Cytoplasmic Male Sterility in Physa!

Editor’s Note – This essay was subsequently published as: Dillon, R.T., Jr. (2023c)  Cytoplasmic male sterility in Physa!  Pp 195 – 206 in The Freshwater Gastropods of North America Volume 7, Collected in Turn One, and Other Essays.  FWGNA Project, Charleston, SC.

Three cheers for our good friend Dr. Patrice David of the CNRS-CEFE, who together with a team from the Universities of Montpellier and Lyon, has opened a new window into the mysteries of sex allocation in pulmonate snails, casting fresh light on mitochondrial evolution across the entire animal kingdom in the process.  Our colleagues from France have discovered the first case of cytoplasmic male sterility ever documented outside the vascular plants [1].  And they have done so in the most underappreciated experimental model of our time, the freshwater gastropod Physa acuta.

Male-fertile & sterile [3]

Genetic phenomena now understood to result from cytoplasmic male sterility were reported in plants as early as 1884 [2]. Many wild populations of the thistle Cirsium oleraceum, for example, are composed of mixtures of (normal) monoecious plants producing monoecious offspring and (male-sterile) female plants producing only female offspring, although always pollinated from monoecious plants. In a series of papers published between 1916 and 1937, the German geneticist Carl Correns demonstrated that the “female C. oleraceum plants contained in their cytoplasms some properties which inhibited the development of male floral parts.”  And in 1939, the obscure Argentinian geneticist E. Gini reported controlled crosses conclusively demonstrating cytoplasmic male sterility in certain varieties of maize, which might, however, be modified by a nuclear gene or genes segregating in the male parent.

Thistles are ordinary dicots, in the sense that their flowers bear both stigma and anther, both pollen and ova.  Maize is a monocot, and among the just 5% of all angiosperms with separate male and female inflorescences; the pollen being produced by the tassel on top, the ovum in the cob below.  What unites maize and thistles is that at least sometimes both demonstrate a mating system called “gynodioecy,”  where both hermaphroditic and female individuals co-exist in a single population.

Between 1940 and 1970, cytoplasmic male sterility was discovered in hundreds of species of (at least occasionally) gynodioecious plants, including many important as crops.  Nuclear genes that moderated the effects of CMS were discovered as well.  In the hands of plant breeders, CMS became a powerful tool to promote hybridization in normally self-pollinating varieties of maize, wheat, rice, carrots, onions, and beets.  And as the beet fields blossomed forth, so too did funding for research into CMS.

By the mid-1980s it was clear that CMS was caused by mitochondrial gene alteration.  By the mid-1990s sterility had been pegged to a variety of genes variably-inserted into otherwise normal plant mitochondria, encoding proteins that interfered with pollen formation [3].  And by the mid-2000s, the sequence data were rolling in.  It materialized that the plant mitochondrial genome is strikingly different from animal mtDNA in its evolutionary dynamics: variably sized, highly repetitive, and characterized by frequent gain, loss, duplication and rearrangement of genes [4].

Now, back to France.  You have heard me sing the praises of Dr. Philippe Jarne, his (1993-96) student Dr. Patrice David, and a bouquet of Montpellier friends and colleagues in many previous essays posted on this blog.  Most recently we featured their collaboration with a host of researchers from across the Americas to work out the evolution of the lymnaeid subgenus Galba, the crappy little amphibious snails serving as intermediate hosts for the sheep liver fluke Fasciola worldwide [5].  Philippe and Patrice also have research interests in Physa, primarily as a model for the study of sex allocation, extending all the way back to 1995.  See note [6] below for a selection of my favorite Physa papers published by Philippe, Patrice, and their colleagues over the years.

Graphical abstract from David et al. [1]

That said.  A phenomenon as startling as cytoplasmic male sterility in little brown trash snails is not something one can design an experiment to discover.  Patrice tells me that the breakthrough was initially made by Dr. Emilien Luquet of the University of Lyon, who has adopted Physa acuta as a model for the study of phenotypic plasticity.  Routinely sequencing the CO1 gene from a sample of Physa collected in a backwater of the Rhone River east of Lyon, Dr. Luquet obtained a couple sequences that were, to use the precise, technical terminology especially developed in Lyon to describe the phenomenon, “weird” [7].

Dr. Luquet, together with a team of four colleagues from the University of Lyon, (Plénet, Lefébure, Konecny and Deglétagne) then developed and characterized 34 isofemale lines of Physa from the Rhone River backwaters, 29 with normal mitochondrial genotypes and 5 weird.  The weird lines bore mitochondria with a median CO1 sequence divergence more than 20% different from the other members of their population, or indeed, any of the hundreds of Physa sequences in GenBank.  Sequences of the mitochondrial 16S gene returned the same remarkable result.

