Annals of Botany 119: 81–93, 2017 doi:10.1093/aob/mcw221, available online at www.aob.oxfordjournals.org
Do floral and niche shifts favour the establishment and persistence of newly arisen polyploids? A case study in an Alpine primrose
Gabriele Casazza1,*, Florian C. Boucher2,3, Luigi Minuto1, Christophe F. Randin4 and Elena Conti2 1DISTAV, University of Genoa, Corso Europa 26, I-16132 Genoa, Italy, 2Department of Systematic and Evolutionary Botany and Botanic Garden, University of Zurich, Zollikerstrasse 107, CH-8008 Zurich, Switzerland, 3Department of Botany and Zoology, University of Stellenbosch, Private Bag X1, Matieland 7602, South Africa and 4Department of Ecology & Evolution, Downloaded from https://academic.oup.com/aob/article-abstract/119/1/81/2730686 by guest on 23 March 2020 University of Lausanne, Biophore, CH-1015 Lausanne, Switzerland *For correspondence. E-mail [email protected]
Received: 18 May 2016 Returned for revision: 29 July 2016 Editorial decision: 10 September 2016 Published electronically: 26 December 2016
Background and Aims Polyploidization plays a key role in plant evolution. Despite the generally accepted ‘mi- nority-cytotype exclusion’ theory, the specific mechanisms leading to successful establishment and persistence of new polyploids remain controversial. The majority of newly formed polyploids do not become established, because they are less common, have fewer potential mates or may not be able to compete successfully with co-occurring progenitors at lower ploidy levels. Changes in floral traits and ecological niches have been proposed as important mechanisms to overcome this initial frequency-dependent disadvantage. The aim of this study was to determine whether dodecaploids of the heterostylous P. marginata differ from their hexaploid progenitors in P. marginata and P. allionii for selected floral traits and ecological preferences that might be involved in establishment and persist- ence, providing a possible explanation for the origin of polyploidized populations. Methods Floral morphological traits and ecological niche preferences among dodecaploids and their hexaploid progenitors in P. marginata and P. allionii, all restricted to the south-western Alps, were quantified and compared Key Results Differences in floral traits were detected between dodecaploids and their closest relatives, but such differences might be too weak to counter the strength of minority cytotype disadvantage and are unlikely to enable the coexistence of different cytotypes. Furthermore, the results suggest the preservation of full distyly and no transi- tion to selfing in dodecaploids. Finally, dodecaploids occur almost exclusively in environments that are predicted to be suitable also for their closest hexaploid relatives. Conclusions In light of the results, P. marginata dodecaploids have probably been able to establish and persist by occupying geographical areas not yet filled by their closest relatives without significant evolution in their climatic and pollination niches. Dispersal limitation and minority-cytotype exclusion probably maintain their current range disjunct from those of its close relatives.
Key words: Ecological niche, minority-cytotype exclusion, pollinator shift, polyploidy, Primula allionii Loisel, Primula marginata Curtis.
INTRODUCTION (Felber-Girard et al.,1996; Lumaret et al., 1987; Husband, 2000; Ramsey and Schemske, 2002). Polyploidy has long been recognized as an important mechan- Given the rare occurrence of unreduced gamete formation ism of genome evolution and as playing a key role in plant spe- (Bretagnolle and Thompson, 1995; Ramsey and Schemske, ciation (Grant, 1981), usually causing immediate reproductive 1998, 2002), which represents the first step in the most common isolation between plants of the original ploidy and the newly mode of polyploidization, newly formed polyploids usually arisen polyploids (e.g. triploid block between diploids and tetra- occur at low frequency in sympatry with their closest relatives, ploids; Husband and Sabara, 2004). However, reproductive iso- hence they may suffer a competitive disadvantage. Most neopo- lation is not sufficient for polyploid speciation (Sobel et al., lyploids are thus expected to disappear quickly (Soltis et al., 2010). In order to become established, newly formed polyploids 2010; Arrigo and Baker, 2012). The ‘minority-cytotype exclu- must increase their fitness in comparison with their progenitors sion’ (MCE) principle (Levin, 1975) states that the proportion either through selfing or through increasing the probability of of the rarer cytotype decreases rapidly as a result of frequency- mating with individuals at the same ploidy level (rather than dependent selection. Indeed, the rarer cytotype is at a reproduct- with individuals of different ploidy) via shifting floral traits or ive disadvantage, because there is a greater likelihood that its blooming period. Additionally, they can also increase their fit- pollen will be transferred to the more frequent cytotype, thus ness through occupying an ecological niche and/or a geographic preventing the formation of fertile seeds. The reproductive space that differ from those of their relatives at lower ploidy, handicap in a given generation leads to a greater handicap in thereby decreasing competition with them. Therefore, the evo- the next, rapidly eliminating the minority cytotype from the lutionary success of polyploids depends on a series of complex population (Levin, 1975; Burton and Husband, 2000; Husband, reproductive, morphological, ecological and geographic factors 2000). Because newly formed polyploids occur in sympatry
