Dr. Rob Dillon, Coordinator





Tuesday, August 3, 2021

What Lymnaea (Galba) schirazensis is not, might be, and most certainly is

The title of last month’s post, “Exactly 3ish American Galba” [6July21], was as accurate as it was imprecise.  The focus of that essay was almost entirely on a set of populations of crappy-little amphibious lymnaeids, previously identified by a variety of Latinate binomina, now firmly united as Lymnaea (Galba) humilis, and a second set of populations of equally-crappy-little amphibious lymnaeids, also previously identified by a variety of Latinate binomina, now loosely united as Lymnaea (Galba) cubensis/viator [1].  To those two sets we added L. truncatula, unconfirmed records of which might reasonably yield either N = 2 or N = 3 American Galba, depending on future research findings in the far Northwest.  Now what is the likelihood of N = 4?

Let me begin this month’s overly-long answer with an anecdote.  Faithful readers of this blog may remember an essay I posted back on [5Aug14], reporting an expedition to the tailwaters of the Wateree Dam about 120 miles north of Charleston to document a spectacular bloom of the invasive viviparid Cipangopaludina japonica [2].  That dam was built where the Catawba/Wateree River tumbles over the broad, rocky fall line about halfway between Columbia, SC and Charlotte, NC.  And it will surprise none of my readership to learn that, although I did not mention it at the time, in addition to the Cipangopaludina, I recorded a variety of other freshwater gastropods on that long summer day in the Wateree River shallows.  I bagged five additional species, including about 6 – 8 individual Lymnaea humilis.

Lymnaea humilis populations are not common in South Carolina.  I have only documented 12, in the 40 years I have been collecting gastropods from the freshwaters of my vastly-triangular home state [3], all in the upstate and midlands, the Wateree Dam population being the southernmost recorded in my database.

So it will be remembered from my essay of [7June21] that in 2015 I struck up a research collaboration with Philippe Jarne and his colleagues in Montpellier, promising to deliver samples from as many populations of North American freshwater pulmonates as possible, focusing on crappy-little amphibious lymnaeids such as Lymnaea (Galba) humilis.  Thus, it came to pass that on 2June 15 I returned to the tailwaters of the Wateree Dam, L. humilis now at the top of my malacological shopping list.

Below the Wateree Dam

I arrived at the fisherman’s access below the dam at 10:25 AM to find the lowest water levels I had ever seen on the Wateree River – damp clay bank exposed everywhere, perfect habitat for L. humilis.  Finding no lymnaeids on the near shore, however, I launched my kayak and paddled through the channel to check the scattered islands, where I seemed to remember bagging my L. humilis the previous summer.  And indeed, I was able to relocate what I remembered as their original habitat, on the exposed bank at the head of one of the smaller islands, and was able to pick up maybe five or ten snails, when the horn at the dam sounded.  Ten sharp blasts.  Crap.

The snails were not abundant in that little habitat patch, and I felt as though my colleagues in Montpellier might be expecting as many as N = 50.  Quickly I waded around the island, scanning the exposed banks, and found no additional lymnaeids.  And I waded through the rapidly-rising waters to a second island nearby and found none.  The little patch of clay bank where I had originally discovered the population in 2014, no more than two meters long and one meter above the water level at present, seemed to be the entire habitat in which I would need to find N = 50 Lymnaea humilis, and very quickly.  I tied my kayak to my left leg and went to work.

I scanned every centimeter of my two-square-meter sample site, lifting sticks, examining leaves and debris, flipping what rocks were scattered about. Interestingly, the rising water seemed to bring the little snails up out of the sandy mud, at least temporarily, and I was able to collect a total of N = 50 in approximately 20 minutes, launch my kayak into the tide now raging around my waist, and paddle to safety.

That evening I preserved my sample of 50 Lymnaea (Galba) humilis in absolute ethanol and the next morning posted them via DHL to Montpellier, along a second sample of L. humilis from North Carolina and 15 other pulmonate populations I had collected from around the Carolinas in the previous couple months.  And in July I sent my colleagues a second batch of pulmonates, including L. humilis from VA, PA, NY, OH and TN.  And in September a third batch, including L. humilis from Michigan.  And in December of 2016, results began to arrive.  And among those results were a couple of very big surprises.

It will be recalled from last month’s post [6July21] that the Montpellier research group adopted a three-step process by which to identify crappy-little amphibious lymnaeids such as the eight populations I had sent from the USA: initially screening by morphology, then by a multiplex microsatellite methodology, then directly sequencing a subset.  So Pili Alda had screened 19 individuals from my sample below the Wateree Dam by multiplex technique [4], and found no amplification in 18 of them, consistent with an identification of L. humilis.  But she found one snail – one single individual – that cross-amplified with primers developed for the infamous Lymnaea (Galba) schirazensis, originally described from Iran, now spread to the new world.  And directly sequencing that single individual, she confirmed an ITS2 sequence match.  What the hell?

