Dr. Rob Dillon, Coordinator





Tuesday, June 22, 2021

The American Galba: Sex, Wrecks, and Multiplex

A couple weeks ago [7June21] we reviewed, in some detail, the worldwide fauna of crappy-little amphibious lymnaeid snails that have been referred to the genus [1] or subgenus Galba or Fossaria, or otherwise lumped together under the adjective, “fossarine.”  Now before we go any further, we really must talk about sex.

Did I catch your attention?  That was a cheap trick, sorry.  What I meant is that the extent to which these populations – any of them  – are selfing or outcrossing is critical to our understanding of their evolutionary relationships.  The FWGNA Project endorses the biological species concept.  Asexual reproduction voids the biological species concept and necessitates a retreat to some sort of typological (usually morphological) species concept fraught with subjectivity.

So in recent years, our colleagues have typically relied on microsatellite DNA analysis to estimate the rate of self-fertilization in pulmonate snails.  I hate to get into the details of the technique here, because it’s a miserably-complicated pain in the ass.  Section 3 of the video above will give you some appreciation.

The important thing to know is that by trial, error, hard work and good technique it is possible to work out sets of primers that will amplify regions of highly-repetitive DNA that vary in their copy number, and hence migrate at different speeds on agarose gels, and hence can serve as genetic markers.  These “microsatellites” are inherited in Mendelian fashion, and can be used to estimate heterozygosity, a measure of self-fertilization.

Microsatellite markers are almost as good as the allozyme variants I resolved by starch gel electrophoresis through most of my career, except microsatellites are much more expensive and time-consuming to develop, and you’ll need at least one or two expendable graduate students.  Microsatellites are also more sensitive.  Highly-repetitive regions of DNA have correspondingly-high mutation rates, and with enough microsatellite loci it is possible to “fingerprint” individual organisms, for paternity testing and so forth.  Such fine-scale genetic resolution may be a good thing.  Or not.

So in 2000 a research group led by Sandrine Trouvé of Lausanne with Sylvie Hurtrez-Boussès and a host of colleagues from Montpellier reported microsatellite analysis of seven populations of Lymnaea (Galba) truncatula sampled from Switzerland [2].  They examined variance at nine microsatellite loci in 7 – 26 individuals per population, with an observed overall heterozygosity Ho = 0.029.  Given the expected heterozygosity He = 0.492, Trouvé and colleagues concluded “a reproduction predominantly through selfing certainly constitutes the main cause.” [3]

In 2017 a second microsatellite analysis was published for snails of the subgenus Galba, this involving 13 populations of Lymnaea cubensis sampled across seven Caribbean and South American countries, for a total of 359 individuals.  A Montpellier research group led by Mannon Lounnas, including Pilar Alda and anchored by Sylvie Hurtrez-Boussès [6] analyzed variance at 15 microsatellite loci, most of which demonstrated only a single allele per population.  But in those five populations of L. cubensis where the hypothesis could be tested, He was very significantly lower than Ho, again strongly suggesting self-fertilization.

Maria Dolores Bargues, Santi Mas-Coma, and our friends in Valencia had previously offered direct, experimental evidence of self-fertilization in laboratory populations of Lymnaea (Galba) schirazensis [7].  And in 2018 Mannon Lounnas, the Montpellier gang and many friends, again anchored by Sylvie Hurtrez-Bousses, published a microsatellite analysis confirming predominant self-fertilization in 18 schirazensis populations sampled from all over the world: Peru, Ecuador, Colombia, Venezuela, USA, Spain and Reunion Island, floating in the Indian Ocean off Madagascar, for heaven sake [8].  Although Lounnas and her colleagues prospected for variance at 22 microsatellite loci, 14 of their 18 schirazensis populations demonstrated only 1 allele per locus.  But in those four populations where any genetic variance was found, no heterozygotes were identified, a significant result.

Worldwide Galba [4] from Hubendick [5]

But wait a minute.  Let’s back up a couple steps.  Didn’t we just learn, in our review of [7June21], that G. schirazensis was described from Iran?  Why did the Montpellier group identify those 18 populations of crappy-little amphibious lymnaeids, sampled from all over the world, with the notable exception of anywhere in the Middle East, as Galba schirazensis?

Indeed, on what authority did the Lounnas group identify the 13 populations they sampled in 2017 as G. cubensis?  Or Trouve’s group identify their G. truncatula way back in 2000?

Both of the Lounnas microsatellite studies were squishy-calibrated by direct sequencing and comparison to sequences in Genbank.  For the schirazensis study, the DNA from some small subsample of individuals from 12 of the 18 populations were sequenced for mitochondrial COI and confirmed by 99-100% homology with Iranian sequences deposited in Genbank.  Not ideal, admittedly, but OK.  For the cubensis study, one or two individuals were sequenced at the (nuclear) ITS-2 region from 9 of the 13 populations (sample size was inadequate for four populations), blasted against GenBank, and confirmed by 99-100% homology to the cloud of previously-deposited cubensis, which may have come from anywhere and everywhere, and were in any case identified by consensus of the clueless.

