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Abstract In light of the current biodiversity crisis, molecular barcoding has developed into an irreplaceable tool. Barcoding has been considerably simplified by developments in high throughput sequencing technology, but still can be prohibitively expensive and laborious when community samples of thousands of specimens need to be processed. Here, we outline an Illumina amplicon sequencing approach to generate multilocus data from large collections of arthropods. We reduce cost and effort up to 50-fold, by combining multiplex PCRs and DNA extractions from pools of presorted and morphotyped specimens and using two levels of sample indexing. We test our protocol by generating a comprehensive, community wide dataset of barcode sequences for several thousand Hawaiian arthropods from 14 orders, which were collected across the archipelago using various trapping methods. We explore patterns of diversity across the Archipelago and compare the utility of different arthropod trapping methods for biodiversity explorations on Hawaii, highlighting undergrowth beating as highly efficient method. Moreover, we show the effects of barcode marker, taxonomy and relative biomass of the targeted specimens and sequencing coverage on taxon recovery. Our protocol enables rapid and inexpensive explorations of diversity patterns and the generation of multilocus barcode reference libraries across whole ecosystems.
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DNA Barcoding of Fresh and Historical Collections of Lichen-Forming Basidiomycetes in the Genera Cora and Corella (Agaricales: Hygrophoraceae): A Success Story?
Lichens collected worldwide for centuries have resulted in millions of specimens deposited in herbaria that offer the potential to assess species boundaries, phenotypic diversification, ecology, and distribution. The application of molecular approaches to historical collections has been limited due to DNA fragmentation, but high-throughput sequencing offers an opportunity to overcome this barrier. Here, we combined a large dataset of ITS sequences from recently collected material and historical collections, obtained through Sanger, 454, or Illumina Sequencing, to test the performance of ITS barcoding in two genera of lichenized Basidiomycota: Cora and Corella. We attempted to generate new sequence data for 62 fresh specimens (from 2016) and 274 historical collections (collected between 1888 and 1998), for a final dataset of 1325 sequences. We compared various quantitative approaches to delimit species (GMYC, bPTP, ASAP, ABGD) and tested the resolution and accuracy of the ITS fungal barcoding marker by comparison with a six-marker dataset. Finally, we quantitatively compared phylogenetic and phenotypic species delimitation for 87 selected Cora species that have been formally described. Our HTS approach successfully generated ITS sequences for 76% of the historical collections, and our results show that an integrative approach is the gold-standard for understanding diversity in this group.
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- Award ID(s):
- 1609022
- PAR ID:
- 10332435
- Date Published:
- Journal Name:
- Diversity
- Volume:
- 14
- Issue:
- 4
- ISSN:
- 1424-2818
- Page Range / eLocation ID:
- 284
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.3390/d14040284
