Major incongruence between plastid, nuclear ribosomal and mitochondrial phylogenies in tribe Malmeeae () Author: Sophia A. Zwartsenberg (920108996040) Supervisor: Dr. Lars W. Chatrou Examiner: Dr. Freek T. Bakker

Abstract Evolutionary biologists have long relied on data for inferring phylogenies. Doing so can however lead to incomplete hypotheses on evolutionary patterns, as using only one genome can obscure incongruence between the three plant genomes. Incongruence can be caused by a suite of technical issues and biological processes. Phylogenetics in the tropical plan family Annonaceae has heavily relied on plastid DNA. The same is true for the genus Oxandra from the tribe Malmeeae. In previous phylogenetic analyses, of the genus was not demonstrated whilst (floral) morphologically it is a homogeneous genus The aim of this study is to resolve phylogenetic relationships between species of Oxandra and of species of Oxandra with closely related genera using data from all genomes (plastid, mitochondrial and nuclear ribosomal DNA) and discover possible incongruence caused by processes such as hybridisation, incomplete lineage sorting and chloroplast capture. We generated high-throughput sequence data for 33 species of the Malmeeae , including 20 species of Oxandra. From the sequence data we obtained a data set of 61136 bp of chloroplast sequences, 5826 bp of nuclear rDNA sequences and 6194 bp of mitochondrial DNA. Based on chloroplast DNA, the genus Oxandra is polyphyletic. However, the genus was inferred to be monophyletic using nuclear ribosomal DNA and mitochondrial DNA. These incongruences between the different phylogenies affect nodes deep in the phylogeny and all receive high support. Species of Oxandra consistently fall into two in every phylogeny made in this study, within these clades the species relationships of Oxandra are congruent across the genomes. Inferring a phylogeny using only the congruent species and a concatenation of data from the three genomes increases support values for the species- relationships in the two Oxandra clades indicating synergy between the genomes. The mechanisms causing this strongly incongruent pattern in Oxandra is unknown but probably involved hybridisation, incomplete lineage sorting, horizontal gene transfer or chloroplast capture. The results of this study show that it might be wise for phylogenetic studies to sample multiple genomes that are available within the plant as these might shed a different light on the organismal evolution.

Introduction been termed reticulate evolution (Aguilar et al., 1999; When the theory of evolution was first presented by Gogarten & Townsend, 2005; Sang et al., 1995). In this Darwin (1859) and Wallace (1858) the described process Anthropocene era the need to understand our own lives appeared to be reasonably simple. Innumerable in connection to life around us is as great as ever. As generations or organisms, subjected to natural Dobzhansky (1973) already said ‘Nothing in biology selection, would finally lead to speciation (a never makes sense except in the light of evolution’ which ending process). Over time evolution has proved to be makes the need to understand evolutionary processes more challenging to understand as evolution does not even greater. Evolutionary biologists therefore have a always move in a bifurcating, tree-like pattern, nor is it responsibility towards all other biologists to advance a linear process. Instead, it often takes difficult winding knowledge on complex evolutionary processes. paths which merge at times through processes such as Over the past decades the field of phylogenetics hybridization causing a network-like pattern which has has evolved rapidly. What started with the use of morphological characters soon became a field that one genome type might obscure important relied mainly on nucleotide data, because of the evolutionary events that led to the extant species development of PCR methods which remained relationships. For example a genus might be inferred mainstream for over two decades (Straub et al., 2012). polyphyletic using nuclear data whilst plastid data Nowadays high-throughput sequencing methods might infer this genus to be monophyletic (Liu et al., enable phylogenetecists to work with genome-scale 2017). data. The usage of such data can help to answer Incongruences are still often seen as a nuisance challenging questions in phylogenetics such as as synergy between the genomes is one of the resolving interspecies or intergeneric relationships of prerequisites for species tree inference. Incongruence difficult clades, shed light on possible incongruences between the three genomes therefore obscures the between different genomes and help adjust species much sought after ‘true species tree’. However, another delimitations (Soltis et al., 2013; Straub et al., 2012; Sun way to perceive these incongruences is that they are et al., 2015). also part of the evolutionary history of the species Plant phylogenetics has largely relied on (assuming the underlying reason for them is a chloroplast DNA (cpDNA), and on nuclear ribosomal biological one) and that therefore these incongruent DNA (nrDNA) to a lesser extent (Olmstead & Palmer, phylogenetic patterns can be as fascinating as finding 1994; Shaw et al., 2005; Soltis & Doyle, 2012; Spooner et the species tree. Following that reasoning, encountering al., 2017; Straub et al., 2012). This preference for the an incongruent phylogenetic pattern can be an excellent plastid genome is not surprising