Phylogenomics and the rise of the angiosperms.

Angiosperms are the cornerstone of most terrestrial ecosystems and human livelihoods1,2. A robust understanding of angiosperm evolution is required to explain their rise to ecological dominance. So far, the angiosperm tree of life has been determined primarily by means of analyses of the plastid genome3,4. Many studies have drawn on this foundational work, such as classification and first insights into angiosperm diversification since their Mesozoic origins5-7. However, the limited and biased sampling of both taxa and genomes undermines confidence in the tree and its implications. Here, we build the tree of life for almost 8,000 (about 60%) angiosperm genera using a standardized set of 353 nuclear genes8. This 15-fold increase in genus-level sampling relative to comparable nuclear studies9 provides a critical test of earlier results and brings notable change to key groups, especially in rosids, while substantiating many previously predicted relationships. Scaling this tree to time using 200 fossils, we discovered that early angiosperm evolution was characterized by high gene tree conflict and explosive diversification, giving rise to more than 80% of extant angiosperm orders. Steady diversification ensued through the remaining Mesozoic Era until rates resurged in the Cenozoic Era, concurrent with decreasing global temperatures and tightly linked with gene tree conflict. Taken together, our extensive sampling combined with advanced phylogenomic methods shows the deep history and full complexity in the evolution of a megadiverse clade.

Strategies of diaspore dispersal investment in Compositae: the case of the Andean highlands.

BACKGROUND AND AIMS: Understanding diaspore morphology and how much a species invests on dispersal appendages is key for improving our knowledge of dispersal in fragmented habitats. We investigate diaspore morphological traits in high-Andean Compositae and their main abiotic and biotic drivers and test whether they play a role in species distribution patterns across the naturally fragmented high-Andean grasslands. METHODS: We collected diaspore trait data for 125 Compositae species across 47 tropical high-Andean summits, focusing on achene length and pappus-to-achene length ratio, with the latter as a proxy of dispersal investment. We analysed the role of abiotic (temperature, elevation and latitude) and biotic factors (phylogenetic signal and differences between tribes) on diaspore traits and whether they are related to distribution patterns across the Andes, using phylogenomics, distribution modelling and community ecology analyses. KEY RESULTS: Seventy-five percent of the studied species show small achenes (length <3.3 mm) and 67% have high dispersal investment (pappus length at least two times the achene length). Dispersal investment increases with elevation, possibly to compensate for lower air density, and achene length increases towards the equator, where non-seasonal climate prevails. Diaspore traits show significant phylogenetic signal, and higher dispersal investment is observed in Gnaphalieae, Astereae and Senecioneae, which together represent 72% of our species. High-Andean-restricted species found across the tropical Andes have, on average, the pappus four times longer than the achene, a significantly higher dispersal investment than species present only in the northern Andes or only in the central Andes. CONCLUSIONS: Small achenes and high diaspore dispersal investment dominate among high-Andean Compositae, traits typical of mostly three tribes of African origin; but traits are also correlated with the environmental gradients within the high-Andean grasslands. Our results also suggest that diaspore dispersal investment is likely to shape species distribution patterns in naturally fragmented habitats.

The complete mitochondrial genome of Trigonisca nataliae (Hymenoptera, Apidae) assemblage reveals heteroplasmy in the control region.

The evolution of mitochondrial genomes in the stingless bees is surprisingly dynamic, making them a model system to understand mitogenome structure, function, and evolution. Out of the seven mitogenomes available in this group, five exhibit atypical characteristics, including extreme rearrangements, rapid evolution and complete mitogenome duplication. To further explore the mitogenome diversity in these bees, we utilized isolated mtDNA and Illumina sequencing to assemble the complete mitogenome of Trigonisca nataliae, a species found in Northern Brazil. The mitogenome of T. nataliae was highly conserved in gene content and structure when compared to Melipona species but diverged in the control region (CR). Using PCR amplification, cloning and Sanger sequencing, six different CR haplotypes, varying in size and content, were recovery. These findings indicate that heteroplasmy, where different mitochondrial haplotypes coexist within individuals, occurs in T. nataliae. Consequently, we argue that heteroplasmy might indeed be a common phenomenon in bees that could be associated with variations in mitogenome size and challenges encountered during the assembly process.