VC The Author 2016. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: [email protected] 82 Casazza et al. — Roles of floral and niche shifts in polyploid establishment and persistence with at least one progenitor, changes in plant morphology, with female (stigmas) and male (anthers) sexual organs placed phenology, physiology and/or ecological preferences are reciprocally in long-styled (L) and short-styled (S) plants deemed necessary to overcome the initial frequency disadvan- (Richards, 2003), a system designed to prevent self-fertilization tage that they inevitably experience and allow their establish- and known as heterostyly (Webb and Lloyd, 1986; Wedderburn ment and persistence (Levin, 2002). There might thus exist and Richards, 1992). In Primula, polyploidization is often asso- strong selective pressures for neopolyploids either to differ in ciated with a shift from self-incompatible heterostyles, charac- their floral traits (thus decreasing the frequency of mating with terized by pronounced anther–stigma separation within each their diploid ancestors), to exploit new niches or to better cope floral morph, to self-compatible homostyles, characterized by with fluctuating environments, in order to outcompete or avoid greatly reduced or no anther–stigma separation in the single flo- competition with their closest relatives (Petit et al.,1999; ral morph. The described shift should favour the establishment Baack, 2004; Leitch and Leitch, 2008; te Beest et al., 2012). of the newly formed polyploids (Wedderburn and Richards, Downloaded from https://academic.oup.com/aob/article-abstract/119/1/81/2730686 by guest on 23 March 2020 Polyploidization is known to alter floral traits (Otto and 1992; Guggisberg et al., 2006). Whitton, 2000; Robertson et al.,2010; Balao et al., 2011), often Primula marginata, described as heterostylous by Richards causing a shift in pollinator preferences (Muchhala and Potts, (2003), comprises populations at either the hexaploid (2n ¼ 6x 2007; Whittall and Hodges, 2007; Blu¨thgen et al.,2008; ¼ 62, 66; hereafter Pm6x) or dodecaploid (2n ¼ 12x ¼ 120– Waterman et al.,2011; Gomez et al.,2014). For example, poly- 128; hereafter Pm12x) levels. No populations with mixed P. ploidization may trigger an increase in organ size, which might marginata cytotypes (Pm12x and Pm6x) have been identified affect floral traits involved in pollinator attraction (e.g. corolla (Casazza et al.,2012), while two mixed populations of Pm12x width; Balao et al.,2011; Tunbridge et al.,2011), flower– and P. allionii individuals (hereafter Pa6x)areknown(Casazza pollinator interactions during nectar probing (e.g. corolla tube et al.,2013a). Hexaploid and dodecaploid populations of length and width; Segraves and Thompson, 1999; Balao et al., P. marginata are geographically differentiated (Fig. 1), with 2011; Tunbridge et al., 2011) or pollen transfer (e.g. positions Pm6x individuals occurring in the western (i.e. from the Cottian of male and female sexual organs in the floral tube; Keller to the south-western Maritime Alps) and Pm12x individuals in et al.,2012, 2014). Shifts of floral traits between diploids and the eastern part of the species’ range (i.e. from the south- polyploids may play an important role in promoting assortative eastern Maritime and Ligurian Alps to the Apennines; Casazza mating within the same cytotype, possibly leading to preferen- et al., 2013b). Recently, Pm12x individuals have been proposed tial pollen transfer among minority-cytotype individuals, des- to be allopolyploids originating from hybridization between pite their lower frequency in a population (Segraves and Pm6x and Pa6x lineages (Casazza et al., 2012). Thompson, 1999; Husband and Sabara, 2004; Kennedy et al., The present study is aimed at characterizing for the first time 2006). selected floral traits in both floral morphs and climatic prefer- Furthermore, polyploids frequently occupy different portions ences that might explain the establishment and persistence of of environmental space and may expand or shift their geograph- polyploidized populations in heterostylous species, focusing on ical ranges relative to the progenitors, sometimes in connection the Alpine P. marginata–P. allionii system. More specifically, with cycles of habitat and range fragmentation driven by cli- we are asking the following questions. (1) Do Pm12x individ- matic oscillations (Stebbins, 1984; Guggisberg et al.,2006, uals differ from Pm6x and Pa6x individuals in floral traits that 2009; Spooner et al.,2010; Fawcett and Van de Peer, 2010; te are involved in pollinator attraction and handling, potentially Beest et al.,2012; Theodoridis et al., 2013). The spatial segre- promoting assortative mating between individuals of the same gation of cytotypes via differentiation of their environmental cytotype? (2) Are Pm12x individuals characterized by de- niches is thought to be one of the primary mechanisms involved creases in floral display traits and anther–stigma distance that in alleviating MCE (Fowler and Levin, 1984; Thompson and might indicate a switch to self-fertilization? (3) Do Pm12x Lumaret, 1992), and it has been suggested as a ‘prerequisite’ populations deviate from full distyly? (4) Do the ecological for polyploid speciation (Levin, 2003). preferences of Pm12x populations differ significantly from While evidence is growing that different mechanisms can those of its progenitors and, if so, could such differences ex- lead to the establishment and persistence of new polyploids, the plain geographic segregation among related lineages at differ- consistency and generality of the proposed mechanisms are still ent ploidy levels? (5) Do Pm12x populations have broader debated (Soltis et al.,2010; Glennon et al., 2014). Therefore, ecological tolerances that would allow them to occupy a analyses of floral and ecological differences between cytotypes broader geographical range than those of their hexaploid rela- at different ploidy levels within and between closely related tives? The results of the study should allow us to better under- species are needed to better understand the mechanisms of stand the mechanisms of polyploid establishment and polyploid evolution. Here, we focus on two species of prim- persistence. roses, Primula marginata Curtis and P. allionii Loisel., which are restricted to the south-western Alps and are closely related, being included in the monophyletic Primula sect. Auricula MATERIALS AND METHODS (Mast ,2001, 2006; Zhang , 2004; Casazza , et al. et al. et al. Study system 2012). This ancestrally hexaploid section comprises species with genomes that are likely to be diploidized (Boucher et al., Individuals of Pa6x form dense evergreen cushions bearing sin- 2016b). Furthermore, allopatric speciation with little niche di- gle pink flowers with a white eye. The species is the only vergence, hence largely non-adaptive, appears to have been the known primrose characterized by petals with a bright surface, most common mode of speciation in this section (Boucher similarly to other plants pollinated by bumble-bees. Individuals et al.,2016b). All species in the section have dimorphic flowers of this species bloom from February to April and are pollinated Casazza et al. — Roles of floral and niche shifts in polyploid establishment and persistence 83 Downloaded from https://academic.oup.com/aob/article-abstract/119/1/81/2730686 by guest on 23 March 2020
02550 Kilometres
