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Home , Allopatric speciation, Primula, Primula marginata

Journal of (J. Biogeogr.) (2016) 43, 591–602

ORIGINAL Allopatric with little niche ARTICLE divergence is common among alpine Florian C. Boucher1*, Niklaus E. Zimmermann2,3 and Elena Conti1

1Institute of Systematic Botany, University of ABSTRACT Zurich,€ 8008 Zurich,€ Switzerland, 2Dynamic Aim Despite the accumulation of cases describing fast radiations of alpine Macroecology, Swiss Federal Research , we still have limited understanding of the drivers of speciation in alpine Institute WSL, 8903 Birmensdorf, Switzerland, 3Department of Environmental floras and of the precise the timing of their diversification. Here, we investi- Systems Science, Swiss Federal Institute of gated spatial and temporal patterns of speciation in three groups of alpine Technology ETH, CH-8092 Zurich,€ Primulaceae. Switzerland Location Mountains of the European Alpine System. Methods We built a new phylogeny of Primulaceae including all in three focal groups: sect. Aretia, sect. Auricula and Soldanella. Combining phylogenetic information with a detailed climatic data set, we investigated patterns of range and ecological overlap between sister-species using an approach that takes phylogenetic uncertainty into account. Finally, we investigated temporal trajectories of diversification in the three focal groups.

Results We found that a large majority of sister-species pairs in the three groups are strictly allopatric and show little differences in substrate and cli- matic preferences, a result that was robust to phylogenetic uncertainty. While rates of diversification have remained constant in Soldanella, both Androsace sect. Aretia and Primula sect. Auricula showed decreased diversification rates in the Pleistocene compared to previous geological epochs. Main conclusions Allopatric speciation with little niche divergence appears to have been by far the most common mode of speciation across the three groups studied. A few examples, however, suggest that ecological and polyploid speciation might also have played a role in the diversification of these three groups. Finally, extensive diversification likely occurred in the late Miocene and Pliocene coinciding with the later phases of the Alpine uplift, while diver- sification slowed down during subsequent glacial cycles of the Pleistocene. Keywords *Correspondence: Florian C. Boucher, Institute alpine plants, Androsace sect. Aretia, diversification rates, European Alpine of Systematic Botany, University of Zurich,€ System, phylogenetic uncertainty, Primula sect. Auricula, sister-species Zollikerstrasse 107, 8008 Zurich,€ Switzerland. Soldanella E-mail: fl[email protected] comparison, , Speciation

revealed that in all mountain ranges on Earth groups of INTRODUCTION alpine plants underwent rapid radiations (reviewed in Understanding why certain groups of and some Hughes & Atchison, 2015), raising two important questions regions contain more species than others is a fundamental on the origin of alpine floras, namely: (1) which factors best question in and one of the major challenges for explain the high rates of diversification in alpine , science in general (Pennisi, 2005). Mountains harbour more and (2) when did this diversity arise? than expected based on the area they occupy Allopatric speciation, either through vicariance or long- (Korner,€ 2004) and plants are no exception to this rule (Kier distance dispersal, has traditionally been considered an et al., 2005). Indeed, recent phylogenetic studies have important driver of alpine plant diversification, for many

