Lineage Sorting Research Articles

Phylogenomics reveals the slow-burning fuse of diatom evolution

Evolution is often uneven in its pace and outcomes, with long periods of stasis interrupted by abrupt increases in morphological and ecological disparity. With thousands of gene histories, phylogenomics can uncover the genomic signatures of these broad macroevolutionary trends. Diatoms are a species-rich lineage of microeukaryotes that contribute greatly to the global cycling of carbon, oxygen, and silica, which they use to build elaborately structured cell walls. We combined fossil information with newly sequenced transcriptomes from 181 diverse diatom species to reconstruct the pattern, timing, and genomic context of major evolutionary transitions. Diatoms originated 270 Mya, and after >100 My of relative stasis in morphology and ecology, a radiation near the Jurassic-Cretaceous boundary led to the diversity of habitats and cell wall architectures characteristic of modern diatoms. This transition was marked by a genome duplication and high levels of gene tree discordance. However, short generation times increase the probability of coalescence between speciation events, minimizing the impacts of incomplete lineage sorting and implicating sequence saturation and gene tree error as the main sources of discordance. Nevertheless, a rigorous tree-based approach to ortholog selection resulted in strongly supported relationships, including some that were uncertain previously. Three pulses of accelerated speciation were detected, two of which were associated with the evolution of novel traits and ecological transitions. The first 100 My of diatom evolution was a slow-burning fuse that led to a burst of innovations in ecology, morphology, and life history that are hallmarks of contemporary diatom assemblages.

Resolving taxonomic uncertainty and exploring evolutionary relationships in the Cymopterus terebinthinus (Apiaceae) species complex

AbstractSpeciation processes in plants can be difficult to evaluate, but are essential to understanding evolutionary processes that lead to diversification. Determining the juncture at which a genetically and/or morphologically divergent population can be reliably considered a separate species is often challenging. This is particularly so with respect to recent divergences amongst closely related taxa wherein factors such as incomplete lineage sorting may yield confounding results. Taxa in the Cymopterus terebinthinus (Apiaceae) species complex have long puzzled botanists. Named entities in this group display similar, yet apparently distinct morphologies that have been classified as varieties under various generic names highlighting long‐standing nomenclatural instability. Previous phylogenetic studies have challenged the monophyly of this complex. This study aims to clarify taxonomic boundaries and infer evolutionary relationships among the four C. terebinthinus varieties and C. petraeus by applying phylogenetic inference and incorporating ecological, morphological, and geographical evidence. We sampled from populations of all varieties of C. terebinthinus and C. petraeus for target capture with the Angiosperms353 bait kit. We performed phylogenetic analyses with maximum likelihood (RAxML and IQ‐TREE) and coalescent‐based phylogenetic analysis (ASTRAL). We also conducted principal component analysis of soil samples and climatic variables. We find that C. terebinthinus and its varietal infrataxa comprise a monophyletic clade that includes C. petraeus. Clade groupings correspond to previous taxonomic assignments and morphology. Clades are often closely associated with geographical variables and at times correlated with ecological variables. Exceptions to this are here attributed to various evolutionary factors that often confound other phylogenetic analyses such as incomplete lineage sorting, introgression, and paralogous loci. Our findings suggests that geographical factors might play a major role in genetic and morphological differentiation in this complex. Despite finding well‐supported clades that correspond to defined morphological characters; further sampling among C. petraeus populations is required to make taxonomic decisions.

The Genus Petunia (Solanaceae): Evolutionary Synthesis and Taxonomic Review.

Many plant groups exhibit complex evolutionary processes, including hybridization, incomplete lineage sorting, and variable evolutionary rates, which make species delimitation challenging. Molecular data have been essential for studying such groups, including Petunia, where local adaptation, allopatric speciation, pollinator interactions, and hybridization shape diversity and population structure. In this study, we produced the first broadly inclusive phylogenetic tree of Petunia using high-throughput DNA sequence data generated by genome complexity reduction-based sequencing (DArT), and incorporating all currently accepted taxa. Additionally, we reviewed previously published phylogenetic and phylogeographic studies on these species to support the taxonomic revision. Phylogenetic analyses based on SNPs were largely congruent, revealing two well-supported clades divided by corolla tube length, consistent with previous studies. These clades likely originated and diversified during the Pleistocene. The phylogenetic trees provided strong support for taxonomic changes, resolving long-standing uncertainties. We recognize P. axillaris, P. parodii, and P. subandina as independent species, elevate P. integrifolia subsp. depauperata to P. dichotoma Sendtn., and resurrect P. guarapuavensis. Additionally, our results highlighted unsolved questions regarding the evolutionary history of the short corolla tube clade, suggesting the need for further investigation into its diversification and genetic structure.

