Incomplete lineage sorting and phenotypic evolution in marsupials.

Incomplete lineage sorting (ILS) makes ancestral genetic polymorphisms persist during rapid speciation events, inducing incongruences between gene trees and species trees. ILS has complicated phylogenetic inference in many lineages, including hominids. However, we lack empirical evidence that ILS leads to incongruent phenotypic variation. Here, we performed phylogenomic analyses to show that the South American monito del monte is the sister lineage of all Australian marsupials, although over 31% of its genome is closer to the Diprotodontia than to other Australian groups due to ILS during ancient radiation. Pervasive conflicting phylogenetic signals across the whole genome are consistent with some of the morphological variation among extant marsupials. We detected hundreds of genes that experienced stochastic fixation during ILS, encoding the same amino acids in non-sister species. Using functional experiments, we confirm how ILS may have directly contributed to hemiplasy in morphological traits that were established during rapid marsupial speciation ca. 60 mya.

Scaphopoda is the sister taxon to Bivalvia: Evidence of ancient incomplete lineage sorting.

The almost simultaneous emergence of major animal phyla during the early Cambrian shaped modern animal biodiversity. Reconstructing evolutionary relationships among such closely spaced branches in the animal tree of life has proven to be a major challenge, hindering understanding of early animal evolution and the fossil record. This is particularly true in the species-rich and highly varied Mollusca where dramatic inconsistency among paleontological, morphological, and molecular evidence has led to a long-standing debate about the group's phylogeny and the nature of dozens of enigmatic fossil taxa. A critical step needed to overcome this issue is to supplement available genomic data, which is plentiful for well-studied lineages, with genomes from rare but key lineages, such as Scaphopoda. Here, by presenting chromosome-level genomes from both extant scaphopod orders and leveraging complete genomes spanning Mollusca, we provide strong support for Scaphopoda as the sister taxon of Bivalvia, revitalizing the morphology-based Diasoma hypothesis originally proposed 50 years ago. Our molecular clock analysis confidently dates the split between Bivalvia and Scaphopoda at ~520 Ma, prompting a reinterpretation of controversial laterally compressed Early Cambrian fossils, including Anabarella, Watsonella, and Mellopegma, as stem diasomes. Moreover, we show that incongruence in the phylogenetic placement of Scaphopoda in previous phylogenomic studies was due to ancient incomplete lineage sorting (ILS) that occurred during the rapid radiation of Conchifera. Our findings highlight the need to consider ILS as a potential source of error in deep phylogeny reconstruction, especially in the context of the unique nature of the Cambrian Explosion.

MAST: Phylogenetic Inference with Mixtures Across Sites and Trees.

Hundreds or thousands of loci are now routinely used in modern phylogenomic studies. Concatenation approaches to tree inference assume that there is a single topology for the entire dataset, but different loci may have different evolutionary histories due to incomplete lineage sorting (ILS), introgression, and/or horizontal gene transfer; even single loci may not be treelike due to recombination. To overcome this shortcoming, we introduce an implementation of a multi-tree mixture model that we call mixtures across sites and trees (MAST). This model extends a prior implementation by Boussau et al. (2009) by allowing users to estimate the weight of each of a set of pre-specified bifurcating trees in a single alignment. The MAST model allows each tree to have its own weight, topology, branch lengths, substitution model, nucleotide or amino acid frequencies, and model of rate heterogeneity across sites. We implemented the MAST model in a maximum-likelihood framework in the popular phylogenetic software, IQ-TREE. Simulations show that we can accurately recover the true model parameters, including branch lengths and tree weights for a given set of tree topologies, under a wide range of biologically realistic scenarios. We also show that we can use standard statistical inference approaches to reject a single-tree model when data are simulated under multiple trees (and vice versa). We applied the MAST model to multiple primate datasets and found that it can recover the signal of ILS in the Great Apes, as well as the asymmetry in minor trees caused by introgression among several macaque species. When applied to a dataset of 4 Platyrrhine species for which standard concatenated maximum likelihood (ML) and gene tree approaches disagree, we observe that MAST gives the highest weight (i.e., the largest proportion of sites) to the tree also supported by gene tree approaches. These results suggest that the MAST model is able to analyze a concatenated alignment using ML while avoiding some of the biases that come with assuming there is only a single tree. We discuss how the MAST model can be extended in the future.

