Pleistocene Climatic Fluctuations Promoted Alternative Evolutionary Histories in Phytelephas Aequatorialis, an Endemic Palm from Western Ecuador

Received: 23 April 2020 | Accepted: 26 November 2020 DOI: 10.1111/jbi.14055

RESEARCH ARTICLE

Pleistocene climatic fluctuations promoted alternative evolutionary histories in Phytelephas aequatorialis, an endemic palm from western Ecuador

1Section for Ecoinformatics and Biodiversity, Department of Biology, Aarhus University, Abstract Aarhus C, Denmark Aim: Pleistocene (2.58 Ma–11.7 ka) climatic fluctuations have shaped intraspecific 2 IRD, UMR DIADE, Université de genetic patterns worldwide; however, their impact on species in many regions re- Montpellier, Montpellier, France 3Center for Biodiversity Dynamics in a mains unknown. In order to determine the impact of Pleistocene climatic fluctuations Changing World (BIOCHANGE), Department on the tropical rain forests of western Ecuador, we explored the evolutionary history of Biology, Aarhus University, Aarhus C, Denmark of the endemic palm Phytelephas aequatorialis. 4Facultad de Ciencias Exactas y Naturales, Location: Western Ecuador, north-western South America. Pontificia Universidad Católica del Ecuador, Taxon: Phytelephas aequatorialis (Arecaceae). Quito, Ecuador Methods: One hundred and seventy-six nuclear genes were sequenced in 91 indi- Correspondence viduals for phylogenomic and population structure analyses. The time of divergence Sebastián Escobar, Section for Ecoinformatics and Biodiversity, between identified genetic lineages was estimated using a coalescent phylogenomic Department of Biology, Aarhus University, analysis. Palaeoecological niche modelling analyses were performed to determine DK 8000 Aarhus C, Denmark. Email: [email protected] areas of historical climatic suitability since the Last Glacial Maximum (LGM; 22 ka) that potentially acted as forest refugia during the Pleistocene. A Wilcoxon test and Funding information SENESCYT; Independent Research Fund Pearson correlations were used to explore how current levels of genetic diversity, in Denmark, Grant/Award Number: 9040- terms of expected heterozygosity (Hs), have been shaped by several palaeoclimatic 00136B; Agence Nationale de la Recherche, Grant/Award Number: ANR-15- CE02- and geographic factors. 0002-01; International Mixed Laboratory Results: Phylogenomic and population structure analyses revealed two main ge- BIO_INCA; International Palm Society netic lineages with a north–south distribution, which diverged 1.14 Ma during the Handling editor: Carina Hoorn Pleistocene. Two potential Pleistocene refugia were identified, one along the Pacific coast of Ecuador and one in the Andean foothills of south-western Ecuador. The location of these refugia agrees with the spatial location of the two genetic lineages. Within the Andean foothills, Hs was lower for the southern lineage than for the northern lineage. Hs significantly increased with decreasing latitude across the species as a whole. Main conclusions: Pleistocene climatic fluctuations promoted intraspecific divergence in P. aequatorialis within the rain forests of western Ecuador. The Andean foothills of south-western Ecuador could be an important area for rain forest evolution

Henrik Balslev and Thomas L. P. Couvreur should be considered joint senior authors.

This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes. © 2021 The Authors. Journal of Biogeography published by John Wiley & Sons Ltd.

Journal of Biogeography. 2021;00:1–15. wileyonlinelibrary.com/journal/jbi | 1 2 | ESCOBAR et al.

because they potentially remained climatically suitable throughout the Pleistocene. Lower genetic diversity in the southern lineage, which apparently remained isolated in the Andean foothills during glacial cycles, adds evidence to the presence of a forest refugium in south-western Ecuador. The geographic pattern in genetic diversity suggests that P. aequatorialis colonized western Ecuador from the north. This study supports the role of Pleistocene climatic fluctuations in promoting intraspecific divergence, and for the first time, we show their impact west of the Andes.

KEYWORDS Arecaceae, ecological niche modelling, genetic diversity, last glacial maximum, north–south genetic discontinuity, phylogenomics, phylogeography, population structure, rain forest