Further, Pili’s multiplex PCR screening of a sample of 27 L. humilis I collected in May of 2015 from below the Deep River dam at Coleridge, North Carolina also yielded 3 individuals cross-amplifying with the schirazensis primers, all 3 of those confirmed by CO1 sequencing.  Pili’s tests on my other six humilis populations from further north returned no surprises.

L schirazensis from Bargues et al [5]

Of course, I wrote to Philippe immediately, expressing my “extreme surprise” to hear about the schirazensis identifications, protesting that “both the NC and the SC samples appeared absolutely homogeneous when I collected them.”  Might these results be a consequence of lab error?  A mix up in sample labelling, perhaps?  Philippe replied that if he were J-P. Pointier, he would say, “Not that surprising.”

In fact, the admixture of schirazensis individuals in Galba populations identified by other specific nomina seems almost the rule, rather than the exception.  Quoting Bargues and colleagues, from their original (2011) resurrection of schirazensis [5]:

"Interestingly, in none of the aforementioned geographical zones (altitudes) did this snail species (L. schirazensis) appear to be the only lymnaeid present in the area. Its populations may appear mixed or close to populations of other… morphologically and ecologically very similar species of the Galba/Fossaria group… Thus, Galba truncatula was found in all the (schirazensis) areas studied in the Old World (Iran, Egypt, Spain). In the New World, Lymnaea cubensis shared the same areas in the Caribbean (the Dominican Republic) and Mexico, L. humilis in Mexico, and G. truncatula, L. cubensis, L. cousini and L. neotropica in South America (Venezuela, Ecuador, Peru).  Worth mentioning was that specimens of L. schirazensis sometimes appeared so mixed or close to one another with specimens of G. truncatula, that one was convinced to deal with a population of only one species."

OK, let’s back up a couple steps and review what we know about the infamous Lymnaea (Galba) schirazensis.  Originally described from Iran by Küster in 1862, the taxon was synonymized under truncatula and forgotten for many years, only to be resurrected by Maria Dolores-Bargues, Santi Mas-Coma, and their Valencia colleagues in 2011 [5].  In my essay of [7June21] we learned that populations of L. schirazensis are broadly indistinguishable from truncatula, humilis, and cubensis/viator in shell morphology, although Bargues noted some radular peculiarities, slight habitat differences, and a resistance to trematode infection.

The primary distinction highlighted by Bargues and her Valencia colleagues, subsequently confirmed by Alda and the Montpellier group [1], is in DNA sequence.  Last month [6July21] we figured four lollipop diagrams showing schirazensis just as genetically distinct as truncatula, humilis, and cubensis/viator on the basis of sequence differences at four genes, both nuclear and mitochondrial.  And in fact, judging by cross-amplification of microsatellite markers, we saw on [22June21] that L. schirazensis appears to be the most genetically distinct crappy-little amphibious lymnaeid in the entire worldwide fauna of crappy-little amphibious lymnaeids.

Bargues and the Valencia group documented populations of L. schirazensis in eight countries, you may recall, including in Mexico and several South American countries, where it seems to have been introduced.  Now Pili Alda and our friends in Montpellier report no fewer than 35 populations of L. schirazensis in the New World, including in both North Carolina and South Carolina, where it seems to be admixed with L. humilis.  And (we come to find out) schirazensis also occurs in Louisiana, where our colleagues have  discovered it mixed with a population of L. cubensis/viator in the little town of Ramah and picked up a pure population in the little town of Bedico.

It does not seem any more likely to me that the two records of schirazensis admixed with humilis in the Carolinas are any more likely to arise as a consequence of lab error than the two records from Louisiana, or the 30+ records from elsewhere in the Americas, for that matter.  The multiplex screening process by which most of these schirazensis records were initially identified is fraught with assumptions, as we saw on [22June21].  But all four US records were subsequently confirmed by direct sequencing, which should be independent [6] of microsatellite genotype.

Detail from Alda et al. [1]

So what was that singleton snail I collected in the rising Wateree Dam tailwaters that Pili Alda subsequently identified as Lymnaea (Galba) schirazensis?  First, I know what it is not.

Lymnaea schirazensis (Küster 1862) is not a specifically distinct element of the North American malacofauna.  It would be absurd to refer that one individual snail to a Latin nomen different from the 18 identical snails with which it shared its tiny, homogeneous habitat patch.  Crazy talk.