I suppose that Trouvé and her colleagues did not feel that their 2000 study needed standardization, since truncatula is the only Galba whose range includes Switzerland.  But for the record, Muller’s 1774 type locality was in central Germany, 500 km to the north.

We interrupt the orderly unfolding of our narrative for a brief but fiery sermon on definitions, standards, and controls.  My faithful readership will already be well-acquainted with my fixation on type localities.  When Trouvé selected a Galba population from some (unspecified) locality in Switzerland to develop her microsatellite primers, she introduced a 500 km error from Germany.  When Lounnas mixed samples from two Cuban Galba populations with a sample from Guadeloupe to develop her primers, she introduced a 0.33 x 2,000 km error from Cuba.  And when she selected a Galba population from Colombia to develop her schirazensis primers, she introduced a 13,000 km error from Iran.  The errors introduced by these decisions affect not just the individual studies in which they were made but multiply through all subsequent studies that may be built upon them, as we shall see.

So now let’s broach the subject of cross-species amplification.  In addition to testing their microsatellite markers on seven populations of nominal G. truncatula, Trouvé and colleagues [2] also tested one population of the (more distantly related) Lymnaea ovata (aka Lymnaea peregra, aka Radix balthica [9]), finding two of their seven primer pairs to amplify PCR products.  Fine.  Lymnaea ovata (or peregra or balthica) is well-characterized biologically (although not taxonomically) and is just as distinct and easy to identify in Switzerland as L. truncatula.  Its type locality is, admittedly, in France (or Prussia, or Sweden).  But the lymnaeid populations that everybody calls truncatula in Switzerland and the populations that everybody calls ovata in Switzerland are two entirely different things.  So, I’m not sure what I expected, but let’s accept a 2/7 = 29% cross-species amplification figure as reasonable.

More recently, here in the New World, Lounnas and colleagues [6] tested their cubensis primers for cross-species amplification with three other nominal species.  Their sample of the recently-described G. neotropica came from its type locality in Peru (Commendable!) and returned 100% amplification using all 15 primer pairs tested.  Their other outcross controls were considerably less controlish, however.  Their sample of viator came from Frias, Argentina, about 1,500 km north of the type locality at Viedma [10], returning 6/15 =  40% cross-species amplification.  And (if you can believe it) their sample of nominal truncatula came from Peru, at a site approximately 10,000 km W of Germany.  This drives me nuts.  And (for what it is worth) the cross-amplification was 7/15 = 47%.

Bandar Anzali, Iran [7]

What, pray tell, makes Mannon Lounnas and her colleagues identify a population of crappy-little amphibious lymnaeids collected in 2012 from the middle of Peru as the same species O. F. Muller collected in 1774 from the middle of Germany?  You guessed it: DNA squishy-calibration.  They directly sequenced one or two individuals from their Peru population at the ITS-2 locus and found 99% similarity to an (unspecified-European) truncatula sequence previously deposited in GenBank.

Lounnas and a slightly-different set of colleagues [8] also tested their schirazensis primers for cross-amplification with three other nominal species, truncatula from France, viator from Argentina, and cubensis from I-don’t-know-where.  Again, all three of these species were identified by DNA squishy-calibration, four genes this time: 18S, ITS-1, ITS-2, and CO1.  And quite surprisingly, no cross-amplification was observed using 22 schirazensis primer-pairs on DNA from any of these three other species tested whatsoever, nadda, goose egg, zip, 3(0/22) = 0.0.  Really?

Let me get this straight.  Primers that Trouvé and colleagues developed for L. truncatula showed 29% cross-amplification with the very-different Lymnaea ovata, which isn’t even in the subgenus Galba.  And 22 schirazensis primers don’t cross-amplify with any other Galba, ever?  A couple weeks ago [7June21] we learned that schirazensis populations have a peculiarly-variable radula morphology and a peculiar resistance to trematode infection.  Evidence seems to be accumulating that snails nominally identified as “Lymnaea schirazensis” may be strange.  More such evidence will be forthcoming.

The photo labelled “B” above was snapped on the bank of the Taleb Abad River at Bandar Anzali, one of the two sites in Iran from which Bargues and colleagues [7] collected their samples of Lymnaea schirazensis.  Neither site was especially near Shiraz, but in the right country, anyway.  Note how high up the bank the arrows point, even in a relatively arid environment.  I’ve inserted the Bargues figure up there in an effort, probably vain, to keep your attention in an essay that is already far too long and shows no promise of shortening any time soon.

Boring Table 1, abridged [11]
Because in 2018 Pilar Alda, Sylvie Hurtrez-Boussès, and a host of coauthors, including yours truly, published “A new multiplex PCR assay to distinguish among three cryptic Galba species [11],” an understanding of which will be necessary to fully appreciate step #2 in the three-step screening process that Pilar Alda and her 19 colleagues, including yours truly, used to screen the 161 Galba populations we’re fixing to analyze next month [12].

