Kathryn E. Theiss 2,4 , Kent E. Holsinger 2 , and Margaret E. K. Evans

American Journal of Botany 97(6): 1031–1039. 2010.

B REEDING SYSTEM VARIATION IN 10 EVENING PRIMROSES ( OENOTHERA SECTIONS ANOGRA AND KLEINIA ; ONAGRACEAE) 1

Kathryn E. Theiss2,4 , Kent E. Holsinger 2 , and Margaret E. K. Evans3,5

2 Department of Ecology and Evolutionary Biology, University of Connecticut, 75 N. Eagleville Road, U-3043, Storrs, Connecticut 06269 USA; and 3 Department of Ecology and Evolutionary Biology, Yale University, New Haven, Connecticut 06511 USA

• Premise of the study: We examined two accounts of the relationship between breeding system and life history variation in a clade of evening primroses ( Oenothera, Onagraceae): (1) selection for reproductive assurance should generate an association between self-compatibility and monocarpy and (2) phylogenetic conservatism leads to retention of breeding system and life history traits among closely related taxa. • Methods: We performed over 4000 hand pollinations under greenhouse conditions to determine the compatibility of 10 Oenothera taxa (sections Anogra [17 taxa] and Kleinia [2 taxa)] for which breeding systems had not previously been reported. We used generalized linear mixed models to evaluate the inﬂ uence of pollination treatment, parents, and population on fruiting success. • Key results: Among the taxa tested, six were self-incompatible, two were variable in compatibility, and two were self-compatible. We combined these data with published studies in Anogra and Kleinia and mapped breeding system and life history onto a published phylogeny. • Conclusions: We found no evidence for phylogenetic conservatism, but detected considerable evolutionary lability in both traits. Additionally, we found no evidence for a consistent relationship between breeding system and life history. Only eight of 19 taxa followed the predicted association between self-incompatibility and polycarpy vs. self-compatibility and monocarpy. Instead, many taxa have retained self-incompatibility, regardless of monocarpy or polycarpy.

Key words: breeding system evolution; Oenothera ; Onagraceae; self-incompatibility.

Understanding the forces underlying the evolution of plant enforced in many angiosperms by a genetically determined reproductive systems, particularly how they determine the bal- self-incompatibility system. Gametophytic self-incompatibility ance between self-fertilization and outcrossing, has been an im- is thought to be ancestral in eudicots, and sporophytic self- portant focus of research since Darwin ( 1877 ). In fl owering incompatibility has arisen multiple times (Holsinger and Stein- plants, the major forces determining that balance are reproduc- bachs, 1997; Igic and Kohn, 2001; Steinbachs and Holsinger, tive assurance, pollen discounting, and inbreeding depression 2002 ; Igic et al., 2008 ). Thus, in eudicots self-compatibility is a ( Holsinger, 2000 ; Johnston et al., 2009 ). Reproductive assur- derived condition in which genetically determined self-incom- ance refers to the fi tness advantage that may be conferred by patibility has been rendered inoperable through mutation. Fol- self-fertilization in the face of unreliable pollinators, while in- lowing Neal and Anderson (2005), we refer to the integrated breeding depression refers to the fi tness disadvantage associ- syndrome of physiological and structural features of plants that ated with reduced fi tness of offspring, and pollen discounting infl uence the extent of outcrossing, including genetically deter- refers to the reduction in success as an outcross pollen parent mined self-incompatibility, as the breeding system. that may be associated with self-fertilization ( Holsinger et al., While life-history correlates of selfi ng vs. outcrossing have 1984 ; see also Charlesworth, 1980 ). Outcrossing may be pro- been discussed for more than 50 years (e.g., Baker, 1955; Steb- moted either by differences in the timing of stigma and anther bins, 1957 ), fewer studies evaluate these correlations in a phy- maturation within fl owers (dichogamy) or by spatial separation logenetic context (Barrett and Graham, 1997; Weller and Sakai, of stigmas and anthers (herkogamy). In addition, outcrossing is 1999 ; Armbruster et al., 2002 ). Of the comparative studies of breeding systems that exist, most focus on the evolution of fl o- 1 Manuscript received 2 September 2009; revision accepted 2 April 2010. ral morphology ( Armbruster, 1993 ; Whittall and Hodges, 2007 ), The authors thank R. Raguso for seeds, K. Skogen and L. Senack for heterostyly (Schoen et al., 1997; Jesson and Barrett, 2003; Graham help with pollination, R. Flynn and S. Smith for sequencing and and Barrett, 2004 ; Barrett and Harder, 2005 ; Mast et al., phylogenetic analyses, C. Morse and M. Opel for greenhouse assistance, S. 2006; Sakai and Wright, 2008; Ferrero et al., 2009), sex expres- Vanderplank for herbarium assistance, M. Donoghue and S. Stearns for sion ( Desfeux et al., 1996 ; Miller and Venable, 2000 ; Vamosi et advice and feedback, and two anonymous reviewers for helpful comments al., 2003; Obbard et al., 2006; Renner et al., 2007), or other as- on an earlier version of this paper. pects of reproductive morphology. Far fewer studies have in- 4 Author for correspondence (e-mail: [email protected]) 5 Present address: Unit of Mathematical Eco-Evolutionary Biology, vestigated the phylogenetic distribution of self-incompatibility Laboratoire d ’ Ecologie & Evolution, CNRS UMR 7625, Ecole Normale (Barrett et al., 1996; Goodwillie, 1999 ; Takebayashi and Morrell, Sup é rieure, 46 rue d ’ Ulm 75230 Paris cedex 05, France 2001 ; Igic et al., 2006 ; Ferrer and Good-Avila, 2007 ), and very few have examined the relationship between self-compatibility doi:10.3732/ajb.0900260 and annual vs. perennial life history (Bena et al., 1998).

