Dr. Rob Dillon, Coordinator

Tuesday, September 7, 2021

Intrapopulation gene flow: King Arthur's lesson

A frequent visitor to the Malacology Department at Academy of Natural Sciences, during the sweet gauzy years of my graduate education in Philadelphia, was a charming scholar of aristocratic bearing named Prof. Arthur J. Cain [1].  Prof. Cain made his reputation on the study of color polymorphism in the European land snail Cepaea, publishing several very influential papers on the subject in the 1950s and early 1960s [2].  He was a wellspring of stories about E.B. Ford and the good old days of “Ecological Genetics,” stomping about the English countryside, collecting data on the frequencies of the myriad color morphs of Cepaea and their correlation with temperature, ground cover, predation, and so forth [3]

Arthur J. Cain (1921 - 1999)
King Arthur once remarked, “Since the term was coined by the founding fathers of the modern synthesis – I don’t know by whom, and it does not matter – nobody has ever imagined that the concept of panmixia might apply to a natural population of gastropods.”

So I had arrived in graduate school knowing that I wanted to focus my research on the phenomenon of speciation in freshwater snails.  And it was crystal clear to me that in order to understand speciation, I had to understand population divergence.  And in order to understand population divergence, I had to understand genetic variation within populations.  And in order to understand genetic variation within populations, I had to understand all those things that Arthur Cain loved to talk about – barriers to dispersal, isolation-by-distance, gene flow and the lack thereof.  In short, why freshwater gastropod populations are not panmictic.  That was the first thing.

Like a good student, I started in the library.  The literature on intrapopulation gastropod dispersal was mind-bendingly huge, even when I first dug into it in the late 1970s.  So I focused on the movement of freshwater gastropods in lotic environments, which was the most directly relevant to my research interest in pleurocerid populations in the Southern Appalachians.  And the first impression I got was the universal tendency for freshwater snails to actively disperse upstream, into currents.  And the second impression I got was the equally-universal likelihood of passive dispersal downstream, by wash-down.  Both of these phenomena are functions of current speed.

The oldest paper in the yellowing folder I still have filed under “Intrapopulation Dispersal” in the cabinet to the right of my desk also turned out to be among the most relevant to my subsequent dissertation research with Pleurocera (“Goniobasis”) proxima – a 1952 study of the Campeloma population inhabiting a small creek in Michigan.  Bovbjerg [4] observed striking aggregations of his large-bodied, burrowing study organism downstream from rocky riffles, which they seemed to have difficulty traversing.  His mark-release study, conducted over six days in a long, sandy region without such obstructions, returned a (surprisingly high) mean upstream dispersal rate of 350 cm per day OTSIWATFA [5], with no downstream dispersal whatsoever.

So the increased population densities Bovbjerg observed downstream from riffles would seem to be consistent with the opposite of dispersal… a barrier, right?  Exactly analogous to the grassy fields and gravel roads that Arthur Cain and the ecological geneticists focused so much of their attention upon in the English countryside, yes?  Back in 1978, I marked Bovbjerg’s paper with a little yellow sticky-tab.
On your mark!  Get set...

Marked with an orange sticky-tab in my intrapopulation dispersal file was a section from the 1978 dissertation of Mancini, involving a Pleurocera (“Goniobasis”) semicarinata population inhabiting a small Indiana stream [6].  Between September 1974 and January 1976, Mancini performed 10 release experiments, each two months in duration, involving 100 marked snails.  The fourth and fifth columns of the author’s Table 7 (reproduced below) show net migration, combining both upstream and downstream movement.  The mean over all five summer values was a modest 4.0 cm per day net upstream movement OTSIWATFA, and the winter mean a very modest +1.9 cm per day.