ª 2015 John Wiley & Sons Ltd http://wileyonlinelibrary.com/journal/jbi 591 doi:10.1111/jbi.12652 F. C. Boucher et al. sister-taxa have allopatric distributions (Ozenda, 1995; dated phylogeny for these three clades. Using an approach to Kadereit et al., 2004). This pattern of allopatry could be consider phylogenetic uncertainty in sister-species compar- explained by the fact that alpine constitute complex ison, we compared patterns of geographical and ecological archipelagos of ‘sky islands’. If allopatric speciation were the overlap over all possible sister-species pairs. We specifically dominant mode of speciation in the alpine flora, we would asked the following questions: (1) Is there general support expect most sister-species pairs to be allopatrically dis- for a particular geographical mode of speciation in these tributed, yet they might show little differen- three groups? (2) Do sister-species strongly differ in their cli- tiation because this would not be needed for speciation to matic or soil preferences, as would be expected if environ- occur (Wiens & Graham, 2005). However, ecological niche mental factors played an important role in speciation? divergence may also have contributed to speciation, regard- Finally, (3) we investigated the timing of diversification for less of the geographical mode of speciation. Indeed, the these three groups and asked whether Pleistocene glacial extreme climatic and edaphic heterogeneity of mountain cycles were associated with increased diversification rates. By regions leads to local in plant investigating the spatial and temporal patterns of diversifica- (Korner,€ 1999) and evidence suggests that this might be tion in the three largest plant clades of the Alpine system, responsible for divergence in the Cape Floristic this study sheds new light on the drivers of speciation in Region (Lexer et al., 2014) and for speciation in the New temperate alpine floras and helps us to understand why they Zealand (Voelckel et al., 2008). are generally so diverse. Although it seems clear that the majority of alpine plant radiations are relatively young and fast, dating to the late MATERIALS AND METHODS Miocene, Pliocene or later (Hughes & Atchison, 2015), the relative importance of late Miocene/Pliocene mountain Study groups uplifts and later Pleistocene climatic fluctuations in driving alpine plant diversification remains unclear. Some studies We chose Androsace sect. Aretia (hereafter /Aretia), Primula suggested that mountain uplifts of the middle and late Ceno- sect. Auricula (hereafter /Auricula) and Soldanella for two zoic (Ollier, 2006) played a major role in stimulating the main reasons. First, these three groups are closely related diversification of alpine floras (e.g. Hughes & Eastwood, within Primulaceae (DeVos et al., 2014), which allowed us to 2006; Joly et al., 2009; Favre et al., 2010; Roquet et al., 2013; reconstruct a single including all of them, Bentley et al., 2014; Linder et al., 2014). Indeed, mountain thereby leading to more consistent estimation of divergence uplifts created both highly fragmented environments and a times across clades (see below). Second, these groups repre- considerable diversity of habitats along steep environmental sent the three most species-rich plant radiations in the gradients, factors that usually promote high speciation rates Alpine system (Ozenda, 1995). All species in these three (Coyne & Orr, 2004). Other studies emphasized the impor- groups are perennials found in the mountains of the Alpine tance of Pleistocene glacial cycles for speciation, as they have system, except Primula palinuri, which grows on coastal cliffs triggered drastic range contractions and expansions in alpine in southern Italy (Fig. 1). /Aretia has been shown to have plants, driving population fragmentation (e.g. Kadereit et al., originated in the late Miocene, hence it has been proposed 2004; Joly et al., 2009). that this group largely diversified during the last active Here, we investigated patterns of speciation in plants of phases of the Alpine uplift (Roquet et al., 2013). In contrast, the Alpine system sensu Ozenda (1995), i.e. the system both /Auricula and Soldanella are thought to have appeared formed by all West-European mountain ranges, from the more recently (i.e. during the Pliocene and Pleistocene Sierra Nevada to the mountains of the Balkans (Fig. 1). In respectively) and it has been suggested that their diversifica- the Alpine system, the young ages inferred for many groups tion largely occurred during Pleistocene glacial cycles (Zhang suggest that the Pleistocene represented a period of active & Kadereit, 2002, 2004). diversification for the Alpine flora (Stebbins, 1984; Vargas, 2003; Kadereit et al., 2004). For the reasons explained above, Phylogenetic inference it is hypothesized that allopatric speciation played a major role in plants of the Alpine system (Ozenda, 1995; Vargas, To identify species pairs and estimate their divergence times, 2003). Seven groups of plants are endemic to the Alpine sys- we needed a dated phylogeny including our three focal tem (Ozenda, 1995). In this article, we used three of these groups. A large dated phylogeny including Androsace s.l., clades as model system and focussed on species from An- Primula s.l. and Soldanella has been recently published drosace sect. Aretia, Primula sect. Auricula, and Soldanella, all (DeVos et al., 2014), but it only includes c. 30% of species of which belong to Primulaceae. Comprising more than 15 in our focal groups. Detailed phylogenetic trees are available described species each, they represent the three largest plant for /Aretia (Boucher et al., 2012), /Auricula (Zhang & Kader- clades in the Alpine system (Ozenda, 1995), hence the best eit, 2004; Crema et al., 2013), and Soldanella (Zhang & possible examples of plant diversification in this region. Kadereit, 2002), but they were obtained using different To determine the relative contribution of geographical and genetic markers and dating techniques, preventing the environmental factors to speciation, we first built a new possibility of combining them into a single dated tree for

592 Journal of Biogeography 43, 591–602 ª 2015 John Wiley & Sons Ltd Speciation in Alpine Primulaceae

(a) (b)

200km

(c) (d)