Phylogenomics and pervasive genome-wide phylogenetic discordance among fin whales (Balaenoptera physalus).

Phylogenomics has the power to uncover complex phylogenetic scenarios across the genome. In most cases, no single topology is reflected across the entire genome as the phylogenetic signal differs among genomic regions due to processes, such as introgression and incomplete lineage sorting. Baleen whales are among the largest vertebrates on Earth with a high dispersal potential in a relatively unrestricted habitat, the oceans. The fin whale (Balaenoptera physalus) is one of the most enigmatic baleen whale species, currently divided into four subspecies. It has been a matter of debate whether phylogeographic patterns explain taxonomic variation in fin whales. Here we present a chromosome-level whole genome analysis of the phylogenetic relationships among fin whales from multiple ocean basins. First, we estimated concatenated and consensus phylogenies for both the mitochondrial and nuclear genomes. The consensus phylogenies based upon the autosomal genome uncovered monophyletic clades associated with each ocean basin, aligning with the current understanding of subspecies division. Nevertheless, discordances were detected in the phylogenies based on the Y chromosome, mitochondrial genome, autosomal genome and X chromosome. Furthermore, we detected signs of introgression and pervasive phylogenetic discordance across the autosomal genome. This complex phylogenetic scenario could be explained by a puzzle of introgressive events, not yet documented in fin whales. Similarly, incomplete lineage sorting and low phylogenetic signal could lead to such phylogenetic discordances. Our study reinforces the pitfalls of relying on concatenated or single locus phylogenies to determine taxonomic relationships below the species level by illustrating the underlying nuances which some phylogenetic approaches may fail to capture. We emphasize the significance of accurate taxonomic delineation in fin whales by exploring crucial information revealed through genome-wide assessments.

Whole Genomes Reveal Evolutionary Relationships and Mechanisms Underlying Gene-Tree Discordance in Neodiprion Sawflies.

Rapidly evolving taxa are excellent models for understanding the mechanisms that give rise to biodiversity. However, developing an accurate historical framework for comparative analysis of such lineages remains a challenge due to ubiquitous incomplete lineage sorting and introgression. Here, we use a whole-genome alignment, multiple locus-sampling strategies, and summary-tree and SNP-based species-tree methods to infer a species tree for eastern North American Neodiprion species, a clade of pine-feeding sawflies (Order: Hymenopteran; Family: Diprionidae). We recovered a well-supported species tree that-except for three uncertain relationships-was robust to different strategies for analyzing whole-genome data. Nevertheless, underlying gene-tree discordance was high. To understand this genealogical variation, we used multiple linear regression to model site concordance factors estimated in 50-kb windows as a function of several genomic predictor variables. We found that site concordance factors tended to be higher in regions of the genome with more parsimony-informative sites, fewer singletons, less missing data, lower GC content, more genes, lower recombination rates, and lower D-statistics (less introgression). Together, these results suggest that incomplete lineage sorting, introgression, and genotyping error all shape the genomic landscape of gene-tree discordance in Neodiprion. More generally, our findings demonstrate how combining phylogenomic analysis with knowledge of local genomic features can reveal mechanisms that produce topological heterogeneity across genomes.

Genomic insights into zokors' phylogeny and speciation in China.

The phylogeny and speciation of subterranean zokors in China are unclear, as previous studies on morphology and limited molecular markers have generated conflicting results. This study unraveled the complex evolutionary history of eight zokor species in China based on de novo assembly at chromosome level and whole-genome sequencing of 23 populations. We found extensive phylogenetic discordances between nuclear and mitochondrial phylogenies, and different coalescent phylogenies, which could be explained by introgression and incomplete lineage sorting (ILS). The recent Qinghai-Tibet Plateau uplift (∼3.60 million y ago; Mya) drove Eospalax to speciate into clade A and clade B (∼3.22 Mya), and discordant phylogenies in this node were mainly attributed to introgression rather than ILS. Clade A rapidly diverged into three lineages due to geographical isolation and glaciation, while glaciation and C4 plant expansion contributed to the speciation of clade B. ILS contributed to the discordances of two rapidly radiated nodes rather than introgression. The effective population sizes (Ne's) of all the species of Eospalax were affected by three glaciations. Ancient polymorphisms and divergence hitchhiking contribute to genomic islands of all the species pairs. Positively selected genes putatively related to specific inhabitation adaptations were identified, such as heart development, neurogenesis, DNA repair, and immune response. Climate, geological tectonism, and C4 vegetation shaped the adaptation and speciation of zokors in China.

