNA sequence data in all three of my previously-published works on this subject have been quite small, all three have focused on pleurocerid populations for which I have tested Hardy-Weinberg equilibrium at multiple polymorphic allozyme loci with sample sizes of at least N = 30, and often much larger.  These are not weak tests, and if cryptic species had been present, I have reason to think that I would have found them [11].  But I have found no evidence of non-random mating in any population from which I have reported mitochondrial superheterogeneity [12].

Whelan & Strong have reinforced this result with some classical (i.e., entirely qualitative) studies of shell and radular morphology, pallial oviduct anatomy, and head-foot coloration in their 12 pleurocerid populations from North Alabama.  Their morphological observations are nicely illustrated and described in quaint, Victorian style (e.g., “Outermost denticle on each side delicate, flimsy, variable in position”).  Although W&S do note some intrapopulation variation in some morphological traits, none seemed to correspond with mtDNA clade.

(5) No evidence of NUMTs.  In some circumstances mitochondrial genomes or portions of genomes have been discovered transposed into the nucleus, yielding “NUclear MiTochodrial DNA” which, being “free to evolve,” can diverge greatly from the bona fide mtDNA genome.  W&S used a next-generation technique to sequence the entire mitochondrial genome of a Leptoxis individual from which they had obtained highly-divergent COI and 16S sequences with their standard (Sanger) methods.  The next-generation sequences matched the Sanger sequences, counter to the NUMT hypothesis.

(6)  No evidence of DUI.  Doubly-uniparental inheritance is the surprising situation where two highly-divergent mtDNA genomes have evolved within a single species, one transmitted through eggs and the other transmitted through sperm.  The phenomenon has been documented in a great variety of bivalve groups, including the freshwater unionids, but is unknown in gastropods.  And indeed, W&S could find no systematic differences in the mtDNA genomes isolated from the male and female samples of pleurocerids they analyzed.

So that’s what we know about mitochondrial superheterogeneity as of March 2016, and what doesn’t cause it.  But what, then, might a reasonable explanation be?  Are data such as these rudely yelling something important to us, in some language we do not understand?  Tune in next time!


[1] Whelan, N.V. & E. E. Strong (2016)  Morphology, molecules and taxonomy: extreme incongruence in pleurocerids (Gastropoda, Cerithiodea, Pleuroceridae). Zoologica Scripta 45: 62 – 87.  Open Access: [html]

[2] Thomaz, D., Guiller, A. & Clarke, B. (1996) Extreme divergence of mitochondrial DNA within species of pulmonate land snails. Proceedings of the Royal Society of London B: Biological Sciences, 263, 363–368.  Goodacre, S. L. & C. M. Wade (2001)  Patterns of genetic variation in Pacific island land snails: the distribution of cytochrome b lineages among Society Island Partula.  Biol. J. Linn. Soc. 73: 131-138.  Guillier, A. et al. (2001)  Evolutionary history of the land snail, Helix aspersa in the western Mediterranean: preliminary results inferred from mitochondrial DNA sequences.  Molecular Ecology 10: 81-87.  Haase, M. et al. (2003) Mitochondrial differentiation in a polymorphic land snail: evidence for Pleistocene survival within the boundaries of permafrost.  J. Evol. Biol. 16: 415-428.

[3] My good friend and colleague Amy Wethington documented a striking case of mitochondrial superheterogeneity (both COI and 16S) in a Charleston population of Physa acuta in 2003.  We never could get a separate paper through the reviewers, but the data were published in her (2004) dissertation.  Andrea Walther and colleagues had better luck with the reviewers in her (2006) paper on Laevapex fuscus.  For more, see:
  • Phylogenetic Sporting and the Genus Laevapex [20July07]
[4] Wilke, T. et al. (2006)  Extreme mitochondrial sequence diversity in the intermediate schistosomiasis host Oncomelania hupensis robertsoni: another case of ancestral polymorphism?  Malacologia 48: 143-157.

