New Phytologist Research Rapid evolutionary divergence and ecotypic diversification of germination behavior in weedy rice populations Han-Bing Xia*,Hui Xia*,Norman C.Ellstrand2,Chao Yang'and Bao-Rong Lu' Department of Ecology and Evolutionary Biology,Key Laboratory for Biodiversity Science and Ecological Engineering,Fudan University,Ministry of Education,Handan Road 220,Shanghai 200433.China;2Department of Botany and Plant Sciences,Center for Conservation Biology,and Center for Invasive Species Research,University of California,Riverside,CA 92521-0124,USA Summary Author for correspondence: Feral plants have evolved from well-studied crops,providing good systems for Bao-Rong Lu elucidation of how weediness evolves.As yet,they have been largely neglected for Tel:+862165643668 this purpose.The evolution of weediness can occur by simple back mutations in Email:brlu@fudan.edu.cn domestication genes(domestication in reverse).Whether the evolutionary steps to Received:17 January 2011 weediness always occur in reverse remains largely unknown. Accepted:5 April 2011 We examined seed germination behavior in recently evolved weedy rice (Oryza sativa f.spontanea)populations and their coexisting cultivars in eastern and north- New Phytologist (2011)191:1119-1127 eastern China to address whether 'dedomestication'is the simple reverse of do:10.1111/0.1469-8137.2011.03766.× domestication. We found that these weedy populations did not diverge from their progenitors Key words:ecotypic differentiation,ferality. by reverting to the pre-domestication trait of seed dormancy.Instead,they have latitudinal variation,local adaptation, evolved a novel mechanism to avoid growing in inappropriate environments via plant-environment interaction,temperature changes in critical temperature cues for seed germination.Furthermore,we found response,weed evolution. evidence for subsequent ecotypic divergence of these populations such that the critical temperature for germination correlates with the local habitat temperature at latitudinal gradients. The origins of problematic plant species,weeds and invasives,have already been studied in detail.These plants can thus be used as systems for studying rapid evo- lution.To determine whether and how that evolution is adaptive,experiments such as those described here can be performed. Introduction model systems for elucidating how weediness or invasive- ness evolves.Furthermore,like invasive species,because Feral plants are descendants of domesticated plants that their histories are generally well known,they can be used as have evolved the ability to persist and reproduce without systems for studying rapid adaptive evolution.As yet,they direct human care.Feral plant evolution,also known as have been largely neglected for this purpose;most studies 'dedomestication',can occur either directly from domesti- have focused on simply documenting plants as feral,rather cated ancestors ('endoferality)or by hybridization of than how 'ferality'evolved (Ellstrand et al,2010). domesticates with wild relatives ('exoferality)(Gressel, Often part of the story is obvious.Many feral plants have 2005a).Dedomestication may produce problematic plants evolved shattering (seed dispersal)from nonshattering crop such as invasives and weeds.Ellstrand et al.(2010)identi- ancestors(Ellstrand et al,2010).However,the trait of shat- fied 13 well-documented cases of problematic plants that tering alone may rarely suffice to permit a seed dispersing evolved from domesticated ancestors.Because such feral 'crop'to persist and reproduce without human intervention. plants evolved from well-studied taxa,they should be good A shattering 'crop'will need more features to persist in the agro-ecosystem,such as the ability to survive in soil seed- These authors contributed equally to this work. banks,to avoid human weeding,and to successfully compete 2011 The Authors New Pbytologist(2011)191:1119-11271119 New Phytologist 2011 New Phytologist Trust www.newphytologist.com
Rapid evolutionary divergence and ecotypic diversification of germination behavior in weedy rice populations Han-Bing Xia1 *, Hui Xia1 *, Norman C. Ellstrand2 , Chao Yang1 and Bao-Rong Lu1 1 Department of Ecology and Evolutionary Biology, Key Laboratory for Biodiversity Science and Ecological Engineering, Fudan University, Ministry of Education, Handan Road 220, Shanghai 200433, China; 2 Department of Botany and Plant Sciences, Center for Conservation Biology, and Center for Invasive Species Research, University of California, Riverside, CA 92521-0124, USA Author for correspondence: Bao-Rong Lu Tel: +86 21 65643668 Email: brlu@fudan.edu.cn Received: 17 January 2011 Accepted: 5 April 2011 New Phytologist (2011) 191: 1119–1127 doi: 10.1111/j.1469-8137.2011.03766.x Key words: ecotypic differentiation, ferality, latitudinal variation, local adaptation, plant–environment interaction, temperature response, weed evolution. Summary • Feral plants have evolved from well-studied crops, providing good systems for elucidation of how weediness evolves. As yet, they have been largely neglected for this purpose. The evolution of weediness can occur by simple back mutations in domestication genes (domestication in reverse). Whether the evolutionary steps to weediness always occur in reverse remains largely unknown. • We examined seed germination behavior in recently evolved weedy rice (Oryza sativa f. spontanea) populations and their coexisting cultivars in eastern and northeastern China to address whether ‘dedomestication’ is the simple reverse of domestication. • We found that these weedy populations did not diverge from their progenitors by reverting to the pre-domestication trait of seed dormancy. Instead, they have evolved a novel mechanism to avoid growing in inappropriate environments via changes in critical temperature cues for seed germination. Furthermore, we found evidence for subsequent ecotypic divergence of these populations such that the critical temperature for germination correlates with the local habitat temperature at latitudinal gradients. • The origins of problematic plant species, weeds and invasives, have already been studied in detail. These plants can thus be used as systems for studying rapid evolution. To determine whether and how that evolution is adaptive, experiments such as those described here can be performed. Introduction Feral plants are descendants of domesticated plants that have evolved the ability to persist and reproduce without direct human care. Feral plant evolution, also known as ‘dedomestication’, can occur either directly from domesticated ancestors (‘endoferality’) or by hybridization of domesticates with wild relatives (‘exoferality’) (Gressel, 2005a). Dedomestication may produce problematic plants such as invasives and weeds. Ellstrand et al. (2010) identi- fied 13 well-documented cases of problematic plants that evolved from domesticated ancestors. Because such feral plants evolved from well-studied taxa, they should be good model systems for elucidating how weediness or invasiveness evolves. Furthermore, like invasive species, because their histories are generally well known, they can be used as systems for studying rapid adaptive evolution. As yet, they have been largely neglected for this purpose; most studies have focused on simply documenting plants as feral, rather than how ‘ferality’ evolved (Ellstrand et al., 2010). Often part of the story is obvious. Many feral plants have evolved shattering (seed dispersal) from nonshattering crop ancestors (Ellstrand et al., 2010). However, the trait of shattering alone may rarely suffice to permit a seed dispersing ‘crop’ to persist and reproduce without human intervention. A shattering ‘crop’ will need more features to persist in the agro-ecosystem, such as the ability to survive in soil seed- *These authors contributed equally to this work. banks, to avoid human weeding, and to successfully compete New Phytologist Research 2011 The Authors New Phytologist 2011 New Phytologist Trust New Phytologist (2011) 191: 1119–1127 1119 www.newphytologist.com
New 1120 Research Phytologist with coexisting crops.These selective pressures probably serve The authors concluded that 'weedy rice populations from as the driving force for the adaptive evolution of feral plants. Liaoning most probably originated from Liaoning rice varie- An example is weedy rice (Oryza sativa f.spontaned),some ties by mutation and intervarietal hybrids' populations of which evolved directly from cultivated rice Given that the evolutionary pathway to weediness in (Oryza sativa)(Cao et al,2006;Reagon et al,2010; these populations is now known,we sought to examine how Thurber et al,2010).Weedy rice is vegetatively very similar weediness evolved in them.Specifically,we sought to deter- to cultivated rice but has some key differences:shattering seed mine the evolution of seed germination behavior in these dispersal,red pericarp pigmentation,and the ability of seeds populations in relation to their responses to germination to persist in the soil (Delouche et al,2007).Weedy rice is a temperatures by comparing them with their cultivated noxious weed of cultivated rice world-wide (chapters 16-21 ancestors,as well as weedy rice populations from elsewhere. in Gressel,2005b;Delouche et al,2007),both causing crop Nearly all mature rice cultivars have no seed dormancy yield losses and degrading the quality of rice when co- and germinate easily under an array of conditions(Grist, harvested(Gressel,2005b;Delouche et al,2007). 