1 | INTRODUCTION decreasing to 2,000 mm to the south (Dodson & Gentry, 1991). In addition to climatic variation, the rain forests of western Ecuador Climatic fluctuations during the Pleistocene (2.58 Ma – 11.7 ka) present a wide altitudinal range from sea level up to 1,500 m.a.s.l. have shaped the current genetic patterns of plant and animal species They are distributed in the lowlands and along the small cordilleras across species-rich tropical rain forests worldwide. The forest refu- of the Pacific coast, as well as along the adjacent western Andean gium hypothesis stipulates that Pleistocene glacial cycles promoted foothills that gradually narrow towards the south (Cerón et al., 1999; retraction and fragmentation of tropical rain forests, isolating popu- Dodson & Gentry, 1991; Sierra, 2013). Thus, the rain forests of lations within climatic suitable areas enhancing allopatric speciation western Ecuador could have responded differently to Pleistocene (Haffer, 1969). Even though the role of the Pleistocene in promoting climatic fluctuations because of their north/south topographic and speciation across the Neotropics has been questioned (Bush, 1994; climatic heterogeneity. Colinvaux et al., 1996, 2000; Hoorn et al., 2010; Leite et al., 2016), The location of rain forest refugia during the Pleistocene in west- it has played a major role in shaping intraspecific genetic diversity ern Ecuador is still poorly understood. Overall, Neotropical rain for- (Hewitt, 2000). Intraspecific genetic lineages have originated in ests are thought to have retracted leading to isolated forest patches areas where tropical species took refuge during the Pleistocene during glacial cycles where temperatures dropped ~5°C compared to because isolated populations restricted gene flow (Cabanne pre-industrial values (Heine, 2000; Hewitt, 2000). Based on current et al., 2016; Carnaval & Bates, 2007; Carnaval et al., 2009; Diniz- patterns of plant distribution, lowland rain forests are hypothesized Filho et al., 2016; Thomas et al., 2012; Vasconcellos et al., 2019). to have survived the Pleistocene within the Chocó refugium, situ- Additionally, higher levels of genetic diversity are usually observed ated along the Pacific coast in north-western Ecuador and western in palaeoclimatically suitable areas where tropical species persisted Colombia (Gentry, 1982; Prance, 1982). Although evidence of the during the Pleistocene (Carnaval et al., 2009; Faye et al., 2016; climatic stability of this region has only been documented for the Mousalli et al., 2009; Walker et al., 2009) because long-term popu- Holocene (10 ka – present; Behling et al., 1998), the high levels of lations tend to accumulate unique genetic diversity when compared precipitation linked to its proximity to the Pacific Ocean may have al- to more recently expanding ones (Excoffier et al., 2009). Within the lowed rain forests to persist in these lowlands during the Pleistocene Neotropics, the intraspecific genetic implications of the Pleistocene (Hooghiemstra & Van Der Hammen, 1998). have not been explored west of the Andes, hampering our under- In addition, the rain or cloud forests that occur on the Andean standing of the recent evolutionary history of this region. foothills of western Ecuador (hereinafter referred as premon- Western Ecuador is a species-rich and heterogeneous region lo- tane rain forests) could have also acted as refugia during the cated west of the Ecuadorian Andes. The rain forests that occur in Pleistocene. Glacial cooling promoted the down-slope displacement western Ecuador form a highly diverse ecoregion in a relatively small of Neotropical premontane and montane rain forests (Colinvaux area of 34,000 km2 (Gentry, 1986; Olson et al., 2001). For instance, et al., 1996; Valencia et al., 2010). However, the moisture of the ad- 870 native plant species have been recorded in just 1 km2 with jacent lowlands could have determined whether they were able to ~20% of endemicity (Dodson & Gentry, 1978). Unfortunately, this displace and spread into the lowlands ('moist forest model"; Ramírez- biodiversity is highly threatened due to widespread deforestation Barahona & Eguiarte, 2013) or whether they contracted into refugia and land conversion for agricultural purposes (Cerón et al., 1999; at mid-elevations by the opposing forces of aridity and cooling ('dry Dodson & Gentry, 1991; Ferrer-Paris et al., 2019; Olson et al., 2001; refugia model'; Ramírez-Barahona & Eguiarte, 2013). The 'moist for- Sierra, 2013). The rain forests of western Ecuador form a limit be- est model' predicts population connectivity in the lowlands, diffuse tween the humid Chocó to the north and the dry Tumbes region to population genetic structure and high genetic diversity because of the south. Therefore, these rain forests present a north/south gra- spatial heterogeneity (Ramírez-Barahona & Eguiarte, 2013). In this dient of annual precipitation, averaging 7,000 mm to the north and sense, the Andean foothills of north-western Ecuador may have not ESCOBAR et al. | 3 acted as Pleistocene refugia because premontane rain forests poten- Ecuador (Table S1). In each locality, we sampled between one and tially moved down-slope into the Pacific coast and remained in the three individuals, which were separated by at least 100 m between Chocó refugium (Hooghiemstra & Van Der Hammen, 1998). The 'dry them to avoid the sampling of closely related individuals. Two pre- refugia model', in contrast, predicts the isolation of populations at viously collected individuals of the sister species P. tumacana from mid-altitude with subsequent genetic structuring and loss of genetic south-western Colombia were used as outgroup in the phylogenomic diversity because of altitudinal contraction and population reduc- analyses. We collected tissue from the youngest leaf of the individu- tion (Ramírez-Barahona & Eguiarte, 2013). Given that the lowlands als, which was then silica gel-dried in plastic bags. We isolated total of south-western Ecuador remained dry during recent glacial cycles genomic DNA from each sample using the MATAB and chloroform (Heusser & Shackleton, 1994), the adjacent Andean foothills could separation methods as described in Mariac et al. (2014), and checked have harboured premontane rain forests during the Pleistocene be- DNA quality and quantity using a NanoDrop™ One spectrophotom- cause they were not able to displace into the arid lowlands. eter (Thermo Scientific™). Here we explore how Pleistocene climatic fluctuations have shaped intraspecific genetic diversity patterns of an endemic plant species from the rain forests of western Ecuador. We reconstruct 2.2 | Library preparation, sequencing, the evolutionary history of the vegetable ivory palm Phytelephas filtering and assembly aequatorialis Spruce, by inferring its intraspecific phylogenomic relationships, population structure, and palaeoclimatic niche dis- Laboratory work was performed at the facilities of the Institut de tribution. This species is an emblematic element of these forests Recherche pour le Développement (IRD) in Montpellier, France. (Acosta-Solís, 1948; Barfod, 1991; Cerón et al., 1999), occurring We prepared genomic libraries using a modified protocol from from sea level to 1,500 m.a.s.l. (Borchsenius et al., 1998). According Rohland and Reich (2012) and captured nuclear markers using to previous molecular dating studies, P. aequatorialis diverged from a bait kit that targets 176 palm-specific nuclear genes (Heyduk its sister species Phytelephas tumacana O.F. Cook approximately 3 et al., 2016). Total DNA for each individual was sheared to a mean – 4 Ma, shortly before the Pleistocene (Barfod et al., 2010; Escobar target size of 350 bp using a Bioruptor® Pico sonication device et al., 2020; Trénel et al., 2007). Speciation in the genus Phytelephas (Diagenode). We then repaired, ligated and nick filled-in the DNA apparently occurred by the action of Andean uplift (Escobar before amplifying it for 10 cycles as a pre-hybridization step. Next, et al., 2020) followed by shifts in pollination mechanisms and vege- after cleaning and quantifying the DNA, we bulked the samples in tative morphology (Barfod et al., 2010). Thus, posterior intraspecific two libraries and added the biotin-labelled baits to the libraries in diversification in Phytelephas species may have occurred during the order to hybridize the targeted regions of the bait kit used (Heyduk Pleistocene. In this context, P. aequatorialis appears as an adequate et al., 2016). We used streptavidin-coated magnetic beads to im- model species for studying how Pleistocene climatic fluctuations mobilize the hybridized biotin-labelled baits applying a magnetic impacted the evolution of rain forest species in western Ecuador. field, and then discarded the unbounded DNA contained in the su- We used a genomic approach by targeting 176 nuclear genes using pernatant. We amplified the enriched DNA fragments eluted from a custom palm baiting kit (Heyduk et al., 2016) sequenced for 91 the beads in a RT-PCR during 15 cycles to complete adapters. Final individuals sampled across the entire distribution of P. aequatorialis. libraries were sequenced with an Illumina® MiSeq (paired-end, We aimed to test whether intraspecific genetic lineages in P. aequa- length 150 bp) at the facilities of the CIRAD (Montpellier, France) torialis are explained by areas of historical climatic suitability that using two lanes. potentially acted as lowland Pleistocene refugia in north-western High-performance computing analyses were done at the South Ecuador and in the Andean foothills of south-western Ecuador. We Green Platform at the IRD. We followed Couvreur et al. (2019) for also explored how several factors have contributed to shape the cleaning and filtering the reads. We controlled the quality using genetic diversity in P. aequatorialis, including the 'moist forest' and FastQC (http://www.bioinformatics.babrah am.ac.uk/projects/ 'dry refugia' models (Ramírez-Barahona & Eguiarte, 2013), palaeo- fastqc), demultiplexed using the 'demultadapt' script (https:// climatic suitability during the Last Glacial Maximum (LGM; 22 ka), github.com/Maillo l/demultadapt) with a 0-mismatch threshold latitude and longitude. With this study we aim to contribute to the and removed the adapters using Cutadapt v.1.2.1 (Martin, 2011). understanding of the recent evolution of Neotropical rain forests Reads with length <35 bp and quality mean values (Q) <30 were west of the Andes. discarded using a custom script for quality filtering (https:// github.com/South G r e e n P l a t f o r m / a r c a d -hts/blob/maste r / s c r i p t s / arcad_hts_2_Filter_Fastq_On_Mean_Quality.pl). Then, we paired 2 | MATERIALS AND METHODS forward and reverse sequences using a comparison script adapted from TOGGLe (Tranchant-Dubreuil et al., 2018), and checked the 2.1 | Geographic sampling and DNA extraction quality again using FastQC. The last 6 bp of the reverse sequences were trimmed to ensure the removal of barcodes in sequences We sampled and georeferenced 91 adult individuals identified in the <150 bp using the 'fastx' script (https://github.com/agordon/ field as P. aequatorialis from 46 different localities across western fastx_toolkit). 4 | ESCOBAR et al.