Navigating phylogenetic conflict and evolutionary inference in plants with target-capture data

Target capture has rapidly become a preferred approach for plant systematic and evolutionary research, marking a step change in the generation of data for phylogenetic inference. Although this advancement has facilitated the resolution of many relationships, phylogenetic conflict continues to be reported and is often attributed to genome duplication, reticulation, incomplete lineage sorting or rapid speciation – common processes in plant evolution. The proliferation of methods for analysing target-capture data in the presence of these processes can be overwhelming for many researchers, especially students. In this review, we break down the causes of conflict and guide researchers through a target-capture bioinformatic workflow, with a particular focus on robust phylogenetic inference in the presence of conflict. Through the workflow, we highlight key considerations for reducing artefactual conflict, managing paralogs and assessing conflict, and discuss current methods for investigating causes of conflict. Although we draw from examples in the Australian flora, this review is broadly relevant for any researcher working with target-capture data. We conclude that conflict is often inherent in plant phylogenomic datasets, and, although further methodological development is needed, when conflict is carefully investigated, target-capture data can provide unprecedented insight into the extraordinary evolutionary histories of plants.

Phylogenomics of the tetraploid Hawaiian lobeliads: Implications for their origin, dispersal history, and adaptive radiation

Hawaiian lobeliads exhibit extensive adaptive radiations and are considered the largest plant clade (143 species) endemic to any oceanic archipelago. Rapid insular radiations are prone to reticulate evolution, yet detecting hybridization is often limited by inadequate sampling of taxa or independent loci. We analyzed 633 nuclear loci (including tetraploid duplications) and whole plastomes for 89% of extant species to derive phylogenies for the Hawaiian lobeliads. Nuclear data provide strong support for nine major clades in both likelihood and ASTRAL analyses. All genera/sections are monophyletic except Clermontia and Cyanea. Nuclear and plastome phylogenies conflict on short, deep branches; the nuclear tree resolves a fleshy-fruited clade of Hawaiian Clermontia/Cyanea-Brighamia/Delissea, sister to Polynesian Sclerotheca, with both sister to a capsular-fruited Hawaiian clade. Incomplete lineage sorting in a rapid radiation starting 8.5-11.3 Ma is sufficient to explain uncertainty and cytonuclear discordance along the backbone. Sequence data support reticulation within Clermontia and especially Cyanea. Nuclear data identify 42 interisland dispersal events: 89% accord with the strict progression rule, involving movement to the next younger island in the hotspot chain, consistent with theory. Plastid data overestimate such events by 17%. Cyanea and Clermontia have undergone parallel adaptive radiations in elevational distribution and flower length on all major islands, with multiple founders and some interisland differences. Hawaiian lobeliad diversification was driven by an early intergeneric divergence in habitat, followed by parallel adaptive radiation and ecological speciation within Clermontia/Cyanea, combined with widespread single-island endemism, frequent interisland dispersal, and occasional hybridization.

CAnDI: a new tool to investigate conflict in homologous gene trees and explain convergent trait evolution.

Phenotypic convergence is found across the tree of life, and morphological similarities in distantly related species are often presumed to have evolved independently. However, clarifying the origins of traits has recently highlighted the complex nature of evolution, as apparent convergent features often share similar genetic foundations. Hence, the tree topology of genes that underlie such traits frequently conflicts with the overall history of species relationships. This conflict, which usually results from incomplete lineage sorting, introgression or horizontal gene transfer, creates both a challenge for systematists and an exciting opportunity to investigate the rich, complex network of information that connects molecular trajectories with trait evolution. Here we present a novel conflict identification program named CAnDI (Conflict And Duplication Identifier), which enables the analysis of conflict in homologous gene trees rather than inferred orthologs. We demonstrate that the analysis of conflicts in homologous trees using CAnDI yields more comparisons than in ortholog trees in six datasets from across the eukaryotic tree of life. Using the carnivorous trap of Caryophyllales, a charismatic group of flowering plants, as a case study we demonstrate that analysing conflict on entire homolog trees can aid in inferring the contribution of standing genetic variation to trait evolution: by dissecting all gene relationships within homolog trees, we find genomic evidence that the molecular basis of the pleisiomorphic mucilaginous sticky trap was likely present in the ancestor of all carnivorous Caryophyllales. We also show that many genes whose evolutionary trajectories group species with similar trap devices code for proteins contributing to plant carnivory and identify a LATERAL ORGAN BOUNDARY DOMAIN transcription factor as a possible candidate for regulating sticky trap development.