1996;also,this study).By contrast,until recently,strong Weedy rice is polyphyletic.Some populations are seed dormancy was thought be the rule for weedy rice descended directly from cultivated rice,both from indica-type (Oard et al,2000;Gu et al,2003,2005a;Gianinetti (mostly pan-tropical)cultivars (Londo Schaal,2007)and Cohn,2008),with a few exceptions (e.g.Schwanke et al, from japonica-type (mostly temperate)cultivars (Cao et al, 2008).What is the nature of dormancy in the new weedy 2006,2009;Vaughan et al,2008),as well as indica x japonica populations?For our purposes,we follow Baskin hybrids(Ishikawa et al,2005).Furthermore,some popula- Baskin's (2004)definition of seed dormancy:'A dormant tions have an exoferal origin as hybrid descendants of hybrids seed (or other germination unit)is one that does not have between cultivated rice and its wild ancestor Oryza rufipogon the capacity to germinate in a specified period of time under (Londo Schaal,2007;Suh,2008),from which cultivated any combination of normal physical environmental factors rice was domesticated from O.rufipogon c.10 000 (8000- (temperature,light/dark,etc.)that otherwise is favourable 11 500)yr ago in the middle and lower parts of the Yangtze for its germination'(similar to that of Finch-Savage River valley (Normile,1997;Zong et al,2007).The exoferal Leubner-Metzger,2006).However,c.60%of plant species lineages regain the strong seed-shattering and seed-dormancy do not have seed dormancy (Baskin Baskin,1998),and traits of the wild ancestor that were lost.However,during the seed germination of these species must be regulated by other domestication process,the wild ancestor lost seed shattering mechanisms.Previous studies indicated that optimal seed and dormancy,resulted in cultivated rice.The former is germination of many nondormant species was closely asso- commonly grown in pan-tropical regions and the latter in ciated with their habitat temperatures.For example,seed temperate regions where no wild ancestors are found germination of spices located at high latitudes requires a (Vaughan et al,2008). higher temperature than that of spices located at low lati- Recent genomic studies (Gross et al,2010;Reagon tudes(Fenner Thompson,2005). et al,2010;Thurber et al.,2010)have focused on some A recent report on nondormant weedy rice (Delouche simple genetic changes that have occurred to account for et al,2007)prompted us to survey populations of weedy rice the evolution of shattering and grain pigmentation in US in China and elsewhere for nondormancy.Our preliminary weedy rice populations.However,the weedy rice material field survey of weedy rice in temperate regions in NEE China examined evolved so long ago that evolutionary insights suggested extremely low or no seed dormancy for nearly all from these studies are limited (Londo Schaal,2007). populations (H.B.Xia et al,pers.obs.).We further studied To better understand the rapid evolution of weediness in these populations and asked the following questions.Does the weedy rice,more recently evolved populations should be nondormancy found in these recently evolved temperate studied.Thus,we focused on some newly evolved popula- populations in NEE China hold true for other,older temper- tions whose origins are better known (Cao et al,2006;Xia ate weedy rice populations,for example in North America and et al,2011).After many years of successful suppression, southern Europe?What fraction of their nondormant seeds within the last 20 yr weedy rice emerged as a problem in the can survive through winter?Given that these populations span rice fields of north-eastern and eastern (henceforth NEE) a considerable latitudinal gradient,have they evolved a corre- China following changes in cropping style and reduced weed sponding range of temperature-dependent germination cues control (Yu et al,2005;Cao et al,2006;Xia et al,2011). matching their eco-geographic distribution?To address these Motivated by this pest's resurgence,Cao et al.(2006)sought questions,we first assessed seed dormancy for weedy rice its evolutionary origin,comparing marker loci from weedy populations collected from different regions,then tested the rice populations collected from China's Liaoning province winter survival of nondormant weedy rice seeds from with those of wild O.rufipogon as well as selected japonica temperate regions,and finally examined germination from and indica accessions.The weedy rice populations had the nondormant temperate weedy rice populations under differ- closest affinity to a local Liaoning cultivar(a japonica type). ent temperatures. New Ph%ytologist(2011)191:1119-1127 ©2011 The Authors www.newphytologist.com New Phytologist 2011 New Phytologist Trust
with coexisting crops. These selective pressures probably serve as the driving force for the adaptive evolution of feral plants. An example is weedy rice (Oryza sativa f. spontanea), some populations of which evolved directly from cultivated rice (Oryza sativa) (Cao et al., 2006; Reagon et al., 2010; Thurber et al., 2010). Weedy rice is vegetatively very similar to cultivated rice but has some key differences: shattering seed dispersal, red pericarp pigmentation, and the ability of seeds to persist in the soil (Delouche et al., 2007). Weedy rice is a noxious weed of cultivated rice world-wide (chapters 16–21 in Gressel, 2005b; Delouche et al., 2007), both causing crop yield losses and degrading the quality of rice when coharvested (Gressel, 2005b; Delouche et al., 2007). Weedy rice is polyphyletic. Some populations are descended directly from cultivated rice, both from indica-type (mostly pan-tropical) cultivars (Londo & Schaal, 2007) and from japonica-type (mostly temperate) cultivars (Cao et al., 2006, 2009; Vaughan et al., 2008), as well asindica · japonica hybrids (Ishikawa et al., 2005). Furthermore, some populations have an exoferal origin as hybrid descendants of hybrids between cultivated rice and its wild ancestor Oryza rufipogon (Londo & Schaal, 2007; Suh, 2008), from which cultivated rice was domesticated from O. rufipogon c. 10 000 (8000– 11 500) yr ago in the middle and lower parts of the Yangtze River valley (Normile, 1997; Zong et al., 2007). The exoferal lineages regain the strong seed-shattering and seed-dormancy traits of the wild ancestor that were lost. However, during the domestication process, the wild ancestor lost seed shattering and dormancy, resulted in cultivated rice. The former is commonly grown in pan-tropical regions and the latter in temperate regions where no wild ancestors are found (Vaughan et al., 2008). Recent genomic studies (Gross et al., 2010; Reagon et al., 2010; Thurber et al., 2010) have focused on some simple genetic changes that have occurred to account for the evolution of shattering and grain pigmentation in US weedy rice populations. However, the weedy rice material examined evolved so long ago that evolutionary insights from these studies are limited (Londo & Schaal, 2007). To better understand the rapid evolution of weediness in weedy rice, more recently evolved populations should be studied. Thus, we focused on some newly evolved populations whose origins are better known (Cao et al., 2006; Xia et al., 2011). After many years of successful suppression, within the last 20 yr weedy rice emerged as a problem in the rice fields of north-eastern and eastern (henceforth NEE) China following changes in cropping style and reduced weed control (Yu et al., 2005; Cao et al., 2006; Xia et al., 2011). Motivated by this pest’s resurgence, Cao et al. (2006) sought its evolutionary origin, comparing marker loci from weedy rice populations collected from China’s Liaoning province with those of wild O. rufipogon as well as selected japonica and indica accessions. The weedy rice populations had the closest affinity to a local Liaoning cultivar (a japonica type). The authors concluded that ‘weedy rice populations from Liaoning most probably originated from Liaoning rice varieties by mutation and intervarietal hybrids’. Given that the evolutionary pathway to weediness in these populations is now known, we sought to examine how weediness evolved in them. Specifically, we sought to determine the evolution of seed germination behavior in these populations in relation to their responses to germination temperatures by comparing them with their cultivated ancestors, as well as weedy rice populations from elsewhere. Nearly all mature rice cultivars have no seed dormancy and germinate easily under an array of conditions (Grist, 1996; also, this study). By contrast, until recently, strong seed dormancy was thought be the rule for weedy rice (Oard et al., 2000; Gu et al., 2003, 2005a; Gianinetti & Cohn, 2008), with a few exceptions (e.g. Schwanke et al., 2008). What is the nature of dormancy in the new weedy populations? For our purposes, we follow Baskin & Baskin’s (2004) definition of seed dormancy: ‘A dormant seed (or other germination unit) is one that does not have the capacity to germinate in a specified period of time under any combination of normal physical environmental factors (temperature, light⁄ dark, etc.) that otherwise is favourable for its germination’ (similar to that of Finch-Savage & Leubner-Metzger, 2006). However, c. 60% of plant species do not have seed dormancy (Baskin & Baskin, 1998), and seed germination of these species must be regulated by other mechanisms. Previous studies indicated that optimal seed germination of many nondormant species was closely associated with their habitat temperatures. For example, seed germination of spices located at high latitudes requires a higher temperature than that of spices located at low latitudes (Fenner & Thompson, 2005). A recent report on nondormant weedy rice (Delouche et al., 2007) prompted us to survey populations of weedy rice in China and elsewhere for nondormancy. Our preliminary field survey of weedy rice in temperate regions in NEE China suggested extremely low or no seed dormancy for nearly all populations (H. B. Xia et al., pers. obs.). We further studied these populations and asked the following questions. Does the nondormancy found in these recently evolved temperate populations in NEE China hold true for other, older temperate weedy rice populations, for example in North America and southern Europe? What fraction of their nondormant seeds can survive through winter? Given that these populations span a considerable latitudinal gradient, have they evolved a corresponding range of temperature-dependent germination cues matching their eco-geographic distribution? To address these questions, we first assessed seed dormancy for weedy rice populations collected from different regions, then tested the winter survival of nondormant weedy rice seeds from temperate regions, and finally examined germination from nondormant temperate weedy rice populations under different temperatures. 1120 Research New Phytologist 2011 The Authors New Phytologist 2011 New Phytologist Trust New Phytologist (2011) 191: 1119–1127 www.newphytologist.com
