Exogenous selection rather than cytonuclear incompatibilities shapes asymmetrical fitness of reciprocal Arabidopsis hybrids.

Reciprocal crosses between species often display an asymmetry in the fitness of F1 hybrids. This pattern, referred to as isolation asymmetry or Darwin's corollary to Haldane's rule, is a general feature of reproductive isolation in plants, yet factors determining its magnitude and direction remain unclear. We evaluated reciprocal species crosses between two naturally hybridizing diploid species of Arabidopsis to assess the degree of isolation asymmetry at different postmating life stages. We found that pollen from Arabidopsis arenosa will usually fertilize ovules from Arabidopsis lyrata; the reverse receptivity being less complete. Maternal A. lyrata parents set more F1 hybrid seed, but germinate at lower frequency, reversing the asymmetry. As predicted by theory, A. lyrata (the maternal parent with lower seed viability in crosses) exhibited accelerated chloroplast evolution, indicating that cytonuclear incompatibilities may play a role in reproductive isolation. However, this direction of asymmetrical reproductive isolation is not replicated in natural suture zones, where delayed hybrid breakdown of fertility at later developmental stages, or later-acting selection against A. arenosa maternal hybrids (unrelated to hybrid fertility, e.g., substrate adaptation) may be responsible for an excess of A. lyrata maternal hybrids. Exogenous selection rather than cytonuclear incompatibilities thus shapes the asymmetrical postmating isolation in nature.

Introduction

In many cases of hybridization, there is an asymmetry in the fitness of reciprocal F1 hybrid crosses (Tiffin et al. 2001; Turelli and Moyle 2007; Bolnick et al. 2008). This asymmetry has been called isolation asymmetry or Darwin's corollary to Haldane's rule (Turelli and Moyle 2007). The pattern cannot be explained by Dobzhansky–Muller incompatibilities (DMIs) between autosomal loci because reciprocal hybrids have the same autosomal genotype (Turelli and Moyle 2007). The nuclear genome is inherited equally from both parents and, aside from interactions between hybrid nuclear genotypes and their environment, is not transmitted differentially. Instead, isolation asymmetry is probably due to DMIs involving uniparentally inherited factors or interactions between the maternal and hybrid progeny's genomes (Turelli and Moyle 2007). These nonnuclear contributions may include cytoplasmic effects (Burton et al. 2013) or genomic imprinting (e.g., unequal contributions to the endosperm; Gehring 2013). Such effects are transmitted differentially (asymmetrically) and thus manifest themselves as fitness differences in reciprocal crosses between species (e.g., Etterson et al. 2007; Martin and Willis 2010; Goodwillie and Ness 2013).

Theory suggests that the direction with the lowest fitness in reciprocal crosses between species (isolation asymmetry) will vary with the relative rates of cytoplasm and nuclear evolution in the parental species (Turelli and Moyle 2007). If these rates differ between parental species, then crosses with the lower rate of offspring viability are those in which the maternal parent originates from the species with a higher comparative rate of cytoplasm evolution (as there is a higher probability of cytonuclear incompatibilities; Bolnick et al. 2008). This theory suggests that the direction of asymmetry might be predictable from the fitness of maternal hybrid species present in natural suture zones, as well as which of the two maternal species accumulates nucleotide substitutions in the cytoplasm at a higher rate.

Here we analyze reciprocal-cross data between two naturally hybridizing species of Arabidopsis (Arabidopsis arenosa and Arabidopsis lyrata) to examine patterns of asymmetry through time at two stages of isolation: seed set and seed germination. These closely related species are self-incompatible hermaphrodites with a sympatric range in parts of central Europe where they hybridize. In these suture zones, both diploid and tetraploid hybrids exhibit A. lyrata maternal backgrounds (Schmickl and Koch 2011; M. Koch, N. Hohmann, G. Muir, unpubl. data), suggesting the presence of isolation asymmetry in fertility (or survivorship) of reciprocal hybrid crosses.

The results of these crosses support the notion that postmating barriers are generally strong and contribute significantly to asymmetrical reproductive isolation in these two species, while never solely leading to complete isolation (for a STRUCTURE analysis, Pritchard et al. 2000; of gene pools and admixture between these two diploids, see Hohmann et al. 2014). Given the split time of these two lineages, based on fossil-calibrated divergence time estimates (1–2 Myr, Hohmann et al. unpubl. data), the potential for accumulating reproductive barriers during speciation has been limited. Together with the direction of asymmetry in the wild, we discuss the factors that shape the evolution and fitness of interspecific Arabidopsis hybrids.

