NewickTreeModifier: A simple web tool to prune and modify Newick trees.

Large-scale selection analyses of protein-coding sequences and phylogenetic tree reconstructions require suitable trees in Newick format. We developed the NewickTreeModifier (NTM), a simple web-based tool to trim and modify Newick trees for such analyses. The users can choose provided master trees or upload a tree to prune it to selected species available in FASTA, NEXUS, or PHYLIP sequence format with an internal converter, a simple species list, or directly determined from a checklist interface of the master trees. Plant, insect, and vertebrate master trees comprise the maximum number of species in an up-to-date phylogenetic order directly transferable to the pruned Newick outfile. NTM is available at https://retrogenomics.uni-muenster.de/tools/ntm.

Homoplasy of Retrotransposon Insertions in Toothed Whales.

Retrotransposon insertion patterns facilitate a virtually homoplasy-free picture of phylogenetic history. Still, a few most likely random parallel insertions or deletions result in rare cases of homoplasy in primates. The following question arises: how frequent is retrotransposon homoplasy in other phylogenetic clades? Here, we derived genome insertion data of toothed whales to evaluate the extension of homoplasy in a representative laurasiatherian group. Among more than a thousand extracted and aligned retrotransposon loci, we detected 37 cases of precise parallel insertions in species that are separated by over more than 10 million years, a time frame which minimizes the effects of incomplete lineage sorting. We compared the phylogenetic signal of insertions with the flanking sequences of these loci to further exclude potential polymorphic loci derived by incomplete lineage sorting. We found that the phylogenetic signals of retrotransposon insertion patterns exhibiting true homoplasy differ from the signals of their flanking sequences. In toothed whales, precise parallel insertions account for around 0.18-0.29% of insertion cases, which is about 12.5 times the frequency of such insertions among Alus in primates. We also detected five specific deletions of retrotransposons on various lineages of toothed whale evolution, a frequency of 0.003%, which is slightly higher than such occurrences in primates. Overall, the level of retrotransposon homoplasy in toothed whales is still marginal compared to the phylogenetic diagnostic retrotransposon presence/absence signal.

A 4-lineage Statistical Suite to Evaluate the Support of Large-Scale Retrotransposon Insertion Data to Reconstruct Evolutionary Trees.

Retrophylogenomics makes use of genome-wide retrotransposon presence/absence insertion patterns to resolve questions in phylogeny and population genetics. In the genomics era, evaluating high-throughput data requires the associated development of appropriately powerful statistical tools. The currently used KKSC 3-lineage statistical test for estimating the significance of retrophylogenomic data is limited by the number of possible tree topologies it can assess in one step. To improve on this, we have extended the analysis to simultaneously compare four lineages, enabling us to evaluate ten distinct presence/absence insertion patterns for 26 possible tree topologies plus 129 trees with different incidences of hybridization or introgression. The new tool provides statistics for cases involving multiple ancestral hybridizations/introgressions, ancestral incomplete lineage sorting, bifurcation, and polytomy. The test is embedded in a user-friendly web R application (http://retrogenomics.uni-muenster.de:3838/hammlet/) and is available for use by the scientific community. [ancestral hybridization/introgression; ancestral incomplete lineage sorting (ILS); empirical distribution; KKSC-statistics; 4-lineage (4-LIN) insertion polymorphism; polytomy; retrophylogenomics.].

Vaccines against the original strain of SARS-CoV-2 provide T cell memory to the B.1.1.529 variant.

BACKGROUND: The SARS-CoV-2 variant B.1.1.529 potentially escapes immunity from vaccination via a heavily mutated Spike protein. Here, we analyzed whether T cell memory towards the B.1.1.529 Spike protein is present in individuals who received two or three doses of vaccines designed against the original Wuhan strain of SARS-CoV-2. METHODS: PBMCs were isolated from two- and three-times vaccinated study participants and incubated in vitro with peptide pools of the Spike protein derived from sequences of the original Wuhan or the B.1.1.529 strains of SARS-CoV-2. Activated antigen-specific T cells were detected by flow cytometry. In silico analyses with NetMHCpan and NetMHCIIpan were used to determine differences in MHC class presentation between the original strain and the B.1.1.529 strain for the most common MHCs in the European-Caucasian population. RESULTS: Here we show, that both CD4 and CD8 responses to the B.1.1.529 Spike protein are marginally reduced compared to the ancestor protein and a robust T cell response is maintained. Epitope analyses reveal minor differences between the two SARS-CoV-2 strains in terms of MHC class presentations for the MHC-alleles being most common in the European-Caucasian population. CONCLUSIONS: The memory T cell response induced via first generation vaccination remains robust and is mostly unaffected by B.1.1.529 mutations. Correspondingly, in silico analyses of MHC presentation of epitopes derived from the B.1.1.529 Spike protein shows marginal differences compared to the ancestral SARS-CoV-2 strain.