By 2018, looking back over three previous studies, Pili, Sylvie, and the Montpellier gang had accumulated a sum total of 9 + 15 + 22 = 46 primer pairs to amplify DNA microsatellites in crappy-little amphibious lymnaeids of the subgenus Galba.  With that list sitting on her desk, knowing which primers cross-amplified bands from other species, and the sizes of the microsatellite bands they amplified, Pili designed 11 candidate “primer mixes,” each mix including one apparently specific primer pair for each of the three previously-characterized species, truncatula, cubensis and schirazensis.

These eleven primer mixes were tested on 11 “known standards,” five representing the three previously-characterized positive species and six test-negatives, as listed in our abridged (but nevertheless still boring) Table 1 above.  Only two of the 11 “standards” were collected from their type localities [13], the other nine being identified by DNA squishy-calibration, blasting ITS-1, ITS-2, CO1, and 18S sequences to GenBank.  This introduces multi-error into an already multi-multiplex technique, but I have already decried this sin from my pulpit, so see previous.  Ultimately a primer mix was identified yielding distinctly-different microsatellite bands for cubensis, schirazensis, and truncatula, and no PCR products whatsoever for our home-grown Lymnaea (Galba) humilis, or South American viator, or South American cousini.

Screening with that “multiplex PCR assay” became step #2 in the three-step process we’re going to talk about next month.  Admittedly, it is rapid and efficient, facilitating the characterization of a much larger sample size of crappy-little amphibious lymnaeids than would otherwise have been practical.  A sample size of 1,722 snails from 161 different populations, to be precise.

Boring gel photo [11]

But a vivid drawback in the technique immediately presents itself.  Columns 7 – 12 in the gel figured  above look exactly like column #6, the negative control.  So how do we know those columns labelled viator, cousini, and humilis aren’t just plain screw-ups?

And in a larger sense, how reliable is our multiplex PCR assay?  How many assumptions is it based upon?  When the schirazensis primers (for example) were developed for a population of crappy-little amphibious lymnaeids sampled 16,000 km west of its type locality, and tested on a nominal cubensis population sampled 1,000 km north of its type locality, might those be second-order assumptions?  Assumptions squared?  Is the error 17,000 km or 1.6 x 10^7 km?

And (to take another example) when we screen by requiring no amplification for viator, we are assuming that viator is specifically distinct from cubensis, schirazensis, and truncatula, am I right?  Is that a good assumption?  Tune in next time.


Notes:

[1] The FWGNA Project has adopted the “Hubendick compromise” model for the classification of the Lymnaeidae, recognizing Galba as a subgenus of the worldwide genus Lymnaea.  In the present series of essays we have often, however, referred to the nomen Galba as though it were a genus, following the usage of the authors whose work we are reviewing.  See:

  • The Classification of the Lymnaeidae [28Dec06]

[2] Trouvé, S., Degen, L., Meunier, C., Tirard, C., Hurtrez-Boussès, S., Durand, P., Guegan, J., Goudet, J., and Renaud, F. (2000) Microsatellites in the hermaphroditic snail, Lymnaea truncatula, intermediate host of the liver fluke, Fasciola hepatica. Molecular Ecology 9(10): 1662–1664. doi:10.1046/j.1365-294x.2000.01040-2.x.

[3] Predominant (although not exclusive) self-fertilization was subsequently confirmed by several excellent studies.  See:

  • Trouve, S., L. Degen, F. Renaud and J. Goudet (2003)  Evolutionary implications of a high selfing rate in the freshwater snail Lymnaea truncatula.  Evolution 57: 2303 – 2314.
  • Meunier C., S. Hurtrez-Bousses, R. Jabbour-Zahab, P. Durand, D. Rondelaud and F. Renaud (2004)  Field and experimental evidence of preferential selfing in the freshwater mollusc Lymnaea truncatula (Gastropoda, Pulmonata).  Heredity 9: 316 – 322.

[4] This figure is a cut-and-paste of four figures from Hubendick [5] rescaled uniformly.  Lymnaea (Galba) truncatula is figure 306f from Denmark, viator is figure 324 from Brazil, cubensis is figure 310c from St. Thomas, V.I., and humilis is figure 308g from Maine.

[5] Hubendick, B. (1951)  Recent Lymnaeidae.  Their variation, morphology, taxonomy, nomenclature and distribution.  Kungliga Svenska Vetenskapsakademiens Handlingar Fjarde Serien 3: 1 - 223.

[6] Lounnas, M., Vázquez, A.A., Alda, P., Sartori, K., Pointier, J.-P., David, P., Hurtrez-Boussès, S. (2017) Isolation, characterization and population-genetic analysis of microsatellite loci in the freshwater snail Galba cubensis (Lymnaeidae). J. Molluscan Stud. 83: 63–68.

[7] Bargues, M.D., P. Artigas, M. Khoubbane, R. Flores, P. Glöer, R. Rojas-Garcia, K. Ashrafi, G. Falkner, and S. Mas-Coma (2011)  Lymnaea schirazensis, an overlooked snail distorting fascioliasis data: Genotype, phenotype, ecology, worldwide spread, susceptibility, applicability.  Plos One 6 (9): e24567.