New Phytologist Research 1121 Materials and Methods Seed dormancy determination Weedy and cultivated rice sampling Testing for primary dormancy The seed collections came from different environments and from different years.To We determined the nature of primary seed dormancy in 35 eliminate maternal effects,environmental effects,and effects weedy rice Oryza sativa f.spontanea L.populations with of varying seed age and storage,we first grew all our weedy various origins (Supporting Information Table S1).We rice collections in a common garden in a standard paddy collected seeds directly from natural populations in China field on Fudan University's campus in Shanghai,China,in (P1-P25)in 2004-2007 by sampling from at least 30 2007.For each of our collected populations,we randomly maternal plants at each site,and placed individual seed chose one seed to plant from each of 15 different maternal families in separate bags.The others were donated by seed families.We randomly chose 15 seeds from each of the various collaborators. donated populations. Of the Chinese populations,we randomly selected four Mature seed families were harvested from 10 to 15 indi- (P1,P10,P11 and P20;Table S1),representing four prov- viduals of each population and used to measure successful inces,to attempt to induce secondary dormancy and for germination fractions.Exactly 6 d after harvest,90 seeds burial experiments.We selected 18 NEE Chinese popula- representing each sampled population were divided into tions from six regions with a range of latitudes(c.32-48N) three replicates and placed on moist filter paper in Petri to examine their seed germination under a temperature dishes.The dishes were put into a growth chamber at a gradient (Fig.1,Table S1).Eighteen rice cultivars (C1- constant temperature of 28C with a light:dark cycle C18;Table S1)coexisting in the same fields as the 18 afore- of 16:8 h;these conditions are known to be ideal for mentioned weedy populations were collected to test their cultivated rice seed germination.Successful germination germination responses to the same temperature gradient. was recorded 18 d later following Baskin Baskin(1998). 50N Testing for secondary dormancy in recently evolved populations Two months after initial collection,weedy rice seeds from P1,P10,P11 and P20(200-800 per population) from NEE China were exposed to shock treatments to P1 China induce secondary dormancy:(1)in soil at 4C for 30 d;(2) P3● ●P2 in soil at-20C for 20 d;(3)in soil at-20C for 100 d and then at 4C for 20 d;(4)under 2-cm-deep soil for 20 d;and (5)on moist filter paper at 4C for 30 d.For the soil treat- 45N P8 ments,we used ordinary moist rice paddy soil transferred P10●0 from a rice field.Following the treatments,seeds were placed P11 on moist filter paper in Petri dishes which were put into a growth chamber at a constant temperature of 28C with a P13 light:dark cycle of 16:8 h to examine dormancy following 40N the procedure of Baskin Baskin(1998). P18 Seed burial experiment To determine seed survival through winter,seeds from four populations were buried in a Fudan University fallow rice P20 35N field which had been allowed to dry to soil moisture contents of 20-40%and subsequently harrowed.Seeds from population 11 were tested from November 2005 to P24.P23 February 2006;seeds from P1,P10 and P20 were tested 100km P25 from December 2006 to March 2007.For each population, 30N eight bags(replicates),each containing 50-80 intact seeds, were buried c.2 cm deep.Two bags from each population Fig.1 Map of the 18 weedy rice(Oryza sativa f.spontanea) were dug up after 20,40,80 and 100 d of burial and populations(circles)and 18 coexisting rice (Oryza sativa)varieties in the same field collected in China and used for the gradient- subjected to germination conditions following Baskin temperature seed germination experiments,representing a Baskin (1998)as detailed above.The ground temperature latitudinal gradient.Six weather stations are represented by was recorded daily during the experiments conducted in the triangles. campus of Fudan University,Shanghai. 2011 The Authors New Phytologist(2011)191:1119-1127 New Phytologist 2011 New Phytologist Trust www.newphytologist.com
Materials and Methods Weedy and cultivated rice sampling We determined the nature of primary seed dormancy in 35 weedy rice Oryza sativa f. spontanea L. populations with various origins (Supporting Information Table S1). We collected seeds directly from natural populations in China (P1–P25) in 2004–2007 by sampling from at least 30 maternal plants at each site, and placed individual seed families in separate bags. The others were donated by various collaborators. Of the Chinese populations, we randomly selected four (P1, P10, P11 and P20; Table S1), representing four provinces, to attempt to induce secondary dormancy and for burial experiments. We selected 18 NEE Chinese populations from six regions with a range of latitudes (c. 32–48N) to examine their seed germination under a temperature gradient (Fig. 1, Table S1). Eighteen rice cultivars (C1– C18; Table S1) coexisting in the same fields as the 18 aforementioned weedy populations were collected to test their germination responses to the same temperature gradient. Seed dormancy determination Testing for primary dormancy The seed collections came from different environments and from different years. To eliminate maternal effects, environmental effects, and effects of varying seed age and storage, we first grew all our weedy rice collections in a common garden in a standard paddy field on Fudan University’s campus in Shanghai, China, in 2007. For each of our collected populations, we randomly chose one seed to plant from each of 15 different maternal seed families. We randomly chose 15 seeds from each of the donated populations. Mature seed families were harvested from 10 to 15 individuals of each population and used to measure successful germination fractions. Exactly 6 d after harvest, 90 seeds representing each sampled population were divided into three replicates and placed on moist filter paper in Petri dishes. The dishes were put into a growth chamber at a constant temperature of 28C with a light : dark cycle of 16 : 8 h; these conditions are known to be ideal for cultivated rice seed germination. Successful germination was recorded 18 d later following Baskin & Baskin (1998). Testing for secondary dormancy in recently evolved populations Two months after initial collection, weedy rice seeds from P1, P10, P11 and P20 (200–800 per population) from NEE China were exposed to shock treatments to induce secondary dormancy: (1) in soil at 4C for 30 d; (2) in soil at )20C for 20 d; (3) in soil at )20C for 100 d and then at 4C for 20 d; (4) under 2-cm-deep soil for 20 d; and (5) on moist filter paper at 4C for 30 d. For the soil treatments, we used ordinary moist rice paddy soil transferred from a rice field. Following the treatments, seeds were placed on moist filter paper in Petri dishes which were put into a growth chamber at a constant temperature of 28C with a light : dark cycle of 16 : 8 h to examine dormancy following the procedure of Baskin & Baskin (1998). Seed burial experiment To determine seed survival through winter, seeds from four populations were buried in a Fudan University fallow rice field which had been allowed to dry to soil moisture contents of 20–40% and subsequently harrowed. Seeds from population 11 were tested from November 2005 to February 2006; seeds from P1, P10 and P20 were tested from December 2006 to March 2007. For each population, eight bags (replicates), each containing 50–80 intact seeds, were buried c. 2 cm deep. Two bags from each population were dug up after 20, 40, 80 and 100 d of burial and subjected to germination conditions following Baskin & Baskin (1998) as detailed above. The ground temperature was recorded daily during the experiments conducted in the campus of Fudan University, Shanghai. Fig. 1 Map of the 18 weedy rice (Oryza sativa f. spontanea) populations (circles) and 18 coexisting rice (Oryza sativa) varieties in the same field collected in China and used for the gradienttemperature seed germination experiments, representing a latitudinal gradient. Six weather stations are represented by triangles. New Phytologist Research 1121 2011 The Authors New Phytologist 2011 New Phytologist Trust New Phytologist (2011) 191: 1119–1127 www.newphytologist.com
