Research Article

The Aquilegia genome provides insight into adaptive radiation and reveals an extraordinarily polymorphic chromosome with a unique history

Vienna BioCenter, Austria
University of California, United States
Masaryk University, Czech Republic
Vienna Graduate School of Population Genetics, Austria
Joint Genome Institute, United States
HudsonAlpha Institute of Biotechnology, United States
Centre of the Region Haná for Biotechnological and Agricultural Research, Czech Republic
Harvard University, United States

Oct 16, 2018

https://doi.org/10.7554/eLife.36426

Open access
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Figures
Tables
Additional files

8 figures, 3 tables and 15 additional files

Figures

Figure 1 with 1 supplement

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Distribution of *Aquilegia* species.

There are ~70 species in the genus *Aquilegia*, broadly distributed across temperate regions of the Northern Hemisphere (grey). The 10 *Aquilegia* species sequenced here were chosen as representatives spanning this geographic distribution as well as the diversity in ecological habitat and pollinator-influenced floral morphology of the genus. *Semiaquilegia adoxoides*, generally thought to be the sister taxon to *Aquilegia* (Fior et al., 2013), was also sequenced. A representative photo of each species is shown and is linked to its approximate distribution.

https://doi.org/10.7554/eLife.36426.002

Figure 1—figure supplement 1

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Origin of species samples used for sequencing.
https://doi.org/10.7554/eLife.36426.003

Figure 2 with 2 supplements

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Polymorphism and divergence in *Aquilegia*.

(a) The percentage of pairwise differences within each species (estimated from individual heterozygosity) and between species (divergence). $F_{ST}$ values between geographic regions are given on the lower half of the pairwise differences heatmap. Both heatmap axes are ordered according to the neighbor joining tree to the left. This tree was constructed from a concatenated data set of reliably-called genomic positions. (b) Polymorphism within each sample by chromosome. Per-chromosome values are indicated by the chromosome number.

https://doi.org/10.7554/eLife.36426.004

Figure 2—figure supplement 1

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Polymorphism across the genome in all ten species samples.
https://doi.org/10.7554/eLife.36426.005

Figure 2—figure supplement 2

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Species and chromosome trees of *Aquilegia*.
https://doi.org/10.7554/eLife.36426.006

Figure 3 with 3 supplements

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Discordance between gene and species trees.

(a) Cloudogram of neighbor joining (NJ) trees constructed in 100 kb windows across the genome. The topology of each window-based tree is co-plotted in grey and the whole genome NJ tree shown in Figure 2a is superimposed in black. Blue numbers indicate the percentage of window trees that contain each of the subtrees observed in the whole genome tree. (b) Genome NJ tree topology. Blue letters a-c on the tree denote subtrees a-c in panel (d). (c) Chromosome four NJ tree topology. Blue letters d and e on the tree denote subtrees d and e in panel (d). (d) Prevalence of each subtree that varied significantly by chromosome. Genomic (black bar) and per chromosome (chromosome number) values are given.

https://doi.org/10.7554/eLife.36426.007

Figure 3—figure supplement 1

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Proportion of significantly-varying subtrees by chromosome.
https://doi.org/10.7554/eLife.36426.008

Figure 3—figure supplement 2

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P-values of proportion tests by chromosome for significantly-different trees.
https://doi.org/10.7554/eLife.36426.009

Figure 3—figure supplement 3

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Subtree prevalence across chromosomes for the nine significantly-different subtrees.
https://doi.org/10.7554/eLife.36426.010

Figure 4 with 1 supplement

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Sharing patterns of derived polymorphisms.

Proportion of derived variants (a) private to an individual species, (b) shared within the geographic region of origin, (c) shared across two geographic regions, and (d) shared across all three geographic regions. Genomic (black bar) and chromosome (chromosome number) values, for all 10 species.

https://doi.org/10.7554/eLife.36426.011

Figure 4—figure supplement 1

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Sharing pattern percentages by pattern type.
https://doi.org/10.7554/eLife.36426.012

Figure 5

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D statistics demonstrate gene flow during *Aquilegia* speciation.