FIG. 1. Distributional ranges of P. marginata (continuous black line) and P. allionii (white-striped areas). Cytotype distribution of P. marginata according to Kress (1969) and Casazza et al.(2012): white circles, P. marginata hexaploids; white triangles, P. marginata dodecaploids. only by Hymenoptera (Minuto et al., 2014). Individuals of ability of flowers to self-pollinate, we quantified the degree of Pm6x and Pm12x form loose cushions, with stems usually bear- anther–stigma separation (i.e. herkogamy) for each flower as ing from five to ten flowers from lilac to pinkish with a mealy the difference between the distance from the middle of anthers eye-ring. Individuals of Pm6x and Pm12x do not show obvious (anther position: AP) to the tip of the stigma (stigma position: morphological differences and they bloom from March to June SP). To check for potential deviations from full distyly in (Richards, 2003). While no study on pollinators of Pm6x and dodecaploid populations of P. marginata, we also calculated Pm12x individuals has been published, according to our per- the sexual reciprocity index of S anchez et al. (2008) in the three sonal observations both taxa are pollinated by Hymenoptera target taxa at different ploidy levels. This index, which we label and Lepidotera. R, compares stigma–stamen height differences for all potential crosses (i.e. stamen position of each morph vs. stigma position of each complementary morph across a population), while ac- Floral traits and sexual organ position counting for the standard deviation of these measurements. This index has the advantage of not being skewed by the most We collected flowers from nine hexaploid and eight dodeca- frequent sex organ (stamens) and can be interpreted as a meas- ploid populations of P. marginata and two populations of the ure of the average population-level deviation from perfect reci- hexaploid P. allionii (Supplementary Data Table S1;seealso procity (R ¼ 0); distyly is usually characterized by R < 0 05. In Casazza et al.,2012, 2013a). The number of analysed flowers the few distylous species of Primula in which R was calculated, was 121 (47 from the L-morph and 74 from the S-morph) for it ranges from 0 007 to 0 020 (Keller et al., 2012, 2014, 2016; Pm6x, 164 (80 L-morph and 84 S-morph) for Pm12x and 100 Aronne et al.,2014). We used an analysis of similarity (48L-morph and 52S-morph) for Pa6x (see Supplementary (ANOSIM; Clarke, 1993) to test the degree and the significance Data Tables S1 and S2 for further details). of differences between floral traits of Pm12x individuals and All flowers were longitudinally dissected and analysed under those of their relatives in all measured floral traits (excluding a Leica M205 C stereomicroscope equipped with a Leica EC3 NF) of each morph. ANOSIM is a non-parametric test that gen- digital colour camera; selected traits were measured using the erates a statistic comparing dissimilarities between and within LAS EZ imaging software (Leica). We focused on floral traits groups and usually ranging from 0 to 1, with 0 indicating com- involved in pollinator attraction and flower–pollinator inter- pletely random grouping and no difference between groups. action (see de Vos et al.,2014). We analysed two traits impli- Negative values may occur, indicating a greater dissimilarity cated in pollinator attraction: flower number and size. among individuals within than between groups. ANOSIM was Specifically, we measured the total number of blooming flow- preferred to parametric tests (e.g. MANOVA) because our data ers per scape during the whole flowering period (NF) (recorded contained a few strong outliers that might have affected the per- only in Pm12x and Pm6x individuals on 94 and 115 scapes, re- formance of the latter. Euclidean distances were used in all spectively, because Pa6x individuals usually bear a single comparisons, and the significance of the statistic was calculated flower per scape; Richards, 2003) and petal lobe length (LL) as with 9999 random permutations. In order to visualize the pat- a proxy for flower size. We analysed two traits implicated in tern of morphological variation among cytotypes, a principal flower–pollinator interaction (Fig. 2): corolla tube length (TL) component analysis (PCA) was carried out. To test whether the and corolla mouth diameter (MD). To assess the potential two morphs of Pm12x individuals differed significantly from 84 Casazza et al. — Roles of floral and niche shifts in polyploid establishment and persistence
Niche differentiation in environmental space Values of environmental variables were extracted for all MD LL MD LL localities of Pm12x, Pm6x and Pa6x populations. We used a non-parametric Kruskal–Wallis test to assess differences be- tween Pm12x and their closest relatives along each climatic TL SP TL AP variable independently. Kernel density plots were also used to visualize the distribution of each variable. AP SP A detectable niche shift between two or more species may be either the result of niche differentiation in environmental space (E-space) that translates into the occupation of different geo- L-morph S-morph graphic space (G-space) or a consequence of differences in the Downloaded from https://academic.oup.com/aob/article-abstract/119/1/81/2730686 by guest on 23 March 2020 FIG. 2. Floral traits measured in individuals of P. marginata (hexaploids and availability of the environment in G-space with no niche differ- dodecaploids) and P. allionii (hexaploid species). Diagrams of long-styled (L) entiation in E-space (Soberon, 2007; Broennimann et al., 2012; and short-styled (S) morphs. Sexual organ traits: AP, anther position; SP, stigma position. Corolla traits: TL, corolla tube length; MD, corolla mouth diameter; Theodoridis et al., 2013). To test for differentiation in E-space, LL, petal limb length. a PCA-based method recently developed by Broennimann et al. (2012) was used to quantify niche breadth and niche overlap of Pm12x, Pm6x and Pa6x populations. Overlap was measured by the respective morphs of their relatives in all measured floral Schoener’s D index, which ranges from 0 (no overlap) to 1 (full traits, we used a Kruskal–Wallis test in the R statistical soft- overlap; Schoener, 1970). All occurrences were then pooled ware v 3.0.1 (R Development Core Team, 2012). and randomly split into two data sets 100 times, maintaining the number of occurrences as in the original data sets to com- pare the niche overlap statistic D with a random distribution. Occurrence data and climatic variables To test whether ecological niches of the taxa being compared are more similar