New 1122 Research Phytologist Seed germination under a temperature gradient 气 80 11℃ To determine how germination varies with temperature, 60 we germinated seeds of the 18 rice varieties collected from 40 20 14℃ varying latitudes in a dark incubator with controlled temper- ature regimes at 11,12,14 and 28C,respectively.To 80 12C estimate the finer scale critical temperature (CT)that might 80 prevent maladaptive premature germination in the soil seed 40 bank,seeds collected directly from 18 weedy rice populations 20 28℃ 0 from varying latitudes(Fig.1)were placed in a dark incuba- 88888988g9998988888988g999g tor with controlled temperature regimes at 8,9,10,11,12 and 14C,respectively.For each treatment,90 seeds divided 60 into three replicates were subjected to germination condi- 40 tions separately,following Baskin Baskin(1998)with a 20 11℃ light:dark cycle of 16:8 h,as described above.CT was arbi- trarily determined as the temperature at which 10%of the seeds germinated.Two-way ANOVA was conducted to ana- 60 40 lyze the effects of temperature and source population latitude 20 12℃ on seed germination.For detailed analysis of differences in germination fraction among the six groups of weedy rice 10°℃ populations from sampling sites over diverse latitudes,one- way ANOVA was conducted using the Duncan model in 20 spss ver.12.0 software(2003;SPSS Inc.,Chicago,IL,USA). 14℃ 388g器8N83888g8器888 48N32N48N一32N Correlation of the critical temperature and habitat Rice cultivars(C)and weedy populations(P) temperature Fig.2 Germination ratios of 18 rice(Oryza sativa)varieties and To test whether CT for seed germination under laboratory their corresponding 18 weedy rice (Oryza sativa f.spontanea) conditions varied with the habitat temperature (HT)of the populations from various latitudes at different temperatures.Each graph represents germination at temperatures of 11,12,14 and weedy rice source locations,the correlation of CT or HT 28C for cultivated rice(upper panels),and 8.9.10,11.12 and with the latitude of the collection site was analyzed using a 14C for weedy rice (lower panels),respectively.The vertical bars linear regression model (Johnson Wichern,1998).We indicate the standard error of the mean.The dashed line in each judged HT to be the average temperature of 18 d following chart indicates the critical temperature(CT),at which 10%of seeds germinated.Cultivars and weedy populations were arranged from rice harvest,because weedy rice seeds require that much time left to right to correspond to north to south.For detailed locations, to ripen to achieve full germination.Thus,we estimated HT refer to Fig.1. by calculating the 10-yr(1995-2004)ground temperature average for the 18 d post-harvest for the sample collection locations.The temperature data was collected from six accordance with previous observations (e.g.Grist,1996),we weather stations of the China Meteorological Data Sharing did not observe primary seed dormancy in cultivated rice. By contrast,the germination behavior at 28C of weedy Service System (http://cdc.cma.gov.cn/)that geographically correspond to the extent of weedy rice source regions rice seeds from 35 geographically diverse populations (Fig.1).Student's t-test was used to analyze the consistency (Table S1)varied from I to 100%(Fig.3).In general, of the two correlation slopes between CT or HT and latitude weedy rice populations from the temperate regions had high (ZhangZhang,2002).In addition,an ANCOVA of HT seed germination percentages,mostly close to 100%,but (used as a covariate)with CT (used as a dependent variance)was with a few as low as c.50%.The average for the temperate conducted to confirm the consistency of HT and CT.All the populations was 86.8%(Fig.3);that is,weak or no primary calculations were performed using the software spss ver.12.0. seed dormancy.By contrast,the tropical weedy rice popula- tions had very low seed germination ratios,ranging from c. 1 to 20%,with an average of 8.9%(Fig.3),indicating rela- Results tively strong primary seed dormancy. Seeds from four randomly selected temperate populations Dormancy of cultivated and weedy rice seeds (P1,P10,P11 and P20)were subjected to treatments to test for induced secondary dormancy.We did not detect All rice cultivars tested germinated at 28C after being col- induced secondary seed dormancy.Average seed germina- lected directly from the field (upper graph,Fig.2).In tion ratios following these treatments were about the same New Ph%ytologist(2011)191:1119-1127 ©2011 The Authors www.newphytologist.com New Phytologist 2011 New Phytologist Trust
Seed germination under a temperature gradient To determine how germination varies with temperature, we germinated seeds of the 18 rice varieties collected from varying latitudes in a dark incubator with controlled temperature regimes at 11, 12, 14 and 28C, respectively. To estimate the finer scale critical temperature (CT) that might prevent maladaptive premature germination in the soil seed bank, seeds collected directly from 18 weedy rice populations from varying latitudes (Fig. 1) were placed in a dark incubator with controlled temperature regimes at 8, 9, 10, 11, 12 and 14C, respectively. For each treatment, 90 seeds divided into three replicates were subjected to germination conditions separately, following Baskin & Baskin (1998) with a light : dark cycle of 16 : 8 h, as described above. CT was arbitrarily determined as the temperature at which 10% of the seeds germinated. Two-way ANOVA was conducted to analyze the effects of temperature and source population latitude on seed germination. For detailed analysis of differences in germination fraction among the six groups of weedy rice populations from sampling sites over diverse latitudes, oneway ANOVA was conducted using the Duncan model in SPSS ver. 12.0 software (2003; SPSS Inc., Chicago, IL, USA). Correlation of the critical temperature and habitat temperature To test whether CT for seed germination under laboratory conditions varied with the habitat temperature (HT) of the weedy rice source locations, the correlation of CT or HT with the latitude of the collection site was analyzed using a linear regression model (Johnson & Wichern, 1998). We judged HT to be the average temperature of 18 d following rice harvest, because weedy rice seeds require that much time to ripen to achieve full germination. Thus, we estimated HT by calculating the 10-yr (1995–2004) ground temperature average for the 18 d post-harvest for the sample collection locations. The temperature data was collected from six weather stations of the China Meteorological Data Sharing Service System (http://cdc.cma.gov.cn/) that geographically correspond to the extent of weedy rice source regions (Fig. 1). Student’s t-test was used to analyze the consistency of the two correlation slopes between CT or HT and latitude (Zhang & Zhang, 2002). In addition, an ANCOVA of HT (used as a covariate) with CT (used as a dependent variance) was conducted to confirm the consistency of HT and CT. All the calculations were performed using the software SPSS ver. 12.0. Results Dormancy of cultivated and weedy rice seeds All rice cultivars tested germinated at 28C after being collected directly from the field (upper graph, Fig. 2). In accordance with previous observations (e.g. Grist, 1996), we did not observe primary seed dormancy in cultivated rice. By contrast, the germination behavior at 28C of weedy rice seeds from 35 geographically diverse populations (Table S1) varied from 1 to 100% (Fig. 3). In general, weedy rice populations from the temperate regions had high seed germination percentages, mostly close to 100%, but with a few as low as c. 50%. The average for the temperate populations was 86.8% (Fig. 3); that is, weak or no primary seed dormancy. By contrast, the tropical weedy rice populations had very low seed germination ratios, ranging from c. 1 to 20%, with an average of 8.9% (Fig. 3), indicating relatively strong primary seed dormancy. Seeds from four randomly selected temperate populations (P1, P10, P11 and P20) were subjected to treatments to test for induced secondary dormancy. We did not detect induced secondary seed dormancy. Average seed germination ratios following these treatments were about the same Rice cultivars (C) and weedy populations (P) Germination ratio (%) 0 20 40 60 80 1000 20 40 60 80 100 0 20 40 60 80 100 P1 P2 P3 P8 P9 P10 P11 P12 P13 P17 P18 P19 P20 P21 P22 P23 P24 P25 P1 P2 P3 P8 P9 P10 P11 P12 P13 P17 P18 P19 P20 P21 P22 P23 P24 P25 8°C 9°C 10°C 48°N 32°N 48°N 32°N C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12 C13 C14 C15 C16 C17 C18 20 40 60 80 C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12 C13 C14 C15 C16 C17 C18 0 20 40 60 80 100 C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12 C13 C14 C15 C16 C17 C18 0 20 40 60 80 100 C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12 C13 C14 C15 C16 C17 C18 12°C 11°C 14°C 28°C 11°C 12°C 14°C Fig. 2 Germination ratios of 18 rice (Oryza sativa) varieties and their corresponding 18 weedy rice (Oryza sativa f. spontanea) populations from various latitudes at different temperatures. Each graph represents germination at temperatures of 11, 12, 14 and 28C for cultivated rice (upper panels), and 8, 9, 10, 11, 12 and 14C for weedy rice (lower panels), respectively. The vertical bars indicate the standard error of the mean. The dashed line in each chart indicates the critical temperature (CT), at which 10% of seeds germinated. Cultivars and weedy populations were arranged from left to right to correspond to north to south. For detailed locations, refer to Fig. 1. 1122 Research New Phytologist 2011 The Authors New Phytologist 2011 New Phytologist Trust New Phytologist (2011) 191: 1119–1127 www.newphytologist.com