I have been wading around the waters of the United States, looking down at the snails, my entire 65 years of life on this earth.  I have seen sibling species, and cryptic species, and subspecies, and semispecies, and intrapopulation variation, and interpopulation variation, and interspecific variation, and ecophenotypic variation, in a tremendous variety of gastropod taxa, in a tremendous variety of environments.  And I have seen pure populations and I have seen mixed populations, and I have seen creek-fulls of crappy little brown snails that did not give a flying rip whether any human being could tell if they were one randomly-breeding population or twenty reproductively-isolated populations.  And that particular sample of 50 crappy-little amphibious lymnaeids I collected on 2June15 from two square meters isolated on an island in the middle of the Wateree River isolated in the middle of South Carolina constituted one, single species – not two, just one.  It is absurd to suggest otherwise.

It might be objected that L. schirazensis is distinct by virtue of its resistance to Fasciola infection.  No, there is no such thing as a “parasitological species concept.”  And don’t get any ideas [7].

Now to understand what Lymnaea schirazensis might be, I would ask my readership to click back to the last essay I posted on Campeloma [7May21], right before we changed subjects to crappy-little amphibious lymnaeids.  And I would ask you all to re-read the second half of that essay, starting with “Not uncommonly, when I am casting about for larger analogies to apply to the messy evolutionary biology of freshwater gastropods, I find myself looking toward the botanical, rather than to the zoological.” Lymnaeids of the subgenus Galba might be dandelions [8].

Botanists hypothesize that dandelions evolved in Eurasia.  They are the archetypical weed – adapted to exploit rich but unstable habitats – demonstrating superior dispersal capabilities, rapid growth, and high reproductive effort.  In May we highlighted their tremendous reproductive diversity, both sexual and asexual, with mixed populations of outcrossers, selfers, and parthenogenetic clones. 

Myriad morphological variants of dandelions, with diverse habitat adaptations and modern distributions, have historically been described under as many as 2,000 Latinate binomina and trinomina.  But in general, the botanical consensus today refers all of them to the simple catch-all nomen Taraxacum officinale, because in the final analysis, all the individual elements of that genetically-byzantine mixed population of weeds poking through the cracks of suburban driveways worldwide pretty much look the same.

I don’t care if some bored geneticist counted 10 self-pollinating lines and 20 parthenogenetic clones of yellow-blooming, blowball-sprouting weed in the school yard Sunday evening; I don’t want to hear that the grounds crew sprayed Roundup on 30 species come Monday morning.  That is crazy talk.

So the worldwide lymnaeid subgenus Galba can be seen as a bouquet of malacological dandelions, to which, over a period of 200 years, we have assigned hundreds of names.  Which, now given powerful genetic tools, we discover to be one sexually-reproducing species and four asexual lineages, the oldest names for which are truncatula, humilis, cubensis/viator, and schirazensis.  The asexual Galba lineages are cast over the face of the earth as gossamer seeds on the wind, settling in transitory habitat patches, reproducing explosively, and disappearing without a trace.

Rapidly turning-over populations of self-fertilizing Galba actually fit a dandelion model better than the perennial populations of parthenogenic Campeloma for which my model was originally proposed.  But in the penultimate sentence of my [7May21] essay on Campeloma, I promised that “We’re going to learn a lot more about phylogenetic systematics in the next few months.”  Those lessons are vividly illustrated by both Campeloma and Galba, considered together.

Here is the first thing that that L. schirazensis most certainly is:  It is a warning to any evolutionary biologist who thinks DNA sequence data will solve his problem.  It will not.  Sequence data might help but can never guide.  Gene trees are dependent variables, not independent variables.  Only if we have developed a strong model of the evolutionary relationships of some set of populations using a good, old-fashioned biological observation can DNA sequence data sets be placed in their context.

The only reason anybody ever thought that sequence data might open any doors not already cracked by old-school biological observation is that originally, from the 1990s through the 2000s, sample sizes were small, and DNA results artificially unambiguous.  The gene tree for the worldwide Viviparidae I reviewed on this blog five months ago [9Mar21] was one vivid example of such artificial unambiguity, as was the Campeloma cytB tree published by Johnson & Bragg in 1999.  Here’s a quote from my [7May21]  review of that work, “It is interesting to notice that at no point in no body of water ever sampled by Dr. Steve Johnson during his entire 16 year career did more than a single nominal species of Campeloma occur sympatrically.”  Why not?  The total sample size for the Johnson & Bragg survey was just N=54 individuals, to represent 31 populations.