[5]  Facon, B. et al. (2003)  A molecular phylogeography approach to biological invasions of the New World by parthenogenetic thiarid snails.  Molecular Ecology 12: 3027-3039.  Lee, T., H. C. Hong, J. J. Kim & D. O’Foighil (2007)  Phylogenetic and taxonomic incongruence involving nuclear and mitochondrial markers in Korean populations of the freshwater snail genus Semisulcospira.  Molecular Phylogenetics and Evolution 43: 386-397.  Miura, O., F. Kohler, T. Lee, J. Li, & D. O’Foighil (2013)  Rare, divergent Korean Semisulcospira spp. mitochondrial haplotypes have Japanese sister lineages.  J. Moll. Stud. 79: 86 – 89.
I reviewed the (2007) paper by Taehwan Lee and his colleagues on this blog in February, 2008:
  • Semisulcospira II: A second message from The East [1Feb08]
[6] 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].  Sides, J. D.  (2005) The systematics of freshwater snails of the genus Pleurocera from the Mobile River Basin. Ph.D. dissertation, University of Alabama.  Lee, T., J. J. Kim, H. C. Hong, J. B. Burch, and D. O’Foighil (2006)  Crossing the contintental 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]
I reviewed the Dillon & Robinson (2009) results in my blog post of March, 2009.  See:
  • The Snails The Dinosaurs Saw [16Mar09]
[7] The note of Dillon & Robinson (2016) is a special case.  John Robinson and I first reported mitochondrial superheterogeneity in East Tennessee populations of Pleurocera clavaeformis and P. simplex in an oral presentation at the 2011 meeting of the American Malacological Society in Pittsburgh.  We uploaded our COI sequences to GenBank shortly thereafter, and drafted a manuscript entitled, “When cometh our reformation?  Molecular typology meets population genetics in the pleurocerid gastropod fauna of east Tennessee,” which we were ultimately unable to publish.  Our 2011 results have just this month become available as a note in Ellipsaria:
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]
More about this little work in a future post.

[8] Well, to be precise, one of the 240 snails was excluded by misidentification.  Then no COI sequence was ultimately obtained for 13 individuals, no 16S sequence obtained for 16 individuals, and no H3 sequence obtained for 15 individuals.  But we can certainly call it 240 for round numbers.

[9] The Genbank accession number of the “clade 14” Shades Creek 16S sequence is KT164167.  Blasting that sequence against the 16S sequence from a random clade 15 Shades Creek snail (KT164165) yielded an identity of 94%.  Blasting KT164167 against a random Clade 14 16S sequence from the Cahaba River at US52 (KT164235) yielded an identity of 99%.

[10] In the first paragraph of their discussion, our good friends Nathan Whelan and Ellen Strong wrote, “We show for the first time in pleurocerids that there is complete congruence in phylogenetic signal between the COI and 16S mitochondrial genes.” They have completely neglected to cite the Dillon & Frankis (2004) paper, either here or anywhere else in their work, even though Dillon & Frankis was heavily-cited by Dillon & Robinson (2009), of which they obviously were aware.  I am sure this was just a simple oversight.

[11] In fact, we did find a cryptic species in the same (2011) allozyme survey that forms the basis of the recent note by Dillon & Robinson [7].  Put another bookmark here – we’ll come back to this later, as well.

[12] The primary reason it has continued to be so difficult (in our experience) to publish reports of mitochondrial superheterogeneity is the immediate (and passionate) objection of reviewers that we have overlooked cryptic species, despite our very good allozyme evidence to the contrary.  That climate now seems to be improving.

Friday, February 5, 2016

Marstonia letsoni, Quite Literally Obscure

Editor’s Note.  This is the second essay of a two-part series on an enigmatic population of hydrobiids in Lake St. Clair, on the US/Canada border east of Detroit.  It will only make sense if you’ve read my post of [19Jan16] first.

In late September of last year I began to mentally outline a short series of essays to catch us up on invasive species news, such as have become a regular feature of this blog.  And I thought to myself, what better way to reintroduce the subject of introduced species than to tell the story of an unidentified hydrobiid snail from Lake St. Clair, which must certainly represent an invasion not heretofore recognized by anybody but yours truly, Rob Dillon?

I don’t know why I picked up Bob Hershler’s (1994) Pyrgulopsis monograph [1] and began leafing through it on Friday morning, 9/25.  I knew that Bob had reviewed 11 Eastern species as well as 54 Western species of Pyrgulopsis in that most thorough and useful work.  I also knew that Bob had teamed up with Fred Thompson in 2002 to “resurrect” Baker’s generic nomen “Marstonia,” transferring those 11 Eastern Pyrgulopsis species into it [2].  In the last 20 years I have continued to find Bob’s 1994 Pyrgulopsis monograph very helpful as I have stomped around Georgia and Tennessee, paying respects to endemic or narrowly-restricted populations of what are now called Marstonia agarhecta, Marstonia arga, Marstonia halcyon, Marstonia ogmorphaphe, and others.

I thought I knew the work pretty well.  But on the morning of 9/25, Bob’s image of a 2.8 mm “Pyrgulopsis” letsoni from a small lake west of Pontiac, Michigan jumped out and bit me like a snake.  There are two species of Marstonia inhabiting Great Lakes drainages, not just the one!  And everything about M. letsoni fit our Lake St. Clair unknowns: the shell morphology, the diminutive adult size, the range, indeed the very obscurity of it.

“Amnicola” letsoni was described by Bryant Walker [3] in 1901 from Pleistocene deposits on the banks of the Niagara River in upstate New York [4].  The first living specimens were not collected until 1943, however, when E. G. Berry dug the silt and mud out of the “honeycombed cavities” in rocks from the bed of a temporarily-dewatered stretch of the Huron River above Ann Arbor, and washed the goop through a sieve [5].  The species first known as Amnicola letsoni, and then later as Pyrgulopsis letsoni, and now as Marstonia letsoni is, quite literally, obscure [6].