2.3 | Phylogenomic inference processed the DNA sequences of 88 individuals with the pipeline SECAPR v.1.14 (Andermann et al., 2018). This pipeline is useful DNA sequences were processed using HybPiper v.1.2 (Johnson when SNPs need to be called because it creates a pseudo refer- et al., 2016) to identify target exonic regions and off-target introns, ence formed by the consensus sequences of the target loci, with which are usually more variable than exons. As a result, we obtained the exception of paralog loci that were identified and automati- 'supercontigs' which are formed by targeted regions and surround- cally removed during the process. The use of the aforementioned ing off-target sequences. We filtered the dataset by keeping only pseudo reference is more efficient than the use of the bait kit ref- the loci with >75% of their length reconstructed in >75% of the in- erence in the phylogenomic analyses because the latter is made dividuals. The remaining supercontigs were aligned using the '—auto' from distantly related palm species (Helmstetter et al., 2020). option in MAFFT v.7.305 (Katoh & Standley, 2013) and were cleaned We used BWA v.0.7.12 (Li & Durbin, 2009) to map this pseudo using Gblocks v.0.91b (Castresana, 2000) with default parameters reference to our paired and cleaned reads. GATK v.4 (McKenna and all allowed gap positions. et al., 2010) was used to remove duplicates and to call SNPs. We

HybPiper also detects potential paralogous loci, which were ver- filtered the data by keeping only biallelic SNPs with a mapping ified by building exon trees in RAxML v.8.2.9 (Stamatakis, 2014), in- quality >40%, depth >25, and quality by depth >2 using BCFtools cluding all putative paralogs. We used a GTR + G substitution model (Li, 2011). Additionally, we removed SNPs with allele frequency because it is the most common and general model for DNA analyses <0.01 and excluded monomorphic sites. (Stamatakis, 2016), producing similar inferences as other models The number of clusters were determined with a Discriminant (Abadi et al., 2019). We also used the option '-f a' to perform 100 Analysis of Principal Components (DAPC; Jombart et al., 2010) rapid bootstraps as node support. For this purpose, we used a refer- using the package 'adegenet' (Jombart, 2008) in R v.3.5.3 (R Core ence where exons are split instead of being merged into genes. We Team, 2019). We used successive K-means with 100,000 iterations examined the exon trees manually to check if the potential paralog per value of K (number of clusters) up to K = 20. We examined the sequences that were flagged by HybPiper clustered together, which change in the Bayesian Information Criterion (BIC) of K = 1–20 to would mean that the potential paralogs are more closely related to determine the number of clusters within P. aequatorialis. We then potential paralogs in other individuals than to the alternative se- did a cross-validation of the DAPC analysis to test that the number quences in the same individual (Helmstetter et al., 2020). In this of principal components chosen was correct. case, we treated the exon as a paralog. If >50% of exons belonging to a certain gene were determined as paralogs, then the gene was marked as 'true' paralog and removed from further steps. 2.5 | Divergence time estimates We used the resulting alignments to infer the phylogenomic relationships within P. aequatorialis using the two individuals of P. tumacana as We estimated the divergence time between the resulting genetic outgroup. We used two approaches to construct the intraspecific tree, lineages by constructing a dated coalescent tree using five random coalescence and maximum likelihood. For the coalescence approach, individuals from each clade/genetic cluster, along with the two in- we used the alignments to infer individual gene trees in RAxML with a dividuals of P. tumacana as outgroup. We calculated the root-to-tip GTR + G model, 100 rapid bootstraps and no partitions. We then used variance of their respective RAxML gene trees and rooted them these gene trees to construct a coalescent tree in ASTRAL-III v.5.6.3 with phyx. We then calculated the variance using SortaDate (https:// (Zhang et al., 2018), collapsing branches with support <10 and annotat- github.com/FePhyFoFum/SortaDate/) and chose the 32 most clock- ing the local posterior probability (pp) as node support. For the maximum like loci (most suitable for dating) to reduce the highly demanding likelihood approach, we first added empty sequences where individuals computational requirements of further steps (Smith et al., 2018). were missing in our alignments and concatenated them with phyx (Brown The best nucleotide substitution model of evolution was deter- et al., 2017), generating one partition per loci. We built the maximum like- mined for each alignment using ModelTest (https://github.com/ lihood tree in RAxML using a GTr + G model and 100 rapid bootstraps. ddarriba/modeltest). The corresponding 32 fasta alignments were Phylogenomic trees were visualized in FigTree v.1.4 (http://tree.bio.ed.ac. converted to nexus format using PGDSpider v.2.1.1.5 (Lischer & uk/software/figtree/). Three individuals of P. aequatorialis showed high Excoffier, 2012). node support with P. tumacana in the two intraspecific trees, suggesting The coalescence analysis was run using Bayesian inference close phylogenomic relatedness between these individuals. Therefore, in BEAST v.2.5.2 (Bouckaert et al., 2019). We used the template we used this mixed clade to root our trees and excluded these three indi- 'StarBeastWithSTACEYops' and assigned the individuals to their viduals of P. aequatorialis from further analyses (see Results). respective lineage. We used a strict clock of 0.001 with the 'estimate' box checked and the best nucleotide substitution model of