Rapid evolution, rearrangements and whole mitogenome duplication in the Australian stingless bees Tetragonula (Hymenoptera: Apidae): A steppingstone towards understanding mitochondrial function and evolution.

The extreme conservation of mitochondrial genomes in metazoans poses a significant challenge to understanding mitogenome evolution. However, the presence of variation in gene order or genome structure, found in a small number of taxa, can provide unique insights into this evolution. Previous work on two stingless bees in the genus Tetragonula (T. carbonaria and T. hockingsi) revealed highly divergent CO1 regions between them and when compared to the bees from the same tribe (Meliponini), indicating rapid evolution. Using mtDNA isolation and Illumina sequencing, we elucidated the mitogenomes of both species. In both species, there has been a duplication of the whole mitogenome to give a total genome size of 30,666 bp in T. carbonaria; and 30,662 bp in T. hockingsi. These duplicated genomes present a circular structure with two identical and mirrored copies of all 13 protein coding genes and 22 tRNAs, with the exception of a few tRNAs that are present as single copies. In addition, the mitogenomes are characterized by rearrangements of two block of genes. We believe that rapid evolution is present in the whole Indo-Malay/Australasian group of Meliponini but is extraordinarily elevated in T. carbonaria and T. hockingsi, probably due to founder effect, low effective population size and the mitogenome duplication. All these features - rapid evolution, rearrangements, and duplication - deviate significantly from the vast majority of the mitogenomes described so far, making the mitogenomes of Tetragonula unique opportunities to address fundamental questions of mitogenome function and evolution.

A Comprehensive Phylogenomic Platform for Exploring the Angiosperm Tree of Life.

The tree of life is the fundamental biological roadmap for navigating the evolution and properties of life on Earth, and yet remains largely unknown. Even angiosperms (flowering plants) are fraught with data gaps, despite their critical role in sustaining terrestrial life. Today, high-throughput sequencing promises to significantly deepen our understanding of evolutionary relationships. Here, we describe a comprehensive phylogenomic platform for exploring the angiosperm tree of life, comprising a set of open tools and data based on the 353 nuclear genes targeted by the universal Angiosperms353 sequence capture probes. The primary goals of this article are to (i) document our methods, (ii) describe our first data release, and (iii) present a novel open data portal, the Kew Tree of Life Explorer (https://treeoflife.kew.org). We aim to generate novel target sequence capture data for all genera of flowering plants, exploiting natural history collections such as herbarium specimens, and augment it with mined public data. Our first data release, described here, is the most extensive nuclear phylogenomic data set for angiosperms to date, comprising 3099 samples validated by DNA barcode and phylogenetic tests, representing all 64 orders, 404 families (96$\%$) and 2333 genera (17$\%$). A "first pass" angiosperm tree of life was inferred from the data, which totaled 824,878 sequences, 489,086,049 base pairs, and 532,260 alignment columns, for interactive presentation in the Kew Tree of Life Explorer. This species tree was generated using methods that were rigorous, yet tractable at our scale of operation. Despite limitations pertaining to taxon and gene sampling, gene recovery, models of sequence evolution and paralogy, the tree strongly supports existing taxonomy, while challenging numerous hypothesized relationships among orders and placing many genera for the first time. The validated data set, species tree and all intermediates are openly accessible via the Kew Tree of Life Explorer and will be updated as further data become available. This major milestone toward a complete tree of life for all flowering plant species opens doors to a highly integrated future for angiosperm phylogenomics through the systematic sequencing of standardized nuclear markers. Our approach has the potential to serve as a much-needed bridge between the growing movement to sequence the genomes of all life on Earth and the vast phylogenomic potential of the world's natural history collections. [Angiosperms; Angiosperms353; genomics; herbariomics; museomics; nuclear phylogenomics; open access; target sequence capture; tree of life.].

Mitochondrial DNA intra-individual variation in a bumblebee species: A challenge for evolutionary studies and molecular identification.