[8] Lounnas, M., Correa, A.C., Alda, P., David, P., Dubois, M-P., Calvopiña, M., Caron, Y., Celi-Erazo, M., Dung, B.T., Jarne, P., Loker, E.S., Noya, O., Rodríguez-Hidalgo, R., Toty, C., Uribe, N., Pointier, J.-P., Hurtrez-Boussès, S. (2018) Population structure and genetic diversity in the invasive freshwater snail Galba schirazensis (Lymnaeidae). Can. J. Zool. 96: 425–435.

[9] Hubendick [5] considered ovatus Draparnaud (1805) a simple junior synonym of peregra Muller (1774) and did not consider that Linne’s (1758) nomen balthica is appropriately applied to a lymnaeid.  Subsequent European authors have disagreed.  I don’t want to get involved.  So since Trouvé identified her snails as “Lymnaea ovata,” that’s what we’ll call them.

[10] D’Orbigny gave the type locality of “Var. A” Lymnoeus viator as “oris Patagonensibus” and “Var. B” Lymnoeus viator as “provincia Limacensi (republica Peruviana).”  This was restricted to the Negro River at Viedma, Argentina by Paraense, W.L.  (1976)  Lymnaea viatrix: a study of topotypic specimens.  Rev. Brasil. Biol. 36: 419 – 428.

[11] Alda, Pilar, M. Lounnas, A. Vázquez, R. Ayaqui, M. Calvopiña, M. Celi-Erazo, R. T. Dillon, P. Jarne, E. Loker, F. Pareja, J. Muzzio-Aroca, A. Nárvaez, O. Noya, L. Robles, R. Rodríguez-Hidalgo, N. Uribe, P. David, J-P. Pointier, & S. Hurtrez-Boussès (2018). A new multiplex PCR assay to distinguish among three cryptic Galba species, intermediate hosts of Fasciola hepatica.  Veterinary Parasitology 251: 101-105.  [html]  [PDF]

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

[13] The two "known standards" we used to calibrate our multiplex PCR test that actually came from their type localities were the Argentinian L. viator and the New York population of Lymnaea (Galba) humilis.  My faithful readership will need no reminder.  But for the rest of you, see:

  • Malacological Mysteries I: The type locality of Lymnaea humilis [25June08]

Monday, June 7, 2021

The American Galba and The French Connection

I do not understand how the United States of America, the richest nation on earth, has fallen so far behind the rest of the world in organismal biology.  Our NASA and our NIH and all those bomb factories run by the DOE are all first-rate, I feel sure.  But when it comes to the biotic majesties of our purple mountains – all the living things that creep under our rocks and crawl through our waters and crowd each other for light from one of our shining seas to the other – we are clueless as Mercedes-full of Kardashians.  Adjusted by GDP, our malacology is shamed by that of Guatemala.

The French National Centre for Scientific Research (CNRS) is the largest basic science agency in Europe, with an annual budget of 3.3 billion euros.  Prominent among their 1,100 research laboratories is the Centre for Functional Ecology and Evolutionary Biology (CEFE) in Montpellier, where a faculty and staff of 282 conduct research on exactly the kinds of scientific questions that we here in America suck at.  Help us, France, you’re our only hope.

I first met Dr. Philippe Jarne of the CEFE at the Society for the Study of Evolution in Snowbird, Utah back in 1993.  But by that point we had already been corresponding for six years.  I still have in my filing cabinet an old-fashioned letter Philippe sent me in 1987, when he was doing his PhD research on the population genetics of Lymnaea peregra [1].  His research was top notch, is top notch, and always has been top notch – using a variety of freshwater pulmonates (e.g., Bulinus, Biomphalaria, Physa) to address questions about mating systems and sex allocation of great generality and importance.  It was Philippe who sent Amy Wethington and me our sample of Physa acuta from France back in 2000 [2].  And I sent him a sample of Lymnaea (Galba) cubensis from here in the Charleston area in 2009, a sample which ultimately played some small role in a paper he and the Montpellier research group published in 2011 [3].

Below is a photo that my daughter snapped at a lunch we enjoyed in Montpellier back in March of 2012, with (from my left) myself, Philippe Jarne, Patrice David, my lovely wife Shary and my French son-in-law Eric.

So on 8Apr15 Philippe emailed me to propose a new collaboration, to “estimate the selfing rate in as many species (of basommatophoran pulmonates) as possible” using a molecular method called RAD-SEQ [4].  Philippe, Tom Janicke, and their collaborators needed about 50 individuals per population from as diverse and as widespread a sample of pulmonates as possible worldwide, the idea being to correlate selfing rates with inbreeding depression.  I told him that I would be happy to help.  I logged many thousand miles on that project through the spring and summer of 2015, covering ten states, ultimately collecting N>50 individuals from 44 populations of 17 pulmonate species [5].  That research effort did not yield results.