Figure 1 Distribution of alpine Primulaceae in the Alpine system. In each panel, the background map shows maximum temperature in June over Western Europe, darker areas being colder. Panel A shows the different mountain ranges that constitute the Alpine system. In the three other panels, occurrence points used in this study are shown for species of /Aretia (b, photo: A. pubescens), /Auricula (c, photo: P. apennina) and Soldanella (d, photo: S. major). Only points that fall at least 20 km apart are shown here (1700 points represented out of the 17,300 used). All photographs were taken by F. Boucher. our target groups. We thus collected ITS2 sequences from employed a GTR+Γ+I model of nucleotide substitution Genbank for all described species in /Aretia, /Auricula, and (Tavare, 1986) and a lognormal relaxed Soldanella, as well as two outgroups (Ardisia crenata Sims. (Drummond et al., 2006). and Anagallis arvensis L.). For some species in /Aretia and / Although the fossil record for Primulaceae is sparse, we Auricula, multiple were included to verify species could calibrate three interior nodes in the phylogeny: An- . We also included additional species in Androsace drosace s.l. was given a minimum age of 5.3 Ma based on s.l. and Primula s.l. to break up long branches leading to our fossil seeds from the Miocene (Dorofeev, 1963a), Primula three groups in the phylogeny, a practice aimed at increasing s.l. was given a minimum age of 15.97 Ma based on fossil phylogenetic accuracy (Poe, 2003). These species were chosen seeds from Primula rosiae from the Middle Miocene (Czaja, to sample all divergences identified in previous studies 2003), and the group formed by Androsace + Primula + Sol- (Boucher et al., 2012; DeVos et al., 2014). To produce a danella was given a minimum age of 28 Ma based on fossil well-resolved phylogenetic hypothesis, sequences of trnL–F seeds of angulata (Primulaceae) from the Early and trnG–R were added for Androsace s.l. and sequences of Oligocene (Dorofeev, 1963b). For these three groups, we trnL and rpl16 were added for Primula s.l. (all sequences fixed an absolute maximum age to 72 Ma, based on a fossil obtained through Genbank, accession numbers available in flower from the , which could be attributed Table S1 in Supporting Information). Sequences were aligned to primuloids (i.e. Maesaceae, Theophrastaceae, Primu- using MUSCLE (Edgar, 2004) and alignments were edited laceae, Myrsinaceae) but not to Primulaceae sensu stricto manually to remove ambiguous sites. The combined data set, (Friis et al., 2010). In each case, stem nodes were calibrated comprising 3301 aligned nucleotide positions in 81 taxa and we applied uniform priors between the minimum and (61% missing data), was analysed in a Bayesian framework maximum bounds because we consider that the paucity of using beast (Drummond et al., 2012), with two partitions: fossil Primulaceae does not allow for more informative ITS2 versus all chloroplast markers. For each partition, we priors.

Journal of Biogeography 43, 591–602 593 ª 2015 John Wiley & Sons Ltd F. C. Boucher et al.

To facilitate analysis, the monophyly of /Aretia, /Auricula, Geographical overlap Soldanella and Primula s.l. + Soldanella was enforced based on previous phylogenetic evidence (DeVos et al., 2014). All If allopatry were the dominant mode of speciation we would other parameters in BEAST were set to their default values expect most species pairs in Alpine Primulaceae to have allo- and two independent MCMC chains were run for 20 million patric distributions. Following Anacker & Strauss (2014), we generations each. One hundred trees were randomly sampled estimated the geographical range of each species by placing a from the posterior distribution generated by the beast 10 km round buffer around each occurrence points and analysis, and were trimmed to species level prior to further merging all overlapping circles obtained. Range overlap was analyses. then measured as the area of overlap between the ranges of two species divided by the range size of the species with the smaller range. Occurrence and environmental data

Distribution data for species in /Aretia,/Auricula and Solda- Environmental overlap nella was obtained from various sources (see Appendix S2). All points with >1 km positional uncertainty were discarded. If selection for different environmental preferences were an If several points were located in the same 100 m pixel we important driver of speciation in alpine Primulaceae, we only kept one presence point for further analyses. The final would expect sister-species to exhibit little environmental data set included 17,300 occurrence points for all species in overlap. Overlap in substrate preferences was measured as the three groups, except for Soldanella angusta, S. mar- 1 if two sister-species had overlapping substrate prefer- marossiensis, S. oreodoxa and S. rugosa for which no occur- ences, and as 0 if the species had divergent substrate pref- rence data could be retrieved (63 spp. in total, Fig. 1). erences. To accurately quantify differences in the climatic niche To quantify niche overlap between sister-species in multi- between species, we used a climatic data set for Europe with dimensional environmental space we used the method of a 100 m spatial resolution, including monthly values for Broennimann et al. (2012). This method compares the den- potential evapotranspiration, moisture index (precipitation – sity of species occurrences against the available environment potential evapotranspiration), solar radiation, precipitation, in a study area along multiple environmental axes in a multi- relative air humidity and temperature. These layers were gen- variate analysis, and measures niche overlap along the axes erated by first downscaling long-term monthly mean, mini- of this multivariate analysis. Here, for each sister-pair we mum, and maximum temperature and monthly precipitation considered the first two axes of a PCA calibrated on the from WorldClim (Hijmans et al., 2005) across Europe. entire environmental space sampled over the ranges of each Downscaling was performed by local regressions in a moving sister-species pair. Climatic niche overlap between the two window (see Zimmermann et al., 2007 for details). Solar taxa was measured using Schoener’s D (Schoener, 1970) in radiation, potential evapotranspiration and moisture index this reduced environmental space. However, two taxa might were calculated following the procedures described in Zim- exhibit differences in their climatic niches only because they mermann et al. (2007). To reduce the number of climatic live in regions where different climates occur (i.e. different variables used (96 initially), we measured all variables over realized niches), not because they have different physiologies 10,000 randomly chosen points in Western Europe, and (i.e. different fundamental niches; see Soberon, 2007; Bou- investigated their correlations (see Appendix S2). From that cher et al., 2014). To control for the potentially confounding we selected six variables to represent the climate of our cho- effect of background climate, we used a niche similarity test sen study area: solar radiation in June, minimum tempera- (Warren et al., 2008), which compares the observed climatic ture in January, maximum temperature in June, relative air niche overlap between two species to the overlap measured humidity in June, and precipitation both in January and in between one of the species’ niche and the randomized niche June. Minimum and maximum temperatures are indeed of its sister. This randomized niche is obtained by randomly more important than mean temperatures for the physiology sampling occurrence points in the region where the species of alpine plants (Korner,€ 1999) and values of climatic vari- occurs. For each sister-pair comparison, we used 99 random- ables from January and June were selected to characterize cli- izations to test whether the niches of the two taxa are more mate in the coldest month of the year (January) and during similar than expected by chance, given their geographical dis- the flowering period of most species from our three focal tributions. groups (June). These six variables were then transformed using principal component analysis (PCA) in each pairwise Integrating phylogenetic uncertainty in sister-pair niche comparison (see below). comparisons Finally, information on species’ substrate preferences was obtained from the literature (Smith & Lowe, 1997; Zhang & To understand patterns of speciation in alpine Primulaceae, Kadereit, 2002, 2004; Richards, 2003) and personal field we first measured the proportion of strictly allopatric sister- observations. Each species was classified as being either species pairs, the proportion of sister-pairs that overlap in calcifuge, calcicole or indifferent to substrate type. substrate preferences, and the proportion of sister-pairs that

