2.6 Genetic analysis
In the laboratory, fin tissue was collected from each specimen and preserved in 95% ethanol, and genetic samples were taken from the planktivorous (N = 36) and benthivorous (N = 33) morphs. Total genomic DNA was isolated using a standard phenol-chloroform method (Sambrook, Fritsch, & Maniatis, 1989). Two mitochondrial gene sequences, the cytochrome b gene (Cyt b, 1,140 bp) and the control region (D-loop, 839 bp), were amplified for all individuals using PCR. Detailed primer information is given in Table S1. PCR was performed in 37 μL reactions containing 1 μL of DNA, 0.375 μL of dNTP mix, 0.3 units of Taq polymerase (TaKaRa), 3.8 μL of 10× reaction buffer, and 0.5 μL of each primer. All reactions were performed under the following thermal cycler conditions: denaturation at 94 °C for 3 min, followed by 30 cycles of 95 °C for 30 s, annealing at 55 °C for 50 s and primer extension at 72 °C for 90 s for Cyt b and 45 s for D-loop and a final extension at 72 °C for 10 min (He & Chen, 2006; Liang et al., 2017). After visualization of the fragments using a 1% agarose gel, the PCR products were sequenced on an ABI 3730 capillary sequencer by Sangon Biotech Company (Shanghai, China).
The chromatograms were visually checked in BioEdit 7.0 (Hall, 1999), and the sequences were aligned with ClustalX (Larkin et al., 2007). Five species in the genus Schizopygopsis (S. younghusbandi ,S. pylzovi , S. stoliczkai , S. bangongensis , andS. malacanthus ) and the closely related speciesHerzensteinia microcephalus were analysed together. Two species,G. eckloni and G. przewalskii, were designated as outgroups. Phylogenetic relationships were constructed under maximum likelihood (ML) and Bayesian inference (BI) in CIPRES (Miller, Pfeiffer, & Schwartz, 2010) using a concatenated sequence of Cyt b and the D-loop, respectively (Zhao et al., 2009). Analysis of molecular variance (AMOVA, Excoffier, Smouse, & Quattro, 1992) was performed using Arlequin 3.5 (Excoffier & Lischer, 2010) to estimate the genetic structure of and differentiation among populations (corresponding to the confirmed morphs).
RESULTS
3.1 Morphological analysis
Across all samples, two morphs were identified with UPGMA cluster analysis on the basis of body shape (Figure 4), referred to here as morph 1 (planktivorous, N = 74) and morph 2 (benthivorous, N = 80). DFA showed that the two morphs differed significantly in terms of body shape (Wilks’ λ = 0.13, N = 154, p < 0.001). A posteriori jackknife cross-validation showed high success, with 98.6 and 98.8% of the samples being correctly allocated to the planktivorous and benthivorous morphs, respectively. According to the MANOVA results shown in Table 1, seven linear traits and two body shape PCs were effective variables for the discriminant analysis. Based on the PCA and reconstructed body shape results (Figure 5), the samples of the planktivorous morph had a more terminal mouth, a longer, more robust head shape and a longer upper jaw, lower jaw and snout length than those of the benthivorous morph. In addition, among all the samples, morphological differences between the sexes were not detected (t-test, df = 152, all p > 0.05, males:females = 1:1.8). With regard to parasites, only cestodes and Caenorhabditis eleganswere identified in a small number of samples (planktivorous, N = 3; benthivorous, N = 9). Parasitism also did not affect the morphology of the specimens (ANCOVA; p = 1.00, > 0.05).
With respect to descriptive traits, the two morphs (planktivorous and benthivorous) significantly differed in head characters. Planktivorous individuals possessed a terminal mouth with a slightly horny edge (width of horny edge: < 0.02 cm) or lacked a horny edge on the lower jaw and had a highly developed mucus cavity in the cheek and chin. The benthivorous individuals were characterized by an inferior or sub-inferior mouth with a sharpened horny edge on the lower jaw (width of horny edge: 0.14-0.42 cm) and the lack of a mucus cavity or only a small mucus cavity in the cheek and chin.
Obvious differences and similarities between morphs were observed in some specialized organs related to ingestion. Although pharyngeal teeth in one or two rows were observed in both morphs (Figure 6), the percentage of individuals with a single row of pharyngeal teeth was larger in the benthivorous morph (30.0%) than in the planktivorous morph (10.8%). The numbers and lengths of gill rakers were similar between morphs (t-test; all p> 0.05). A summary of basic information regarding the specimens of the two morphs captured in the field is given in Table S2.