Lessons from assembling UCEs: A comparison of common methods and the case of Clavinomia (Halictidae).

Sequence data assembly is a foundational step in high-throughput sequencing, with untold consequences for downstream analyses. Despite this, few studies have interrogated the many methods for assembling phylogenomic UCE data for their comparative efficacy, or for how outputs may be impacted. We study this by comparing the most commonly used assembly methods for UCEs in the under-studied bee lineage Nomiinae and a representative sampling of relatives. Data for 63 UCE-only and 75 mixed taxa were assembled with five methods, including ABySS, HybPiper, SPAdes, Trinity and Velvet, and then benchmarked for their relative performance in terms of locus capture parameters and phylogenetic reconstruction. Unexpectedly, Trinity and Velvet trailed the other methods in terms of locus capture and DNA matrix density, whereas SPAdes performed favourably in most assessed metrics. In comparison with SPAdes, the guided-assembly approach HybPiper generally recovered the highest quality loci but in lower numbers. Based on our results, we formally move Clavinomia to Dieunomiini and render Epinomia once more a subgenus of Dieunomia. We strongly advise that future studies more closely examine the influence of assembly approach on their results, or, minimally, use better-performing assembly methods such as SPAdes or HybPiper. In this way, we can move forward with phylogenomic studies in a more standardized, comparable manner.

A globally synthesised and flagged bee occurrence dataset and cleaning workflow.

Species occurrence data are foundational for research, conservation, and science communication, but the limited availability and accessibility of reliable data represents a major obstacle, particularly for insects, which face mounting pressures. We present BeeBDC, a new R package, and a global bee occurrence dataset to address this issue. We combined >18.3 million bee occurrence records from multiple public repositories (GBIF, SCAN, iDigBio, USGS, ALA) and smaller datasets, then standardised, flagged, deduplicated, and cleaned the data using the reproducible BeeBDC R-workflow. Specifically, we harmonised species names (following established global taxonomy), country names, and collection dates and, we added record-level flags for a series of potential quality issues. These data are provided in two formats, "cleaned" and "flagged-but-uncleaned". The BeeBDC package with online documentation provides end users the ability to modify filtering parameters to address their research questions. By publishing reproducible R workflows and globally cleaned datasets, we can increase the accessibility and reliability of downstream analyses. This workflow can be implemented for other taxa to support research and conservation.

The evolutionary history of bees in time and space.

Bees are the most significant pollinators of flowering plants. This partnership began ca. 120 million years ago, but the uncertainty of how and when bees spread across the planet has greatly obscured investigations of this key mutualism. We present a novel analysis of bee biogeography using extensive new genomic and fossil data to demonstrate that bees originated in Western Gondwana (Africa and South America). Bees likely originated in the Early Cretaceous, shortly before the breakup of Western Gondwana, and the early evolution of any major bee lineage is associated with either the South American or African land masses. Subsequently, bees colonized northern continents via a complex history of vicariance and dispersal. The notable early absences from large landmasses, particularly in Australia and India, have important implications for understanding the assembly of local floras and diverse modes of pollination. How bees spread around the world from their hypothesized Southern Hemisphere origin parallels the histories of numerous flowering plant clades, providing an essential step to studying the evolution of angiosperm pollination syndromes in space and time.

Phylogenetic relationships and the evolution of host preferences in the largest clade of brood parasitic bees (Apidae: Nomadinae).

Brood parasites (also known as cleptoparasites) represent a substantial fraction of global bee diversity. Rather than constructing their own nests, these species instead invade those of host bees to lay their eggs. Larvae then hatch and consume the food provisions intended for the host's offspring. While this life history strategy has evolved numerous times across the phylogeny of bees, the oldest and most speciose parasitic clade is the subfamily Nomadinae (Apidae). However, the phylogenetic relationships among brood parasitic apids both within and outside the Nomadinae have not been fully resolved. Here, we present new findings on the phylogeny of this diverse group of brood parasites based on ultraconserved element (UCE) sequence data and extensive taxon sampling with 114 nomadine species representing all tribes. We suggest a broader definition of the subfamily Nomadinae to describe a clade that includes almost all parasitic members of the family Apidae. The tribe Melectini forms the sister group to all other Nomadinae, while the remainder of the subfamily is composed of two sister clades: a "nomadine line" representing the former Nomadinae sensu stricto, and an "ericrocidine line" that unites several mostly Neotropical lineages. We find the tribe Osirini Handlirsch to be polyphyletic, and divide it into three lineages, including the newly described Parepeolini trib. nov. In addition to our taxonomic findings, we use our phylogeny to explore the evolution of different modes of parasitism, detecting two independent transitions from closed-cell to open-cell parasitism. Finally, we examine how nomadine host-parasite associations have evolved over time. In support of Emery's rule, which suggests close relationships between hosts and parasites, we confirm that the earliest nomadines were parasites of their close free-living relatives within the family Apidae, but that over time their host range broadened to include more distantly related hosts spanning the diversity of bees. This expanded breadth of host taxa may also be associated with the transition to open-cell parasitism.