New Phytologist Research 1123 100T 00904至 12C;at 14C all varieties showed some germination (but only half reached 50%germination ratios).At temperatures 80 between no germination(11C)and complete germination (28C),rice cultivars had similar seed germination behavior 60 which did not co-vary with their latitude of origin. Similar to cultivated rice,weedy rice seed germination 40 responses were more or less uniform at the most extreme 20 temperatures tested.At the lowest temperature(8C),the sam- ples showed very little (P2 and P3)or no (the others) 0+ germination;at the highest temperature (14C),all samples 38卫易品品8B3器品3马H君8338图8838图8型3888 showed very high germination (>90%)(lower graph,Fig.2). Weedy rice population (P1-P35) In contrast to cultivated rice,weedy rice seed germination Fig.3 Germination ratios at 28C of 35 weedy rice(Oryza sativa f. was significantly influenced by the experimental tempera- spontanea)populations from temperate (circles)and tropical ture (P<0.001),the latitude of the sampling site (P< (triangles)regions 6 d after harvest used to evaluate primary seed 0.001),and their interaction (P<0.001),based on the dormancy.Refer to Supporting Information Table S1 for the specific origin of each population.Vertical bars indicate SE of the mean. two-way ANOVA.Weedy rice seed germination varied significantly and systematically under the intermediate as those observed for primary germination for these popula- temperature regimes (9,10,11 and 12C)(Table 2)such tions (Table S2). that the populations from the higher latitudes(P1-P3 and P8-P10)germinated more readily at lower temperatures Survival of weedy rice seeds through winter in the soil (starting at 9C)than the populations from the mid or lower latitudes.Indeed,germination for these high-latitude Seed burial experiments revealed that seeds from temperate populations increased with increasing temperature to a weedy rice populations survived and germinated through maximum ratio at 14C.In contrast,populations from the Shanghai's winter conditions at high enough ratios to lowest latitudes collected (P17-P25)showed substantial sustain their populations.In the first-year experiment(P11 germination at 11C and above;their germination ratios only),c.30%of the weedy rice seeds survived after burial in also increased to a maximum at 14C.Populations from soil for 100 d (Table 1).The average temperature was the mid range of samples(P11-P13)showed a germination 7.1C with a minimum of-4.0C during this experiment response intermediate between those of the others (lower (17 November 2005 to 25 February 2006).Similarly,in graph,Fig.2).Overall,as a population's latitude increased, the second-year experiment(P1,P10 and P20),c.32-84% its ability to germinate at lower temperatures also increased. of the weedy rice seeds survived to germinate after burial for The following section examines this relationship more closely a maximum of 100 d (Table 1).During this experiment (15 December 2006 to 23 March 2007),the average tem- perature was 8.3C with a minimum of-2.0C.Most Correlations among CT,latitude and HT in weedy rice remaining nongerminating seeds at the end of the experi- Linear regression analysis revealed a significant negative cor- ments were rotten. relation between CT(the temperature at which there was 10%seed germination)determined in the temperature Seed germination under different temperatures gradient experiment (lower graph,Fig.2)and the source latitude for the weedy rice populations (r=0.672, Not one rice cultivar in this study germinated at 11C P<0.001)(Fig.4).The same relationship was found (upper graph,Fig.2).Most showed some germination at between HT at the weedy rice collection sites and source Table 1 Seed survival and subsequent germination ratios of four randomly selected weedy rice(Oryza sativa f.spontanea)populations after varying times buried in the soil Seed germination ratio (%)after different burial periods(d) Population 20 40 80 100 P1 97.5(96.9-98.1) 83.1(80.9-85.1) 88.2(83.3-93.1) 83.7(80.0-89.4) h P10 58.9(53.4-64.4) 45.0(44.3-45.7) 31.3(30.4-32.5) P11 64.0(46.9-81.1) 37.2(31.0-43.4) 38.1(32.0-44.2) 29.8(12.0-47.6⑤ P20 66.6(64.0-69.2) 56.1(55.6-56.5) 55.7(53.7-57.7) 32.0(29.4-34.5) Numbers in parentheses indicate ranges;most nongerminating seeds had rotted in the soil. PData not available because of insufficient seeds for this population. ©20l1 The Authors New Phytologist(2011)191:1119-1127 New Phytologist 2011 New Phytologist Trust www.newphytologist.com
as those observed for primary germination for these populations (Table S2). Survival of weedy rice seeds through winter in the soil Seed burial experiments revealed that seeds from temperate weedy rice populations survived and germinated through Shanghai’s winter conditions at high enough ratios to sustain their populations. In the first-year experiment (P11 only), c. 30% of the weedy rice seeds survived after burial in soil for 100 d (Table 1). The average temperature was 7.1C with a minimum of )4.0C during this experiment (17 November 2005 to 25 February 2006). Similarly, in the second-year experiment (P1, P10 and P20), c. 32–84% of the weedy rice seeds survived to germinate after burial for a maximum of 100 d (Table 1). During this experiment (15 December 2006 to 23 March 2007), the average temperature was 8.3C with a minimum of )2.0C. Most remaining nongerminating seeds at the end of the experiments were rotten. Seed germination under different temperatures Not one rice cultivar in this study germinated at 11C (upper graph, Fig. 2). Most showed some germination at 12C; at 14C all varieties showed some germination (but only half reached 50% germination ratios). At temperatures between no germination (11C) and complete germination (28C), rice cultivars had similar seed germination behavior which did not co-vary with their latitude of origin. Similar to cultivated rice, weedy rice seed germination responses were more or less uniform at the most extreme temperatures tested. At the lowest temperature (8C), the samples showed very little (P2 and P3) or no (the others) germination; at the highest temperature (14C), all samples showed very high germination (> 90%) (lower graph, Fig. 2). In contrast to cultivated rice, weedy rice seed germination was significantly influenced by the experimental temperature (P < 0.001), the latitude of the sampling site (P < 0.001), and their interaction (P < 0.001), based on the two-way ANOVA. Weedy rice seed germination varied significantly and systematically under the intermediate temperature regimes (9, 10, 11 and 12C) (Table 2) such that the populations from the higher latitudes (P1–P3 and P8–P10) germinated more readily at lower temperatures (starting at 9C) than the populations from the mid or lower latitudes. Indeed, germination for these high-latitude populations increased with increasing temperature to a maximum ratio at 14C. In contrast, populations from the lowest latitudes collected (P17–P25) showed substantial germination at 11C and above; their germination ratios also increased to a maximum at 14C. Populations from the mid range of samples (P11–P13) showed a germination response intermediate between those of the others (lower graph, Fig. 2). Overall, as a population’s latitude increased, its ability to germinate at lower temperatures also increased. The following section examines this relationship more closely. Correlations among CT, latitude and HT in weedy rice Linear regression analysis revealed a significant negative correlation between CT (the temperature at which there was 10% seed germination) determined in the temperature gradient experiment (lower graph, Fig. 2) and the source latitude for the weedy rice populations (r 2 = 0.672, P < 0.001) (Fig. 4). The same relationship was found between HT at the weedy rice collection sites and source Fig. 3 Germination ratios at 28C of 35 weedy rice (Oryza sativa f. spontanea) populations from temperate (circles) and tropical (triangles) regions 6 d after harvest used to evaluate primary seed dormancy. Refer to Supporting Information Table S1 for the specific origin of each population. Vertical bars indicate ± SE of the mean. Table 1 Seed survival and subsequent germination ratios of four randomly selected weedy rice (Oryza sativa f. spontanea) populations after varying times buried in the soil Population Seed germination ratio (%) after different burial periods (d)a 20 40 80 100 P1 97.5 (96.9–98.1) 83.1 (80.9–85.1) 88.2 (83.3–93.1) 83.7 (80.0–89.4) P10 58.9 (53.4–64.4) 45.0 (44.3–45.7) 31.3 (30.4–32.5) b P11 64.0 (46.9–81.1) 37.2 (31.0–43.4) 38.1 (32.0–44.2) 29.8 (12.0–47.6) P20 66.6 (64.0–69.2) 56.1 (55.6–56.5) 55.7 (53.7–57.7) 32.0 (29.4–34.5) a Numbers in parentheses indicate ranges; most nongerminating seeds had rotted in the soil. b Data not available because of insufficient seeds for this population. New Phytologist Research 1123 2011 The Authors New Phytologist 2011 New Phytologist Trust New Phytologist (2011) 191: 1119–1127 www.newphytologist.com