At this point Dr. Luquet sent samples to our mutual friend Patrice, saying “I apparently have Physa that came from outer space, look at them and tell me if this is really Physa acuta.”  Patrice, aware that here in North America we have several species of Physa that are indeed rather cryptic, began a series of experimental crosses with an albino line of P. acuta developed in the CNRS-CEFE laboratory.

When mounted by albino controls mating in the male role, the weird lines of Physa outcrossed readily, and actually showed some evidence of fecundity improved over the normal lines from Lyon.  But it materialized that the weird lines were male-sterile.  They showed much-reduced behavioral tendency to mount partners in the male role, their seminal vesicles apparently shrunken, containing very little sperm.  They could not self-fertilize.  This is the first demonstration of cytoplasmic male sterility in the animal kingdom…

… but Amy Wethington and I had a shot at it 20 years ago.

Amy and I started working with Physa when she was an undergraduate student at the College of Charleston in the late 1980s.  And our first samples came from a pond in the state park just around the corner from my house, Charles Towne Landing.  That population of Physa became a standard for 20 papers published over the 20 years that followed [8].  We called it population C.

Amy at Charles Towne Landing

Almost immediately, Amy and I became interested in the evolutionary relationships between our population C, which we initially identified as “Physa heterostropha pomilia,” and all the other populations of trash Physa worldwide, identified with a wide variety of other names.   And my readership with admirably long memory may recall that it was from Philippe Jarne back in 2000 that Amy and I received the batch of French Physa acuta we used in our experiments demonstrating no reproductive isolation between population C and five other populations of Physa that had been known by three different names: Physa acuta, Physa heterostropha, and Physa integra [9].

So given that all six of these populations of Physa from four states and two European countries were conspecific, Amy and I were next curious to estimate the amount of genetic divergence among them.  A CofC undergraduate named Matt Rhett and I ran the allozyme gels [10], and Amy (now in the PhD program at the University of Alabama) sequenced two mitochondrial genes (CO1 and 16S) for approximately 20 snails from each of those six populations: C = Charleston, P = Philadelphia, D = Douglas Lake, Michigan, N = New Harmony, Indiana, F = France, I = Ireland.  The gigantic 16s+CO1 neighbor-joining gene tree that resulted is shown below.

Let’s look at that tree from the bottom up.  At the bottom you see two other good biological species of Physa: three individual Physa gyrina, and three individual Physa pomilia, which we were calling Physa hendersoni at that time [11].  The gigantic middle branch of the tree shows all six of our Physa acuta populations all mixed up: C, P, D, N, F, and I, with a little bit of geographic structure but not much.  Now what the heck is that branch sticking way out at the top?

That top branch, labelled C10, C13, C15 and C18, shows four snails from Charles Town Landing bearing mitochondria with sequences approximately 30% different from all other Physa acuta from four states and two European countries.  Amy called those sequences “whacky.”

So in 2004 Amy drafted a manuscript by Wethington, Rhett, and Dillon to report all our allozyme data and all our sequence data across all six Physa populations.  Meanwhile, I recruited another undergraduate student, Nick DiNitto, and together Nick and I went back to the pond at Charles Town Landing, collected 26 adult Physa, and started isofemale lines.  Once the original mothers had laid eggs, our plan was to enlist the aid of my colleague Bob Frankis to sequence their CO1 genes, hoping to found pure whacky [12] cultures for breeding experiments.

From FWGNA Circular #5 [pdf]

But alas.  Working with Bob, Nick was able to sequence just six of the original mothers before the semester came to a close and the project foundered.  None of those six bore whacky mitotypes [12].  Nick’s poster for the 2004 meeting of the American Malacological Society in Sanibel, Florida, reporting just N=6 unremarkable CO1 sequences, is available for download from note [13] below.

Meanwhile Amy was having no success whatsoever interesting journal editors in publishing the Wethington, Rhett & Dillon manuscript.  In the eyes of reviewers, the whacky result depicted at the top of our Figure 1 invalidated the entire paper.  This must be some sort of lab accident!  Or cryptic speciation?  Either Amy’s technique was hopelessly sloppy, or our premise was hopelessly wrong.  Amy ultimately gave up trying to publish the separate paper, cut the four whacky sequences, and folded the rest of the data into her 2004 dissertation [14].  Her amazing discovery was forgotten.

Until today.  Today I am making the original 2004 manuscript of A. R. Wethington, J. M. Rhett and R. T. Dillon, entitled “Allozyme, 16S, and CO1 sequence divergence among populations of the cosmopolitan freshwater snail, Physa acuta” available for download as FWGNA Circular #5, here: 

-  Wethington, Rhett & Dillon (2022)  -

And perhaps I should have written “almost forgotten” two paragraphs above.  For in footnote (3) of my [15Mar16] essay on mitochondrial superheterogeneity, I made passing reference to “a striking case of mtSH (both COI and 16S) in a Charleston population of Physa acuta.”  Which leads me to offer two final hypotheses, ask two final questions, and make an appeal.

