Inferring hypothesis-based transitions in clade-specific models of chromosome number evolution in sedges (Cyperaceae)

Depósito de investigación de la Universidad de Sevilla https://idus.us.es/ “This is an Accepted Manuscript of an article published by Elsevier in Molecular Phylogenetics and Evolution on Márquez-Corro, J.I., Martín-Bravo, S., Spalink, D., Luceño, M., Escudero, M., (2019) Inferring hypothesis-based transitions in clade-specific models of chromosome number evolution in sedges (Cyperaceae), Molecular Phylogenetics and Evolution, 135, pp. 203 - 209., available at: https://doi.org/10.1016/j.ympev.2019.03.006 Original article Running head: Chromosome number evolution in the Cyperaceae Title: Inferring hypothesis-based transitions in clade-specific models of chromosome number evolution in the Cyperaceae José Ignacio Márquez-Corro1, Santiago Martín-Bravo1, Daniel Spalink2, Modesto Luceño1 & Marcial Escudero3 1. Department of Molecular Biology and Biochemical Engineering, Universidad Pablo de Olavide, Carretera de Utrera km 1, ES-41013 Seville, Spain. 2. Department of Biology, University of Utah, Salt Lake City, UT 84112, USA. 3. Department of Plant Biology and Ecology, University of Seville, Reina Mercedes sn, ES- 41012 Seville, Spain. Corresponding author: José Ignacio Márquez-Corro, e-mail: [email protected]. Abstract Large-scale changes in chromosome number have been associated with diversification shifts in many lineages of plants. For instance, several ancient rounds of polyploidization events have been inferred to promote genomic differentiation and/or isolation and, consequently, angiosperm diversification. Dysploidy, although less studied, has been suggested to play also an important role in angiosperm diversification. In this article, we aim to elucidate the role of chromosomal rearrangements on lineage diversification by analyzing a 1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 2 new comprehensive sedge (Cyperaceae) phylogenetic tree. Mode and tempo of chromosome evolution were inferred to be homogeneous in rate and process across the complete phylogeny as the null hypothesis. In order to discern patterns of diversification shifts and chromosome number changes within the family tree, we tested clade-specific chromosome evolution models for several subtrees according to previously reported increments of diversification rates. Results show that alternative hypotheses of different clade-specific models of chromosome evolution are significantly supported against the null hypothesis of a model with no transition events along the phylogeny. This could suggest a link between diversification and changes in chromosome number evolution. Our methodological approach may allow identifying different patterns of chromosome evolution, as found for Cyperaceae, for other lineages at different evolutionary levels. Key words ChromEvol, chromosome evolution, Cyperaceae, diversification rates, holocentric chromosomes, phylogeny 1. Introduction Chromosomal rearrangements are frequent in eukaryotes and are in many cases correlated with differentiation and speciation (Coghlan et al., 2005). These rearrangements can be produced by a sole mechanism or a combination of translocations, aneuploidy, dysploidy and polyploidy (whole genome duplication; WGD) (Coghlan et al., 2005). Whereas some of these events could produce changes in the genome structure and linkage of genes (Butlin, 2005), others could affect directly the gene content through either deletions or duplications of DNA (Coghlan et al., 2005). These events may promote speciation by 3 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 4 provoking changes in species fitness, adaptability to new habitats, reproductive isolation and/or shifts in recombination rates (Butlin, 2005; Coghlan et al., 2005; Coyne and Orr, 2004; Navarro and Barton, 2003a, 2003b; Otto and Whitton, 2000; Rieseberg, 2001; Soltis et al., 2009). In angiosperms, the role of polyploidy and its consequences on speciation have been intensely studied, with a particular interest in ancient polyploid events in some of the most species-rich lineages (Debodt et al., 2005; Smith et al., 2018; Soltis et al., 2009; Soltis and Soltis, 2016). This has led to an understanding of polyploidization as a possible driver for lineage radiation (Comai, 2005; Hegarty and Hiscock, 2007, 2008; Levin, 1983; Otto, 2007; Otto and Whitton, 2000; Soltis and Soltis, 2016, 2000; Van de Peer, 2011). On