considering the opportunity for evolutionary biologist to deepen the advantages. The plastid genome is easy to extract and understanding of the wide variety of complex processes sequence because of the high copy number (up to 1000 that underlie speciation and evolution. The placement per cell) and relatively small size (120-160 kbp). It is of COM (Celastrales-Oxalidales-Malpighiales) within useful for studies at different phylogenetic levels as the Rosidae is an example of such a of such a complex rates of evolution vary over the different regions of the evolutionary question, Sun et al. (2015) found that the genome. Because of low recombination rates and placement is uncertain due to deep phylogenetic uniparental inheritance, plastid data sets containing incongruence which is most probably caused by ancient multiple markers hardly ever produce incongruent hybridisation or incomplete lineage sorting. On a lower gene trees (Raubeson & Jansen, 2005; Spooner et al., taxonomic level, Liu et al. (2017) found that the genus 2017). Phylogenetic signal, i.e. the number of variable Hedysarum L. of the family Fabaceae, of which the characters, present in plastid DNA however is limited, systematic position was still uncertain, probably and to gain good resolution at the species level large experienced chloroplast capture via introgression. amounts of data would need to be concatenated. Incongruent phylogenetic patterns are known Moreover, using only a single type of genomic to have a suite of possible underlying causes with both data, such as the plastid genome, can in some cases lead technical and biological background. Technical to incomplete hypotheses on evolutionary patterns. explanations include sample contamination, taxon Incongruences between the different genomes were sampling error and analytical uncertainties (Wendel & already observed when only a few markers of nuclear Doyle, 1998). Biological explanations for incongruence ribosomal DNA and chloroplast DNA were sequenced include hybridization, incomplete lineage sorting using PCR (Soltis & Kuzoff, 1995). High-throughput (Spooner et al., 2017; Sun et al., 2015; Zhang et al., 2017), sequencing methods can yield data on the three chloroplast capture (Acosta & Premoli, 2010; Liu et al., genomes in one single run namely plastid, nuclear 2017; Nauheimer et al., 2012; Soltis & Kuzoff, 1995), ribosomal repeat regions and mitochondrial data gene duplication and horizontal gene transfer (Wendel (Steele & Pires, 2011; Straub et al., 2012). The growing & Doyle, 1998). Even if technical causes can be availability of nucleotide data on public data banks and completely ruled out, recognizing the underlying phylogenomics studies seem to have led to more causes for incongruence can be challenging. Gene discoveries of incongruences between the genomes in duplication and incomplete lineage sorting followed by different plant families (García et al., 2014; Liu et al., random extinctions is expected to give a chaotic 2017; Spooner et al., 2017; Sun et al., 2015). It is beneficial incongruent pattern with markers giving many to our understanding of evolution that the usage of different conflicting topologies. A single hybridisation nucleotide data from the multiple genomes is becoming event on the other hand is expected to give only two more commonplace as the inference of evolutionary conflicting topologies, each of which is consistent over patterns of plant species, genera and families using only different markers (Frajman et al., 2009). Chloroplast capture is complicated as explanatory mechanism as it The latter would seem less probable as the floral can be both caused by hybridisation events (Acosta & morphology of the genus is constant, and different from Premoli, 2010) as well as horizontal gene transfer closely-related genera, suggesting synapomorphic (Stegemann et al., 2012). It is however unclear how floral traits (Junikka et al., 2016). Another genus of the likely horizontal transfer of the chloroplast is in natural Annonaceae, Dasymaschalon (Hook. f. & Thomson) environments. Horizontal gene transfer in angiosperms Dalla Torre & Harms, was inferred to be polyphyletic is not very common in general and if it happens it when analysed with chloroplast DNA (Wang et al., mostly involves the mitochondrion (Adams et al., 2002; 2012). The genus was however retrieved as Richardson & Palmer, 2006). A phylogeny based on monophyletic when analysed with nuclear DNA (Guo plastid DNA after chloroplast capture is found to et al., 2014). They hypothesised that the incongruence resemble the geography of the studied group better was caused by chloroplast capture. than the actual species relationships in some cases. This The aim of this study is to resolve phylogenetic means that species that are geographically in close relationships between species of Oxandra and of species proximity are also inferred to be closely related in the of Oxandra with closely related genera using data from phylogeny (Acosta & Premoli, 2010). To distinguish all genomes (plastid, mitochondrial and nuclear between hybridisation and incomplete lineage sorting ribosomal DNA) and discover possible incongruence is challenging. The most frequently used test to caused by processes such as hybridisation, incomplete differentiate between the two is coalescence simulation, lineage sorting and chloroplast capture. We use high- the first problem with this test is that it assumes throughput sequencing methods to sequence twelve knowledge on effective population size, generation