evolution for each locus as determined in ModelTest. The tree was 2.4 | Population structure generated with a Coalescent Constant Population prior because it is particularly suitable for describing intraspecific relationships (Ritchie We studied the population structure of P. aequatorialis by deter- et al., 2017). The tree was constrained with a secondary calibra- mining the number of genetic clusters within its distribution. We tion placed at the stem node between P. aequatorialis and its sister ESCOBAR et al. | 5 species P. tumacana. We used a uniform prior and constrained the was <0.7 (Dormann et al., 2013). We identified variables that could age of the node between the 95% highest posterior density heights potentially limit the distribution of the species. Potential evapo- (95% HPD) of the speciation time between P. aequatorialis and P. tu- transpiration (PET) is one of the strongest predictors of plant diver- macana (see Escobar et al., 2020 for details), which were used as sity patterns at broad scales (Eiserhardt et al., 2011 and references the minimum and maximum values of the prior (3.01–3.76 Ma). We therein). More specifically, low precipitation and low temperatures ran three independent Markov Chain Monte Carlo (MCMC) analyses can constrain the distribution of palms (Eiserhardt et al., 2011). We with 100 million generations sampling every 10,000 trees in BEAST. therefore chose PET of the wettest quarter and PET of the driest We checked that the MCMC runs reached convergence using Tracer quarter from ENVIREM, and precipitation of the driest month (BIO v.1.7 (Rambaut et al., 2018), ensuring that all effective sample sizes 14) and temperature annual range (BIO 7) from WorldClim as our (ESS) >200. The log and tree files of the three runs were combined predictor variables. We did not choose minimum temperature of the with LogCombiner v.2.5.2 (Bouckaert et al., 2019) with a burn-in coldest month (BIO 6) as a predictor variable because it was highly of 10%. TreeAnnotator v.2.5.2 (Bouckaert et al., 2019) was used to correlated with PET of the driest quarter (>0.7). generate a maximum clade credibility (MCC) tree by summarizing the To model the potential current and LGM distributions of P. aequa- remaining 27,003 trees, and to calculate 95% HPD. The tree was vi- torialis, we used the new 'maxnet' R-package to fit a maximum en- sualized in FigTree. tropy (Maxent) model (Phillips et al., 2017). To reduce the effects of sampling bias with this presence-only algorithm, we used a weighted-target group approach (Phillips et al., 2009). This approach uses 2.6 | Palaeoecological niche modelling a background sample likely to contain the same sampling bias as the occurrence records for the focal species, allowing the bias to be can- We modelled the ecological niche of the species under current celled out of the fitting and thus more accurate presence-only mod- conditions, and then projected it to the LGM to determine the els can be produced (Baumgartner et al., 2018; Merow et al., 2017; potential effect of Pleistocene glaciations on the distribution of Phillips et al., 2009). For this purpose, we used 1,149 occurrence P. aequatorialis. In addition to occurrence records collected from records of palm species within a 200 km buffer around the records our fieldwork, we downloaded records from four online reposito- of P. aequatorialis. The palm records were downloaded from the ries: the Global Biodiversity Information Facility (GBIF.org, 2020), same sources listed above and cleaned in the same way. The buffer the Berkeley Ecoengine (Ecoengine), the Botanical Information size selected includes environments that are probably accessible to and Ecological Network (BIEN v.4.1) and the Latin American the species given its dispersal limitations and the configuration of Seasonality Dry Tropical Forest Floristic Network (DRYFLOR). natural barriers (Barve et al., 2011). We used the linear, quadratic We accessed occurrence records from the repositories using the and product features in the Maxent model to produce smoother R-packages 'BIEN' (Maitner et al., 2018) and 'spocc' (Chamberlain & response curves that reduce overfitting, which is especially rele- Boettiger, 2017), and direct download (http://www.dryflor.info/). vant when projecting to novel conditions (Merow et al., 2013). The We cleaned the downloaded occurrence data before use with the performance of the model was assessed by calculating the average package 'CoordinateCleaner' (Zizka et al., 2019), removing records Continuous Boyce Index (CBI; Hirzel et al., 2006) through a fivefold that met any of the following criteria: recorded prior to 1950; du- systematic spatial blocking technique using the package 'blockCV' plicated records; coordinate uncertainty greater than 5 km; impos- (Valavi et al., 2019). CBI values range between −1 and 1, where sible or unlikely coordinate; within 10 km of a country capital or values >0 indicate the model's output is positively correlated with political unit centroids; and within 100 m of biodiversity institu- the true probability of presence and values <0 indicate it is nega- tions. All occurrence records were spatially thinned at a distance tively correlated with the true probability of presence (Fielding & of at least 10 km to reduce spatial sampling bias using the pack- Bell, 1997). Model fit was inspected by the average omission rate age 'spThin' (Aiello-Lammens et al., 2015). We retained and used based on the minimum training presence value (ORMTP). ORMTP val- a total of 86 records after cleaning and filtration of data, which ues range from 0 for models that are not overfit to 1 for models were transformed to a Behrmann projection to be used along with that are overfit. For the LGM, we averaged the projections of the resampled climatic variables. three General Circulation Models continuous range maps to form a The current (average 1950–2000) and LGM (22 ka) variables were consensus continuous map. Current and LGM continuous suitability downloaded at a spatial resolution of 2.5 arc-minute from WorldClim maps were converted to binary maps indicating suitable/unsuitable 1.4 (https://worldclim.org; Hijmans et al., 2005) and ENVIREM habitat. For this purpose, we used a threshold that maximizes the (http://envirem.github.io; Title & Bemmels, 2018). Variables were sum of sensitivity and specificity, a frequently recommended ap- then resampled to 5 km resolution using bilinear interpolation with a proach that tends to reflect the prevalence of the modelled species Behrmann projection. Climatic variables from the LGM were down- well (Liu et al., 2013, 2016). We then overlaid the two binary maps loaded for the three General Circulation Models CCSM4 (Gent to show areas of historical climatic suitability that potentially worked et al., 2011), MIROC-ESM (Watanabe et al., 2011) and MPI-ESM-P as refugia during the LGM. We used this overlaid map as an approx- (Giorgetta et al., 2013). To minimize potential issues with collinearity imation of the potential refugia location during previous Pleistocene between predictor variables, we ensured the Pearson's R correlation glacial cycles. 6 | ESCOBAR et al.