Amnicola/Pyrgulopsis/Marstonia letsoni is so obscure that all the index entries under “letsoni” in Burch’s [6] North American Freshwater Snails direct the user to incorrect pages.  (Burch’s little line drawing of a Pyrgulopsis letsoni shell is hidden on the bottom of page 238.)  Arthur Clarke [7] did mention (but did not figure) Pyrgulopsis letsoni under Marstonia decepta (page 60), only venturing so far as to suggest that letsoni “may live in Canada.”

But returning to the morning of 9/25, especially compelling to me was the penial morphology.  In his message of 8/28, Bob had described the penis of the Lake St. Clair unknowns as “bifurcate.”  But I have never personally seen a Marstonia penis I would describe in that fashion.  Nymphophiline hydrobiids, such as Marstonia, bear a penis with a single duct plus a glandular lobe.  Typically the glandular lobe is larger than the duct that transmits the sperm, so that the overall nymphophiline penial morphology is bladelike, with a little “penial filament” hanging out along the margin.  E. G. Berry’s figure of the Marstonia lustrica penis at left below is typical.  The adjective “bifurcate” is not called to mind.

But Berry’s [5] figure of the penis of Marstonia letsoni illustrated a glandular lobe much smaller than any I have ever seen in a Marstonia – smaller than the duct that bears the sperm.  The overall morphology of the letsoni penis shown at right above might indeed be described as “bifurcate.”

So on 9/26 I swapped a couple fresh emails with Bob Hershler, asking for clarification on the penial morphology of the Lake St. Clair unknowns, and suggesting Marstonia letsoni as a possible identification.  And here is what Bob said: “I did think of letsoni in this context, but, again, I will need to see more specimens of the mystery snail to nail it down.”  A cautious worker is our good buddy Bob.

Well, those of you who know me, know that I think of evolutionary biology as a science – the construction of testable hypotheses about the natural world.  And the names I assign to populations of freshwater gastropods are hypotheses.  Sometimes they are weak hypotheses, and sometimes they are strong hypotheses, but Rob Dillon’s identifications are never “nailed down.”

So I composed the present two-part essay series in early October, firmly and boldly hypothesizing that the Lake St. Clair unknowns are Marstonia letsoni (Walker 1901), and prepared to go to press.  But there is yet one final twist in this story.  Our good buddy Ron Griffith asked me to delay release of the news as he was (even at that moment, in October) writing up a note for formal publication.

Ron reported that he had access to historical data from Lake St. Clair benthic samples, suggesting that the rise and spread of our M. letsoni population seems to be a fairly recent phenomenon.  In other words, M. letsoni may be a native invasive.  Might its range currently be demonstrating a significant expansion?  Ron also wanted time to send additional samples to Bob H., to try again for that "nailing down" thing.

So as most of you will have subsequently noticed, I wrote essays on three other invasive species topics for the months of October, November, and December.  And in the interim, Ron G. and Bob H. have been busy with the malacological hammer, confirming our identification even to Bob’s exacting standards.  And now at long last, nine months after that first little sample of tiny snails spilled under my dissecting scope in Milwaukee, our six-member team has reached consensus on an identification of M. letsoni.  Cradled in the metropolitan Detroit/Windsor area, bathed in one of the world’s busiest waterways, and yet remaining quite surprisingly, quite literally, obscure.


[1]  Hershler, R. (1994)  A review of the North American freshwater snail genus Pyrgulopsis (Hydrobiidae).  Smithsonian Contributions to Zoology 554: 1-115.

[2] Thompson, F. G. & R. Hershler (2002)  Two genera of North American freshwater snails: Marstonia Baker, 1926, resurrected to generic status, and Floridobia, new genus (Prosobranchia: Hydrobiidae: Nymphophilinae).  The Veliger 45: 269 - 271.

[3] For my tribute to Bryant Walker, see:
  • Bryant Walker’s Sense of Fairness [9Nov12]
[4]  Walker, B. (1901)  A new Amnicola.  Nautilus 14: 113-114.

[5] Berry, E. G. (1943)  The Amnicolidae of Michigan: Distribution, ecology, and taxonomy.  Misc. Publ. Mus. Zool. Univ. Mich. 57: 1 – 68.

[6] Burch, J. B. (1989)  North American Freshwater Snails.  Malacological Publications, Hamburg, MI.  365 pp.

[7] Clarke, A. H. (1981)  The Freshwater Molluscs of Canada.  National Museum of Natural Sciences, Ottawa.  446 pp.

Tuesday, January 19, 2016

A New Invasive Gastropod in the Great Lakes?

“I have absolutely no idea what this is.  I got nuthin.  Where did you say this sample came from, again?”