The correlation between life history and mating system, the MATERIALS AND METHODS balance between self-fertilization and outcrossing, has been discussed for many years (Stebbins, 1950; Morgan, 2001). In Study system— Within the Onagraceae, the genus Oenothera comprises 145 plants that have only one chance to reproduce, i.e., annuals or species in 18 sections native to North and South America (Wagner et al., 2007). monocarpic perennials, reproductive assurance may provide a Members of sections Anogra and Kleinia are morphologically similar, bearing substantial fi tness benefi t, favoring self-fertilization and there- large, white fl owers that open at dusk. In the wild, fl owers are visited by both hawkmoths and bees ( Gregory, 1963 ; Linsley et al., 1963a , b ), and they wilt as fore self-compatibility. Conversely, polycarpic perennials have temperatures rise the morning after opening. Under greenhouse conditions, many opportunities to reproduce, so inbreeding depression may fl owers can remain open up to 72 h. Members of these taxa occur in western favor outcrossing and therefore self-incompatibility (Morgan, North America on loose, coarse-grained soils in a wide variety of open habitats 2001). Thus self-incompatibility is expected in perennials and such as dunes, washes, grasslands, and alpine meadows (Evans et al., 2005, self-compatibility in annuals (Morgan et al., 1997; Morgan 2009 ; Wagner et al., 2007 ). Most of the taxa are diploid, but O. californica 2001). An alternative possibility is that patterns of variation in subsp. californica is tetraploid, and both O. coronopifolia and O. nuttallii have diploid and tetraploid populations ( Wagner et al., 2007 ). life history and/or breeding system may be best accounted for by phylogenetic relationships, i.e., either by the conserved in- Plant propagation —Seeds were collected in natural populations of all taxa heritance of traits from a common ancestor or by phylogenetic between 1996 and 2005, with an additional commercial seed source for Oeno- niche conservatism ( Felsenstein, 1985 ). In either case, if phylo- thera pallida Lindley subsp. latifolia (Rydberg) Munz (Appendix 1). In January genetic relationships account for patterns of variation in breed- 2006, seeds were surface sterilized with a 5% bleach solution (10 min), then ing system and life history, we would expect combinations of soaked overnight in distilled water to soften the seed coat. Embryos were dis- characters to be typical of entire clades. These patterns may be sected from the seed coat and placed on moistened fi lter paper in a petri dish. similar to the reproductive assurance results where all annual Petri dishes were kept in a growth chamber (16-h day, 22 ° C day and night) and watered daily. Once a suffi cient root system and the fi rst set of leaves devel- taxa are self-compatible and in one clade and all perennials are oped, seedlings were planted in 2 : 1 sand to potting soil mixture. After a few self-incompatible and in another clade. However, there are weeks of growth, plants were transplanted to larger containers and transferred to other possible patterns (self-incompatible annuals in one clade a greenhouse at Marsh Botanic Garden (Yale University). We watered plants and self-compatible annuals in another clade) that may account daily but minimally, applied nutrients (a solution of 20-20-20 N-P-K) approxi- for deviations from the reproductive assurance hypothesis. In mately every 2 wk, and used supplemental lighting to stimulate fl owering. this paper, we present results from a comparative analysis of Plants were relocated to the University of Connecticut greenhouses in June 2006 and transferred to larger pots. Once the plants arrived at the University of breeding system and annual vs. perennial life-history evolution Connecticut, the nutrient solution was applied once per week during fl owering. in a clade of evening primroses (Oenothera L., Onagraceae With the exception of O. albicaulis Pursh and O. nuttallii Sweet, taxa grew Juss.). well in the greenhouses, and most populations were represented by more than For 70 years, the genus Oenothera has served as a model for fi ve individuals with a range of 6– 34 plants per taxon (Appendix 1). One day of the study of plant evolution, particularly for the study of repro- severe drought at the Marsh Botanic Garden (31 March 2006) affected O. pal- ductive systems (e.g., Raven, 1979, 1988 ; Wagner et al., 2007 ; lida subsp. latifolia , O. pallida subsp. pallida Lindley, and O. pallida subsp. runcinata (Rydberg) Munz, but pollination treatments were repeated after Johnson et al., 2009 ). Classic papers on the population genetics plants recovered from the drought. A few individuals from select taxa (O. cali- of self-incompatibility are based on studies of Oenothera or- fornica (S. Watson) S. Watson subsp. californica , O. pallida subsp. pallida , O. ganensis (Emerson, 1938, 1939 ), and detailed studies of the nuttallii) produced a small number of fl owers in 2006, and pollinations were genus have revealed extensive variability in breeding system repeated in 2007 where possible. (e.g., Hecht, 1944 ; Crowe, 1955 ; Klein, 1970 ; Straley, 1977 ; Wagner and Lammers, 2005) and pollination (e.g., Gregory, Pollination— During the fl owering season, plants were hand-pollinated be- 1963; Linsley et al., 1963a, b ; Wolin et al., 1984). Although tween the hours of 0600 and 1100 on the morning after they opened. Each recent molecular work has suggested that the genus is paraphyl- fl ower was pollinated with one to two anthers of a single donor of the appropriate cross type (self or outcross). Because the experiment was designed to deter- etic ( Levin et al., 2003, 2004 ), several of the recognized sec- mine whether individuals were self-compatible and not to estimate the frequency tions are strongly supported as monophyletic (Evans et al., with which self-fertilization occurs, fl owers were not emasculated; Oenothera 2005 ). pollen is notoriously easy to see, particularly because of the viscin threads Our analyses focus on the 19 taxa in Oneothera section (Wagner et al., 2007), so we were able to visually confi rm the lack of contami- Anogra (Spach) W. L. Wagner & Hoch and section Kleinia nation by self pollen. In addition, herkogamy seems to prevent self-fertilization Munz, which, when combined, form a well-supported clade in all but one taxon that we tested (see Discussion). Moreover, only if contam- ination by self pollen reduced fruit set in outcross pollinations would it affect (Levin et al., 2004; Evans et al., 2005). Previous studies in these interpretation of our results. sections have reported both self-incompatible and self-compat- We allocated available fl owers to pollination type (self vs. outcross) and ible taxa, and in some taxa, both self-compatible and self- fathers as evenly as possible with the goal of equal sample sizes (ideally 15 self incompatible individuals (Hecht, 1944; Crowe, 1955; Klein, and 15 outcross) with respect to these factors. However, due to variation in 1964; Wagner et al., 2007). In addition, the taxa vary in life his- fl owering, some plants had as few as two or as many as 60 fl owers pollinated. tory: 10 are perennial, seven are annual, and two have both an- For taxa with multiple populations, we attempted to perform roughly equal numbers of within and between population crosses. Outcross pollinations nual and perennial populations ( Klein, 1970 ; Evans et al., 2005 ; served two purposes: (1) they acted as a control to show that the maternal plant Wagner et al., 2007). We combined existing reports on breeding was capable of producing fruit throughout fl owering, and (2) they were used in systems in this clade with results from new hand-pollination ex- calculating Bawa’ s index of self-incompatibility (see Statistical analyses). periments to answer the following questions: (1) What are the We marked each ovary with either a colored string or a coin tag for fruit breeding systems (self-compatible or self-incompatible) of the collection. 10 taxa in Oenothera sections Anogra and Kleinia for which data are not currently available? (2) Is self-compatibility more Fruit collection— Fruits were collected 4 – 6 wk after pollination. Ovaries strongly associated with annuals than perennials, and is self-in- that showed no sign of swelling were scored as not producing a fruit and were discarded. Ovaries showing any sign of swelling were collected and stored in compatibiity more strongly associated with perennials? (3) Are coin envelopes under refrigeration. Fruits were scored as successful if we found life history and breeding system evolutionarily labile, or are at least one seed upon dissection. Seeds of questionable quality were cut open particular character states characteristic of entire clades? to confi rm or deny the presence of a viable (white and fl eshy) embryo. June 2010] Theiss et al. — OENOTHERA breeding system 1033