A phylogenomic perspective on interspecific competition.

Evolutionary processes may have substantial impacts on community assembly, but evidence for phylogenetic relatedness as a determinant of interspecific interaction strength remains mixed. In this perspective, we consider a possible role for discordance between gene trees and species trees in the interpretation of phylogenetic signal in studies of community ecology. Modern genomic data show that the evolutionary histories of many taxa are better described by a patchwork of histories that vary along the genome rather than a single species tree. If a subset of genomic loci harbour trait-related genetic variation, then the phylogeny at these loci may be more informative of interspecific trait differences than the genome background. We develop a simple method to detect loci harbouring phylogenetic signal and demonstrate its application through a proof-of-principle analysis of Penicillium genomes and pairwise interaction strength. Our results show that phylogenetic signal that may be masked genome-wide could be detectable using phylogenomic techniques and may provide a window into the genetic basis for interspecific interactions.

A Phylogenomic Assessment of Processes Underpinning Convergent Evolution in Open-Habitat Chats.

Insights into the processes underpinning convergent evolution advance our understanding of the contributions of ancestral, introgressed, and novel genetic variation to phenotypic evolution. Phylogenomic analyses characterizing genome-wide gene tree heterogeneity can provide first clues about the extent of ILS and of introgression and thereby into the potential of these processes or (in their absence) the need to invoke novel mutations to underpin convergent evolution. Here, we were interested in understanding the processes involved in convergent evolution in open-habitat chats (wheatears of the genus Oenanthe and their relatives). To this end, based on whole-genome resequencing data from 50 taxa of 44 species, we established the species tree, characterized gene tree heterogeneity, and investigated the footprints of ILS and introgression within the latter. The species tree corroborates the pattern of abundant convergent evolution, especially in wheatears. The high levels of gene tree heterogeneity in wheatears are explained by ILS alone only for 30% of internal branches. For multiple branches with high gene tree heterogeneity, D-statistics and phylogenetic networks identified footprints of introgression. Finally, long branches without extensive ILS between clades sporting similar phenotypes provide suggestive evidence for the role of novel mutations in the evolution of these phenotypes. Together, our results suggest that convergent evolution in open-habitat chats involved diverse processes and highlight that phenotypic diversification is often complex and best depicted as a network of interacting lineages.

Phylogenomics Reveals High Levels of Incomplete Lineage Sorting at the Ancestral Nodes of the Macaque Radiation.

The genus Macaca includes 23 species assigned into 4 to 7 groups. It exhibits the largest geographic range and represents the most successful example of adaptive radiation of nonhuman primates. However, intrageneric phylogenetic relationships among species remain controversial and have not been resolved so far. In this study, we conducted a phylogenomic analysis on 16 newly generated and 8 published macaque genomes. We found strong evidence supporting the division of this genus into 7 species groups. Incomplete lineage sorting (ILS) was the primary factor contributing to the discordance observed among gene trees; however, we also found evidence of hybridization events, specifically between the ancestral arctoides/sinica and silenus/nigra lineages that resulted in the hybrid formation of the fascicularis/mulatta group. Combined with fossil data, our phylogenomic data were used to establish a scenario for macaque radiation. These findings provide insights into ILS and potential ancient introgression events that were involved in the radiation of macaques, which will lead to a better understanding of the rapid speciation occurring in nonhuman primates.

New Approaches for Inferring Phylogenies in the Presence of Paralogs.

The availability of whole genome sequences was expected to supply essentially unlimited data for phylogenetics. However, strict reliance on single-copy genes for this purpose has drastically limited the amount of data that can be used. Here, we review several approaches for increasing the amount of data used for phylogenetic inference, focusing on methods that allow for the inclusion of duplicated genes (paralogs). Recently developed methods that are robust to high levels of incomplete lineage sorting also appear to be robust to the inclusion of paralogs, suggesting a promising way to take full advantage of genomic data. We discuss the pitfalls of these approaches, as well as further avenues for research.

