Genomics of Ferns and Lycophytes

1. Genomics of Ferns and Lycophytes

2. Chapter 6: Structure and evolution of fern plastid genomes Paul G. Wolf and Jessie M. Roper

3. Question: What is the inheritance of the chloroplast genome in ferns? Marchantia cp genome • ca. 150 kb, circular molecule • large and small single copy regions separated by inverted repeat • gene number and order +/- conserved across land plants

4. Generally in land plants: maternal (via the egg, excluded via sperm) • maternal with some biparental in Angiosperms • paternal in Gymnosperms • ferns? Phyllitis (Aspleniaceae) – biparental Osmunda (Osmundaceae)– maternal Polystichum (Drypoteridaceae) – maternal Pteridium Dennstaedtiaceae) – maternal Pellaea (Pteridaceae) – maternal “During insemination in Ceratopteris richardii [Pteridaceae], the sperm cytoskeleton and flagella rearrange, and the coils of the cell extend while entering the neck canal. . . . All cellular components, except plastids, enter the egg cytoplasm” Lopez-Smith and Renzagalia, 2008 (Sexual Plant Reproduction)

5. 1992 Marchantia 30kb inversion tobacco Marchantia cp genome • ca. 150 kb • large and small single copy regions separated by inverted repeat • gene number and order +/- conserved across land plants

6. 1992 30kb inversion Lycopodium Equisetum Psilotum Lycopodium = Marchantia order Osmunda ferns = tobacco order

7. Fern and lycophyte total chloroplast genomes sequenced • Huperzia • Isoetes • Selaginella • Equisetum (basal fern) • Psilotum (basal fern) • Angiopteris (basal fern) • Adiantum (polypod) • Alsophila (polypod • - 2009 paper)* Gao et al. (2009) Complete chloroplast genome sequence of a tree fern Alsophila spinulosa

8. Fern and lycophyte total chloroplast genomes sequenced • few advanced ferns sequenced • but, Fern Tree of Life project will do many more

9. Rearrangements in fern chloroplast genomes loss of some tRNA and other protein coding genes Gao et al. 2009

10. Rearrangements in fern chloroplast genomes loss of some tRNA and other protein coding genes 2 inversions in the Inverted Repeat (IR) of some ferns Gao et al. 2009

11. Rearrangements in fern chloroplast genomes IR inversion 2 loss of some tRNA and other protein coding genes 2 inversions in the Inverted Repeat (IR) of some ferns IR inversion 1 ? [also using PCR assays for these inversions in other genera] 30kb inversion

12. Chapter 7: Evolution of the nuclear genome of ferns and lycophytes Takuya Nakazato, Michael S. Barker, Loren H. Rieseberg, and Gerald J. Gastony Unfurling fern biology in the genomics age (BioScience, 2010) Michael S. Barker and Paul G. Wolf

13. Academic family tree of Gerald J. Gastony

14. Rolla and Alice Tryon 1950s and 1990s Is there an “Alice Tryon Women in Science” bequest for Botany Department?

15. Academic family tree of Gerald J. Gastony Rieseberg Nakazato Barker

16. The neglected fern and lycophyte nuclear genomes - or the “crying ferns” 1 genetic linkage map - Ceratopteris 4 EST libraries – Selaginella (2), Ceratopteris, Adiantum 3 BAC libraries - Selaginella (2), Ceratopteris 1 nuclear genome sequencing project in the works - Selaginella

17. The neglected fern and lycophyte nuclear genomes - or the “crying ferns” Why? large genome size (>2X) lack of funding for low economically important plants

18. The neglected fern and lycophyte nuclear genomes - or the “crying ferns” Why? large genome size (>2X) lack of funding for low economically important plants But ! 2nd largest land plant group sister to seed plants diverse land plant lineages need to be compared homologs of important seed plant genes occur in ferns

19. A short history of the study of the fern genome Haploid chromosome number • 57 in ferns vs. 16 in angiosperms • [ > 14 = polyploid (Grant, 1981) ] Ophioglossum (adder’s-tongue fern) - 2n = 1440 (96 ploid) in O. reticulatum

20. A short history of the study of the fern genome Haploid chromosome number • 57 in ferns vs. 16 in angiosperms • [ > 14 = polyploid (Grant, 1981) ] Questions: How does this fern choreograph meiosis with an n > 600? Has it ever been observed? Do large n's lead to more aborted or nonviable spores?