the other hand, although dysploidy (translocations, fusions and fissions that lead to changes in chromosome number) is more frequent than polyploidy and especially aneuploidy (duplication or deletion of an entire chromosome) in angiosperms (Grant, 1981), its consequences in diversification have been largely unexamined (though, see Gitaí et al., 2014; Lee and Namai, 1993, 1992; Orellana et al., 2007; Vallès et al., 2012; Vickery, 1995; Weiss‐Schneeweiss et al., 2009). Dysploidy has recently been suggested to not represent a dead end through evolutionary time (Escudero et al., 2014). Probabilistic models have been recently formulated for chromosome. These models vary in their complexity, with the simplest ones calculating the rate of gains and losses of chromosomes and changes in ploidy level along a phylogeny. More complex models allow identifying linear dependency between the current number of chromosomes and the rate of increasing and decreasing chromosome numbers. More recently, Freyman and Höhna (2018) expanded ChromEvol functions (Glick and Mayrose, 2014; Mayrose et al., 2010) with the ChromoSSE package in revBayes (Höhna et al., 2014). This software allows not only 3 5 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 6 7 detecting shifts in the mode of chromosome evolution during anagenetic processes but also during cladogenesis, that can be associated with diversification rate shifts. Moreover, BiChroM type models (correlated rates of phenotype and chromosome evolution; Zenil- Ferguson et al. 2017, 2018) can be integrated with the classic ChromEvol models. However, none of these new approaches considers the possibility of more complex models of chromosome evolution, with different parameters throughout the phylogeny. Here, we expand these studies by applying different models of karyotypic evolution to different clades. This approach is crucial to identify changes in the mode of chromosomal evolution as innovations that may be related to shifts in diversification rates. The cosmopolitan family of sedges (Cyperaceae, ca. 5500 species; Govaerts et al., 2017) is the tenth most species-rich angiosperm family. It has mainly diversified in the tropics, although genus Carex L., the most diversified genus of the family (ca. 2200 spp., 40% of species richness; Govaerts et al. 2017), and several other lineages are distributed mostly in temperate regions (Reznicek, 1990). Cyperaceae has the highest known chromosome number variation among all angiosperm families (2n=4–224; Roalson, 2008). Because of its high species richness and wide range of chromosome numbers, Cyperaceae constitutes a model taxon for incorporating studies of biodiversity with evolution and systematics (e.g. Hipp, 2007). This is especially true of the genus Carex, which alone displays a wide variation of chromosome number (2n=12–124; Hipp, 2007; Roalson, 2008) . Variation in the number of chromosomes and changes in the mode of evolution have been suggested as a possible driver of diversification in Carex (Escudero et al., 2012b, 2014). The huge continuous variation in chromosome number of this family is explained by the presence of holocentric chromosomes, which means that the kinetochoric activity is present along the chromosomes. By contrast, monocentric chromosomes have a clear primary constriction in which kinetochoric activity is 8 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 9 concentrated (Hipp et al., 2013; Melters et al., 2012; Mola and Papeschi, 2006). In lineages with holocentric chromosomes (see review in Márquez-Corro et al. 2017), fusions and fissions (termed symploidy and agmatoploidy, respectively; Escudero et al. 2014) are more common (Grant, 1981). This occurs even within species level, due to the characteristics of the kinetochoric plate (Hipp et al., 2013; Melters et al., 2012; Mola and Papeschi, 2006) that allows more or less constant C-values despite chromosome number variation (Escudero et al., 2014). Four main shifts in diversification rate have been detected in Cyperaceae. Escudero et al. (2012b) found an increase in diversification rates in the non-Siderostictae clade (that comprises Core Carex, Caricoid Carex and Carex subgenus Vignea), which has been confirmed in a recent study by Spalink et al. (2016b). Escudero and Hipp (2013) used Hinchliff and Roalson's (2013) phylogeny to infer an additional shift in diversification rates in the clade including the tribes Scirpeae, Dulichieae, and Cariceae