Cytoplasmic Male Sterility in the Snake River Physa

Editor’s Note – This is the fourth installment of my three part series on the Snake River Physa controversy.   So if you’re interested in all the public policy craziness surrounding the nominally endangered Physa natricina, I would recommend that you back up to my posts of [14May24], [11June24], and [2July24] before proceeding.  If you’re just interested in the science, on the other hand, you might want to refresh your memory with  my [9June22] essay on cytoplasmic male sterility.

Last month we reviewed a mtDNA sequence dataset collected by Mike Young and colleagues [1] from “The Twelve Phascinating Physa of Bliss,” a small sample taken by IPC/FWS biologists from the tailwaters of the Bliss Dam in the Snake River of Idaho in 2019.  That sample included N = 4 snails bearing a CO1 sequence so strange and divergent that our buddy Mike hesitated even to assign it to the genus Physa.  He called the four snails bearing that sequence “Snake River Candidate Species 3,” or SRCS3 for short.

Blasting Mike’s SRCS3 sequences against the entire worldwide NCBI GenBank, we confirmed that OK510774 (taken as typical for the set of four) was more similar to a planorbid from Bangladesh than to any other physid sequence ever reported, “with five phascinating exceptions.”  SRCS3 was approximately 93% similar to two Physa sequenced in Singapore [2], and three Physa sequenced in South Africa [3].

With those little scraps of data in front of us last month, I speculated that SRCS3 might be a strain of Physa acuta demonstrating cytoplasmic male sterility (CMS).  And at that point I abruptly shifted focus to a remarkable paper published in 2022 by my good friend Patrice David and colleagues, reporting very similar levels of mtDNA sequence divergence in a French strain of Physa acuta unable to mate in the male role, but nevertheless fully fertile as female [4].  Could the Snake River – Singapore – South Africa sequence signal the existence of a second case of CMS, a mitotype strikingly different both from the normal, simultaneously hermaphroditic (N) strain and the divergent, male-sterile (D) discovered by Patrice and his team?

Yes.  Two days after I posted last month’s essay I sent an email to Patrice, subject line “Shout-out on the FWGNA blog,” the entire body of which simply read, “I thought you might be interested to see how your 2022 paper on cytoplasmic male sterility could have an impact on conservation biology here in the USA.”  And Patrice replied with a pdf reprint that knocked my socks off [5].

The paper was published in Evolution by Patrice’s student Fanny Laugier, working with a team of nine coauthors from Montpellier and Villeurbanne [6].  Although available in preprint since March, it just appeared in the physical journal last month.  But let’s back up two steps before we leap forward three.

Cytoplasmic male sterility is well studied in plants.  And given the accelerated rates of mutation demonstrated by mitochondria hosting CMS genes, I do not suppose it is surprising that natural populations of gynodioecious plants often host multiple, independently evolving CMS lines.

And as I mentioned in my essay of [9June22] natural populations of gynodioecious plants hosting CMS mitochondria have also often evolved nuclear genes that restore male fertility.  This results from the selective advantage that a fully functional hermaphrodite has over other members of its population reproducing strictly as a single sex [7].

So, reasoning from numerous well-documented cases in plants, Fanny and her team returned to the Physa populations around Lyon originally studied by Patrice and his colleagues to look for additional CMS strains.  And sure enough, prospecting around with a clever PCR test, they found seven individual snails in two populations bearing a “mitotype K,” startlingly different from both normal hermaphroditic N and the CMS mitotype D they had reported in 2022.

My readership might also remember from [9June22] that CMS mitotype D was, around the entire mitochondrial genome, approximately 44% different from the normal Physa acuta mitotype N.  The 20% CO1 sequence divergence between those two mitotypes was actually less than average.  So the newly discovered mitotype K ultimately proved to be 35% - 57% divergent from N and 35% - 57% divergent from D (ten genes), with CO1 sequence divergences 23% and 28%, respectively.  This is the phenomenon I have termed “mitochondrial superheterogeneity” in many previous posts on this blog [8].

And can you smell what our French chefs are cooking?  Focusing now on the CO1 sequence, the French CMS strain bearing the newly discovered mitotype K matched Ting Hui Ng’s [2] Singapore sequence and Molaba’s [3] South Africa sequence 100%, almost identically.  And the match with Mike Young’s [1] SRCS3 sequence from the Bliss Rapids was 93%, just as I reported last month.  Fanny Languier, Patrice David, and their colleagues had discovered a strain of Physa acuta bearing a wildly divergent mitochondrion worldwide in its distribution.

Laugier [6] Figs. 1B and 1C, modified.

But that is not the headline news.  Here is the headline.  Mitotype K Physa acuta were not male-sterile!  Our French colleagues cultured up a big batch of Mitotype K Physa and were easily able to cross them with their albino N laboratory line [9], both lines copulating readily in both the male and the female role.