Mitochondrial DNA (mtDNA) regions have been widely used as molecular markers in evolutionary studies and species identification. However, the presence of heteroplasmy and NUMTs may represent obstacles. Heteroplasmy is a state where an organism has different mitochondrial haplotypes. NUMTs are nuclear pseudogenes originating from mtDNA sequences transferred to nuclear DNA. Evidences of heteroplasmy were already verified in the bumblebee Bombus morio in an earlier study. The present work investigated in more detail the presence of intra-individual haplotypes variation in this species. Heteroplasmy was detected in individuals from all the ten sampled locations, with an average of six heteroplasmic haplotypes per individual. In addition, some of these heteroplasmic haplotypes were shared among individuals from different locations, suggesting the existence of stable heteroplasmy in B. morio. These results demonstrated that heteroplasmy is likely to affect inferences based on mtDNA analysis, especially in phylogenetic, phylogeographic and population genetics studies. In addition, NUMTs were also detected. These sequences showed divergence of 2.7% to 12% in relation to the mitochondrial haplotypes. These levels of divergence could mislead conclusions in evolutionary studies and affect species identification through DNA barcoding.

Conserved numts mask a highly divergent mitochondrial-COI gene in a species complex of Australian stingless bees Tetragonula (Hymenoptera: Apidae).

Tetragonula carbonaria, Tetragonula davenporti, Tetragonula hockingsi and Tetragonula mellipes comprise a species complex of Australian stingless bee species known as the 'Carbonaria' group. The species are difficult to distinguish morphologically and the major species-defining characters relate to comb architecture and nest entrance ornamentation. The taxonomy of the group is further complicated by likely nuclear mitochondrial pseudogenes (numts) and inter-specific hybrids. Here we demonstrate the existence of COI numts and isolate and characterize the 'true' mt-COI gene in T. carbonaria and T. hockingsi. Numts were isolated from enriched-nuclear DNA extraction followed by PCR amplification and Sanger sequencing, and were recognized by the presence of deletions and/or premature stop codons in the translated sequences. The mt-COI sequences were obtained from NGS sequencing using purified mtDNA. In T. carbonaria, two numts (numt1 and numt2) were identified and a third (numt3) was identified in T. hockingsi. Numt2 and numt3 are similar (1.2% sequence divergence), indicating a recent common origin. The genetic distance between the mt-COI of the two Tetragonula species was higher than might be expected for closely related species, 16.5%, corroborating previous studies in which T. carbonaria and T. hockingsi were regarded as separate species. The three numts are more similar to the COI of other stingless bee species, including Australian Austroplebia australis and South American Melipona bicolor (81.7-83.9%) than to the mt-COI of their own species (70-71.4%). This is because the mt-COI of T. carbonaria and T. hockingsi differ greatly from other Meliponinae. Our findings explain some formerly puzzling aspects of Carbonaria biogeography, and misinterpreted amplifications.

Fidelity of DNA polymerases in the detection of intraindividual variation of mitochondrial DNA.

Here we investigated the consequences of PCR amplification errors in the identification of intraindividual mtDNA variation. The bumblebee Bombus morio was chosen as model for the COI gene amplification tests with two DNA polymerases (Taq and Q5) presenting different error rates. The amplifications using Taq resulted in a significant increase of singleton haplotypes per individual in comparison to Q5. The sequence characteristics indicated that Taq resulted haplotypes are mostly due to amplification errors. Studies focusing on intraindividual variability should address special attention to the DNA polymerase fidelity to avoid overestimation of heteroplasmic haplotypes.

Diapause in a tropical oil-collecting bee: molecular basis unveiled by RNA-Seq.

BACKGROUND: Diapause is a natural phenomenon characterized by an arrest in development that ensures the survival of organisms under extreme environmental conditions. The process has been well documented in arthropods. However, its molecular basis has been mainly studied in species from temperate zones, leaving a knowledge gap of this phenomenon in tropical species. In the present study, the Neotropical and solitary bee Tetrapedia diversipes was employed as a model for investigating diapause in species from tropical zones. Being a bivoltine insect, Tetrapedia diversipes produce two generations of offspring per year. The first generation, normally born during the wet season, develops faster than individuals from the second generation, born after the dry season. Furthermore, it has been shown that the development of the progeny, of the second generation, is halted at the 5th larval instar, and remains in larval diapause during the dry season. Towards the goal of gaining a better understanding of the diapause phenomenon we compared the global gene expression pattern, in larvae, from both reproductive generations and during diapause. The results demonstrate that there are similarities in the observed gene expression patterns to those already described for temperate climate models, and also identify diapause-related genes that have not been previously reported in the literature. RESULTS: The RNA-Seq analysis identified 2275 differentially expressed transcripts, of which 1167 were annotated. Of these genes, during diapause, 352 were upregulated and 815 were downregulated. According to their biological functions, these genes were categorized into the following groups: cellular detoxification, cytoskeleton, cuticle, sterol and lipid metabolism, cell cycle, heat shock proteins, immune response, circadian clock, and epigenetic control. CONCLUSION: Many of the identified genes have already been described as being related to diapause; however, new genes were discovered, for the first time, in this study. Among those, we highlight: Niemann-Pick type C1, NPC2 and Acyl-CoA binding protein homolog (all involved in ecdysteroid synthesis); RhoBTB2 and SASH1 (associated with cell cycle regulation) and Histone acetyltransferase KAT7 (related to epigenetic transcriptional regulation). The results presented here add important findings to the understanding of diapause in tropical species, thus increasing the comprehension of diapause-related molecular mechanisms.

