Evolutionary Significance Of

Polyploidy In The New Zealand Flora

Brian MurraySchool of Biological SciencesThe 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

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)

Many NZ angiosperm species are:

Polyploidy in the 10 largest NZ angiosperm genera

Genus No. polyploid/Total counted%polyploid

Veronica 35/104 34Carex holocentric so excluded -Celmisia 70/70 100Coprosma 16/58 28Epilobium 0/45 0Ranunculus 36/38 95Myosotis 11/11 100Poa 34/34 100Olearia 34/34 100Aciphylla 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

Autoploids –• Multiple copies of same genome• Random chromosome synapsis• Multivalent-forming

Are the NZ polyploids autoploids or alloploids?

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

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?

• 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

Plantago molecular cytogenetics

Genome size in Plantago

Species Ploidy C-value Cx-value

P. lanigera 2x 3.87 1.94P. spathulata 8x 9.03 1.13P. picta 8x 9.35 1.16 P. raoulii 8x 9.91 1.24P. triandra 8x 10.52 1.32P. unibracteata 10x 14.13 1.41P. unibracteata 12x 17.51 1.46P. n. sp. 16x 17.95 1.12

Significant genome downsizing in the transition from 2x to 8x but not 10x to 12x unibracteata.

DAPI

lanigera probe

triandra probe (left)obconica probe (right)

GISH analyses of P. spathulata

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 16xLL/OO and TT LLTT???? LL?????????? LLLLTTTT????????

DAPI stained chromosomes of P. spathulata 8x

The same cell probed with 45S rDNA (green) and 5S rDNA (red) probes

FISH analysis of P. spathulata

P. lanigera 2x

P. n. sp. 16x

P. spathulata 8x

P. picta 8x

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).

•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.

Conclusions

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.

Acknowledgements