Species Identification in Malaise Trap Samples by DNA Barcoding Based on NGS Technologies and a Scoring Matrix

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 pre-sorting process will yield approximately 30% more high score BINs compared to the nonsorted 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

Flight over the Proto-Caribbean seaway: Phylogeny and macroevolution of Neotropical Anaeini leafwing butterflies

Our understanding of the origin and evolution of the astonishing Neotropical biodiversity remains somewhat limited. In particular, decoupling the respective impacts of biotic and abiotic factors on the macroevolution of clades is paramount to understand biodiversity assemblage in this region. We present the first comprehensive molecular phylogeny for the Neotropical Anaeini leafwing butterflies (Nymphalidae, Charaxinae)and, applying likelihood-based methods, we test the impact of major abiotic (Andean orogeny, Central American highland orogeny, Proto-Caribbean seaway closure, Quaternary glaciations)and biotic (host plant association)factors on their macroevolution. We infer a robust phylogenetic hypothesis for the tribe despite moderate support in some derived clades. Our phylogenetic inference recovers the genus Polygrapha Staudinger, [1887]as polyphyletic, rendering the genera Fountainea Rydon, 1971 and Memphis Hübner, [1819]paraphyletic. Consequently, we transfer Polygrapha tyrianthina (Salvin & Godman, 1868)comb. nov. to Fountainea and Polygrapha xenocrates (Westwood, 1850)comb. nov. to Memphis. We infer an origin of the group in the late Eocene ca. 40 million years ago in Central American lowlands which at the time were separated from South America by the Proto-Caribbean seaway. The biogeographical history of the group is very dynamic, with several oversea colonization events from Central America into the Chocó and Andean regions during intense stages of Andean orogeny. These events coincide with the emergence of an archipelagic setting between Central America and northern South America in the mid-Miocene that likely facilitated dispersal across the now-vanished Proto-Caribbean seaway. The Amazonian region also played a central role in the diversification of the Anaeini, acting both as a museum and a cradle of diversity. We recover a diversification rate shift in the Miocene within the species-rich genus Memphis. State speciation and extinction models recover a significant relationship between this rate shift and host plant association, indicating a positive role on speciation rates of a switch between Malpighiales and new plant orders. We find less support for a role of abiotic factors including the progressive Andean orogeny, Proto-Caribbean seaway closure and Quaternary glaciations. Miocene host plant shifts possibly acted in concert with abiotic and/or biotic factors to shape the diversification of Anaeini butterflies.This work was supported by grant BA2152/20-1 (DFG), and by the grants PR2015-00305(MINECO) and CGL2016-76322-P (AEI/FEDER, UE) to R.V. FMSD, OHHM and MMC thank Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Edital 15/2014 CAPES/EMBRAPA) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, 141143/2009-6, 150542/2013-5, 308247/2013-2, 304639/2014-1), for financial support