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.

Sample collection

The specimens of host R. lagowskii 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). Gyrodactylus 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, Gyrodactylus 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 G. konovalovi 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 of G. konovalovi 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 Gyrodactylus specimen was amplified and sequenced for molecular identification according to the method of previous study (You et al., 2014), Gyrodactylus species display the threshold of 1.39% between intraspecific and interspecific variation using ITS sequences (Vanhove et al., 2013). The values of the mean distance of ITS sequences among 21 populations of Gyrodactylus species were within the threshold of 1.39% (Table 2), then it identified Gyrodactylus specimen as G. konovalovi. Subsequently, the cox1 gene sequences of G. konovalovi 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 Gyrodactylus brachymystacis and Gyrodactylus parvae (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 MgCl₂, 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^® 3730 automated sequencer (Applied Biosystems, Foster City, USA).

GenBank under accession number MN379691- MN379736.