But as the number of sequenced genes has increased, and the number of populations has increased, and the number of individuals has increased, and the number of tree-generating algorithms has increased, and elaborate pre-screening techniques (such as multiplex PCR) have been developed, and sample sizes have reached 1,722, we find ourselves knee-deep in the Wateree River, kayaks tangled around our legs, bent over two square meters of mud, imagining that we have discovered two species of crappy little amphibious lymnaeids, when any biologist with a high school education and three days of experience in the field can clearly see are just one.

So second, Lymnaea schirazensis is a vivid demonstration of the pitfalls of all recent efforts to redefine the word “species.” Carl von Linne’, Jean Baptist de Lamarck, Charles Darwin, Louis Agassiz, Ernst Haeckel, and ten generations of the best scientific minds the world has ever known defined the word “species” to mean an organism or group of organisms they thought distinct.  That definition was subjective, but it worked.  Science advanced.  When the architects of the Modern synthesis combined Darwin + Mendel, the definition of the word “species” was improved to an objective concept, based on reproductive isolation.  Science advanced faster.

Now the community of evolutionary science labors under the combined weight of at least five ill-conceived, typological, and embarrassingly-subjective species concepts based on DNA sequence data [7].  And lazy thinking about the evolutionary significance of sequence data has led 20 perfectly competent scientists [9] to put our names on a paper hypothesizing that there are two species of crappy-little amphibious lymnaeids on a homogeneous mud bank in the tailwaters of the Wateree Dam, 18 individuals of one and a singleton of the other.

Repent!  When the biological species concept is voided, as it is in the case of Galba by asexual reproduction, we must return to the firm foundation of morphology, not blunder onward into the slough of DNA.

Now finally, in summary.  North America is inhabited by two species of the subgenus Galba, to which we will refer from this day forward as Lymnaea humilis and Lymnaea cubensis/viator.  Those two species are distinguishable by old-fashioned shell and radular morphology.

Well, maybe threeish.  The range of L. truncatula may have extended over the Bering Land bridge into Alaska and NW Canada, but that needs confirmation, and not by DNA.  With that single exception noted, however, the benediction has been spoken, the preacher is walking back up the aisle to the portico, the choir is singing a seven-fold amen, we are done.


Notes

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

  • Exactly 3ish American Galba [6July21]

[2] In 2014 the FWGNA was referring those big invasive viviparids to the genus Bellamya, and my 5Aug14 post described a large population of “Bellamya japonica.”  Earlier this year, however, the FWGNA reassigned those populations to the genus Cipangopaludina.  See:

  • A Gene Tree for the Worldwide Viviparidae [9Mar21]

[3] "Too small for a republic, too large for an insane asylum."  South Carolina Unionist James L. Petigru, 1860.

[4] 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].  For a review, see:

  • The American Galba: Sex, Wrecks, and Multiplex [22June21]

[5] 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.

[6] Well, independentish might better describe it.  Almost all the lymnaeid populations involved in the development of the multiplex technique were initially identified by DNA sequence.  So the microsatellite data and the sequence data are not, strictly speaking, independent.  But arguable, I suppose.

[7] De Queiroz listed 11 species concepts in his Table 1, five of which are commonly applied to molecular phylogenies.  To quote Mary Poppins, “That will be quite enough of that.”  See:

  • De Queiroz, K. (2007)  Species concepts and species delimitation.  Syst. Zool. 56: 879 – 886.

[8] Scholarly journals bulge with thousands of papers on the biology of dandelions.  Here is a small selection that I found especially useful composing my two paragraphs on the subject:

  • Hughes, J. & Richards, A. (1988) The genetic structure of populations of sexual and asexual Taraxacum (dandelions). Heredity 60: 161–171.
  • Lyman JC & Ellstrand NC (1984). Clonal diversity in Taraxacum officinale (Compositae), an apomict. Heredity. 53 (1): 1–10.
  • Mogie M & Ford H. (1988) Sexual and asexual Taraxacum species. Biol J Linn Soc. 35:155–168.
  • VerDuijn, MJ, VanDijk, PJ & VanDamme, JMM (2003) Distribution, phenology and demography of sympatric sexual and asexual dandelions (Taraxacum officinale s.l.): geographic parthenogenesis on a small scale. Biol J Linn Soc 82: 205–218.

Or hell, you could just google-up the Wikipedia article, which looks fine.

[9] Including myself.  I am a sinner, saved but by Darwin.

Tuesday, July 6, 2021

Exactly 3ish American Galba

Editor’s Note – This is the third in a four-part series prompted by the recent publication of our paper on genetic relationships in the worldwide lymnaeid genus Galba [1].  You will certainly find it helpful to review my essay of [7June21] before proceeding.  My essay of [22June21] is optional, depending upon how deep into this justifiably-obscure topic you actually want to dive.