Materials and Methods

Source material, artificial crosses, and experimental design

We conducted reciprocal crosses in the greenhouse between a diploid member of the Arabidopsis arenosa group (A. carpatica, hereafter A. arenosa) and A. lyrata subsp. petraea (hereafter A. lyrata). Material for the crosses was raised from open-pollinated seeds collected in Nízke Tatry and Vel'ká Fatra, central Slovakia (A. arenosa), and from the foothills of the eastern Austrian limestone Forealps (A. lyrata). Eight reciprocal crosses (between couples) were made between these two species; each couple producing full-sib offspring. A further six and four (half-sib) conspecific crosses were conducted within A. arenosa and A. lyrata, respectively, to control for differences in the receptivity or fecundity of the parental taxa. Ten to fifteen pollinations were made by hand for each parental cross. All pollinations were performed in a pollinator-free environment and conducted without competition; pollen from only a single paternal parent was placed on each stigma. Measures for crossing success were seed set and the proportion of seeds that were viable (F1 seed viability). Seed viability was measured for >20 seeds for each cross performed and assessed by germination ability (% germination of seeds sown).

Prior to germination, seeds were washed three times for 10 min in a 10% sodium hypochlorite solution and washed thoroughly in sterile water. After partially drying, seeds were plated on agar plates containing half-strength salts and vitamins, 1.5% sucrose, and 0.8% agar (Murashige and Skoog 1962). The plates were placed for 2 days at 4°C, and then seedlings were planted in medium containing a 3:1 mixture of a peat-based compost and 1–3 mm grit. Potted seedlings were raised under short-day conditions (8 h of light/16 h of dark) at 22°C.

Seed traits, fitness, and statistical analysis

Reproductive isolation was defined separately for each fitness-related parameter: seed set and F1 seed viability. Fitness was measured as the total number of fully mature seeds produced per maternal plant for each cross performed, assessed over an extended 5-month period (May–September).

Likelihood ratio chi-square tests were used to test whether the success of a cross was significantly affected by which species was the pollen parent and which species was the seed parent. Separate tests were conducted for each of the stages at which isolation was measured.

The following morphological traits for each cross and their parents were measured from a sample of five siliques (seeds included) per individual: silique length, seed width and length (to the nearest 0.1 μm), and ratios of seed and wing size (minimum and maximum length ÷ width). Images of these traits were analyzed with WinFolia image analysis software (Regent Instrument Inc., Quebec, Canada). A principal component analysis was conducted on these five values in SPSS Statistics for Windows v19.0 (IBM, Armonk, NY) to identify key components of the fruiting structure that explained the greatest possible variance in the data, and to group and/or separate parents/F1 progeny visually.

Results

Hybrid fitness depends on the direction of the cross

Significant asymmetries in the strength of reproductive isolation between A. arenosa and A. lyrata were found at both stages of isolation (Fig.1A and B). All eight heterospecific crosses performed to generate the hybrid F1 generation were successful in at least one direction. However, the success of the crosses was dependent on the species of the maternal and the paternal parent, that is, the direction of the cross affected either seed production or germination and thus success rates. For crosses between A. lyrata (as the maternal parent, ♀) × A. arenosa (as the pollen donor, ♂), seeds were produced in higher quantities than the reciprocal cross (Fig.1A), with a twofold reduction when A. arenosa was the maternal parent. A. lyrata maternal parents produce on average almost twice as many seeds as A. arenosa maternal parents (Fig.1A; Mann–Whitney U-test, P = 0.029).

Figure 1

Relative mean fitness of F1 individuals from intra- and interspecific experimental pollinations. Seed production (A) was defined as the total number of seeds collected from viable siliques for each cross performed. Germination (B) was defined by the number of germinating seeds per 100 seeds sown. Maternal parents are grouped by species. For each parental cross (intraspecific/interspecific), four to eight F1 families (replicates), respectively, were generated.

