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.

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