It was day four of the Society for Freshwater Science meeting in Milwaukee this past spring, and I had spent the afternoon enthroned under the “Gastropoda” sign at the Taxonomy Fair [1], identifying freshwater gastropod samples from all corners of the continent, from all baskets of molluscan biodiversity.  Well, juvenile Physa, mostly.

So up walked Adam Frankiewicz and Jerry Shepard from the EPA lab in Duluth, bearing several small vials of small hydrobiid snails from benthic grab samples taken on the Canadian side of Lake St. Clair, maybe 25 miles east of Detroit.  Some of their samples were collected from sand bottoms as little as 0.9 m deep, others from muck/clay as deep as 5.6 m.

Of course, I was expecting Potamopyrgus.  The invasive “New Zealand Mud Snail” was first reported from Lake Ontario in 1991, and has since popped up in four of the five Great Lakes [2].  But the snail sample that spilled out under my scope on that May afternoon immediately struck me as too small in adult size, even by hydrobiid standards.  Although apparently mature, I saw no specimen in excess of 3.0 mm standard shell length, and most were closer to 2.0 mm.  And the shell morphology simply did not match Potamopyrgus.  The example specimens shown in the growth series above (from Ron Griffiths) range from 2.0 mm to 2.8 mm only.

Moreover, there really weren’t a whole lot of other candidate hydrobioid species in the Great Lakes section of my mental database.  Marstonia lustrica is vaguely similar, but bigger and fatter.  None of the other familiar hydrobioids are even close - Cincinnatia, Amnicola, Lyogyrus, Bithynia?  No way.  I have never, in eight years of service to the SFS/NABS Taxonomy Fair, been so completely stumped as I was that May afternoon in Milwaukee.

But strange and unexpected as this episode was, it became stranger.  Our good friend Ron Griffiths, who works as a consultant in Ontario, happened to be passing by the Gastropoda table at that very moment.  And Ron said, “Oh, I’ve seen those little snails too.”  Ron had also been working on benthic samples from the Canadian side of Lake St. Clair, in the vicinity of the Thames River mouth, and had been similarly puzzled by the tiny hydrobiid snails they contained.  He did not have a sample with him in Milwaukee, however.

“Gentlemen,” said I, “We are not going to solve this mystery today.”  I suggested to the working group assembled that if Adam or Ron could take some good jpeg images of our little critters, I would forward the images to my buddy Bob Hershler at the USNM.  And I further suggested that if, as seemed likely, Bob wanted to see some actual specimens, our little group should stand ready to send samples to Washington directly.  And I closed with a request that I be kept on the CC line.  I was fascinated by my own ignorance.

Lake St. Clair ain’t the Peruvian Amazon!  It is one of the most heavily-traveled commercial waterways in the world.  Cradled in the greater Detroit/Windsor metropolitan area, the gentle waters of Lake St. Clair lap upon shores of great wealth, great power, and great learning.  Could we North American biologists be utterly ignorant of our own commonplace biota?

So Adam and Ron did indeed follow up with several jpeg images within the week, Adam’s comprising the top half of the figure below.  I myself photographed the juvenile Potamopyrgus specimens shown in the bottom half of the figure at the same scale, pasted the two images together for comparative purposes, and forwarded the montage onward to Bob Hershler in early June, with a cover message.  Sure enough, Bob wanted to examine some actual specimens, which Ron promptly posted to Washington.

And this is what our buddy Bob said (6/10): “These are not the New Zealand mudsnail (Potamopyrgus antipodarum), nor are they (juvenile) Elimia [3].  They are phallate and oviparous, and probably are ‘hydrobioids’ but I will need to examine additional material to get a better handle on their identity.  If your colleague is sequencing specimens then the resulting data could also be used toward this end.”    

So yes, quite fortunately, Jerry did indeed have connections with the Molecular Ecology Research Branch of the EPA lab in Cincinnati.  And even as Bob, Ron, Adam and I had been fumbling around with the old fashioned morphology, Jerry was packing a sample of our Lake St. Clair unknowns off to Dr. Erik Pilgrim in Cincinnati for sequencing.

And by late August Erik, the newest member of our working group, had been able to amplify “nice, clean CO1 and 16S sequences for two specimens.”  He reported (8/26): 
“Searching the COI barcode sequences places these snails in Marstonia, and they appear most closely related to, but not the same species as, M. pachyta (95% match), M. lustrica (95%), M. comalensis (95%), M. hershleri (94%), M. agarhecta (94%), M. halcyon (93%), and M. castor (93%). The 16S sequences also place them as close relative of M. agarhecta, M. hershleri, and M. halcyon (apparently not as many 16S sequences are in the database). So, bottom line, those snails are some species of Marstonia.”
 I will confess that Erik’s conclusion seemed highly improbable to me.  Marstonia is a North American genus, with approximately 10-15 species, most of which are endemic to the southeastern United States.  And although I may not have expressed my opinion openly prior to August, it had been my strong impression, from the very moment I laid eyes on these little critters in Milwaukee, that the Lake St. Clair unknowns must represent a heretofore unrecognized alien invasion. 