During the 2015 field season I did my best to keep the malacological pipeline between Charleston and Montpellier stuffed with samples of basommatophoran pulmonate snails, especially the crappy-little amphibious lymnaeids that we here in the USA often call “Fossaria,” but most of the rest of the world calls Galba [2].  In July I returned to the type locality of Thomas Say’s (1822) L. humilis (and L. modicella, oddly) on the bank of the Susquehanna River in the little town of Owego, NY [4].  I also visited Philadelphia to collect a topotypic sample of Lymnaea obrussa Say 1825, Cincinnati to sample topotypic Lymnaea parva Lea 1841, and Tennessee to sample topotypic Lymnaea exigua Lea 1841.  Most of the other specific nomina one occasionally sees in the literature for these little lymnaeids are more difficult to pin down geographically.  But I felt as though if I added samples from South Carolina, North Carolina, Virginia, and Michigan, genetic variation in North American populations of tricuspid fossarines should be adequately surveyed.

Topotypic L. (Galba) humilis [5]

I also sent a fresh sample of the bicuspid L. cubensis from Charleston to Montpellier, again harkening back to my [25June08] essay on our particularly-interesting local population.

And I very much enjoyed working with Dr. Pilar (“Pili”) Alda, the post-doc in the Hurtrez-Boussès lab spearheading the Galba survey.  In addition to her obvious scientific and technical skills, she turned out to be relentlessly cheery and outgoing, character traits that would serve her well dealing with the likes of me, to say nothing of her team of 18 other co-authors recruited from all over the world. According to my long-time friend Philippe Jarne, who often served as liaison, most of the field work was done by Jean-Pierre Pointier guided by colleagues “generally accompanying him for days to weeks in places where he would have a hard time going on his own, and also providing facilities (e.g. car, mules and lodging).”

Five years passed.  I knew that Pili’s analysis would involve our brand new multiplex PCR test [6], as well as more conventional morphological and DNA sequence data.  But regarding the myriad analytical details I was actually rather pleased to be out of the loop.  Finally, at long last, the paper appeared this spring in Molecular Phylogenetics and Evolution [1].

My first impression was one of marvel at the scope of the study I had imposed myself into.  Holy trash snails, Batman!  1,722 individual Galba analyzed from 161 sites in ten countries and territories, over a period of twenty years!  In addition to the 9 populations I myself had contributed (MI, OH, PA, NY, TN, VA, NC, SC-humilis, SC-cubensis), I was especially gratified to discover in the database 5 populations from New Mexico, 6 from Louisiana, 1 from Florida, 1 from Texas, and 2 from way up near Montreal.

This huge sample was first characterized by “shell morphology and reproductive anatomy,” but not by radula, which has been a source of chronic irritation to me for at least ten years now [7], but it’s beginning to look like I’ll have to let that go.  The sample of 1,420 snails not distinguished morphologically was then analyzed using our multiplex PCR technique, about which I also have misgivings, but having dwelled upon those at considerable length last month [22June21], will also set aside at this point.

From Bargues et al. [8]

A subset of the samples passing through multiplex PCR screening were analyzed by a third level of genetic characterization, direct sequencing.  Pili ultimately sequenced 151 individual Galba, mostly the nuclear ITS2 gene and the COI mitochondrial gene, with a few ITS1 and 16S sequences as well, for a total of 300 sequences.  To these 300 sequences she added 517 sequences fished from GenBank “apparently attributable to lymnaeids of the Genus Galba” as follows: 166 COI, 163 ITS2, 118 ITS1, and 70 16S sequences, from 132 New World sites and 45 Old World sites.  The Old World sample was sequenced from 14 countries, the New World sample included 7 countries not sampled in our original coverage.  This stupendous dose of sequence data – which ultimately (setting aside obvious misidentifications) totaled 796 sequences, formed the basis of the analysis we will review this month.

And I feel as though my influence was felt, to some extent, in the priority given to type localities by Pili and her research group.  In Table 1 we listed, in some cases designating for the first time ever, type localities for all eight nomina ultimately playing some role in this research: cousini, cubensis, humilis, meridensis, neotropica, schirazensis, truncatula and viator.  Do look back at the first post in this series [7June21] to refresh your memory on the biology of these eight taxa in particular.  Then for each of these eight nominal species an effort was made to designate type sequences for ITS1, ITS2, 16S and COI, to the extent possible.

But now might be a good opportunity to emphasize that my contribution to the overall research project under review here was much, much less than 1/20 = 0.05 of the effort that ultimately brought the paper to publication.  And as must inevitably be the case in any collaboration of this breadth and complexity, there are elements of the final product of which I am proud, and elements of which I am not-so-proud.