D statistics for tests with (**a–c**) all North American species, (d) both European species, (e) Asian species other than *A. oxysepala*, and (f) *A. oxysepala* as H3 species. All tests use *S. adoxoides* as the outgroup. D statistics outside the green shaded areas are significantly different from zero. In (**a–e**), each individual dot represents the D statistic for a test done with a unique species combination. In (f), D statistics are presented by chromosome (chromosome number) or by the genome-wide value (black bar). In all panels, E = European and A = Asian without *A. oxysepala*. In some cases, individual species names are given when the geographical region designation consists of a single species. Right hand panels are a graphical representation of the D statistic tests in the corresponding left hand panels. Trees are a simplified version of the genome tree topology (Figure 2b), in which the bold sub tree(s) represent the bifurcation considered in each set of tests. H3 species are noted in blue while the H1 and H2 species are specified in black. (Figure 5—source data 1).

https://doi.org/10.7554/eLife.36426.013

Figure 5—source data 1 (D statistics).: https://doi.org/10.7554/eLife.36426.014
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Figure 6 with 2 supplements

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The effect of differences in coalescence time and gene flow on tree topologies.

(a) The observed proportion of informative derived variants supporting each possible Asian tree topology genome-wide and on chromosome four. Species considered include *A. oxysepala* (oxy), *A. japonica* (japon), and *A. sibirica* (sib). (b) The coalescent model with bidirectional gene flow in which *A. oxysepala* diverges first at time t2, but later hybridizes with *A. japonica* between t = 0 and t1 at a rate determined by per-generation migration rate, m. The population size (N) remains constant at all times. (c) The proportion of each tree topology and estimated D statistic for simulations using four combinations of m and Nvalues (t1 = 1 in units of N generations). The combination presented in the first row (m = 2x10^-5and N = 11667) generates tree topology proportions that match observed allele sharing proportions genomewide. Simulations with increased m and/or N (rows 3–4) result in proportions which more closely resemble those observed for chromosome four. Colors in proportion plots refer to tree topologies in (a), with black bars representing the residual probability of seeing no coalescence event. While this simulation assumes symmetric gene flow, similar results were seen for models incorporating both unidirectional and asymmetric gene flow (Figure 6—figure supplements 1 and 2).

https://doi.org/10.7554/eLife.36426.015

Figure 6—figure supplement 1

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Model output for all three gene flow scenarios.
https://doi.org/10.7554/eLife.36426.016

Figure 6—figure supplement 2

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Tree topology proportions simulated under assymmetric and unidirectional models.
https://doi.org/10.7554/eLife.36426.017

Figure 7 with 2 supplements

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Recombination and selection on chromosome four (a) Physical vs.

genetic distance for all chromosomes calculated in an *A. formosa* x *A. pubescens* mapping population. High nucleotide diversity on chromosome four was also observed in parental plants of this population (Figure 7—figure supplement 1. (b) Relationship between gene density (proportion exonic) and recombination rate (main effect p-value < 2 x 10^-16, chromosome four effect p-value < 2 x 10^-16, interaction p-value < 1.936 x 10^-11, adjusted R² = 0.8045). (c) Relationship between gene density and D statistic for *A. oxysepala* and *A. japonica* gene flow. (d) Relationship between gene density and mean neutral nucleotide diversity. Figure 7—source data 1.

https://doi.org/10.7554/eLife.36426.018

Figure 7—source data 1 (Physical and genetic distance for A.formosa x A.pubescens markers).: https://doi.org/10.7554/eLife.36426.021
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Figure 7—figure supplement 1

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Polymorphism in the *A. formosa x A. pubescens* mapping population.
https://doi.org/10.7554/eLife.36426.019

Figure 7—figure supplement 2

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Distribution of gene expression values by chromosome.
https://doi.org/10.7554/eLife.36426.020

Figure 8 with 1 supplement

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Cytogenetic characterization of chromosome four in *Semiaquilegia* and *Aquilegia* species.