than expected at random from their geograph- Locality data were extracted from Faverger (1965), Kress ical ranges, we use niche similarity tests (Warren et al.,2010). (1969, 1989)andCasazza et al. (2012) for hexaploid and Niche similarity tests compare the set of environmental condi- dodecaploid populations of P. marginata and from Casazza tions occupied by different taxa via taking into account the et al. (2013a) for P. allionii. The final georeferenced data set background environmental conditions that are available in the consisted of all populations of P. marginata for which the geographic area occupied by each taxon. Briefly, the observed ploidy level is known (19 for Pm6x and17forPm12x) and all climatic niche overlap between two species was compared with known populations of P. allionii (54 for Pa6x). the overlap measured between one of the species’ niche and the Climatic variables related to temperature and precipitation randomized niche of the other species. This randomized niche are thought to be important for physiological limitations and was obtained by randomly sampling occurrence points in the re- adaptation in Alpine plants (Ko¨rner, 2003). We thus down- gion where the species occurs (the ‘background area’). We re- loaded 19 bioclimatic variables from the WorldClim database peated this randomization procedure 100 times. We created (http://www.worldclim.org) at a 30 s (i.e. about 1 1km)spa- different background areas following two different approaches. tial resolution (Hijmans et al.,2005) for the south-western A first approach used the outputs of the SDMs for Pm12x, Alps. Because (micro-)topographic features can strongly affect Pm6x and Pa6x separately (see below). Probabilities of pres- climate in Alpine habitats (Ko¨rner, 2003) and because the reso- ences from SDMs were converted into binary predictions using lution of the environmental layer used in modelling may affect a baseline threshold based on the point in the receiver operating model prediction (Guisan et al.,2007; Randin et al.,2009; characteristic (ROC) curve where the sum of sensitivity and Kirchheimer et al.,2016), we employed a finer spatial reso- specificity is maximized (Manel et al., 2001; Liu et al., 2005), lution. The coarse-scale (1 km resolution) climatic grids were and background maps of predicted presences were generated. statistically down-scaled to 100 m resolution, following the ap- In addition, we also used a geographic background obtained proach detailed in Zimmermann et al. (2007).Toreducemulti- from combining the SDMs of all three taxa (common back- collinearity between predictors and minimize model overfitting, ground). A second approach used 10, 20 and 30 km buffer we measured pairwise Pearson’s correlation coefficients among zones around the occurrence points of each species (Warren values of all climatic predictors extracted by occurrences points et al.,2010). and retained a set of predictors with a high relative contribu- Niche breadth in E-space of each species was assessed by tion to the species distribution models (SDMs), but not highly randomly sampling 100 pixels in the niches of Pm12x, Pm6x correlated with each other (r j0.70j;seerecommendation and Pa6x populations (i.e. pixels were sampled according to the from Elith et al., 2006). The five variables used in further ana- density of the species occurrences), extracting their scores along lyses were: Mean Diurnal Range of Temperature (BIO2); the two PCA axes, and calculating the standard deviation of the Temperature Seasonality (BIO4); Mean Temperature of scores along each axis (Broennimann et al., 2012). This proced- Wettest Quarter (BIO8), Mean Temperature of Driest Quarter ure was repeated 1000 times and the distribution of the breadth (BIO9), Precipitation Seasonality (BIO15); Precipitation of values was compared using boxplots for each PCA axis. Warmest Quarter (BIO 18); and Precipitation of Coldest All analyses of niche overlap, niche similarity and niche Quarter (BIO19). breadth were performed in R using the set of functions provided Casazza et al. — Roles of floral and niche shifts in polyploid establishment and persistence 85 in Broennimann et al. (2012) and implemented in the ‘ecospat’ morph) and from 8 93 mm for Pm12x to 8 24 for Pm6x and package (Broennimann et al.,2015). 8 50 for Pa6x (S-morph). The corolla tubes of Pm12x individ- uals were significantly longer and more herkogamous than those of their closest relatives for both floral morphs (Fig. 3C, Niche differentiation in geographical space E; Supplementary Data Table S3), while corolla mouth diam- Occurrence data and bioclimatic variables were used for the eter of Pm12x individuals was intermediate between those of construction of SDMs for each taxon with Maxent 3.3.3e Pa6x and Pm6x for both morphs (Fig. 3D; Table S3). Values of (Phillips et al., 2006). Maxent is a very flexible modelling algo- the sexual reciprocity index were always lower than 0 05, indi- rithm widely used because of its high predictive performance cating distyly, and did not differ significantly among Pm12x (Elith et al., 2006) even with low sample sizes (down to as few (0 0424), Pa6x (0 0434) and Pm6x (0 0433). To summarize, as ten points; Hernandez et al., 2006; Pearson et al.,2007). We Pm12x individuals differed only weakly from their closest rela- Downloaded from https://academic.oup.com/aob/article-abstract/119/1/81/2730686 by guest on 23 March 2020 used 70 % of the occurrence records for each taxon to calibrate tives in pollinator attraction traits, but differed more markedly the model and 30 % to test it, as recommended (Phillips et al., in traits involved in flower–pollinator interaction and were 2006). All other parameters were set to default. Model perform- characterized by a significantly higher degree of herkogamy. ance was assessed using the area under the ROC curve (AUC; Hanley and McNeil, 1982). The niches of different taxa were also compared in G-space Niche differentiation in environmental space using a niche similarity test implemented in ENMtools (Warren et al.,2010). This test compares the observed overlap (again, When comparing the climatic preferences of Pm12x and measured using Schoener’s D) between niches of Pm12x popu- Pm6x, the values of both Mean Diurnal Range of Temperature lations and their relatives with a distribution of simulated over- (BIO2) and Precipitation of Warmest Quarter (BIO18) differed lap values generated by comparing the SDM of one species significantly, while only the values of BIO2 were significantly with an SDM created from random