Statistical analyses— For each taxon, we used a generalized linear mixed- these characters onto the consensus tree using the program Mesquite v.2.4 effects regression (LMER) to examine the effects of pollination treatment, ma- ( Maddison and Maddison, 2007 ) using parsimony optimization. ternal and paternal plant, maternal and paternal population (where applicable), and year (where applicable) on fruit set. Pollination treatment was considered a fi xed effect, while the remaining factors were treated as random effects. For this RESULTS analysis, each pollinated fl ower was considered a unique datum. We fi t LMER models (version 0.99875-9) including all explanatory variables (fruit ~ treat- Compatibility tests— Of the 10 taxa we examined, only two ment + (1 | maternal) + (1 | paternal) + (1 | maternal population) + (1 | paternal were consistently self-compatible: O. neomexicana (Small) population) + year) on a per-taxon basis using the statistical software R (version 2.6.0; R Development Core Team, 2007 ). We then removed individual factors Munz ( N = 1 population, 16 plants, 479 pollinations) and O. and tested for signifi cance by performing a likelihood ratio test comparing the nuttallii ( N = 2 populations, 7 plants, 45 pollinations). We de- full (all variables) and reduced (single variable removed) model. Factors were tected no difference in percentage fruit set between self and considered signifi cant if the full model fi t the data signifi cantly better than the outcross pollination treatments in either species (P = 0.84 and reduced model (P < 0.05). P = 0.92 respectively, Table 1 ). The ratio of fruit set after self In addition, we calculated a descriptive, continuous measure of self-incom- pollination compared to outcross pollination (Bawa ’ s index of patibility: the ratio of fruit set following self pollination compared to outcross pollination (following Bawa, 1974 ). We calculated this index of self-incompat- self-incompatibility, ISI) was 1.00 and 0.97 for these two taxa, ibility (ISI) at the taxon level, when we detected no maternal or paternal effects respectively ( Table 1 ). Four of the remaining taxa were clearly on fruit set in the mixed-effects regression. Where we detected signifi cant ma- self-incompatible (O. albicaulis, N = 3 populations, 16 plants, ternal effects, ISI was calculated for each mother. Values of ISI below 0.1 or 105 pollinations; O. coronopifolia Torrey & A. Gray, N = 1 0.2 are considered clear evidence of self-incompatibility (Igic et al., 2008), population, 13 plants, 274 pollinations; O. engelmannii (Small) while larger values indicated some degree of self-compatibility. Because calcu- Munz, N = 1 population, 6 plants, 182 pollinations; and O. pal- lation of ISI requires data on the number of fruit set per number of attempts, not just success or failure of any cross, we calculated ISI only for the taxa that we lida subsp. trichocalyx (Nuttall) Munz & W. M. Klein, N = 3 hand-pollinated and not for taxa whose breeding system was derived from our populations, 14 plants, 673 pollinations). Very few fruits de- literature survey. veloped in response to self pollination in any of these taxa, even though outcross pollinations were highly successful (P < Phylogenetics and character mapping— We used a previously developed 0.01, Table 1 ). Bawa ’ s ISI ranged from zero to only 0.13 ( Ta- phylogeny of Anogra and Kleinia ( Evans et al., 2009 ) to explore the evolution- ble 1) in these taxa. In fact, the data from O. pallida subsp. ary history of breeding systems and life histories. This phylogeny is the result trichocalyx were so uniformly indicative of self-incompatibil- of a Bayesian analysis of DNA sequence data from six gene regions, one nu- ity that we found only one fruit in 315 self crosses vs. 305 clear (ITS) and fi ve chloroplast ( trnH-trnK , trnL-trnF, rpoB-trnC, trnD-trnT, fruits in 358 outcrosses ( χ 2 = 483.45, P < 0.001). Two more and trnS-trnG), from all currently delimited taxa in Anogra and Kleinia (spe- taxa were mostly self-incompatible, but fruiting success varied cies and subspecies listed in Wagner et al., 2007), with Oenothera xylocarpa Coville (Sect. Contortae W. L. Wagner) as the outgroup. signifi cantly among mothers (O. pallida subsp. latifolia , N = 2 We built a character matrix for breeding system and life history for all taxa populations, 23 plants, 688 pollinations; O. pallida subsp. in sections Anogra and Kleinia , using data collected from our own experiments runcinata, N = 1 population, 18 plants, 696 pollinations; Table and the primary literature. We chose an ordered character model with three 1 ). Most mothers were strongly self-incompatible (ISI of 0), states as the most appropriate coding scheme for both breeding system and life and the mean ISI among mothers was very low (0.04) for both history. This allowed us to treat the taxa that are variable in life history or of these taxa. However there were four individuals of O. pal- breeding system as distinct, rather than combining them with one of the other character states. Because the ancestor for these taxa was most likely a peren- lida subsp. latifolia and one individual of O. pallida subsp. nial, self-incompatible taxon (Raven, 1979, and Results), we coded breeding runcinata that had ISI values greater than 0.1. These individu- system as self-incompatible = 0, variable = 1, self-compatible = 2, and life als typically set fi ve or fewer seeds (whereas fruits potentially history as perennial = 0, variable = 1, annual = 2. We allowed forward transi- contain 50– 100 seeds) in less than fi ve of their self-crossed tions from self-incompatible to variable to self-compatible and from perennial fruits, and sometimes, as in the case of the O. pallida subsp. to variable to annual, but did not allow the reverse transitions. These constraints runcinata individual, they had low fruit set among the out- on character state transitions are compatible with the lack of evidence for a self- compatible taxon giving rise to a gametophytic self-incompatible taxon (Igic et crossed pollinations. al., 2006) and that, throughout the Onagraceae, annuals have been frequently The remaining two taxa, O. californica subsp. californica derived from perennials, but the reverse transition is unknown. We mapped ( N = 3 populations, 34 plants, 672 pollinations) and O. pallida

