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.