I said this to the group (8/27): “Geeze, there is NOTHING about those little hydrobiids that speaks Marstonia to me.  I’m not the expert, but they don’t look like Marstonia or smell like Marstonia – they’re too small, they’re in the wrong habitat, in the wrong part of the world.”  I then fussed and fumed for a couple paragraphs, advancing my invasive species hypothesis to no effect, and concluded, “When I first looked at those snails way back in May, I told you I got nuthin.  And nuthin is what I still got.”  To which Bob Hershler replied (8/28):
“As I said previously, I would have to examine a well preserved anatomical series to confidently identify this snail.  The single male that I dissected had a bifurcate penis consistent with Marstonia, which is widely distributed in the Great Lakes region.  Although Ron's shells are more elongate than what I am used to seeing in Marstonia, I would not dismiss the mtCOI results out of hand given that (in my experience) this bar code is almost always spot on in pinpointing identities of hydrobioid snails.”
So that’s where the question sat, when our little Lake St. Clair hydrobiid working group adjourned sine die on the morning of 28Aug15.  In retrospect, I understood Bob to be referring to Marstonia lustrica, which is indeed “widely distributed in the Great Lakes region.”  But I myself had ruled out Marstonia lustrica immediately, way back in May, because (as Bob observed) the shell morphology of our unknowns is much “more elongate.”  And lustrica has a larger adult size as well.  The Lake St. Clair unknowns don’t look any more like Marstonia lustrica than they look like Potamopyrgus.

Ah, but there is a second Great Lakes species of Marstonia for which the descriptor “widely distributed” does not come to mind, but which fits the modifier “obscure” as well as any creature on Noah’s Ark.  What diminutive denizen of the benthos might lurk forgotten in the inky darkness of Lake St. Clair?  Tune in next time!


[1] See You In Milwaukee? [30Mar15]

[2]  Potamopyrgus has not been reported from Lake St. Clair as of this writing, however.  See the USGS Nonindigenous Aquatic Species Database.  [USGS-NAS]

[3] I am sure that our good buddy Bob meant to write Pleurocera here, rather than the obsolete nomen “Elimia.”  And surely Bob’s observations in this regard must have been addressed to our EPA colleagues, not to yours truly, who has dedicated much of his career to the Pleuroceridae, and has 19 papers published in the peer-reviewed literature on the subject, and would know a juvenile Pleurocera if he saw one.  Surely.

Wednesday, December 16, 2015

The Many Invasions of Hilton Head

The history of the oblong, 109 kmpatch of South Carolina coastland now identified as “Hilton Head Island” is brief, but eventful.  During the glacial cycles of the last million years, the pricey patch of beachfront real estate between Beaufort and Savannah has been alternately inundated and dewatered well inland, only upon rare and fleeting occasions, evolutionarily speaking, presenting itself as an island.  But by 1663, at initial entry into the logbook of Capt. William Hilton [1], it had acquired the topography typical of a Carolina-Georgia “sea island,” separated from the mainland by winding estuaries and extensive Spartina marshes.

The front of such a sea island is typically decorated with a broad beach of white sand, backed by parallel dunes between which water collects, yielding pools of varying persistence and freshness.  Grasses and palmettos on the front dunes yield to live oaks decked with Spanish moss in the island interior, which together collect every photon of light, leaving very little understory vegetation.  The gastropod fauna, both terrestrial and freshwater, can be surprisingly diverse.

Bombardment of Ft Walker
But between 1700 and 1860 Hilton Head Island was entirely deforested and converted to intensive row-crop agriculture [2].  The first successful crop of long-staple “sea island cotton,” legendary for its silky texture, was harvested from Hilton Head in 1790.  At the outbreak of the Recent Unpleasantness, there were over 20 working plantations on Hilton Head Island, and all surface water on the island ditched, diked, dammed, and carefully controlled.

The Union seized Confederate Fort Walker, located at the northern end of Hilton Head Island, in the Battle of Port Royal on November 7, 1861.  The massive amphibious invasion was co-commanded by Gen. Thomas W. (Tim) Sherman and Admiral Samuel F. Du Pont.  During four years of military occupation, the island population swelled to exceed 40,000 troops [3], camp followers, and freed slaves.  After the war the economy of the entire region was shattered, and the island gradually returned to forest.  By the 1950s the population of Hilton Head had dipped as low as 300 residents, essentially all the descendants of freedmen [4].

If the first invasion of Hilton Head was agricultural, and the second military, the invasion that stepped off in the summer of 1956 was motivated by the allure of cheap real estate.  For on May 19, 1956 the James F. Byrnes Bridge was dedicated to admit automobile traffic from the mainland.  And in 1957, real estate developer Charles E. Fraser formed “Sea Pines Company” and began subdividing residential properties on the island for sale.