Comparative phylogeography in the Atlantic forest and Brazilian savannas: pleistocene fluctuations and dispersal shape spatial patterns in two bumblebees.

BACKGROUND: Bombus morio and B. pauloensis are sympatric widespread bumblebee species that occupy two major Brazilian biomes, the Atlantic forest and the savannas of the Cerrado. Differences in dispersion capacity, which is greater in B. morio, likely influence their phylogeographic patterns. This study asks which processes best explain the patterns of genetic variation observed in B. morio and B. pauloensis, shedding light on the phenomena that shaped the range of local populations and the spatial distribution of intra-specific lineages. RESULTS: Results suggest that Pleistocene climatic oscillations directly influenced the population structure of both species. Correlative species distribution models predict that the warmer conditions of the Last Interglacial contributed to population contraction, while demographic expansion happened during the Last Glacial Maximum. These results are consistent with physiological data suggesting that bumblebees are well adapted to colder conditions. Intra-specific mitochondrial genealogies are not congruent between the two species, which may be explained by their documented differences in dispersal ability. CONCLUSIONS: While populations of the high-dispersal B. morio are morphologically and genetically homogeneous across the species range, B. pauloensis encompasses multiple (three) mitochondrial lineages, and show clear genetic, geographic, and morphological differences. Because the lineages of B. pauloensis are currently exposed to distinct climatic conditions (and elevations), parapatric diversification may occur within this taxon. The eastern portion of the state of São Paulo, the most urbanized area in Brazil, represents the center of genetic diversity for B. pauloensis.

A protocol for isolating insect mitochondrial genomes: a case study of NUMT in Melipona flavolineata (Hymenoptera: Apidae).

Nuclear mitochondrial DNA insertions (NUMTs) are mitochondrial DNA sequences that have been transferred into the nucleus and are recognized by the presence of indels and stop codons. Although NUMTs have been identified in a diverse range of species, their discovery was frequently accidental. Here, our initial goal was to develop and standardize a simple method for isolating NUMTs from the nuclear genome of a single bee. Subsequently, we tested our new protocol by determining whether the indels and stop codons of the cytochrome c oxidase subunit I (COI) sequence of Melipona flavolineata are of nuclear origin. The new protocol successfully demonstrated the presence of a COI NUMT. In addition to NUMT investigations, the protocol described here will also be very useful for studying mitochondrial mutations related to diseases and for sequencing complete mitochondrial genomes with high read coverage by Next-Generation technology.

Very low mitochondrial variability in a stingless bee endemic to cerrado.

Partamona mulata is a stingless bee species endemic to cerrado, a severely threatened phytogeographical domain. Clearing for pasture without proper soil treatment in the cerrado facilitates the proliferation of termite ground nests, which are the nesting sites for P. mulata. The genetic consequences of these changes in the cerrado environment for bee populations are still understudied. In this work, we analyzed the genetic diversity of 48 colonies of P. mulata collected throughout the species' distribution range by sequencing two mitochondrial genes, cytochrome oxidase I and cytochrome B. A very low polymorphism rate was observed when compared to another Partamona species from the Atlantic forest. Exclusive haplotypes were observed in two of the five areas sampled. The sharing of two haplotypes between collection sites separated by a distance greater than the flight range of queens indicates an ancient distribution for these haplotypes. The low haplotype and nucleotide diversity observed here suggests that P. mulata is either a young species or one that has been through population bottlenecks. Locally predominant and exclusive haplotypes (H2 and H4) may have been derived from local remnants through cerrado deforestation and the expansion of a few colonies with abundant nesting sites.