Vaccination against SARS-CoV-2 results in the production of proteins called antibodies, that bind and inactivate the virus, and cells that help to eliminate it from the body in a future encounter, such as memory T cells. Both antibodies and memory T cells remain in the body after vaccination with memory T cells being present for longer than antibodies. Here, we determined that even though most of the first generation vaccines were created to prevent infection with the original SARS-CoV-2 virus, the memory T cells generated by this vaccination can also detect the omicron variant.

ExoPLOT: Representation of alternative splicing in human tissues and developmental stages with transposed element (TE) involvement.

Advances in RNA high-throughput sequencing and large-scale functional assays yield new insights into the multifaceted activities of transposed elements (TE) and many other previously undiscovered sequence elements. Currently, no tool for easy access, analysis, quantification, and visualization of alternatively spliced exons across multiple tissues or developmental stages is available. Also, analysis pipelines demand computational skills or hardware requirements, which often are hard to meet by wet-lab scientists. We developed ExoPLOT to enable simplified access to massive RNA high throughput sequencing datasets to facilitate the analysis of alternative splicing across many biological samples. To demonstrate the functonality of ExoPLOT, we analyzed the contributon of exonized TEs to human coding sequences (CDS). mRNA splice variants containing the TE-derived exon were quantified and compared to expression levels of TE-free splice variants. For analysis, we utilized 313 human cerebrum, cerebellum, heart, kidney, liver, ovary, and testis transcriptomes, representing various pre- and postnatal developmental stages. ExoPLOT visualizes the relative expression levels of alternative transcripts, e.g., caused by the insertion of new TE-derived exons, across different developmental stages of and among multiple tissues. This tool also provides a unique link between evolution and function during exonization (gain of a new exon) and exaptation (recruitment/co-optation) of a new exon. As input for analysis, we derived a database of 1151 repeat-masked, exonized TEs, representing all prominent families of transposons in the human genome and the collection of human consensus coding sequences (CCDS). ExoPLOT screened preprocessed RNA high-throughput sequencing datasets from seven human tissues to quantify and visualize the dynamics in RNA splicing for these 1151 TE-derived exons during the entire human organ development. In addition, we successfully mapped and analyzed 993 recently described exonized sequences from the human frontal cortex onto these 313 transcriptome libraries. ExoPLOT's approach to preprocessing RNA deep sequencing datasets facilitates alternative splicing analysis and significantly reduces processing times. In addition, ExoPLOT's design allows studying alternative RNA isoforms other than TE-derived in a customized - coordinate-based manner and is available at http://retrogenomics3.uni-muenster.de:3838/exz-plot-d/.

paPAML: An Improved Computational Tool to Explore Selection Pressure on Protein-Coding Sequences.

Evolution is change over time. Although neutral changes promoted by drift effects are most reliable for phylogenetic reconstructions, selection-relevant changes are of only limited use to reconstruct phylogenies. On the other hand, comparative analyses of neutral and selected changes of protein-coding DNA sequences (CDS) retrospectively tell us about episodic constrained, relaxed, and adaptive incidences. The ratio of sites with nonsynonymous (amino acid altering) versus synonymous (not altering) mutations directly measures selection pressure and can be analysed by using the Phylogenetic Analysis by Maximum Likelihood (PAML) software package. We developed a CDS extractor for compiling protein-coding sequences (CDS-extractor) and parallel PAML (paPAML) to simplify, amplify, and accelerate selection analyses via parallel processing, including detection of negatively selected sites. paPAML compiles results of site, branch-site, and branch models and detects site-specific negative selection with the output of a codon list labelling significance values. The tool simplifies selection analyses for casual and inexperienced users and accelerates computing speeds up to the number of allocated computer threads. We then applied paPAML to examine the evolutionary impact on a new GINS Complex Subunit 3 exon, and neutrophil-associated as well as lysin and apolipoprotein genes. Compared with codeml (PAML version 4.9j) and HyPhy (HyPhy FEL version 2.5.26), all paPAML test runs performed with 10 computing threads led to identical selection pressure results, whereas the total selection analysis via paPAML, including all model comparisons, was about 3 to 5 times faster than the longest running codeml model and about 7 to 15 times faster than the entire processing time of these codeml runs.