It is difficult to overstate the importance of Sea Pines Plantation as a model for commercial
"Harbour Town" at Sea Pines
development, both elsewhere on Hilton Head and all along the Carolina-Georgia coast.  In the next thirty years the island was partitioned into a dozen gated communities with private beaches, golf courses, yacht harbors and tennis clubs.  Elaborate networks of roads, thousands of lavish houses, and the services to support them cannot be constructed without extensive recourse to the bulldozer.  But to the extent possible, Charles Fraser and the developers who followed him endeavored to maintain at least the appearance of nature, as that noun had come to be locally understood, given invasions #1 and #2.  They left some trees, anyway [5].

So last month I made passing reference to the interest of my colleagues at the Department of Natural Resources in the recent invasion of South Carolina by Pomacea apple snails [6].  I was first contacted by Ms. Elizabeth Gooding, a Wildlife Biologist I working on the SCDNR Pomacea project back in February of 2015.  And I was most gratified to discover, even at that early date, that Ms. Gooding and her colleagues were interested in our entire freshwater gastropod fauna, not just the Pomacea.  The study design called for a stratified random sample of 100 ponds across the five coastal counties of South Carolina [7].  Given that only four populations of Pomacea have (to this date) been documented in the Palmetto State, Ms. Gooding and her colleagues realized that they were setting themselves up to shoot a lot of blanks.  Thus the study plan that matured as a solution to the evils of invasion-biased oversampling may have been born as an antidote for boredom.

Ms. Gooding and I kept in touch as the 2015 field season progressed.  I also enjoyed getting to know Ms. Tiffany Brown, an undergraduate who worked on the project during the summer.  It was upon a 7/27 email from Ms. Brown that our story now turns: 
Good afternoon Dr. Dillon, I request your assistance one more time in identifying these freshwater snails. The snail labeled K (in the attached jpeg) is interesting because referring back to your guide and dichotomous key, this doesn't seem to be a South Carolina snail and was found in a Beaufort County pond.
Tiffany’s “Snail K” is the old world thiarid Melanoides tuberculata, of course, invasions of which are well-documented in Florida, Texas, and scattered about the American West, but heretofore unknown in South Carolina [8].  I wondered immediately whether her collection might represent an established population or a singleton aquarium refugee.  So I replied requesting more complete locality data, and inquiring about her sample size.  Ms. Brown answered that her sample size was N = 1.  And her sample pond was on Hilton Head Island.

M. tuberculata, Hilton Head
Most of the private developments on Hilton Head today garrison both an outer checkpoint to protect the well-to-do from the unwashed masses, and inner checkpoints, to protect the genuinely wealthy from the merely well-to-do.  Although the coordinates sent to me by Ms. Brown showed the SCDNR sample sited deep within the second line defenses of a jealously-guarded enclave called “Palmetto Dunes,” satellite imagery suggested a connection through a series of ditches and moats to a sample point that might be more lightly defended.  I resolved to attempt an invasion of my own.

Saturday, 22Aug15, was S-day.  I was unwittingly waved through the first line of defenses by young men wearing orange vests, apparently assuming I was attending some public event of which I knew nothing.  I then parked in the complex of members-only tennis courts and club houses, donned my camo, and proceeded by footpath to a moat running between the parking-for-guests-only Marriott and a machine gun nest guarding the Palmetto Dunes keep.

As my eyes adjusted to the dim light in the ditch under the jungle of bayberry and greenbrier, I was able to distinguish three things: water, sand, and Melanoides tuberculata.  The snails were grazing at densities around 100-200/m2 through a fine layer of organic sediment all over the coarse sand bottom.  All ages and size classes represented – clearly an old and well established introduction.  The water was brackish to the taste, although I had no equipment to measure salinity with me that particular afternoon [9].  I also found hydrobiids very common on the dead leaves and woody debris, which I took to be Littoridinops.  I cast about for perhaps 10 – 15 minutes looking or other freshwater gastropods, but given the salinity, was not surprised by the absence of any additional species.  I was in and out unscathed in 30 minutes.

But any hope I might have harbored that a single pinpoint strike could adequately sample so complex a biota as that of Hilton Head was doomed to disappointment.  The hydrobiids I collected from the organic debris in that brackish ditch were not Littoridinops.  The little sample that spilled into the dish under my dissecting scope Monday morning demonstrated a Promethean diversity of shell morphology – short & fat, tall & skinny, dark & pale.  Some shells even bore crenulations or short spines on the whorl shoulders, reminiscent of (even surpassing!) the shell polymorphism one sometimes sees in Potamopyrgus.  The females bore embryos in a brood pouch, again as in Potamopyrgus.  But males were well-represented, bearing a cochliopine penial morphology, like Littoridinops.  I had stumbled upon a population of the hydrobiid genus Pyrgophorus.
Pyrgophorus parvulus, Hilton Head

The natural range of Pyrgophorus is usually given as the Caribbean rim: Cuba, the Lesser Antilles, Venezuela, Mexico, Texas and Florida [10].  As one might expect for a population of snails bearing such diverse shell morphology, the literature includes over 40 specific nomina assigned to Pyrgophorus, Hershler & Thompson [11] “uncertain if more than just a few of these are valid or if only one should be recognized.”  The oldest name available on the list is Pyrgophorus parvulus, described by Guilding from the Caribbean Island of St. Vincent in 1828.