So again, reasoning from many years of accumulated research on cytoplasmic male sterility in plants, Fanny and her team suspected that nuclear genes might have evolved in the mitotype K line to restore male function.  And they launched a 17 – generation introgression experiment to insert mitochondrial lineages derived from their wild-caught (pigmented) K line into the “naïve” nuclear background of their (albino) laboratory line N.

And sure enough!  After just 5 generations of introgression, 69% of the naïve snails bred to bear K-type mitochondria had lost their ability to mate as males, with no such loss whatsoever in control lines bearing the N-type mitochondria.  After 11 generations, the proportion of naïve male-sterile K snails stabilized at around 60%.  No loss in female function was ever detectable in any line.  In my 5July24 email back to Patrice, I wrote:

“Wow, nuclear restorers!  It's a shame we work with crap-brown little trash snails.  If snails were maize, wheat, or rice [10], we'd be rich.”

What wonderful science!  Deductive reasoning, tested by rigorous experimentation carefully designed to proceed from the known to the unknown.  Conducted for the joy of it, for the exploration of evolutionary mechanisms, for the pushing back of the darkness.  The construction of testable hypotheses about the natural world, period, full stop, nothing more and nothing less.  Pure and unsullied science, utterly useless and wonderful!

Laugier [6] Fig. S1, modified.

And yet, it turns out, completely by accident, useful.  Ting Hui’s study in Singapore was directed toward invasive species, the Molaba study in South Africa was parasitological, and Mike Smith’s study on the Snake River directed toward conservation.  Fanny’s results contribute toward an understanding of all those results, and more.

And here is Lesson Number One.  Gene sequences are not species.  A species is a population or group of populations reproductively isolated from all others [11].  Sequence divergence is typically correlated with reproductive isolation [12], of course, no different from morphological divergence.  But gene trees are not species trees, and genes are not species.

So, as I pointed out in last month’s essay [2July24], the Snake River line of Physa acuta that Mike Young and colleagues called SRCS3 is not the same as the French mitotype K.  The two lines are 7% different, as though they arose from a single mutation in some mitochondrial DNA repair gene sometime in the past and have subsequently diverged.  Could such a mutation have occurred more than once in the evolutionary history of the Physidae, here in North America, where Physa seem to have first evolved?  You betcha.

There is no reason that the CO1 gene sequence that Gates, Kerans and colleagues [13] recovered from the stunted Physa below the Minidoka Dam, roughly 15% different from the N line at Bliss (OK510580), couldn’t be evidence of yet another CMS strain in Physa acuta.  Gates and Kerans identified that sequence as “Physa natricina.”  But gene sequences are not species.  Species are defined by reproductive isolation.  And we have no data on reproductive relationships between the Minidoka population and the dirt-common Physa acuta downstream whatsoever.  And plenty of evidence otherwise [14].

Nor is there any reason in the world that the peculiar mitotype shared by Mike Young’s SRF14, the Owyhee Wet Rock Physa of eastern Oregon [15] and scattered in odd lot Physa populations from California to British Columbia couldn’t be evidence of a CMS strain in Physa gyrina.  MtDNA sequence data, and the gene trees we make with them, are (at best) weak null models of population relationships, correlated with speciation, nothing more [16].

Do not misunderstand me.  I am not demanding controlled breeding studies between every pair of the 2.2 x 10^6 species described from Planet Earth.  But for nominally endangered species, such as Physa natricina, before we enact pages of Federal regulations and spend millions of dollars on conservation, we could at least run a couple of $12 allozyme gels and test for evidence of assortative mating [17]  with a nuclear polymorphism or two, am I right?

Notes

[1] Young, M.K., R. Smith K.L. Pilgrim, and M.K. Schwartz (2021)  Molecular species delimitation refines the taxonomy of native and nonnative physinine snails in North America.  Scientific Reports 11: 21739. https://doi.org/10.1038/s41598-021-01197-3

[2] Ng, T.H., Tan, S.K. & Yeo, D.C. (2015) Clarifying the identity of the long-established, globally-invasive Physa acuta Draparnaud, 1805 (Gastropoda: Physidae) in Singapore. BioInvasions Rec. 4, 189–194.