Pachytene chromosome spreads were probed with probes corresponding to oligoCh4 (red), 35S rDNA (yellow), 5S rDNA (green) and two (peri)centromeric tandem repeats (pink). Chromosomes were counterstained with DAPI. Scale bars = 10 μm.

https://doi.org/10.7554/eLife.36426.025

Figure 8—figure supplement 1

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Immunodetection of anti-5mC antibody.
https://doi.org/10.7554/eLife.36426.026

Tables

Table 1

GO term enrichment on chromosome four

https://doi.org/10.7554/eLife.36426.022

GO	Corrected P-value	Number on Chr_04		Percent of Chr_04 genes	GO term
GO	Corrected P-value	Observed	Expected	Percent of Chr_04 genes	GO term
0043531	$5.61 \times 10^{- 79}$	140	9	7.57	ADP binding
0016705	$4.40 \times 10^{- 48}$	179	39	9.68	Oxidoreductase activity, actingon paired donors, withincorporation or reduction of molecular oxygen
0004497	$7.19 \times 10^{- 46}$	158	32	8.55	Monooxygenase activity
0005506	$2.73 \times 10^{- 41}$	181	46	9.79	Iron ion binding
0020037	$2.57 \times 10^{- 37}$	186	53	10.06	Heme binding
0010333	$1.72 \times 10^{- 15}$	39	4	2.11	Terpene synthase activity
0016829	$2.08 \times 10^{- 13}$	39	5	2.11	Lyase activity
0055114	$9.53 \times 10^{- 10}$	247	149	13.36	Oxidation-reduction process
0016747	$6.66 \times 10^{- 5}$	44	16	2.38	Transferase activity,transferring acyl groups other than amino-acyl groups
0000287	$1.23 \times 10^{- 4}$	42	15	2.27	Magnesium ion binding
0008152	$2.56 \times 10^{- 4}$	137	83	7.41	Metabolic process
0006952	$3.60 \times 10^{- 4}$	32	10	1.73	Defense response
0004674	$4.52 \times 10^{- 4}$	23	5	1.24	Protein serine/threoninekinase activity
0016758	$1.35 \times 10^{- 3}$	44	18	2.38	Transferase activity, transferringhexosyl groups
0005622	$4.14 \times 10^{- 3}$	14	42	0.76	Intracellular
0008146	$2.68 \times 10^{- 2}$	9	1	0.49	Sulfotransferase activity
0016760	$3.72 \times 10^{- 2}$	12	2	0.65	Cellulose synthase(UDP-forming) activity

Table 2

Content of the A. coerulea v3.1 reference by chromosome

https://doi.org/10.7554/eLife.36426.023

	Chromosome							Genome
	1	2	3	4	5	6	7	Genome
Number of genes	5041	4390	4449	3149	4786	3292	4443	29550
Genes per Mb	112	102	104	69	107	108	102	100
Mean gene length (bp)	3629	3641	3689	3020	3712	3620	3708	3580
Percent repetitive	38.9	41.1	39.1	54.2	39.4	39.3	40.6	42.0
Percent genes withHIGH effect variant	25.3	23.8	23.6	32.3	24.1	22.1	23.6	24.7
Percent GC	36.8	37.0	36.9	37.0	37.1	36.8	36.8	37.0

Table 3

Population genetics parameters for Semiaquilegia by chromosome

https://doi.org/10.7554/eLife.36426.024

	Percent pairwise differences
	Chromosome							Genome
	1	2	3	4	5	6	7	Genome
Polymorphism within Semiaquilegia	0.079	0.085	0.081	0.162	0.076	0.078	0.071	0.082
Divergence between Aquilegia and Semiaquilegia	2.46	2.47	2.47	2.77	2.48	2.47	2.47	2.48