Standing genetic variation and introgression shape the cryptic radiation of Aquilegia in the mountains of Southwest China

Cryptic diversity in evolutionary radiation offers an excellent system for investigating the intricacies of evolutionary progress. Understanding the evolution of cryptic diversity is imperative for unraveling the hidden complexities of biodiversity. However, empirical evidence elucidating the mechanisms behind cryptic radiation remains limited, particularly in plants. Here, we focus on a monophyletic group of Aquilegia species mainly distributed in the mountains of Southwest China, one of the world’s biodiversity hotspots. Using whole-genome resequencing of 158 individuals from 23 natural populations, we identify three to four paraphyletic lineages within each morphological species. Our findings reveal that 39 out of 43 detected instances of introgression occurred post-lineage formation. Identifying shared genomic regions indicates that the divergence of fixed singletons in lineages from morphological species A. kansuensis and A. rockii predates lineage formation, supporting a scenario where incomplete lineage sorting of standing variation contributes to morphological parallelism. Furthermore, strong positive correlations among genomic differentiation, divergence, and introgression suggest that standing variations and introgression from non-sister lineages contribute to the rapid genetic divergence. Our study illuminates the important roles of standing variations and introgression in plant cryptic radiation, advancing our understanding of the complex mechanisms behind the evolution of biodiversity in recent radiation events.

Incomplete lineage sorting and introgression among genera and species of Liliaceae tribe Tulipeae: insights from phylogenomics

BackgroundPhylogenetic research in Tulipa (Liliaceae), a genus of significant economic and horticultural value, has relied on limited nuclear (mostly nuclear ribosomal internal transcribed spacer, nrITS) and plastid DNA sequences, resulting in low-resolution phylogenetic trees and uncertain intrageneric classifications. The genus, noted for its large genome, presents discordant relationships among Amana, Erythronium, and Tulipa, likely due to incomplete lineage sorting (ILS) and/or reticulate evolution. Thus, phylogenomic approaches are needed to clarify these relationships and the conflicting signals within the tribe Tulipeae.ResultsWe newly sequenced 50 transcriptomes of 46 species of tribe Tulipeae (including multiple accessions of all four genera) and one outgroup species of the sister tribe Lilieae (Notholirion campanulatum), and downloaded 15 previously published transcriptomes of tribe Tulipeae to supplement the sampling. One plastid dataset (74 plastid protein-coding genes, PCGs) and one nuclear dataset (2594 nuclear orthologous genes, OGs) were constructed, with the latter used for species tree inference based on maximum likelihood (ML) and multi-species coalescent (MSC) methods. To investigate causes of gene tree discordance, “site con/discordance factors” (sCF and sDF1/sDF2) were calculated first, after which phylogenetic nodes displaying high or imbalanced sDF1/2 were selected for phylogenetic network analyses and polytomy tests to determine whether ILS or reticulate evolution best explain incongruence. Key relationships not resolved by this technique, especially those among Amana, Erythronium, and Tulipa, were further investigated by applying D-statistics and QuIBL.ConclusionsWe failed to reconstruct a reliable and unambiguous evolutionary history among Amana, Erythronium, and Tulipa due to especially pervasive ILS and reticulate evolution, likely caused either by obscured minority phylogenetic signal or differing signals among genomic compartments. However, within Tulipa we confirmed the monophyly of most subgenera, with the exception of two species in the small subgenus Orithyia, of which Tulipa heterophylla was recovered as sister to the remainder of the genus, whereas T. sinkiangensis clustered within subgenus Tulipa. In contrast, most traditional sections of Tulipa were found to be non-monophyletic.