2.7 | Determinants of genetic diversity 3 | RESULTS

We explored how palaeoclimatic and geographic factors may 3.1 | Phylogenomic inference have contributed to shape current levels of genetic diversity in P. aequatorialis. For each individual, we obtained the expected het- We obtained a total of 47.167 million reads from the 91 adult in- erozygosity (Hs) using the R-package 'adegenet' (Jombart, 2008) dividuals of P. aequatorialis. We identified 157 loci with >75% of and the index of palaeoclimatic suitability during the LGM its length reconstructed in >75% of individuals. Sixteen loci were from the consensus continuous map using the package 'raster' considered as paralogs and therefore removed, resulting in 141 (Hijmans et al., 2020). We first explored how the 'moist forest' supercontigs. The resulting ASTRAL coalescent tree separated and 'dry refugia' models have potentially shaped genetic diver- most of the P. aequatorialis individuals from the two individuals of sity in the Andean foothills. To do so, we tested whether individ- P. tumacana (Figure 1a). Three individuals sampled in the north- uals from the Andean foothills that acted as Pleistocene refugia ern part of the distribution of P. aequatorialis formed a clade with present lower Hs than the individuals from the foothills that did P. tumacana with high local posterior probability as node support not act as refugia using a Wilcoxon rank sum test in R. We also (pp = 1). The other individuals of P. aequatorialis separated in two performed Pearson's R correlations to regress Hs against values major clades, suggesting the presence of two distinct evolution- of palaeoclimatic suitability during the LGM, latitude and longi- ary units within the species. One of the clades was formed by 79 tude for all individuals. Additionally, genetic differentiation (FST) individuals sampled from northern and central populations (north- between the clusters was calculated with the package 'hierfstat' ern clade), whereas the other clade was formed by nine individu- (Goudet, 2005) using the Nei's method (Thomas et al., 2012). als sampled from southern populations (southern clade). Only the

(a) (b)

FIGURE 1 Intraspecific genetic lineages of the palm Phytelephas aequatorialis in the rain forests of western Ecuador. (a) ASTRAL coalescent tree showing phylogenomic relationships between individuals with local posterior probabilities (pp) as node support. (b) Spatial distribution of the genetic clusters at K = 2 as determined by the DAPC analysis using 88 individuals, and of the individuals used as outgroup in the phylogenomic analyses. The shading represents the Andes and smaller cordilleras in the Pacific coast. The Chimbo River is represented by the light blue line. (c) Membership probability of the 88 individuals at K = 2 as determined by the DAPC analysis ESCOBAR et al. | 7

3.3 | Palaeoecological niche modelling

The mean CBI value was 0.91, indicating that the model performance is better than a random model and therefore adequate. The

model did not over fit as indicated by the low ORMTP value of 0.02. The model accurately predicted the current known distribution of the species with exception of the northernmost part of the range (Figure 3a). The lowlands of north-western Ecuador appeared unsuitable even though P. aequatorialis has been reported in that area, whereas an area in the Andean foothills of Colombia, where the species has not been reported from, appeared as suitable. Overall, climatic suitability of P. aequatorialis decreased during the LGM and its potential distribution remained in the Pacific coast while decreasing on the Andean foothills (Figure 3b). The overlaid map formed FIGURE 2 Dated coalescent tree inferred to estimate the time by the two suitable/unsuitable maps for current and past conditions of divergence between the two intraspecific genetic lineages of the palm Phytelephas aequatorialis in the rain forests of western during the LGM showed two areas of historical climatic suitability Ecuador. The tree was obtained through Bayesian inference (Figure 3c). The first one is located in the Pacific coast while a second in BEAST using a Coalescent Constant Population prior and a smaller area lies on the Andean foothills of south-western Ecuador. StarBeastWithSTACEYops template. Five random individuals of The location of the larger area of historical climatic suitability agrees P. aequatorialis from each genetic lineage were used along with with the spatial distribution of the northern clade/genetic cluster, two individuals of the sister species Phytelephas tumacana as whereas the smaller area agrees with the southern clade/genetic outgroup. The 95% highest posterior density heights (95% HPD) of the time of speciation between P. aequatorialis and P. tumacana cluster. ( 3.01 – 3.76 Ma; Escobar et al., 2020) were used to set a uniform prior to constrain the tree where the arrow indicates. The blue bar represents the 95% HPD of the time estimation and the shade 3.4 | Determinants of genetic diversity represents the time frame of the Pleistocene (2.58 Ma – 11.7 ka)

The nine individuals from the Andean foothills of south-western southern clade showed high node support. The RAxML maximum Ecuador that belong to the southern lineage (Figure 1; Table S1) likelihood tree recovered the same two major clades in P. aequa- showed lower Hs than the 19 individuals from the Andean foot- torialis (Figure S1). Nevertheless, one individual of P. aequatorialis hills of north-western Ecuador that belong to the northern lineage from the northern part of its distribution resolved as sister of the (Wilcoxon rank sum test: W = 11, p = 0.0000278; Figure 4a). We two major clades. The same three individuals of P. aequatorialis did not detect a significant correlation between Hs and the index of 2 from the north formed a clade with P. tumacana with high boot- palaeoclimatic suitability during the LGM (R = 0.0019, p = 0.6854, strap as node support (93). Again, only the southern clade showed t = −0.4064, df = 86; Figure 4b). A significant but weak increase 2 high node support (bootstrap = 100; Figure S1). in Hs with decreasing latitude was inferred (R = 0.099, p = 0.0028, t = 3.0753, df = 86; Figure 4c). Hs was not correlated with longitude 2 (R = 0.0115, p = 0.3183, t = 1.0037, df = 86; Figure 4d). Genetic dif-

3.2 | Population structure and divergence ferentiation (FST) between the two genetic clusters was 0.023. time estimates

A total of 1,394 SNPs were identified from 118 different loci with 4 | DISCUSSION 0% of missing data. We kept 50 principal components after cross- validation, as it was showed appropriate for inferring population Our phylogenomic and population structure analyses revealed structure in DAPC analyses (Figure S2). Two genetic clusters were the presence of two distinct latitudinally oriented evolutionary determined within P. aequatorialis (Figure 1b) based on the values of lineages in the palm species Phytelephas aequatorialis within the BIC and on the analyses of principal components (Figures S3, S4). All rain forests of western Ecuador (Figure 1). Although our intraspe- individuals showed a membership probability to their corresponding cific trees were in general poorly supported, the southern clade cluster >0.9 (Figure 1c). The two resulting clusters are composed by showed high support under the two phylogenomic approaches used the same individuals forming the two major clades in the ASTRAL (Figure 1a; Figure S1). In addition, the individuals from the southern coalescent tree (Figure 1a). The division between the two genetic clade formed a distinct genetic cluster (Figure 1b, 1c), adding more lineages lies around −2.4° in latitude. We estimated that the two ge- evidence to their distinct evolutionary history. A similar pattern of netic clusters diverged during the Pleistocene 1.14 Ma (95% HPD: phylogeographic structure was detected for the widely distributed 1.01 – 2.48; Figure 2). Neotropical palm Oenocarpus bataua, in which two genetic clusters 8 | ESCOBAR et al.