Plastid phylogenomics and fossil evidence provide new insights into the evolutionary complexity of the 'woody clade' in Saxifragales.

BACKGROUND: The "woody clade" in Saxifragales (WCS), encompassing four woody families (Altingiaceae, Cercidiphyllaceae, Daphniphyllaceae, and Hamamelidaceae), is a phylogenetically recalcitrant node in the angiosperm tree of life, as the interfamilial relationships of the WCS remain contentious. Based on a comprehensive sampling of WCS genera, this study aims to recover a robust maternal backbone phylogeny of the WCS by analyzing plastid genome (plastome) sequence data using Bayesian inference (BI), maximum likelihood (ML), and maximum parsimony (MP) methods, and to explore the possible causes of the phylogenetic recalcitrance with respect to deep relationships within the WCS, in combination with molecular and fossil evidence. RESULTS: Although the four WCS families were identically resolved as monophyletic, the MP analysis recovered different tree topologies for the relationships among Altingiaceae, Cercidiphyllaceae, and Daphniphyllaceae from the ML and BI phylogenies. The fossil-calibrated plastome phylogeny showed that the WCS underwent a rapid divergence of crown groups in the early Cretaceous (between 104.79 and 100.23 Ma), leading to the origin of the stem lineage ancestors of Altingiaceae, Cercidiphyllaceae, Daphniphyllaceae, and Hamamelidaceae within a very short time span (∼4.56 Ma). Compared with the tree topology recovered in a previous study based on nuclear genome data, cytonuclear discordance regarding the interfamilial relationships of the WCS was detected. CONCLUSIONS: Molecular and fossil evidence imply that the early divergence of the WCS might have experienced radiative diversification of crown groups, extensive extinctions at the genus and species levels around the Cretaceous/Paleocene boundary, and ancient hybridization. Such evolutionarily complex events may introduce biases in topological estimations within the WCS due to incomplete lineage sorting, cytonuclear discordance, and long-branch attraction, potentially impacting the accurate reconstruction of deep relationships.

Phylogenomics of Afrotherian mammals and improved resolution of extant Paenungulata.

Molecular investigations have gathered a diverse set of mammals-predominantly African natives like elephants, hyraxes, and aardvarks-into a clade known as Afrotheria. Nevertheless, the precise phylogenetic relationships among these species remain contentious. Here, we sourced orthologous markers and ultraconserved elements to discern the interordinal connections among Afrotherian mammals. Our phylogenetic analyses bolster the common origin of Afroinsectiphilia and Paenungulata, and propose Afrosoricida as the closer relative to Macroscelidea rather than Tubulidentata, while also challenging the notion of Sirenia and Hyracoidea as sister taxa. The approximately unbiased test and the gene concordance factor uniformly recognized the alliance of Proboscidea with Hyracoidea as the dominant topology within Paenungulata. Investigation into sites with extremly high phylogenetic signal unveiled their potential to intensify conflicts in the Paenungulata topology. Subsequent exploration suggested that incomplete lineage sorting was predominantly responsible for the observed contentious relationships, whereas introgression exerted a subsidiary influence. The divergence times estimated in our study hint at the Cretaceous-Paleogene (K-Pg) extinction event as a catalyst for Afrotherian diversification. Overall, our findings deliver a tentative but insightful overview of Afrotheria phylogeny and divergence, elucidating these relationships through the lens of phylogenomics.

Phylogenomic analyses revealed widely occurring hybridization events across Elsholtzieae (Lamiaceae).

