DOI: 10.5061/dryad.dz08kprtq
title: Measuring protected-area effectiveness using vertebrate distributions from leech iDNA

description: Protected areas are key to meeting biodiversity conservation goals, but
 direct measures of effectiveness have proven difficult to obtain. We
 address this challenge by using environmental DNA from leech-ingested
 bloodmeals to estimate spatially-resolved vertebrate occupancies across
 the 677 km2 Ailaoshan reserve in Yunnan, China. From 30,468 leeches
 collected by 163 park rangers across 172 patrol areas, we identify 86
 vertebrate species, including amphibians, mammals, birds and squamates.
 Multi-species occupancy modelling shows that species richness increases
 with elevation and distance to reserve edge. Most large mammals (e.g.
 sambar, black bear, serow, tufted deer) follow this pattern; the
 exceptions are the three domestic mammal species (cows, sheep, goats) and
 muntjak deer, which are more common at lower elevations. Vertebrate
 occupancies are a direct measure of conservation outcomes that can help
 guide protected-area management and improve the contributions that
 protected areas make towards global biodiversity goals. Here, we show the
 feasibility of using invertebrate-derived DNA to estimate
 spatially-resolved vertebrate occupancies across entire protected areas.
description: These are the input fastq files to our metabarcoding processing
 pipeline.  The raw samples are tubes of leeches
 collected from a subtropical forest in southwestern China, preserved in
 the equivalent of RNALater solution. Each sample replicate is a small
 ziploc baggie of tubes with leeches, collected by one ranger in one site
 along a stretch of trail.  <strong>Excerpt from
 methods section</strong>:  We extracted DNA from each replicate, and
 then PCR-ampliﬁed two mitochondrial markers: one from the 16S rRNA
 (MT-RNR2) gene, and the other from the 12S rRNA (MT-RNR1) gene. We
 hereafter refer to these two markers as LSU (16S) and SSU (12S),
 respectively, referring to the ribosomal large subunit and small subunit
 that these genes code for. The LSU primers are designed to target mammals,
 and the SSU primers to amplify all vertebrates. A third primer pair
 targeting the standard cytochrome c oxidase I marker [36] was tested but
 not adopted in this study as it co-ampliﬁed leech DNA and consequently
 returned few vertebrate reads. Primers were ordered with
 sample-identifying tag sequences, and we used a fully-redundant
 twin-tagging strategy to identify and remove ‘tag jumping’ errors [60]
 using the DAMe protocol [81]. From our 893 replicate tubes, we
 successfully PCR-ampliﬁed in triplicate 661 samples using our LSU primers
 and 745 samples using our SSU primers. Successful ampliﬁcations were sent
 to Novogene (Beijing, China) for PCR-free library construction and 150 bp
 paired end sequencing on an Illumina HiSeq X Ten.
description: We have three pipelines, reflecting the successive contributions
 of YJ, DWY, and CCMB, respectively, to the three stages of the
 analysis (1) Pipeline for processing raw Illumina
 HiSeq/MiSeq files https://github.com/jiyinqiu/ailaoshan_leeches_method_code https://zenodo.org/badge/latestdoi/238595182 (2) Bioinformatic scripts for processing pipeline output, including taxonomic reference datasets https://github.com/dougwyu/screenforbio-mbc-ailaoshan/releases/tag/1.3 https://doi.org/10.5281/zenodo.6971096 (3) Code for all statistical analysis and figures https://github.com/bakerccm/leeches2018-public https://doi.org/10.5281/zenodo.5914708 For a list of software needed, please refer to pipeline 1:  https://zenodo.org/badge/latestdoi/238595182