Linked by Ancestral Bonds: Multiple Whole-Genome Duplications and Reticulate Evolution in a Brassicaceae Tribe

Abstract

Pervasive hybridization and whole-genome duplications (WGDs) influenced genome evolution in several eukaryotic lineages. Although frequent and recurrent hybridizations may result in reticulate phylogenies, the evolutionary events underlying these reticulations, including detailed structure of the ancestral diploid and polyploid genomes, were only rarely reconstructed. Here, we elucidate the complex genomic history of a monophyletic clade from the mustard family (Brassicaceae), showing contentious relationships to the early-diverging clades of this model plant family. Genome evolution in the crucifer tribe Biscutelleae (∼60 species, 5 genera) was dominated by pervasive hybridizations and subsequent genome duplications. Diversification of an ancestral diploid genome into several divergent but crossable genomes was followed by hybridizations between these genomes. Whereas a single genus (Megadenia) remained diploid, the four remaining genera originated by allopolyploidy (Biscutella, Lunaria, Ricotia) or autopolyploidy (Heldreichia). The contentious relationships among the Biscutelleae genera, and between the tribe and other early diverged crucifer lineages, are best explained by close genomic relatedness among the recurrently hybridizing ancestral genomes. By using complementary cytogenomics and phylogenomics approaches, we demonstrate that the origin of a monophyletic plant clade can be more complex than a parsimonious assumption of a single WGD spurring postpolyploid cladogenesis. Instead, recurrent hybridization among the same and/or closely related parental genomes may phylogenetically interlink diploid and polyploid genomes despite the incidence of multiple independent WGDs. Our results provide new insights into evolution of early-diverging Brassicaceae lineages and elucidate challenges in resolving the contentious relationships within and between land plant lineages with pervasive hybridization and WGDs.

Keywords: chromosome rearrangements; diploidization; dysploidy; hybridization; phylogenetics; polyploidy; reticulate evolution; whole-genome duplication.

Fig. 1.

Genome structure of Megadenia pygmaea and Heldreichia bupleurifolia based on CCP analysis. (A) Comparative cytogenomic map of M. pygmaea (n = 6; Mp1–Mp6) and the purported origin of the Megadenia genome from the ancestral Proto-Calepineae Karyotype (ancPCK, n = 8; see supplementary fig. 2, Supplementary Material online, for more details). (B) The extant autotetraploid Heldreichia genome (n = 5; Hb1–Hb5’) originated by a WGD of an ancestral n = 5 genome derived from ancPCK-like genome (n = 8) by descending dysploidy (see supplementary fig. 3, Supplementary Material online, for more details). The different colors correspond to the eight chromosomes of Ancestral Crucifer Karyotype (ACK), whereas capital letters refer to 22 genomic blocks (A–X). Bacterial artificial chromosome (BAC) clones of Arabidopsis thaliana defining each genomic block are listed along the chromosomes. Centromeres are indicated by black hourglass symbols.

Fig. 2.

Comparative cytogenomic map and genome origin of Lunaria annua and L. rediviva (n = 14; Lu1–Lu14) based on CCP analysis. The ancestral allotetraploid Lunaria genome (n = 15) originated through hybridization between ancPCK (n = 8) and PCK (n = 7) genomes followed by descending dysploidy (see supplementary fig. 4, Supplementary Material online, for more details). The different colors correspond to the eight chromosomes of ACK, whereas capital letters refer to 22 genomic blocks (A–X). Bacterial artificial chromosome (BAC) clones of Arabidopsis thaliana defining each genomic block are listed along the chromosomes. Centromeres are indicated by black hourglass symbols.

Fig. 3.

Comparison of paralog divergence using distribution of synonymous substitutions per synonymous site (Ks). Paralogous gene pairs were identified in transcriptomes of seven Biscutelleae species. An arrow indicates the recent n-WGD in Heldreichia. The three mesopolyploid WGD events in Biscutella, Lunaria, and Ricotia are indicated as m-WGDs, whereas α-WGD refers to the ancient duplication shared by all Brassicaceae.

Fig. 4.