Contradictory Phylogenetic Signals in the Laurasiatheria Anomaly Zone.

Relationships among laurasiatherian clades represent one of the most highly disputed topics in mammalian phylogeny. In this study, we attempt to disentangle laurasiatherian interordinal relationships using two independent genome-level approaches: (1) quantifying retrotransposon presence/absence patterns, and (2) comparisons of exon datasets at the levels of nucleotides and amino acids. The two approaches revealed contradictory phylogenetic signals, possibly due to a high level of ancestral incomplete lineage sorting. The positions of Eulipotyphla and Chiroptera as the first and second earliest divergences were consistent across the approaches. However, the phylogenetic relationships of Perissodactyla, Cetartiodactyla, and Ferae, were contradictory. While retrotransposon insertion analyses suggest a clade with Cetartiodactyla and Ferae, the exon dataset favoured Cetartiodactyla and Perissodactyla. Future analyses of hitherto unsampled laurasiatherian lineages and synergistic analyses of retrotransposon insertions, exon and conserved intron/intergenic sequences might unravel the conflicting patterns of relationships in this major mammalian clade.

Euarchontoglires Challenged by Incomplete Lineage Sorting.

Euarchontoglires, once described as Supraprimates, comprise primates, colugos, tree shrews, rodents, and lagomorphs in a clade that evolved about 90 million years ago (mya) from a shared ancestor with Laurasiatheria. The rapid speciation of groups within Euarchontoglires, and the subsequent inherent incomplete marker fixation in ancestral lineages, led to challenged attempts at phylogenetic reconstructions, particularly for the phylogenetic position of tree shrews. To resolve this conundrum, we sampled genome-wide presence/absence patterns of transposed elements (TEs) from all representatives of Euarchontoglires. This specific marker system has the advantage that phylogenetic diagnostic characters can be extracted in a nearly unbiased fashion genome-wide from reference genomes. Their insertions are virtually free of homoplasy. We simultaneously employed two computational tools, the genome presence/absence compiler (GPAC) and 2-n-way, to find a maximum of diagnostic insertions from more than 3 million TE positions. From 361 extracted diagnostic TEs, 132 provide significant support for the current resolution of Primatomorpha (Primates plus Dermoptera), 94 support the union of Euarchonta (Primates, Dermoptera, plus Scandentia), and 135 marker insertion patterns support a variety of alternative phylogenetic scenarios. Thus, whole genome-level analysis and a virtually homoplasy-free marker system offer an opportunity to finally resolve the notorious phylogenetic challenges that nature produces in rapidly diversifying groups.

Reunion of Australasian Possums by Shared SINE Insertions.

Although first posited to be of a single origin, the two superfamilies of phalangeriform marsupial possums (Phalangeroidea: brushtail possums and cuscuses and Petauroidea: possums and gliders) have long been considered, based on multiple sequencing studies, to have evolved from two separate origins. However, previous data from these sequence analyses suggested a variety of conflicting trees. Therefore, we reinvestigated these relationships by screening $\sim$200,000 orthologous short interspersed element (SINE) loci across the newly available whole-genome sequences of phalangeriform species and their relatives. Compared to sequence data, SINE presence/absence patterns are evolutionarily almost neutral molecular markers of the phylogenetic history of species. Their random and highly complex genomic insertion ensures their virtually homoplasy-free nature and enables one to compare hundreds of shared unique orthologous events to determine the true species tree. Here, we identify 106 highly reliable phylogenetic SINE markers whose presence/absence patterns within multiple Australasian possum genomes unexpectedly provide the first significant evidence for the reunification of Australasian possums into one monophyletic group. Together, our findings indicate that nucleotide homoplasy and ancestral incomplete lineage sorting have most likely driven the conflicting signal distributions seen in previous sequence-based studies. [Ancestral incomplete lineage sorting; possum genomes; possum monophyly; retrophylogenomics; SINE presence/absence.].

