Looking back over my essays of the last two months [13Feb25, 11Mar25], as well as my essay of February a year ago [13Feb24], I feel as though my efforts to review the historical and technical background behind my Oregon field trip for L. bulimoides last summer have been largely successful. But there is a human context as well, to which I fear I have given short shrift.
My faithful readership will (I trust) remember our old friend Dr. Phillippe Jarne of the French National Centre for Scientific Research (CEFE-CNRS) in Montpellier [1], yes? It was Philippe who introduced me electronically to Dr. Pilar (Pili) Alda back in 2017, when she was working as a postdoc in the laboratory of Dr. Sylvie Hurtrez-Bousses at the Universite’ de Montpellier. Pili was the lead author on both our (2018) multiplex PCR paper on Galba [3] and that huge (2021) molecular phylogenetic review of the subgenus Galba worldwide [4]. She is currently at CONICET-CERZOS, the Argentinian National Research Agency in Bahia Blanca.
So, I have long harbored suspicions about the lymnaeid fauna of Western North America. Those suspicions compounded when I began working with Bruce Stephen on the Freshwater Gastropods of the Great Plains project in 2020, expecting to find Lymnaea (Galba) bulimoides in that region, finding L. cubensis/viator and L. cockerelli but nothing that matched Isaac Lea’s 1840 bulimoides types from Oregon [5] whatsoever. I promoted my suspicions to the status of an hypothesis in a pair of essays posted on this blog in February [13Feb24] and March [12Mar24] of last year, ultimately concluding that while “huge international teams” have published “mountains of research” on the evolutionary relationships among all the other hosts of Fasciola worldwide, here in the USA we have “zero authentic DNA sequences” for any population of our own L. bulimoides.
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| Lymnaea (Galba) of the American West [6] |
Shortly after that, I began swapping emails with Philippe and Pili. And over the course of the following months, from mid-March to early May of last year, a miniature online symposium developed on evolutionary relationships among the Galba populations of the western USA, ultimately growing to include Patrice David (CEFE-CNRS), Jean-Pierre Pointier (Universite de Perpignan), and Sam Loker and Martina Laidemitt of the University of New Mexico. I confess that the main item on my agenda [7] was to find somebody else – anybody else – to fly out to Oregon and collect us a bona fide, gold-seal sample of L. bulimoides. But ultimately, I found myself volunteering. And Pili already had plans to return to Montpellier in September, and volunteered to do the sequencing and analysis.
And so it came to pass that my wife and I boarded Delta Flight 954 bound for Portland on July 31, 2024, to return, five days later [8], defeated and humiliated by a basommatophoran pulmonate. See my essays of February[13Feb25] and March [11Mar25] for a painful recounting. But tucked into my shirt pocket were those N = 2 individual L. bulimoides that Ms. Courtney Hendrickson had preserved from Gahr Pond in the spring of 2023. And those I forwarded onward to Philippe and Pili in August.
And in early November, Pili sent us her results. She sequenced the same four genes that we had analyzed in our 2021 collaboration: the mitochondrial CO1 and 16S, and the nuclear ITS1 and ITS2. And her results looked like a phylogenetic shotgun blast.
For some reason I always look at CO1 first, and the closest CO1 match to our two Gahr Pond bulimoides anywhere in GenBank was 90 – 91% to Galba cousini from Ecuador. That strikingly low similarity with anything else supported my primary hypothesis, that all DNA sequences previously deposited into GenBank as “bulimoides” were spurious [13Feb24]. The result did not support my speculation (based on shell morphology) that South American L. cousini might be conspecific with North American L. bulimoides – 10% CO1 divergence is very significant in pulmonate phylogenetics [9]. But it left me with a fig leaf. The evolutionary relationship between bulimoides and cousini appeared to be closer than any other.
Pili’s 16S mtDNA results also supported my primary hypothesis. Our pair of Gahr Pond 16S sequences were around 96% similar to maybe 50 – 60 sequences already deposited in GenBank, including truncatula and “humilis” from the former Soviet Republic of Georgia, Brazilian “truncatula,” Japanese “pacifica,” junk from everywhere. Simply no bona fide matches.
One of Pili’s ITS1 sequences was too short to be informative, but the other was wildly unique – no match closer than 82% in all of GenBank.
Our Gahr Pond ITS2 results, by striking contrast, did hit close matches to a pair of sequences already in GenBank, uploaded in 2020 by the English researchers A. J. Saadi and colleagues in connection with a molecular phylogenetic study of the freshwater pulmonates [10]. Working from their laboratory at the University of Nottingham, Saadi et al. sequenced 4000 nucleotides from the rRNA gene cluster of 39 individual snails, including part of the 5.8S gene, the complete ITS2 region, and almost the entire 28S gene. They missed the ITS1 region (just upstream from 5.8S) and didn’t touch the mitochondria at all.