The asymmetry was prevalent in all eight crosses for seed production and six of eight crosses for viable seeds. Moreover, for both directions, the asymmetries were significant at P < 0.0001. Note that these data are corrected for differences in the potential of parental taxa to set seed or in the proportion of viable seeds produced under experimental conditions.

Interestingly, the direction of asymmetry at germination was reversed (Fig.1B). Germination rates of the fewer F1 hybrid seeds produced when A. arenosa was the maternal parent were significantly higher than germination rates of A. lyrata maternal F1 hybrid seeds, despite producing more seeds (Mann–Whitney U-test, P = 0.027).

Expected development failure of low germinating A. lyrata maternal F1 seeds not apparent from PCA

The low germination of A. lyrata maternal F1's may be a result of endosperm development failure (Haig 2013), which would be evident in the size and/or appearance of nongerminating seeds. In the absence of dissecting fertilized ovules to check for incomplete development, we used a PCA of seed morphology in the parents and the F1, as a proxy for endosperm overgrowth (large seeds). Interestingly, the low germinating F1 A. lyrata seeds are subsumed in the same cloud as their parents, that is, the expected development failure is not apparent (Fig.2). On the other hand, the higher germinating F1 maternal A. arenosa seeds are noticeably smaller. They sit outside the main cloud containing both parents/F1 maternal A. lyrata. This suggests that development failure might not be a factor in the low germination of A. lyrata maternal F1 hybrids. However, endosperm dissection data would be required to confirm this.

Figure 2

PCA projection of seed morphology (as a proxy of endosperm development) measured in A. arenosa, A. lyrata, and their F1 offspring. First two principal components from a PCA analysis of seed morphology measured from F1 seeds of conspecific crosses (A. arenosa, blue and A. lyrata, green circles, respectively) and heterospecific crosses (F1 maternal A. arenosa, orange and F1 maternal A. lyrata, purple crosses, respectively).

An analysis of variance on germination rates (as the response variable) indicated that the source of the seed (parent as a fixed factor) was statistically significant (P = 0.013) while seed size (covariate) and the interaction between the two were not significant (P = 0.105 and P = 0.193, respectively), suggesting that while seed size appears to have no effect on seed viability, the maternal parent is a significant component of seed fitness in our experiment.

Discussion

Several prezygotic mechanisms may account for the asymmetries in seed set observed in this study. Self-incompatible species may be less receptive to foreign pollen than self-compatible species, for example, leading to significant asymmetries in hybrid seed set between the two mating types (Lewis and Crowe 1958). Both species, however, are self-incompatible (SI), leaving this explanation unlikely. In addition, although detailed analysis of the maternal component of the SI system (S receptor kinase, SRK) and its segregation in our F1 families (Appendix 1, TableA1) showed some evidence for segregation distortion, this was often due to selection against homozygotes, which is expected for loci involved in self-incompatibility (due to strong inbreeding depression). Similar distortion was found for conspecific as well as heterospecific crosses and so there is no evidence that the S locus might be involved in selection against hybrids.

Table A1

Segregation tests of S locus genotypes against the null expectation of equal probability of transmission

Family/cross	Parental genotypes	Progeny1					χ2	P-value
						Total
Interspecific2
H		Ah18 x	Ah18 3	x x	x 3
A. lyrata ♀ × A. arenosa ♂	Ah18 x ♀ × x 3 ♂	9 (5.5)	4 (5.5)	0 (5.5)	9 (5.5)	22	10.36	0.016
L			25 16	x 25 or 25 25	x 16
A. lyrata ♀ × A. arenosa ♂	25 x ♀ × 25 16 ♂		0 (2.75)	3 (5.5)	8 (2.75)	11	13.91	0.001
Intraspecific
M			18a 25	x 18a or 18a18a	x 25
A. arenosa	18a x ♀ × 18a 25 ♂		3 (5)	11 (10)	6 (5)	20	0.57	0.577
	18a 25 ♀ × 18a x ♂		3 (4)	8 (8)	5 (4)	16	0.50	0.779
	Total		6 (9)	19 (18)	11 (9)	36	1.50	0.472
O3			25 x or 25 25	16 25	16 x
A. arenosa	25 16 ♀ × 25 25 ♂		7 (4.5)	2 (4.5)	0	9	2.78	0.100
	25 25 ♀ × 25 16 ♂		3 (5)	7 (5)	0	10	1.60	0.206
	Total		10 (9.5)	9 (9.5)	0	19	0.05	0.819
R		16 18a	16 16	8 18a	8 16
A. arenosa	16 8 ♀ × 18a 16 ♂	6 (3.25)	0 (3.25)	4 (3.25)	3 (3.25)	13	5.77	0.123
	18a 16 ♀ × 16 8 ♂	4 (2.5)	0 (2.5)	5 (2.5)	1 (2.5)	10	6.80	0.079
	Total	10 (5.75)	0 (5.75)	9 (5.75)	4 (5.75)	23	11.26	0.010
A			Cg5 14	x Cg5 or Cg5 Cg5	x 14
A. lyrata	Cg5 x ♀ × Cg5 14 ♂		1 (2.25)	4 (4.5)	4 (2.25)	9	2.11	0.348
	Cg5 14 ♀ × Cg5 x ♂		0 (1.75)	2 (3.5)	5 (1.75)	7	8.43	0.015
	Total		1 (4)	6 (8)	9 (4)	16	9.0	0.011