herbarium accessions of Oxandra. Another 21 accessions time and clade age (all of which can often only be were already sequenced for another study (Lopes, 2016) roughly estimated) (De Kuppler et al., 2015). The including eight more Oxandra species and also species second challenge of this test is that if incongruence can of the genera Ephedranthus, Klarobelia, Pseudomalmea, be explained by coalescence it does not exclude Ruizodendron, Pseudoxandra R. E. Fr., Cremastosperma R. hybridisation as explanation (Villiers et al., 2013). E. Fr., Mosannona Chatrou, Unonopsis R. E. Fr. and The plant family Annonaceae consists of Malmea R. E. Fr. (outgroup). tropical trees and lianas and it comprises ca 108 genera and ca 2400 species (Rainer & Chatrou, 2006) and Materials and Methods contributes significantly to tree diversity in tropical Taxon sampling forests (Chatrou et al., 2012; Valencia et al., 1994). In this study high-throughput data for 33 accessions Annonaceae phylogenetics has relied heavily on the use was used which was either sequenced for this study or of plastid DNA (Chatrou et al., 2012; Erkens et al., 2014; was already available (Lopes, 2016). The accessions Pirie et al., 2006; Scharaschkin et al., 2005) and studies represent 30 species of tribe Malmeeae, subfamily using nuclear or mitochondrial markers have not been , of the Annonaceae. Of these 30 species, published to date. An example of a genus that has only 17 belong to Oxandra, resulting in 63% of the species been studied using plastid data is Oxandra A. Rich. diversity of this genus being included (Junikka et al., (Chatrou et al., 2012; Pirie et al., 2006). The of 2016). Three species are represented by two accessions, the genus has recently been revised. Within Oxandra 27 as some species were still in the process of identification species are recognized, most of which occur in tropical and were only identified after sequencing. All other South America. Only six species occur in different areas, genera of tribe Malmeeae were also sampled (except for viz. Mexico, Central America and the West Indies Pseudephedranthus as no NGS data was available). For (Greater and Lesser Antilles) (Junikka et al., 2016). In each of these genera two species were used if available, previous phylogenetic studies that incorporated only one species was sampled for monotypic genera. Oxandra (Chatrou et al., 2012; Pirie et al., 2006) the The following genera were used Ephedranthus (2 relationships of the genus with other genera in tribe accessions), Klarobelia (1), Pseudomalmea (1), Malmeeae (Klarobelia Chatrou, Pseudomalmea Chatrou, Ruizodendron (1), genera Pseudoxandra (2), Pseudephedranthus Aristeg., Ruizodendron R. E. Fr. and Cremastosperma (2), Mosannona (2), Unonopsis (2) and Ephedranthus S. Moore) were poorly resolved and did Malmea (2) (Table 2). not exclude possible of Oxandra. This raised the question whether Oxandra is actually a DNA extraction monophyletic genus but poorly resolved due to DNA was extracted from herbarium material and from insufficient data, or whether the genus is polyphyletic. one silica gel sample (see Table 1) using a modified cetyltrimethylammonium bromide-polyvinyl stopped by adding 5 µL of 0,5M EDTA. Upon diluting pyrrolidone (CTAB) DNA extraction protocol the sample with water in an 1:1 ratio, purification was (Doyle & Doyle, 1987). The following modifications done following the Agencourt AMPure XP PCR were made: a) the leaf material was ground using a Purification protocol. pestle and mortar that were (pre-) cooled using liquid Library preparation and Illumina® nitrogen instead of grinding with metal beads as leaf sequencing material of Annonaceae tends to be too tough for this Library preparation for Illumina® sequencing was method and: b) DNA precipitation was performed in done using NEBNext® Ultra™ DNA Library Prep Kit isopropanol at -20 °C for two weeks instead of for Illumina® following the producer’s protocol. Size overnight as heavily fragmented DNA tends to selection was done for the samples that contained more precipitate slowly and increased precipitation time than 50 ng of DNA fragments, selecting for ~350-bp gains yield (personal communication Rita Brandão). length (see Table 1). The concentration was determined After DNA extraction, secondary metabolites using Qubit High sensitivity dsDNA kit. The number of were still present in some samples as can be recognized PCR cycles was determined by the amount of DNA in by the brown colour of the precipitate (Table 1). DNA the sample following recommendations in the protocol. purification was done using the Promega Wizard® For 300-500 ng six cycles, for 100-200 ng seven cycles, DNA Clean-Up System (Dong et al., 2013) according to for 40-50 ng nine cycles and for 5-18 ng twelve cycles the manufacturer’s protocol, with the exception of the (see Table 1). The size profile was determined on a waiting time at step 7, which was increased to 1,5 Bioanalyzer 2100 using a High Sensitivity DNA Assay. minutes. The elution was done with 30 µL of water. This This data was used to make an equimolar pooling step was repeated twice to increase the recovery of (every sample with a final concentration of 20 nM). DNA. Sequencing was performed at Maastricht UMC+ on an After purification the concentration of all Illumina NextSeq 500 sequencing system using the samples was measured using the Qubit High sensitivity Illumnia NextSeq 500/550 Mid Output Kit. All samples dsDNA kit. If concentration of a sample was too low for were run on one