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  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 . 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.” 
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  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 . 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 . 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  from Hubendick |
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  also tested one population of the (more distantly related) Lymnaea ovata (aka Lymnaea peregra, aka Radix balthica ), 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  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 , 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 |
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  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  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 |
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 one of the 11 “standards” was collected from its type locality , the other ten 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 |
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.
 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]
 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.
 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.
 This figure is a cut-and-paste of four figures from Hubendick  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.
 Hubendick, B. (1951) Recent Lymnaeidae. Their variation, morphology, taxonomy, nomenclature and distribution. Kungliga Svenska Vetenskapsakademiens Handlingar Fjarde Serien 3: 1 - 223.
 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.
 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.
 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.
 Hubendick  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.
 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.
 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]
 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]
 That would be the New York population of Lymnaea (Galba) humilis, of course. My faithful readership will need no reminder. But for the rest of you, see:
- Malacological Mysteries I: The type locality of Lymnaea humilis [25June08]