594 Journal of Biogeography 43, 591–602 ª 2015 John Wiley & Sons Ltd Speciation in Alpine Primulaceae have significantly similar climatic niches. In addition, we also the robustness of these metrics to uncertainty in sister-pair investigated whether these metrics are correlated with time estimation. since divergence of the sister-pairs. We expect that, if specia- tion is allopatric, range overlap should be zero for recently Temporal patterns of diversification diverged species pairs and may increase with time since divergence if the two sister-species shift their ranges (Barra- To test whether diversification rates of alpine Primulaceae clough et al., 1998). Similarly, if divergence in ecological increased during the Quaternary glacial cycles, we compared preferences is an important driver of speciation, we expect two different diversification models: (1) a constant birth- recently diverged species pairs to show little ecological over- model during the entire history of each group and (2) a birth- lap, but ecological overlap may increase with time. death model where rates of speciation and can differ We first evaluated all of these metrics over the species before and after the Pliocene/Pleistocene boundary (i.e. 2.4 Ma pairs that received > 85% posterior probability in BEAST’s ago). As extinction is difficult to estimate from molecular phy- maximum credibility tree (Table 1, Fig. 2). This pro- logenies (Rabosky, 2010) we also compared models without cedure considers phylogenetic uncertainty, as sister-species extinction, i.e. pure-birth models. All models were fitted on are inferred from the posterior distribution of trees. Yet, if the three groups separately. We note here that /Aretia also groups are poorly resolved, very few sister-pairs may be includes the North American Douglasia Lindley, which was not recovered with high posterior probability, reducing sample included in our phylogeny. However, Douglasia is mono- size for comparative analyses. In addition, sister-pairs pre- phyletic (Boucher et al., 2012), hence our analysis may only sent in trees sampled from the posterior distribution may miss one speciation event in the Alpine system (the one leading show clear patterns of geographical or environmental over- to the stem lineage of Douglasia), while it includes the remain- lap. For example, all species in a clade may be allopatric, ing 19 speciation events. Models were fitted on 100 different strongly supporting allopatric speciation irrespective of the trees from BEAST trimmed to the species level and their fit was precise sister-species relationships. To account for phyloge- compared using the Akaike Information Criterion (AIC). netic uncertainty in our analyses, we calculated metrics of Unless otherwise specified, all analyses were run in R (R range and ecological overlap for all species pairs retrieved Core Team, 2014) using packages ade4 (Dray & Dufour, in each of the 100 trees randomly sampled from the pos- 2007), raster (Hijmans & van Etten, 2012) and TreePar terior distribution. This provides a complete assessment of (Stadler, 2011).

Table 1 Range and niche differences in the 11 sister-species pairs from the MCC tree with more than 85% posterior support. For each sister-species pair, columns indicate: the posterior probability that the two species are sister, the median divergence time inferred from the posterior of BEAST, range overlap, overlap in climatic niches, the P of the niche similarity test and overlap in substrate preferences respectively.

Posterior Divergence Geographical Climatic P value Species pair probability time (Ma) overlap overlap similarity Substrate overlap

Androsace wulfeniana 0.87 2.98 0% 27% 0.02 Same substrate A. brevis A. hausmanii 0.99 4.07 0% 0.1% 0.52 Same substrate A. komovensis A. cylindrica 0.98 5.31 2% 10% 0.1 Different substrates A. vandellii A. pubescens 0.91 1.56 44% 79% 0.01 Same substrate A. helvetica A. laggeri 1 0.88 7% 35% 0.01 Same substrate A. halleri A. lactea 0.86 4.72 0% 10% 0.01 Different substrates A. mathildae A. adfinis 1 3.29 0% 4% 0.01 Same substrate A. cantabrica Primula latifolia 0.99 2.04 20% 49% 0.01 Same substrate P. marginata P. daonensis 1 0.52 4% 41% 0.01 Same substrate P. hirsuta P. glutinosa 0.97 4.05 3% 48% 0.01 Different substrates P. spectabilis P. glaucescens 0.99 3.08 0% 0.3% 0.02 Different substrates P. deorum

Journal of Biogeography 43, 591–602 595 ª 2015 John Wiley & Sons Ltd F. C. Boucher et al.

