Hybridization in the depths of the last glacial period created a world-conquering clover

White clover, Trifolium repens, in a Los Angeles city park, a hemisphere away from the glacial refugia where it originated. (jby)

Plants’ flexibility with the structure of their genome — able to cope with proliferating transposons, whole-genome duplications, or even acquisition of complete sets of chromosomes from another species — is a big source of evolutionary novelty. Duplication of a single gene allows the duplicate and its template to evolve new functions; adding a whole additional genome provides that much more raw material. That may be the secret of the success of one worldwide weed we’ve seen featured on this blog before, Trifolium repens, or common white clover. A new paper in The Plant Cell delineates two fairly intact progenitor genomes within the T. repens genome, and reconstructs the history of an evolutionary mashup that created a wildflower you can very likely find by simply stepping outside and walking to the nearest well-watered lawn.

White clover’s natural range stretches from Gibraltar to western Siberia, and it’s been successfully introduced — intentionally as livestock forage, and otherwise — on every other continent. It’s been known for awhile that T. repens is tetraploid, having four homologous copies of each chromosome instead of the baseline two, and that the two sub-genomes came from different ancestral Trifolium lineages closely related to pale clover (T. pallescens) and western clover (T. occidentale). Where white clover grows in just about any climate that supports grassland, those ancestors probably had much narrower niches; pale clover grows in alpine conditions between 1800 and 2700m of elevation, and western clover is restricted to the shorelines of western Europe. Pale and western clover don’t co-occur anywhere in their current distributions, so it’s been hypothesized that they came together to hybridize and create white clover during the last glacial maximum, when glaciation and colder climates would have pushed them into the same refugial habitats.

The authors of the new paper, a team from institutions in New Zealand and Denmark led by Andrew Griffiths, reconstruct that history of hybridization by assembling genomes for T. repens, T. pallescens, and T. occidentale. The genomes involved are relatively small — about 500 million base pairs for the progenitor species, and a bit more than a gigabase for T. repens — the authors used a "synthetic long-read" method based on Illumina technology, and they used linkage disequilibrium estimates and a previously constructed linkage map for T. repens to inform assembly. They also resequenced four T. repens lines to provide some information about variation within the species.

(A) A conceptual model the histories of Trifolium pallescens (Tp), T. occidentale (To) and the portions of the T. repens (Tr) genome coming from each. (B) Estimates for the timing to most recent common ancestor for each pairing in (A). (C) The time of the hybridization event (blue) and the speciation of T. occidentale and T. pallescens (gray) overlaid on the history of global temperature anomalies. Detail of Figure 3, Griffiths et al. (2019)

Very high sequence similarity between the T. pallescens chloroplast genome and that of T. repens confirmed T. pallescens as the maternal parent species. And then, more interestingly, they quantified variation shared between T. repens chromosomes and the parental species from which they came: there were far more — about 50x as many — single-nucleotide polymorphisms shared between the parental species and the subgenomes they’ve contributed to T. repens than SNPs that were unique to the parents. That’s a strong signal that the hybridization event, or more likely events, that created T. repens is (are) quite recent.

The authors analyzed a formal model of introgression between the two progenitor species, parameterized with a range of mutation rates informed by rescaling the estimate from Arabidopsis for a larger genome, and by comparison to another legume, the domestic peanut. That gave them an estimate of the time to most recent common ancestor for the two progenitor species, and then T<sub>MRCA</sub> for each progenitor and the T. repens subgenomes it had contributed to the hybrid species.

This placed the speciation of the two progenitors about 192 thousand years ago, and the hybridization that formed T. repens about about 16 thousand years ago. That’s consistent with T. pallescens and T. occidentale splitting during a climate in which their habitats would have been separate; but then hybridizing to form T. repens during the last glacial maximum. In that era, high altitudes would have been too cold for T. pallescens (indeed, buried under glaciers), and it would have moved into lower elevations where it could intermingle with T. occidentale.

White clover in its native range … the lawn of Hyde Park, London. (jby)

Griffiths et al. found the subgenomes retain extensive synteny with the progenitor genomes, and they found that, across four different types of tissue (roots, stolons, leaves, and flowers), biases in the expression of genes from one subgenome or the other were quite consistent. That first result indicates there’s been relatively little rearrangement toward "diploidization" of the two subgenomes T. repens received from its progenitor species. The second result suggests that, at a broad scale, genes contributed from the two genomes aren’t taking on specialized functions within different parts of the plant.

Among genes that did show unusually high tissue-specific differences in bias towards one subgenome or the other, secondary chemistry was overrepresented — particularly genes in the flavonoid biosynthesis pathway. Flavonoids come into play in a lot of interactions with other species — both mutualists like the rhizobia bacteria that fix nitrogen for clovers, and antagonists like herbivores and pathogens. Griffiths et al. suggest this has been a big contribution to the global success of T. repens: the tetraploid clover has had a larger suite of flavonoid biosynthesis genes to draw on as it adapts to biological communities from Alaska to Australia.

References

Ellison NW, A Liston, JJ Steiner, WM Williams, and NL Taylor. 2006. Molecular phylogenetics of the clover genus (Trifolium – Leguminosae). Mol. Phylogen. Evol. 39, 688-705. doi: 10.1016/j.ympev.2006.01.004

Griffiths AG, BA Barrett, D Simon, AK Khan, P Bickerstaff, CB Anderson, BK Franzmayr, KR Hancock, and CS Jones. 2013. An integrated genetic linkage map for white clover (Trifolium repens L.) with alignment to Medicago. BMC Genomics 14, 388. doi: 10.1186/1471-2164-14-388

Griffiths AG, R Moraga, M Tausen, V Gupta, TP Bilton, MA Campbell, RL Ashby, I Nagy, A Khan, A Larking, C Anderson, B Franzmayr, K Hancock, A Scott, NW Ellison, M Cox, T Asp, T Mailund, M H Schierup, and SU Andersen. 2019. Genomics of allopolyploidy in white clover. The Plant Cell doi: 10.1105/tpc.18.00606

About Jeremy Yoder

Jeremy Yoder is an Assistant Professor of Biology at California State University, Northridge. He also blogs at Denim and Tweed, and tweets under the handle @jbyoder.
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