Comparative Analysis of the Chloroplast Genomes of Cypripedium: Assessing the Roles of SSRs and TRs in the Non-Coding Regions of LSC in Shaping Chloroplast Genome Size.

Cypripedium is renowned for its high morphological diversity and complex genetic and evolutionary characteristics. The chloroplast genome serves as a valuable tool for investigating phylogenetic relationships and evolutionary processes in plants. Currently, research on the evolution of the chloroplast genome within the Cypripedium genus is limited due to insufficient large-scale sampling and a lack of comprehensive understanding. Consequently, the mechanisms underlying the significant differences in chloroplast genome size among Cypripedium species remain poorly understood. In this study, we conducted a comprehensive comparative analysis of the chloroplast genomes of 29 Cypripedium species. The lengths of these genomes ranged from 162,092 to 246,177 base pairs (bp) and contained between 127 and 134 genes. Our results indicate that, while the overall structure of the chloroplast genomes in Cypripedium species is relatively conserved, significant differences were observed among the large single-copy (LSC), small single-copy (SSC), and inverted repeat (IR) regions. Several genes, including psaC, rpl32, ycf1, and psbK, exhibited higher levels of variability and may serve as molecular markers in taxonomic studies. The results of our correlation analysis suggest that the expansion of the LSC region, the increase in simple sequence repeats (SSRs), and tandem repeats (TRs) have significantly enlarged the size of the chloroplast genome in Cypripedium species. Phylogenetic signal testing supports the notion that genetic variation has driven species divergence within the genus. Overall, our findings provide insights into the substantial differences in chloroplast genome length observed among Cypripedium species. However, the relationship between diversification and the evolutionary mechanisms affecting Cypripedium, including ecological adaptive evolution, incomplete lineage sorting (ILS), hybridization, and reticulate events, requires further investigation.

Reconciling Gene Tree Discordance and Biogeography in European Crows.

Reconstructing the evolutionary history of young lineages diverging with gene flow is challenging due to factors like incomplete lineage sorting, introgression, and selection causing gene tree discordance. The European crow hybrid zone between all-black carrion crows and grey-coated hooded crows exemplifies this challenge. Most of the genome in Western and Central European carrion crow populations is near-identical to hooded crows, but differs substantially from their Iberian congeners. A notable exception is a single major-effect colour-locus under sexual selection aligning with the 'species' tree. To understand the underlying evolutionary processes, we reconstructed the biogeographic history of the species complex. During the Pleistocene carrion and hooded crows took refuge in the Iberian Peninsula and the Middle East, respectively. Allele-sharing of all-black Western European populations with likewise black Iberian crows at the colour-locus represents the last trace of carrion crow ancestry, resisting gene flow from expanding hooded crow populations that have homogenised most of the genome. A model of colour-locus introgression from an Iberian ancestor into hooded crow populations near the Pyrenées was significantly less supported. We found no positive relationship between introgression and recombination rate consistent with the absence of genome-wide, polygenic barriers in this young species complex. Overall, this study portrays a scenario where few large-effect loci, subject to divergent sexual selection, resist rampant and asymmetric gene exchange. This study underscores the importance of integrating population demography and biogeography to accurately interpret patterns of gene tree discordance following population divergence.

Generic delimitation in the Cyphotheca-Plagiopetalum-Sporoxeia clade.

Generic delimitation in the Cyphotheca-Plagiopetalum-Sporoxeia clade.

Phylotranscriptomic analyses reveal the reticulate evolution in Dennstaedtiaceae

AbstractDennstaedtiaceae, a heterogeneous family of ferns with 11 genera and about 270 species, has a global distribution. While substantial progress has been made in elucidating the intergeneric relationships within Dennstaedtiaceae, certain nodes, particularly within Hypolepidoideae, remain controversial. To date, no phylogenomic investigation of nuclear genes has been conducted for Dennstaedtiaceae, and the biological processes underlying its complex evolution remain largely unknown. In this study, we generated transcriptome sequences from nine species in Dennstaedtiaceae and combined them with publicly available data sets from 13 species and one outgroup. By utilizing the 23 transcriptomic data sets, representing nine out of the 11 genera in Dennstaedtiaceae, we successfully resolved the intergeneric relationships within the family and established a fundamental phylogenetic framework to investigate its evolutionary history. By combining the analyses of rate‐adjusted Ks‐based age distributions and phylogenetic reconciliation approaches, we found evidence of at least one round of whole‐genome duplication (WGD) that is shared by all Dennstaedtiaceae species prior to their divergence. Extensive gene tree discordance was found across the backbone of Dennstaedtiaceae, with the most significant discordance within Hypolepidoideae. The results of incomplete lineage sorting (ILS) simulation revealed that ILS is a substantial contributor to these conflicts. Evidence from phylogenetic networks and introgression tests indicates the occurrence of gene flow among the clades of Paesia, Hiya, and Histiopteris, potentially explaining the observed cytonuclear discordance in Hypolepidoideae. Our phylotranscriptomic study of Dennstaedtiaceae provides novel insights into its complex reticulate evolutionary history, paving the way for future studies aimed at unraveling the mechanisms underlying its diversification and adaptation.

