Climatic Niche Comparison Among Ploidal Levels in the Classic Autopolyploid System, Galax Urceolata

RESEARCH ARTICLE

Climatic niche comparison among ploidal levels in the classic autopolyploid system, Galax urceolata

Michelle L. Gaynor1,4 , D. Blaine Marchant2,3, Douglas E. Soltis2,3, and Pamela S. Soltis3

Manuscript received 8 March 2018; revision accepted 4 July 2018. PREMISE OF THE STUDY: Autopolyploidy, or whole-genome duplication within a species, 1 Department of Biology, University of Central Florida, Orlando, leads to closely related cytotypes in one geographic location. One hypothesized Florida 32816, USA mechanism by which autopolyploids become established is climatic niche divergence from 2 Department of Biology, University of Florida, Gainesville, Florida their diploid progenitor. Here we tested this hypothesis in diploid, triploid, and tetraploid 32611, USA Galax urceolata (Diapensiaceae) and predicted the effects of climate change on the relative 3 Florida Museum of Natural History, University of Florida, distributions of these cytotypes. Gainesville, Florida 32611, USA 4 Author for correspondence (e-mail: [email protected]) METHODS: We investigated whether climatic niche divergence has shaped the current Citation: Gaynor, M. L., D. B. Marchant, D. E. Soltis, and P. S. Soltis. distributions of Galax urceolata cytotypes in eastern North America using climatic niche 2018. Climatic niche comparison among ploidal levels in the classic modeling, multivariate analyses of environmental space, and geographic range analyses. autopolyploid system, Galax urceolata. American Journal of Botany We then projected the models of the three cytotypes onto an ensemble of future climate 105(10): 1631–1642. maps to determine how the distributions might be altered over time. doi:10.1002/ajb2.1161 KEY RESULTS: All cytotypes are geographically sympatric; however, climatic niche contraction and a slight niche shift of the tetraploids was observed relative to that of the diploids. Climate projections for all diploid and tetraploid cytotypes showed substantial range contraction without much, or any, range shift, suggesting that Galax urceolata will likely go extinct in nature as mountain refugia become warmer.

CONCLUSIONS: Galax urceolata tetraploids occupy a slightly wetter habitat than that of their diploid progenitors. While we cannot take into account future adaptation, our models suggest extensive decreases in range distributions for both diploid and tetraploid G. urceolata based on climate change projections. Galax urceolata may therefore be under extreme threat due to loss of suitable habitat, and conservation efforts will be needed.

KEY WORDS Autopolyploidy; climate change; climatic niche modeling; Galax urceolata; genome duplication; polyploidy.

Polyploidy, or whole-genome duplication (WGD), is an impor- Polyploids are often classified into two major categories, al- tant evolutionary force, particularly in angiosperms (e.g., Leitch lopolyploids and autopolyploids. While allopolyploids are derived and Bennett, 1997; Soltis et al., 2009). During much of the 20th from interspecific hybridization (Stebbins, 1950), autopolyploids century, estimates of the frequency of polyploidy in angiosperms arise within a species and were long considered very rare in nature ranged from approximately 30% to 70% (see Stebbins, 1971), but and maladapted due to both meiotic irregularities and the genetic, recent data suggest that all angiosperms share an ancestral WGD morphological, physiological, and ecological similarity between the event (Jiao et al., 2011; Amborella Genome Project, 2013; but see established diploid populations and new autopolyploid (Clausen Ruprecht et al., 2017). Additionally, WGD is an important speciation et al., 1945; Stebbins, 1947; Grant, 1981). However, expanded in- mechanism and a key factor in the diversification of angiosperms. corporation of genomic and cytometric techniques has greatly However, much is still unknown about the mechanisms associated increased recognition of the frequency and importance of autopoly- with the initial establishment and persistence of polyploids (Fowler ploids in nature (Soltis et al., 2007, Soltis et al., 2014a; Parisod et al., and Levin, 1984; Levin, 1975; Lewis, 1980; Ramsey and Schemske, 2010; Barker et al., 2015; Spoelhof et al., 2017). 1998; Levin, 2002; Ramsey and Schemske, 2002; Rieseberg and The formation of unreduced gametes is estimated to occur Willis, 2007; Soltis et al., 2010). at a rate of 0.5–2% in angiosperms; therefore, it is predicted that

autopolyploids form at a relatively high frequency within natural occupied a subset of the range of the diploids. Diploids occupy populations (Ramsey and Schemske, 1998, 2002). Formation of au- more xeric habitats, while the tetraploids are more often found in topolyploids can be an instant sympatric speciation mechanism as mesic sites. This habitat preference was later tested by Johnson et al. the novel autopolyploids are reproductively isolated from their dip- (2003), who found evidence for habitat differentiation of the cyto- loid progenitors; however, the evolutionary success of an autopoly- types based on the surrounding vegetation. ploid can vary greatly. Nascent autopolyploid individuals are often Servick et al. (2015), investigating both cytotype prevalence and much fewer in number than their established diploid progenitors the genetic diversity of populations of Galax urceolata, identified a and are thus considered the minority cytotype within their popu- clear pattern of genetic differentiation among ecoregions, with sim- lations. As a result, few compatible mates are available, and a high ilar patterns observed among cytotypes. Different genetic clusters of rate of futile pollination exchanges occurs with their more preva- diploid populations were detected, and many were associated with lent diploid counterparts. These negative frequency-dependent se- distinct tetraploid populations that originated from that diploid lection pressures, known as the minority cytotype exclusion (MCE) genotype. These results suggest that the occurrence of Galax is a principle (Levin, 1975; Fowler and Levin, 1984; Felber 1991; Rausch complex function of both genetic and ecological variables in that and Morgan, 2005), can lead to reduced fitness and eventual extinc- the tetraploid cytotype has formed numerous times across the range tion of the less prevalent cytotype unless the polyploid outcompetes of Galax and tetraploid populations of independent origin typically the progenitor or a niche shift is achieved. Due to MCE, it has been occur in close proximity to their respective diploid progenitor pop- inferred that autopolyploids have a high rate of failure to establish ulations (Servick et al., 2015). and thus a high extinction rate (Rausch and Morgan, 2005). While the ecology and population dynamics of Galax urceolata Fowler and Levin (1984) modeled nascent autopolyploids and have been investigated using a variety of approaches (Baldwin, 1941; demonstrated that an autopolyploid population may persist and Nesom, 1983; Soltis et al., 1983; Burton and Husband, 1999; Johnson become established by outcompeting and replacing the diploid et al., 2003; Servick et al., 2015), the cytotypes have not yet been the progenitors in a population, stochastic colonization of recently subject of detailed investigations of climatic niche space. Due to the open habitat, and/or niche separation of the two cytotypes. Several overall limited range of G. urceolata (Appendix S1; see Supplemental studies have since investigated these mechanisms of survival in Data with this article) and of each cytotype in particular, it is also natural populations of autopolyploids and their diploid progeni- important to identify how climate change will affect the future dis- tors, revealing varying mechanisms of establishment of autopoly- tribution of the species and of each cytotype. Using a variety of ge- ploids. Niche divergence has often accompanied autopolyploidy in ographical and climatic analyses, we aimed to: (1) compare current diploid-autopolyploid systems that remained sympatric or became climatic niche space and geographic extent of the three cytotypes allopatric (Glennon et al., 2012; McIntyre, 2012; Laport and Ramsey, of G. urceolata; and (2) determine how climate change will affect 2015; Visger et al., 2016). However, niche divergence is not neces- the geographic ranges of each cytotype. Given the lack of genetic sarily a requirement for the establishment of an autopolyploid. For differentiation among cytotypes of G. urceolata, likely due to inde- example, diploid and autotetraploid Heuchera cylindrica Douglas ex pendent origins of the polyploids from different diploid populations Hook. (Saxifragaceae) share similar niche requirements, yet the two (Servick et al., 2015), we hypothesized that tetraploids and triploids cytotypes differ in geographic distribution (Godsoe et al., 2013). In will occupy niches that are similar to that of their diploid progenitor. contrast, niche differentiation between diploid and autotetraploid In addition, climate change effects are predicted to be detrimental to Ranunculus adoneus A.Gray (Ranunculaceae) was not observed, endemic species with highly limited ranges (Schwartz et al., 2006). and the cytotype distributions overlap (Baack, 2005; Baack and Given the restricted range of G. urceolata, we predicted that climate Stanton, 2005). To improve our understanding of the role of niche change would reduce the amount of suitable habitat available for divergence in the establishment and persistence of autopolyploids, each cytotype within the next century. additional autopolyploid systems should be investigated. Galax urceolata (Poiret) Brummitt (Diapensiaceae) is an ideal polyploid system in which to investigate the role of niche diver- MATERIALS AND METHODS gence in the establishment of an autopolyploid. Galax urceolata is endemic to the Appalachian mountain range in the southeastern Locality data collection United States (Gleason, 1952). The single described species in- cludes three cytotypes: (1) diploid (2n = 12); (2) triploid (2n = 18); We obtained locality data of Galax urceolata from previous studies and (3) tetraploid (2n = 24) (Nesom, 1983; Burton and Husband, of the species for which ploidal level was clearly indicated (Servick 1999; Servick et al., 2015). Because Galax is morphologically and et al., 2015; Barringer and Galloway, 2017). Digitized specimen re- phylogenetically distinct from all other genera in Diapensiaceae, cords from herbaria and iDigBio (www.idigbio.org) were examined especially in the eastern United States, the tetraploid cytotype has but could not be incorporated into our data set due to the lack of long been considered an autotetraploid (see Nesom, 1983) and was, information on ploidy and the morphological similarity among the in fact, the only unambiguous case of autopolyploidy accepted by three cytotypes of G. urceolata (Baldwin, 1941). Locality coordi- Stebbins (1950). Although autopolyploidy is now considered much nates were checked for appropriate precision (<1 km), and duplicate more common than previous estimates (Soltis et al., 2007; Soltis and coordinates were removed. Soltis, 2009; Barker et al., 2015; Spoelhof et al., 2017), Galax remains as one of the classic examples of autopolyploidy. Ecological Niche Modeling (ENM) layers The three cytotypes of Galax overlap across most of their broad geographic range, lacking coarse ecological and geographic barriers We used all 19 environmental layers of the current (1950-present) (Baldwin, 1941). However, Nesom (1983), studying the geographic BioClim dataset from the WorldClim database with spatial resolu- distribution of G. urceolata cytotypes, found that the tetraploids tion of 30 arcsec (Hijmans et al., 2005; Fick and Hijmans, 2017). The October 2018, Volume 105 • Gaynor et al.—Climatic niche comparison among Galax cytotypes • 1633

layers were trimmed tightly around all of the locality records (here- climatic tolerances (Levins, 1968). In addition, a niche identity test after referred to as the “full extent”) of Galax urceolata to the extent was performed in ENMTools (Warren et al., 2010) by comparing 39.73° N, –86.42° W to 32.64° N, –75.12°W using the raster clipping niche models with the same number of occurrence records as the feature in Quantum GIS version 2.18.2 (QGIS Development Team, original models, but made from randomly distributed localities 100 2009). Because tetraploids occupy only a subset of the current times. The results of this test are plotted on a histogram to calculate range of the diploid cytotype, we circumscribed the locality records the null distribution of niche overlap values to which the observed of each cytotype separately using a convex hull and buffer of 1°, niche overlap values are compared. which is about 100 km at this latitude (hereafter referred to as the “convex hull extent”), with the rgeos R package (Bivand et al., 2017) Statistical analysis and trimmed the BioClim layers based on those distinct extents (Appendix S2). Each layer was converted to ASCII format. The full Using the ‘Research tool’ in Quantum GIS version 2.18.2 (QGIS extent layers were checked for high (> |0.80|) pairwise correlation Development Team, 2009), we generated 10,000 random points in- values using ENMTools (Warren et al., 2010), and only layers lack- side the full extent layers. We set a minimum suitability threshold of ing high correlation values (annual mean temperature, mean diur- 0.25 for both the full extent and convex hull extent models projected nal range, mean temperature of wettest quarter, mean temperature onto the full extent layers to signify an area as suitable for each cyto- of driest quarter, annual precipitation, precipitation seasonality, type, while a value under 0.25 was designated unsuitable, for pres- precipitation of driest month, and precipitation of coldest quarter) ent and future distributions. We then used the ‘Point sampling tool’ were retained. plugin in Quantum GIS version 2.18.2 (QGIS Development Team, Additionally, we obtained matching future projected BioClim 2009) to determine whether a point is within or outside the present layers from the WorldClim database. Specifically, we used five and future ranges. Geographic overlap (G) was then calculated for commonly implemented climate models from the Coupled Model each cytotype as the percentage of points found in the present dis- Intercomparison Project Phase 5 (CCSM4, GFDL-CM3, GISS- tribution relative to those in the future distribution. Additionally, G E2-R, HadGEM2-ES, and MIRCO5) (Sheffield et al., 2013) for 2070 was calculated using this method among the present distributions (average 2061-2080) for the two greenhouse gas representative of the three cytotypes. concentration pathway (rcp) trajectories of 2.6 and 8.5 (Riahi et al., To explore future altitudinal shifts, the current and future dis- 2011; Villaverde et al., 2017). Each future layer also had the spatial tributions were similarly calculated relative to altitude for each resolution of 30 arcsec and was trimmed to the full extent. cytotype. Altitude was not included in ENM due to high correlation values with included layers. Using the ‘Research tool’ in Ecological Niche Modeling Quantum GIS version 2.18.2 (QGIS Development Team, 2009), we generated 10,000 random points inside the full extent. We used Using BioClim full extent layers and the collected cytotype local- the same suitable/unsuitable model layers for the present and fu- ity data, we produced separate ecological niche models (ENMs) ture distributions of the cytotypes upon the altitude layer from for diploid, triploid, and tetraploid Galax urceolata in MAXENT the WorldClim dataset with the spatial resolution of 30 arcsec version 3.3 using standard parameters (Phillips et al., 2006). In ad- (Hijmans et al., 2005). The altitude value for each random point dition, we also made separate models using the convex hull extent was determined as well as whether or not the point was within layers for each cytotype to produce ENMs for diploid, triploid, and each model distribution using the ‘Point sampling tool’ plugin in tetraploid G. urceolata with projections of these models onto the Quantum GIS v. 2.18.2 (QGIS Development Team, 2009). We then full extent in MAXENT version 3.3 using standard parameters compared the average altitude for the points within the present (Phillips et al., 2006). We projected both the full extent and con- and future distributions of each cytotype. Altitude data were not vex hull extent models onto the five commonly used climatic layers normally distributed based on a Shapiro-Wilk test in R version at the two rcp trajectories to predict the future distributions of the 3.2.1 (R Development Core Team, 2015); therefore, we used a three cytotypes with projected climate change. Five replicates were Wilcoxon rank sum test in R version 3.2.1 (R Development Core run for each model, and 75% of the occurrence data were randomly Team, 2015) for comparing the average present and future altitude selected to calculate the models and 25% to test each (Phillips et al., values of each cytotype. 2006). The area under the receiver operating characteristic curve The values of the nine climatic layers were extracted for each (AUC) was used to assess each model’s ability to differentiate be- locality record using the ‘Point sampling tool’ plugin in Quantum tween suitable and unsuitable area. GIS version 2.18.2 (QGIS Development Team, 2009). The climatic The mean output of the five replicates for each model was used layers were then used as loading factors to analyze the niches of for all subsequent analyses. Pairwise niche overlap was calculated the three cytotypes in multivariate space with a principal compo- among the mean models of the three cytotypes, using Schoener’s nent analysis (PCA) in R version 3.2.1 (R Development Core Team, D similarity index in ENMTools (Warren et al., 2010). Schoener’s 2015). In addition, analysis of the niches of the three cytotypes was D ranges from 0 to 1, where zero represents no niche similarity conducted using a multivariate analysis of variance (MANOVA) in between the models and one represents completely identical niches R version 3.2.1. Following the MANOVA, post hoc tests were con- (Schoener, 1968; Warren et al., 2008). Levins’ inverse concentration ducted using the Tukey Honest Significant Differences (TukeyHSD) measure of niche breadth (Levins, 1968) for each model was calcu- method in R version 3.2.1. The population structure analysis in lated in ENMTools (Warren et al., 2010). Niche breadth is a means Servick et al. (2015) found six distinct population clusters (K = 6) of of calculating the breadth of suitable climatic factors for a species, diploid Galax urceolata. To determine whether the climatic niches providing a value ranging from 0 to 1, where larger values repre- of the six population clusters differed, we coded the locality records sent more generalist species with wider climatic tolerances and by population cluster and re-ran the PCA for the diploids, using smaller values represent more specialized species with more narrow the climatic values pulled in the previous analysis. Low sample size 1634 • American Journal of Botany

precluded statistical analysis of the genetic clusters of triploids and AUC score was 0.711 ± 0.315. This low AUC score is likely due to tetraploids identified in Servick et al. (2015). the small number of triploid samples used to construct this model. In both the full extent and convex hull extent models, we found that diploids and tetraploids had significantly differentiated climatic RESULTS niches, despite relatively high levels of niche overlap and geographic sympatry. Models for the diploid and triploid showed the largest We retrieved 1042 georeferenced points from Servick et al. (2015), pairwise niche overlap for both those constructed with the full ex- and 8 additional georeferenced points were retrieved from Barringer tent (Schoener’s D = 0.63, Fig. 2A) and convex hull extent (Schoener’s and Galloway (2017). After the removal of duplicates, representing D = 0.60, Fig. 2B); lower pairwise niche overlap values were found populations within 1 km of each other, the total number of occur- between the diploid and tetraploid models for both model extents rence points was reduced to 95. These points represented 48 diploid, (Schoener’s D = 0.48, Schoener’s D = 0.49, Fig. 2), as well as between 12 triploid, and 35 tetraploid localities (Fig. 1). the triploid and tetraploid models (Schoener’s D = 0.42, Schoener’s All AUC scores from the full extent models fell above 0.75, i.e., D = 0.38, Fig. 2). The niche identity test demonstrated that climatic diploid model 0.873 ± 0.024, triploid model 0.980 ± 0.030, and niche overlap was not significantly different from the null distribu- tetraploid model 0.978 ± 0.025. These AUC values indicate that our tion between triploids and diploids for both sets of models, while models are sufficient for predicting suitable and unsuitable area. Schoener’s D was significantly lower than expected for diploids and The convex hull extent models for diploids and tetraploids had AUC tetraploids for both sets of models (Appendix S3). For the com- scores above 0.75, i.e., diploid model 0.758 ± 0.043 and tetraploid parison of triploids and tetraploids, Schoener’s D was not signifi- model 0.922 ± 0.042; however, the triploid convex hull extent model cantly different than expected for models based on the full extents;

Diploid Triploid Tetraploid

0170 40 280 Kilometers

FIGURE 1. Map depicting sampled Galax urceolata populations (Servick et al., 2015; Barringer and Galloway, 2017). Pie charts within locality points indicate when two or more cytotypes occur in the same location. October 2018, Volume 105 • Gaynor et al.—Climatic niche comparison among Galax cytotypes • 1635

A Diploid Triploid Tetraploid 0.71 0.28

1.00 0.17 0.63 0.42

0 0 0 0.25 0.25 0.25 0.5 0.5 0.5 0.75 0.25 0.75 0.60 0.75 1 1 1 Breadth = 0.48 Breadth = 0.29 Breadth = 0.11 0.48

B DiploidTriploidTetraploid 0.55 0.24 0.52 0.17

0.60 0.38

0 0 0 0.25 0.25 0.25 0.5 0.5 0.5 0.75 0.17 0.75 0.25 0.75 1 1 1 Breadth = 0.58 Breadth = 0.32 Breadth = 0.28 0.49 FIGURE 2. Modeled average niche suitability utilizing both (A) the full extent and (B) cytotype-specific convex hull extents, shown on each map for each cytotype; increasing color intensity shows a higher predicted niche suitability as indicated by scale. “Breadth” indicates Levins’ inverse concentration measure of niche breadth for each cytotype; gold represents Schoener’s D similarity index, or niche overlap; blue represents geographic overlap. however, Schoener’s D was significantly different than expected for Analysis of geographic overlap among cytotypes revealed that the models based on convex hull extents (Appendix S3). diploids currently occupy a larger geographic area than both tetra- The MANOVA analysis of the niches of the three cytotypes re- ploids and triploids when the full extent models are used (Fig. 2A). vealed significant differences among cytotypes for a subset of layers The range of the tetraploids is completely sympatric within the range (Appendix S4). Based on post hoc tests, we found that tetraploids of the diploids (G = 1.00), but is only a small subset of the diploid and diploids had significantly different mean diurnal temperature range (G = 0.17). Similarly, the triploids were largely sympatric with range (P = 0.041), annual precipitation (P = 0.012), precipitation of the diploids (G = 0.71), while the range of the diploids expanded well driest month (P = 0.004), and precipitation seasonality (P = 0.006). beyond that of the triploids (G = 0.25). Triploids occupied about half Additionally, post hoc tests revealed that triploids and diploids had of the range of the tetraploids (G = 0.6), while the tetraploids occu- significantly different temperature annual range (P = 0.002), an- pied only a small subset of the triploids’ range (G = 0.28). Models nual precipitation (P = 0.007), precipitation of the driest month (P constructed with convex hull extents support these findings, although = 0.012), and precipitation during the warmest quarter (P = 0.004). lower amounts of geographic overlap were observed (Fig. 2B). The multivariate analysis of the three cytotypes showed high overlap The geographic overlap and average niche suitability between but slight variation and sub-partitioning of the tetraploid and trip- present and future projected ranges varied for the models based on loid niche relative to the broader diploid niche (Fig. 3A; Table 1). The both the full extent and convex hull extent layers (Table 2; Fig. 4; highest-loading variables on PC1 for the PCA of the three cytotypes Appendices S5 and S6). We found geographic overlap to be low for were temperature annual range (bio7), mean temperature of the diploids using both the full extent (Table 2, G = 0.00 – 0.40) and wettest quarter (bio8), annual precipitation (bio12), precipitation of convex hull extent (Table 2, G = 0.00 – 0.44) models. Low to mod- the driest month (bio14), and precipitation of the warmest quarter erate geographic overlap between the present and future projected (bio18) (Table 1; Fig. 3A). For PC2, annual mean temperature (bio1), ranges of the tetraploids was observed using the full extent (Table 2, mean temperature of the driest quarter (bio9), and precipitation sea- G = 0.07 – 0.64) and convex hull extent (Table 2, G = 0.06 – 0.54) sonality (bio15) were the highest-loading variables (Table 1; Fig. 3A). layers. For both cytotypes, the low geographic overlap seemed to be Using the full extent models, we observed the largest niche due to an overall decrease in range, rather than to a geographic shift breadth in diploids (breadth = 0.48) and the lowest niche breadth (Appendices S5 and S6). The geographic overlap between the pres- in tetraploids (breadth = 0.11), while triploids had an intermedi- ent and future ranges of the triploid using both the full extent and ate niche breadth (breadth = 0.29), suggesting that niche breadth convex hull extent models was highly variable (Table 2, G = 0.00 decreased as ploidy increased. A similar trend was observed in the – 0.99, G = 0.34 – 1.00). Diploids are predicted to move into signif- convex hull extent models with larger niche breadths observed in icantly higher altitudes for all projected models, while tetraploids diploids (breadth = 0.58) followed by triploids (breadth = 0.32) and are predicted to move into higher altitudes for all future climate tetraploids (breadth = 0.28). change models except for GISS-E2- R, at 2.6 rcp using the convex 1636 • American Journal of Botany

A B

bio1 bio9 bio1 bio9 5 ) 2.5 bio1 ) 2 bio1 bio18 bio1 bio8 8

bio1 bio12 2 bio2 bio8 0.0 bio2

0 bio14 PC2 (27.4% PC2 (19.9% bio7 bio14 bio7

−2 −2.5 −6 −4 −2 024 −5.0 −2.5 0.02.5 PC1 (49.4%) PC1 (60.9%) A C E groups groups diploidtetraploid triploid B D F

FIGURE 3. (A) Principal component analysis (PCA) of the climatic layers including annual mean temperature (bio1), mean diurnal range (bio2), temperature annual range (bio7), mean temperature of the wettest quarter (bio8), mean temperature of the driest quarter (bio9), annual precipitation (bio12), precipitation of the driest month (bio14), precipitation seasonality (bio15), and precipitation of warmest quarter (bio18) for diploids (blue), triploids (orange), and tetraploids (purple) based on current layers. (B) Principal component analysis of climatic layers for the six diploid populations identified in Servick et al. (2015); cluster A = red, cluster B = blue, cluster C = yellow, cluster D = purple, cluster E = red, cluster F = green. hull extent (Fig. 5; Appendix S7). In contrast, triploids are predicted of climate change for the three cytotypes of Galax urceolata, to move into lower altitudes; however, not all models predict statis- long considered the classic example of autopolyploidy in nature tically significant altitude shifts (Fig. 5; Appendix S7). (Stebbins, 1950). Previous studies have identified general trends be- The PCA of the six diploid population clusters identified in tween autopolyploid species and their diploid progenitors in terms Servick et al. (2015) based on genetic data showed minor niche of climatic niche (Glennon et al., 2014). First, the niche of the au- differentiation among populations (Fig. 3B; Table 1). However, low topolyploid could overlap and engulf that of the diploid, indicating sample size per cluster limits the statistical power of this analysis. climatic niche expansion in the polyploid. For example, tetraploid The highest-loading variables for PC1 were temperature annual Centaurea stoebe L. (Asteraceae) was found to have a significantly range (bio7), annual precipitation (bio12), precipitation of the dri- wider niche breadth than that of the diploid progenitor despite est month (bio14), and precipitation of the warmest quarter (bio18) considerable overlap in niche (Glennon et al., 2014). The second (Table 1; Fig. 3B). For PC2, the highest-loading variables were an- general trend is that of climatic niche contraction, in which the nual mean temperature (bio1), mean temperature of the driest quar- autopolyploid is restricted to a subset of the range of the diploid. ter (bio9), and precipitation seasonality (bio15) (Table 1; Fig. 3B). This contraction was seen in Ranunculus kuepferi Greuter & Burdet (Ranunculaceae), where fine-grained analyses revealed that autotetraploids occupied a niche that was a subset of the diploid pro- DISCUSSION genitor’s niche (Kirchheimer et al., 2016). The third trend is that of climatic niche conservatism where there is no discernable difference Using niche, geographic, and multivariate analyses, we examined between the niches of the diploid and autotetraploid. This result has climatic niche differentiation and the broad ecological ramifications been seen in autopolyploid Lilium lancifolium Thunb. (Liliaceae), in

TABLE 1. Geographic overlap between each future projection and the current Ecological Niche Model (ENM) for each cytotype. cc26 cc85 gf26 gf85 gs26 gs85 he26 he85 mc26 mc85 Full extent 2x 0.24 0.11 0.06 0.01 0.40 0.09 0.13 0.00 0.38 0.26 3x 0.83 0.46 0.31 0.29 0.69 0.32 0.42 0.00 0.76 0.99 4x 0.64 0.39 0.14 0.07 0.56 0.45 0.44 0.56 0.56 0.56 Convex hull 2x 0.23 0.10 0.05 0.03 0.39 0.10 0.16 0.01 0.44 0.20 extent 3x 0.87 0.77 0.54 0.34 1.00 0.77 0.79 0.38 0.99 0.99 4x 0.42 0.28 0.29 0.06 0.54 0.38 0.41 0.10 0.31 0.14 Notes: cc indicates CCSM4, gf indicates GFDL-CM3, gs indicates GISS-E2-R, he indicates HadGEM2-ES, and mc indicates MIRCO5. Representative Concentration Pathways (RCPs) 2.6 is represented by 26, and rcp8.5 is represented by 85. October 2018, Volume 105 • Gaynor et al.—Climatic niche comparison among Galax cytotypes • 1637

which the diploid and autotetraploid niche breadths did not differ their progenitors is possible if there is fine-scale climatic niche dif- even though the breadth of the triploid cytotype had slightly ex- ferentiation or differentiation in biotic interactions (e.g., phenology, panded beyond that of the diploid progenitor (Chung et al., 2015). pollinator interactions, parasitism), as these more subtle niche shifts Minimal climatic niche differentiation between polyploids and can reduce competition among cytotypes. Moreover, fine-scale habitat differentiation, as observed among cytotypes of the Cardamine TABLE 2. Relative contribution of the climatic layers including annual mean schulzii Urbanska-Worytkiew (Brassicaceae) complex over a mat- temperature (bio1), mean diurnal range (bio2), temperature annual range (bio7), ter of meters (Zozomová-Lihová et al., 2014), may be impossible mean temperature of the wettest quarter (bio8), mean temperature of the driest to detect using modeling approaches such as those applied here for quarter (bio9), annual precipitation (bio12), precipitation of the driest month Galax and other diploid-polyploid species groups (e.g., Theodoridis (bio14), precipitation seasonality (bio15), and precipitation of warmest quarter (bio18) to principal components (PC) displayed in Fig. 3A and 3B. et al., 2013; Glennon et al., 2014; Chung et al., 2015; Kirchheimer et al., 2016). 3A 3B Even without a climatic niche shift, diploids and autotetraploids PC1 PC2 PC1 PC2 may still occupy different geographic ranges. For example, diploid bio1 8.0 26.4 5.6 22.4 and tetraploid Heuchera cylindrica have different geographic ranges, bio2 7.4 3.7 10.1 2.1 yet occupy similar climatic niches, consistent with niche conserva- bio7 12.8 5.1 14.5 7.4 tion between the two cytotypes (Godsoe et al., 2013). This trend bio8 13.1 2.6 11.4 9.9 was also seen in diploid, tetraploid, and hexaploid Larrea tridentata bio9 8.5 23.4 6.9 17.1 (DC.) Coville (Zygophyllaceae), in which distribution models indi- bio12 14.5 3.4 16.5 0.9 cate that each cytotype occupies a different geographic range, while bio14 14.3 0.8 15.8 5.1 niche values were conserved among cytotypes (Laport et al., 2013). bio15 8.0 23.1 5.0 22.9 bio18 13.3 11.6 14.2 12.1 This pattern of niche conservatism, but distinct geographic ranges, can occur when the polyploids, rather than the diploids, become

DiploidTriploidTetraploid t Curren 2 c c 5 8 c c6

FIGURE 4. Modeled average niche suitability using convex hull extents is shown for each cytotype with current and climate change projections; increasing color intensity shows a higher predicted niche suitability. CCSM4 is used as a representative climate change model. Representative Concentration Pathways (RCP) 2.6 is represented by “cc26,” and RCP 8.5 is represented by “cc85.” Additional future projections are available in Appendices S4 and S5. 1638 • American Journal of Botany

A B C 1500 1500 50 0 1000 1000 100 01 00 00 500 05 05

DE F 1500 1500 1500 1000 1000 1000 00 00 500 05 05 0

FIGURE 5. Box plots of altitude (in meters) within the present and future distributions of each cytotype. The box plots show the median (thickened central bar), first and third quartiles (bottom and top of the boxes), 1.5× the interquartile range (whiskers), and outliers. Black represents the current model, blue represents CCSM4, green represents GFDL-CM3, red represents GISS-E2- R, orange represents HadGEM2-ES, and purple represents MIRCO5. Representative Concentration Pathways (RCP) 2.6 is separated from RCP 8.5 by a solid black line on each box plot. (A-C) are models based on the full extent; (D-F) are models based on the convex hull extents. (A) Diploid model projected at current, RCP 2.6, and RCP 8.5. (B) Triploid projected at current, RCP 2.6, and RCP 8.5. (C) Tetraploid projected at current, RCP 2.6, and RCP 8.5. (D) Diploids projected at current, RCP 2.6, and RCP 8.5. (E) Triploids projected at current, RCP 2.6, and RCP 8.5. (F) Tetraploids projected at current, RCP 2.6, and RCP 8.5. Overall, shift to higher altitudes is projected with future climatic conditions. established by chance in unoccupied or recently disturbed habi- precipitation-related. Combined, these results suggest the tetraploid tat (Fowler and Levin, 1984). Competition due to similar climatic has undergone niche contraction relative to the ancestral niche of niches and MCE would maintain the geographic barrier between the diploid progenitor. In comparison, the triploid overlaps consid- the two cytotypes without the need for niche differentiation. erably with both the tetraploids and diploids in terms of geographic For Galax urceolata, we found relatively high degrees of geo- distribution and niche. graphic sympatry and climatic niche overlap among the three cyto- Based on microsatellite data, the tetraploid cytotype of Galax types with a general decrease in range and niche breadth as ploidy has originated independently an estimated 46 times, while triploid increased. Even though the diploid and tetraploid distributions and formation is estimated to have occurred 31 times independently climatic niches seemed to overlap considerably, niche overlap was (Servick et al., 2015). Moreover, individuals within an ecoregion significantly lower than that of the null distribution. When viewed were more genetically similar to each other regardless of ploidy in multivariate space, it becomes readily apparent that the tetra- than were individuals of the same cytotype on a broad geographic ploids occupy a subset of the diploid climatic niche with a slight scale (Servick et al., 2015). The data suggest greater gene flow among shift towards wetter habitats. This finding is further supported by neighboring populations regardless of cytotype than among more the MANOVA results, which identified four climatic layers as dif- geographically distant populations within each cytotype. Local ad- fering significantly between the two cyotypes, of which three were aptation also seems to play a role in the genetic and environmental October 2018, Volume 105 • Gaynor et al.—Climatic niche comparison among Galax cytotypes • 1639