Species tree inference and phylogenetic signal analyses based on 1,545 single-copy orthologs. (A) Species phylogeny of Brassicaceae, or topology T1. All branches were supported by posterior probabilities of 1.0. Branch lengths indicate numbers of substitution per site estimated from concatenation method. Pie chart at each node indicates ASTRAL quartet scores for the three possible arrangements (q1–q3) for the respective branch leading to the node, with q1 representing the displayed topology. Numbers indicate concordance factors (CF) estimated from Bayesian concordance analysis (BUCKy). Nodes receiving inconclusive support were labeled with lowercase letters. (B) Alternative phylogenetic hypotheses (T2–T5) used to test distribution of phylogenetic signals together with the species tree obtained in this study (referred to as T1). T2 and T5 were recovered by Nikolov et al. (2019) and Mandáková et al. (2018), respectively. T3 and T4 were adapted from Huang et al. (2016) by placing Arabis alpina to two possible positions due to the lacking information in the original paper. (C) Number of genes supporting each hypothesis. Colors indicate the range of △GSL scores. (D) Number of sites supporting each hypothesis within the single-copy genes. Strong sites were defined as those having △SSL scores larger than 0.5.

Fig. 5.

Reticulate species divergence revealed by molecular dating and HyDe analyses. (A) Matrix of mean age estimates for the most recent common ancestor (MRCA) between a pair of species pairs. Only divergence times between 21.5 and 23.5 Ma are shown. (B) Network analysis using MRCA estimates between species pairs as distances. (C) Summary of significant four-taxon tests for interclade hybridizations involving the Biscutelleae tribe. (D) Summary of significant four-taxon tests in each of the Biscutelleae species. Branches representing different clades were colored following figure 4A. L1, L2, and L3 indicate species of Lineages I, II, and III, respectively. Aal, Arabis alpina. Aar, Aethionema arabicum. Bis, Biscutelleae.

Fig. 6.

Subgenome assignment for mesopolyploid Biscutelleae genomes. (A) Pipeline classifying duplicated genes according to local topologies within gene trees. For each pair of “perfect-copy” genes in Biscutella, one of the genes is labeled as subgenome A if it is sister to Heldreichia, and the other is labeled as subgenome B; in Lunaria/Ricotia, one of the genes is labeled as subgenome A if it forms a monophyletic clade with Heldreichia and Megadenia, the other is labeled as subgenome B with additional requirement that it should not be directly sister to the clade including subgenome A. (B) Contribution of different types of duplicated genes in Biscutelleae species. (C) Distribution of divergence times between duplicated genes. (D) The species tree inferred by ASTRAL with “perfect-copy” genes. Branches representing different Brassicaceae clades were colored following figure 4A.

Fig. 7.

The origin and evolution of the Biscutelleae diploid–polyploid genome complex. (A) A simplified phylogenetic scheme showing the position of the Biscutelleae in the family Brassicaceae (based on Nikolov et al. 2019). Red- and blue-labeled branches indicate evolutionary trajectory of ancestral ancPCK (n = 8) and PCK (n = 7) genomes, respectively. Star symbols indicate the genus-specific WGDs. (B) Reconstructed origin and genome evolution for individual genera of Biscutelleae. All extant Biscutelleae genomes have descended from the ancestral ancPCK-like genome (red contours and arrows). Its divergence led to the origin of ancestral diploid genomes of Heldreichia (n = 5) and Megadenia (n = 6). The extant Heldreichia genome originated via autopolyploidization. An ancestral Biscutella genome (n = 16) was formed by hybridization between two ancPCK-like genomes followed by a WGD. Allotetraploid genomes of Lunaria and Ricotia (n = 15) originated through recurrent hybridizations between ancPCK (n = 8) and PCK genome (n = 7; blue contours and arrows). WGDs in Biscutella, Lunaria, and Ricotia were followed by genus- and species-specific descending dysploidies mediated by nested chromosome insertions (NCI) and end-to-end translocations (EET). Nondysploidal rearrangements included translocations (T), as well as paracentric (I^pa) and pericentric (I^pe) inversions. The different colors correspond to chromosomes and genomic blocks in ancPCK and PCK genomes, centromeres are indicated by black hourglass symbols (see figs. 2 and 3 for details).