Collection and morphological analysis

Fishes of the families Carangidae and Sparidae were collected from Moreton Bay (Queensland) by line fishing and seining, as well as being sourced from commercial tunnel-netting fisheries. Fishes were euthanized with AquiS® and examined following the recommendations of Cribb & Bray (Reference Cribb and Bray2010). Trematodes were fixed by pipetting into near-boiling saline and were preserved in 70% ethanol for parallel morphological and molecular characterization. Specimens for morphological analysis were washed in fresh water, stained with Mayer's haematoxylin, destained in a solution of 1.0% hydrochloric acid and neutralized in 0.5% ammonium hydroxide solution. Specimens were then dehydrated through a graded ethanol series, cleared in methyl salicylate and mounted in Canada balsam. Measurements were made using an Olympus SC50 digital camera mounted on an Olympus BX-53 compound microscope using cellSens Standard imaging software (Olympus, Tokyo, Japan). Measurements are in micrometres (μm) unless otherwise stated, and are presented as a range, followed by the mean in parentheses. Where length is followed by width, the two measurements are separated by ‘×’. Drawings were made using an Olympus BX-53 compound microscope and drawing tube. Type and voucher specimens are lodged in the Queensland Museum (QM), Brisbane, Australia.

We tested the significance of differences in the prevalence of monorchiids in Moreton Bay sparids with the Unconditional Exact Test in the software Quantitative Parasitology 3 (Rozsa et al., Reference Rozsa, Reiczigel and Majoros2000; Reiczigel et al., Reference Reiczigel, Abonyi-Toth and Singer2008).

Molecular sequencing

Total genomic DNA was extracted using phenol/chloroform extraction techniques (Sambrook & Russell, Reference Sambrook and Russell2001). Sequence data for two markers of ribosomal DNA (rDNA) were generated for this study, the complete ITS2 rDNA and the partial (D1–D3 region) 28S rDNA region. Cycle sequencing of these regions was carried out following the protocols of Cutmore et al. (Reference Cutmore, Diggles and Cribb2016) using the primers LSU5 (5′-TAG GTC GAC CCG CTG AAY TTA AGC A-3′; Littlewood, Reference Littlewood1994) and 1500R (5′-GCT ATC CTG AGG GAA ACT TCG-3′; Snyder & Tkach, Reference Snyder and Tkach2001) for the 28S amplification and 3S (3S: 5′-GGT ACC GGT GGA TCA CGT GGC TAG TG-3′; Morgan & Blair, Reference Morgan and Blair1995) and ITS2.2 (5′-CCT GGT TAG TTT CTT TTC CTC CGC-3′; Cribb et al., Reference Cribb, Anderson, Adlard and Bray1998) for the ITS2 amplification. Amplified DNA was purified using a Bioline ISOLATE II PCR and Gel Kit according to the manufacturer's protocol (Bioline, London, UK). Sanger sequencing of purified DNA was conducted at the Australian Genome Research Facility using ABI Big Dye™ v.3.1 chemistry (Applied Biosystems, Carlsbad, California, USA) following the manufacturer's recommendations, using the same primers as those used for polymerase chain reaction (PCR) amplification as well as the additional 28S rDNA primers 300 F (5′-CAA GTA CCG TGA GGG AAA GTT G-3′; Littlewood et al., Reference Littlewood, Curini-Galletti and Herniou2000) and ECD2 (5′-CCT TGG TCC GTG TTT CAA GAC GGG-3′; Littlewood et al., Reference Littlewood, Rohde and Clough1997). Sequencher™ version 4.5 (GeneCodes Corp., Ann Arbor, Michigan, USA) was used to assemble and edit contiguous sequences. The start and the end of the ITS2 region were determined by annotation through the ITS2 Database (Keller et al., Reference Keller, Schleicher, Schultz, Muller, Dandekar and Wolf2009; Ankenbrand et al., Reference Ankenbrand, Keller, Wolf, Schultz and Forster2015) using the ‘Metazoa’ model. GenBank accession numbers for the sequences generated during this study are presented in table 1.

Table 1.

28S rDNA sequence data used in phylogenetic analyses.

Phylogenetic analysis

The partial 28S rDNA sequences generated during this study were analysed together with sequences of monorchiid species available from GenBank (table 1). Sequences were aligned using MUSCLE version 3.7 (Edgar, Reference Edgar2004) with ClustalW sequence weighting and UPGMA clustering for iterations 1 and 2. The resultant alignments were refined by eye using MESQUITE (Maddison & Maddison, Reference Maddison and Maddison2017) and the ends of each fragment were trimmed to match the shortest sequence in the alignment.

Bayesian inference and Maximum Likelihood analyses of the 28S dataset were conducted to explore relationships among monorchiid taxa. Bayesian inference analysis was performed using MrBayes version 3.2.6 (Ronquist et al., Reference Ronquist, Teslenko, van der Mark, Ayres, Darling, Hohna, Larget, Liu, Suchard and Huelsenbeck2012), run on the CIPRES portal (Miller et al., Reference Miller, Pfeiler and Schwartz2010). Maximum Likelihood analysis was performed using RAxML version 7.7.1 (Stamatakis et al., Reference Stamatakis, Hoover and Rougemont2008). The software jModelTest version 2.1.10 (Darriba et al., Reference Darriba, Taboada, Doallo and Posada2012) was used to estimate the best nucleotide substitution model for the dataset. Both the Akaike Information Criterion (AIC) and Bayesian Information Criterion (BIC) in jModelTest predicted the GTR + Γ model as the best estimator. Nodal support in the Maximum Likelihood analysis was estimated by performing 100 bootstrap pseudoreplicates. Bayesian inference analysis was run over 10,000,000 generations (ngen = 10,000,000) with two runs each containing four simultaneous Markov Chain Monte Carlo (MCMC) chains (nchains = 4) and every 1000th tree was saved (samplefreq = 1000). Bayesian inference analysis used the following parameters: nst = 6, rates = gamma, ngammacat = 4 and the priors parameters of the combined dataset were set to ratepr = variable. Samples of substitution model parameters, and tree and branch lengths were summarized using the parameters ‘sump burnin = 3000’ and ‘sumt burnin = 3000’. Species of the family Lepocreadiidae were designated as the functional outgroup.