Table 1. Results from mixed effects regressions testing fruiting success in 10 evening primroses (Oenothera ) and calculations of Bawa ’ s index of self-incompatibility. Likelihood ratio tests were used to evaluate the signiﬁ cance of effects (pollination treatment, maternal and paternal plant, and population), with the exception of O. pallida subsp. trichocalyx , where we performed a χ 2 test due to lack of variability in the data.

Maternal Paternal Pollination Taxon Maternal plant population Paternal plant population treatment Breeding system Bawa SI index

O. albicaulis ns ns ns ns *** SI 0 O. californica subsp. californica *** ns ns ns *** V 0.25 (0 – 1.3) O. coronopifolia ns NA ns NA ** SI 0.13 O. engelmannii ns NA ns NA *** SI 0 O. neomexicana ns NA ns NA ns SC 1.00 O. nuttallii ns ns ns ns ns SC 0.97 O. pallida subsp. latifolia ** ns ** ns ** SI 0.04 (0 – 0.21) O. pallida subsp. pallida *** ns ns ns *** V 0.16 (0 – 0.93) O. pallida subsp. runcinata *** NA *** NA *** SI 0.04 (0 – 0.39) O. pallida subsp. trichocalyx *** SI 0 Notes: NA = not applicable, ns = not signifi cant at the 0.05 level, ** = signifi cance at the 0.01 level, *** = signifi cance at the 0.001 level, SI = self- incompatible, SC = self-compatible, V = variable. 1034 American Journal of Botany [Vol. 97 subsp. pallida ( N = 3 populations, 13 plants, 459 pollinations), showed evidence of substantial within-taxon variability in breeding system. In the case of O. californica subsp. californica, both self-compatible and self-incompatible mothers were found in all populations tested ( Fig. 1 ), and we detected no differences among populations in the frequency of self-compatible mothers as refl ected in the nonsignifi cant maternal population effect (P = 1, Table 1). Bawa’ s ISI varied from zero to 1.30 among O . californica subsp. californica mothers ( Table 1 ). We were unable to test for a maternal population effect on self- compatibility in O. pallida subsp. pallida because of very small sample sizes in two populations. In the remaining population, plants were highly variable in compatibility ( Fig. 1 ), and the calculated Bawa ’ s ISI varied from zero to 0.93 among these mothers ( Table 1 ). Individuals from the Salt Lake City population that were determined to be self-compatible in 2006 were tested again in 2007, and we detected no difference in compatibility between years (P > 0.05).

Evolutionary pattern— In the consensus tree ( Evans et al., 2009), the following taxa form very strongly supported group- ings (posterior probability ≥ 0.98): Oenothera coronopifolia and O. nuttallii as distinct from all other taxa; O. albicaulis and O. engelmannii as distinct from the remaining taxa; O. arizonica (Munz) W. L. Wagner, O. californica (including all three subspecies), and O. neomexicana (hereafter referred to as the O. californica clade); and the four subspecies of O. pallida . The rest of the taxa, O. deltoides Torrey & Fr é mont (fi ve subspecies) and O. wigginsii W. M. Klein may or may not form a clade (Fig. 2). Because many of the internal nodes do not have strong support, we conservatively focus our analysis on those key nodes that are well supported. When we combined our breeding system data with information from the primary literature, we found that six of the perennial taxa were self-incompatible, two were self-compatible, and two were variable, while four of the annual species were self-incompatible, two were self-compatible, and one was variable ( Table 2 ). Of the two taxa that are variable in life history, one was self-compatible [Oenothera deltoides subsp. cognata (Jepson) W. M. Klein] and the other was self-incompatible (O. pallida subsp. trichocalyx ) with respect to breeding system. As expected from the discordance of character states in these traits, reconstruction of their evolutionary history was inconsistent both with a scenario of phylogenetic conservatism and with a scenario of tight life-history breeding system coevolu- tion ( Fig. 2 ). If selection for reproductive assurance was modi- fi ed by life history as expected, the colors in each tree in Fig. 2 would match, white with white, gray with gray, and black with black. This would also be evident in Table 2 where only the top left cell and bottom right cell would be populated; instead, we Fig. 1. Percentage of hand pollinations that were successful (produc- see taxa with all possible combinations of the two traits. Many ing a fruit) for (A, B) Oenothera californica subsp. californica and (C) O. annual taxa have retained self-incompatibility, and many pe- pallida subsp. pallida. Each pair of bars (one dark gray, one light gray) rennials are either self-compatible or have a variable breeding represents a single plant. Population delineations are made on the x -axis. system. Dark gray = outcross, light gray = self pollinations. Error bars measure standard deviation. Neither is the pattern of variation in breeding system and life history compatible with phylogenetic conservatism. If we confi ne our attention to well-supported clades (i.e., collapsing all nodes with > 0.98 posterior probability), we can conclude contrast, life history is weakly conservative on the phylogeny. that self-compatible taxa and variable taxa have evolved a Annuals are concentrated within the O. deltoides group and in minimum of fi ve times (Oenothera nuttallii , O. deltoides the group O. engelmannii + O. albicaulis. The phylogenetic subsp. cognata , O. pallida subsp. pallida , O. neomexicana , O. uncertainty underlying the relationships of the subspecies O. californica subsp. californica; Fig. 2). Only the O. californica deltoides and O. wigginsii do not allow us to reach more pre- clade is predominantly self-compatible or variable (Fig. 2). In cise conclusions. June 2010] Theiss et al. — OENOTHERA breeding system 1035

