DOI: 10.5061/dryad.rjdfn2z9b
title: Dataset for: Molecular diversity of dragonflies in high altitude Andean lakes through DNA barcoding

description: Genetic and morphological identification of dragonflies’ larvae species in
 three high elevation Andean tropical lakes was done using DNA barcoding of
 the cytochrome oxidase 1 gene (COI). Phylogeny allowed inferring the
 evolutionary relationships of at least 5 species (from 74 samples) that
 belong to two different families within the Odonata order.
description: <b>1. Study
 area</b> Three Andean lakes of high
 elevation; Yahuarcocha, San Pablo and Caricocha were selected as sampling
 areas to collect the samples. These lakes are located in the Northern part
 of Ecuador. While, Yahuarcocha lake has a surface area of 2.61 Km² with an
 elevation of 2200 meters above sea level (<i>m.a.s.l</i>)
 00°22´300 N, 78°06´100 W , San Pablo has more than the double in area with
 6.4 km<sup>2</sup> with an elevation of 2660
 <i>m.a.s.l</i> 0°13'0" N  78°12'0" W .
 Finally the largest lake, the Caricocha (belonging to the Mojanda lake
 system), with an area of 13.29 Km<sup>2 </sup>at 3713
 <i>m.a.s.l</i> 00°08´389 N, 78°15´397 W, with the lowest human
 influence but more than 1000 meters higher than the other two lakes
 (Cabrera, 2015; Casallas & Gunkel, 2002; Van Colen et al.,
 2017). <strong>2. Specimen
 sampling and identification</strong> A total of 74 macroinvertebrates
 larvae organisms were collected near the shore of each lake between July
 and September 2016. On each sampling site (see <i>Supplementary file
 Table 1</i> for the exact coordinate positions of each sampling
 point), the samples were collected using professional hand net with wooden
 handle (2mm mesh Efe & GB nets) for macroinvertebrates (Alba et
 al., 2005). All samples were preserved in the field using 95% ethanol in a
 cooler with cooling blocks and stored at the Environmental Research
 Laboratory (LABINAM) in the North Technical University, Ecuador.
 For sample processing and
 taxonomic identification all individuals were enumerated and identified to
 the lowest practical taxonomic level using a stereomicroscope LEICA M165C
 and using available species identification keys (Heckman, 2006).
 <b>3. DNA extraction,
 amplification and sequencing</b> Total genomic DNA was isolated
 from head and legs from each sample using the kit PureLinkTM Genomic DNA
 and following the protocol supplied by the company. The primers COI-F:
 5’ATAATTGGRGGRTTYGG RAAYTG-3’ (forward) y COI–R: 5’CCAAARAATCAAA
 ATAARTGTTG-3’ (reverse) were used for the amplification of 450 bp amplicon
 of the COI region (Hayashi, Dobata, & Futahashi, 2005; Yong et
 al., 2014). DNA extract concentrations were measured in a <i>Colibri
 Microvolume Spectrometer</i>, ranging from 5 to 10 ng/µl in average
 per sample. DNA region was amplified using Go Taq® Green Master Mix, 2X
 (Promega Corporation, 2012). The 10 μL PCR reaction mix was composed of
 3.7 μL of ultrapure water, 5 μL of Go Taq Green, 0.4 μL of each primer (4
 μM) and 0.5 μL f DNA template. The PCR amplification protocol used was:
 95°C for 5 min, 35 cycles of denaturation at 95°C for 1 min, Primer
 annealing at 50°C for 1 min and extension at 72°C for 1:30 min, with a
 final extension at 72°C for 5 min. The PCR products were visualized on
 agarose 1.7% gels and checking that the fragments were the correct ones
 and purified using the kit Wizard® SV Gel and PCR Clean-Up System (Promega
 Corporation, 2010). The company Macrogen in South Korea carried out DNA
 sequencing of the amplified fragments. <b>4. Data
 analysis</b> A combination of different
 strategies were applied to search for most similar sequences in two public
 databases: GenBank (NCBI, National Centre for Biotechnology Information)
 and BOLD (Barcode life Data Systems). The identification was made based on
 the most similar matches (&gt; 99% similarity) (Sonet et al., 2013).
 The most similar sequences are called "best match" (Meier et
 al., 2006). In GenBank, we used Basic Local Alignment Search Tool (BLAST)
 (McClean, 2004). In BOLD, the in-built Identification System (IDS) was
 applied (Ratnasingham & Hebert, 2007). The obtained sequences of
 COI were aligned using Geneious Prime ® 2020.2.2 (Kearse et al., 2012) and
 the online tool (https://mafft.cbrc.jp/alignment/server/index.html) for
 the MAFFT 7 alignment method. Phylogenetic reconstruction was performed
 with the Markov Chain Monte Carlo (MCMC) Bayesian approach implemented in
 BEAST version 2.6.3. Phylogenetic analysis was carried out allowing BEAST
 package to detect the best substitution model for the dataset alignment.
 Additionally, the phylogenetic analysis was simultaneously carried out
 with the GTR+Invariable+Gamma as the best substitution models suggested by
 the software jModelTest for DNA sequences alignments (Darriba, Taboada,
 Doallo, & Posada, 2012), which was in agreement with the best
 substitution model detected by BEAST. A non-parametric Bayesian Skyline
 (Piecewise-constant) coalescent model with a was used with a Strict
 molecular clock method. MCMC was developed with 10 million generations,
 subsampled every 1000 generations by applying a Random as the starting
 tree.The output of the MCMC analysis was summarized using TreeAnnotator
 software included in the Beast package. Maximum Clade Credibility tree was
 produced after discarding 10% of burn-in. The final tree was visualized
 using FigTree version 1.4.2. (Rambaut, 2012). Maximum Clade Credibility
 tree was produced after discarding 10 % of burn-in. The final tree was
 visualized through FigTree version 1.4.4.
description: A phylogeny of the
 cytochrome C oxidase subunit 1 (COI) gene was reconstructed including
 reference sequences from GenBank (NCBI) and BOLD as well as the samples
 generated de novo in this study. The sequences of the COI gene of the
 macroinvertebrates of the order Odonata are grouped in five clades next to
 the reference sequence of which the name of the identified species has
 been assigned (see Figure 2). The clades are supported by bootstrap values
 ​​of 99.92% for group 1 (G1) belonging to the species
 <i>Rhionaeschna marchali </i>99.88% for group 2 (G2) of the
 <i>Rhionaeschna elsia</i> species 99.98% for group 3 (G3)
 consisting of 2 individuals from <i>Remartinia luteipennis</i>
 99.98% for group 4 (G4) that were grouped with the reference sequence
 belonging to the species <i>Ischnura ramburii</i> and 99.97%
 of bootstrap group 5 (G5) that could not be grouped to any
 reference.