points drawn from the geo- different between Pm12x and Pa6x (Table 1). In particular, ker- graphical background area of the other species (Warren et al., nel density plots (Supplementary Data Fig. S2) showed that the 2008). Significant results suggest that the ecological niches of niche of Pm12x populations was characterized by lower values sister species are either more different or more similar than ex- of Mean Diurnal Range (BIO2) and by a wider range of values pected, and for this reason this test is treated as a two-tailed for Precipitation of the Warmest Quarter (BIO18) than those of test. The software ENMtools was used to perform niche simi- both its closest relatives. In general, Pm12x populations seemed larity tests in G-space using the same backgrounds as previ- to occur under slightly more oceanic conditions than their clos- ously presented for E-space. Even if this approach is common, est relatives. the results of niche differentiation tests in G-space should be The PCA (Fig. 4A, B) identified two components that collect- handled with caution, because measured differences between ively explained 66 85 % of the total climatic variation between niches could represent differences in the environmental charac- the cytotypes (PC1 ¼ 43 1%;PC2¼ 23 8 %) and 68 1%of teristics of the study area rather than real differences between the total variation between Pm12x and Pa6x populations species (Broennimann et al.,2012). (PC1 ¼ 41 5%;PC2¼ 26 7 %). Three variables [Mean Niche breadth in G-space was estimated by applying the in- Temperature of Wettest Quarter (BIO8), Mean Temperature of verse concentration metric of Levins (1968) as implemented in Driest Quarter (BIO9) and BIO18] showed almost equal contri- ENMTools to the resulting sets of suitability scores, standar- butions to both axes, two variables [BIO2 and Precipitation dized so that minimum possible niche breadth within this space Seasonality (BIO15)] were more strongly associated with PC1, is 0 (indicating that only one grid cell in the geographical space while Temperature Seasonality (BIO4) and Precipitation of has a non-zero suitability) and maximum niche breadth is 1 Coldest Quarter (BIO19) were more strongly associated with (where all grid cells are equally suitable). PC2. The niche of Pm12x largely overlapped with the niches of its closest relatives in E-space (Fig. 4C, D). The niche of Pm6x RESULTS was slightly wider than that of Pm12x and was characterized by a wider diurnal and seasonal range of temperature (BIO2 and Floral traits and sexual organ positions BIO4). In contrast, the niche of Pa6x was narrower than that of Multivariate analysis of floral traits using ANOSIM (Pm12x vs. Pm12x populations (Fig. 4D). These results were in accordance Pm6x: R ¼ 0 29, P < 0 01; Pm12x vs. Pa6x: R ¼ 0 30, P < with univariate analyses obtained with the Kruskal–Wallis test. 0 01) and PCA (Supplementary Data Fig. S1)suggestedthat Niche breadth of Pm12x populations along PC1 was equal to Pm12x individuals are weakly but significantly different from that of Pm6x populations, while Pa6x populations had a weakly their closest relatives. Considering all traits separately, individ- narrower niche breadth than Pm12x populations (Fig. 5A). uals of Pm12x did not differ significantly from their closest Along PC2, the niche breadth of Pm12x populations was nar- relatives in number of flowers per scape for both L- and S- rower than that of Pm6x, but wider than that of Pa6x popula- morphs (Fig. 3A; Table S3). Petal lobes of Pm12x flowers were tions (Fig. 5B). According to the classification scheme provided significantly longer than those of Pm6x and Pa6x for L-morphs, by Ro¨dder and Engler (2011), average niche overlap in andthanthoseofPm6x, but not Pa6x, for S-morphs (Fig. 3B; E-space was very limited between Pm12x and Pa6x populations Table S3), with average petal lobe length ranging from (D ¼ 0 117 in Table 2) and moderate or low between Pm12x 9 85 mm for Pm12x to 8 85 for Pm6x and 8 64 for Pa6x (L- and Pm6x populations (D ¼ 0 534 in Table 2). 86 Casazza et al. — Roles of floral and niche shifts in polyploid establishment and persistence
ABL-morph S-morph L-morph S-morph
16 12 14 10 12 8 10
6 8
4 Downloaded from https://academic.oup.com/aob/article-abstract/119/1/81/2730686 by guest on 23 March 2020 Petal lobe length (mm) 6 Number of flowers per scope 2 4
= = ≠≠ ≠ = Pm6x Pm6x Pm6x Pa6x Pm6x Pa6x Pm12x Pm12x Pm12x Pm12x C L-morph S-morph D L-morph S-morph
6 18
5 16
14 4
12 3 10
Corolla tube length (mm) 2 8 Corolla mouth diameter (mm)
6 1 ≠≠ ≠≠ ≠≠ ≠≠ Pm6x Pa6x Pm6x Pa6x Pm6x Pa6x Pm6x Pa6x Pm12x Pm12x Pm12x Pm12x E L-morph S-morph
Kruskal–Wallis statistical test: 10 = P ≥ 0·05; ≠ P < 0·05 Pm12x: P. marginata dodecaploids Pm6x: P. marginata hexaploids Pa6x : P. allionii hexaploids 8
6
4 Stigma–anther distance (mm)
≠≠ ≠≠ Pm6x Pa6x Pm6x Pa6x Pm12x Pm12x
FIG. 3. Boxplots of measurements of number of flowers per scape (A), petal limb length (B), corolla tube length (C), corolla mouth diameter (D) and stigma–anther distance (E). Data are reported for long-styled (L-morph) and short-styled (S-morph) morphs separately. Pm6x, P. marginata hexaploids; Pm12x, P. marginata dodecaploids; Pa6x, P. allionii. Results of Kruskal–Wallis tests for statistical differences are reported: a lack of significant difference (P 0 05) is indicated by ¼, while significant differences (P < 0 05) are indicated by 6¼.
Niche differentiation in geographical space 0 972, 0 944 and 0 995 in Pm12x, Pm6x and Pa6x populations, respectively). Niche breadth analyses based on the inverse The AUC values of models produced by Maxent and inter- concentration metric revealed that Pm12x populations have preted using the classification of Araujo et al. (2005) indicated roughly the same niche width as Pm6x populations (0 708 vs. excellent model performance (cross-validated AUC values of 0 675), but a larger niche than Pa6x (0 708 vs. 0 177). Casazza et al. — Roles of floral and niche shifts in polyploid establishment and persistence 87
TABLE 1. Results of Kruskal–Wallis statistical tests of differentiation for each climatic variable
Climatic variable Kruskal–Wallis Kruskal–Wallis Pm6x vs. Pm12x Pa6x vs. Pm12x
BIO2: Mean Diurnal Range 7 278** 7 037** BIO4: Temperature Seasonality 0 308n.s. 1 148n.s. BIO8: Mean Temperature of Wettest Quarter 0 603n.s. 0 744n.s. BIO9: Mean Temperature of Driest Quarter 0 145n.s. 0 445n.s. BIO15: Precipitation Seasonality 0 798n.s. 1 614n.s. BIO18: Precipitation of Warmest Quarter 4 784* 0 291n.s. BIO19: Precipitation of Coldest Quarter 0 84n.s. 2 099n.s.