The closest of the close matches to our Gahr Pond bulimoides was 98.2% to a sequence from a snail that Saadi and colleagues identified as “Polyrhtis apicina,” but was subsequently emended to Stagnicola or “Ladislavella” apicina, with collection location unknown and collector unknown. Absolute garbage. Sometimes I think the NCBI should rename its GenBank to GenDump.
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| The Willamette Valley of Oregon |
But the other close match, 97.6%, was to a sequence from a snail that Saadi identified as “Bakerilymnaea bulimoides (= Fossaria bulimoides),” subsequently listed in GenBank as Galba bulimoides [11], from “Corner of Alvadore Road, Lane County, OR, USA” with the collector identified as our old buddy Dwight Taylor [13].
The corners of Alvadore Road – there are two of them about 500 m apart, actually – are just 40 km south of the Bellfountain Road site I had targeted back in August as the ideal type locality for Lymnaea (Galba) bulimoides. How a sample of bona fide L. bulimoides, correctly identified with good locality data, found its way from Dwight Taylor’s 1970s-era collection bucket into the buzzing thermal cyclers of Nottingham, England, I cannot imagine.
So, my hypothesis was confirmed, with an asterisk. In my post of [13Feb24] I had written:
“Meanwhile here in the USA, the richest country on earth, the leader of the free world, we have zero authentic sequences for any population of our own Lymnaea (Galba) bulimoides, known to be an important host of livestock fluke across the Pacific Northwest since 1929.”
That turned out to be sort-of true, but I should have footnoted, “except two rRNA sequences contributed by Saadi et al, one of which was misidentified and utterly useless.” All the other (really rather few) sequences labelled bulimoides in GenBank were spurious, most notably the 16S/CO1 pair from Oklahoma uploaded by Remigio [14, 15] that caused Correa [16] and Alda [4] and me myself a sinner [7Aug12] to hypothesize that bulimoides might be a junior synonym of cubensis/viator.
I taught freshman biology at the College of Charleston for many years, although I was never any good at it, to judge from student evaluations, which were the only criterion that mattered when the time came for my own annual evaluation. And the first lecture of the semester, which most of the students missed, because they were all still standing in line at registration or lost on campus, was “The Nature of Science, and The Science of Nature.” I kicked it off with a blanket statement that this was to be the most important lecture of the semester, and the material I intended to review was so complex and so other-worldly and so difficult to grasp that even most professional scientists never figure it out in our entire careers.
Science is the construction of testable hypotheses about the natural world. The process by which science moves forward is called “the scientific method.” But please do not confuse the product with the process. It is entirely possible to use the scientific method to determine (for example) what color box of detergent is most attractive to housewives shopping in the grocery store. Just because some guy is wearing a white lab coat, conducting experiments and collecting data in a systematic fashion, does not mean that he is a scientist. The subject matter must be the natural world.
The scientific method begins with a question, like “What is Lymnaea bulimoides?” [13Feb24] Then, good scientists do library research – a lot of it – which together with experience and intuition from a fundamental understanding of the foundations of their discipline, enable them to precisely frame a testable hypothesis about the natural world, like “there is not a worker in the field today – not malacologist, nor parasitologist, nor veterinarian, nor guardian of the public health – who could identify a bona fide Lymnaea (Galba) bulimoides if it bit him on the boot toe” [13Feb25].
Then comes the gathering of data to test that hypothesis, such the expedition to Oregon and subsequent DNA sequencing I have now spent three blog posts detailing. Then data analysis – GenBank fishing in our case – and conclusion. Hypothesis true.
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| The Scientific Method [17] |
As I delivered my lecture I was, of course, scrawling the steps of the scientific method on the chalkboard in some grossly graphic fashion. I would then suddenly turn to my students and assert that in actual practice, most of what passes for science of the present day is done backwards. Huge research groups and agencies – I usually mentioned NASA, for example – typically begin with data collection, then try to find an hypothesis within those data somehow, and then finally, maybe, go to the library to see if anybody had published anything on an hypothesis like theirs before.
It has now gotten to the point, I used to wag my finger at my half-empty lecture hall of freshmen, that my colleagues often segregate science conducted according to the diagram I have outlined on the chalkboard as “hypothesis-driven research.” But if your research is not driven by a precisely-framed, rigorously-testable hypothesis about the natural world, you are not doing science, you are just screwing around with test tubes.
Now turning to you, my present readership. The construction of a gene tree is not and cannot be the beginning of a research effort! You cannot derive an hypothesis from a gene tree. The only appropriate application for a gene tree is to test an hypothesis that you have clearly stated a priori on the basis of library research, field experience, and biological intuition.