Illumnia lane generating paired-end fragmentation, the sample was concentrated using a 150-bp reads. vacuum desiccator (Table 1). Even though the DNA from herbarium material DNA assembly and annotation is by itself already fragmented, a fragmentation was For all 33 accessions the raw Illumina data was used to done to standardize fragment length. This was done make assemblies using exactly the same procedure, to using NEBNext® dsDNA Fragmentase® following the make sure there were no differences between accessions protocol “Digestion with NEBNext dsDNA in data treatment. DNA was assembled using IOGA Fragmentase (M0348)”. The samples were incubated at (Bakker et al., 2016) the Iterative Organelle Genome 37 °C. The desired fragment size was 200-1000 bp Assembly pipeline which is designed to assemble therefore the incubation time was 35 minutes (this is the organellar data but can also be used to assemble other normal incubation time increased by 10 minutes as the nucleotide data when mapped to the right reference starting material was less than 100 ng). The reaction was genome. Assemblies were made for chloroplast, nuclear

Table 1. Sample preparation details

Species (sample #) Herbarium accesion # Purification Concentrated Size selection PCR cycles # Huber, O. & Canales, H. O. espintana (1) 406/16 No Yes No 12 O. bolivarensis (2) Cuadros V. 3155 No Yes Yes 7 O. sphaerocarpa (3) Pennington, T.D. et al. 17022 Yes Yes No 9 O. unibracteata (4) Silva, I. A. 272 Yes No Yes 6 O. espintana (5) Maas et al. 8820 No Yes No 12 O. panamensis (6) Hammel et al. 16334 Yes Yes No 12 O. guianensis (7) Ek, R.C. et al. 805 Yes Yes No 9 O. riedeliana (8) Mori, S.A. et al. 9052 Yes Yes Yes 7 O. sessiliflora (9) Taylor, E.L. et al. E1125 No Yes No 12 O. krukoffii (10) Oldenburger et al. 581 Yes Yes Yes 7 O. surinamensis (11) Bordenave et al 8149 Yes Yes No 9 O. asbeckii (12)1 Maas 10565 No Yes Yes 6

1Silica gel sample. All other samples are derived from herbarium material. ribosomal and mitochondrial DNA. The assemblies examined in Mesquite v3.2 (Maddison & Maddison, were annotated using Geneious (Kearse et al., 2012). If 2001). Regions that were poorly aligned were either needed the data used to make the assembly was manually re-aligned in Mesquite or deleted. The length subsampled until the right sample size was reached to of the sequences was made similar by trimming the achieve an average sampling depth in the final tails. In-dells were deleted if they were only present in assembly of around 200 to avoid introduction of one of the taxa, inserts were left in the alignment as long sequencing error. The assembly with the highest ALE as two or more species shared the insert. All gaps were (Assembly Likelihood Estimation) score was used as set to missing. The coding genes all were given a final assembly except when the assembly differed a lot reading frame minimizing the stop codons. The introns from expectations, in this case the second best assembly and spacers sequences as well as nuclear ribosomal was used. For the chloroplast assembly the standard DNA were all set to non-coding. reference of IOGA was used which consists of sixteen Phylogenetics complete chloroplast genomes of plant species Per marker a parsimony analysis was done using distributed over het whole plant kingdom. For nuclear PAUP* (Swofford, 2003) to (visually) assess congruence ribosomal DNA a complete ribosomal DNA sequence between genes within one genome type. Markers of the of Brassica rapa was used and for mitochondrial DNA same genome were found to be either congruent or too the markers ATP1 and matR of two Annonaceae species poorly resolved to be identified as incongruent due to (Annona muricata L. and Asimina triloba Adans.) were lack of phylogenetic signal. Therefore all markers were used as reference (for details see table S1). Plastid concatenated per genome. All plastid markers were assemblies were made for 33 taxa, assembly of concatenated to form a matrix of 61136 bp (1740 mitochondrial DNA and nuclear ribosomal DNA was parsimony informative sites, 3.58% missing data). The only possible for 32 and 30 of these taxa respectively as nuclear matrix consisted of 5826 bp (215 parsimony most of the DNA extracted from plant leaves is informative sites, 2.44% missing data) and the matrix of chloroplast based, nuclear and mitochondrial DNA mitochondrial DNA accounted to 6194 bp (55 take up less of the total extracted bp meaning it is also parsimony informative sites, 25.03% missing data), less present in the Illumina output which in turn leads (Table S2 for details on markers per species). The to lower success rates when assembling these genomes. supermatrices were separately analysed under The chloroplast DNA assembly was annotated maximum likelihood and using Bayesian statistics. To using the complete plastid genome of Liriodendron determine the best partitioning scheme every data set tulipifera (GenBank acc nr. DQ899947.1). Nuclear rDNA was analysed using PartitionFinder (v2.1.1) (Lanfear et was annotated using the complete ribosomal sequence al., 2012). The input data blocks were all genes of Brassica rapa (GenBank acc nr. KM538956.1) and for separated by codon positions, spacers and introns. mitochondrial DNA the complete mitochondrion of Branch lengths were set to linked, models of evolutions Liriodendron tulipifera (GenBank acc nr. NC_021152.1) were set to GTR+G for chloroplast (to make analysis was used. Similarity for annotation was set to 75%, with PartitionFinder possible) and to ‘MrBayes’ (this