DOI: 10.5061/dryad.37pvmcvgv
title: Low levels of genetic differentiation with isolation by geography and environment in populations of Drosophila melanogaster from across China

description: The fruit fly Drosophila melanogaster is a model species in evolutionary
 studies. However, the population processes of this species in East Asia
 are poorly studied, even though this area was one of the first regions
 colonized outside of its native distribution range. Here we examined the
 population genetic structure of D. melanogaster across China. There were
 14 mitochondrial haplotypes with ten unique ones out of 23 known from
 around the globe. Pairwise FST values estimated from 15 novel
 microsatellites ranged from 0 to 0.11, with geographically isolated
 populations showing the highest level of genetic uniqueness. STRUCTURE
 analysis identified high levels of admixture at both the individual and
 population levels. Mantel tests indicated a strong association between
 genetic distance and geographical distance as well as environmental
 distance. Full RDA analysis showed that independent effects of
 environmental conditions and geography accounted for 62.10% and 31.58% of
 the total explained genetic variance, respectively. When geographic
 variables were constrained in a partial RDA analysis, three environmental
 variables of bio2 (mean diurnal air temperature range), bio13
 (precipitation of the wettest month), and bio15 (precipitation
 seasonality) were correlated with genetic distance. Our study suggests
 that a high level of gene flow, geographical isolation, and environmental
 factors have together shaped the population genetic structure of D.
 melanogaster after its introduction into China.
description: <b>Sample collection and DNA
 extraction</b> Specimens of <i>D.
 melanogaster</i> were collected from 16 locations in China were
 trapped by using rotten fruits (e.g., grapes, watermelon and banana) in
 the year 2019 (Table 1; Fig. 1). The sampled flies<i> </i>were
 firstly checked morphologically using an anatomical lens, followed by
 molecular identification by BLAST searching of the mitochondrial
 cytochrome oxidase subunit I (<i>cox1)</i> gene (see below)
 against the nucleotide database (nt) and in the BOLD system
 (http://www.boldsystems.org) to ensure the reliability of morphological
 identification. All samples were stored in 98% alcohol, and frozen at −80
 °C before DNA extraction. A DNeasy Blood and Tissue Kit (Qiagen, Germany)
 was used to extract total genomic DNA individually for 330 samples
 randomly selected from 16 populations. <b>Mitochondrial gene
 amplification and sequencing</b> To
 characterize the mitochondrial variation and validate correct
 identification of the specimens, a fragment of the <i>cox1</i>
 gene involving the DNA barcoding region of insects was sequenced using the
 universal primer pairs AF (5’ GGTCAACAAATCATAAAGATATTGG 3’) and AR (5’
 TAAACTTCAGGGTGACCAAAAAATCA 3’) (Folmer<i> et
 al</i>, 1994). Polymerase chain reaction
 (PCR) was conducted using the Mastercycler Pro system (Eppendorf, Germany)
 under standard conditions with an annealing temperature of 52 °C. PCR
 components were added as recommended by the manufacturer of Takara LA Taq
 (Takara Biomedical, Japan). Amplified products were purified and sequenced
 on an ABI 3730xl DNA Analyzer by TianYi HuiYuan Biotechnology Co. Ltd
 (Beijing, China). <b>Microsatellite marker
 development and genotyping</b> We developed
 genome-wide microsatellites from the<i> D. melanogaster</i>
 genome (accession PGRM00000000). The QDD3 program (Meglecz<i> et al</i>,
 2010) was used to extract microsatellites along
 with their flanking sequences (300 bp each) from the scaffolds, and to
 design the primers. According to previous reports, dinucleotide
 microsatellites are prone to polymerase slippage during the amplification
 process, which may lead to mistyping, so muti- (including tri-, tetra-,
 penta- and hexa-) nucleotide loci were preferentially selected in this
 study. The criteria and parameters of primer design for the isolated
 microsatellite markers followed Cao<i> et
 al</i> (2016a). Next, the output primers were
 further filtered manually using more stringent criteria: (i) only perfect
 (without repeat region interruptions) loci were retained; (ii) the minimum
 distance between the 3′ end of two primer pairs and their target region
 had to be &gt;10 bp; and (iii) primers using the ‘A’ design strategy
 that do not have multiple microsatellites, nanosatellites, or homopolymers
 in the amplicon were retained. A total of 60 primer pairs were ultimately
 screened out, which were used for further isolation of polymorphic
 microsatellite loci. The sixty primer pairs were
 initially chemically synthesized by Tsingke Biotechnology Co. Ltd
 (Beijing, China), with a universal primer (CAGGACCAGGCTACCGTG) added to
 the 5′ end of the forward primers based on the method of Blacket<i> et al</i>
 (2012). One individual <i>D.
 melanogaster</i> from each of the 16 populations was randomly
 selected to test the polymorphism level of target microsatellite sequences
 and the amplification efficiency of synthesized primers. The PCR
 amplification reaction was carried out in a 10 μL volume consisting of 0.5
 µL of template DNA (5-20 ng/µL), 5 µL of Master Mix (Promega, Madison, WI,
 USA), 0.08 µL of PC tail modified forward primer (10 mM), 0.16 µL of
 reverse primer (10 mM), 0.32 µL of fluorescence-labeled PC tail (10 mM),
 and 3.94 µL of ddH<sub>2</sub>O. The thermal profiles for DNA
 amplification were as follows: 4 min at 94 °C; 35 cycles of 30 s at 94 °C,
 30 s at 56 °C, and 45 s at 72 °C, followed by a final 10-min extension at
 72 °C. The amplified PCR fragments were analyzed on an ABI 3730xl DNA
 Analyzer (Applied Biosystems) using the GeneScan 500 LIZ size standard
 (Applied Biosystems). Genotyping was conducted using GENEMAPPER 4.0
 (Applied Biosystems, USA). Primer pairs that failed in the PCR
 amplification for more than two individuals, that were monomorphic in
 testing individuals, or that produced more than two peaks were discarded
 (Table 2).
description: Genetic diversity analysis of <i>Drosophila</i>
 <i>melanogaster.</i>