My SCDNR colleagues were most interested to hear the news of not just the one, but two exotic freshwater gastropod populations on Hilton Head.  And together we began to plan additional expeditions, more heavily-reinforced than my commando raid of 22Aug15.  In an SCDNR vehicle, one can (generally) obtain access to even the holiest sanctums of Hilton Head with a simple declaration of intent.

On S2-day, 5Nov15, Elizabeth Gooding and I established that the Melanoides and Pyrgophorus populations extended throughout most of the brackish ditches and ponds of the Palmetto Dunes development.  We also discovered that the ponds and ditches of the Shipyard Plantation and Long Cove developments neighboring to the immediate South and West were fresh – apparently isolated from the Palmetto Dunes system by low dikes of some older vintage.

It was in a shallow pond by the main road through Shipyard Plantation that Ms. Gooding and I discovered a large and dense population of yet another freshwater gastropod invader, Biomphalaria havanensis.  The natural range of Biomphalaria (listed as either obstructa or as havanensis, see note 12 below) was given by Malek [13] as Texas, Louisiana, Florida, Mexico, Puerto Rico, and Cuba.  The FWGNA database contained but two previous records of Biomphalaria in southern Atlantic drainages, a Charleston population I documented [14] in 1992 (now perhaps extinct?) and a 1960 record from McIntosh County, Georgia, that I have been unable to confirm.  Hilton Head Island may today be home to the only viable Biomphalaria population north of Florida.

Ms. Gooding samples Biomphalaria
The Biomphalaria were crawling at about 50 - 100/m2 on leaves, detritus, and sparse aquatic macrophytes in shallow water uniformly across the bottom of our shallow pond.  Also present was a large population of Physa acuta and lesser densities of Helisoma trivolvis, Hebetancylus excentricus and Laevapex fuscus.  Ms. Gooding and I determined that the Biomphalaria population extended through canals and roadside ditches at least 1 – 2 km beyond Shipyard Plantation into the community.

And in the Long Cove Subdivision, just across William Hilton Parkway from Palmetto Dunes and Shipyard Plantation, we discovered a dense and apparently healthy population of Bellamya japonica.  Good grief!  Bellamya introductions are not uncommon in the larger impoundments and reservoirs of the Carolina mainland, but this is the first record of a sea island population, to my knowledge.  The serpentine system of ponds we sampled in a residential section of Long Cove was also inhabited by large populations of Physa acuta, Helisoma trivolvis, and Hebetancylus, and we picked up a couple Lymnaea columella as well.

I don’t think it has appeared on anybody’s radar screen as yet, but it is my impression that the range of Hebetancylus has been expanding up from the south significantly in recent years [15].  And if you asked anybody with any knowledge of freshwater gastropods in Europe, Asia, Africa or South America, he’d tell you that our North American Physa acuta, Helisoma trivolvis, and Lymnaea columella can be spectacularly invasive everywhere else in the rest of the world.

On S3-day, 14Dec15, Elizabeth, Amy Fowler and I expanded our survey south to include Sea Pines Plantation and the Wexford subdivision.  Although we did not confirm any of our (now four!) nonindigenous freshwater gastropod populations in the quarter of the island occupied by Sea Pines, we were most impressed by the locally heavy infestations of the dreissenid mussel Mytilopsis leucophaeata, in ponds of salinity as low as 1.2 ppt.

Riding back to Charleston on the evening of 14Dec15, it occurred to me that I had spent three full field days sampling a freshwater benthic community comprised entirely of invasive species.  At some time scale, this insight is trivial.  Hilton Head didn’t even exist at the last interglacial period, so its entire freshwater and terrestrial biota must be invasive at a scale of 105 years [16].  But the gastropod community my SCDNR colleagues and I have been sampling this fall looks 102 invasive to me, and might even be 101 invasive.  If Capt. William Hilton, Gen. Tim Sherman, or Mr. Charles Fraser had left us any freshwater gastropod data, we’d have a better estimate.

It never hurts to remind ourselves occasionally that all biotas are dynamicMelanoides tuberculata and Pyrgophorus parvus turned out to be species #68 and #69 on the list of freshwater gastropods documented from the nine-state Atlantic drainage region that has been the focus of FWGNA activities thus far.  Which means that the old 67-species “Synthesis” of the distribution of commonness and rarity we published back in 2013 [17] is already obsolete, just two years later.

