Reliable, verifiable, and efficient monitoring of biodiversity via metabarcoding: Supporting Information

To manage and conserve biodiversity, one must know what is being lost, where, and why, as well as which remedies are likely to be most effective. Metabarcoding technology can characterise the species compositions of mass samples of eukaryotes or of environmental DNA. Here, we validate metabarcoding by testing it against three high-quality standard data sets that were collected in Malaysia (tropical), China (subtropical) and the United Kingdom (temperate) and that comprised 55,813 arthropod and bird specimens identified to species level with the expenditure of 2,505 person-hours of taxonomic expertise. The metabarcode and standard data sets exhibit statistically correlated alpha- and beta-diversities, and the two data sets produce similar policy conclusions for two conservation applications: restoration ecology and systematic conservation planning. Compared with standard biodiversity data sets, metabarcoded samples are taxonomically more comprehensive, many times quicker to produce, less reliant on taxonomic expertise and auditable by third parties, which is essential for dispute resolution.

Motivation. -The rainforest of south-western China is a biodiversity hotspot, with high levels of endemism; it is also an area that is subject to extensive human impact. This area is predicted to be impacted by climate change, resulting in increased average temperature and precipitation and more frequent extreme weather events (Ding et al. 2007;IPCC 2007). In order to understand the impacts of future climate change on the biodiversity of this region, we first need to establish baseline information on the current distributions of taxa. Altitudinal gradients are an ideal study system with steep shifts in environmental variables in a small geographical area. Using mountain systems as surrogates for changes in temperature and precipitation, we are able to examine the current distribution patterns of taxa, and make predictions about future shifts, based on each species' current climate envelope. Little attention, in general, has been paid to invertebrates in conservation assessments of the impacts of climate change, yet terrestrial arthropods are, on the one hand, key drivers of ecological processes (Wilson 1987) and, on the other, excellent predictors of environmental change (Basset et al. 1998). Understanding how arthropod assemblages respond to climate (and the associated vegetation assemblages) is key in understanding likely future changes to diversity and distribution of arthropods. We established a set of three permanent altitudinal transects in Yunnan province, China (tropical, sub-tropical and temperate forest). Here we present the results of the first transect, in subtropical rainforest at Ailaoshan.  Kitching et al. 2005). These light traps employ a vertical actinic tube, run from dusk to dawn, using 12 V lead acid batteries. The Pennsylvania light trap design has been modified, with a larger hole in the trap opening for large moths, light-weight large tin lids to keep out rain, and automatic timers. A Dicholorvos-impregnated strip, wrapped in paper towelling, is placed in the trap to kill moths in situ, along with cardboard material to reduce damage to moth specimens by beetles.
At each of the four altitudes (2000, 2200, 2400, and 2600 m a.s.l.), moths were sampled in five blocks. Two traps were run simultaneously in each block for three nights, with one trap approximately 2 m above the ground, and one raised into the canopy. Canopy lines were placed as high as possible in the canopy. The average height of the canopy drops with increasing altitude, and therefore the average height of the canopy traps was higher at the lower altitude sites. The total number of collections was 40 (4 altitudes X 5 blocks X 2 strata), but one sample was lost and so the final metabarcoding dataset consists of 39 samples. For some of the 39 samples, moth numbers were great enough that half the sample could be set aside in 100% EtOH for subsequent metabarcoding and the other half used for morphospecies assignment. For the smaller-volume samples, moths were extracted from the collection, identified to morphospecies, and then placed in 100% EtOH, along with the non-indicatortaxa specimens, for subsequent metabarcoding. For each pinned moth (see next paragraph), two legs were removed and placed in the corresponding metabarcoding sample.
All moths with a wing length greater than 1 cm were sorted to morphospecies. In the case of moths belonging to the superfamily Pyraloidea, moths with a wing length less than 1 cm were also processed. During the field session, at least 5 representatives from each morphospecies were pinned and dried. Moths were sorted into approximate morphospecies groups by volunteers, and then sorted and identified to family by Roger Kitching and Louise Ashton.
Moths that could be confidently identified as belonging to an existing morphospecies were then recorded and discarded. Any moths where the identification was ambiguous were pinned for later identification.

Thetford
Motivation. -In Europe, much conservation concern is for species now dependent on semi- and ephemeral open habitat elements within the forest landscape, and across the forest to reconnect external heathland remnants. We examined whether physical disturbance treatments enhanced the value of this ecological network. Response to treatments was examined in terms of assemblage composition, species richness and abundance of early-successional specialist and generalist forest species; invertebrate assemblages were also compared to reference heath sites.
Study site. -Thetford Forest was planted in the early 20th century and occupies 185 km 2 of Breckland in eastern England (0°40' E, 52°27' N). Breckland is characterised by semicontinental climate, sandy, nutrient-poor soil and a long history of grazing and episodic cultivation supporting a regional biota that includes coastal, continental and Mediterranean elements. Physically disturbed heathland and ruderal habitats support at least 542 priority species (rare, scarce, range-restricted or UK Biodiversity Action Plan species)! (Dolman!et!al.! 2012). The forest is dominated by conifer plantations, with 80% comprised of Corsican (Pinus nigra) and Scots (P. sylvestris) pine, managed by clear-felling (typically at 60-80 years) and replanting of even-aged coupes (mean area 9.0 ha ± 8.6 SD). Coupes are subdivided by a network of forestry trackways that provide management access. Trackways comprised two elements: central wheelings with sparse vegetation and exposed substrate, flanked by vegetated verges that are cut annually to facilitate access but lack bare substrate. Indicator taxa were first sorted to carabids, ants, and spiders using paid undergraduates. The ants were identified by the Norfolk county ant recorder, the carabids were identified by the Norfolk beetle recorder and an amateur coleopterist, and Scott Pedley identified all spiders.
Adult spiders were identified to species following Roberts (1987Roberts ( , 1996; juveniles and subadults were not identified due to the lack of developed reproductive structures. Identification carabids, and ants) plus the unidentified bycatch, which included Insecta (Orthoptera, Diptera, non-carabid Coleoptera, Plecoptera, Lepidoptera, and Hemiptera), Isopoda, Collembola, and Myriapoda. After metabarcoding, one control (undisturbed trackway) site was deleted from both the STD and MBC datasets because it was discovered that it had been inadvertently mowed before sampling, leaving 67 sites for analysis.

Danum Valley
Motivation. -We need an improved understanding of how tropical production forests fit into conservation agendas both ecologically and financially. Understanding the trade-offs between biodiversity conservation and financial returns from logging is critical for developing effective conservation strategies. Efficiency is an important goal for conservation, given the increasing isolation of protected areas, increasing habitat loss, and limited conservation funding. We use biodiversity and financial datasets to provide an empirical analysis of the trade-offs between human modification of a landscape via logging and biodiversity protection.
In doing so, it allows us to explore the role of costs in identifying efficient conservation agendas.