Fig. 2. Character mapping of two traits, breeding system (left) and life history (right), for the 19 taxa in Oenothera sections Anogra and Kleinia, using an ordered character parsimony model. For breeding system, self-incompatibility is white, variable compatibility is gray and self-compatability is black. For life history, perenniality is white, variable is gray and annual is black. The topology is based on the consensus tree from a Bayesian analysis of six gene regions (reported in Evans et al., 2009 ).

DISCUSSION Stubbe and Raven, 1979). Further sampling from the more southern populations would allow us to determine the geo- Breeding system— Breeding system variation occurs at all graphical extent of the breakdown. We detected similar varia- levels in the Oenothera clade Anogra + Kleinia: among species tion in the degree of self-compatibility among individuals in or subspecies, among populations, and within populations. This only one population of the other variable taxon, O. pallida variation, in a group of closely related and recently derived taxa subsp. pallida (Bawa ’ s ISI: 0 – 0.93). Limited ﬂ ower production (Evans et al., 2005, 2009 ), suggests that breeding system evolu- from the two additional populations of O. pallida subsp. pallida tion is highly dynamic. Of the 19 taxa in Oenothera sections sampled prevents us from determining if this taxon is also expe- Anogra and Kleinia, breeding systems for nine taxa had previ- riencing widespread breakdown of self-incompatibility. ously been reported ( Crowe, 1955 ; Klein, 1964 ; Klein, 1970 ; Oenothera neomexicana was the only taxon we studied that Wagner et al., 2007). Our experiments, involving over 4000 consistently set fruit in greenhouses even in the absence of ex- hand-pollinations on the remaining 10 taxa, revealed six self- perimental manipulation (K. Theiss, personal observation). The incompatible taxa, two self-compatible taxa, and two that are lack of evidence of dichogamy in Oenothera ( Raven, 1979) variable within populations ( Table 1 ). suggests a breakdown of herkogamy in O. neomexicana , for Of the two taxa with variable breeding systems, we found a example, by the stigma falling onto the anthers. Autonomous wide range of self-compatibility among individuals in all three self fertilization (Lloyd and Schoen 1992) has also been re- populations of Oenothera californica subsp. californica stud- ported in O. wigginsii where the style is only slightly exserted ied: Bawa’ s index of ISI ranged from 0 to 1.3. These popula- ( Gregory, 1963 ). Oenothera nuttallii , the other self-compatible tions are all found in the northern part of the range of O. taxon we studied, did not set fruit autonomously. californica subsp. californica , suggesting a breakdown in self- The remaining six taxa were all determined to be self-incom- incompatibility across part of its range, similar to the pattern patible. Nonetheless, previous studies suggest that breeding found in O. grandiﬂ ora L’ Her (Steiner and Stubbe, 1984; system may vary across the ranges of at least O. coronopifolia . 1036 American Journal of Botany [Vol. 97

Table 2. Breeding system and life history for the 19 taxa of Oenothera , sections Anogra and Kleinia , reﬂ ecting results from the present study and data from primary literature.