We do not need any more DNA sequences from random freshwater gastropods shoveled into the NCBI GenDump. We do not need any more odd-lot molecular phylogenies of random freshwater gastropod genera or families – we don’t understand the ones we’ve got now. We do not need more data; we do not need more analysis. What we need are more precisely-framed, rigorously-testable hypotheses. Which can only come from a thorough education in evolutionary science, a firm understanding of the biology of the remarkable creatures to which we have dedicated our careers, rigorous scholarship and field experience, lots of both.
So finally, returning to our research program on Lymnaea (Galba) bulimoides. We have proceeded in orderly fashion from question to hypothesis to data to analysis to conclusion. But there remains yet one final box in the flow chart I scrawled on the blackboard in front of my doe-eyed freshmen – a box that the International Bulimoides Study Team has not as yet checked. And there will be a fourth essay in this series, eventually. But next month we are moving on to other topics. So, stay tuned.
Notes
[1] See the brief bio and cute little 2012 photo of Philippe and his postdoc Patrice David [2] in:
- The American Galba and The French Connection [7June21]
[2] Patrice would subsequently rocket to malacological stardom by discovering:
- Cytoplasmic male sterility in Physa! [9June22]
- Cytoplasmic male sterility in the Physa of the Snake River [7Aug24]
[3] 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]
[4] 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]
[5]
Isaac Lea initially described Lymnaea bulimoides in brief Latinate form in
1841, with more complete English description in 1844:
- Lea, I (1841) On fresh water and land shells (continued). Proceedings of the American Philosophical Society 2(17): 30 – 34.
- Lea, I. (1844/46) Continuation of Mr. Lea’s paper on fresh water and land shells. Transactions of the American Philosophical Society 9(1): 1 – 31.
[6] Lymnaea bulimoides from Alvadore Road, L. viator from Alda [4], L. cockerelli from Bosque del Apache, NM. Photo credits J-P. Pointier.
[7]
Actually, the evolutionary relationships of the mysterious “Bosque del Apache”
population from New Mexico may have been a topic of greater discussion than
Lymnaea bulimoides. Originally
identified as Galba cubensis in the gigantic 2021 paper we had all coauthored
with Pili [4], molecular data kicked the Bosque del Apache population out as
specifically separate. Ultimately the
research I reported in my essay of [12Mar24], together with the shell morphology,
convinced the entire working group that “Galba sp Bosque del Apache” must have
been Lymnaea (Galba) cockerelli. Grist,
perhaps, for a future post.
[8] My
exceptionally keen-eyed readership might note that my essays of February and
March covered three days only. Shary and
I toured the Oregon Coast on Aug 3, and the Columbia Gorge on Aug 4. It was the least I could do for her. She was very patient with me.
[9] As a
rule of thumb, a CO1 sequence divergence of 5% or more suggests speciation in
the freshwater pulmonates. For my
rationale, see:
- The Lymnaeidae 2012: stagnalis yardstick [4June12]
[10]
Saadi, A.J., A. Davison and C.M Wade (2020) Molecular phylogeny of freshwater
snails and limpets (Panpulmonata: Hygrophila)
Zoological Journal of the Linnean Society 190: 518 – 531.
[11]
Here, writ in flickering pixels on the face of an obscure blog, is the
Embarrassment of International Malacology.
Six generic names – Stagnicola, Ladislavella, Polyrytis, Bakerilymnaea,
Fossaria, and Galba – for a single biological species that ought never to have
been split from Lamarck’s (1799) Lymnaea [12].
Please stop making up ridiculous names for random subsets of mollusks,
everybody! You make us look like a bunch
of clueless hacks. Which we are. But must we broadcast that sad fact to the world?
[12] For
a bracing splash of 1951 common sense in the rouged face of 21st century
systematic malacology, see
- The Classification of the Lymnaeidae [28Dec06].
[13] My faithful readership will certainly remember Dwight Taylor as the author of the great Snake River Physa scandal [12Mar08, 14May24, 11June24]. Early in his career he (correctly) recognized the specific status of Lymnaea cockerelli [12Mar24] and (cavalierly) detonated the genus Helisoma [11Apr08].
[14]
Remigio, E.A. and Hebert, P.D. (2003) Testing the utility of partial COI
sequences for phylogenetic estimates of gastropod relationships. Molecular Phylogenetics and Evolution 29 (3): 641-647.
[15]
Remigio, E.A. (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 (7), 687-696.
[16]
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. I reviewed that paper in my
post:
- The Lymnaeidae 2012: Fossarine Football [7Aug12]
[17] Graphic from The Biologos Forum.
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