New 1124 Research Phytologist Table 2 Differences in the germination ratios of different groups of weedy rice(Oryza sativa f.spontanea)populations along a latitudinal gra- dient over different temperature regimes as indicated by the one-way ANOVA(Duncan model) Experimental temperature (C) Weedy rice population 9 10 11 12 14 Group 1:P1-P3 NS A A A A NS Group 2:P8-P10 A B AB A Group 3:P11-P13 B C Group 4:P17-P19 B D B AB Group 5:P20-P22 CD AB B Group 6:P23-P25 B D P1-P25:P value 0.123 0.000 0.000 0.035 0.016 0.062 (Fvalue) (2.191) (12.818) (76.036 (3.498) (4.404) (1.953) Different capital letters indicate significant differences among populations at different latitudes.A significant difference among all populations is indicated by the P-values(last row).The degrees of freedom between groups and within groups and the total degrees of freedom were 5, 12 and 17,respectively. (from China,South Korea and Italy)had little or no pri- 14- mary dormancy.One previous study also found that certain weedy rice populations from temperate rice planting regions 11 have either extremely weak or no seed dormancy (Delouche etal,2007). 巴 Differences in seed dormancy in tropical vs temperate weedy rice may have to do with their different evolutionary origins.The temperate populations examined here (Cao et al,2006)and others (e.g.Ishikawa et al,2005;Londo Schaal,2007;Reagon et al,2010;Thurber et al,2010) are known to have evolved directly from domesticated rice 30 40 50 (without hybridization with a wild ancestor).Like most Latitude (N) cereals,cultivated rice typically has no seed dormancy Fig.4 Changes in critical seed germination temperature (CT;red (Chang Yen,1969;Cai Morishima,2000;Gu et al, circles)and habitat temperature(HT;blue triangles)for 18 weedy 2004,2005b).By contrast,the seed dormancy of tropical rice (Oryza sativa f.spontanea)populations across a latitudinal weedy rice may be attributable to a hybrid ancestry involv- gradient.The y-axis represents the temperature:either CT (y=-0.156x+16.330:=0.672)orHT(y=-0.220x+15.682: ing a wild species that donated genes for dormancy (Cai 2=0.798).Bars indicate SE. Morishima,2000;Gu et al,2003;Veasey et al,2004). Our germination experiments on recently evolved tem- latitude (=0.798,P=0.017)(Fig.4).Based on these perate weedy rice populations from NEE China regressions,the CT required for weedy rice seed germination demonstrated that a variety of treatments did not induce was 2-3C higher than the HT(Fig.4),indicating that HT secondary dormancy in these weedy rice seeds.Similarly, in every location immediately after rice harvesting was too our burial experiments revealed that seeds from these popu- cold to allow germination of the local weedy rice population. lations survived to germinate through the winter at high The ANCOVA in which HT was used as a covariate and enough ratios to sustain their populations.This finding is CT as a dependent variable showed that HT had no signifi- supported by those of other studies in northeastern China, cant interaction with CT(P=0.339),indicating that HT where 20%of weedy rice seeds (probably,but not defi- and CT had the same tendency to change with latitude. nitely,nondormant)survived after winter burial (Meng, Further analysis demonstrated that the slopes generated 2004;Yuan et al,2006).Clearly,nondormant temperate from CT and HT vs latitude were essentially consistent weedy rice seeds can survive through winter in soil seed- (Student t-test:t=0.252,df 20,P>0.05)(Fig.4). banks until they receive appropriate germination cues. Further experiments revealed that weedy rice seeds rely Discussion on a CT as a germination cue.Specifically,our results show that,at the time at which these weedy rice seeds disperse, We confirmed that there was strong seed dormancy for the local temperature is too low for germination.The diverse weedy rice populations from tropical regions.By local average temperature gradually decreases after rice is contrast,we found that temperate weedy rice populations harvested in autumn.The temperature remains below the New Phytologist (2011)191:1119-1127 ©2011 The Authors www.newphytologist.com New Phytologist 2011 New Phytologist Trust
latitude (r 2 = 0.798, P = 0.017) (Fig. 4). Based on these regressions, the CT required for weedy rice seed germination was 2–3C higher than the HT (Fig. 4), indicating that HT in every location immediately after rice harvesting was too cold to allow germination of the local weedy rice population. The ANCOVA in which HT was used as a covariate and CT as a dependent variable showed that HT had no signifi- cant interaction with CT (P = 0.339), indicating that HT and CT had the same tendency to change with latitude. Further analysis demonstrated that the slopes generated from CT and HT vs latitude were essentially consistent (Student t-test: t = 0.252, df = 20, P > 0.05) (Fig. 4). Discussion We confirmed that there was strong seed dormancy for diverse weedy rice populations from tropical regions. By contrast, we found that temperate weedy rice populations (from China, South Korea and Italy) had little or no primary dormancy. One previous study also found that certain weedy rice populations from temperate rice planting regions have either extremely weak or no seed dormancy (Delouche et al., 2007). Differences in seed dormancy in tropical vs temperate weedy rice may have to do with their different evolutionary origins. The temperate populations examined here (Cao et al., 2006) and others (e.g. Ishikawa et al., 2005; Londo & Schaal, 2007; Reagon et al., 2010; Thurber et al., 2010) are known to have evolved directly from domesticated rice (without hybridization with a wild ancestor). Like most cereals, cultivated rice typically has no seed dormancy (Chang & Yen, 1969; Cai & Morishima, 2000; Gu et al., 2004, 2005b). By contrast, the seed dormancy of tropical weedy rice may be attributable to a hybrid ancestry involving a wild species that donated genes for dormancy (Cai & Morishima, 2000; Gu et al., 2003; Veasey et al., 2004). Our germination experiments on recently evolved temperate weedy rice populations from NEE China demonstrated that a variety of treatments did not induce secondary dormancy in these weedy rice seeds. Similarly, our burial experiments revealed that seeds from these populations survived to germinate through the winter at high enough ratios to sustain their populations. This finding is supported by those of other studies in northeastern China, where > 20% of weedy rice seeds (probably, but not defi- nitely, nondormant) survived after winter burial (Meng, 2004; Yuan et al., 2006). Clearly, nondormant temperate weedy rice seeds can survive through winter in soil seedbanks until they receive appropriate germination cues. Further experiments revealed that weedy rice seeds rely on a CT as a germination cue. Specifically, our results show that, at the time at which these weedy rice seeds disperse, the local temperature is too low for germination. The local average temperature gradually decreases after rice is harvested in autumn. The temperature remains below the Fig. 4 Changes in critical seed germination temperature (CT; red circles) and habitat temperature (HT; blue triangles) for 18 weedy rice (Oryza sativa f. spontanea) populations across a latitudinal gradient. The y-axis represents the temperature: either CT (y = )0.156x + 16.330; r 2 = 0.672) or HT (y = )0.220x + 15.682; r 2 = 0.798). Bars indicate ± SE. Table 2 Differences in the germination ratios of different groups of weedy rice (Oryza sativa f. spontanea) populations along a latitudinal gradient over different temperature regimes as indicated by the one-way ANOVA (Duncan model) Weedy rice population Experimental temperature (C) 8 9 10 11 12 14 Group 1: P1–P3 NS A A A A NS Group 2: P8–P10 A B AB A Group 3: P11–P13 B C A A Group 4: P17–P19 B D B AB Group 5: P20–P22 B CD AB B Group 6: P23–P25 B D B B P1–P25: P value (F value) 0.123 (2.191) 0.000 (12.818) 0.000 (76.036) 0.035 (3.498) 0.016 (4.404) 0.062 (1.953) Different capital letters indicate significant differences among populations at different latitudes. A significant difference among all populations is indicated by the P-values (last row). The degrees of freedom between groups and within groups and the total degrees of freedom were 5, 12 and 17, respectively. 1124 Research New Phytologist 2011 The Authors New Phytologist 2011 New Phytologist Trust New Phytologist (2011) 191: 1119–1127 www.newphytologist.com