At the top of my intrapopulation dispersal stack, marked with a red sticky-tab, was the 1966 study of Crutchfield [8] on the Pleurocera proxima population of a North Carolina stream – the same study organism I had targeted for my own dissertation research, in exactly the same environment.  His was a single-release study of 15 week’s duration, between late December and early April of 1958.  Of the 53 snails Crutchfield marked in December, only 5 could be relocated in April, a result the author correlated to rains and high water.  All five snails had moved upstream, at a median distance of 55 feet (16.0 cm/d OTSIWATFA), ranging from 20 feet (5.8 cm/d) to “>100” feet (>29 cm/d)

Perhaps unsurprisingly, however, the best studies of freshwater gastropod dispersal published (as of my years in graduate school) focused on the medically-important planorbid Biomphalaria [9].  Adult Biomphalaria are adapted for lentic waters, not lotic, bearing clunky, bulbous shells typically enfolding an air bubble.  But the mark-release experiments of Paulini (for example), conducted in a ditch with a gentle current of 10 cm/sec, yielded a mean upstream dispersal rate of 130 cm/day OTSIWATFA.  After 6 days, the population mode was 15 meters upstream from Paulini’s release point.

In higher currents, the likelihood of dislodgement seems to become quite significant in Biomphalaria.  Pimentel’s mark-release experiment in a rapid Puerto Rican stream returned a net downstream transport that initially averaged 55 cm/day OTSIWATFA, as against only 36 cm/day upstream, for his first week of observation.  The same downstream-bias continued until observations ended at day 42, although at about half the initial rate.

The most refined study of freshwater gastropod dislodgement of which I am aware is Dussart’s [10] comparison of Biomphalaria with six other pulmonates, conducted in a transparent pipe.  He introduced each snail into a gentle current, allowing them to crawl directly on the PVC walls, and (of course) they all oriented upstream.  Gradually increasing the flow rate, he recorded the time at which each snail became dislodged and calculated a peculiar statistic (cubic centimeter minutes) measuring the total flow to which each snail had been exposed.  The most important variable predicting mean detachment flow (in cc.m) turned out to be profile area of the shell, rather than overall mass or foot size.

From Mancini [6]

One of the more memorable field experiences of my undergraduate education at Virginia Tech took place over the 24 hours I spent with a graduate student named Jim Kennedy sampling macroinvertebrate drift from the New River bridge in Fries, Virginia.  Jim had rigged a battery of plankton nets to suspend in the water column under the bridge, and we checked them every hour through one very cold winter afternoon in 1977, the night, and morning that followed. 

Freshwater gastropods are recovered from such nets at a surprisingly high frequency.  The most spectacular case of which I am aware was documented by P. C. Marsh [11], working in a small Minnesota Creek draining 17.6 square kilometers of extensively-ditched agricultural lands.  Marsh reported collecting at least a few Physa [12] in his drift nets at each of 10 sampling periods over the course of 20 months.  Heavy rains and high discharge seem to have been responsible for a peak mean of 326 snails/net/day on one sample Marsh collected in early April. 

But in late May a second peak in snail count was observed, this unaccompanied by rain.  Marsh’s average catch of 1,398 snails/net/day translated to an eye-popping 533,000 snails/cubic meter discharge/second, averaged over the entire day of observation.  This figure represented at that time (and may still be) the highest macroinvertebrate biomass ever documented from stream drift sampling.  Marsh speculated that this drift even may have been triggered by crowding and competition in the rapidly-growing Physa population upstream.

Marsh’s literature review found three studies of macroinvertebrate drift published between 1944 – 1980 containing quantitative data on gastropods [13].  Sitting in my carrel at the University of Pennsylvania Library, I was able to add a 1981 paper by Waters [14] and an excellent study by McKillop and Harrison [15] on the Caribbean Island of St Lucia, published in 1982.

New River Bridge at Fries, VA [16]

McKillop and Harrison positioned standard dip nets at the outfalls of several small, enclosed dasheen (or taro) marshes, recording total captures at 06:00 and 18:00 hours for 11 days during the month of April [18].  Summing all 11 samples taken at 18:00 hours, the authors recorded 29 individual Biomphalaria glabrata and 40 individuals of the little hydrobioid Pyrgophorus parvulus.  Totaled across all samples drawn at 06:00, McKillop and Harrison tallied 59 B. glabrata and 114 P. parvulus.  The authors compared the size frequency distributions of drifting Biomphalaria and Pyrgophorus to those of the resident populations and found that drift contained significantly increased frequencies of the smallest size classes.