Obtaining a robust phylogeny proves challenging due to the intricate evolutionary history of species, where processes such as hybridization and incomplete lineage sorting can introduce conflicting signals, thereby complicating phylogenetic inference. In this study, we conducted comprehensive sampling of Elsholtzieae, with a particular focus on its largest genus, Elsholtzia. We utilized 503 nuclear loci and complete plastome sequences obtained from 99 whole-genome sequencing datasets to elucidate the interspecific relationships within the Elsholtzieae. Additionally, we explored various sources of conflicts between gene trees and species trees. Fully supported backbone phylogenies were recovered, and the monophyly of Elsholtzia and Keiskea was not supported. Significant gene tree heterogeneity was observed at numerous nodes, particularly regarding the placement of Vuhuangia and the E. densa clade. Further investigations into potential causes of this discordance revealed that incomplete lineage sorting (ILS), coupled with hybridization events, has given rise to substantial gene tree discordance. Several species, represented by multiple samples, exhibited a closer association with geographical distribution rather than following a strictly monophyletic pattern in plastid trees, suggesting chloroplast capture within Elsholtzieae and providing evidence of hybridization. In conclusion, this study provides phylogenomic insights to untangle taxonomic problems in the tribe Elsholtzieae, especially the genus Elsholtzia.

Incomplete lineage sorting and hybridization underlie tree discordance in Petunia and related genera (Petunieae, Solanaceae).

Despite the overarching history of species divergence, phylogenetic studies often reveal distinct topologies across regions of the genome. The sources of these gene tree discordances are variable, but incomplete lineage sorting (ILS) and hybridization are among those with the most biological importance. Petunia serves as a classic system for studying hybridization in the wild. While field studies suggest that hybridization is frequent, the extent of reticulation within Petunia and its closely related genera has never been examined from a phylogenetic perspective. In this study, we used transcriptomic data from 11 Petunia, 16 Calibrachoa, and 10 Fabiana species to illuminate the relationships between these species and investigate whether hybridization played a significant role in the diversification of the clade. We inferred that gene tree discordance within genera is linked to hybridization events along with high levels of ILS due to their rapid diversification. Moreover, network analyses estimated deeper hybridization events between Petunia and Calibrachoa, genera that have different chromosome numbers. Although these genera cannot hybridize at the present time, ancestral hybridization could have played a role in their parallel radiations, as they share the same habitat and life history.

A new nuclear phylogeny of the tea family (Theaceae) unravels rapid radiations in genus Camellia.

Molecular analyses of rapidly radiating groups often reveal incongruence between gene trees. This mainly results from incomplete lineage sorting, introgression, and gene tree estimation error, which complicate the estimation of phylogenetic relationships. In this study, we reconstruct the phylogeny of Theaceae using 348 nuclear loci from 68 individuals and two outgroup taxa. Sequence data were obtained by target enrichment using the recently released Angiosperm 353 universal probe set applied to herbarium specimens. The robustness of the topologies to variation in data quality was established under a range of different filtering schemes, using both coalescent and concatenation approaches. Our results confirmed most of the previously hypothesized relationships among tribes and genera, while clarifying additional interspecific relationships within the rapidly radiating genus Camellia. We recovered a remarkably high degree of gene tree heterogeneity indicative of rapid radiation in the group and observed cytonuclear conflicts, especially within Camellia. This was especially pronounced around short branches, which we primarily associate with gene tree estimation error. Our analysis also indicates that incomplete lineage sorting (ILS) contributed to gene-tree conflicts and accounted for approximately 14 % of the explained variation, whereas inferred introgression levels were low. Our study advances the understanding of the evolution of this important plant family and provides guidance on the application of target capture methods and the evaluation of key processes that influence phylogenetic discordances.

Phylogenomics of mulberries (Morus, Moraceae) inferred from plastomes and single copy nuclear genes.

Mulberries (genus Morus), belonging to the order Rosales, family Moraceae, are important woody plants due to their economic values in sericulture, as well as for nutritional benefits and medicinal values. However, the taxonomy and phylogeny of Morus, especially for the Asian species, remains challenging due to its wide geographical distribution, morphological plasticity, and interspecific hybridization. To better understand the evolutionary history of Morus, we combined plastomes and a large-scale nuclear gene analyses to investigate their phylogenetic relationships. We assembled the plastomes and screened 211 single-copy nuclear genes from 13 Morus species and related taxa. The plastomes of Morus species were relatively conserved in terms of genome size, gene content, synteny, IR boundary and codon usage. Using nuclear data, our results elucidated identical topologies based on coalescent and concatenation methods. The genus Morus was supported as monophyletic, with M. notabilis as the first diverging lineage and the two North American Morus species, M. microphylla and M. rubra, as sister to the other Asian species. In the Asian Morus species, interspecific relationships were completely resolved. However, cyto-nuclear discordances and gene tree-species tree conflicts were detected in the phylogenies of Morus, with multiple evidences supporting hybridization/introgression as the main cause of discordances between nuclear and plastid phylogenies, while gene tree-species tree conflicts were mainly caused by ILS.