New Phytologist Research 1125 CT from autumn until the next spring.Weedy rice seeds ferality before their introduction to North America. germinate when high enough temperatures return at that Confirming that these populations are descended from time. Asian cultivated rice,they found that 'reversion of domesti- We found evidence for ecotypic differentiation for germi- cation alleles does not account for the pigmented grains of nation temperature cues with positive correlations among a weed accessions,'nor does introgression from a wild relative population's CT,its local HT and its latitude.Populations Instead,they concluded that novel allelic change must from cooler,more northerly latitudes tended to germinate at account for the red pigmentation.Examining the evolution lower temperatures than those from warmer,more southerly of shattering in the same populations,Thurber et al.(2010) latitudes.By contrast,we did not find such a relationship for came to a similar conclusion.They found that the non- rice cultivars from corresponding latitudes. shattering allele for cultivated rice that distinguishes it from Interestingly,all weedy populations germinated at its shattering wild progenitors was present in its weedy temperatures too low for substantial germination of the descendants,despite the ability of those descendents to easily cultivars.Most weedy populations had high germination disperse their seeds.Clearly,a mutation at another locus is ratios at 12C,a temperature at which most cultivars responsible for the evolution of shattering in the weedy showed low germination ratios.At 14C,all weedy rice populations.Therefore,weedy rice pigmentation and shat- populations had >90%germination ratios,whereas not tering evolved via phenotypic convergence with the wild one tested cultivar had a germination ratio as high as 90% ancestor without genotypic convergence.In our case,weedy at that temperature.These weedy rice populations have rice evolved a wholly novel seed germination behavior diverged evolutionarily from their cultivated ancestors with phenotype,different from those of both its immediate culti- regard to seed-germination temperature cues.With a lower vated rice progenitors and its more distant wild rice CT than rice culitvars,weedy rice germinates early and has ancestors.In all three cases,dedomestication proceeded a head start on directly seeded cultivated rice,resulting in a without evolution at the loci involved in domestication. competitive advantage over rice in the field. Our results not only demonstrate the evolutionary diver- The nondormant weedy rice populations that we studied gence of weedy rice from cultivated rice,but also reveal evolved an adaptive mechanism different from those of ecological diversification of temperature cues for individual other weedy rice populations to regulate their seed germina- weedy rice populations to match local climatic conditions. tion to avoid growing in inappropriate environments.This We found that CT covaried with both HT and the latitude evolution occurred rapidly.According to local agricultural at which the weedy rice populations were sampled.The extension services,weedy rice was first found in the sampled significant correlation between CT and HT across a wide regions 20 yr after rice farmers started to move away from range of recently evolved weedy rice populations indicates transplanting to direct seeding and other no-till technolo- the rapid evolution of local adaptive differentiation of this gies.With a novel mechanism to inhibit germination under mechanism across temperate rice-planting regions in China. unfavorable conditions,it is not clear that continued evolu- Because our seed germination data from cultivars collected tion to acquiring true dormancy would present any further from geographically corresponding sites did not show any adaptive advantage. geographic or temperature-dependent pattern,introgression Our data challenge the view that the evolution of endo- from local rice varieties into weedy populations can be ruled ferality involves back mutation at domestication loci out as an evolutionary mechanism for this diversification. (Gressel,2005a);that is,domestication in reverse.During Previous studies have documented high levels of variation rice domestication,the evolutionary loss of seed dormancy within and among weedy rice populations(Delouche et al, represents the key seed germination difference between 2007),but we believe ours is the first to demonstrate that cultivated rice and its wild progenitors(Gu et al,2004, adaptive differentiation has occurred rapidly.In a review of 2005b;Veasey et al,2004;Vaughan et al,2008). ferality,Warwick Stewart(2005)called for studies on the However,the weedy rice populations that we examined speed of dedomestication.In the case of weedy rice,the evolved weediness without reverting to the seed dormancy close relationship between CT and local HT evolved in less of their wild ancestors.It appears that the immediate pro- than two decades. genitors of weedy rice first obtained seed shattering and Evidence is accumulating suggesting that rapid local adap- subsequently evolved other key traits that enhanced their tive evolution may be a common feature of both weeds and ability to survive under natural conditions. invasives (e.g.Maron et al,2004;Bossdorf et al,2005; Recent genomic work in weedy rice also supports our con- Keller et al,2009;but see Keller Taylor,2008). clusion that the evolutionary pathway to ferality need not be Common garden experiments have demonstrated that two domestication in reverse.Most rice cultivars have white other plants with crop ancestry evolved ecotypic differentia- grains;nearly all weedy rice populations exhibit red pigmen- tion in less than a century:weedy rye (Secale cereale)of the tation.Gross et al(2010)examined the genetic basis of red western USA,and California's wild radish (Raphanus pigmentation in US weedy rice populations that had evolved satiuus),known as both agricultural weeds and invasives of ©20l1 The Authors New Phytologist(2011)191:1119-1127 New Phytologist 2011 New Phytologist Trust www.newphytologist.com
CT from autumn until the next spring. Weedy rice seeds germinate when high enough temperatures return at that time. We found evidence for ecotypic differentiation for germination temperature cues with positive correlations among a population’s CT, its local HT and its latitude. Populations from cooler, more northerly latitudes tended to germinate at lower temperatures than those from warmer, more southerly latitudes. By contrast, we did not find such a relationship for rice cultivars from corresponding latitudes. Interestingly, all weedy populations germinated at temperatures too low for substantial germination of the cultivars. Most weedy populations had high germination ratios at 12C, a temperature at which most cultivars showed low germination ratios. At 14C, all weedy rice populations had > 90% germination ratios, whereas not one tested cultivar had a germination ratio as high as 90% at that temperature. These weedy rice populations have diverged evolutionarily from their cultivated ancestors with regard to seed-germination temperature cues. With a lower CT than rice culitvars, weedy rice germinates early and has a head start on directly seeded cultivated rice, resulting in a competitive advantage over rice in the field. The nondormant weedy rice populations that we studied evolved an adaptive mechanism different from those of other weedy rice populations to regulate their seed germination to avoid growing in inappropriate environments. This evolution occurred rapidly. According to local agricultural extension services, weedy rice was first found in the sampled regions < 20 yr after rice farmers started to move away from transplanting to direct seeding and other no-till technologies. With a novel mechanism to inhibit germination under unfavorable conditions, it is not clear that continued evolution to acquiring true dormancy would present any further adaptive advantage. Our data challenge the view that the evolution of endoferality involves back mutation at domestication loci (Gressel, 2005a); that is, domestication in reverse. During rice domestication, the evolutionary loss of seed dormancy represents the key seed germination difference between cultivated rice and its wild progenitors (Gu et al., 2004, 2005b; Veasey et al., 2004; Vaughan et al., 2008). However, the weedy rice populations that we examined evolved weediness without reverting to the seed dormancy of their wild ancestors. It appears that the immediate progenitors of weedy rice first obtained seed shattering and subsequently evolved other key traits that enhanced their ability to survive under natural conditions. Recent genomic work in weedy rice also supports our conclusion that the evolutionary pathway to ferality need not be domestication in reverse. Most rice cultivars have white grains; nearly all weedy rice populations exhibit red pigmentation. Gross et al. (2010) examined the genetic basis of red pigmentation in US weedy rice populations that had evolved ferality before their introduction to North America. Confirming that these populations are descended from Asian cultivated rice, they found that ‘reversion of domestication alleles does not account for the pigmented grains of weed accessions,’ nor does introgression from a wild relative. Instead, they concluded that novel allelic change must account for the red pigmentation. Examining the evolution of shattering in the same populations, Thurber et al. (2010) came to a similar conclusion. They found that the nonshattering allele for cultivated rice that distinguishes it from its shattering wild progenitors was present in its weedy descendants, despite the ability of those descendents to easily disperse their seeds. Clearly, a mutation at another locus is responsible for the evolution of shattering in the weedy populations. Therefore, weedy rice pigmentation and shattering evolved via phenotypic convergence with the wild ancestor without genotypic convergence. In our case, weedy rice evolved a wholly novel seed germination behavior phenotype, different from those of both its immediate cultivated rice progenitors and its more distant wild rice ancestors. In all three cases, dedomestication proceeded without evolution at the loci involved in domestication. Our results not only demonstrate the evolutionary divergence of weedy rice from cultivated rice, but also reveal ecological diversification of temperature cues for individual weedy rice populations to match local climatic conditions. We found that CT covaried with both HT and the latitude at which the weedy rice populations were sampled. The significant correlation between CT and HT across a wide range of recently evolved weedy rice populations indicates the rapid evolution of local adaptive differentiation of this mechanism across temperate rice-planting regions in China. Because our seed germination data from cultivars collected from geographically corresponding sites did not show any geographic or temperature-dependent pattern, introgression from local rice varieties into weedy populations can be ruled out as an evolutionary mechanism for this diversification. Previous studies have documented high levels of variation within and among weedy rice populations (Delouche et al., 2007), but we believe ours is the first to demonstrate that adaptive differentiation has occurred rapidly. In a review of ferality, Warwick & Stewart (2005) called for studies on the speed of dedomestication. In the case of weedy rice, the close relationship between CT and local HT evolved in less than two decades. Evidence is accumulating suggesting that rapid local adaptive evolution may be a common feature of both weeds and invasives (e.g. Maron et al., 2004; Bossdorf et al., 2005; Keller et al., 2009; but see Keller & Taylor, 2008). Common garden experiments have demonstrated that two other plants with crop ancestry evolved ecotypic differentiation in less than a century: weedy rye (Secale cereale) of the western USA, and California’s wild radish (Raphanus sativus), known as both agricultural weeds and invasives of New Phytologist Research 1125 2011 The Authors New Phytologist 2011 New Phytologist Trust New Phytologist (2011) 191: 1119–1127 www.newphytologist.com