I was disappointed by the failure of the Janicke/Jarne project, of course, and I confess, a little bit sore.  But thank heaven, at the opening of the initiative I was able to negotiate along a related line of research that ultimately did yield publishable results, which have now cast considerable light into one of the darker corners of North American freshwater malacology, those crappy little amphibious lymnaeids we here often called “Fossaria,” but which the rest of the world usually calls Galba [6].

In my enthusiastic reply of 8Apr15 I referenced Lymnaea (Galba) humilis, which I offered as a perfect example of North American ignorance regarding pulmonate self-fertilization.  Philippe then let it drop in his follow-up of 10Apr15 that “a PhD student under the supervision of S. Hurtrez here in Montpellier” (who turned out to be Pilar Alda) was even at that date working on “these small Lymnaea, including the infamous schirazensis.”  And so in an email of 13April15, I seized the opportunity to “brainstorm on a tangent.”

The Alda/Hurtrez project to which Philippe was referring was an extension of the 2011 research I mentioned five paragraphs above [3], focused on the Galba of medical and veterinary importance, primarily sampled from South and Central America and the Caribbean.  So I suggested an expansion through North America to include humilis and all the taxa that Hubendick [7] thought synonymous under humilis, but that Burch [8], following Baker [9], had considered separate.  In addition to collecting humilis/modicella from its type locality in New York, I volunteered to collect obrussa from its type locality in Philadelphia, parva from its type locality in Cincinnati, exigua from its type locality in Tennessee, and similar-looking crappy-little amphibious lymnaeid populations from a broad swath of additional muddy riverbanks across the eastern USA.  And on 5May15, Philippe, together with his colleagues Patrice David and Sylvie Hurtrez, agreed.

L. humilis on the margins of Lake Pymatuning, PA

The results of that research have just been published in the April issue of Molecular Phylogenetics and Evolution – a massive work involving 161 populations of Galba and almost as many coauthors [10].  But before I review our findings, I feel as though I should back up and introduce, or in some cases re-introduce, a cast of amphibious little crap-brown snails that has now grown worldwide in scope.  Most of the specific nomina that have been assigned to the lymnaeid subgenus Galba over the last 200 years will be unfamiliar to my North American readership.  Quite a few of those unfamiliar names will, however, become important in the essay I am planning to post next month.

Buccinum truncatulum was described by O.F. Müller in 1774 from Thangelstedt, a village in central Germany.  Populations of Muller’s crappy-little amphibious lymnaeid, which we now classify as Lymnaea (Galba) truncatula, are native to the muddy margins of ponds, rivers, and lakes across Northern Europe and Asia, reportedly extending over the Bering Land Bridge to Alaska, Yukon, and British Columbia.  The species also seems to have been introduced to South America, currently ranging from Chile through Brazil and Peru to Venezuela.  Lymnaea (Galba) truncatula serves as the intermediate host of the sheep liver fluke, Fasciola, and has been the focus of a great deal of research interest, for many years.  The first-marginal teeth on their radular ribbons bear three cusps.  Reproduction is primarily by self-fertilization [11].

The second crappy-little amphibious lymnaeid to reach description worldwide was our own Lymnaeus humilis, authored by Thomas Say (1822).  Populations of L. humilis inhabit the same sorts of marginal habitats as Old World L. truncatula, ranging throughout the United States and Canada.  I was first drawn into the bigtime world of international Galba research in early 2008, when Prof. Dr. Santiago (“Santi”) Mas-Coma of the University of Valencia contacted me about collecting some L. humilis from their type locality here in South Carolina.  Santi and his wife, Maria Dolores Bargues, together with many collaborators, had even by that early date published over 20 papers on fascioliasis and its gastropod vectors worldwide.

Thomas Say did, indeed, receive samples of crappy-little amphibious lymnaeids from the Charleston area in the early nineteenth century, and it does indeed seem likely that samples from Sullivan’s Island (at the mouth of Charleston Harbor) were before him when he described his Lymnaeus humilis.  But it turns out that all our crappy-little amphibious lymnaeids here in the Charleston area have bicuspid first lateral radular teeth, rather than tricuspid, and would today conventionally be identified as Lymnaea cubensis.  So since Say also mentioned “a variety of” his L. humilis at “Oswego” (Owego), New York, I suggested in an essay published on this blog in the summer of 2008 that Say’s humilis type locality be restricted to New York, where the crappy-little amphibious lymnaeid populations are tricuspid [12].

From Bargues et al [13]
Shortly after posting that 2008 essay I drove up to Owego, collected a topotypic batch of L. humilis, and packed them off to my buddy Santi Mas-Coma. The Valencia group then sequenced five DNA markers (CO1, 16S, 18S, ITS-1 and ITS-2) for eight of the snails I collected at that type locality, plus six cubensis individuals I collected from the Charleston area, and developed a manuscript [13], which was never published [14], which was even at that early date becoming a theme of my experience with European research groups.

But let’s back up a couple hundred years and get a fresh start at our historical narrative.  In 1825 Thomas Say redescribed the “Oswego” population as “Lymnaeus modicelles,” and added obrussa from Philadelphia and the subfossil galbana from New Jersey.  Baker [9] respelled that first nomen “modicella” and shifted it to subspecific status under humilis.