Figure 2 Maximum clade credibility tree of Primulaceae. The tree includes all species in /Aretia (shown in blue), /Auricula (red) and Soldanella (purple), as well as other taxa used for rooting or to improve estimation of branch lengths (black). Black stars show branches leading to clades with >85% posterior support, red stars show posterior support >95%. See Fig. S1 for support at other nodes. Time is shown in Ma on the x-axis, based on median node heights estimated in the posterior of BEAST. Placement of fossils is shown using large circles at the nodes which were calibrated: Primulaceae in black, Androsace s.l. in blue, Primula s.l. in red. The three groups of alpine Primulaceae likely appeared prior to the Pleistocene (see Fig. S2 for uncertainty in divergence times).

(see Fig. 2). Previous relationships in /Aretia were mostly RESULTS confirmed, and the reciprocal monophyly of the two sub- sections of /Auricula (Zhang & Kadereit, 2004) was recov- Phylogenetic inference ered with more than 97% posterior support. In Soldanella, Convergence of the two BEAST runs was verified and the the western-central group (Zhang & Kadereit, 2002) was first 10 millions generations of each run were discarded as confirmed to be monophyletic with 94% support, while the burn-in. The two runs were combined to produce a maxi- eastern group was poorly resolved and inferred to be para- mum clade credibility tree (hereafter MCC). Most nodes in phyletic (Fig. 2). In the MCC, 11 sister-species pairs were the phylogeny outside of the three focal groups received inferred with more than 85% posterior probability and 100% posterior support (Fig. 2). In /Aretia, 17 out of 25 could be used for sister-species comparisons (hereafter nodes received >85% posterior support, while this was the ‘main sister-pairs’, see Table 1, Fig. S1). The choice of this case for 16 out of 29 nodes in /Auricula.InSoldanella, threshold represents a compromise between the level of however, only four of the 15 nodes received > 85% support support required and the number of sister-pairs retained in

596 Journal of Biogeography 43, 591–602 ª 2015 John Wiley & Sons Ltd Speciation in Alpine Primulaceae the MCC. All species for which multiple subspecies were Temporal patterns of diversification included (i.e. Androsace adfinis, A. cylindrica, Primula auric- ular and P. lutea) were found to be monophyletic with In the three groups, pure-birth models provided a better fit more than 88% posterior support. Dating yielded a median than birth-death models. In Soldanella, a time-constant crown age of 8.2 Ma (95% HPD: 4.4–14.5 Ma) for /Aretia, model and a model with a shift in diversification rates at the 6.6 Ma (3.4–12.0) for /Auricula and 4.3 Ma (1.4–9.5) for Pleistocene had very similar AIC scores (median Soldanella (see Fig. 2 and Fig. S2 for uncertainty in diver- DAIC = 0.74 over the 100 trees). Under the constant pure- gence times). birth model, the net diversification rate in Soldanella was 0.48 species/Myr on average over the 100 trees (range: 0.23– 0.93, see Table 2). In /Aretia and /Auricula, however, the Geographical overlap model with the lowest AIC was a pure-birth model with dif- Most sister-pairs from the three groups were allopatric: ferent diversification rates in different epochs (median among the 11 main sister-pairs, five were strictly allopatric DAIC> 3.71 for /Aretia, median DAIC> 3.38 for /Auricula). and four had less than 10% range overlap (Table 1). Only In both clades a decrease in diversification rates was observed two sister-pairs showed substantial range overlap: An- in the Pleistocene (Table 2): in /Aretia the net diversification drosace helvetica and A. pubescens (44%), and Primula rate before the Pleistocene was 0.34 sp./Myr (range: 0.11– latifolia and P. marginata (20%). The same pattern was 0.71), and dropped to 0.11 sp./Myr (range: 0.001–0.29) dur- found when considering phylogenetic uncertainty: on ing the Pleistocene; in /Auricula, this rate dropped from 0.51 average over the 100 trees, 57% of sister-pairs were sp./Myr (range: 0.17–1.18) to 0.19 sp./Myr (range: 0.05– strictly allopatric (range: 47–68% of sister-pairs), and up 0.37). to 85% of sister-pairs had less than 10% geographical overlap (range: 75–91% of sister-pairs). Range overlap DISCUSSION was not correlated with time since divergence of the two sister-species. We investigated the three largest plant radiations in the European Alpine system to gain insights into the relative contributions of geographical barriers versus ecological pref- Environmental overlap erences to their diversification. Our analyses largely relied on In contrast, environmental overlap was high between sister- comparing current ranges and ecological preferences in sis- species. Indeed, over the 11 main sister-pairs, only four spe- ter-species pairs. This methodology has known limitations, cies pairs did not overlap in their substrate preferences since it ignores the possibility that species’ ranges and niches (Table 1). These were: Androsace cylindrica (calcicole) and have evolved since speciation. We attempted to control for A. vandellii (calcifuge), A. lactea (calcicole) and A. mathil- possible post-speciation range movement and niche evolu- dae (calcifuge), Primula glutinosa (calcifuge) and P. spect- tion by measuring the correlation between time since diver- abilis (calcicole), and P. glaucescens (calcicole) and P. gence and range or niche overlap (Barraclough et al., 1998). deorum (calcifuge). Results were the same over the 100 However, range overlap was always very low between sister- trees: on average 71% of sister-species shared the same sub- species pairs while their niches were most often similar, strate preferences (range: 53–83% of sister-pairs). We found regardless of time since divergence. Although we remain that sister-pairs differing in their substrate preferences aware of the potential caveats listed above, this finding diverged earlier than sister-pairs sharing the same substrate: increases our confidence in our results. In addition, we have this was true for the 11 main sister-pairs (mean difference in divergence times = 1.8 Ma, P = 0.017, R2 = 43.2%), as well as over each of the 100 trees (range: 0.3–5.0 Ma, Table 2 Diversification rates in the three studied groups. For significant in 59 trees). each group, net diversification rates (estimated using a pure- Overlap in climatic niches was on average 28% over the birth model, see Material and Methods) are presented separately for the periods before and during the Pleistocene. The average 11 main sister-pairs, and climatic niches were significantly < net diversification rate estimated over the 100 trees from the similar in all sister-pairs (niche similarity test: P 0.05, see BEAST posterior is presented along with its standard deviation. Table 1) except Androsace cylindrica and A. vandellii Values are identical in both periods for Soldanella as a model (P = 0.1) and A. hausmanii and A. komovensis (P = 0.52). with no rate shift in the Pleistocene had an AIC score very close This tendency was confirmed when taking phylogenetic to a model with a shift (see Results). All rates are expressed in uncertainty into account: on average 77% of sister-species species/Myr. – had similar climatic niches (range: 58 90% of sister-pairs). Net diversification Net diversification rate None of the 61 possible sister-pairs found across the 100 Clade rate before the Pleistocene in the Pleistocene trees had significantly divergent climatic niches. Overlap in Æ Æ climatic niches was neither correlated with time since /Aretia 0.34 0.14 0.11 0.06 Æ Æ divergence of the two sister-species, nor with overlap in /Auricula 0.51 0.21 0.19 0.09 Soldanella 0.48 Æ 0.14 0.48 Æ 0.14 substrate.