Peering through the hedge: Multiple datasets yield insights into the phylogenetic relationships and incongruences in the tribe Lilieae (Liliaceae).

The increasing use of genome-scale data has significantly facilitated phylogenetic analyses, contributing to the dissection of the underlying evolutionary mechanisms that shape phylogenetic incongruences, such as incomplete lineage sorting (ILS) and hybridization. Lilieae, a prominent member of the Liliaceae family, comprises four genera and approximately 260 species, representing 43% of all species within Liliaceae. They possess high ornamental, medicinal and edible values. Yet, no study has explored the validity of various genome-scale data in phylogenetic analyses within this tribe, nor have potential evolutionary mechanisms underlying its phylogenetic incongruences been investigated. Here, transcriptome, Angiosperms353, plastid and mitochondrial data, were collected from 50 to 93 samples of Lilieae, covering all four recognized genera. Multiple datasets were created and used for phylogenetic analyses based on concatenated and coalescent-based methods. Evolutionary rates of different datasets were calculated, and divergence times were estimated. Various approaches, including coalescence simulation, Quartet Sampling (QS), calculation of concordance factors (gCF and sCF), as well as MSCquartets and reticulate network inference, were carried out to infer the phylogenetic discordances and analyze their underlying mechanisms using a reduced 33-taxon dataset. Despite extensive phylogenetic discordances among gene trees, robust phylogenies were inferred from nuclear and plastid data compared to mitochondrial data, with lower synonymous substitution detected in mitochondrial genes than in nuclear and plastid genes. Significant ILS was detected across the phylogeny of Lilieae, with clear evidence of reticulate evolution identified. Divergence time estimation indicated that most of lineages in Lilieae diverged during a narrow time frame (ranging from 5.0 Ma to 10.0 Ma), consistent with the notion of rapid radiation evolution. Our results suggest that integrating transcriptomic and plastid data can serve as cost-effective and efficient tools for phylogenetic inference and evolutionary analysis within Lilieae, and Angiosperms353 data is also a favorable choice. Mitochondrial data are more suitable for phylogenetic analyses at higher taxonomic levels due to their stronger conservation and lower synonymous substitution rates. Significant phylogenetic incongruences detected in Lilieae were caused by both incomplete lineage sorting (ILS) and reticulate evolution, with hybridization and "ghost introgression" likely prevalent in the evolution of Lilieae species. Our findings provide new insights into the phylogeny of Lilieae, enhancing our understanding of the evolution of species in this tribe.

Phylogenomic Analyses Reveal an Allopolyploid Origin of Core Didymocarpinae (Gesneriaceae) Followed by Rapid Radiation.

Allopolyploid plants have long been regarded as possessing genetic advantages under certain circumstances due to the combined effects of their hybrid origins and duplicated genomes. However, the evolutionary consequences of allopolyploidy in lineage diversification remain to be fully understood. Here, we investigate the evolutionary consequences of allopolyploidy using 138 transcriptomic sequences of Gesneriaceae, including 124 newly sequenced, focusing particularly on the largest subtribe Didymocarpinae. We estimated the phylogeny of Gesneriaceae using concatenated and coalescent-based methods based on five different nuclear matrices and 27 plastid genes, focusing on relationships among major clades. To better understand the evolutionary affinities in this family, we applied a range of approaches to characterize the extent and cause of phylogenetic incongruence. We found that extensive conflicts between nuclear and chloroplast genomes and among nuclear genes were caused by both incomplete lineage sorting (ILS) and reticulation, and we found evidence of widespread ancient hybridization and introgression. Using the most highly supported phylogenomic framework, we revealed multiple bursts of gene duplication throughout the evolutionary history of Gesneriaceae. By incorporating molecular dating and analyses of diversification dynamics, our study shows that an ancient allopolyploidization event occurred around the Oligocene-Miocene boundary, which may have driven the rapid radiation of core Didymocarpinae.