(a) (b) (c)

Climatic suitability Palaeoclimatic suitability Historical climatic suitability

1.0 1.0 2 0.8 0.8 1 0.6 0.6 0 0.4 0.4 0.2 0.2 North 0.0 0.0 South Occurrence records Occurrence records

FIGURE 3 Palaeoecological niche modelling of the palm Phytelephas aequatorialis in the rain forests of western Ecuador using Maxent and a weighted-target group approach for (a) current conditions, and then projected to (b) the Last Glacial Maximum (LGM; 22 ka). Palaeoecological niche during the LGM was modelled under three different General Circulation Models (CCSM4, MIROC-ESM, MPI- ESM-P), which were averaged to construct a consensus continuous map. Dotted line represents the Pacific coastline during the LGM while continuous line represents the current coastline. (c) Areas of historical climatic suitability determined by overlaying binary suitable/ unsuitable maps for current and past conditions (LGM) ranging from 0 (species not present in any of the two periods) to 2 (species present in the two periods). Binary maps were obtained by applying a threshold of maximum specificity and sensitivity to continuous suitability maps. Coordinates of analysed individuals were transformed to Behrmann projection to plot them over the map. Note the overlap of the individuals of the two lineages with areas of historical climatic suitability were also identified in western Ecuador: one located in north-west- The two evolutionary lineages detected for P. aequatorialis di- ern Ecuador/south-western Colombia, and a second one in south- verged 1.14 Ma (95% HPD: 1.01 – 2.48) during the Pleistocene western Ecuador and extending to the east of the Andes (Escobar (Figure 2), a period when glacial cycles impacted the distribution and et al., 2018). Although the genetic structure patterns of both species genetic diversity of species (Hewitt, 2000, 2004). Based on our ge- are not completely identical due to the cross-Andean distribution of netic and palaeoecological niche analyses (Figure 3c), P. aequatorialis O. bataua, they nevertheless suggest the existence of a north–south potentially took refuge in two areas of historical climatic suitability in phylogeographic break in south-western Ecuador. The Chimbo River western Ecuador during the Pleistocene. The spatial distribution of has been proposed as a geographic barrier for some reptiles and am- the recovered genetic lineages overlaps with that of the two inferred phibians in south-western Ecuador (Arteaga et al., 2016), and it lies potential refugia (Figure 3c), indicating that these areas of historical close to the phylogeographical break of P. aequatorialis (Figure 1b). climatic suitability could have played a vicariant role in P. aequato- Nevertheless, individuals that belong to the northern lineage were rialis. Populations from northern and southern lineages apparently sampled south of the river, indicating that the intraspecific diver- became isolated along the narrow strips of the Andean foothills of gence in P. aequatorialis is not caused by this geographic barrier. south-western Ecuador ~1 Ma during a glacial cycle and developed Such north–south phylogeographic breaks have been reported in distinct genetic footprints. Gene flow between northern and south- other tropical rain forests because of differences in past and cur- ern populations could have occurred during the following intergla- rent climate and in geological history, including those from west- cial cycles, hindering speciation between the two genetic lineages as ern Amazonia (Escobar et al., 2018; Jimenez-Vasquez et al., 2017; suggested by their low level of genetic differentiation (FST = 0.023). Reis et al., 2020; Roncal et al., 2015; Symula et al., 2003), eastern The low admixture detected for the three northernmost individuals Brazil (Mousalli et al., 2009), central Africa (Bryja et al., 2014; Faye of the southern lineage (Figure 1c) partially supports the existence et al., 2016; Hardy et al., 2013; Helmstetter et al., 2020; Heuertz of gene flow between the two lineages. It is possible that stronger et al., 2014; Ley et al., 2017; Piñeiro et al., 2017) and Australia secondary contact has occurred within areas not sampled here. (Schneider et al., 1998). Analysing the phylogeographic patterns of Therefore, detailed analyses of the populations located between more plant species will help to elucidate whether a phylogeographic the recovered distributions of the two genetic lineages will help break in south-western Ecuador is common for plants in the region. to elucidate whether gene flow between the lineages has impeded ESCOBAR et al. | 9

(a) (b)

0.45

0.40

0.35 Expected heterozygosity ( Hs ) 0.30 W = 11 R2 = 0.0019 P < 0.0001 P = 0.6854

Andean foothills Andean foothills 0.1 0.200.30.4 .5 0.6 South North Palaeoclimatic suitability