which worked well for conserved regions and genes. To setting includes all models available in MrBayes) for find non-coding spacer DNA blastn in Geneious was nuclear and mitochondrial data, the metric of selection used, after which the sequences were annotated by was set to BIC and the search algorithm was set to hand. Genes containing introns and exons where greedy. The best partitioning scheme from manually separated into intron and exon sequences. PartitionFinder was used in the RAxML and MrBayes Supermatrix construction analyses. The chloroplast data was divided into ten The obtained markers were aligned separately in partitions, the nuclear ribosomal into three partitions Geneious using MAFFT v7.017 (Katoh & Standley, and the two mitochondrial markers comprised a single 2013) with a slow accurate (G-INS-i) algorithm. If the partition. number of species for which the marker was missing Maximum likelihood was below a certain value the alignments were exported A maximum likelihood analysis was done using as Nexus file. The amount of missing sequences allowed RAxML v8.2.9 (Stamatakis, 2014), run at the CIPRES for chloroplast data was 3 out of 33, for nuclear science gateway (Miller et al., 2010). The rapid heuristic ribosomal DNA this was only a single marker out of 32, bootstrap search was done with 1000 bootstrap and for mitochondrial DNA this was 15 out of 30 as replicates. The rapid bootstrap analysis and search for mitochondrial DNA proved difficult to assemble (see the best scoring ML tree were conducted in a single table 2). Every separate alignment was visually program run. Table 2. Taxa used for phylogeny inference Species Herbarium collection Chloroplast Nuclear Mitochondrial ribosomal Cremastosperma cauliflorum R. E. Fr. Chatrou, L. W. 224 v v v Cremastosperma leiophyllum (Diels) R. E. Fr. Pirie, M. 2 v v v Ephedranthus guianensis R. E. Fr. Scharf, U. 64 v v v Ephedranthus sp. Chatrou, L. W. 173 v v v Klarobelia inundata Chatrou Chatrou, L. W. 205 v v v Malmea dielsiana R. E. Fr. Chatrou, L. W. 122 v v v Malmea dimera Chatrou Croat, T. 34626 v v v Mosannona discolor (R. E. Fr.) Chatrou Jansen-Jacobs, M. J. 6000 v v v Mosanonna costaricensis (R. E. Fr.) Chatrou Chatrou, L. W. 90 v v v Oxandra asbeckiia (Pulle) R. E. Fr. Maas, P. J. M. 10565 v v v Oxandra asbeckiib (Pulle) R. E. Fr. UGNB 55 v v v Oxandra bolivarensis (Maas & Junnika) Cuadros, H. V. 3155 (U) v v v Oxandra espintanaa (Spruce ex Benth.) Baill. Huber, O. 406/16 (U) v v - Oxandra espintanab (Spruce ex Benth.) Baill. Maas, P. J. M. 8820 v v - Oxandra euneura Diels Chatrou, L. W. 235 v - - Oxandra guianensisa R. E. Fr. Ek, R. C. 805 v v v Oxandra guianensisb R. E. Fr. Scharf, U. 67 v v v Oxandra krukoffii R. E. Fr. Oldenburger., F.H.F 581 v v v Oxandra laurifolia (Sw.) A. Rich. Maas, P. J. M. 8375 v v v Oxandra longipetala R. E. Fr. Chatrou, L. W. 114 v v v Oxandra macrophylla R. E. Fr. Chatrou, L. W. 204 v v v Oxandra martiana (Schltdl.) R. E. Fr. Lopes, J. C. 363 v v v Oxandra panamensis R. E. Fr. Hammel, B. 16334 v v v Oxandra polyantha R. E. Fr. Chatrou, L. W. 215 v v v Oxandra riedeliana R. E. Fr. Mori, S. A. 9052 v v v Oxandra sessiliflora R. E. Fr. Taylor, E. L. E1125 v v v Oxandra sphaerocarpa R. E. Fr. Pennington, T. D. 17022 v v v Oxandra surinamensis Jans.-Jac. Bordenave, B. G. 8149 v v v Oxandra unibracteata J. C. Lopes, Junikka & Mello-Silva Silva, I. A. 272 (T) v v v Pseudomalmea diclina (R. E. Fr.) Chatrou Chatrou, L. W. 136 v v v Pseudoxandra obscurinervis Maas Webber, A.C. no number v v v Pseudoxandra spiritus-sancti Maas Lopes, J. C. 436 v v v Ruizodendron ovale (Ruiz & Pav.) R. E. Fr. Maas, P. J. M. 8600 v v v

Bayesian Inference For Bayesian inference the program MrBayes v3.2.6 Results (Ronquist et al., 2012) was used at the CIPRES science Phylogenetics gateway. For each of the three data sets, four runs with Sequencing and subsequent assembly and annotation four chains each were set to run for 20 million led to three datasets: a chloroplast dataset (61136 bp), a generations. The temperature was set to 0.05, nuclear ribosomal dataset (5826 bp) and a substitution models were allowed to vary across the mitochondrial dataset (6194 bp). From these datasets partitions, and rates were set to gamma. After the three genome trees were inferred using raxML and analysis a burn-in fraction of 0.25 was deleted. MrBayes. The Bayesian analyses converged with all ESS Convergence was assessed by checking the ESS values values above 200. using Tracer v1.6 (Rambaut et al., 2014). A 50% majority rule consensus tree was obtained for every genomic DNA type separately including the posterior probabilities of every node. The separate analyses clearly show two incongruent patterns of the relationships of the genus Geographic distribution Oxandra and related genera. The mitochondrial and The geographic location of all Oxandra accessions used nuclear ribosomal DNA infer the genus Oxandra to be in this study are shown on the map (Figure 3), the monophyletic with high posterior probabilities (>0.99; colours represent the two Oxandra clades. The Figure 5 and Figure 6). The chloroplast data, however, geographic range of both clades is completely unambiguously indicate polyphyly of the genus with overlapping. The two O. espintana accessions used for maximum support (Figure 5 and Figure 6). In the this study are placed very far apart with one on the chloroplast tree Oxandra is split into two distinct clades, coast of Brazil and the other in Venezuela crossing the Brazilian savannah (Cerrado).