Journal of Biogeography 43, 591–602 597 ª 2015 John Wiley & Sons Ltd F. C. Boucher et al. used an approach to sister-pair comparisons that fully of the Cape Floristic Region (Van der Niet & Johnson, accounts for phylogenetic uncertainty. This showed that our 2009), suggesting that alpine floras might differ from lowland results are robust to uncertainty in the estimation of sister- floras in their main speciation mechanisms. species, which further increases our confidence in the main patterns of speciation found in the study groups. Although Possible processes driving speciation in alpine these all belong to Primulaceae, patterns of diversification, Primulaceae range and niche overlap are so clear that they enable us to make strong inferences and provide insights into the drivers Although biogeographical and phylogenetic data alone do of speciation in temperate alpine floras in general. not provide enough information to fully elucidate the pro- cesses driving speciation in alpine Primulaceae, they allow us to make hypotheses regarding possible drivers of speciation. Evidence for allopatric speciation with little Allopatric speciation does not require divergent selection to divergence in ecological niches act on the ecological characteristics of incipient species, and Considered together, our results allow us to make robust alone can drive speciation. Drift is especially inferences about the main mode of speciation in alpine likely to be an important driver of speciation in alpine plants Primulaceae. First, a majority (i.e. 57% on average) of sister- since they often have relatively small population sizes and/or species pairs had strictly allopatric distributions, regardless of probably underwent strong bottlenecks during glacial cycles, the time since their divergence. Another 28% of species pairs as suggested for several groups of high-elevation plants from had range overlap between 0 and 10%, which indicates either the Cape Floristic Region (Verboom et al., 2015). However, peripatric, parapatric or again allopatric distributions, three the onset of between incipient species situations that we cannot distinguish since we placed buffers via genetic drift might be rather slow, while selection might of 10 km around occurrence points to calculate range over- speed up the process (Coyne & Orr, 2004). In /Aretia and / lap. These results support the conclusion that geographical Auricula, the occurrence of many naturally occurring hybrids separation played a key role in the diversification of these suggests that reproductive isolation between closely related three groups. The clearest examples are the divergence species is often incomplete, providing circumstantial evidence between Androsace adfinis, distributed in the Alps, and A. that genetic drift might have played an important role in cantabrica, endemic of the Cantabrian mountains, or the speciation. Indeed, eight such hybrids have been described in split between Primula glaucescens, restricted to the Dolo- /Aretia (Smith & Lowe, 1997) and as many as 32 in /Auricula mites, and P. deorum, endemic of the Rila mountains. Sec- (Kadereit et al., 2011). ond, our measurements of ecological niche overlap show However, genetic drift need not be the sole driver of speci- that sister-species most often grow on the same substrate ation in allopatry and divergent selection on ecological attri- (71% of species pairs on average) and have similar climatic butes might also be involved in the building of reproductive niches (77% on average). This similarity in climatic niches is isolation between incipient species, as recently suggested in a especially noticeable since we used many more climatic vari- study of cryptic speciation the high-elevation cape sedge ables and a better spatial resolution than commonly done. Tetraria triangularis (Britton et al., 2014). In plants of the These two results suggest that allopatric speciation with little European Alpine system, substrate type has been shown to ecological niche divergence has been the main mode of spe- be a major driver of genetic structure (Alvarez et al., 2009). ciation in /Aretia,/Auricula, and Soldanella, making these Since we found that on average 29% of sister-species pairs in groups classic examples of non-adaptive radiations (Gitten- alpine Primulaceae grow on different substrates, substrate berger, 1991). A similar scenario has been suggested for specialization could have contributed to speciation in the many other groups of alpine plants (Ozenda, 1995; Vargas, groups studied here. Indeed, different substrates require dif- 2003; Kadereit et al., 2004; Gehrke & Linder, 2011; Verboom ferent physiological adaptations to cope with pH or chemical et al., 2015). One possible reason for this widespread pattern elements (e.g. excess of calcium and deficiency in phospho- is that allopatric speciation, which is facilitated by the frag- rus and iron in alkaline soils; Zohlen & Tyler, 2004). Hybrids mentation of alpine habitats, does not require that the cli- between plants adapted to two different substrates might matic niches of sister-species differ (Wiens & Graham, 2005). thus show poor performance on either substrate, as shown Furthermore, failure of climatic niches to evolve over short for hybrids between Primula hirsuta (silicicole) and P. lutea time-scales might indeed explain why some populations of a (calcicole, Kadereit et al., 2011). In addition, since different species become isolated from each other when the environ- substrates are often spatially separated in mountains, diver- ment changes quickly and some environments become gence in substrate preferences would increase spatial isolation unsuitable in the middle of a species range, thus initiating between diverging populations. However, since we found allopatric speciation (Wiens & Graham, 2005). Interestingly, that sister-species pairs growing on different substrates tend opposite results involving frequent niche divergence between to be significantly older than pairs growing on the same sub- sister-pairs has been found in floras from non-alpine regions, strate, divergence in substrate preferences might not be initi- like the California Floristic Province (Anacker & Strauss, ating speciation in alpine Primulaceae, but rather occur after 2014), the Arctic (Theodoridis et al., 2013), or the lowlands divergence. Finally, since our study only considered a few