So if I must re-run the entire overall synthesis, it occurred to me that I might as well add the 740 fresh records that have accumulated in the FWGNA database over the last two years.  And so the bottom line for the present essay is that in the last couple months I have uploaded an almost entirely fresh “v11/15” of the FWGNA website, with new state and regional totals, new line maps, a new synthesis, and new incidence ranks [18].  I’d like to blame Hilton, Sherman, or Fraser for all this additional data churn.  But I suppose it’s just inevitable.


[1] “The Lands are laden with large tall Oaks, Walnut and Bayes, except facing on the Sea, it is most Pines tall and good.”  Read more at the Heritage Library of Hilton Head Island, here: [html]

[2] The best historical chronology I’ve found on the web is available from the Town of Hilton Head Island, here: [html]

[3] The 54th Massachusetts Infantry Regiment, made famous by the Academy-award-winning film “Glory,” was posted on Hilton Head for several months in 1863, prior to their ill-fated attack on Battery Wagner.

[4] Although set on adjacent Daufuskie Island, Pat Conroy’s (1972) memoir “The Water Is Wide” (and its Hollywood adaptation, “Conrack”) are especially evocative of this era.

[5] I met Charles Fraser in Washington in 1983, when I was a AAAS fellow, working on Section 404 of the Clean Water Act.  I remember his rising, as the first speaker at our first advisory panel meeting, to recite a verse from Sidney Lanier’s “The Marshes of Glynn.”  I don’t remember his doing of much else.

[6] I have three previous posts on our local Pomacea invasion:
  • Pomacea spreads to South Carolina [15May08]
  • Two dispatches from the Pomacea front [14Aug08]
  • Pomacea News [25July13]
[7] Nothing published as yet, but here’s a flavor of the project:
Gooding E., Brown T., Kingsley-Smith P., Knott D., Dillon R., and Fowler A. (abstract) The spread and potential impacts of freshwater invasive island snails (Pomacea maculata) in coastal South Carolina, USA.  Nineteenth International Conference on Aquatic Invasive Species, Winnipeg, CA.  (Upcoming April 10 – 14, 2016) [pdf]
[8] My regular readership will remember, however, that the USGS Nonindigenous Aquatic Species Database does contain a 2001 report of Melanoides tuberculata in coastal North Carolina.  See:
And my regular readership may also begin to perceive, dimly, what brought me to poking around in the especially untidy corner of the internet occupied by the USGS-NAS earlier this fall.  And prompted me to launch this entire series on invasive species, which shows no signs of ending, here three months later.

[9] Measurements that Ms. Gooding and I took in November from the ditch inhabited by the Melanoides and Pyrgophorus populations returned a (remarkably high) salinity of 14.5 ppt.  To the north and east, both populations extend into salinities as high as 17.9 ppt.  Zowie!

[10] Harrison, A. D. (1984)  Redescription of Pyrgophorus parvulus (Gastropoda: Hydrobiidae) from St. Vincent, St. Lucia, and Grenada, West Indies.  Proc. Acad. Natl. Sci. Phila. 136: 145-151.

[11] Hershler, R. & F. G. Thompson (1992)  A review of the aquatic gastropod subfamily Cochliopinae (Prosobranchia: Hydrobiidae).  Malacological Review Supplement 5: 1 - 140.

[12]  For many years there was a great deal of uncertainty regarding the identity of Biomphalaria havanensis at its type locality in Cuba, and hence no consensus on the identity of populations here in the USA.  But see:
  • Yong, M, Pointier J-P. & Perera,  G. (1997)  The type locality of Biomphalaria havanensis (Pfeiffer 1839).  Malacological Review 30: 115-117.
  • Yong, M., Gutierrez, A., Perera G., Durand P. & Pointier J-P. (2001)  The Biomphalaria havanensis complex (Gastropoda: Planorbidae) in Cuba: A morphological and genetic study.  Journal of Molluscan Studies 67: 103 - 111.
[13] Malek, E. (1985)  Snail hosts of schistosomiasis and other snail-transmitted diseases in tropical America: A manual. Washington, D.C., Pan American Health Organization.  325 pp.

[14] Dillon, R. T., Jr. & A. Dutra-Clarke (1992)  Biomphalaria in South Carolina. Malacological Review, 25: 129-130.

[15] My old colleague, the late Julian Harrison, reported the first South Carolina population in:
Harrison, J. R. (1989) The freshwater limpet Hebetancylus excentricus (Morelet) in South Carolina (Abstract).  ASB Bulletin 36(2): 110.
[16] Amy Wethington and I demonstrated this phenomenon more rigorously in:
Dillon, R. T., Jr., and A. R. Wethington (1995) The biogeography of sea islands: clues from the population genetics of the freshwater snail, Physa heterostropha. Syst. Biol. 44: 400-408.  [pdf]
[17] I posted two essays describing the 2013 FWGNA “Synthesis” in considerable detail:
The 11/15 version of this analysis (currently online) features a somewhat enlarged data set, but is otherwise identical in approach.

[18] See last month’s formal announcement for additional details: