The German Barcoding initiatives BFB and GBOL have generated a reference library of more than 16,000 metazoan species, which is now ready for applications concerning next generation molecular biodiversity assessments. To streamline the barcoding process, we have developed a meta-barcoding pipeline: We pre-sorted a single malaise trap sample (obtained during one week in August 2014, southern Germany) into 12 arthropod orders and extracted DNA from pooled individuals of each order separately, in order to facilitate DNA extraction and avoid time consuming single specimen selection. Aliquots of each ordinal-level DNA extract were combined to roughly simulate a DNA extract from a non-sorted malaise sample. Each DNA extract was amplified using four primer sets targeting the CO1-5’ fragment. The resulting PCR products (150-400bp) were sequenced separately on an Illumina Mi-SEQ platform, resulting in 1.5 million sequences and 5,500 clusters (coverage ≥10; CD-HIT-EST, 98%). Using a total of 120,000 DNA barcodes of identified, Central European Hymenoptera, Coleoptera, Diptera, and Lepidoptera downloaded from BOLD we established a reference sequence database for a local CUSTOM BLAST. This allowed us to identify 529 Barcode Index Numbers (BINs) from our sequence clusters derived from pooled Malaise trap samples. We introduce a scoring matrix based on the sequence match percentages of each amplicon in order to gain plausibility for each detected BIN, leading to 390 high score BINs in the sorted samples; whereas 268 of these high score BINs (69%) could be identified in the combined sample. The results indicate that a time consuming presorting process will yield approximately 30% more high score BINs compared to the non-sorted sample in our case. These promising results indicate that a fast, efficient and reliable analysis of next generation data from malaise trap samples can be achieved using this pipeline.

Weevils (Curculionoidea) comprise one of the most diverse groups of organisms on earth. There is hardly a vascular plant or plant part without its own species of weevil feeding on it and weevil species diversity is greater than the number of fishes, birds, reptiles, amphibians and mammals combined. Here, we employ ultraconserved elements (UCEs) designed for beetles and a novel partitioning strategy of loci to help resolve phylogenetic relationships within the radiation of Australasian smurf-weevils (Eupholini). Despite being emblematic of the New Guinea fauna, no previous phylogenetic studies have been conducted on the Eupholini. In addition to a comprehensive collection of fresh specimens, we supplement our taxon sampling with museum specimens, and this study is the first target enrichment phylogenomic dataset incorporating beetle specimens from museum collections. We use both concatenated and species tree analyses to examine the relationships and taxonomy of this group. For species tree analyses we present a novel partitioning strategy to better model the molecular evolutionary process in UCEs. We found that the current taxonomy is problematic, largely grouping species on the basis of similar color patterns. Finally, our results show that most loci required multiple partitions for nucleotide rate substitution, suggesting that single partitions may not be the optimal partitioning strategy to accommodate rate heterogeneity for UCE loci.

Among vertebrates, turtles have many unique characteristics providing biologists with opportunities to study novel evolutionary innovations and processes. We present here a high-quality, partially phased, and chromosome-level Red-Eared Slider (Trachemys scripta elegans, TSE) genome as a reference for future research on turtle and tetrapod evolution. This TSE assembly is 2.269 Gb in length, has one of the highest scaffold N50 and N90 values of any published turtle genome to date (N50 = 129.68 Mb and N90 = 19 Mb), and has a total of 28,415 annotated genes. We introduce synteny analyses using BUSCO single-copy orthologs, which reveal two chromosome fusion events accounting for differences in chromosome counts between emydids and other cryptodire turtles and reveal many fission/fusion events for birds, crocodiles, and snakes relative to TSE. This annotated chromosome-level genome will provide an important reference genome for future studies on turtle, vertebrate, and chromosome evolution.

The simplicity and cost efficiency of Illumina amplicon sequencing has greatly contributed to the advancement of DNA barcoding and metabarcoding applications. However, current amplicon sequencing‐based barcoding approaches are usually restricted to short, single‐locus fragments, limiting their taxonomic and phylogenetic resolution. Here, we establish a cost efficient and simple multiplex PCR protocol for arthropod systematics by Illumina amplicon sequencing. We introduce primer sets, including several new, generic primers, to reliably amplify nine loci across a wide range of arthropods. Using a diverse collection of arthropod species from 19 orders, we test loci for amplification efficiency and estimate the effect of cross‐species amplification bias on taxon recovery from bulk community samples. We then explore the taxonomic and phylogenetic utility of the primer sets, focusing on a dataset of spiders that includes both deep and recent divergences. The set of loci provides good phylogenetic support across a wide taxonomic spectrum, making it a useful addition to COI for resolving lineages within a comparative context. All loci recover sequences for the majority of arthropod taxa in separate PCRs. However, cross‐species amplification bias in some primers prevents an exhaustive taxon recovery from bulk community samples. Our protocol makes it possible to generate multilocus datasets for large numbers of arthropod taxa for a fraction of the price and workload of Sanger sequencing. This opens up the possibility for parallel phylogenetic and taxonomic analysis of large collections of arthropods, but also enables rapid exploratory analyses of target lineages. Primers for metabarcoding applications should be carefully evaluated for their performance in bulk community samples and chosen to minimize cross‐species amplification bias.

Aim We studied the gecko genus Ebenavia to reconstruct its colonization history, test for anthropogenic versus natural dispersal out of Madagascar, and correlate divergence date estimates of our phylogeny with geological age estimates of islands in the region.Location Madagascar and surrounding islands of the Western Indian Ocean (Comoros, Mayotte, Mauritius, Pemba). MethodsWe reconstructed the phylogeny of Ebenavia covering its entire geographical range using a molecular data set of three mitochondrial and two nuclear markers. We estimated divergence times based on calibrations using (1) previously calculated mutation rates of mitochondrial markers, (2) a combination of these rates with old or (3) young geological age estimates for some of the islands inhabited by the genus, and (4) an independent data set with fossil outgroup calibration points.Results Ebenavia inunguis, one of two recognized species of the genus, comprises multiple ancient evolutionary lineages. The earliest divergence within this complex (Miocene, 13-20 Ma; 95% credibility interval [CI]: 4-29 Ma) separates the population of the Comoros Islands, excluding Mayotte, from all other lineages. The age estimates for island lineages coincide with the geological age estimates of the islands except for Grand Comoro, where the age of the local clade (3-5 Ma; 95% CI: 2-7 Ma) significantly predates the estimated island age (0Á5 Ma). A clade from north Madagascar + Mayotte + Pemba is estimated to have diverged from an eastern Malagasy clade in the Miocene. Main ConclusionsOur results suggest that Grand Comoro Island is geologically older than previously estimated. The islands of the Comoros and Pemba were probably colonized via natural dispersal out of Madagascar (> 1000 km in the case of Pemba). Mauritius was most likely colonized only recently from eastern Madagascar via human translocation.