598 Journal of Biogeography 43, 591–602 ª 2015 John Wiley & Sons Ltd Speciation in Alpine Primulaceae dimensions of species’ ecological niches, it is still possible Pleistocene following after glacial retreats that divergent selection on other niche dimensions, like (Stebbins, 1984), at least for our three study groups. This is interactions with pollinators, micro-, or finer charac- further supported by the fact that only three recently derived teristics of the substrate used by each species, might have polyploid taxa have been reported in these groups: Androsace been involved in speciation. adfinis subsp. brigantiaca, Primula clusiana and some popula- tions of P. marginata. Polyploidization events are relatively few, compared to all speciation events in /Aretia,/Auricula and Timing of diversification of alpine Primulaceae Soldanella, suggesting that polyploid speciation following sec- The three studied groups of alpine Primulaceae probably origi- ondary contact took place (see Casazza et al., 2012), but did nated before the Pleistocene (Fig. 2, Appendix S1). Contrary not play a major role in the history of alpine Primulaceae. to our expectations, the Pleistocene was probably not a period We acknowledge that our interpretation is influenced by of accelerated diversification, for we found that diversification the details of molecular dating analyses, which yielded actually slowed down at this time in both /Aretia and /Auricula older crown ages for both /Auricula and Soldanella than (Table 2). This conforms with results from earlier phyloge- earlier studies (see detailed discussion in Appendix S1). netic analyses in /Aretia (Roquet et al., 2013) and /Auricula Indeed, a Pleistocene origin for these groups would yield (Kadereit et al., 2004), which documented diversification higher support to the secondary contact model of specia- slowdowns using phylogenetic methods that differ from those tion, as suggested previously for /Auricula (Kadereit et al., used in our study. In light of these findings, it appears that the 2004, Casazza et al., 2012). Although our phylogenetic general hypothesis that plants of the Alpine system diversified study of Primulaceae includes more fossil calibration mostly during the glacial cycles of the Pleistocene may not rep- points than previous ones and should thus produce the resent the only possible explanation to the origin of species most accurate age estimates possible, uncertainty is still diversity in these environments. Rather, extensive diversifica- high (see Appendix S1). Given the scarcity of fossils in tion may have taken place in the late Miocene and Pliocene Primulaceae, it is, however, unlikely that higher accuracy during the last phases of the Alpine uplift (Ozenda, 1995), can soon be reached. Furthermore, we would like to while the Pleistocene might have been mostly marked by dif- emphasize that the diversification slowdowns detected in ferentiation within species (Vargas, 2003). These results are both /Aretia and /Auricula do not depend on absolute consistent with evidence from the fossil record suggesting that nodal age estimates per se. few speciation events took place in response to Pleistocene glacial cycles (Bennet, 2004). CONCLUSION Slowdowns in diversification are often interpreted as evi- dence for diversity-dependence via intra-clade competition In this study we investigated patterns of diversification of leading to the filling of geographical and/or ecological space the three largest plant clades of the European Alpine sys- (Moen & Morlon, 2014). However, many species in /Aretia tem: Androsace sect. Aretia, Primula sect. Auricula and Sol- and /Auricula grow in rock crevices that generally contain soil danella. Two clear patterns emerged across the three with reasonable levels of nutrients (Korner,€ 1999), but host a groups. First, we found strong evidence for allopatric very low density of plant individuals. Hence, it is unlikely that speciation with little ecological divergence as the main plants in these groups experience strong competition for light mode of speciation, suggesting that these groups represent or nutrients, although they might compete for pollinators. On non-adaptive radiations. Second, the origins of these three the contrary, facilitative interactions often dominate in alpine clades likely trace back to the late Miocene or Pliocene and plant communities (Callaway et al., 2002). We thus believe they had already started to diversify significantly before the that diversity-dependence is not responsible for the diversifica- Pleistocene. Although specific to the three studied groups, tion slowdowns observed in these two groups. Rather, the our results bear important implications for our understand- deceleration of diversification might be due to increased ing of diversification in plants of the Alpine system, as well extinction during the Pleistocene following drastic range con- as in temperate alpine floras in general. Finally, we note tractions imposed by glacial cycles, or to a progressive reduc- that two out of the three groups that we investigated (/Are- tion in species’ range size as events of allopatric speciation tia and /Auricula) are of polyploid origins (Kress, 1963), as occurred, reducing opportunities for further allopatric specia- is the case for several other fast radiations of plants and tion events (Moen & Morlon, 2014). Our finding that pure- vertebrates (Seehausen, 2004). Whether or not these groups birth models had the best fit in the three groups should not be are of origins and whether or not this influenced interpreted as evidence that extinction did not occur, as it is diversification rates, as suggested by Seehausen (2004), difficult to estimate extinction rates from molecular phyloge- remains to be explored. nies (Rabosky, 2010). Therefore’, distinguishing between the two alternative explanations for diversification slowdowns pre- ACKNOWLEDGMENTS sented above is not possible. Our results, however, allow us to reject the alternative We are indebted to Y. Bouchenak-Khelladi and Y. Xing model in which speciation would have increased during the for help with phylogenetic inference. We thank T. Karakiev,