Breeding system Life history Self-incompatible Variable Self-compatible

Perennial O. californica subsp. avita O. californica subsp. californica O. neomexicana O. californica subsp. eurekensis O. pallida subsp. pallida O. nuttalli O. coronopifolia O. deltoides subsp. howellii O. pallida subsp. latifolia O. pallida subsp. runcinata

Variable O. pallida subsp. trichocalyx O. deltoides subsp. cognata

Annual O. albicaulis O. deltoides subsp. deltoides O. arizonica O. deltoides subsp. ambigua O. wigginsii O. deltoides subsp. piperi O. engelmanii

Raven (1979) reported unpublished data indicating a self-com- whereas self-incompatibility should be more common in poly- patible population of O. coronopifolia . The single population carpic perennials than in annuals or monocarpic perennials. we examined was putatively SI (Bawa ISI 0.13, Table 1 ). Be- Parsimony mapping on a consensus tree indicated little cor- cause this taxon has a broad geographic range (Evans et al., respondence between the inferred evolutionary history of 2009) and has both diploid and tetraploid populations (Wagner breeding system and life history, in spite of the fact that both of et al., 2007), further sampling across its range and across the these traits have been evolutionary labile: self-compatibility chromosomal variation is necessary to better determine the evolved a minimum of fi ve times, and the annual habit evolved breeding system of this taxon. a minimum of three times ( Fig. 2 ). Instead, annual taxa often It is interesting to note that the three tetraploid taxa, O. cali- retain self-incompatibility and many perennials are self-com- fornica subsp. californica (all populations), O. coronopifolia patible. Five of 10 perennials are self-incompatible, and four of and O. nuttallii (some populations; Wagner et al., 2007 ), each seven annuals are self-incompatible ( Table 2 ). In short, we demonstrate some break down in self-incompatibility. Gameto- failed to detect an association between breeding system and life phytic self-incompatibility systems, as found in Oenothera history. ( Crowe, 1955 ; Raven, 1979 ), have been shown to weaken or break down after polyploid events ( Mable, 2004 ). This may ex- Trait evolution— Breeding system appears to be more evolu- plain why O. coronopifolia has been reported as both self-in- tionarily labile than life history in this group. We estimate that compatible (our results) and self-compatible ( Raven, 1979 ), if there have been at least four transitions from self-incompatibil- diploid populations retain the self-incompatibility system and ity to self-compatibility and one transition from self-incompat- tetraploid populations are self-compatible or variable. A more ibility to polymorphism for breeding system, while there have complete understanding of the variability of the breeding sys- been at least two transitions from perennial life habit to annual tem within a taxon will obviously require both more extensive life habit. As found in other studies (e.g., Bena et al., 1998; sampling of individuals within populations and a larger sample Goodwillie, 1999), self-compatibility is more strongly associ- of populations. ated with tips than with interior branches. In the Oenothera The patterns in breeding system both within and between californica clade, for example, each subclade has one member taxa that we found in Anogra + Kleinia , are similar to those that is self-incompatible and one that is not. Similarly, two of found in other sections of Oenothera. Sections Contortae , Er- the fi ve subspecies of O. deltoides and one of the four subspe- emia , and Ravenia have all been reported to have self-incom- cies of O. pallida exhibit some level of self-compatibility (Fig. patible, variable, and self-compatible species ( Wagner and 2 ). Lammers, 2005). Straley (1977) also found both self-incompat- In contrast, life history states are less variable within clades. ible and self-compatible taxa in section Kneiffi a, although he In spite of breeding system differences across the O. californica did not report any variable taxa. clade, all the taxa retain the ancestral character state of perenniality except for O. arizonica . The same pattern holds true for Association between breeding system and life history — Given the O. pallida clade with only one of four taxa variable in life the variation in breeding system in Anogra + Kleinia , the varia- history. The group containing O. deltoides and O. wigginsii is tion in annual vs. perennial life history ( Evans et al., 2005 ) and the only grouping containing all three life history states, al- the expected correlation between these two traits ( Stebbins 1950 ), though this conclusion must be regarded as tentative because of we explored the relationship between breeding system and life the weak support for this clade. history in this group. The reproductive assurance hypothesis for the evolution of self-fertilization is that “ [i]t is better to have re- Conclusion— The variation in breeding systems within and produced by selfi ng than never to have reproduced at all ” ( Hols- among members of sections Anogra and Kleinia in Oenothera inger, 1992, p. iii ). 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Appendix 1. Complete list of all reports for breeding system used in this study, broken down by taxon and population. We also report the number of plants and ﬂ owers sampled where known and the author ’ s determination of breeding system.