So by all means, feel free to read our introduction, which is the usual boilerplate about how the study that follows will revolutionize worldwide understanding of evolutionary science and cure a plague on the health of the third world.  And by all means, continue through methods sections 2.1 – 2.3, Figure 1 and Table 1.  Then as a personal favor to me, set our paper aside.  Please do not read the remainder of the methods section, or any of the results, or any of the discussion.  Please, I’m begging you.

From Alda et al [1].  Click for larger.

Instead, just look at the four unrooted networks above, inferred by a Baysian technique aptly called “Beast2.”  This figure was composed for a May 2019 draft of our manuscript, but ultimately split into four separate figures for publication [9].  Six colorful blobs have been marked in all four of the networks, as follows:

  • The green cluster contains the type population of L. truncatula and 102 other populations collected from Europe and South America.  Good, that’s clear.
  • The blue cluster contains the type population of L. humilis from New York, the type populations of the later-described modicella, obrussa, parva, and exigua, and 33 other fossarine populations from the United States and Canada.  These populations are united by shells bearing higher spires and more incised whorls, and by tricuspid first lateral teeth on their radula.  Super!  We're done with all that ancient Baker/Burch mess [10], finally.
  • The purple cluster contains the type populations of L. cousini, the more recently-described L. meridensis, and 35 other wide-apertured, sexually-reproducing populations of South America.  Fine, that’s clear as well.
  • The yellow cluster contains the type population of L. schirazensis and 84 other fossarine populations from Asia and The Americas.  That meaning of that result is not clear at all, and we will return to it next month.
  • The salmon-colored cluster contains the type population of L. cubensis, the more recently-described L. neotropica, and 114 other fossarine populations collected throughout the Americas.  The population of L. cubensis I collected here in the Charleston area is in that salmon blob, as well as a bunch collected from Florida, Louisiana, Texas and New Mexico.  Also included was one 16S sequence from Oklahoma labeled “Fossaria bulimoides” fished from Genbank [11].  These Galba populations bear bicuspid first marginals (as far as is known) and shells with lower spires and more convex whorls.  Well, okay, but…
  • The fuchsia cluster contains the type population of L. viator from Argentina and 17 other South American populations.  And good grief.  That fuchsia cluster is contained entirely within the salmon-colored L. cubensis cluster in three of the four networks.

There is no difference of any sort – genetic, morphological, or ecological – between the populations of crappy-little amphibious lymnaeids that d’Orbigny described as Lymnoeus viator in 1835 and the populations of crappy-little amphibious lymnaeids that Pfeiffer described as Limnaea cubensis in 1839.  All have shells bearing more rounded whorls with less incised sutures and bicuspid first marginal teeth on the radula.  The minor ITS1 sequence distinction might simply reflect geography.  Our multiplex PCR results [6] are of no help because they assume the result.  Pili selected her primer mix to amplify bands on a population of nominal cubensis but not on a population of nominal viator.  So our multiplex PCR results cannot subsequently be used to test the hypothesis that cubensis and viator are distinct.

The biological species concept is voided here by asexual reproduction.  The morphological species concept would demand that Pfeiffer’s 1839 cubensis be synonymized under d’Orbigny’s 1835 viator, since the two taxa are indistinguishable.  And from the standpoint of service to humanity, which is a very unfamiliar point for me to stand, some medical and veterinary benefit might be gained by calling all those populations of crappy-little fascioliasis vectors the same thing, rather than two different things, implying that there might be some difference in the best way to control them, which there ain’t.

But darn it.  As I have pointed out on this blog at least a couple times a year for 20 years, the binomial system of nomenclature was not proposed to reflect biological, reproductive, or evolutionary relationships among organisms.  It was proposed for the cataloguing and retrieval of information, a sort-of Dewey Decimal system for critters.  And the first commandment in the Worldview of Information is, Thou Shalt Not Lose Information [12].  And large and important literatures are now indexed under both the names “cubensis” and “viator,” which science now calls us to unite, without losing information under either tab.

North American detail from Fig 2 of [1]

OK, I’m going rogue.  Or roguer than normal, anyway.  In Figure 2 of our MP&E paper (the Galba distribution maps, modified above) we referred to the entire cluster ranging from Carolina to Argentina as “cubensis/viator.”  Henceforth, therefore, the FWGNA Project will refer to crappy-little amphibious lymnaeids with bicuspid first laterals and less-incised shell whorls with the slash-name: “Lymnaea (Galba) cubensis/viator Pfeiffer 1839/d’Orbigny 1835” [12.5].  I am not aware of any law in the international code of zoological nomenclature explicitly prohibiting such an admittedly awkward/vivid Latinate amalgam, but if there is, the FWGNA don’t need no stinking badges [13].  Never have.