Complete supermatrix The plastid phylogeny is in general well resolved on species-level, the phylogeny based on nuclear rDNA is slightly less resolved and the one based on mitochondrial DNA is hardly resolved at the species- level. Even though plastid DNA exhibits deep incongruence with nuclear rDNA and mitochondrial

Figure 1 Oxandra Clade I. Part of chloroplast tree on the left, on the right part of DNA, the two clades of Oxandra do not show any the rDNA tree incongruence between plastid and nuclear rDNA (Figure 1 andFigure 2). Support values for the mitochondrial phylogeny are too low to reveal any incongruence. Because the pattern of evolution is the same within both clades of Oxandra there is no reason to believe that these clades are incongruent with each other. Therefore, another tree was made using all DNA data and only those species that do not hold an with Oxandra clade I being more closely related to incongruent position. Excluding species that cause an Klarobelia, Pseudomalmea, Ruizodendron and incongruence from the data set results in a data set with Figure 2 Oxandra clade II. Left side: tree based on chloroplast DNA, right side: tree based on rDNA all species of Oxandra (minus O. sessiliflora and O. unibracteata). Two Cremastosperma species were added Ephedranthus than to Oxandra clade II. Two species of as outgroup. The supermatrix constructed included 20 Oxandra (O. sessiliflora and O. unibracteata) do not fall species and a total of 73137 characters. The MrBayes within either of the Oxandra clades (Figure 5 Figure 6). analyses were well converged with all ESS values above The phylogenetic position of these two species varies 200. Concatenating data from the three genomes between the genome trees, often with low support. The increased the support values of the nodes, with two Oxandra clades are consistent over the chloroplast maximum support values for both RAxML and and nuclear ribosomal trees (Figure 1 and Figure 2). MrBayes analyses on most nodes indicating synergism Oxandra clade I encompasses: O. espintana, O. between the genomes for these clades (Figure 4). bolivarensis, O. krukoffii, O. riedeliana, O. euneura, O. Concatenation three genomes also increases resolution, polyantha, O. asbeckii, O. surinamensis and O. longipetala. only one polytomy remains in the phylogeny making Oxandra clade II encompasses: O. guianensis, O. the placement of sister species O. sphaerocarpa and O. laurifolia, O. panamensis, O. macrophylla, O. sphaerocarpa macrophylla, O. panamensis and the clade of O. guianensis and O. martiana. The mitochondrial tree is too poorly and O. laurifolia uncertain. In the RAxML analysis this resolved at the species level to conclude if these clades polytomy was resolved placing O. sphaerocarpa, O. are present or not. Oxandra espintanaa is inferred to be macrophylla and O. panamensis in a clade as sister to the more closely related to Oxandra bolivarensis than to O. guianensis and O. laurifolia clade with O. panamensis Oxandra espintanab. The other two species for which two accession were used (Oxandra asbeckii and Oxandra guianensis) are inferred monophyletic (Figure 1 and Figure 2). Complete phylogenies for all three genome types can be found in the supplementary files. sister to O. sphaerocarpa and O. macrophylla (72% Deep phylogenetic incongruences such as the bootstrap support). ones found in this study can have multiple causes including both biological and technical explanations. Discussion Biological explanations include incomplete lineage The data from the three genomes show a deep sorting, gene duplication, horizontal gene transfer and incongruence in the relationships among species of hybridisation. Technical explanations include sample Oxandra and related clades, with nuclear rDNA and contamination, taxon sampling error and analytical mitochondrial DNA supporting monophyly of Oxandra uncertainties (Wendel & Doyle, 1998). and cpDNA showing the genus to be polyphyletic. Possible reasons for incongruent pattern Because of the deep and well supported incongruence When looking into incongruences between genomes an between the three genomes the phylogenetic important question is whether the incongruence reflects relationships of species of Oxandra with other genera actual evolutionary history. To be able to conclude this, remain unclear. However, the event causing the the possibility of technical errors needs to be incongruence seems to be a singular one, only affecting eliminated. In this study utmost caution was taken to a single node in the plastid phylogeny. The prevent contamination of the samples. Even if sample evolutionary patterns within the two clades of Oxandra contamination would have occurred it can be ruled out after this one event show no evidence of incongruence as a cause for the observed incongruence in this study across the three genomes. Therefore a tree was made by as the assemblies of the three genomes were based on concatenating data from all three genomes and the same DNA extract. Taxon sampling in this study excluding all species involved in the incongruence. The covers 63% of the genus Oxandra. In addition it includes all genera that were previously found to be related to Oxandra (Chatrou et al., 2012; Pirie et al., 2006) except for Pseudephedranthus as NGS data was not available for this one genus. All phylogenetic analyses done in this study resulted in mostly well resolved phylogenies. Both chloroplast and nrDNA analyses resulted in well resolved phylogenies with high support values, the mtDNA phylogeny is mostly unresolved except for the deeper nodes and basal nodes involving Oxandra which is probably due to insufficient character sampling. For the mtDNA phylogeny the bootstrap values of the RAxML analysis returned lower than the posterior probabilities of the Bayesian analyses for many nodes.