No. of plants Accession Taxon Population (ﬂ owers) Compatibility Year collected information

Oenothera albicaulis Pursh Buena Vista, CO 1 (2) SI 1995 ARIZ-44317 Moab, UT 13 (91) SI 2005 ARIZ-RAR05-03 Sells, AZ 2 (12) SI 1998 ARIZ-44319

O. arizonica (Munz) W. L. Wagner Hope I, AZ 5 SC Klein 1964

O. californica (S. Wats.) S. Wats. subsp. avita W.M. Klein Amboy I, CA 2 SI Klein 1964 Austin, NV 1 SI Klein 1964 Cedar Canyon I, CA 4 SI Klein 1964 Cedar Canyon II, CA 1 SI Klein 1964 Jean, NV 1 SI Klein 1964 Pine Valley, UT 1 SI Klein 1964

O. californica (S. Wats.) S. Wats. subsp. californica Helendale, CA 16 (461) SC/SI 2005 ARIZ-MEKE-21 Onyx, CA 8 (192) SC/SI 2005 ARIZ-MEKE-22 Tehachapi, CA 10 (19) SC/SI 2005 ARIZ-MEKE-23 Castaic, CA 3 SC/SI Klein 1964 Morongo Valley, CA 1 SI Klein 1964 San Bernadino, CA 1 SI Klein 1964 Yucca Valley, CA 1 SI Klein 1964

O. californica (S. Wats.) S. Wats. subsp. eurekensis (Munz & Eureka Valley I, CA 3 SI Klein 1964 J. C. Roos) W. M. Klein Eureka Valley II, CA 2 SI Klein 1964

O. coronopifolia Torr. & A. Gray Estes Park, CO 13 (274) SI 1999 ARIZ-52684 June 2010] Theiss et al. — OENOTHERA breeding system 1039

Appendix 1. Continued.

No. of plants Accession Taxon Population (ﬂ owers) Compatibility Year collected information O. deltoides Torr. & Fr é m. subsp. ambigua (S. Wats.) Boulder City, NV 3 SI Klein 1964 W. M. Klein Logandale, NV 1 SI Klein 1964

O. deltoides Torr. & Fr é m. subsp. cognata (Jeps.) W. M. Klein Merced River, CA 7 SC Klein 1964 Merced, CA 1 SC Klein 1964

O. deltoides Torr. & Fr é m. subsp. deltoides Barstow, CA 2 SI Klein 1964 Bermuda Dunes, CA 4 SC Klein 1964 Blythe, CA 1 SI Klein 1964 Bouse, AZ 2 SI Klein 1964 Cronese, CA 5 SI Klein 1964 Daggett, CA 8 SI Klein 1964 Essex, CA 1 SI Klein 1964 Hinkley, CA 4 SI Klein 1964 Kelso, CA 5 SI Klein 1964 Las Vegas, NV 1 SI Klein 1964 Yermo, CA 4 SI Klein 1964 California SI Crowe 1955

O. deltoides Torr. & Fr é m. subsp. howellii (Munz) W. M. Klein Antioch I, CA 5 SC/SI Klein 1964 Antioch II, CA 4 SI Klein 1964

O. deltoides Torr. & Fr é m. subsp. piperi (Munz) W. M. Klein Carson City, NV 2 SI Klein 1964 Fallon, NV 1 SI Klein 1964

O. engelmannii (Small) Munz Portales, NM 6 (182) SI 1999 ARIZ-352692

O. neomexicana (Small) Munz Pinaleños, AZ 16 (479) SC 1996 ARIZ-114850

O. nuttallii Sweet Estes Park, CO 4 (27) SC 2004 ARIZ-RAR04-13 Larimer Co., CO 3 (18) SC 1999 ARIZ-352651

O. pallida Lindl. subsp. latifolia (Rydb.) Munz Pike ’ s Peak, CO 4 (111) SI 1998 ARIZ-49717 Western Native Seed 19 (577) SI No herbarium accession Wyoming SI Crowe 1955

O. pallida Lindl. subsp. pallida Bogus Basin, ID 2 (44) SI 1998 ARIZ-44419 St. Anthony Dunes, ID 3 (29) SI 1998 ARIZ-345302 Salt Lake City, UT 8 (386) SC/SI 1998 ARIZ-345300 Washington SI Crowe 1955

O. pallida Lindl. subsp. runcinata (Engelm.) Munz & Flagstaff, AZ 18 (696) SI 1996 ARIZ-RAR96-5 W. M. Klein New Mexico SI Crowe 1955 O. pallida Lindl. subsp. trichocalyx (Nutt.) Munz & Carbon Co., UT 6 (285) SI 1998 ARIZ-44428 W. M. Klein Moab, UT 3 (121) SI 2004 ARIZ-RAR04-04 Tuba City, AZ 5 (267) SI 1998 ARIZ-114651 Washington a SC Crowe 1955

O. wigginsii W. M. Klein San Quint í n, Mexico 10 SC Klein 1964 Notes: AZ = Arizona, CA = California, CO = Colorado, ID = Idaho, NM = New Mexico, NV = Nevada, SC = self-compatible, SI = self-incompatible, UT = Utah a Although Crowe (1955) reports that this example of O. pallida subsp. trichocalyx is self-compatible, on the basis of locality information and lack of herbarium accession, we believe that it is actually an example of O. pallida subsp. pallida and therefore do not include it in our evolutionary character mapping.