Sliding window analyses for optimal selection of mini-barcodes, and application to 454-pyrosequencing for specimen identification from degraded DNA

DNA barcoding remains a challenge when applied to diet analyses, ancient DNA studies, environmental DNA samples and, more generally, in any cases where DNA samples have not been adequately preserved. Because the size of the commonly used barcoding marker (COI) is over 600 base pairs (bp), amplification fails when the DNA molecule is degraded into smaller fragments. However, relevant information for specimen identification may not be evenly distributed along the barcoding region, and a shorter target can be sufficient for identification purposes. This study proposes a new, widely applicable, method to compare the performance of all potential 'mini-barcodes' for a given molecular marker and to objectively select the shortest and most informative one. Our method is based on a sliding window analysis implemented in the new R package SPIDER (Species IDentity and Evolution in R). This method is applicable to any taxon and any molecular marker. Here, it was tested on earthworm DNA that had been degraded through digestion by carnivorous landsnails. A 100 bp region of 16 S rDNA was selected as the shortest informative fragment (mini-barcode) required for accurate specimen identification. Corresponding primers were designed and used to amplify degraded earthworm (prey) DNA from 46 landsnail (predator) faeces using 454-pyrosequencing. This led to the detection of 18 earthworm species in the diet of the snail. We encourage molecular ecologists to use this method to objectively select the most informative region of the gene they aim to amplify from degraded DNA. The method and tools provided here, can be particularly useful (1) when dealing with degraded DNA for which only small fragments can be amplified, (2) for cases where no consensus has yet been reached on the appropriate barcode gene, or (3) to allow direct analysis of short reads derived from massively parallel sequencing without the need for bioinformatic consolidation.     

Big and aerial invaders: dominance of exotic spiders in burned New Zealand tussock grasslands

As post-disturbance community response depends on the characteristics of the ecosystem and the species composition, so does the invasion of exotic species rely on their suitability to the new environment. Here, we test two hypotheses: exotic spider species dominate the community after burning; and two traits are prevalent for their colonisation ability: ballooning and body size, the latter being correlated with their dispersal ability. We established spring burn, summer burn and unburned experimental plots in a New Zealand tussock grassland area and collected annual samples 3 and 4 years before and after the burning, respectively. Exotic spider abundance increased in the two burn treatments, driven by an increase in Linyphiidae. Indicator analysis showed that exotic and native species characterised burned and unburned plots, respectively. Generalised linear mixed-effects models indicated that ballooning had a positive effect on the post-burning establishment (density) of spiders in summer burn plots but not in spring plots. Body size had a positive effect on colonisation and establishment. The ability to balloon may partly explain the dominance of exotic Linyphiidae species. Larger spiders are better at moving into and colonising burned sites probably because of their ability to travel longer distances over land. Native species showed a low resilience to burning, and although confirmation requires longer-term data, our findings suggest that frequent fires could cause long lasting damage to the native spider fauna of tussock grasslands, and we propose limiting the use of fire to essential situations.     

Spider: an R package for the analysis of species identity and evolution, with particular reference to DNA barcoding

Spider: SPecies IDentity and Evolution in R is a new R package implementing a number of useful analyses for DNA barcoding studies and associated research into species delimitation and speciation. Included are functions essential for generating important summary statistics from DNA barcode data, assessing specimen identification efficacy, and for testing and optimizing divergence threshold limits. In terms of investigating evolutionary and taxonomic questions, techniques for assessing diagnostic nucleotides and probability of reciprocal monophyly are also provided. Additionally, a sliding window function offers opportunities to analyse information across a gene, essential for marker design in degraded DNA studies. Spider capitalizes on R's extensible ethos and offers an integrated platform ideal for the analysis of both nucleotide and morphological data. The program can be obtained from the comprehensive R archive network (CRAN, http://cran.r-project.org) and from the R-Forge package development site (http://spider.r-forge.r-project.org/).     

Gauging megadiversity with optimized and standardized sampling protocols: A case for tropical forest spiders

Characterizing and monitoring biodiversity and assessing its drivers require accurate and comparable data on species assemblages, which, in turn, should rely on efficient and standardized field collection. Unfortunately, protocols that follow such criteria remain scarce and it is unclear whether they can be applied to megadiverse communities, whose study can be particularly challenging. Here, we develop and evaluate the first optimized and standardized sampling protocol for megadiverse communities, using tropical forest spiders as a model taxon. We designed the protocol COBRA‐TF (Conservation Oriented Biodiversity Rapid Assessment for Tropical Forests) using a large dataset of semiquantitative field data from different continents. This protocol combines samples of different collecting methods to obtain as many species as possible with minimum effort (optimized) and widest applicability and comparability (standardized). We ran sampling simulations to assess the efficiency of COBRA‐TF (optimized, non‐site‐specific) and its reliability for estimating taxonomic, phylogenetic, and functional diversity, and community structure by comparing it with (1) commonly used expert‐based ad hoc protocols (nonoptimized, site‐specific) and (2) optimal protocols (optimized, site‐specific). We then tested the performance and feasibility of COBRA‐TF in the field. COBRA‐TF yielded similar results as ad hoc protocols for species (observed and estimated) and family richness, phylogenetic and functional diversity, and species abundance distribution. Optimal protocols detected more species than COBRA‐TF. Data from the field test showed high sampling completeness and yielded low numbers of singletons and doubletons. Optimized and standardized protocols can be as effective in sampling and studying megadiverse communities as traditional sampling, while allowing data comparison. Although our target taxa are spiders, COBRA‐TF can be modified to apply to any highly diverse taxon and habitat as long as multiple collecting techniques exist and the unit effort per sample is comparable. Protocols such as COBRA‐TF facilitate studying megadiverse communities and therefore may become essential tools for monitoring community changes in space and time, assessing the effects of disturbances and selecting conservation areas.     

The invertebrate fauna of epiphyte mats in the canopy of northern rata (Myrtaceae: Metrosideros robusta A. Cunn.) on the West Coast of the South Island,New Zealand

Abstract

The range of microhabitats and microclimatic conditions provided by epiphytes has been linked to the high diversity of invertebrates in many forest canopies worldwide, but comparably little is known about the invertebrate fauna in this habitat in New Zealand. This study compiled an inventory of the invertebrate fauna of epiphyte mats in the canopy of northern rata (Myrtaceae: Metrosideros robusta A. Cunn.) at two study sites on the West Coast of the South Island. A total of 242 069 invertebrate specimens was collected over one year, representing 4 phyla, 9 classes and more than 160 families, 225 genera and 446 species. At least 10 new species and 3 new genera were identified, while 5 species were recorded outside their known geographical range. Epiphyte mats provided habitat for an invertebrate fauna, highly diverse and abundant both taxonomically and functionally, dominated in terms of abundance by Acari, Collembola and Hymenoptera (largely ants), and in terms of feeding guilds by epiphyte grazers and ants. As the first inventory of this taxonomic depth and breadth compiled for New Zealand forest epiphyte habitats, this study provides important baseline data for the conservation of biodiversity in New Zealand's indigenous forests.