Observed number of F1 individuals within each full-sib (heterospecific)/half-sib (conspecific) cross and genotype class. Expected values are shown in parentheses. x, missing allele; Cg, SRK allele similar to Capsella grandiflora; Ah, SRK allele similar to A. halleri.

For each reciprocal cross, each genotype was used both as a female (♀) and a male (♂) parent. Note that one allele is frequently missing from either (or both) parent because of inconclusive genotyping.

Although only a single allele was resolved in one parent for this family (O), sample sizes were sufficient to obtain a robust test of whether this was due to homozygosity or the presence of a null allele. Assuming homozygosity in one parent fit the data much better (χ2 = 0.05; P = 0.819, see also main text).

Differential fruit abortion may account for the asymmetry in seed set. Variation in reproductive success may occur because pollen competes for access to ovules or because seed parents differentially exclude pollen phenotypes (Moore and Pannell 2011). The asymmetry we observe for seed set is consistent with Kaneshiro's (1980) hypothesis of asymmetrical mate choice, predicting that pollen from an ancestral taxon (A. arenosa) may fertilize ovules from a derived taxon (A. lyrata), but not vice versa. While plants do not choose their mates in the same way female animals may actively choose (as envisaged originally by Kaneshiro 1980), discrimination among pollen grains based on the genotype expressed at SI loci, for example, is of course possible. Kaneshiro's prediction can thus be tested in plants (Tiffin et al. 2001). We observed that the isolation asymmetry between A. arenosa and A. lyrata was not (100%) complete, however, suggesting that barriers to gene flow between these two species may be reversed over the course of species divergence (e.g., Fuller 2008).

Arabidopsis lyrata maternal hybrids are more successful in the wild – contra predictions based on chloroplast evolution

Asymmetries in postzygotic incompatibility between plant species are less well documented particularly the asymmetry reversals between life history stages reported here. Postzygotic isolation may result from several types of nuclear–cytoplasmic interactions (Burton et al. 2013). Cytoplasmic male sterility elements may be responsible for the asymmetric hybrid viabilities if male sterility in the maternal parent is not restored by nuclear genes in the F1 (hybrid) background. In reciprocal crosses between species, the maternal parent with faster cytoplasm evolution will tend to produce less viable F1 hybrids (lower germination rates) owing to an increased probability of cytonuclear incompatibilities (Turelli and Moyle 2007). We tested this prediction using whole chloroplast genome data and molecular evolution rates from a clade of Arabidopsis close relatives including A. arenosa and A. lyrata (Appendix 2; Fig.A1, TableA2). As predicted, the species which tended to be the inferior maternal parent for F1 hybrids (A. lyrata; seed viability) exhibited accelerated chloroplast genome evolution, providing comparative evidence for a systematic basis to Darwin's corollary. This result is consistent with the hypothesis that cytonuclear incompatibilities can play an important role in reproductive isolation in our reciprocal crosses. However, such asymmetrical reproductive isolation does not explain the direction of asymmetrical chloroplast introgression observed between A. arenosa and A. lyrata in natural suture zones, where A. lyrata tends to be the hybrid maternal parent (Schmickl and Koch 2011; Koch et al. unpubl. data). This suggests that there may be further delayed hybrid breakdown of fertility at later developmental stages or that later-acting selection against A. arenosa maternal hybrids (unrelated to hybrid fertility, e.g. substrate adaptation Schmickl and Koch 2011) is responsible for the apparent excess of A. lyrata maternal hybrids in the wild.