First hypothesis.  It seems quite likely to me that mtSH = CMS in pulmonate snails broadly.  The first report of the phenomenon I subsequently dubbed “mitochondrial superheterogeneity” came in the 1996 paper of Thomaz and colleagues, working with the land snail Cepaea nemoralis [15].  Cases of mtSH have been reported at least three additional times in the land snails that I know of, and one other time in a freshwater pulmonate, the limpet Laevapex fuscus.  See footnotes (2) and (3) of my [15Mar16] essay.

Second hypothesis. It seems possible that CMS -> DUI in bivalves.  Doubly-uniparental inheritance of mitochondria was first reported in the marine mussel Mytilus in the early-1990s and has subsequently been documented in a wide variety of bivalves, including the unionid mussels of American freshwaters.  As the name implies, researchers have found that mitochondria can be passed by sperm as well as by egg in these bivalve groups.  And most intriguingly, the genomes of the male mitochondria and the female mitochondria are always found to be strikingly divergent.  Sophie Breton of the University of Quebec and a team of bivalve researchers including our friend Randy Hoeh have just very recently published a nice paper making this argument in compelling fashion [16].

First question.  Could CMS -> MtSH in pleurocerids and other prosobranch snails?  That is a stretch I am not intellectually limber enough to make.  Sexes are separate in the pleurocerids, as in almost all other prosobranch groups, mechanism of sex determination unknown.  If you wade out into the creek, fish up a nice sample of adult pleurocerids and crack them open, you will almost always find sex ratios significantly biased toward the female [17].  This is a secondary sex ratio, of course.  I don’t think anybody knows the primary sex ratio for any prosobranch population.

I would love to hypothesize that the mitochondrial superheterogeneity so often demonstrated by freshwater prosobranch populations – ten cases listed in footnotes (4), (5), (6), and (7) in my essay of [15Mar16] alone – might be related to such sex ratio biases.  But Whelan & Strong [18] did not find any relationship between sex and mt haplotype in their superheterogeneous populations of Leptoxis from Alabama, and I don’t know where else to look for evidence.

And second question.  What is the origin of CMS in the Physa population of the Rhone River?  Patrice and colleagues ultimately sequenced the weird mitochondrial genome in its entirety, documenting extreme divergence everywhere they looked, summing to an eye-popping 44.4% median deviation from the normal mitotype.  Yet no divergence in the nuclear genome was apparent.  Sequencing the (nuclear) 28S gene returned no significant difference across their entire sample of 34 lines, nor were any differences in microsatellite gene frequencies apparent.  The Mendelian genetics confirm that the Rhone River at Lyon is inhabited by a single randomly interbreeding population of Physa acuta.

Which brings us to my appeal.  To all of our colleagues who persist in defining species with mtDNA gene trees, please stop.  You’re embarrassing us.

Patrice went on to offer several compelling lines of evidence suggesting that the weird mitotype results from an acceleration of mutation rate, perhaps due to some defect in the mitochondrial DNA repair mechanism, rather than selection on mitochondrial functions more broadly.  He further observed that high mutation rates might both produce male-sterility genes, and help them persist over evolutionary time, counter to the evolution of nuclear genes restoring male fertility.  There are all sorts of selfish-DNA-type directions one could go from here.

And so, to conclude.  None of the thoughts, questions, hypotheses and speculations outlined above could have flickered through my brain before the marvelous paper by Patrice David and his colleagues appeared last month.  Job well done, all of you!  La reussite scientifique de la France est intimement lie au bonheur de l’Amerique [19].

Notes

[1] 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.

[2] The historical review in this paragraph is extracted from:

• Edwardson, J.R. (1956) Cytoplasmic male-sterility.  Botanical Review 22: 696-738.
• Edwardson, J.R. (1970) Cytoplasmic male-sterility.  Botanical Review 36: 341 – 420.

[3] Schnable, P.S., and R.P. Wise (1998)  The molecular basis of cytoplasmic male sterility and fertility restoration.  Trends in Plant Science 3: 175 – 180.