New 1126 Research Phytologist less managed ecosystems (Burger,2006;Burger et al,2006; Burger JC,Lee S,Ellstrand NC.2006.Origin and genetic structure of Hegde et al,2006;Ridley Ellstrand,2009).Both of these feral rye in the western United States.Molecular Ecology 15:2527- evolved local adaptation in less than a century.Furthermore, 2539. Cai HW,Morishima H.2000.Genomic regions affecting seedshattering common garden experiments that compare invasive plants and seed dormancy in rice.Theoretical and Applied Genetics 100: to those collected from the native range have revealed the 840-846. evolution of latitudinal differentiation for some invasives. Cao QJ,Lu BR,Xia H,Rong J,Sala F,Spada A,Grassi F.2006.Genetic The invasive populations of the shrub Hypericum canariense diversity and origin of weedy rice (Onza sativa f.spontanea)populations in the USA have been shown to be derived from a single found in North-eastern China revealed by simple sequence repeat(SSR) markers.Annals of Botany 98:1241-1252. source population in the Canary Islands but,despite genetic Cao QJ,Xia H,Yang X,Lu BR.2009.Performance of hybrids between bottlenecks,have still evolved adaptive latitudinal differences weedy rice and insect-resistant transgenic rice under field experiments: in flowering phenology in less than half a century(Dluglosch implication for environmental biosafety assessment./ournal of Parker,2008).Only a handful of experimental studies Integrative Plant Biology 51:1138-1148. report no evidence for adaptive evolution for invasive popu- Chang TT,Yen ST.1969.Inheritance of grain dormancy in four rice crosses.Botanical Bulletin Academia Sinica 10:1-9. lations relative to their ancestral populations(e.g.Brodersen Delouche JC,Burgos NR,Gealy DR,de San Martin GZ,Labrada R. etal,2008). 2007.Weedy rices:origin.biology.ecology and conrol Rome,Italy:FAO. Because the origins of problematic plants species have Dluglosch K,Parker IM.2008.Invading populations of an ornamental already been studied in detail,these plants can be used as shrub show rapid life history evolution despite genetic bottlenecks. systems for studying rapid evolution.Feral plants are partic- Ecology Letters 11:701-709. Ellstrand NC,Heredia SM,Leak-Garcia JA,Heraty JM,Burger JC,Yao ularly good systems for this purpose if they diverged in L Nohzadeh-Malakshah S,Ridley CE.2010.Crops gone wild: sympatry with their progenitor,but caution must be evolution of weeds and invasives from domesticated ancestors. employed when determining whether that evolutionary Evolutionary Applications 3:494-504. divergence is adaptive or not (Keller Taylor,2008). Fenner M,Thompson K.2005.The ecology ofseeds.Cambridge,UK: History,contingency,chance,and gene flow can be alter- Cambridge University Press. Finch-Savage WE,Leubner-Metzger G.2006.Seed dormancy and the nate explanations to apparent adaptation.Experimental control of germination.New Pbytologist 171:501-523. approaches such as those described for weedy rice and the Gianinetti A,Cohn MA.2008.Seed dormancy in red rice.XIIl: other examples given above can help distinguish among the Interaction of dry-afterripening and hydration temperature.Seed Science alternatives. Research18:151-159. Gressel J.2005a.Introduction-the challenges of ferality.In:Gressel J,ed. Crop ferality and volunteerism.Boca Raton,FL,USA:CRC Press,1-7. Acknowledgements Gressel J.2005b.In:Gressel ].ed.Crop ferality and volunteerism.Boca Raton,FL,USA:CRC Press,257-367. This study was supported by the Chinese Ministry of Grist DH.1996.Rice,6th edn.London,UK:Longman. Science and Technology (Grants.2011CB100401)and Gross BL,Reagon M,Hsu SC,Caicedo AL,Jia Y,Olsen KM.2010. the Nature Science Foundation of China(grants 30871503 Seeing red:the origin of grain pigmentation in US weedy rice.Mlecar Ecol0w19:3380-3393. and 30730066),as well as a John Simon Guggenheim Gu XY,Chen ZX,Foley ME.2003.Inheritance of seed dormancy in Memorial Fellowship and a United States National weedy rice.Crop Science 43:835-843. Science Foundation OPUS Grant awarded to N.C.E.(DEB Gu XY,Kianian SF,Foley ME.2004.Multiple loci and epistases control 1020799).We thank Prof.H.S.Suh of Yeungnam genetic variation for seed dormancy in weedy rice (Oryza satiua) University,South Korea and Dr B.Basso of the University Genetics166:1503-1516. Gu XY,Kianian SF,Foley ME.2005a.Phenotypic selection for dormancy of Milan,Milan,Italy,for their donations of some weedy introduced a set of adaptive haplotypes from weedy into cultivated rice. rice accessions. Genetics171:695-704. Gu XY,Kianian SF,Hareland GA,Hoffer BL,Foley ME.2005b.Genetic analysis of adaptive syndromes interrelated with seed dormancy in References weedy rice (Oryza sativa).Theoretical and Applied Genetics 110. 1108-1118. Baskin CC.Baskin JM.1998.Seeds:ecology,biogeography,and evolution of Hegde SG,Nason JD,Clegg J,Ellstrand NC.2006.The evolution of dormancy and germination.San Diego,CA,USA:Academic Press. Baskin IM,Baskin CC.2004.A classification system for seed dormancy California's wild radish has resulted in the extinction of its progenitors. Evolution60:1187-1197. Seed Science Research 14:1-16. BossdorfO,Auge H,Lafuma L Rogers WE,Siemann E,Prati D.2005. Ishikawa R,Toki N,Imai K,Sato YI,Yamagishi H,Shimamoto Y,Ueno K,Morishima H,Sato T.2005.Origin of weedy rice grown in Bhutan Phenotypic and genetic differentiation between native and introduced plant populations.Oecologia 14:1-11. and the force of genetic diversity.Genetic Resources and Crop Evolution 52:395-403. Brodersen C,Lavergne S,Molofsky J.2008.Genetic variation in Johnson RA,Wichern DW.1998.Applied multivariate statistical analysis. photosynthetic characteristics among invasive and native populations of reed canarygrass(Phalaris arundinaced).Biological Inasions10: Upper Sadle River,New Jersey,USA:Pearson Prentice Hall. Keller SR,Sowell DR,Neiman M,Wolfe LM,Taylor DR.2009. 1317-1325. Burger JC.2006.Genetic correlates to weediness in feral rye(Secale cereale Adaptation and colonization history affect the evolution of clines in two L).PhD dissertation,University of California,Riverside,CA,USA. introduced species.New Pbytologist 183:678-690. New Phytologist(2011)191:1119-1127 ©2011 The Authors www.newphytologist.com New Phytologist 2011 New Phytologist Trust
less managed ecosystems (Burger, 2006; Burger et al., 2006; Hegde et al., 2006; Ridley & Ellstrand, 2009). Both of these evolved local adaptation in less than a century. Furthermore, common garden experiments that compare invasive plants to those collected from the native range have revealed the evolution of latitudinal differentiation for some invasives. The invasive populations of the shrub Hypericum canariense in the USA have been shown to be derived from a single source population in the Canary Islands but, despite genetic bottlenecks, have still evolved adaptive latitudinal differences in flowering phenology in less than half a century (Dluglosch & Parker, 2008). Only a handful of experimental studies report no evidence for adaptive evolution for invasive populations relative to their ancestral populations (e.g. Brodersen et al., 2008). Because the origins of problematic plants species have already been studied in detail, these plants can be used as systems for studying rapid evolution. Feral plants are particularly good systems for this purpose if they diverged in sympatry with their progenitor, but caution must be employed when determining whether that evolutionary divergence is adaptive or not (Keller & Taylor, 2008). History, contingency, chance, and gene flow can be alternate explanations to apparent adaptation. Experimental approaches such as those described for weedy rice and the other examples given above can help distinguish among the alternatives. Acknowledgements This study was supported by the Chinese Ministry of Science and Technology (Grants. 2011CB100401) and the Nature Science Foundation of China (grants 30871503 and 30730066), as well as a John Simon Guggenheim Memorial Fellowship and a United States National Science Foundation OPUS Grant awarded to N.C.E. (DEB 1020799). We thank Prof. H. S. 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