Figure 3

Comparison of chloroplast substitution rates between A. arenosa and A. lyrata, using Arabidopsis cebennensis as an outgroup. The maximum likelihood tree depicts rates of synonymous and nonsynonymous substitution based on ∼127 kbp from whole chloroplast genome sequences. Values indicate node support; bootstrap values estimated using RAxML (Stamatakis 2014). The rate differences between A. arenosa and A. lyrata for whole genome sequences were highly significant based on the relative rate test (Tajima 1993) implemented in MEGA (Tamura et al. 2013). TableA2 summarizes the results of relative rate tests performed for all pairwise heterospecific comparisons in MEGA (including both synonymous and nonsynonymous sites). All comparisons exhibited a significant difference in rates between the two species at the P = 0.01 threshold (without corrections for multiple testing).

Table A2

Pairwise relative rate tests for Arabidopsis arenosa1 and A. lyrata chloroplast genome lineages

Sequence A	Sequence B	Identical sites in all three sequences	Divergent sites in all three sequences	Unique differences in Sequence A	Unique differences in Sequence B	Unique differences in Sequence C	χ2	P-value
Acarpatica3	Alyratapetraea1	127091	0	69	134	209	20.81	0.00001
Acarpatica3	Alyratapetraea2	127110	0	69	115	209	11.5	0.0007
Acarpatica3	Alyratapetraea3	127108	0	69	117	209	12.39	0.00043
Acarpatica3	Alyratapetraea7	127109	0	68	116	210	12.52	0.0004
Acarpatica3	Alyratapetraea9	127105	0	68	120	210	14.38	0.00015
Acarpatica3	Alyratapetraea10	127106	0	69	119	209	13.3	0.00027
Acarpatica3	Alyratapetraea11	127103	0	68	122	210	15.35	0.00009
Acarpatica3	Alyratapetraea12	127110	0	68	115	210	12.07	0.00051
Acarpatica4	Alyratapetraea1	127087	0	73	134	209	17.98	0.00002
Acarpatica4	Alyratapetraea2	127106	0	73	115	209	9.38	0.00219
Acarpatica4	Alyratapetraea3	127104	0	73	117	209	10.19	0.00141
Acarpatica4	Alyratapetraea7	127105	0	72	116	210	10.3	0.00133
Acarpatica4	Alyratapetraea9	127101	0	72	120	210	12	0.00053
Acarpatica4	Alyratapetraea10	127102	0	73	119	209	11.02	0.0009
Acarpatica4	Alyratapetraea11	127099	0	72	122	210	12.89	0.00033
Acarpatica4	Alyratapetraea12	127106	0	72	115	210	9.89	0.00166
Acarpatica6	Alyratapetraea1	127088	0	72	134	209	18.66	0.00002
Acarpatica6	Alyratapetraea2	127107	0	72	115	209	9.89	0.00166
Acarpatica6	Alyratapetraea3	127105	0	72	117	209	10.71	0.00106
Acarpatica6	Alyratapetraea7	127106	0	71	116	210	10.83	0.001
Acarpatica6	Alyratapetraea9	127102	0	71	120	210	12.57	0.00039
Acarpatica6	Alyratapetraea10	127103	0	72	119	209	11.57	0.00067
Acarpatica6	Alyratapetraea11	127100	0	71	122	210	13.48	0.00024
Acarpatica6	Alyratapetraea12	127107	0	71	115	210	10.41	0.00125
Acarpatica7	Alyratapetraea1	127091	0	69	134	209	20.81	0.00001
Acarpatica7	Alyratapetraea2	127110	0	69	115	209	11.5	0.0007
Acarpatica7	Alyratapetraea3	127108	0	69	117	209	12.39	0.00043
Acarpatica7	Alyratapetraea7	127109	0	68	116	210	12.52	0.0004
Acarpatica7	Alyratapetraea9	127105	0	68	120	210	14.38	0.00015
Acarpatica7	Alyratapetraea10	127106	0	69	119	209	13.3	0.00027
Acarpatica7	Alyratapetraea11	127103	0	68	122	210	15.35	0.00009
