1. Evolutionary Significance Of Polyploidy In The New Zealand Flora
Brian Murray
School of Biological Sciences
The University of Auckland
NEW ZEALAND

2. NZ chromosome numbers are high
High incidence of polyploidy:
-90.9% have n > % 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.

3. 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.

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

5. 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

6. 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

7. 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)

8. 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?

9. 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.

10. 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

11. Genome size in Plantago
Species Ploidy C-value Cx-value
P. lanigera 2x
P. spathulata 8x
P. picta 8x
P. raoulii 8x
P. triandra 8x
P. unibracteata 10x
P. unibracteata 12x
P. n. sp. 16x
Significant genome downsizing in the transition from 2x to 8x but not 10x to 12x unibracteata.

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

13. 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????????

14. 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

15. 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

16. 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).

17. 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.

18. 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.