Could seasonally deteriorating environments favour the evolution of autogamous selfing and a drought escape physiology through indirect selection? A test of the time limitation hypothesis using artificial selection in Clarkia

Abstract

Background and aims: The evolution of selfing from outcrossing may be the most common transition in plant reproductive systems and is associated with a variety of ecological circumstances and life history strategies. The most widely discussed explanation for these associations is the reproductive assurance hypothesis - the proposition that selfing is favoured because it increases female fitness when outcross pollen receipt is limited. Here an alternative explanation, the time limitation hypothesis, is addressed, one scenario of which proposes that selfing may evolve as a correlated response to selection for a faster life cycle in seasonally deteriorating environments.

Methods: Artificial selection for faster maturation (early flowering) or for low herkogamy was performed on Clarkia unguiculata (Onagraceae), a largely outcrossing species whose closest relative, C. exilis, has evolved higher levels of autogamous selfing. Direct responses to selection and correlated evolutionary changes in these traits were measured under greenhouse conditions. Direct responses to selection on early flowering and correlated evolutionary changes in the node of the first flower, herkogamy, dichogamy, gas exchange rates and water use efficiency (WUE) were measured under field conditions.

Key results: Lines selected for early flowering and for low herkogamy showed consistent, statistically significant responses to direct selection. However, there was little or no evidence of correlated evolutionary changes in flowering date, floral traits, gas exchange rates or WUE.

Conclusions: These results suggest that the maturation rate and mating system have evolved independently in Clarkia and that the time limitation hypothesis does not explain the repeated evolution of selfing in this genus, at least through its indirect selection scenario. They also suggest that the life history and physiological components of drought escape are not genetically correlated in Clarkia, and that differences in gas exchange physiology between C. unguiculata and C. exilis have evolved independently of differences in mating system and life history.

Fig. 1.

(A) Experimental design for creation of the baseline generation. Numbers indicate seed families from field collections: plants sharing a number are maternal half-sibs assuming different pollen parents. Letters refer to the two independent replicates created from each field population. × = reciprocal cross-pollination following emasculation of recipient flowers. Grey boxes indicate which of the two potential maternal plants within each cross was chosen at random to produce seed for the first generation of selection. Pairs are numbered sequentially for clarity of presentation only: in reality, pair membership was assigned at random (see the Materials and Methods for details). (B) Experimental design for the first generation of selection. Dashed arrows indicate control plants that were chosen randomly from the set above. Solid arrows indicate selected plants chosen from the set above using the criteria described in the Materials and Methods. Families in the early flowering outcrossed group could also potentially be represented in the control selfed or low herkogamy selected group, although different individual plants were used in the different lines. In subsequent generations of selection, families were chosen randomly (controls) or selected from within each of the four groups. See the Materials and Methods for details.

Fig. 2.

Days to first flowering, herkogamy scores and autogamous fruit set of greenhouse-raised plants after two generations of selection. (A) Direct selection on early flowering. (B) Direct selection on low herkogamy. (C) Indirect selection on early flowering due to direct selection on low herkogamy. (D) Indirect selection on low herkogamy due to direct selection on early flowering. (E) Autogamous fruit set of early flowering lines and their controls. (F) Autogamous fruit set of low herkogamy lines and their controls (not measured for the M population). For (A–D), bars show the mean ±1 s.e.; for (E) and (F) they show medians and interquartile ranges. In (F), medians for all lines of the J population were zero; the interquartile range for the control line of replicate A was also zero. n = 15 families for all groups. ***P < 0.001, **P < 0.01, *P < 0.05, +P < 0.1; one-tailed tests for direct selection comparisons; two-tailed tests for indirect selection comparisons (t-tests for A–D; Kruskal–Wallis tests for E and F). All significant differences remained significant at P < 0.05 or lower after adjustment for multiple comparisons using the false discovery rate (FDR) criterion (Benjamini and Hochberg, 1995; García, 2004), except for those in parentheses.

Fig. 3.

Days to first flower, node of first flower, anther–stigma distance and protandry of field-raised plants after three generations of selection for early flowering. Bars show the least squares means ± 1 s.e. of residual values from plot means (A–C) or from a model incorporating plot mean and days to first flower (D). n = 6 families for all groups except M Rep A and Rep B control lines, for which n = 5 families. See the Materials and Methods for details. Raw data are provided in Supplementary Data Fig. S1 and Table S2. ***P < 0.001, **P < 0.01, *P < 0.05, +P < 0.1; one-tailed t-tests for direct selection comparisons (days to first flower); two-tailed tests for indirect selection comparisons (node of first flower, anther–stigma distance, protandry). All significant differences remained significant at P < 0.05 or lower after adjustment for multiple comparisons using the false discovery rate (FDR) criterion (Benjamini and Hochberg, 1995; García, 2004), except for those in parentheses.

Fig. 4.

Gas exchange rates and water use efficiencies (WUEs) of field-raised plants after three generations of selection for early flowering. Early season measures are shown on the left-hand side, late season measures on the right. δ¹³C is a measure of integrated WUE over the lifetime of sampled leaves (see the Materials and Methods for details). Units of measurement are: A (μmol CO₂ m^–2 s^–1); E (mol H₂O m^–2 s^–1); WUE (μmol CO₂ mol^–1 H₂O × 10^–4); δ¹³C (‰). Bars show the least squares means ± 1 s.e. of residual values after controlling for variation among plots and (for gas exchange rates and WUE) air temperature or measurement date. See the Materials and Methods for details. Raw values are provided in Supplementary Data Fig. S2 and Table S3. For gas exchange rates and WUE, n = 6 families for all groups except the M Rep A and Rep B control lines, for which n = 5 families. For δ¹³C, n = 4–6 families for all groups except the M Rep A early flowering line, for which n = 1 family.