Acarpatica7	Alyratapetraea12	127110	0	68	115	210	12.07	0.00051
Acarpatica8	Alyratapetraea1	127087	0	73	134	209	17.98	0.00002
Acarpatica8	Alyratapetraea2	127106	0	73	115	209	9.38	0.00219
Acarpatica8	Alyratapetraea3	127104	0	73	117	209	10.19	0.00141
Acarpatica8	Alyratapetraea7	127105	0	72	116	210	10.3	0.00133
Acarpatica8	Alyratapetraea9	127101	0	72	120	210	12	0.00053
Acarpatica8	Alyratapetraea10	127102	0	73	119	209	11.02	0.0009
Acarpatica8	Alyratapetraea11	127099	0	72	122	210	12.89	0.00033
Acarpatica8	Alyratapetraea12	127106	0	72	115	210	9.89	0.00166
Apetrogenapetrogena1	Alyratapetraea1	127090	0	70	132	211	19.03	0.00001
Apetrogenapetrogena1	Alyratapetraea2	127108	0	71	114	210	9.99	0.00157
Apetrogenapetrogena1	Alyratapetraea3	127106	0	71	116	210	10.83	0.001
Apetrogenapetrogena1	Alyratapetraea7	127107	0	70	115	211	10.95	0.00094
Apetrogenapetrogena1	Alyratapetraea9	127103	0	70	119	211	12.7	0.00036
Apetrogenapetrogena1	Alyratapetraea10	127105	0	70	117	211	11.81	0.00059
Apetrogenapetrogena1	Alyratapetraea11	127101	0	70	121	211	13.62	0.00022
Apetrogenapetrogena1	Alyratapetraea12	127108	0	70	114	211	10.52	0.00118
Apetrogenapetrogena2	Alyratapetraea1	127088	1	72	133	209	18.15	0.00002
Apetrogenapetrogena2	Alyratapetraea2	127106	0	73	115	209	9.38	0.00219
Apetrogenapetrogena2	Alyratapetraea3	127104	0	73	117	209	10.19	0.00141
Apetrogenapetrogena2	Alyratapetraea7	127106	1	71	115	210	10.41	0.00125
Apetrogenapetrogena2	Alyratapetraea9	127102	1	71	119	219	12.13	0.0005
Apetrogenapetrogena2	Alyratapetraea10	127103	1	72	118	209	11.14	0.00085
Apetrogenapetrogena2	Alyratapetraea11	127100	1	71	121	210	13.02	0.00031
Apetrogenapetrogena2	Alyratapetraea12	127107	1	71	114	210	9.99	0.00157
Aneglectaneglecta3	Alyratapetraea1	127082	0	78	134	209	14.79	0.00012
Aneglectaneglecta3	Alyratapetraea2	127100	0	79	116	208	7.02	0.00806
Aneglectaneglecta3	Alyratapetraea3	127098	0	79	118	208	7.72	0.00546
Aneglectaneglecta3	Alyratapetraea7	127099	0	78	117	209	7.8	0.00522
Aneglectaneglecta3	Alyratapetraea9	127095	0	78	121	209	9.29	0.0023
Aneglectaneglecta3	Alyratapetraea10	127097	0	78	119	209	8.53	0.00349
Aneglectaneglecta3	Alyratapetraea11	127093	0	78	123	209	10.08	0.0015
Aneglectaneglecta3	Alyratapetraea12	127100	0	78	116	209	7.44	0.00637

Diploid representatives of the Arabidopsis arenosa aggregate, namely A. carpatica, A. petrogena, and A. neglecta (Schmickl et al. 2012). Assuming that chloroplast (cp) lineages in both species have maintained their function and are exposed to similar evolutionary constraints, then they should show similar rates of evolution. If some lineages have experienced accelerated cp evolution, then these lineages are expected to show elevated rates of evolution. To discriminate between these two hypotheses, we conducted a relative-rate test (Tajima 1993) between all pairwise heterospecific sequences (denoted as “A” and “B”) using a whole cp genome sequence from Arabidopsis cebennensis as an outgroup. The results consistently indicate a significantly lower substitution rate for A. arenosa than for A. lyrata. Rate constancy can thus be rejected at the 1% level for the whole cp genome between these two species.