Figure 3 Geographic locations of all Oxandra accessions used for the phylogenetic inference. Green markers refer to Oxandra clade I, yellow markers to Oxandra clade II and grey markers to Oxandra species that fall outside of either clade.

support values of this analysis are higher than in any of the separate analyses. The synergy of the three genome types, together with the fact that both mitochondrial DNA and nuclear rDNA support monophyly of Oxandra, promote the idea that the clades in this tree represents the species tree of Oxandra given the current taxon sampling (Chase & Cox, 1998). A highly similar (though opposite) result was found by Liu et al. (2017), they found that the genus Hedysarum (Fabaceae) showed a biphyletic pattern of evolution for nuclear genes opposed to monophyly when inferred with plastid DNA. They concluded that chloroplast capture was the most likely cause and that therefore the genus delimitations should be reconsidered. Figure 4 Tree based on concatenation of all genome types with removal of all species that cause incongruence. Topologies is a Bayesian 50% majority consensus trees with posterior probabilities and raxML boostrap support values on nodes. Asterisk means maximum support for both Bayesian and maximum likelihood statistics. Bootstrap analyses are known to require relatively large the inverted repeat is stacked as if being a single copy amounts of data in comparison to Bayesian posterior part of the chloroplast. The effect of this potential probabilities to attain high confidence values on short erroneous stacking on the quality of the assembly and internodes (Alfaro et al., 2003). This probably phylogenetic inference is unclear. The inverted repeats influenced the support values of the mtDNA phylogeny are known to be very similar (Shaw et al., 2007) and to as the amount of mtDNA informative nucleotide data accumulate mutations at a much lower rate than the was quite low (55 potentially informative sites). Because single copy regions (Perry & Wolfe, 2002). Therefore, of the above mentioned it is not very probable that the the effect on phylogenetic inference is probably incongruence found in this study is due to technical negligible. Similar concerns exist for the nrDNA errors though it cannot be completely ruled out. assembly as many very similar copies are present in the The quality of the genome assembly may also genome which programs in the IOGA pipeline influence the quality of the phylogenetic analyses. In recognize as being the same. This may causes this study the high throughput sequencing data was paralogues copies to be erroneously regarded as assembled using the IOGA pipeline (Bakker et al., 2016). reflecting organismal phylogenetic relationships. The The IOGA pipeline was designed to be able to assemble amount of variation between multiple copies of the large part or even complete but depending ribosomal repeat is however expected to be low due to on the used reference can also assemble different concerted evolution (Ganley & Kobayashi, 2007). Only genome types. A notable aspect we observed during a few SNPs were observed when mapping the primary this assembly is that the inverted repeat receives double reads back to the final assembly of the ribosomal repeat. values for assembly coverage which might mean that The presence of these SNPs might influence the quality

Figure 5 Comparison between chloroplast and nuclear ribosomal phylogeny of Oxandra and related genera. Topologies are Bayesian 50% majority consensus trees with posterior probabilities and raxML boostrap support values on nodes. Asteriks means maximum support for both Bayesian and maximum likelihood statistics.

Figure 6 Comparison between chloroplast and mitochondrial DNA phylogeny of Oxandra and related genera. Topologies are Bayesian 50% majority consensus trees with posterior probabilities and raxML boostrap support values on nodes. Asteriks means maximum support for both Bayesian and maximum likelihood statistics. of the phylogeny although a large influence is unlikely. over the genes (Frajman et al., 2009). This seems to be The presence of SNPs when assembling using IOGA is the case in this study. This is not enough, however, to probably comparable with introduction of some be able to differentiate between incomplete lineage sequencing error in the final assembly. Sequencing sorting and hybridisation. Many studies try to error is only known to cause problems for phylogenetic differentiate between reticulation and incomplete inference when the amount of potentially informative lineage sorting using coalescent simulations (Joly et al., sites is very small (Wendel & Doyle, 1998) which is not 2009; Pelser et al., 2010; Pirie et al., 2009; Willyard et al., the case with 215 potentially informative sites in the 2009). This approach assumes knowledge on effective nuclear rDNA matrix. Even so, in future studies it population size, generation time and clade ages (De would be interesting to see if different methods for Kuppler et al., 2015), parameters that are often treating these SNPs (coding them as missing data or unknown and can only be estimated with wide giving them a IUPAC code) would influence the confidence intervals. Another problem with phylogeny. coalescence simulation is that if the incongruences can Assuming that the incongruent pattern found be explained by ILS, following the simulation results, it in Oxandra is biologically meaningful, what would be does not exclude hybridisation as a possible the reason behind the incongruence? Horizontal gene explanation (Villiers et al., 2013). Other methods to transfer, gene duplication, incomplete lineage sorting differentiate between ILS and hybridisation are and hybridisation / introgression are the known therefore being developed and include isolation-with- mechanisms behind incongruent phylogenetic patterns migration analyses (Hey, 2009), filtered supernetwork (Wendel & Doyle, 1998). Horizontal gene transfer is not approaches (Holland et al., 2008), usage of maximum very common in angiosperms, and is known to usually likelihood networks (Yu et al., 2014) and morphological involve the mitochondrion (Adams et al., 2002; shifts to identify reticulation in the ‘coalescent Richardson & Palmer, 2006). It is