clustering of Galax; the six diploid population clusters from Servick Soltis et al., 2014b; te Beest et al., 2012). Polyploidy substantially al- et al. (2015) also clustered in niche space based on multivariate ters the genomic composition (Buggs et al., 2012, 2014; Henry et al., analyses of the climatic layers. 2014) and patterns of gene regulation (see Yoo et al., 2014, and ref- Galax urceolata commonly reproduces via clonal propagation, erences therein) in the newly derived species, potentially yielding an thereby reducing the need for sexual reproduction and the threat array of novel phenotypes (e.g., Levin, 1983; Soltis et al., 2014b, 2015; of MCE in the short term. Barringer and Galloway (2017) found Soltis and Soltis, 2016) that may allow polyploids to undergo local that both diploids and tetraploids are self-incompatible and have adaptation or niche shifts relative to those of their diploid progeni- overlapping flowering phenology and shared pollinators. The lack tors. Whereas the effects of genome duplication on gene expression of differences in the reproductive ecology of these two cytotypes, and genome rearrangement are highly complex in allopolyploids coupled with local genetic similarity, indicates that gene flow among (e.g., Doyle et al., 2008; Buggs et al., 2014; Soltis et al., 2015; Wendel, cytotypes may occur (Servick et al., 2015), despite the potential 2015), very little is known about the impact of these processes in post-zygotic barriers predicted for diploid-tetraploid crosses. Such autopolyploids (Parisod et al., 2010; Spoelhof et al., 2017). Given the interploidal crosses could provide a “triploid bridge” for gene flow morphological and apparent ecological similarities of the cytotypes between diploids and tetraploids (Ramsey and Schemske, 1998; in Galax urceolata and the many repeated formations of the trip- Burton and Husband, 2001). Further study of inter-cytotype mating loid and tetraploid populations, the ongoing genetic and ecological and triploid fertility is needed to evaluate the role of triploids as a changes and selective pressures of each cytotype are unclear. It is long-term source of genetic and ecological diversity in Galax. possible that the tetraploids will disperse to a novel range or adapt Our results are of particular interest when the niche contrac- to a novel niche and become reproductively isolated from the dip- tion and slight niche shift of the tetraploids, as demonstrated here loids; however, these factors cannot be incorporated into projected and by Johnson et al. (2003), are taken into account. Because there future distributions. To understand the adaptability of the cytotypes is limited, if any, gene flow between tetraploids of different pop- of G. urceolata, additional investigation of the genomic and physi- ulations, a recurrent and parallel niche shift may have occurred ological implications of polyploidy and the multiple origins of the with each tetraploid origin. Recurrent gene flow with local diploids triploids and tetraploids is necessary. could further prevent local adaptation of each tetraploid popula- Galax urceolata is just one of many species exhibiting a disjunct tion. Detailed physiological comparisons of diploids and tetraploids distribution between the southern Appalachian Mountains and of independent formation may help clarify the niche differences of the coastal plain ravines of Virginia and North Carolina (Schafale diploids and tetraploids. and Weakley, 1990; Spira, 2011). Other species having this dis- The different climate change models for Galax urceolata showed junction include Kalmia latifolia L. (mountain laurel, Ericaceae), variation in the predicted future ranges of the three cytotypes; how- Erythronium umbilicatum C.R.Parks & Hardin (dimpled trout lily, ever, many models indicated drastic range contraction without Liliaceae), Mitella diphylla L. (two-leaf miterwort, Saxifragaceae), range shifts for all three cytotypes. Triploids are projected to con- and Cypripedium candidum Muhl. ex Willd. (white lady-slipper, tinue to occur in eastern coastal habitat; in contrast, tetraploids and Orchidaceae) (Schafale and Weakley, 1990). There are also numerous diploids are projected to occur only at higher altitudes. We expected endemics to the southern Appalachians, including Calamagrostis all cytotypes to move upslope where temperature is predicted to be cainii A.S. Hitchc. (Poaceae), Carex misera Buckl. (Cyperaceae), colder than at lower elevations, as the temperature increases (cf. Geum radiatum Michx. (Rosaceae), and Trichophorum cespito- Parmesan, 2006). The projections suggest that triploids will contra- sum Michx. (Cyperaceae) (Godt et al., 1996). Galax urceolata can dict this hypothesis and instead move into lower altitudes towards therefore serve as a model for the fate of many Appalachian en- the coastal plain ravines. More fine-scale ecological studies should demics and the mountain-coastal plain disjunct flora. Our results be conducted to determine if triploid G. urceolata may persist in for Galax suggest that with projected climate change aspects of this these coastal habitats or at other microrefugial sites. Importantly, it unique Appalachian flora will only persist at higher altitudes and is doubtful that the triploid populations could be sustained, except with limited overall survival. Similarly, the predicted future range via clonal reproduction, without sympatric diploid or tetraploid of the Appalachian species Geum radiatum represents a dramatic populations hybridizing and producing new triploids due to the shift to higher altitudes due to climate change (Ulrey et al., 2016). meiotic instability and sterility that typically accompany triploidy Compounding these habitat limitations, refuge sites at higher alti- (Levin, 1975; Ramsey and Schemske, 1998; Barringer and Galloway, tudes are expected to become more limited due to human land use 2017). as climate change progresses (Beniston, 2003). Therefore, mountain A key concern for floras as climate conditions change is the abil- refugia are projected to provide only short-term sanctuaries for this ity of plants to disperse to suitable habitat on the time scale of in- high-elevation flora (Ulrey et al., 2016). Due to the projected lack creasing temperature (Thuiller, 2004). Rapid range shifts of Galax of natural habitats available to G. urceolata in the future, we recom- urceolata to habitats in higher altitudes may be restricted by their mend Galax (all ploidal levels) be conserved in botanical gardens. limited dispersal. While the dispersal abilities of G. urceolata have Although ecological niche modeling has been applied to numer- not been quantified, their seeds are very small with at least the ous systems and questions regarding biodiversity, important caveats potential for some long-distance transport (Cain, 1930). Despite must be reiterated (Wiens et al., 2009). First and foremost is the rel- small, potentially transportable seeds, genetic variation is locally atively coarse resolution (~1 km) of the climatic layers with which clustered (Servick et al., 2015), indicating that seed movement is these models are formed. This coarse resolution can overlook the indeed limited and that pollen limitation may further inhibit gene effects of microclimates, such as those found in ravines, small val- flow (Barringer and Galloway, 2017). leys, or rock outcrops (Ashcroft et al., 2009; Franklin et al., 2013). The adaptability of polyploid populations is shaped by their In addition, these models do not take into account biotic interac- ecological, phenotypic, and genetic characteristics and diversity tions (e.g., pollination, dispersal, herbivory, parasitism) or other (Adams and Wendel, 2005; Soltis and Soltis, 2009; Ramsey, 2011; abiotic factors (e.g., soil pH, soil type, available nutrients) so that 1640 • American Journal of Botany

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