Then here on the mudbanks of North American fresh waters, we host two common and widespread species of crappy-little amphibious (“fossarine”) lymnaeids, the tricuspid Lymnaea (Galba) humilis Say 1822 and the bicuspid Lymnaea (Galba) cubensis/viator Pfeiffer 1839/d’Orbigny 1835. Junior synonyms of L. humilis include, but are not limited to: cyclostoma Walker 1908, exigua Lea 1841, galbana Say 1825, modicella Say 1825, obrussa Say 1825, peninsulae Walker 1908, parva Lea 1841, rustica Lea 1841, and tazwelliana Wolf 1869.  Junior synonyms of cubensis/viator include, but are not limited to: alberta Baker 1919, bulimoides Lea 1841 [11], cockerelli Pilsbry & Ferriss 1906, dalli Baker 1907, hendersoni Baker 1909, perplexa Baker & Henderson 1929, perpolita Dall 1905, sonomaensis Hemphill 1906, techella Haldeman 1867, and vancouverensis Baker 1939.

Now before we go any further, I feel called to introduce a boxed essay on the concept of “cryptic species.”  Almost all freshwater gastropods are cryptic to almost everybody, I suppose.  Crypsis is in the eye of the beholder.

So the human population of the world is roughly 8 x 10^9.  And the total number of beholders who will ever read the present essay, if I am optimistic, might approach 4 x 10^1.  I am here to re-assure you, the  0.000000005 fraction of the world’s population who constitute my readership, that Lymnaea humilis and Lymnaea cubensis/viator are not entirely cryptic.

The whorls of the shells borne by cubensis/viator are typically more rounded with suture lines less impressed than the whorls of the shells borne by humilis.  Look at Santi Mas-Coma’s figure of the (tricuspid) L. humilis from its New York type locality way up at the top of this essay [5].  Now look at his figure of L. cubensis/viator further down [8].  You, my N = 4 x 10^1 readership, will not usually get populations of those two subsets confused, I feel confident.

Is the shell morphology demonstrated by every snail in every humilis population always distinctive from the shell morphology demonstrated by every snail in every cubensis/viator population?  No, certainly not.  Refer back to Anna Correa’s figure [7] in my essay of [7June21].  But the two Galba species are just as distinctive as Physa acuta and Physa gyrina, or (sometimes) Lymnaea elodes and Lymnaea catascopium [14].  If you, my readership, know what to look for, you will probably make the right identification.

From Baker [15] and Clarke [16]
The reason I am making such a big deal of the shell morphological distinction between humilis and cubensis/viator here is that there is traditionally understood to be a third species of crappy-little amphibious lymnaeid ranging across a narrow, frigid strip of North America, Lymnaea (Galba) truncatula.  The data are shell-morphological only.  We have no genetic evidence one way or the other.

Baker/Burch [10] gave the North American range of L. truncatula as, “portions of Alaska and Yukon Territory.”  Baker himself, however, never laid eyes on an American truncatula.  The shells figured on his plate XXVII were collected from England.

Baker’s American truncatula records seem to have come almost entirely from William Healey Dall [17].  And the challenge is that Dall also recorded populations he identified as Lymnaea (Galba) galbana from Alaska and The Yukon, which we now understand to be a junior synonym of humilis.

Similarly, Arthur Clarke [16] identified truncatula from a small patch of the southern Yukon, as well as from a second patch of southern British Columbia [18] about 1,000 miles south.  But again, Clarke also reported populations of crappy-little amphibious lymnaeids he identified as exigua, ferruginea, modicella, and parva widespread and common throughout the entirety of western Canada, all of which Clarke distinguished minor aspects of shell morphology.  Here’s how Clarke characterized L. truncatula:

“the strongly rounded whorls (shouldered in many specimens), solid shell, deep and partly obscured umbilicus, and general appearance are such distinctive features that identification of this species [truncatula], once seen, can confidently be made.”

To the extent we credit the finely-honed conchological perspicacity of illustrious forefathers such as Dall, Baker, and Clarke, we must admit a third species of crappy-little amphibious lymnaeid to the North American fauna, Lymnaea (Galba) truncatula (Muller 1774).  Viewed from the other side of the Bering Sea, however, Larisa Prozorova [19] listed L. truncatula as a “question mark” in North America.  Her assessment of the situation sounds fair to me.

So here at the end of yet another overly-long essay on yet another justifiably-obscure topic, we find ourselves circling back to the title.  Lymnaea humilis plus L. cubensis/viator certainly sums to two species of Galba inhabiting North America.  Confirmation of Lymnaea truncatula would add a third.