also possible for the stochasticity zone’ (Villiers et al., 2013). All of these chloroplast to be transferred completely via horizontal methods have their own drawbacks and combination of gene transfer which can be a mechanism underlying multiple methods might give the clearest insight. Many chloroplast capture (Stegemann et al., 2012). How likely of these methods operate at least partly on the this is to happen in a natural environment is unclear but population level. To implement these methods for it could explain the observed pattern. Chloroplast Oxandra, taxon sampling would have to be more capture can also be caused by hybridisation (Acosta & exhaustive both on species level and on population Premoli, 2010), which assumes species of Oxandra are level. able to cross. In the event of chloroplast capture the An interesting result in relation to possible phylogeny can resembles the geographic pattern hybridisation in this study is the congruence between instead of the actual species relationships. The species the mitochondrion and the nuclear ribosomal repeat with a different (captured) chloroplast genome are in and the incongruence of those two with the chloroplast. that case expected to inhabit distinct areas (Acosta & In the majority of angiosperms, the chloroplast and the Premoli, 2010). In case of Oxandra the two clades mitochondrion are maternally inherited although many currently inhabit a similar overlapping area. This exceptions, mainly for the chloroplast, exist (Corriveau however does not mean that the historical pattern of & Coleman, 1988; Mogensen, 1996; Reboud & Zeyl, distribution of the two Oxandra clades was not distinct. 1994). For example in Magnolia and Liriodendron trace Gene duplication as well as incomplete lineage amounts of paternal inheritance of the chloroplast was sorting (ILS) followed by random extinctions is observed (Sewell et al., 1993) although the majority was expected to cause a chaotic pattern of incongruence still maternally inherited. Presuming that in with many different conflicting topologies (Frajman et Annonaceae the plastid and mitochondrion are both al., 2009). This does not seem to be the case in this study inherited maternally it is surprising that these are as the separate markers within the genome (given that incongruent assuming the incongruent patterns are there was enough phylogenetic signal) gave a very caused by hybridisation. similar pattern. The trees were only visually inspected The genus Oxandra so using an incongruence test such as the Incongruence Morphological division of Oxandra Length Difference (ILD) (Planet, 2006) test would In every phylogeny made in this study Oxandra is provide an objective measure for this observation. A consistently divided into two clades. When comparing single hybridisation event on the other hand is expected the morphology of Oxandra (using the recently to give two conflicting topologies that are consistent published review of Junikka et al. (2016) for morphological characters) with the phylogenies prediction needs verification using nucleotide data and produced in this study only a few characters seem to phylogenetics. differ between the two Oxandra clades. The primary Oxandra phylogenetics venation differs between both clades although not with In the plastid DNA, nrDNA as well as the overall great consistency. Species in Oxandra clade I mostly Oxandra phylogeny O. bolivarensis is placed together have raised primary veins except for O. bolivarensis with O. espintana. This might indicate that one of the O. which has impressed to flat primary veins and O. espintana individuals is wrongly identified. It could also espintana in which primary venation varies from mean that the species delimitation of O. espintana would slightly impressed to slightly raised. The species in need to be revised. One of the Oxandra espintana Oxandra clade II all have impressed to flat veins. The individual might for instance represent another species, flower bud shape varies from globose, subglobose to as the amount of substitutions separating the ellipsoid in Oxandra clade I whereas in Oxandra clade II individuals is quite large for individuals belonging to this is consistently ellipsoid. O. unibracteata and O. the same species (see branch lengths of O. asbeckii and sessiliflora both have globose flower buds. The last O. guianensis for comparison). It might be good to study character that seems to differ between the two clades is the collections of O. espintana and O. bolivarensis in the maximum number of monocarps with Oxandra detail to see if splitting up O. espintana might be better. clade I this varies between 3 and 10 with 7 monocarps Conclusions on average, for Oxandra clade II the number varies This study is yet another example of a group of species between 7 and 25 with an average of 15 monocarps. in which evolution is not as simple and straightforward Seed rumination often is a distinguishing character for as we hoped or expected. The incongruence we many Annonaceous genera, but it does not separate discovered in Oxandra will no doubt be part of a long Oxandra clade I and II. In both clades the rumination is line of uncovered incongruences as the usage of high- consistently spiniform but can vary within a species to throughput sequencing data and multiple genome peg-shaped. It does however seem to separate O. types for phylogeny inference is becoming more sessiliflora and O. unibracteata from the other Oxandra commonplace. The lack of a well resolved species tree species as both have lamellate rumination in four parts, is however not something to bemoan, it is rather a although in O. sessiliflora this can vary to flattened pegs. challenge to us as evolutionary biologists to gain better The mating system varies in Oxandra with most species understanding of the complex mechanisms underlying being bisexual and four species that are androdioecious speciation. To do so we will need development of more (both male and bisexual ). This study included sophisticated methods for phylogeny inference that two of these androdioecious species (O. panamensis and take into account processes such as hybridisation, gene O. martiana) which are both placed in clade II. 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