[3] Molaba, G.G. et al. (2023) Molecular detection of Fasciola, Schistosoma and Paramphistomum species from freshwater snails occurring in Gauteng and Free State provinces, South Africa.  Veterinary Parasitology 320: 109978.  https://doi.org/10.1016/j.vetpar.2023.109978

[4] David, Patrice, Cyril Degletagne, Nathanaëlle Saclier, Aurel Jennan, Philippe Jarne, Sandrine Plénet, Lara Konecny, Clémentine François, Laurent Guéguen, Noéline Garcia, Tristan Lefébure, Emilien Luquet (2022) Extreme mitochondrial DNA divergence underlies genetic conflict over sex determination.  Current Biology 32: 2325 - 2333.  https://doi.org/10.1016/j.cub.2022.04.014.  For a review, see:

• Cytoplasmic Male Sterility in Physa! [9June22]

[5] Patrice went on to apologize “I told my PhD student Fanny a dozen times to send this to you, that you would probably make good use of it because it was solving a controversy, apparently she didn’t, so I do it myself.” Yes, please send me your reprints!  I lost my library privileges when I was banned from campus in 2016.  I really haven’t had access to most of the scientific literature published since.

[6] Laugier, Fanny, Nathanaëlle Saclier, Kévin Béthune, Axelle Braun, Lara Konecny, Tristan Lefébure, Emilien Luquet, Sandrine Plénet, Jonathan Romiguier, and Patrice David (2024) Both nuclear and cytoplasmic polymorphisms are involved in genetic conflicts over male fertility in the gynodioecious snail, Physa acuta.  Evolution 78 (7): 1227–1236. https://doi.org/10.1093/evolut/qpae053

[7] Yes, mitochondria bearing CMS genes are “selfish” in the sense of the Richard Dawkins (1976) classic.   Don’t get me started on Richard Dawkins.

[8] Mitochondrial superheterogeneity (mtSH), where two or more of the members of a single population demonstrate greater than 10% divergence in any single-copy mtDNA gene, not sex linked, seems to be remarkably common in freshwater gastropods.  In pulmonate populations, I wouldn’t be surprised if most or all mtSH is ultimately traceable to CMS.  In prosobranch populations, however, I think mtSH is a signature of great age, plus low-frequency long distance dispersal, the “Jetlagged Wildebison Model.”  Here is a sample of my previous posts on mtSH:

• The Snails the Dinosaurs Saw [16Mar09]
• Mitochondrial superheterogeneity: What we know [15Mar16]
• Mitochondrial superheterogeneity: What it means [6Apr16]
• Mitochondrial superheterogeneity and speciation [3May16]
• Mitochondrial heterogeneity in Marstonia lustrica [3Aug20]

[9] Dillon, R.T. and A.R. Wethington (1992) The inheritance of albinism in a freshwater snail, Physa heterostropha. Journal of Heredity 83:208-210. [pdf]  For nice review, see:

• Albinism and sex allocation in Physa [5Nov18]

[10] The clever manipulation of cytoplasmic male sterility, together with nuclear restorers, has been one of the more important methods by which plant breeders have achieved the outcrossing of normally self-pollinating crop plants.  I am quite sure that a lot of money has been made with that genetic technology.  With Physa acuta, however, the prospects are not as lucrative.

[11] This is the biological species concept, most closely associated with the work of Ernst Mayr.  It should need no restatement, much less a defense.  But the best paper Jerry Coyne ever wrote was a defense of both Mayr and his species concept, here:

• Coyne, J. A. (1994) Ernst Mayr and the origin of species.  Evolution 48: 19 – 30.

[12] Dillon, R. T., A. R. Wethington, and C. Lydeard (2011)  The evolution of reproductive isolation in a simultaneous hermaphrodite, the freshwater snail Physa.  BMC Evolutionary Biology 11:144. [html] [pdf].  For a review, see:

• What is a species tree? [12July11]

[13] Gates, K. K., B. L. Kerans, J. L. Keebaugh, S. K. Kalinowski & N. Vu (2013) Taxonomic identity of the endangered Snake River physa, Physa natricina (Pulmonata: Physidae) combining traditional and molecular techniques.  Conserv. Genet. 14: 159-169.  For a review, see:

• The Mystery of the SRALP: No Physa acuta were found [2May13]

[14] Rogers, D.C. & A.R. Wethington (2007) Physa natricina Taylor 1988, junior synonym of Physa acuta Draparnaud, 1805 (Pulmonata: Physidae).  Zootaxa 1662: 45 - 51.  For a review, see:

• Red flags, water resources, and Physa natricina [12Mar08]

[15] Moore, A.C., J.B. Burch, and T.F. Duda Jr. (2015) Recognition of a highly restricted freshwater snail lineage (Physidae: Physella) in southeastern Oregon: convergent evolution, historical context, and conservation considerations.  Conservation Genetics 16: 113 – 123.

[16] I have made this argument as many times as the number of essays listed under the label “Gene trees” above.  For an overview, see:

• Gene trees and species trees [13July08]

[17] For an explanation of gametic phase disequilibrium, its power to distinguish species, and its extension to character phase disequilibrium, see:

• What is character phase disequilibrium? [4Jan22]
• Character phase disequilibrium in the Gyraulus of Europe [4Feb22]
• Just 125 Species of Pyrgulopsis in the American West [7Sept22]