Unraveling the extensive phylogenetic discordance and evolutionary history of spurless taxa within the Aquilegia ecalcarata complex.

Parallel evolution of the same, or at least very similar, phenotype(s) in different lineages is often interpreted as evidence for the action of natural selection. However, caution is required when inferring parallel evolution based on uncertain or potentially incorrect phylogenetic frameworks. Here, by conducting extensive phylogenomic and population genetic analyses, we aim to clarify the evolutionary history of spurless taxa within the Aquilegia ecalcarata complex. We observed substantial discordance in the phylogenetic patterns across the entire genome, primarily attributed to ancient introgression and incomplete lineage sorting. Additionally, we identified several spurless lineages whose phylogenetic positions were distorted by admixture events. Using a backbone tree and demographic modeling, we determined that these spurless taxa independently originated twice within this group. Intriguingly, our investigation revealed that the spurless taxa experienced population expansion during global cooling, while their spurred sister groups underwent population contraction. The parallel losses of petal spurs, therefore, may be linked to adaptations for low-temperature conditions. These findings emphasize the importance of comprehensive population-level analyses in phylogenetic inference and provide valuable insights into the dynamics of trait loss and its implications for the adaptive strategies.

Misspecification Strikes: ASTRAL can Mislead in the Presence of Hybridization, even for Nonanomalous Scenarios.

ASTRAL is a powerful and widely used tool for species tree inference, known for its computational speed and robustness under incomplete lineage sorting. The method has often been used as an initial step in species network inference to provide a backbone tree structure upon which hybridization events are later added to such a tree via other methods. However, we show empirically and theoretically, that this methodology can yield flawed results. Specifically, we demonstrate that under the network multispecies coalescent model-including nonanomalous scenarios-ASTRAL can produce a tree that does not correspond to any topology displayed by the true underlying network. This finding highlights the need for caution when using ASTRAL-based inferences in suspected hybridization cases.

Phylogenomic analyses re-evaluate the backbone of Corylus and unravel extensive signals of reticulate evolution.

Phylogenomic analyses re-evaluate the backbone of Corylus and unravel extensive signals of reticulate evolution.

A thorny tale: The origin and diversification of Cirsium (Compositae).

A thorny tale: The origin and diversification of Cirsium (Compositae).

Phylogenomics of marine angelfishes: diagnosing sources of systematic discordance for an iconic reef fish family (F: Pomacanthidae).

Genome-scale datasets are resolving challenging nodes across the tree of life. These datasets however, characterized by inherent heterogeneity, often push traditional phylogenetic reconstruction methods to their limits. By integrating multiple phylogenomic approaches, we can identify the causes of topological discordance within genomic partitions while accounting for various sources of heterogeneity and systematic errors. Here we conduct the first in-depth analysis of discordance for the reef family Pomacanthidae (marine angelfishes) using target enrichment data of ~1,000 ultraconserved elements from 45 pomacanthid species. Our combined phylogenomic approach resolved the systematics of the family at the base of the pomacanthid tree. Despite this resolution, our analyses also highlight discordance in ancestral nodes associated with the paraphyletic Centropyge genus and monotypic Pygoplites lineage, and the impact of incomplete lineage sorting in the evolutionary history pomacanthids. Species network searches and model selection supported a reticulated evolutionary history suggesting three ancient gene flow events between ghost (or unsampled) lineages at the root of the Pomacanthidae tree and ancestors of Genicanthus, Centropyge, Chaetodontoplus and Pomacanthus lineages. This study advances our understanding of diagnosing topological discordance in genome-scale phylogenies and provide an analytical pathway for limiting systematic errors. In the process of diagnosing discordance, we identify key evolutionary processes involved in the complex evolution of marine angelfishes. While often inconvenient in phylogenetic analyses, patterns of discordance can shed light on underlying biological and evolutionary processes that shape the evolution of biodiversity.