But wait.  Can we back up to that fourth bullet-point way up above?  What’s this business about “The yellow cluster contains the type population of L. schirazensis and 84 other fossarine populations from Asia and The Americas?”  The Americas, pleural?  Might “the infamous schirazensis” complicate our math yet further?  Tune in next time.

Postscript added 9July21 - Our colleague Sam Loker has just published an excellent paper online (open access), figuring shells from 14 North American populations of Galba humilis, including all 8 of the populations I contributed to the Alda et al effort.  See:

  • Loker, Dolignow, Pape, Topper, Alda, Pointier, Ebbs, Sanchez, Verocai, DeJong, Brant, and Laidemitt (2021) An outbreak of canine schistosomiasis in Utah: Acquisition of a new snail host (Galba humilis) by Heterobilharzia americana, a pathogenic parasite on the move.  One Health 13: 100280. [html]


Notes:

[1] 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]

[2] 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 [3].  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]

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

[4] Long-time readers will remember the humilis/cubensis confusion that I resolved by restricting Say’s type locality to New York.  For the rest of you, see:

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

[5] This figure is taken from my unpublished collaboration with Prof. Dr. Santiago Mas-Coma and his colleagues in Valencia:

  • 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.

[6]  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].  For a review, see:

  • The American Galba: Sex, Wrecks, and Multiplex [22June21]

[7] 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]

[8] This is figure 7 from Bargues, M.D., J.B. Malandrini, P. Artigas, C.C. Soria, J.N. Velasquez, S. Camevale, L. Mateo, M. Khoubbane, and S. Mas-Coma (2016)  Human fascioliasis endemic areas in Argentina: multigene characterization of the lymnaeid vectors and climatic-environmental assessment of the transmission pattern.  Parasites and Vectors 9:306.  DOI 10.1186/s13071-016-1589-z

[9] Figures S4 – S7 in the supplementary material ultimately published show painfully-detailed tree-form phylogenies for each of these four genes separately if you are curious about the fine structure.

[10] 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).

[11] Our synonymization of bulimoides Lea 1841 under cubensis/viator is especially tentative.  Hubendick [3] considered the nomen valid, to distinguish a rotundly-shelled fossarine ranging from the Mississippi River broadly across the American west.  I have no personal observations one way or the other.  I am quite certain, however, that the single 16S sequence uploaded to GenBank by Remigio, labelled “Fossaria bulimoides” but collected 2,000 miles from the bulimoides type locality in Oregon, is weak evidence, indeed.

  • Remigio, E., 2002. Molecular phylogenetic relationships in the aquatic snail genus Lymnaea, the intermediate host of the causative agent of fascioliasis: insights from broader taxon sampling. Parasitol. Res. 88, 687–696.

[12] I developed the idea of duality between the Worldview of Information and the Worldview of Science here:

  • When Worlds Collide: Lumpers and splitters [4Sept12]

[12.5] Shortly after this essay was posted, I received a comment from our good friend Bryan England inquiring why I listed cubensis before viator in the slash-construction.  The short answer is that Pfeiffer’s cubensis is the more familiar of the two names here in North America.  But if other workers would prefer to list d’Orbigny’s viator first, they should feel free.  Or by all means, use the single-slash construction that Bryan suggested, rather than the double-slash.  I am not advocating the addition of any additional rules to the code!

[13] Although an optional FWGNA badge has just recently become available.  See:

[14] The shell morphology demonstrated by populations of the two big "stagnicoline" lymnaeids we host here in North America, L. elodes and L. catascopium, is very much a function of ecophenotypic plasticity: skinny in the weeds, fat in exposed environments.  Sometimes the two species look different, and sometimes they do not.  This was the subject of an elaborate series on posts from April to July of 2012, and again from June to September of 2015.  Here's the bottom line:

  • The Lost Thesis of Samantha Flowers [3Sept15]

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

[16] Clarke, A. (1981) The Freshwater Mollusks of Canada. Ottawa: The National Museums of Canada

[17] Dall, W. (1905) Land and Fresh Water Mollusks of Alaska and Adjoining Regions. Harriman Alaska Expeditions 1899, no. 13. Washington, D.C.: Smithsonian Institution.

[18] Jacquie Lee did not confirm L. truncatula in British Columbia, however, identifying only galbana, modicella, and parva.  See:

  • Lee, J.S. (2000)  The distribution and ecology of the freshwater molluscs of Northern British Columbia.  M.Sc. thesis, University of Northern British Columbia, 238 pp.

[19] Prozorova, L. A., 1998. Annotated list of Beringian freshwater mollusks. The Bulletin of the Russian Far Eastern Malacological Society 2: 12–28.  See her Appendix 1.

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.