MACSima imaging cyclic staining (MICS) technology reveals combinatorial target pairs for CAR T cell treatment of solid tumors.

Many critical advances in research utilize techniques that combine high-resolution with high-content characterization at the single cell level. We introduce the MICS (MACSima Imaging Cyclic Staining) technology, which enables the immunofluorescent imaging of hundreds of protein targets across a single specimen at subcellular resolution. MICS is based on cycles of staining, imaging, and erasure, using photobleaching of fluorescent labels of recombinant antibodies (REAfinity Antibodies), or release of antibodies (REAlease Antibodies) or their labels (REAdye_lease Antibodies). Multimarker analysis can identify potential targets for immune therapy against solid tumors. With MICS we analysed human glioblastoma, ovarian and pancreatic carcinoma, and 16 healthy tissues, identifying the pair EPCAM/THY1 as a potential target for chimeric antigen receptor (CAR) T cell therapy for ovarian carcinoma. Using an Adapter CAR T cell approach, we show selective killing of cells only if both markers are expressed. MICS represents a new high-content microscopy methodology widely applicable for personalized medicine.

Gene Conversion amongst Alu SINE Elements.

The process of non-allelic gene conversion acts on homologous sequences during recombination, replacing parts of one with the other to make them uniform. Such concerted evolution is best described as paralogous ribosomal RNA gene unification that serves to preserve the essential house-keeping functions of the converted genes. Transposed elements (TE), especially Alu short interspersed elements (SINE) that have more than a million copies in primate genomes, are a significant source of homologous units and a verified target of gene conversion. The consequences of such a recombination-based process are diverse, including multiplications of functional TE internal binding domains and, for evolutionists, confusing divergent annotations of orthologous transposable elements in related species. We systematically extracted and compared 68,097 Alu insertions in various primates looking for potential events of TE gene conversion and discovered 98 clear cases of Alu-Alu gene conversion, including 64 cases for which the direction of conversion was identified (e.g., AluS conversion to AluY). Gene conversion also does not necessarily affect the entire homologous sequence, and we detected 69 cases of partial gene conversion that resulted in virtual hybrids of two elements. Phylogenetic screening of gene-converted Alus revealed three clear hotspots of the process in the ancestors of Catarrhini, Hominoidea, and gibbons. In general, our systematic screening of orthologous primate loci for gene-converted TEs provides a new strategy and view of a post-integrative process that changes the identities of such elements.

Evolution of Renal-Disease Factor APOL1 Results in Cis and Trans Orientations at the Endoplasmic Reticulum That Both Show Cytotoxic Effects.

The recent and exclusively in humans and a few other higher primates expressed APOL1 (apolipoprotein L1) gene is linked to African human trypanosomiasis (also known as African sleeping sickness) as well as to different forms of kidney diseases. Whereas APOL1's role as a trypanolytic factor is well established, pathobiological mechanisms explaining its cytotoxicity in renal cells remain unclear. In this study, we compared the APOL family members using a combination of evolutionary studies and cell biological experiments to detect unique features causal for APOL1 nephrotoxic effects. We investigated available primate and mouse genome and transcriptome data to apply comparative phylogenetic and maximum likelihood selection analyses. We suggest that the APOL gene family evolved early in vertebrates and initial splitting occurred in ancestral mammals. Diversification and differentiation of functional domains continued in primates, including developing the two members APOL1 and APOL2. Their close relationship could be diagnosed by sequence similarity and a shared ancestral insertion of an AluY transposable element. Live-cell imaging analyses showed that both expressed proteins show a strong preference to localize at the endoplasmic reticulum (ER). However, glycosylation and secretion assays revealed that-unlike APOL2-APOL1 membrane insertion or association occurs in different orientations at the ER, with the disease-associated mutants facing either the luminal (cis) or cytoplasmic (trans) side of the ER. The various pools of APOL1 at the ER offer a novel perspective in explaining the broad spectrum of its observed toxic effects.