The data of McKillop and Harrison demonstrate one of the best-documented characteristics of macroinvertebrate drift, diel periodicity.  At night, especially on the new moon, stream organisms actively enter the water column at an increased frequency.  So as was the case with the Physa population in the Minnesota ditches, the data of McKillop & Harrison suggest that at least some of the Biomphalaria and Pyrgophorus inhabiting St. Lucia marshes intentionally release into the water currents.

For eleven years, ecology students at the University of Cape Town, South Africa [19] sampled five sites along the 11 km Liesbeek River nearby.  The first two sites were near the mountainous source of the river, showing average March current velocities of 61 cm/sec and 47 cm/sec.  Sites 3, 4, and 5 were approaching the coast, with average March current speeds of 41, 30, and 13 cm/s, respectively.  The students first collected invasive Physa acuta at site 5 in 1979.  By 1980 the population had spread 3.1 km upstream to site 4, and in 1981 it first appeared at site 3, another 1.8 km upstream.  Thus, over two years, P. acuta displayed a minimum net upstream movement of 4.9 km, or 671 cm/d.  Active dispersal cannot account for such a pace.

The Physa population became established at sites 3, 4, and 5 over the next seven years, in some places reaching densities in the hundreds per square meter, but as of 1988 had not spread upstream to site 2.  These observations are all consistent with an hypothesis that the Liesbeek River Physa population has relied on avian transport, bait bucket hitchhiking, or some similarly passive agency for upstream dispersal.  My readership is referred back to my 2016-17 series of essays on the arial dispersal of freshwater gastropods [20] for further development of this interesting theme.

The bottom line for this month is that the gastropod populations inhabiting lotic environments move.  Their movement upstream is slow but measurable, while their movement downstream is rapid and episodic.  What are the evolutionary consequences?  Might these two processes be sufficient to maintain panmixia, contrary to King Arthur’s lesson?  Tune in next time.


[1] For more about this “polymath, and one of Britain’s leading evolutionary biologists,” see:

  • Clarke, B.C. (2008) Arthur James Cain, 25 July 1921 – 20 August 1999.  Biographical Memoirs of Fellows of the Royal Society 54: 47 – 57.

 [2] My favorites from Arthur Cain’s bibliography:

  • Cain, A.J. and P.M. Sheppard (1954) Natural selection in Cepaea.  Genetics 39: 89 – 116.
  • Cain, A.J. and P.M. Sheppard (1954) The theory of adaptive polymorphism.  American Naturalist 88: 321 – 326.
  • Cain, A.J. and P.M. Sheppard (1956) Adaptive and selective value.  American Naturalist 90: 202-203.
  • Cain, A.J. and J.D. Currey (1963) Area effects in Cepaea.  Philosophical Transactions of the Royal Society Series B 246: 1 – 81.

[3] The masterful review of J.S. Jones and colleagues might fairly be said to sum up the entire research corpus of the British school of ecological genetics:

  • Jones, J.S., B.H. Leith, and P. Rawlings (1977) Polymorphism in Cepaea: A problem with too many solutions?  Annual Review of Ecology and Systematics 8: 109 – 143.

[4] Bovbjerg, R.V. (1952)  Ecological aspects of dispersal of the snail Campeloma decisum.  Ecology 33: 169 – 176.

[5] OTSIWATFA = Of The Snails I Was Able To Find Again.

[6] Eugene Mancini’s 1978 dissertation was an old-school gem packed with great observations on pleurocerid biology.  I first heard about it from Steve Chambers [7], and in March of 1981 wrote a long letter to Dr. Mancini, then working at Woodward-Clyde Associates in California, to request a copy.  He cordially complied in April.  I was so impressed by the 93 page work that I showed it to my mentor George Davis, at that time editor of Malacologia.  George agreed with me that it should be published, and so I wrote a second letter to Mancini, thanking him for his kindness, complimenting him on his work, and offering a prescription of modest edits by which we felt it could be published in Malacologia.  I never heard from him again.

