Towards holomorphology in entomology: rapid and cost‐effective adult–larva matching using NGS barcodes

Abstract

In many taxa the morphology of females and immatures is poorly known because species descriptions and identification tools have a male bias. The root causes are problems with matching life-history stages and genders belonging to the same species. Such matching is time-consuming when conventional methods are used (e.g. rearing) and expensive when the stages are matched with DNA barcodes. Unfortunately, the lack of associations is not a trivial problem because it renders a large part of the phenome of insects unexplored, although larvae and females are useful sources of characters for descriptive and phylogenetic purposes. In addition, many collectors intentionally avoid females and immature stages, which skews survey results, interferes with collecting life-history information, and makes it less likely that rare species are discovered. These problems even exist for well-studied taxa like Odonata, where obtaining adult-larva matches relies largely on rearing. Here we demonstrate how the matching problem can be addressed with cost-effective tagged amplicon sequencing of a 313-bp segment of cox1 with next-generation sequencing (NGS) ('NGS barcoding'). We illustrate the value of this approach based on Singapore's odonate fauna which is of a similar size as the European fauna (Singapore, 122 extant species; Europe, 138 recorded species). We match the larvae and adults of 59 species by first creating a barcode database for 338 identified adult specimens representing 83 species. We then sequence 1178 larvae from a wide range of sources. We successfully barcode 1123 specimens, which leads to adult-larva matches for 59 species based on our own barcodes (55) and online barcode databases (4). With these additions, 84 of the 131 species recorded in Singapore have now been associated with a species name. Most common species are now matched (83%), and good progress has been made for vulnerable/near-threatened (55%), endangered (53%), and critically endangered species (38%). We used nondestructive DNA extraction methods in order to be able to use high-resolution imaging of matched larvae to establish a publicly available digital reference collection for odonates which is incorporated into 'Biodiversity of Singapore' (https://singapore.biodiversity.online/). We suggest that the methods described here are suitable for many insect taxa because NGS barcoding allows for fast and low-cost matching of well-studied life-history stages with neglected semaphoronts (eggs, larvae, females). We estimate that the specimen-specific amplicons in this study (c. 1500 specimens) can now be obtained within eight working days and that the laboratory and sequencing cost is c. US$600 (< US$0.40 per specimen).

High-throughput sequencing for community analysis: the promise of DNA barcoding to uncover diversity, relatedness, abundances and interactions in spider communities

“…Here, specimens are dropped directly into the PCR buffer, and the traces of DNA they release are sufficient for barcode amplification. This method has been tested and established in different insect groups (Thongjued et al 2019;Yeo et al 2018) but has yet to be optimized for spiders.…”

MinION sequencing of seafood in Singapore reveals creatively labelled flatfishes, confused roe, pig DNA in squid balls, and phantom crustaceans

“…The equipment and maintenance 98 cost for Illumina and PacBio instruments are so high that these sequencers are mostly found 99 in sequencing centres that have fairly long turnaround times for submitted samples. In 100 addition, due to the high cost of flowcells, the cost per DNA barcode is high unless 101 thousands of products are sequenced at a time (Ho, Foo, Yeo, & Meier, 2019;Kutty et al, 102 2018;Srivathsan et al, 2018;Wang, Srivathsan, Foo, Yamane, & Meier, 2018;Yeo, 103 Puniamoorthy, Ngiam, & Meier, 2018). 104 Fortunately, these issues can now be addressed with Oxford Nanopore sequencing which is 105 implemented on small and portable MinION™ sequencers.…”

Mini-barcodes are equally useful for species identification and more suitable for large-scale species discovery in Metazoa than full-length barcodes

“…The proportion of successful identification differs depending 527 on how well a particular fauna has been barcoded. This is illustrated by our recent work on 528 dragon-and damselflies (Odonata), ants (Formicidae), and non-biting midges (Chironomidae) 529 of Singapore , Yeo et al, 2018Baloğlu et al, 2018). These studies also 530 document why it is important to distinguish between species-and specimen-level identification 531 because the results differ widely.…”

“…Due to these constraints, very few studies have utilized DNA barcodes to 112 sort entire samples into putative species (but see Fagan-Jeffries et al, 2018). Instead, most 113 studies use a mixed approach where species-level sorting is carried out based on morphologyWe would thus argue that the initial species-level sorting can be achieved using barcodes that 126 can be obtained via "tagged amplicon sequencing" on next-generation sequencing platforms 127 ("NGS barcodes";Wang et al, 2018;Yeo et al, 2018). Until recently, obtaining full-length NGS 128 barcodes via tagged amplicon sequencing was difficult because the reads of most next-129 generation-sequencing platforms were too short for sequencing the full-length barcode.…”

“…We would thus argue that the initial species-level sorting can be achieved using barcodes that 126 can be obtained via "tagged amplicon sequencing" on next-generation sequencing platforms 127 ("NGS barcodes";Wang et al, 2018;Yeo et al, 2018). Until recently, obtaining full-length NGS 128 barcodes via tagged amplicon sequencing was difficult because the reads of most next-129 generation-sequencing platforms were too short for sequencing the full-length barcode.…”