SARS-CoV-2-reactive T cells in healthy donors and patients with COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the rapidly unfolding coronavirus disease 2019 (COVID-19) pandemic1,2. Clinical manifestations of COVID-19 vary, ranging from asymptomatic infection to respiratory failure. The mechanisms that determine such variable outcomes remain unresolved. Here we investigated CD4+ T cells that are reactive against the spike glycoprotein of SARS-CoV-2 in the peripheral blood of patients with COVID-19 and SARS-CoV-2-unexposed healthy donors. We detected spike-reactive CD4+ T cells not only in 83% of patients with COVID-19 but also in 35% of healthy donors. Spike-reactive CD4+ T cells in healthy donors were primarily active against C-terminal epitopes in the spike protein, which show a higher homology to spike glycoproteins of human endemic coronaviruses, compared with N-terminal epitopes. Spike-protein-reactive T cell lines generated from SARS-CoV-2-naive healthy donors responded similarly to the C-terminal region of the spike proteins of the human endemic coronaviruses 229E and OC43, as well as that of SARS-CoV-2. This results indicate that spike-protein cross-reactive T cells are present, which were probably generated during previous encounters with endemic coronaviruses. The effect of pre-existing SARS-CoV-2 cross-reactive T cells on clinical outcomes remains to be determined in larger cohorts. However, the presence of spike-protein cross-reactive T cells in a considerable fraction of the general population may affect the dynamics of the current pandemic, and has important implications for the design and analysis of upcoming trials investigating COVID-19 vaccines.

The multicomparative 2-n-way genome suite.

To effectively analyze the increasing amounts of available genomic data, improved comparative analytical tools that are accessible to and applicable by a broad scientific community are essential. We built the "2-n-way" software suite to provide a fundamental and innovative processing framework for revealing and comparing inserted elements among various genomes. The suite comprises two user-friendly web-based modules. The 2-way module generates pairwise whole-genome alignments of target and query species. The resulting genome coordinates of blocks (matching sequences) and gaps (missing sequences) from multiple 2-ways are then transferred to the n-way module and sorted into projects, in which user-defined coordinates from reference species are projected to the block/gap coordinates of orthologous loci in query species to provide comparative information about presence (blocks) or absence (gaps) patterns of targeted elements over many entire genomes and phylogroups. Thus, the 2-n-way software suite is ideal for performing multidirectional, non-ascertainment-biased screenings to extract all possible presence/absence data of user-relevant elements in orthologous sequences. To highlight its applicability and versatility, we used 2-n-way to expose approximately 100 lost introns in vertebrates, analyzed thousands of potential phylogenetically informative bat and whale retrotransposons, and novel human exons as well as thousands of human polymorphic retrotransposons.

The impact of transposable elements in adaptive evolution.

The growing knowledge about the influence of transposable elements (TEs) on (a) long-term genome and transcriptome evolution; (b) genomic, transcriptomic and epigenetic variation within populations; and (c) patterns of somatic genetic differences in individuals continues to spur the interest of evolutionary biologists in the role of TEs in adaptive evolution. As TEs can trigger a broad range of molecular variation in a population with potentially severe fitness and phenotypic consequences for individuals, different mechanisms evolved to keep TE activity in check, allowing for a dynamic interplay between the host, its TEs and the environment in evolution. Here, we review evidence for adaptive phenotypic changes associated with TEs and the basic molecular mechanisms by which the underlying genetic changes arise: (a) domestication, (b) exaptation, (c) host gene regulation, (d) TE-mediated formation of intronless gene copies-so-called retrogenes and (e) overall increased genome plasticity. Furthermore, we review and discuss how the stress-dependent incapacitation of defence mechanisms against the activity of TEs might facilitate adaptive responses to environmental challenges and how such mechanisms might be particularly relevant in species frequently facing novel environments, such as invasive, pathogenic or parasitic species.

The Volcano Rabbit in the Phylogenetic Network of Lagomorphs.

The order Lagomorpha unifies pikas (Ochotonidae) and the hares plus rabbits (Leporidae). Phylogenetic reconstructions of the species within Leporidae based on traditional morphological or molecular sequence data provide support for conflicting hypotheses. The retroposon presence/absence patterns analyzed in this study revealed strong support for the broadly accepted splitting of lagomorphs into ochotonids and leporids with Pronolagus as the first divergence in the leporid tree. Furthermore, the retroposon presence/absence patterns nested the rare volcano rabbit, Romerolagus diazi, within an unresolved network of deeper leporid relationships and provide the first homoplasy-free image of incomplete lineage sorting and/or ancestral hybridization/introgression in rapidly radiated Leporidae. At the same time, the strongest retroposon presence/absence signal supports the volcano rabbit as a separate branch between the Pronolagus junction and a unified cluster of the remaining leporids.