The majority of diploid A. lyrata populations in the wild grow on calcareous outcrops in the east Austrian Forealps, but populations also grow on siliceous bedrocks, for example, the Bohemian Massif in the Czech Republic (Schmickl et al. 2010), suggesting either the presence of local edaphic adaptation or extreme physiological plasticity within this species. Similarly, within diploid A. arenosa, calcicole populations occur exclusively in the Carpathians and the Balkan Peninsula, while siliceous populations are mainly restricted to the High Tatras (Schmickl et al. 2012). This substrate specialization has led to spatial separation of ecological populations within both species. The role of substrate adaptation, however, in shaping both this diversification and the fitness of heterospecific hybrids is unknown.

One way to test whether exogenous, rather than endogenous, selection shapes hybrid fitness would be to investigate the sensitivity of germination and early seedling growth to substrate of origin by comparing the performance of F1 hybrids with their parents (“home versus away” contrast sensu Kawecki and Ebert 2004). Genotype × genotype interactions (endogenous selection) should result in deviations from expectation under additive genetic architecture (Lynch and Walsh 1998). We therefore expect that if intergenomic (or cytonuclear) incompatibilities are weak (or absent), trait values for the F1 hybrids will equal the pooled average of the parents (Rhode and Cruzan 2005). This is indeed what we observe. Seed set and germination of artificially generated F1 hybrids do not exceed the worst performing parent (Fig.1).

Given that the predicted accelerated rates of chloroplast genome evolution in A. lyrata are not accompanied by an asymmetrical fitness of maternal F1 A. lyrata in the wild, we suggest that divergence in local substrate adaptation may be subject to parent–offspring coadaptation and that isolation barriers are likely to be environmentally dependent (exogenous) rather than endogenous.

Substrate treatments were not included in our experiment, however, and so future garden experiments will need to include heterospecific crosses (also between substrate ecotypes) to investigate how selection (exogenous vs. endogenous) could offset the decreased fitness of any new migrate allele both in a new hybrid genetic background (Dobzhansky 1937; Barton and Hewitt 1985; Barton 2001) and the substrate in which new migrant alleles are expressed (genotype × environment interactions; Barton and Gale 1993).

Directional asymmetry at seed set is reversed in predicted direction at seed viability

The reversal in asymmetry between life stages is curious, because nuclear cytoplasmic interactions should be apparent in both seed set and their inherent viability. One could argue that as the patterns for seed set and germination are diametrically opposite, the effects cancel each other out. On the other hand, germinating at a low frequency (despite high abundance) for the long(er)-lived perennial A. lyrata may be a better life history strategy than germinating at high frequency to produce founding populations, as is evident for the colonizer A. arenosa (Donohue 2009; Rajon et al. 2009).

Seed germination rates are notoriously variable across environments even within species (for Arabidopsis, see Donohue et al. 2005; Montesinos-Navarro et al. 2012) and so broader population sampling is required to capture all of the variance among sites between species in this biological system. That we detected no significant difference in seed mass between the two cross types may argue against any viability interpretation based on germination. The artificial environment used in our experiment may not have been conducive to germination for A. lyrata, commensurate with the contradictory results from the field where A. lyrata maternal hybrids prevail. Finally, germination is of course a difficult fitness trait to interpret because failure to germinate may actually be the best strategy (Simons and Johnston 2006; Childs et al. 2010). Different maternal effects between the two species, whatever their ultimate basis, may not be surprising in this sense, and those effects should not necessarily go in the same direction for all traits – not least because the directionality of traits is difficult to define, particularly for germination (Donohue 2009).

Conclusion

In Arabidopsis (A. arenosa and A. lyrata), the direction of isolation asymmetry between hybridizing species in the wild does not vary predictably with the relative rate of chloroplast and nuclear evolution in parental species detected here; a pattern that is not consistent with theoretical predictions (Turelli and Moyle 2007). Our data do not allow us to test whether differences in seed viability (having used a proxy), or dormancy, contribute to isolation asymmetry between these two species. If dormancy is misregulated, preventing germination, then many interesting questions regarding maternal versus embryonic control of dormancy arise (Donohue 2009) beside related issues of parent–offspring conflict (Ellner 1986) and bet-hedging (Slatkin 1974; Simons and Johnston 2006; Childs et al. 2010).