plus Khaosokia caricoides (SDC clade) and the tribes Fuireneae, Abildgaardieae, Eleocharideae, and Cypereae (FAEC clade). Spalink et al. (2016b) showed instead shifts in three different lineages inside the SDC+FAEC clade reported by Escudero and Hipp (2013). Thus, in addition to the shift in the non-Siderostictae clade (as in Escudero et al. 2012b), Spalink et al. (2016b) also found a shift in the FAEC clade and in the represented taxa of the C4 photosynthetic pathway Cyperus within Cypereae 2 clade (within FAEC). Different modes of chromosomal evolution are present in Cyperaceae. For example, Carex karyotype evolves mainly via agmatoploidy and symploidy (Heilborn 1924; Davies 1956), whereas polyploidy is more common in the rest of sedges (Escudero et al., 2012b). Thus, this hyperdiverse family and its wide range of karyotypic variation constitute an ideal lineage to study shifts in chromosome evolution and how they could be related with changes 5 10 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 11 12 in diversification rates. We hypothesize that some shifts in lineage diversification could be related, at least in part, with changes in the mode of chromosome evolution. This could be explained by the fact that chromosome evolution may lead to different mechanisms of adaptation (e.g. adaptive mutation perpetuated by fission events) and/or reproductive isolation that could drive differentiation and speciation (Butlin, 2005; Coghlan et al., 2005; Coyne and Orr, 2004; Navarro and Barton, 2003a, 2003b; Otto and Whitton, 2000; Rieseberg, 2001; Soltis et al., 2009). However, there is still possible that diversification rates shifts within the family are related with others characteristic rather than mode of chromosome number evolution, so further studies must be carried out. The aims of this study are (i) to elucidate the role of chromosome evolution in the diversification of the sedge family using probabilistic models, and (ii) to evaluate the utility of nested models for studying chromosome evolution in diverse lineages. We hypothesize that transitions in the mode of chromosome evolution are closely preceded or followed by a shift in diversification rates in Cyperaceae. Our null hypothesis, by contrast, is that chromosome numbers change in the family at a constant rate, regardless of the diversification rate of independent clades. 2. Materials and Methods 2.1. Family Tree and Chromosome Counts A new comprehensive phylogeny of Cyperaceae was created from NCBI GenBank database sequences of previous studies (e.g. Hinchliff and Roalson, 2013; Spalink et al., 2016b; Jiménez-Mejías et al. 2016a). This analysis included 1058 species out of the ca. 5500 circumscribed to Cyperaceae (Govaerts et al. 2017), and was based on a supermatrix alignment of the nuclear ribosomal genes ETS and ITS, the plastid genes matK, ndhF, rbcL, 13 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 14 ycf6, and the chloroplast spacer region trnC-ycf6. Though we used the GTRCAT model in RAxML (Stamatakis, 2006) for computational purposes, the model parameters were individually calculated for five different partitions identified using PartitionFinder v2 (Lanfear et al., 2016). We converted the resulting maximum likelihood phylogeny to ultrametric using treePL (Smith and O’Meara 2012; see Fig.1, Appendix A). A total of eleven calibrations were placed on key nodes throughout the phylogeny based on fossil evidence (Jiménez-Mejías et al. 2016b; Spalink et al. 2016a, 2016b; Appendix B). Species haploid numbers were collected from online databases IPCN (Index to Plant Chromosome Numbers, Goldblatt and Johnson 2017), CCDB (Chromosome Counts Database, Rice et al. 2015), and some chromosome number reports (see Appendix B). Chromosomes counts were downloaded for a total of 825 taxa that were included in the phylogeny (Appendix B). Due to the holocentric characteristic of sedge chromosomes, counts can vary within single species (Roalson, 2008). Because we aimed to detect shifts in chromosome number evolution along the family tree, we assigned to the tips the most frequent number in the species dominated by symploidy/agmatoploidy series, and the record with the lowest chromosome number for species presenting polyploidy (see Appendix B). 