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F. Prosser, B. Vres, W. Willner, the CBNA, CBNMED and Casazza, G., Granato, L., Minuto, L. & Conti, E. (2012) Poly- InfoFlora for sharing occurrence data with us and various ploid and Pleistocene glacial cycles: A case study members of the Institute of Systematic Botany of the Univer- from the alpine primrose (Primula- sity of Zurich€ for insightful comments on previous versions ceae). BMC , 12, 56. of this manuscript. This work was supported by a Plant Fel- Coyne, J.A. & Orr, H.A. (2004) Speciation. Sinauer Associates lows grant to F.B. Inc, Sunderland, Massachusetts, USA. Crema, S., Kadereit, J.W. & Cristofolini, G. (2013) Phyloge- netic Insights into Primula Sect. Auricula in the Apennine REFERENCES Peninsula. Flora Mediterranea, 23, 157–172. Alvarez, N., Thiel-Egenter, C., Tribsch, A., Holderegger, R., Czaja, A. 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and broadleaf tree species in Utah. Journal of Applied Ecol- BIOSKETCH ogy, 44, 1057–1067. Zohlen, A. & Tyler, G. (2004) Soluble inorganic tissue phos- Florian Boucher is a post-doctoral researcher at the Institute phorus and calcicole-calcifuge behaviour of plants. Annals of Systematic Botany of the University of Zurich.€ He is inter- 94 – of Botany, , 427 32. ested in the evolution of species’ ranges and climatic niches, and in their influence on diversification, especially in alpine plants.

SUPPORTING INFORMATION Author contributions: F.B., N.Z. and E.C. conceived the Additional Supporting Information may be found in the ideas; F.B. built the phylogeny and analysed the data; F.B. led online version of this article: the writing with substantial contributions from N.Z. and E.C.

Appendix S1 Phylogenetic inference and dating. Appendix S2 Occurrence data and climatic quantification. Editor: Stephen Cavers

DATA ACCESSIBILITY

All data used in this article are available upon request via e-mail from the corresponding author.

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