The asterisks indicate a significant difference: **P 0 01; *P 0 05; n.s.P 0 05. Downloaded from https://academic.oup.com/aob/article-abstract/119/1/81/2730686 by guest on 23 March 2020 Pm6x, P. marginata hexaploids; Pm12x, P. marginata dodecaploids; Pa6x, P. allionii.
ABPm12x vs. Pm6x Pm12x vs. Pa6x bio4 bio4
bio8 bio18 bio8 bio18 bio2 bio2 bio15
bio15
bio9 bio9
bio19 bio19
CD6
4 4
2 2
0
PC2 (23·78 %) PC2 (26·69 %) 0
–2 –2
–4 –2 0 24 –2 0 24 PC1 (43·07 %) PC1 (41·52 %)
FIG. 4. Niches of the dodecaploids of P. marginata (Pm12x), hexaploids of P. marginata (Pm6x)andP. allionii (Pa6x) in climatic space. (A and B) The contribution of climatic variables on the two PCA axes and the percentage of variation explained by these axes in Pm12x vs. Pm6x (A) and Pm12x vs. Pa6x (B), respectively. (C and D) The niche of two pairs of taxa, Pm12x (blue) vs. Pm6x (red) and Pm12x (blue) vs. Pa6x (green), respectively. The solid and dashed lines represent 100 and 50 % of the entire available environmental space (background), respectively. Colour shadings illustrate the density of the occurrences of Pm6x, Pm12x and Pa6x in each climatic cell. Notice the general overlap between niches but also the differences in mean niche position (areas with more intense colour), which result in moder- ate overlap measures. 88 Casazza et al. — Roles of floral and niche shifts in polyploid establishment and persistence
A PC1 B PC2 floral traits of Pm12x individuals significantly exceed the mean values of their closest relatives in three (i.e. petal lobe length, 2·0 2·2 corolla tube length, stigma–anther distance) out of five cases, while the remaining two traits are equal or intermediate be- 2·0 tween those of their closest relatives (i.e. number of flowers per 1·8 1·8 scape and corolla mouth diameter, respectively; Fig. 3). These results are counter to the current lack of taxonomic distinction 1·6 between dodecaploids and hexaploids of P. marginata. 1·6 The comparisons of floral traits between the dodecaploids of 1·4 P. marginata and their two hexaploid relatives showed that Pm12x individuals have slightly larger petal lobes in three out 1·4 1·2 Downloaded from https://academic.oup.com/aob/article-abstract/119/1/81/2730686 by guest on 23 March 2020 of four comparisons (Fig. 3B), but do not differ in the number
Pm6x Pm12x Pa6x Pm6x Pm12x Pa6x of flowers per scape (Fig. 3A). Are such small differences in floral size likely to affect pollinator attraction? It has been pro- FIG. 5. Boxplots showing the distribution of niche breadth values of dodeca- posed that changes in floral traits induced by polyploidy may ploids and their progenitors along the two PCA axes. Niche breadth was as- promote a decrease in the frequency of interploidy pollination, sessed by randomly sampling 100 pixels in the niche of each taxa, extracting their scores along the two PCA axes and calculating the standard deviation of thus allowing for the establishment of newly formed polyploids the scores along the two PCA axes. This procedure was repeated 1000 times. (Husband and Sabara, 2004; Kennedy et al.,2006; Godsoe Pm6x, P. marginata hexaploids; Pm12x, P. marginata dodecaploids; Pa6x, et al.,2013). A few studies have provided evidence supporting P. allionii. the above prediction (Husband and Schemske, 2000; Kennedy et al.,2006). However, traits related to pollinator attraction are not universally larger in polyploids than in diploids, and differ- Schoener’s D was higher for the pair Pm12x vs. Pm6x popula- ences in floral size may be important for polyploid establish- tions (0 797 in Table 2) and lower for the pair Pm12x vs. Pa6x ment only under certain conditions, i.e. when pollinators are populations (0 451 in Table 2). choosy, scarce or unpredictable (Vamosi et al., 2007). Additionally, the importance of floral display in insect attrac- tion is affected by phenology and vegetation context (Sletvold Niche similarity tests in E- and G-space et al., 2013). In our case, competition for pollinators is low dur- ing the partially overlapping flowering periods of the three Consistent with the observed overlap between Pm12x and its studied primroses (Richards, 2003), because few other species closest relatives, tests of niche similarity in E-space suggested flower simultaneously in the same plant communities (Minuto that the ecological niches are more similar than expected given et al., 2014). As a result, small differences in visual display be- their environmental backgrounds (Table 2). Results of back- tween taxa may be relatively unimportant for early-blooming ground similarity tests in G-space were partially congruent with taxa like the primroses investigated here. results in E-space, showing that the niches of Pm12x and Pm6x In contrast to pollinator attraction, we detected greater differ- are more similar than expected by chance (Table 2). Results of ences between dodecaploids of P. marginata and their progeni- background similarity tests between Pm12x and Pa6x popula- tors for traits related to flower–pollinator interaction. Increment tions showed that the niche of Pm12x was less similar to the in length and reduction in mouth width of the floral tube have niche of Pa6x than expected by chance, while the niche of been observed to reduce the number of species of pollinator in Pa6x was more similar to the niche of Pm12x (Table 2). To flowers pollinated by bumble-bees (Suzuki et al., 2007)and summarize, the niche of Pm12x is usually significantly more hawkmoths (More´ et al., 2007). Flowers of Pm12x have corolla similar to the niche of Pm6x and it is significantly different tubes that are longer, but corolla mouth diameters that are inter- from the niche of Pa6x in G-space only. mediate to those of the progenitors (Fig. 3C, D). The dimen- sions of floral tubes suggest that Pm12x flowers may be DISCUSSION pollinated by smaller than or the same range of pollinators as Pa6x and Pm6x. In fact, the wider and shorter tubes of Pa6x in- Do dodecaploids of P. marginata differ from their progenitors dividuals allow them to be pollinated by a large number of pol- in floral traits? linators irrespective of proboscis length (Minuto et al.,2014). In general, autopolyploidy is expected to generate pheno- The longer tubes of Pm12x plants may hinder the advantage of types that are extreme or novel relative to parental lines (Levin, having tubes that are wider than those of Pm6x