Five years later, way down south, Captain P. P. King [16] described Limnaea diaphana from collections made in the Straits of Magellan area, on the first voyage of The Beagle.  That little snail bears a shell indistinguishable from truncatula and humilis, according to Hubendick [7] and Paraense [17].  But the sequence data seem to suggest that L. diaphana is not a Galba, but rather an “archaic relict” stagnicoline [18].  So, let’s set King’s nomen aside.

The next name published in the worldwide literature of crappy-little amphibious lymnaeids was Lymnoeus viator, described by d’Orbigny in 1835 from two places simultaneously, Patagonia (exact locality unspecified) and Peru (Lima), subsequently restricted to the Negro River at Viedma, Argentina by Paraense [19].  Very similar biologically to all the other lymnaeid populations we have reviewed in this essay, the nominal range of L. viator (as conventionally understood) ranges across the bottom of South America from Chile through Argentina to Uruguay. This seems to be the oldest name [20] attached to any population of the subgenus Galba bearing bicuspid first marginal teeth on the radula.  Populations of L. viator can also serve as the hosts of Fasciola which, in some parts of the New World at least, can infect humans as well as livestock.

Shortly thereafter Pfeiffer (1839) described Limnaea cubensis from Cuba, exact locality unspecified.  Populations of this nominal species are conventionally considered to range across the entirety of South and Central America and the Caribbean, overlapping with L. viator in the south, extending into the southern United States, as touched upon five paragraphs above.  Lymnaea (Galba) cubensis is the most important intermediate host of Fasciola in the New World.  Reproduction seems almost exclusively by self-fertilization [11].  The essay I wrote reviewing Charleston-area lymnaeid populations back in 2008 featured a history of L. cubensis, both natural and otherwise, figuring shell and bicuspid radula [12], if you’re hungry for more.

So welcome back to the USA.  In 1841 our old buddy Isaac Lea [21] described Lymnaea parva from Ohio (Cincinnati), L. rustica from Ohio (Poland), L. exigua from Tennessee (unspecified) and L. bulimoides from Oregon (unspecified).  The first three bear three cusps on their first marginal radular teeth and are indistinguishable from humilis in all respects.  But Lea’s nomen bulimoides seems to be the oldest name homegrown here in the USA for crappy little amphibious lymnaeids that have turned out to bear bicuspid first laterals.
From Fig 3 of Correa et al [3]

The dawn of the 20th century saw an explosion of taxonomic activity in the American Lymnaeidae, with Pilsbry, Dall, Baker and Walker adding dozens of nomina, including perpolita, cockerelli, sonomaensis, dalli, cyclostoma, peninsulae, hendersoni, alberta, perplexa, and vancouverensis.  Baker’s (1911) review listed 30 species and subspecies of crappy-little amphibious lymnaeids from North and Middle America,  which he accumulated into Shrank’s (1803) large and inclusive genus Galba [9].  In 1928 Baker [22] split the littlest Galbas into Fossaria (Westerlund 1885), which he divided into tricuspid and bicuspid subgenera, the system essentially adopted by Burch [8].

Back to the Old World one last time, we cannot overlook, as many subsequent authorities most certainly have, Limnaeus schirazensis, described by Küster in 1862 from Shiraz, Iran.  The “infamous” schirazensis, as Philippe termed it, was resurrected in 2011 by Maria Bargues, Santi Mas-Coma, and their coauthors [23] on the basis of DNA sequence distinctions at 18S, ITS-1, ITS-2, 16S and CO1.  Our good friends in Valencia identified schirazensis populations inhabiting eight countries: Iran, Egypt, Spain, Mexico, Venezuela, Ecuador, Peru, and The Dominican Republic, the last five presumably resulting from artificial introduction.

Lymnaea schirazensis populations seem to reproduce almost exclusively by self-fertilization [11].  In habitat, the Valencia group noted that they seem to demonstrate an unusual preference for the land-side of amphibious over the water-side of amphibious.  The situation with the first marginal radular teeth also seems peculiar, “usually bicuspid” but with a “faint tendency” to appear tricuspid in some populations.  And most importantly, from a human standpoint, populations of L. schirazensis are apparently entirely refractory to Fasciola infection.  The Valencia group concluded by suggesting that confusion between refractory schirazensis and susceptible populations of truncatula, cubensis, and viator might have “distorted” data on the incidence of fascioliasis worldwide.

While Küster’s nomen schirazensis was being merely forgotten, Limnaea pictonica, described from Tierra Del Fuego by Rochebraune & Mabille in 1885, was being really most sincerely forgotten [24].  But continuing on to the present day is Jousseaume’s Limnaea cousini, proposed in 1887 to describe a population of crappy-little amphibious lymnaeids in Quito, Ecuador.  Populations of Lymnaea (Galba) cousini bear shells with an unusually-large body whorl and are not generally considered “cryptic” underneath the other crappy-little amphibious lymnaeid populations of South America.  They are also unusual in that they seem to reproduce primarily by outcrossing.  Their radular ribbons demonstrate tricuspid first laterals.  For morphology see Paraense [25], for genetics ask our friends in Valencia [26].