0.45

0.40

0.35 Expected heterozygosity ( Hs ) 0.30 R2 = 0.099 R2 = 0.0115 P = 0.0028 P = 0.3183

−3 −2 −1 0 1 −80.5−80.0−79.5 −79.0 Latitude Longitude

FIGURE 4 Determinants of genetic diversity of the palm Phytelephas aequatorialis in the rain forests of western Ecuador. (a) Expected heterozygosity (Hs) in the individuals from the Andean foothills of south-western Ecuador, which potentially acted as a Pleistocene refugium, is significantly lower than in the individuals from the northern Andean foothills (Table S1) according to a Wilcoxon rank sum test. Blue and red lines show the median values of Hs. (b) Hs of all individuals is not significantly correlated with the index of palaeoclimatic suitability during the Last Glacial Maximum (22 ka). (c) Hs increases with decreasing latitude, (d) but does not with longitude. Blue circles represent individuals from the northern lineage, whereas red circles represent individuals from the southern lineage, as in Figure 1. Dotted lines show the trend of the correlations speciation. Continuous isolation and secondary contact between populations from the Andean foothills of north-western Ecuador the two lineages probably happened several times during the last migrated down-slope during glacial cycles (Figure 3), in agreement 1 Ma. These contraction/expansion dynamics have promoted intra- with the 'moist forest model' of montane and premontane rain specific divergence in P. aequatorialis during the Pleistocene. This has forest evolution (Ramírez-Barahona & Eguiarte, 2013). Given that been observed in plants and animals from other tropical (Cabanne the Pacific coast remained moist at the time (Hooghiemstra & Van et al., 2016; Carnaval & Bates, 2007; Carnaval et al., 2009; Diniz- Der Hammen, 1998), the populations from the Andean foothills of Filho et al., 2016; Faye et al., 2016; Hardy et al., 2013; Helmstetter north-western Ecuador were able to spread into the lowlands and et al., 2020; de Lima et al., 2014; Thomas et al., 2012; Vasconcellos potentially maintain connectivity with the populations from the et al., 2019) and temperate regions (Blanco-Pastor et al., 2019; Park Chocó refugium. Similarly, palynological data showed that plants & Donoghue, 2019). from the eastern Ecuadorian Andean foothills descended ~1,000 m The northern genetic lineage apparently took refuge along the during glacial cycles (Brunschön & Behling, 2010; Cárdenas humid lowlands and on the small mountain ranges of the Pacific et al., 2011) when western Amazonia remained humid and covered coast, but not along the Andean foothills (Figure 3c). The inferred by rain forest (Colinvaux et al., 2000). High palaeoclimatic suitability refugium for the northern lineage overlaps with the southern sec- in coastal regions has also been detected in the Brazilian Atlantic re- tion of the suggested Chocó refugium (Gentry, 1982; Prance, 1982), gion (Carnaval et al., 2009; Mousalli et al., 2009) and in central Africa which could have extended further south to the Pacific coast of (Faye et al., 2016; Helmstetter et al., 2020), indicating that coastal western Ecuador. Our palaeoclimatic analyses suggest that the areas might have been important refugia for tropical rain forests. 10 | ESCOBAR et al.