individuals, 1983), whereas allopolyploidy is expected to generate pheno- allowing Pm12x flowers to be pollinated by the same or a sub- types that are either intermediate between the parents set of the pollinators that visit Pm6x flowers (Fig. 3C, D). (Thompson, 1991; Johnston et al.,2001; Schranz and Osborn, Considered together, our results suggest that the differences be- 2004), similar to one or the other parent (Abbot and Lowe, tween Pm12x individuals and their progenitors in floral traits 2004) or a mosaic of parental traits (Ramsey and Schemske, that are known to affect pollinator attraction and flower–pollin- 2002; Schranz and Osborn, 2004; Murren and Pigliucci, 2005). ator interaction may not promote preferential mating among In our system, both the ANOSIM and the PCA suggest that, Pm12x individuals through a shift in their pollinator prefer- even if some differences occur in single traits, Pm12x individ- ences. In conclusion, while we have detected differences in flo- uals do not markedly differ from their closest relatives when ral traits between Pm12x individuals and their closest relatives, considering all floral traits together (Fig. S1). In particular, the these differences might be too weak to counter the strength of Casazza et al. — Roles of floral and niche shifts in polyploid establishment and persistence 89
TABLE 2. Results of niche similarity tests in environmental and geographical spaces among dodecaploids of P. marginata and their progenitors
Background
Test Comparison Niche overlap D SDM CB SDM 10 km buffer 20 km buffer 30 km buffer
Environmental space Pm12x Pm6x 0 269 More* More* More* More* More* Pm6x Pm12x More* More* More* More* More* Pm12x Pa6x 0 193 More* n.s. More* More* More* Pa6x Pm12x More* More* More* More* More* Geographical space Pm12x Pm6x 0 797 More** More** More** More** More** Pm6 Pm12x More** n.s. More** More** More** Pm12x Pa6x 0 451 Less** Less** Less** Less** Less** Downloaded from https://academic.oup.com/aob/article-abstract/119/1/81/2730686 by guest on 23 March 2020 Pa6x Pm12x n.s. More* n.s. More** More**
Backgrounds are defined by each taxon’s ecological niche model set to a baseline threshold that maximizes the sum of sensitivity and specificity of the test data (SDM), by the combination of SDM of each taxa (SDM CB), and by applying 10, 20 and 30 km buffer zones around the occurrence points of each species. Significant results are indicated by ‘less’ for significant divergence or ‘more’ for significant similarity between test and comparison cytotypes. **P 0 01; *P 0 05; n.s. P 0 05. Pm6x, P. marginata hexaploids; Pm12x, P. marginata dodecaploids; Pa6x, P. allionii. minority-cytotype disadvantage and are unlikely to enable the of active debate. Divergence of habitat preferences between coexistence of different cytotypes. Additional field studies will related diploid and polyploid taxa was detected in several stud- be required to test explicitly whether any differences in pollin- ies (Parisod et al.,2010; Glennon et al.,2012; Manzaneda ator assemblages and fidelity exist between Pm12x individuals et al.,2012; McIntyre, 2012; Kol a r et al.,2013; Sonnleitner and their closest relatives. et al.,2016), including in Primula sect. Aleuritia (Theodoridis Polyploidization may be associated with a shift to self- et al., 2013). Conversely, other studies, including reciprocal fertilization (Barringer, 2007). While polyploidization is often transplantation experiments, reported no niche differentiation associated with increases in organ size, as discussed above, self- between polyploids and their related diploids (Baack and ing renders investment in pollinator attraction unnecessary, and Stanton, 2005; Godsoe et al., 2013; Glennon et al., 2014; hence is often associated with smaller flowers (Vamosi et al., Lopez-Alvarez et al.,2015). Therefore, other mechanisms, 2007; Barringer and Geber, 2008). In Primula, polyploidization including limited seed and pollen dispersal (Baack, 2005)and is usually concomitant with a shift from distyly to homostyly the capability of polyploids to colonize recently deglaciated and from self-incompatibility to self-compatibility, concur- areas (Stebbins, 1984; Brochmann et al.,2004; Godsoe et al., rently with a marked reduction of anther–stigma distance 2013; Theodoridis et al., 2013), have been invoked to explain (Richards, 2003; Guggisberg et al.,2006; de Vos et al., 2014). the establishment of polyploids. In fact, the degree of herkogamy correlates negatively with the In line with these latter studies, our results show that the degree of self-fertility (de Vos et al.,2012) and it may be con- niche occupied by Pm12x populations does not significantly sidered a reliable proxy of selfing rate. We detected an differ from those of their closest relatives (Table 2; Fig. 3), increased herkogamy in Pm12x individuals and virtually no dif- even though, as suggested by the moderate niche overlap val- ferences in levels of distyly between dodecaploid populations ues, the dodecaploids differ in their optimal niche position of P. marginata and their progenitors (Fig 3B, E). Taken to- (indicated by areas with more intense colour in Fig. 4; Table 1), gether, these results suggest preservation of full distyly and no preferring slightly more oceanic conditions than their hexaploid transition to selfing in Pm12x populations. Similarly, distyly is relatives (Table 1; Figs S1 and S2). As suggested by Glennon also preserved in P. clusiana, the only other dodecaploid taxon et al. (2014), this difference in optimal niche positions without of P. section Auricula (Zhang and Kadereit, 2004). The high global niche shift may be the result of limited dispersal and herkogamy and the conservation of heterostyly in Pm12x indi- competition between cytotypes for space. This suggests that viduals corroborates the idea that polyploidization itself does Pm12x populations may have taken advantage of the geograph- not necessarily trigger the switch from heterostyly to homostyly ical space not yet filled by their parents where more oceanic (Shore et al.,2006; Naiki, 2012). Furthermore, the lack of tran- conditions exist without shifting their climatic niche. This inter- sition to selfing is in line with the increase of petal lobe length pretation is congruent with the nearly allopatric range of Pm12x in Pm12x individuals, suggesting that flower size in polyploids populations (Fig. 1) and the previously proposed conclusion may be related to the importance of pollinators for plant repro- that allopatric speciation without ecological divergence is the duction (Vamosi et al., 2007). main mode of speciation in P. sect Auricula (Boucher et al., Do dodecaploids of P. marginata differ from their progeni- 2016a). The lack of niche shift supports the idea that, for some tors in ecological preferences? species, mechanisms other than niche shift (e.g. limited disper- According to theories on polyploid speciation, spatial segre- sal capabilities or biogeographic history) may be responsible gation of new polyploids via differentiation of their environ- for the success of polyploid establishment and persistence. mental niches may allow them to escape MCE and become Finally, theory suggests that newly formed allopolyploids established (Levin, 1975; Fowler and Levin, 1984; Thompson should have broader ecological tolerances than their progenitors and Lumaret, 1992). However, the role of niche shift in poly- through an increase in heterozygosity (Stebbins, 1971; Otto and ploid establishment and persistence continues to be the subject Whitton, 2000; Levin, 2002). However, evidence for the 90 Casazza et al. — Roles of floral and niche shifts in polyploid establishment and persistence presumed broader ecological tolerances of polyploids remains scenario is the most likely explanation for the persistence of controversial, and support for the opposite pattern is accumulat- Pm12x, as proposed for speciation in the entire section Auricula ing (Stebbins and Dawe, 1987; Petit and Thompson, 1999; (Boucher et al., 2016a). Our current results, combined with evi- Martin and Husband, 2009; Theodoridis et al.,2013; Glennon dence from previous molecular phylogenetic analyses (Casazza et al., 2014; Sonnleitner et al., 2016). In the current study, we et al., 2012), suggest that Pm12x individuals originated from found mixed evidence for the two alternatives above, because interspecific hybridization in a glacial refugium shared between the climatic niche of Pm12x was significantly wider than that Pm6x and Pa6x individuals, and later persisted by shifting their of Pa6x, but narrower than that of Pm6x (Fig. 5). range in geographical areas not yet filled by their closest rela- These differences in climatic niche breadth closely match tives without significant evolution of their climatic niche. differences in range size among the three taxa: Pa6x is a narrow Dispersal limitations in the rugged south-western Alps and
endemic restricted to two small areas of the Maritime Alps, MCE probably maintain the current range of Pm12x largely Downloaded from https://academic.oup.com/aob/article-abstract/119/1/81/2730686 by guest on 23 March 2020 Pm12x has an intermediate range extending from the south- allopatric from those of its progenitors. eastern Maritime and Ligurian Alps to the Apennines, and Pm6x has the widest range of the three taxa, since it occurs SUPPLEMENTARY DATA from the Cottian to the south-western Maritime Alps (Casazza et al.,2012; see also Fig. 1). A similar relationship between Supplementary data are available online at www.aob.oxfordjour niche breadth and range size has been observed in other species, nals.org and consist of the following. Table S1: sampling of but the direction of causality remains uncertain (Slatyer et al., dodecaploids and hexaploids of Primula marginata and 2013). On the one hand, having a wider niche allows a species Primula allionii. Table S2: sampling of dodecaploids and hexa- to occupy a larger geographical range than species with a nar- ploids of Primula marginata and Primula allionii for popula- rower niche. On the other hand, species with a small geographic tions, number of individuals, number of scapes, number of range are usually exposed to a narrower range of climatic con- flowers per scape and flower morphology. Table S3: results of ditions than species with a larger range, resulting in a narrower Kruskal–Wallis test used to determine whether each floral trait realized niche (Soberon, 2007; Dullinger et al., 2012). differed significantly between dodecaploids of P. marginata Moreover, other factors, including MCE, dispersal limitation and its closest relatives. Figure S1: PCA of P. marginata hexa- and lineage age, might affect the relationship between niche ploids, P. marginata dodecaploids and P. allionii based on four breadth and range size (Peterson et al.,2011; Theodoridis et al., floral traits. Figure S2: kernel density plots of the five climatic 2013; Glennon et al.,2014). In our study group, the narrowest variables at 100 m resolution for the two cytotypes of P. mar- realized niche of Pa6x populations may be the result of post- ginata and P. allionii. glacial dispersal limitation (Casazza et al.,2013a; Minuto et al.,2014), as observed in other Alpine endemics (Essl et al., ACKNOWLEDGEMENTS 2011). The narrower realized niche of Pm12x compared with Pm6x populations may be due to the younger age of the allopo- We are grateful to Diletta Ghiglione, Ivano Moraldo and the lyploids. Thus, it is likely that the differences in realized niche staff of the Parco Naturale Alpi Maritime (in particular Paolo breadth detected among the studied taxa are largely due to dis- Fantini, Valderia Botanical Garden manager) for their tech- persal limitation, rather than to differences in fundamental nical support. We would like to thank the editor and three an- niche breadth. onymous reviewers for their constructive suggestions for manuscript improvement. CONCLUSIONS LITERATURE CITED In this study, we detected minor differences in floral traits related to pollinator attraction, and stronger differences in traits Abbot RJ, Lowe AJ. 2004. Origins, establishment and evolution of new poly- related to pollinator handling between Pm12x individuals and ploid species: Senecio cambrensis and S. eboracensis in the British Isles. Biological Journal of the Linnean Society 82: 467–474. their closest relatives. Although this remains to be properly as- Araujo MB, Pearson RG, Thuiller W, Erhard M. 2005. Validation of spe- sessed experimentally, such differences do not seem to be suffi- cies–climate impact models under climate change. Global Change Biology ciently large to have promoted a consequential decrease in 11: 1504–1513. interploidy mating. 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