Evolutionary Significance Of Polyploidy In The New Zealand Flora Brian Murray School of Biological Sciences The University of Auckland NEW ZEALAND http://nzprn.otago.ac.nz/wiki/bin/view/NZPRN/WebHome

NZ chromosome numbers are high High incidence of polyploidy: -90.9% have n > 10 -73.0% have n > 14 Even numbers more frequent than odd [polyploids Counts available for c. 83% of NZ angiosperms, 100% of gymnosperms, most ferns but relatively few bryophytes. Majority of species have high chromosome numbers. Even numbers more frequent than odd numbers. Haploid numbers >10 to 14 have been considered to be polyploid.

Many NZ angiosperm species are: Palaeopolyploids – Polyploids of more ancient origin History is obscured by the absence of obvious diploid relatives Identification via genome sequencing Arabidopsis, Oryza, Vitis, etc. Others are: Neopolyploids – Polyploids of relatively recent origin Similar/closely related to extant diploids (or lower polyploids) Need to differentiate between neo and palaeopolyploids.

Polyploidy in the 10 largest NZ angiosperm genera Genus No. polyploid/Total counted %polyploid Veronica 35/104 34 Carex holocentric so excluded - Celmisia 70/70 100 Coprosma 16/58 28 Epilobium 0/45 0 Ranunculus 36/38 95 Myosotis 11/11 100 Poa 34/34 100 Olearia 34/34 100 Aciphylla 0/21 0

Have the neopolyploids arisen autochthonously? Half of genera analyzed contained neopolyploids 40 genera: all species have the same chromosome number 42 genera: two or more ploidy levels Intraspecific polyploid variation in NZ endemic species Veronica (V. diosmifolia, V. odora and V. pinguifolia) Lobelia angulata Crassula ruamahanga Plantago lanigera, P. unibracteata Analysis of 82 genera with known chromosome numbers for 5+ species

Are the NZ polyploids autoploids or alloploids? Multiple copies of same genome Random chromosome synapsis Multivalent-forming Alloploids – Multiple copies of genomes that are sufficiently different to prevent pairing between them Bivalent-forming

Most NZ polyploid species are bivalent-forming Meiotic metaphase I is illustrated for 201 polyploid species in 77 genera 191 species are bivalent-forming 10 species are multivalent- forming Veronica (6), Pomaderris (2), Myoporum (1), and Myosotis (1)

Is the frequency of NZ autoploidy being underestimated? Does meiotic chromosome pairing give the ‘true’ story? Some NZ species radiations such as Veronica have diversified from a single colonizing genotype Low interspecific sequence variation Expect homoploid (diploid) hybrids rather than polyploid hybrids Examples where morphology and phylogeny suggest autoploidy But bivalent-forming, e.g. 20x Lobelia angulata Or multivalent-forming at very low frequency e.g. 4x Veronica odora. Low multivalent frequency in Lobelia, Myoporum and Myosotis is not a consequence of a low chiasma frequency Suggests almost all neopolyploids are alloploid, but is the frequency of autoploidy being underestimated? Several reasons to question whether the observations on meiotic chromosome pairing are giving the ‘true’ story. Sequence variation between species of radiating groups is characteristically low. Some examples such as Veronica are proposed to have diversified from a single colonizing genotype. Where genetic divergence of parental species is low they tend to form homoploid hybrids rather than polyploid ones. Examples where morphology and phylogeny suggest autoploidy yet they are bivalent-forming e.g. 20x Lobelia angulata multivalent-forming at very low frequency e.g. 4x Veronica odora. Low multivalent frequency in Lobelia, Myoporum and Myosotis is not a consequence of a low chiasma frequency as they all show many bivalents with two chiasmata. Has there been widespread genomic differentiation in examples of autochthonous polyploids?

Has there been widespread genomic differentiation in examples of autochthonous polyploids? NZ Plantago Six ploidy levels, 2x, (4x), 8x, 10x, 12x and 16x Autoploid series? But both 8x and 16x species are bivalent-forming = alloploidy? Currently working on the NZ species of Plantago.

Plantago molecular cytogenetics Genome size measurements Fluorescence in situ hybridization (mitotic and meiotic) Homologous sequences hybridize to the probe, allowing identification of specific sequences and similar genomes Two approaches: GISH and FISH GISH: Use whole genomic DNA of diploids to probe/paint the chromosomes of related polyploids FISH: Use specific sequences (rDNA) to locate 45S and 5S rDNA sites as a proxy for genome reorganization or differentiation

Genome size in Plantago Species Ploidy C-value Cx-value P. lanigera 2x 3.87 1.94 P. spathulata 8x 9.03 1.13 P. picta 8x 9.35 1.16 P. raoulii 8x 9.91 1.24 P. triandra 8x 10.52 1.32 P. unibracteata 10x 14.13 1.41 P. unibracteata 12x 17.51 1.46 P. n. sp. 16x 17.95 1.12 Significant genome downsizing in the transition from 2x to 8x but not 10x to 12x unibracteata.

GISH analyses of P. spathulata DAPI lanigera probe triandra probe (left) obconica probe (right)

Summary of GISH analysis The diploids P. lanigera and P. obconica have similar genomes but different to P. triantha All 8x species are alloploids containing 2 copies of P. lanigera/ P. obconica, 2 copies of P. triantha plus 4 copies of unidentified genome(s) 12x unibracteata is an alloploid containing 2 copies of the P. lanigera genome 16x P. n. sp. Seems to be an alloploid with 4 copies of the P. lanigera and 4 copies of the P. triantha genome 2x 8x 12x 16x LL/OO and TT LLTT???? LL?????????? LLLLTTTT????????

FISH analysis of P. spathulata DAPI stained chromosomes of P. spathulata 8x The same cell probed with 45S rDNA (green) and 5S rDNA (red) probes

P. lanigera 2x P. spathulata 8x P. picta 8x P. n. sp. 16x P. raoulii 8x P. triandra 8x FISH localization of 45S (green) and 5S (red) rDNA loci in Plantago

Meiotic metaphase 1 in 8x P. spathulata The ‘lanigera’ genome painted green in ‘B’ with GISH. Neither of the rDNA probes (red and green in ‘C’) hybridizes to the ‘lanigera’ chromosomes (arrowed).

Conclusions Polyploidy is widespread in the NZ flora with evidence for autochthonous origins in many cases. Polyploidy is associated with many of the species radiations in NZ angiosperms, but some have radiated at the diploid level. There is a predominance of alloploids (bivalent-pairing) despite low levels of sequence differentiation and possible origins from a single colonizer. Molecular cytogenetic analysis of the endemic Plantago species strongly suggests that they are alloploids and that there has been significant genomic reorganization associated with speciation. Genome reorganization has been important for the diploidization process.

Acknowledgements Charles Wong for the beautiful GISH and FISH images. Ross Ferguson and Jingli Li (The NZ Institute for Plant and Food Research) for help with the flow cytometry. Heidi Meudt (Museum of New Zealand Te Papa Tongarewa) Mei Lin Tay and Phil Garnock-Jones (Victoria University of Wellington) for plant material and for sharing their knowledge of the genus Plantago.