Detail from Baker [8] Plate VII

There were also a bunch of 20th-century nomina proposed by Pilsbry and colleagues for South American populations of crappy-little amphibious lymnaeids that I am simply not going to mention, synonymized by Hubendick [7], rest in peace, all of them.  Two more recently-described South American taxa, neotropica [27] from Lima, Peru and meridensis [26] from Merida State, Venezuela will however play minor rolls in our review next month.

In 1951 Bengt Hubendick, bless his heart, tried to impose some modern order upon this classical mess, lowering hundreds of lymnaeid nomina into synonymy worldwide [7].  In addition to the Holarctic truncatula he recognized humilis, cubensis, and bulimoides from North and Middle America, plus viator, pictonica and cousini from South America, for a total worldwide fauna of seven species of crappy-little amphibious lymnaeids that anybody today might assign to Galba.  And it was Hubendick’s clean, simple model that the FWGNA project adopted in [28Dec06].

Alas, Hubendick’s signal contribution to our understanding of the worldwide Lymnaeidae has been widely ignored.  Burch [8] advocated a seven-species, ten-subspecies, two-subgenus model for North American “Fossaria” based on the work of F. C. Baker [9, 22], and it has been under the Baker/Burch model that most of the USA has labored for 40 years.

Until now, with the help of our French brethren across the seas.  “Le bonheur de l'Amérique est intimement lié au bonheur de toute l'humanité [28].”  Tune in next time.


Notes:

[1] More recently many of our colleagues in Europe have begun referring to Lymnaea peregra as “Radix balthica.”  That the malacofauna of Europe is better known than ours here in North America does not imply that their taxonomy is more stable.  Just exactly the opposite.

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

  • To Identify a Physa, 2000 [6Dec18]

[3] Correa, A.C., J.S. Escobar, O. Noya, L.E. Velasquez, C. Gonzalez-Ramirez, S. Hurtrez-Bousses & J-P. Pointier (2011)  Morphological and molecular characterization of Neotropic Lymnaeidae (Gastropoda: Lymnaeoidea), vectors of fasciolosis.  Infection, Genetics and Evolution 11: 1978-1988.  For a review, see:

  • The Lymnaeidae 2012: Fossarine Football [7Aug12

[4] Rubin, B.E.R., R.H. Ree, and C.S. Moreau (2012)  Inferring phylogenies from RAD sequence data.  Plos One 7(4): e33394. 

[5] Not including Physa acuta!  No audience better than you, the faithful readership of my footnotes, are equipped to appreciate what a challenge it was to collect N>50 individuals from 44 populations of pulmonate snails across 10 American states, representing 17 species, not to include Physa acuta.  And to understand my frustration when that effort yielded nothing, not even an oral presentation, not even an acknowledgment.  So you want to be a scientist, kid? 

[6] The FWGNA Project has adopted the “Hubendick compromise” model for the classification of the Lymnaeidae, recognizing Galba as a subgenus of the worldwide genus Lymnaea [7].  In the series of essays that follows we will often, however, refer to the nomen Galba as though it were a genus, following the usage of the authors whose work we are reviewing.  See:

  • The Legacy of Frank Collins Baker [20Nov06]
  • The Classification of the Lymnaeidae [28Dec06]

[7] Hubendick, B. (1951)  Recent Lymnaeidae.  Their variation, morphology, taxonomy, nomenclature and distribution.  Kungliga Svenska Vetenskapsakademiens Handlingar Fjarde Serien 3: 1 - 223.

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

[9] Baker, F. C. (1911) The Lymnaeidae of North and Middle America, Recent and Fossil. Special Publication, no. 3. Chicago: Chicago Academy of Natural Sciences.

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

[11] I am currently drafting a separate essay on the subject of asexual reproduction in crappy-little amphibious lymnaeids of the subgenus Galba, to be posted in the near future.  For now, here are the key references:  Trouvé, S. et al. (2000) Microsatellites in the hermaphroditic snail, Lymnaea truncatula, intermediate host of the liver fluke, Fasciola hepatica. Molecular Ecology 9(10): 1662–1664.  Trouvé, S. et al. (2003)  Evolutionary implications of a high selfing rate in the freshwater snail Lymnaea truncatula.  Evolution 57: 2303 – 2314.  Meunier C. et al. (2004)  Field and experimental evidence of preferential selfing in the freshwater mollusc Lymnaea truncatula (Gastropoda, Pulmonata).  Heredity 9: 316 – 322.  Lounnas, M. et al. (2017) Isolation, characterization and population-genetic analysis of microsatellite loci in the freshwater snail Galba cubensis (Lymnaeidae). J. Molluscan Stud. 83: 63–68.  Lounnas, M. et al. (2018) Population structure and genetic diversity in the invasive freshwater snail Galba schirazensis (Lymnaeidae). Can. J. Zool. 96: 425–435.

