DOI: 10.5061/dryad.j6q573n8p
title: Phylogeography of Gyrodactylus konovalovi (Monogenoidea: Gyrodactylidae) in Qinling Mountains in Central China

description: Gyrodactylus konovalovi is an ectoparasite on East Asia minnow
 Rhynchocypris lagowskii that is widely distributed in cold freshwater, the
 dispersal of this ectoparasite depends on host switching, and it is
 therefore a good model species to study parasite and fish phylogeographic
 patterns. We examined the phylogeography of G. konovalovi along with the
 distribution of its host in the Qinling Mountains in Central China. A
 total of 109 individuals collected from 21 localities were subsequently
 sequenced for 536 bp of a mitochondrial cytochrome oxidase subunit I
 (cox1) gene and 46 haplotypes were obtained. The ratio of substitution
 sites (dN/dS) was 0.015 and showed purifying selection. Haplotype
 diversity (h) was highest in the YP population, while nucleotide diversity
 ( π ) was highest in the TTG population. While h and π were lowest in the
 XYB population, except for ten populations. Phylogenetic trees based on
 BI, ML and MP methods and network analysis revealed that all haplotypes
 were consistently supported in four different lineages, indicating a
 significant geographic distribution pattern. There was a significant
 positive correlation between genetic differentiation (Fst) and geographic
 distance. The results of mismatch distribution, neutrality test and
 Bayesian skyline plot analyses showed that all lineages were stable
 without expansion during the Pleistocene, while the total population
 underwent population contraction during the late Pleistocene. The
 molecular clock calibration inferred that the most common ancestor was
 estimated to have emerged in the early to middle Pleistocene. Therefore,
 our study suggests that clearly phylogeographic distribution of G.
 konovalovi may be related to geological events such as orogenesis,
 drainage capture changes and vicariance, during the Pleistocene in the
 Qinling Mountains in Central China, while the population subdivision was
 hardly affected by climate fluctuation during the Pleistocene.
description: Sample collection The specimens of host
 <i>R. lagowskii</i> were sampled from 48 localities covered
 the most Qinling Mountains from May to October in 2016 and 2017 and stored
 in 96% ethanol (Figure 1). <i>Gyrodactylus </i>species were
 collected from the skin and fins of unique host under a stereomicroscope
 in the laboratory, and one parasite specimen per host individual was used
 to avoid pseudoreplication. Subsequently, <i>Gyrodactylus</i>
 specimens were examined microscopically, and species identification was
 based on the morphology of the opisthaptor (Ergens, 1976) and molecular
 ITS sequences. A total of 109 samples of
 <i>G</i><i>.</i><i> konovalovi</i>
 from 21 localities were identified (Figure 2 and Table 1). Finally, each
 parasite specimen and host specimen were individually stored in 96%
 ethanol at 4°C. Voucher specimens of parasites and hosts were deposited in
 the Fish Disease Laboratory, College of Life Sciences, Shaanxi Normal
 University, Xi’an, China, 710062. DNA extraction, PCR
 amplification and direct sequencing The DNA
 extraction, PCR amplification and direct sequencing were followed the
 method of previous study with slight modification (Ye et al., 2017). Total
 genomic DNA<i> </i>of<i> G. konovalovi</i> was
 extracted from a single individual using the TIANamp Micro DNA kit
 (Tiangen Biotech, Beijing, China) following the manufacturer's
 protocol. The 518 bp ITS2 rDNA (Internal Transcribed Spacer) fragment from
 each <i>Gyrodactylus</i> specimen was amplified and sequenced
 for molecular identification according to the method of previous
 study<sup> </sup>(You et al., 2014),
 <i>Gyrodactylus</i> species display the threshold of 1.39%
 between intraspecific and interspecific variation using ITS
 sequences<sup> </sup>(Vanhove et al., 2013). The values of the
 mean distance of ITS sequences among 21 populations of
 <i>Gyrodactylus</i> species were within the threshold of 1.39%
 (Table 2), then it identified <i>Gyrodactylus</i> specimen as
 <i>G</i><i>.</i><i> konovalovi</i>.
 Subsequently, the cox1 gene sequences of <i>G.</i><i>
 </i><i>konovalovi</i> from the 21 populations with 109
 individuals were amplified by polymerase chain reaction (PCR)
 amplification using a newly specific primers designed based on some genus
 species <i>Gyrodactylus</i><i> brachymystacis</i>
 and <i>Gyrodactylus</i><i> parvae</i> (Ye et al.,
 2017), the forward primer gkcox1-F
 (5'-TAAAGTGGGTAATATAGGAAAA-3') and reverse primer gkcox1-R
 (5'-CTAAAGAGAAGACATAGTGGAA-3') were used to amplify a segment of
 536 bp. Each PCR amplification was performed in a total volume of 25 µl,
 containing 3.0 mM MgCl<sub>2</sub>, 10 mM Tris-HCl (pH 8.3),
 50 mM KCl, 0.25 mM of each dNTP, 1.25 U rTaq polymerase (TaKaRa, Dalian,
 China), 0.4 μM of each primer, 45 ng gDNA, tapped with Milli-Q water. The
 cycling conditions were applied: initial denaturation for 1 min at 93°C
 followed by 35 cycles of denaturation for 10 s at 92°C, annealing for 1.5
 min at 48°C and extension for 2 min at 60°C with a final extension for 6
 min at 72°C. All PCR fragments were initially purified with a PCR
 purification kit (BGI Biotech, Shenzhen, China), subsequently subjected to
 electrophoresis in a 1% agarose gel and finally sequenced with PCR forward
 primer with an ABI Prism<sup>®</sup> 3730 automated sequencer
 (Applied Biosystems, Foster City, USA).
description: GenBank under accession number MN379691- MN379736.