  • Mancini, E.R. (1978)  The biology of Goniobasis semicarinata (Say) in the Mosquito Creek drainage system, southern Indiana.  Ph.D. Dissertation, University of Louisville.  93 pp.

[7] Steve was an important influence on my young career, and a good friend.   See

  • Fred Thompson, Steve Chambers, and the pleurocerids of Florida [15Feb17]

[8] Crutchfield, P.J. (1966)  Positive rheotaxis in Goniobasis proxima.  Nautilus 79:80 -86.

[9] My favorite references on intrapopulation dispersal in Biomphalaria:

  • Pimentel, D., P.C. White & V. Idelfonso (1957) Vagility of Australorbis glabratus intermediate host of Schistosoma mansoni in Puerto Rico.  Am J Trop Med Hyg 12: 191 – 196.
  • Scorza, J.V., J. Silva, L. Gonzalez, & R. Machado (1961) Stream velocity as a gradient in Australorbis glabratus (Say, 1818).  Zeitschrift fur Tropenmedizin und Parasitologie 12: 191-196.
  • Paulini, E. (1963) Field observation on the upstream migration of Australorbis glabratus.  Bull WHO 29: 838 – 841.
  • Etges, F.J. & L.P. Frick (1966) An experimental field study of chemoreception and response in Australorbis glabratus (Say) under rheotactic conditions.  Am J Trop Med Hyg 15(3):434-438.

[10] Dussart. G.B.J. (1987) Effects of water flow on the detachment of some aquatic pulmonate gastropods.  American Malacological Bulletin 5: 65 – 72.

[11] Marsh. P.C. (1980) An occurrence of high behavioral drift for a stream gastropod.  American Midland Naturalist 104: 410 – 411.

[12] Marsh referred to his study organism as “Physa gyrina,” but I’m sure my readership will agree with me that Physa acuta is a much more likely identification.

[13] Papers documenting freshwater gastropod drift, from Marsh [11]:

  • Dendy, J.S. (1944) The fate of stream animals in stream drift when carried into lakes.  Ecological Monographs 14: 333-357.
  • Logan, S.M. (1963)  Winter observations on bottom organisms and trout in Bridger Creek, Montana.  Trans Am Fish Soc 92: 140 -145.
  • Clifford, H.F. (1972) Drift of invertebrates in an intermittent stream draining marshy terrain of west-central Alberta.  Can J Zool 50: 985 – 991.

[14] Waters, T.F. (1981) Drift of stream invertebrates below a cave source.  Hydrobiologia 78: 169 – 175.

[15] McKillop. W. & A. Harrison (1982)  Hydrobiological studies of eastern Lesser Antillean Islands.  VII. St. Luca: Behavioral drift and other movements of freshwater marsh mollusks.  Archiv fur Hydrobiologie 94: 53 – 69.

[16] You are looking at the only straight patch of road in all of Grayson County, Virginia.  Jim and I spent 24 hours living in a van, parked down by the river at left.  About 3-4:00 PM a good old boy, driving a jacked-up Plymouth, stopped by for a chin-wag.  As he departed, he boasted that he could “get rubber in four gears” across that bridge, in the roughly 300 yards from the store at left to the mountain wall at right.  I’m not sure how he knew he could do it [17], but he did it.

[17] But I’ve got a pretty good hunch.

[18] McKillop & Harrison sampled other months, but the diel periodicity is not as dramatic, primarily because of the confounding effects of rain.  Refer to their paper directly for the entire data set.

[19] Appleton, C.C. & G.M. Branch (1989)  Upstream migration by the invasive snail, Physa acuta, in Cape Town, South Africa.  South African Journal of Science 85: 189 – 190.

[20] The phenomenon of aerial dispersal in freshwater gastropods has been reviewed on four occasions in the long history of this blog:

  • Freshwater gastropods take to the air, 1991 [15Dec16]
  • A previously unrecognized symbiosis? [11Jan17]
  • Accelerating the snail’s pace, 2012 [24Apr17]
  • Freshwater snails and passerine birds [26May17]

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.


[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]


[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.


[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]