Gene Tree Estimation Error with Ultraconserved Elements: An Empirical Study on Pseudapis Bees.

Summarizing individual gene trees to species phylogenies using two-step coalescent methods is now a standard strategy in the field of phylogenomics. However, practical implementations of summary methods suffer from gene tree estimation error, which is caused by various biological and analytical factors. Greatly understudied is the choice of gene tree inference method and downstream effects on species tree estimation for empirical data sets. To better understand the impact of this method choice on gene and species tree accuracy, we compare gene trees estimated through four widely used programs under different model-selection criteria: PhyloBayes, MrBayes, IQ-Tree, and RAxML. We study their performance in the phylogenomic framework of $>$800 ultraconserved elements from the bee subfamily Nomiinae (Halictidae). Our taxon sampling focuses on the genus Pseudapis, a distinct lineage with diverse morphological features, but contentious morphology-based taxonomic classifications and no molecular phylogenetic guidance. We approximate topological accuracy of gene trees by assessing their ability to recover two uncontroversial, monophyletic groups, and compare branch lengths of individual trees using the stemminess metric (the relative length of internal branches). We further examine different strategies of removing uninformative loci and the collapsing of weakly supported nodes into polytomies. We then summarize gene trees with ASTRAL and compare resulting species phylogenies, including comparisons to concatenation-based estimates. Gene trees obtained with the reversible jump model search in MrBayes were most concordant on average and all Bayesian methods yielded gene trees with better stemminess values. The only gene tree estimation approach whose ASTRAL summary trees consistently produced the most likely correct topology, however, was IQ-Tree with automated model designation (ModelFinder program). We discuss these findings and provide practical advice on gene tree estimation for summary methods. Lastly, we establish the first phylogeny-informed classification for Pseudapis s. l. and map the distribution of distinct morphological features of the group. [ASTRAL; Bees; concordance; gene tree estimation error; IQ-Tree; MrBayes, Nomiinae; PhyloBayes; RAxML; phylogenomics; stemminess].

Two new species of Pseudapis Kirby, 1900 (Hymenoptera: Halictidae: Nomiinae) from Africa.

Two previously unknown species of the genus Pseudapis Kirby, 1900 are described and illustrated: Pseudapis neumayeri Bossert Pauly, sp. nov. (♂, ♀, Kenya, Tanzania), and P. crassivertex Bossert Pauly, sp. nov. (♂, ♀, Mali, Mauritania, Niger, Senegal). We provide diagnoses for the new species and report all currently available records. Lastly, we present a revised identification key for the African species of the genus.

Combining transcriptomes and ultraconserved elements to illuminate the phylogeny of Apidae.

Two increasingly popular approaches to reconstruct the Tree of Life involve whole transcriptome sequencing and the target capture of ultraconserved elements (UCEs). Both methods can be used to generate large, multigene datasets for analysis of phylogenetic relationships in non-model organisms. While targeted exon sequencing across divergent lineages is now a standard method, it is still not clear if UCE data can be readily combined with published transcriptomes. In this study, we evaluate the combination of UCEs and transcriptomes in a single analysis using genome-, transcriptome-, and UCE data for 79 bees in the largest and most biologically diverse bee family, Apidae. Using existing tools, we first developed a workflow to assemble phylogenomic data from different sources and produced two large nucleotide matrices of combined data. We then reconstructed the phylogeny of the Apidae using concatenation- and coalescent-based methods, and critically evaluated the resulting phylogenies in the context of previously published genetic, genomic, and morphological data sets. Our estimated phylogenetic trees are robustly supported and largely congruent with previous molecular hypotheses, from deep nodes to shallow species-level phylogenies. Moreover, the combined approach allows us to resolve controversial nodes of the apid Tree of Life, by clarifying the relationships among the genera of orchid bees (Euglossini) and the monophyly of the Centridini. Additionally, we present novel phylogenetic evidence supporting the monophyly of the diverse clade of cleptoparasitic Apidae and the placement of two enigmatic, oil-collecting genera (Ctenoplectra and Tetrapedia). Lastly, we propose a revised classification of the family Apidae that reflects our improved understanding of apid higher-level relationships.

Pollinivory and the diversification dynamics of bees.