Introgression underlies phylogenetic uncertainty but not parallel plumage evolution in a recent songbird radiation.

Instances of parallel phenotypic evolution offer great opportunities to understand the evolutionary processes underlying phenotypic changes. However, confirming parallel phenotypic evolution and studying its causes requires a robust phylogenetic framework. One such example is the "black-and-white wagtails", a group of five species in the songbird genus Motacilla: one species, Motacilla alba, shows wide intra-specific plumage variation, while the four others form two pairs of very similar-looking species (M. aguimp + M. samveasnae and M. grandis + M. maderaspatensis, respectively). However, the two species in each of these pairs were not recovered as sisters in previous phylogenetic inferences. Their relationships varied depending on the markers used, suggesting that gene tree heterogeneity might have hampered accurate phylogenetic inference. Here, we use whole genome resequencing data to explore the phylogenetic relationships within this group, with a special emphasis on characterizing the extent of gene tree heterogeneity and its underlying causes. We first used multispecies coalescent methods to generate a "complete evidence" phylogenetic hypothesis based on genome-wide variants, while accounting for incomplete lineage sorting (ILS) and introgression. We then investigated the variation in phylogenetic signal across the genome, to quantify the extent of discordance across genomic regions, and test its underlying causes. We found that wagtail genomes are mosaics of regions supporting variable genealogies, because of ILS and inter-specific introgression. The most common topology across the genome, supporting M. alba and M. aguimp as sister species, appears to be influenced by ancient introgression. Additionally, we inferred another ancient introgression event, between M. alba and M. grandis. By combining results from multiple analyses, we propose a phylogenetic network for the black-and-white wagtails that confirms that similar phenotypes evolved in non-sister lineages, supporting parallel plumage evolution. Furthermore, the inferred reticulations do not connect species with similar plumage coloration, suggesting that introgression does not underlie parallel plumage evolution in this group. Our results demonstrate the importance of investigation of genome-wide patterns of gene tree heterogeneity to help understanding the mechanisms underlying phenotypic evolution.

Phylotranscriptomic analyses reveal the evolutionary complexity of Paris L. (Melanthiaceae), a morphologically distinctive genus with significant pharmaceutical importance.

BACKGROUND AND AIMS: Previous phylogenetic studies on the pharmaceutically significant genus Paris (Melanthiaceae) have consistently revealed substantial cytonuclear discordance, yet the underlying mechanism responsible for this phenomenon remains elusive. This study aims to reconstruct a robust nuclear backbone phylogeny and elucidate the potential evolutionarily complex events contributing to previously observed cytonuclear discordance within Paris. METHODS: Based on a comprehensive set of nuclear low-copy orthologous genes obtained from transcriptomic data, the intrageneric phylogeny of Paris, along with its phylogenetic relationships to allied genera were inferred, using coalescent and concatenated approaches. The analysis of gene tree discordance and reticulate evolution, in conjunction with an incomplete lineage sorting (ILS) simulation, was conducted to explore potential hybridization and ILS events in the evolutionary history of Paris and assess their contribution to the discordance of gene trees. KEY RESULTS: The nuclear phylogeny unequivocally confirmed the monophyly of Paris and its sister relationship with Trillium, while widespread incongruences in gene trees were observed at the majority of internal nodes within Paris. The reticulate evolution analysis identified five instances of hybridization events in Paris, indicating that hybridization events might have recurrently occurred throughout the evolutionary history of Paris. In contrast, the ILS simulations revealed that only two internal nodes within sect. Euthyra experienced ILS events. CONCLUSIONS: Our data suggest that the previously observed cytonuclear discordance in the phylogeny of Paris can primarily be attributed to recurrent hybridization events, with secondary contributions from infrequent ILS events. The recurrent hybridization events in the evolutionary history of Paris not only drove lineage diversification and speciation but also facilitated morphological innovation, and enhanced ecological adaptability. Therefore, artificial hybridization has great potential for breeding medicinal Paris species. These findings significantly contribute to our comprehensive understanding of the evolutionary complexity of this pharmaceutically significant plant lineage, thereby facilitating effective exploration and conservation efforts.