[4] Galtier, N. (2011) The intriguing evolutionary dynamics of plant mitochondrial DNA. BMC Biol. 9, 61. http://www.biomedcentral.com/1741-7007/9/64

[5] 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:

• The American Galba and the French Connection [7June21]
• The American Galba: Sex, Wrecks, and Multiplex [22June21]
• Exactly 3ish American Galba [6July21]
• What Lymnaea (Galba) schirazensis is not, might be, and most certainly is [3Aug21]

[6] Here are a few of my favorite Physa papers from Philippe and colleagues:

• Jarne, P., M-A Perdieu, A-F Pernot, B. Delay, and P. David (2000)  The influence of self-fertilization and grouping on fitness attributes in the freshwater snail Physa acuta: population and individual inbreeding depression. J. Evol. Biol. 13:645-655.
• Bousset, L., P-Y. Henry, P. Sourrouille, & P. Jarne (2004)  Population biology of the invasive freshwater snail Physa acuta approached through genetic markers, ecological characterization and demography. Molec. Ecol., 13: 2023-2036.
• Tsitrone A, Jarne P, David P: Delayed selfing and resource reallocations in relation to mate availability in the freshwater snail Physa acuta. Amer Natur 2003, 162:474-488.
• Henry PY, Bousset L, Sourrouille P, Jarne P: Partial selfing, ecological disturbance and reproductive assurance in an invasive freshwater snail. Heredity 2005, 95:428-436
• Noel, E., Chemtob, Y., Janicke, T., Sarda, V., Pelissie, B., Jarne, P., and David, P. (2016). Reduced mate availability leads to evolution of self-fertilization and purging of inbreeding depression in a hermaphrodite. Evolution 70, 625–640.

[7] Quoting Patrice, “Initially we called them W for weird; we later opted for D as in Divergent which sounds more serious and doesn't induce confusion with W chromosomes.”

[8] I have reviewed the many years of fruitful collaboration that Amy and I enjoyed in quite a few essays previously posted on this blog.  Here are my favorites:

• To identify a Physa, 1989 [3Oct18]
• Albinism and sex allocation in Physa [5Nov18]
• To identify a Physa, 2000 [6Dec18]
• TRUE CONFESSIONS: I described a new species [7Apr10]
• What is a Species Tree? [12July11]

[9] Dillon, R. T., A. R. Wethington, J. M. Rhett and T. P. Smith (2002) Populations of the European freshwater pulmonate Physa acuta are not reproductively isolated from American Physa heterostropha or Physa integra.  Invertebrate Biology 121: 226-234.  [pdf]

[10]  I know that an allozyme survey sounds hopelessly outdated.  A postcard from 1982.  But it was allozyme markers that allowed us to do all our breeding studies.  We could not have verified outcrosses without baseline allozyme surveys such as my students and I labored over for years.  There are a lot of research groups who wish they had that hopelessly-outdated 1982 technology today.

[11] Dillon, R. T., J. D. Robinson, and A. R. Wethington (2007)  Empirical estimates of reproductive isolation among the freshwater pulmonates Physa acuta, P. pomilia, and P. hendersoni.  Malacologia 49: 283 - 292.  [pdf]

[12] Actually, Nick preferred to call the weird sequences “zany” rather than “whacky.”  So that’s what we called them on his poster…

[13] DeNitto, N. W., R. C. Frankis and R. T. Dillon (2004) Extensive mitochondrial CO1 sequence diversity in a population of the freshwater snail, Physa: Admixture or cryptic speciation?  American Malacological Society, Sanibel Island, FL. [Poster, pdf]

[14] Amy’s dissertation was ultimately published as: Wethington, A.R., & C. Lydeard (2007) A molecular phylogeny of Physidae (Gastropoda: Basommatophora) based on mitochondrial DNA sequences. Journal of Molluscan Studies 73: 241 - 257. [pdf]  For a review, see:

• The classification of the Physidae [12Oct07]

[15] Thomaz, D., Guiller, A. & Clarke, B. (1996) Extreme divergence of mitochondrial DNA within species of pulmonate land snails. Proc. of the Royal Society of London B: Biological Sciences, 263, 363–368.

[16] Breton, Sophie, Donald T. Stewart, Julie Brémaud, Justin C. Havird, Chase H. Smith, and Walter R. Hoeh (2022)  Did doubly uniparental inheritance (DUI) of mtDNA originate as a cytoplasmic male sterility (CMS) system?  Bioessays 44: 2100283. https://doi.org/10.1002/bies.202100283

[17] Cipiaris, S., W. F. Henley and J.R. Voshell (2012)  Population sex ratios of pleurocerid snails (Leptoxis spp.): Variability and relationships with environmental contaminants and conditions.   Amer. Malac. Bull. 30: 287 - 298.

[18] Whelan, N.V. & E. E. Strong (2016)  Morphology, molecules and taxonomy: extreme incongruence in pleurocerids (Gastropoda, Cerithiodea, Pleuroceridae). Zoologica Scripta 45: 62 – 87.  For a review, see:

• Mitochondrial superheterogeneity: What we know [15Mar16]
• Mitochondrial superheterogeneity: What it means [6Apr16]
• Mitochondrial superheterogeneity and speciation [3May16]

[19] The scientific success of France is closely tied to the good fortune of America.  I offer this benediction as an echo of the Marquis de Lafayette quote with which I ended my essay of [7June21].