2.2. Selecting the Best Scenario of Chromosome Evolution We used ChromEvol v.2.0 (Glick and Mayrose, 2014; Mayrose et al., 2010) to model the mode of chromosome evolution. This software determines the likelihood of a model to explain the given data along the phylogeny, based on the combination of two or more of the following parameters: (i) gain or (ii) loss of a single chromosome, (iii) polyploidization, (iv) demi-polyploidization (half increment of the chromosome number) and (v) incremental 7 15 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 16 17 changes to the base number with regard to a rate of multiplication that is different from a regular duplication. Two additional parameters detect linear dependency between the current haploid number and the rate of (vi) gain and (vii) loss of chromosomes. Shifts in diversification have been previously detected in four main nodes (1-4; Fig. 2) of Cyperaceae (SDC+FAEC, FAEC, non-Siderostictae Carex and C4 Cyperus; Escudero et al., 2012b; Escudero and Hipp, 2013; Spalink et al., 2016b), so analyses were conducted independently not only for the complete phylogeny but also for the same phylogeny split in several combinations of subtrees (see below). These included clades that exhibit diversification rates shifts, the background phylogeny of these clades (i.e. pruned tree without the corresponding clade), and further combinations of clades and backgrounds. A similar methodology, but not with models of chromosome number evolution, has been previously used to infer transitions in continuous character evolution using Brownian and Ornstein- Uhlenbeck models (see Escudero et al., 2012a, 2010; Hipp, 2007; O’Meara et al., 2006). Specifically, we used the censored approach described by O’Meara et al. (2006). This approach breaks up the original tree in several subtress and the branches that connect the subtrees are excluded from the analyses. The main advantage of this approach is that assumptions are not made about when and how the trait shift occurs in the missing branch. We developed models ranging from the simplest (one model) to most complex (five models) scenarios, identifying the models that best fit the data by calculating the Akaike information criterion score with ChromEvol (AIC, Mayrose et al. 2010). In order to compare the simplest (one model) with the more complex scenarios (two to five models), the branches connecting the subtrees were removed in both the single model and two to five model cases. AIC weights (Wagenmakers and Farrell, 2004) were calculated and summed to infer the importance weights of a transition occurring on each specific clade. 18 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 19 transitions and shifts in diversification rates. The results of these studies could provide new insight into the macroevolutionary processes that explain these patterns. 4.2. Final Remarks Summing up, this study proposes (i) the use of single model vs. complex models (i.e. two to five different models) of chromosome evolution as a feasible approach to the study of chromosome evolution; (ii) that, for Cyperaceae, the statistical support for a complex transition scenario was much higher than a simple model of chromosome number evolution; (iii) a clear pattern of high rate of duplications, and possibly fusions and fissions, as the main mean of chromosome evolution for, at least, part of the lineage of C4 Cyperus species, (iv) very high rate of agmatoploidy and symploidy in genus Carex (except Siderostictae clade), (v) karyotype stability (low rates of chromosome evolution) through most FAEC clade lineages. Figure captions FIGURE 1. Summarize infographic of the methodology followed in the study. FIGURE 2. Best-fitting scenarios of chromosome evolution for the Cyperaceae phylogeny. Numbered clades correspond to those in which a shift in diversification rate have been detected (1, FAEC clade; 2, Carex lineage; 3, C4 Cyperus lineage; 4, SDC+FAEC clade). Akaike information criterion (AIC) of the bestfitting scenario (AIC1) appear next to the phylogeny, compared (ΔAIC) to the null hypothesis AIC score (AIC0). Acknowledgements This work was carried out with financial support from the Spanish Ministry of Economy and Competitiveness through a research project (CGL2016-77401-P) and a Ph.D. scholarship to J.I. Márquez-Corro (BES-2017-079621). The authors thank the help provided 15 35 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 36 37 by Brian O’Meara on the analytical approach. The authors also thank the reviewers for their helpful comments that improved the previous version of this manuscript. 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