Pollinivory-the consumption of pollen rather than arthropod prey-is a defining feature of bees (Anthophila; the flower lovers). In virtually all bee species, larvae consume a diet composed of pollen mixed with nectar or floral oils. Bees arose from within a group of solitary, carnivorous, apoid wasps in the Early to Mid-Cretaceous, coincident with the rapid rise of flowering plants. It is assumed that the switch from carnivory to pollen-feeding was a key innovation that led to the rapid diversification of bees, but this has never been examined empirically. Here, we explore the hypothesis that pollinivory led to the increased diversification of bees. In contrast to common perception, we find that the switch to pollen-feeding per se does not explain their extensive diversification. Rather, our results indicate that pollinivory was a necessary but not sufficient condition for diversification, and that other complementary innovations, such as a broadening of host-plant diet, allowed the diversification of the major bee lineages. Our results have broad implications for understanding tempo and mode of bee diversification dynamics in light of their floral resources.

The impact of GC bias on phylogenetic accuracy using targeted enrichment phylogenomic data.

The field of sequence based phylogenetic analyses is currently being transformed by novel hybrid-based targeted enrichment methods, such as the use of ultraconserved elements (UCEs). Rather than analyzing relationships among organisms using a small number of genes, these methods now allow us to evaluate relationships with many hundreds to thousands of individual gene loci. However, the inclusion of thousands of loci does not necessarily overcome the long-standing challenge of incongruence among phylogenetic trees derived from different genes or gene regions. One factor that impacts the level of incongruence in phylogenomic data sets is the level of GC bias. GC rich gene regions are prone to higher recombination rates than AT rich regions, driven by a process referred to as "GC biased gene conversion". As a result, high GC content can be negatively associated with phylogenetic accuracy, but the extent to which this impacts incongruence among UCEs is currently unstudied. We investigated the impact of GC content on phylogeny reconstruction using in silico captured UCE data for the corbiculate bees (Hymenoptera: Apidae). The phylogeny of this group has been the subject of extensive study, and incongruence among gene trees is thought to be a source of phylogenetic error. We conducted coalescent- and concatenation-based analyses of 810 individual gene loci from all 13 currently available bee genomes, including 8 corbiculate taxa. Both coalescent- and concatenation-based methods converged on a single topology for the corbiculate tribes. In contrast to concatenation, the coalescent-based methods revealed significant topological conflict at nodes involving the orchid bees (Euglossini) and honeybees (Apini). Partitioning the loci by GC content reveals decreasing support for the inferred topology with increasing GC bias. Based on the results of this study, we report the first evidence that GC biased gene conversion may contribute to topological incongruence in studies based on ultraconserved elements.

The Cryptic Bombus lucorum Complex (Hymenoptera: Apidae) in Austria: Phylogeny, Distribution, Habitat Usage and a Climatic Characterization Based on COI Sequence Data.

Silas Bossert, Barbara-Amina Gereben-Krenn, Johann Neumayer, Bernhard Schneller, and Harald W. Krenn (2016) The Bombus lucorum complex represents a group of three distinct but cryptic bumblebee species in Europe. With the advent of DNA-based identification methods, their species status was confirmed and the use of COI barcoding proved to be an especially useful tool for species identification within the group. Meanwhile, the identification based on morphology remains difficult and recent studies challenged the general distinguishability by revealing an important character to be unreliable. This has consequences for our understanding of the distribution and ecology of the species in Europe and aggravates our patchy knowledge of the situation in Austria and the whole area of the European Alps. In this study, we investigate the exact species composition and distribution of the Bombus lucorum complex in Austria based on the reliable species identification with COI sequence data. The habitat usage is studied and the first extensive investigation of altitudinal and climatic differentiation is provided. The results support three distinct genotypic groups in the Bombus lucorum complex. B. lucorum and B. cryptarum co-occur in several areas across the country, with B. lucorum being the most common and most widespread species. The study provides no evidence for the presence of B. magnus in Austria. The less common species, B. cryptarum, mainly occurs in the high mountains and is the predominant species of the complex above altitudes of 2100 m a.s.l. Further, B. cryptarum is almost absent from woodlands and is relatively more abundant in habitats with colder climate than B. lucorum in Austria. Additionally, the results indicate a very low intraspecific genetic variation within B. lucorum and B. cryptarum. This study confirms previous findings of three distinct species within the species complex. Based on reliable COI identification, the first coherent overview of the species complex in Austria can be achieved. The climatic data allows us to explain the differences in the distribution patterns. Moreover, the low intraspecific variation may indicate past bottleneck conditions for B. lucorum and B. cryptarum.

The high alpine bee fauna (Hymenoptera: Apoidea) of the Zillertal Alps, Austria.

Bees from the Zemmgrund area in the Zillertal Alps (Austria, Tyrol) were collected and determined to investigate the species composition of the area. A total of 61 specimens were collected over a two year period; they represent 24 species from 8 genera. Building on these records, the first commented checklist for the area is presented, with notes on habitats and visited flowers.