21. A short history of the study of the fern genome Haploid chromosome number • 57 in ferns vs. 16 in angiosperms • [ > 14 = polyploid (Grant, 1981) ] • 13.6 in heterosporous ferns is exception • heterosporous lycophytes << homosporous lycophytes • heterosporous seed plants << homosporous ferns & allies Therefore, homosporous ferns acquire high chromosome number to select for increased heterozygosity via polyploidy Hypothesis of Klekowski & Baker (1966)

22. A short history of the study of the fern genome Two lines of evidence did not support this hypothesis Isozyme analysis indicated widespread silencings of genes – diploid numbers of copies nn Most homosporous ferns are outcrossing Therefore, homosporous ferns acquire high chromosome number to select for increased heterozygosity via polyploidy Hypothesis of Klekowski & Baker (1966)

23. A short history of the study of the fern genome Homosporous ferns acquired high chromosome numbers with diploid gene expression via repeated cycles of polyploidization and subsequent gene silencing without chromosome loss Hypothesis of Chris Haufler (1987)

24. A short history of the study of the fern genome Many lines of evidence support this as the working hypothesis in ferns Pseudogenes in nuclear genes in Polystichum FISH detection of multiple dispersed chromosomal locations of rDNA in Ceratopteris +/- Genetic linkage map analysis in Ceratopteris Homosporous ferns acquired high chromosome numbers with diploid gene expression via repeated cycles of polyploidization and subsequent gene silencing without chromosome loss Hypothesis of Chris Haufler (1987)

25. The future of fern genomics? Ceratopteris has emerged as the “model” organism for fern genomics Study of the origin of polyploidy (neo- and paleo-) Correlating genomic changes to speciation and development • Two examples using Ceratopteris • Nakazato et al. (2006) genetic linkage analysis • Barker (2010) EST analysis

26. The future of fern genomics? Ceratopteris genetic linkage analysis • 700 genetic markers • 85% multiple copies • 24% single copy – low! • large numbers of duplicate genes on different chromosomes

27. The future of fern genomics? Ceratopteris genetic linkage analysis surprises! Maize linkage map • Expect clusters of linked duplicate genes on different chromosomes in recent (neo-) polyploids

28. Oxford plot of polyploid cotton’s A & D genomes Rong et al. 2004 • Expect clusters of linked duplicate genes on different chromosomes in recent (neo-) polyploids

29. Duplicated gene copies are hyper-dispersed across the genome of Ceratopteris Indicates ancient polyploid event and many subsequent chromosomal changes • Expect clusters of linked duplicate genes on different chromosomes in recent polyploids

30. The future of fern genomics? Ceratopteris EST analysis • expressed sequence tags • examines transcriptome • mRNA is extracted

31. The future of fern genomics? Ceratopteris EST analysis • cDNA is made with reverse transcriptase • ds cDNA is cloned into vector – library formed • cDNA sequenced from 5’ and 3’ ends (= Tags) • 400-800 bp ESTs can be contiged

32. The future of fern genomics? Ceratopteris EST analysis • synonymous substitution (silent) rate – Ks – obtained for duplicate genes • most duplications young and placed in ‘zero’ class • peak in duplications at 0.96 – 1.84 Ks or showing paleopolyploidy

33. The future of fern genomics? Ceratopteris EST analysis • synonymous substitution (silent) rate – Ks – obtained for duplicate genes • most duplications young and placed in ‘zero’ class • peak in duplications at 0.96 – 1.84 Ks or showing paleopolyploidy • using molecular clocks and phylogenetic trees, paleopolyploidy linked to early polypod diversification

34. Question Set 1 Question Set 2 • Ferns and fern allies are diverse and old; is it really appropriate to expect that all have their nuclear genomes evolving by same “rules”? • You have been given a blank check to sequence the fern genome of yourchoice. Which would you choose and why? What methods would you use? • Why is the fate of most duplicate genes to eventually become silenced? Could mutations accumulate in both copies at the same rate causing subfunctionalization, where mutations cause the two copies to functionally be diminished to one over time? • If you are really interested in understanding the process of speciation, would ferns be the better choice relative to angiosperms? • What are the justifications for selecting Ceratopteris richardii as a model organism for ferns? Do the “idiosyncratic” features of its genome affect generalization to ferns? • Could maintaining large amounts of physical genetic material be disadvantageous for fern evolution? Could it be related to slow speciation rates, compared to angiosperms? Or, on the other hand, could the silenced genes hold the key to the long history of fern evolution? • Can high chromosome numbers in ferns and lycophytes simply be an outcome of the ‘stringent bivalent pairing’ that is known in the group? How might that idea be further examined or tested?