Phylogenetic relationships and the repeated loss of traits associated with sicklebill pollination in Centropogon subgenus Centropogon (Campanulaceae).

Centropogon subgenus Centropogon comprises 55 species found primarily in midelevation Andean forests featuring some of the most curved flowers among angiosperms. Floral curvature is linked to coevolution with the sicklebill hummingbird, which pollinates most species. Despite charismatic flowers, there is limited knowledge about the phylogenetic relationships and floral evolution. We conducted the first densely sampled phylogenomic analysis of the clade using methods that account for incomplete lineage sorting on a sequence capture dataset generated with a lineage-specific probe set. Using phylogenetic comparative methods, we test for correlated evolution of two traits central to sicklebill pollination. We improve understanding of species relationships by more than doubling past taxon sampling. We confirm the monophyly of the subgenus and two sections, and the non-monophyly of remaining sections. The subgenus is characterized by high gene tree discordance. Three widespread species display contrasting phylogenetic dynamics, with C. cornutus forming a clade and C. granulosus and C. solanifolius forming non-monophyletic, biogeographically clustered lineages. Correlated evolution of floral curvature and inflorescence structure has led to multiple putative losses of sicklebill pollination. Centropogon subgenus Centropogon adds to a growing body of literature of Andean plant clades with high gene tree discordance. This phylogeny serves as a foundational framework for further macroevolutionary investigations into the environmental and biogeographic factors shaping the evolution of pollination-related traits.

Phylogenomic Analysis Reveals Evolutionary Relationships of Tropical Drosophilidae: From Drosophila to Scaptodrosophila.

Species radiation in the family Drosophilidae has led to a diversity of species occupying a wide range of ecological niches. Despite the high diversity within this family, with over thousands of species and more than a hundred species recorded in Thailand and the Malay Peninsula, taxonomic classifications remain complicated due to morphological plasticity and inconsistent phylogenetic reconstructions based on limited genetic data. In this study, we assemble new mitochondrial genomes from Drosophila and Scaptodrosophila species collected in Thailand, expanding the genomic resources for these underexplored tropical regions. Phylogenetic analyses of 13 mitochondrial protein-coding genes revealed well-supported evolutionary relationships, with Scaptodrosophila forming a distinct lineage and several Drosophila subgroups, such as ananassae, montium, and melanogaster, exhibiting monophyly. Notable discrepancies were observed in the placement of the suzukii subgroup, which was not recovered as monophyletic, and the position of the punjabiensis subgroup, reflecting the complexities of lineage sorting and hybridization events. This comprehensive genomic analysis provides a more accurate understanding of evolutionary relationships within the family Drosophilidae's diversification in the tropics.

Coalescent-based branch length estimation improves dating of species trees.

Species trees need to be dated for many downstream applications. Typical molecular dating methods take a phylogenetic tree with branch lengths in substitution units as well as a set of calibrations as input and convert the branch lengths of the species tree to the unit of time while being consistent with the pre-specified calibrations. When dating species trees from multi-locus genome-scale datasets, the branch lengths and sometimes the topology of the species tree are estimated using concatenation. However, concatenation does not address gene tree heterogeneity across the genome. While Bayesian dating methods can address some forms of gene tree heterogeneity, such as incomplete lineage sorting, they are not scalable to large datasets. In this paper, we introduce a new scalable pipeline for dating species trees that addresses gene tree discordance for both topology and branch length estimation. The pipeline uses discordance-aware methods that account for incomplete lineage sorting for estimating the topology and branch lengths and maximum likelihood-based methods for the dating step. Our simulation study on datasets with gene tree discordance shows that this pipeline produces more accurate and less biased dates than pipelines that use concatenation or unpartitioned Bayesian methods. Furthermore, it is substantially more scalable and can handle datasets with thousands of species and genes. Our results on two biological datasets show that this new pipeline improves the inference of node ages and branch lengths for some nodes, in particular extant taxa, and improves the downstream diversification analysis.