True Homoplasy of Retrotransposon Insertions in Primates.

How reliable are the presence/absence insertion patterns of the supposedly homoplasy-free retrotransposons, which were randomly inserted in the quasi infinite genomic space? To systematically examine this question in an up-to-date, multigenome comparison, we screened millions of primate transposed Alu SINE elements for incidences of homoplasious precise insertions and deletions. In genome-wide analyses, we identified and manually verified nine cases of precise parallel Alu insertions of apparently identical elements at orthologous positions in two ape lineages and twelve incidences of precise deletions of previously established SINEs. Correspondingly, eight precise parallel insertions and no exact deletions were detected in a comparison of lemuriform primate and human insertions spanning the range of primate diversity. With an overall frequency of homoplasious Alu insertions of only 0.01% (for human-chimpanzee-rhesus macaque) and 0.02-0.04% (for human-bushbaby-lemurs) and precise Alu deletions of 0.001-0.002% (for human-chimpanzee-rhesus macaque), real homoplasy is not considered to be a quantitatively relevant source of evolutionary noise. Thus, presence/absence patterns of Alu retrotransposons and, presumably, all LINE1-mobilized elements represent indeed the virtually homoplasy-free markers they are considered to be. Therefore, ancestral incomplete lineage sorting and hybridization remain the only serious sources of conflicting presence/absence patterns of retrotransposon insertions, and as such are detectable and quantifiable. [Homoplasy; precise deletions; precise parallel insertions; primates; retrotransposons.].

Genome of the Tasmanian tiger provides insights into the evolution and demography of an extinct marsupial carnivore.

The Tasmanian tiger or thylacine (Thylacinus cynocephalus) was the largest carnivorous Australian marsupial to survive into the modern era. Despite last sharing a common ancestor with the eutherian canids ~160 million years ago, their phenotypic resemblance is considered the most striking example of convergent evolution in mammals. The last known thylacine died in captivity in 1936 and many aspects of the evolutionary history of this unique marsupial apex predator remain unknown. Here we have sequenced the genome of a preserved thylacine pouch young specimen to clarify the phylogenetic position of the thylacine within the carnivorous marsupials, reconstruct its historical demography and examine the genetic basis of its convergence with canids. Retroposon insertion patterns placed the thylacine as the basal lineage in Dasyuromorphia and suggest incomplete lineage sorting in early dasyuromorphs. Demographic analysis indicated a long-term decline in genetic diversity starting well before the arrival of humans in Australia. In spite of their extraordinary phenotypic convergence, comparative genomic analyses demonstrated that amino acid homoplasies between the thylacine and canids are largely consistent with neutral evolution. Furthermore, the genes and pathways targeted by positive selection differ markedly between these species. Together, these findings support models of adaptive convergence driven primarily by cis-regulatory evolution.

De-novo emergence of SINE retroposons during the early evolution of passerine birds.

BACKGROUND: Passeriformes ("perching birds" or passerines) make up more than half of all extant bird species. The genome of the zebra finch, a passerine model organism for vocal learning, was noted previously to contain thousands of short interspersed elements (SINEs), a group of retroposons that is abundant in mammalian genomes but considered largely inactive in avian genomes. RESULTS: Here we resolve the deep phylogenetic relationships of passerines using presence/absence patterns of SINEs. The resultant retroposon-based phylogeny provides a powerful and independent corroboration of previous sequence-based analyses. Notably, SINE activity began in the common ancestor of Eupasseres (passerines excluding the New Zealand wrens Acanthisittidae) and ceased before the rapid diversification of oscine passerines (suborder Passeri - songbirds). Furthermore, we find evidence for very recent SINE activity within suboscine passerines (suborder Tyranni), following the emergence of a SINE via acquisition of a different tRNA head as we suggest through template switching. CONCLUSIONS: We propose that the early evolution of passerines was unusual among birds in that it was accompanied by de-novo emergence and activity of SINEs. Their genomic and transcriptomic impact warrants further study in the light of the massive diversification of passerines.