[12] For a detailed review of the cubensis/humilis situation here in the Charleston area, with notes on morphology and natural history, see:

  • Malacological Mysteries I: The type locality of Lymnaea humilis [25June08]

[13] Bargues, M.D., P. Artigas, R.T. Dillon, Jr., and S. Mas-Coma (unpubl) Fascioliasis in North America: Multigenic characterization of a major vector and evaluation of the usefulness of rDNA and mtDNA markers for lymnaeids.

[14] Here’s a quote from a Mas-Coma email of 26may11: “You cannot imagine the problems originated by your sending of L. cubensis from Sullivan island to the French Jean- Pierre Pointier!!! He did publish his results before we did! […] They even used our sequences of L. humilis taken from GenBank even if we did not yet publish them. And of course they never mention that these sequences are ours, but write the article in a manner [15] that the reader believes that these sequences were made by them!!”

[15] To be fair.  Correa, Hurtrez-Boussès, Pointier and the Montpellier group did cite the GenBank accession numbers for our humilis sequences from Owego, which if one refers to GenBank, are attributed to Bargues/Mas-Coma.  It is my impression that this is a general problem with the GenBank system – secondary researchers mining and publishing research based on sequences which the primary authors may not have as yet published.

[16] King, P.P. (1830) Description of the Cirripeda, Conchifera and Mollusca in a collection formed by the Officers of the HMS Adventure and Beagle employed between the years 1826 and 1830 in surveying the southern coasts of South America, including the Straits of Magalhaens and the Coast of Tierra del Fuego Zoological Journal 5, Article 47: 332- 349.

[17] Paraense WL 1984. Lymnaea diaphana: a study of topotypic specimens (Pulmonata: Lymnaeidae). Mem Inst Oswaldo Cruz 79: 75-81.

[18] Bargues, M.D., R. L.M. Sierra, P. Artigas, and S. Mas-Coma (2012)  DNA multigene sequencing of topotypic specimens of the fascioliasis vector Lymnaea diaphana and phylogenetic analysis of the genus Pectinidens (Gastropoda).  Mem Inst Oswaldo Cruz 107: 111 – 124.

[19] Paraense, W.L. (1976)  Lymnaea viatrix: a study of topotypic specimens.  Rev. Brasil. Biol. 36: 419 - 428.

[20] Paraense suggested that d’Orbigny’s 1835 nomen be respelled as “viatrix” to agree in gender with the feminine “Lymnaea.”  In March of 2019 I asked our good friend Harry Lee for a ruling on this usage.  Harry replied that d’Orbigny’s specific epithet is not an adjective, rather “it is clear from the Latin (and French; see d'Orbigny, 1837: 340) that ‘viator’ is an appositive (an unambiguous noun; like viatrix) and should not be declined to agree with the gender of any combining generic epithet.”  Harry concluded that Paraense’s respelling is an “unjustified emendation in the language of the Code, and an unavailable name under its provisions. By coincidence, I think the same relationship holds for Lymnaea and Lymnoeus!”

[21] Lea, Isaac (1841) On fresh water and land shells.  Proceedings of the American Philosophical Society 2: 30 – 34.  Proc. Amer. Philos. Soc. II, 30 – 34.

[22] Baker, F.C. (1928)  The Fresh Water Mollusca of Wisconsin, Part I, Gastropoda.  Bulletin 70 of the Wisconsin Geological and Natural History Survey.  507 pp.

[23] Bargues, M.D., P. Artigas, M. Khoubbane, R. Flores, P. Glöer, R. Rojas-Garcia, K. Ashrafi, G. Falkner, and S. Mas-Coma (2011)  Lymnaea schirazensis, an overlooked snail distorting fascioliasis data: Genotype, phenotype, ecology, worldwide spread, susceptibility, applicability.  Plos One 6 (9): e24567.

[24] Lymnaea pictonica (Rochebraune & Mabille 1885) may be a synonym of L. diaphana.  See Bargues et al. (2012) from note [18].

[25] Paraense W.L. 1995. Lymnaea cousini Jousseaume, 1887, from Ecuador (Gastropoda: Lymnaeidae). Mem Inst Oswaldo Cruz 90: 605-609.

[26] Bargues, M.D., Artigas, P., Khoubbane, M., Mas-Coma, S., 2011. DNA sequence characterisation and phylogeography of Lymnaea cousini and related species, vectors of fascioliasis in northern Andean countries, with description of L. meridensis n. sp. (Gastropoda: Lymnaeidae). Parasites & Vectors. 4 (132).

[27] Bargues, M.D., Artigas, P., Mera y Sierra, R., Pointier, J.-P., Mas-Coma, S., 2007. Characterisation of Lymnaea cubensis, L. viatrix and L. neotropica n. sp., the main vectors of Fasciola hepatica in Latin America, by analysis of their ribosomal and mitochondrial DNA. Ann. Tropical Med. Parasitology 101:621–641.

[28] Usually translated as, “The good fortune of America is closely tied to the good fortune of all humanity.”  From a letter written home by the Marquis de Lafayette, as he sailed toward blockaded Charleston in 1777.