We also inferred that the southern genetic lineage potentially climatic fluctuations on intraspecific diversity during the LGM varied took refuge in a narrow strip of Andean foothills in south-western between regions and even was different among closely related spe- Ecuador over 1,000 m.a.s.l. (Figure 3c; Table S1). These populations cies within a same region. Thus, the current levels of genetic diver- could have survived the Pleistocene isolated on the slopes of the sity in P. aequatorialis have been potentially shaped by the isolation Andes because they were unable to migrate down-slope into the of individuals in the Andean foothills during glacial cycles and by the lowlands of south-western Ecuador, which remained arid during re- species' colonization history. cent glacial cycles (Heusser & Shackleton, 1994). Thus, the southern Phylogenomic relationships and geographical limits between P. lineage evolved into a distinct genetic group following isolation as aequatorialis and its sister species, P. tumacana, remain to be elu- shown by our phylogenomic (Figure 1a; Figure S1) and population cidated. Phytelephas tumacana occurs in south-western Colombia structure analyses (Figure 1b, 1c). In contrast with the northern (Bernal & Galeano, 2010) at ~50 km from the three individuals lineage, these results support the 'dry refugia model' (Ramírez- of P. aequatorialis with which it formed a highly supported clade Barahona & Eguiarte, 2013) in the premontane rain forests of (Figure 1a; Figure S1). This suggests that P. tumacana could also south-western Ecuador. Thus, the Pleistocene climatic fluctuations occur in north-western Ecuador and we could have sampled it incor- could have affected contiguous rain forests differently depending rectly as P. aequatorialis. There is also the possibility that we sampled on their latitudinal location. A similar response of the Andes may a hybrid population between the two species, given the close prox- have occurred next to the dry inter-Andean valleys of Colombia imity of their geographic ranges. One of the northernmost individu- where palynological data suggested that the Andean foothills con- als of P. aequatorialis resolved with high support as a sister clade of stituted safe havens for rain forests (Bush et al., 2011; Hooghiemstra the two intraspecific genetic lineages in the maximum likelihood tree & Van Der Hammen, 2004). Rain forests also have remained stable (Figure S1), which adds uncertainty to the identity of P. aequatorialis throughout the Pleistocene in the Eastern Arc Mountains of Kenya populations in north-western Ecuador. In addition, the inability of and Tanzania where the lowlands were suggested to be dry during our palaeoecological niche modelling to predict the current distribu- glacial cycles (Finch et al., 2009; Mumbi et al., 2008). Additional tion of P. aequatorialis in north-western Ecuador suggests that differ- studies on the genetic composition and palaeoclimatic niche of plant ent climatic conditions could be involved in its differentiation with P. and animal species are necessary to determine whether the Andean tumacana. Detailed genetic, morphological and phenological studies foothills of south-western Ecuador were indeed a refugium for rain must be performed to these populations to confirm their taxonomic forest species. identity. The reduced genetic diversity detected in the individuals from the Andean foothills of south-western Ecuador (Figure 4a), an area that potentially acted as a Pleistocene refugium, provides further 5 | CONCLUSIONS evidence for the 'dry refugia model' acting on the Andean foothills of south-western Ecuador. These individuals may present lower This is the first demonstration of the potential effects of genetic diversity compared to the individuals from the northern Pleistocene climatic fluctuations on the distribution and genetic Andean foothills, which did not act as forest refugia, because of patterns of a plant species endemic to the rain forests of west- geographic isolation and potential population reduction during the ern Ecuador using nuclear genomic data. Our results support the Pleistocene (Ramírez-Barahona & Eguiarte, 2013). Nevertheless, it role of Pleistocene climatic fluctuations and areas of historical cli- is also possible that this decrease in genetic diversity is due to dif- matic suitability in promoting intraspecific divergence in tropical ferences in the number of individuals analysed and, therefore, these species. Phytelephas aequatorialis shows a discrete north–south results should remain as exploratory. In addition, genetic diversity distribution of its intraspecific genetic lineages estimated to have follows a geographic pattern by increasing jointly with decreasing originated ~1 Ma. This divergence was potentially caused by the latitude (Figure 4c). This suggests that P. aequatorialis could have col- isolation of populations into two rain forest refugia during glacial onized western Ecuador from the north because genetic diversity cycles, as suggested by the palaeoecological niche of P. aequato- tends to decrease when species expand their ranges (Hewitt, 1999, rialis during the LGM. The Chocó refugium could have extended 2000, 2004; Park & Donoghue, 2019; Petit et al., 2003; Zinck further south to the Pacific coast of western Ecuador and could & Rajora, 2016). In contrast, the palaeoclimatic suitability index have included the populations of P. aequatorialis that descended during the LGM did not influence genetic diversity in P. aequatorialis from the northern Andean foothills. The Andean foothills of (Figure 4b), indicating that areas with more suitable palaeoclimatic south-western Ecuador may be an important area for rain forest conditions do not necessarily harbour higher current genetic diver- evolution because they could have remained climatically suitable sity. A similar pattern was observed in the African rain forests for through the Pleistocene. This promoted intraspecific divergence the palm Podococcus acaulis using unique haplotypes as a measure and decreased genetic diversity in the populations of P. aequatori- of genetic diversity (Faye et al., 2016). However, the sister species alis from that region. Therefore, this study also provides phylogeo- Podococcus barteri showed higher levels of unique genetic diversity graphic and palaeoclimatic evidence for the 'moist forest' and 'dry in areas with high palaeoclimatic suitability during the LGM in the refugia' models acting on the Andean foothills of western Ecuador same rain forests (Faye et al., 2016). This suggests that the impact of (Ramírez-Barahona & Eguiarte, 2013), showing that contiguous ESCOBAR et al. | 11 premontane rain forests could have been affected differently by processing of targeted enriched Illumina sequences, from raw reads to alignments. PeerJ, 2018(7), 1–15. https://doi.org/10.7717/ Pleistocene climatic fluctuations. Our study shows that the rain peerj.5175 forests of south-western Ecuador should be further studied and Arteaga, A., Pyron, R. A., Peñafiel, N., Romero-Barreto, P., Culebras, J., protected to preserve its unique intraspecific genetic diversity Bustamante, L., Yánez-Muñoz, M. H., & Guayasamin, J. M. (2016). that has potentially originated during the Pleistocene. Comparative phylogeography reveals cryptic diversity and repeated patterns of cladogenesis for amphibians and reptiles in Northwestern Ecuador. PLoS One, 11(4), e0151746. https://doi.org/10.1371/journ ACKNOWLEDGEMENTS al.pone.0151746 We thank Ecuador's Ministerio del Ambiente for providing the sam- Barfod, A. S. (1991). A monographic study of the subfamily pling permit to undertake this research (MAE-DNB-CM-2018-0082). Phytelephantoideae (Arecaceae). Opera Botanica, 105, 1–73. We also thank Rodrigo Bernal for providing leaf material. This re- Barfod, A. S., Trénel, P., & Borchsenius, F. (2010). Drivers of diversification in the vegetable ivory palms (Arecaceae: Ceroxyloideae, s e a r c h w a s f i n a n c i a l l y s u p p o r te d by t h e S EN E S C Y T (P h D s c h o l a r s h i p Phytelepheae) – Vicariance or adaptive shifts in niche traits? In O. to S.E.), the Independent Research Fund Denmark (9040-00136B Seberg, G. Peterson, A. Barfod, & J. Davis (Eds.), Diversity, Phylogeny, to H.B.), the Agence Nationale de la Recherche (ANR-15- CE02- and Evolution in the Monocotyledons (pp. 225–243). 0002-01 to T.L.P.C.) and the International Mixed Laboratory (LMI) Barve, N., Barve, V., Jiménez-Valverde, A., Lira-Noriega, A., Maher, S. P., Peterson, A. T., Soberón, J., & Villalobos, F. (2011). The crucial role of BIO_INCA. We are also very grateful to the generous financial sup- the accessible area in ecological niche modeling and species distribu- port provided by the International Palm Society (IPS) for funding a tion modeling. Ecological Modelling, 222(11), 1810–1819. https://doi. field trip to western Ecuador without which part of the sampling org/10.1016/j.ecolmodel.2011.02.011 would not have been possible (field grant to S.E.). We dedicate Baumgartner, J. B., Esperón-Rodríguez, M., & Beaumont, L. J. (2018). Identifying in situ climate refugia for plant species. Ecography, 41(11), this article to Dr. Calaway H. Dodson (1928-2020), botanist and 1850–1863. https://doi.org/10.1111/ecog.03431 orchidologist, who worked for many years on the flora of Ecuador Behling, H., Hooghiemstra, H., & Negret, A. J. (1998). Holocene history and authored the five volumes of 'Orchids of Ecuador'. He was also of the Chocó rain forest from Laguna Piusbi, southern Pacific low- the first Honorary Director of the Herbario Nacional del Ecuador lands of Colombia. Quaternary Research, 50(3), 300–308. https://doi. org/10.1006/qres.1998.1998 (QCNE), now part of the Museo Ecuatoriano de Ciencias Naturales Bernal, R., & Galeano, G. 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and basic results of CMIP5-20c3m experiments. Geoscientific SUPPORTING INFORMATION Model Development, 4(4), 845–872. https://doi.org/10.5194/ Additional supporting information may be found online in the gmd-4-845-2011 Supporting Information section. Zhang, C., Rabiee, M., Sayyari, E., & Mirarab, S. (2018). ASTRAL-III: Polynomial time species tree reconstruction from partially resolved gene trees. BMC Bioinformatics, 19(S6), 153. https://doi.org/10.1186/ s12859-018-2129-y How to cite this article: Escobar S, Helmstetter AJ, Jarvie S, Zinck, J. W. R., & Rajora, O. P. (2016). Post-glacial phylogeography and Montúfar R, Balslev H, Couvreur TLP. Pleistocene climatic evolution of a wide-ranging highly-exploited keystone forest tree, fluctuations promoted alternative evolutionary histories in eastern white pine (Pinus strobus) in North America: Single refugium, multiple routes. BMC Evolutionary Biology, 16(1), 1–17. https://doi. Phytelephas aequatorialis, an endemic palm from western org/10.1186/s12862-016-0624-1 Ecuador. J Biogeogr. 2021;00:1–15. https://doi.org/10.1111/ Zizka, A., Silvestro, D., Andermann, T., Azevedo, J., Duarte Ritter, C., jbi.14055 Edler, D., Antonelli, A., Farooq, H., Herdean, A., Ariza, M., Scharn, R., Svantesson, S., Wengström, N., Zizka, V., & Antonelli, A. (2019). CoordinateCleaner: Standardized cleaning of occurrence records from biological collection databases. Methods in Ecology and Evolution, 10(5), 744–751. https://doi.org/10.1111/2041-210X.13152

BIOSKETCH Sebastián Escobar conducted this research as part of his PhD program at the Department of Biology in Aarhus University. He is interested in unravelling the evolutionary history and phylogenetic relationships of tropical plants to understand the processes that have shaped current patterns of biodiversity.

Author contributions: R.M., H.B., and T.L.P.C conceived the study; S.E., S.J., R.M. and T.L.P.C. collected the data; S.E. performed laboratory work; S.E. analysed the data with assistance from A.J.H. and S.J.; S.E. led the writing with contribution from all authors.