A natural hybrid of a probable intergeneric mating between the striped shiner
As in plants, hybridization and introgression play important roles in the evolution of fish (Hubbs, 1955; Dowling and DeMarais, 1993; Scribner et al., 2001). They represent the driving force behind the evolution of new lineages with the potential to rapidly adapt to new habitats (DeMarais et al., 1992; Gerber et al., 2001; Grant and Grant, 1992). However, hybridization can also lead to extinction of rare indigenous species and presents challenges for the formulation of a biological species concept and definition, the reconstruction of phylogenetic trees and the practice for biological conservation (Grant and Grant, 1992; Rhymer and Simberloff, 1996; Allendorf et al., 2001). Natural hybridization occurs across diverse piscine taxa (Verspoor and Hammar, 1991) and is quite common in cyprinid fishes (Hubbs, 1955; Gerber et al., 2001; Scribner et al., 2001; Ünver et al., 2008). Hybridization between fish species frequently occurs in nature resulting from the intrinsic properties of the species, external fertilization and weakly developed reproductive isolating mechanisms, as well as natural or man-made causes, such as disparity in the number of parent species, habitat disturbance and fragmentation, the introduction of non-native species, and climate change (Hubbs, 1955; Rhymer and Simberloff, 1996; Allendorf et al., 2001; Scribner et al., 2001; Šorić, 2004; Ünver et al., 2008; Kwan et al., 2014).
Morphological analysis is a conventional approach to identification of naturally occurring hybrids because of the intermediate phenotypes. However, morphology alone is not always sufficient to determine the hybrid status and to trace the parent species (Avise and Saunders, 1984; Allendorf et al., 2001; Scribner et al., 2001). Analyses of genetic markers with different evolutionary histories provide convincing evidence for hybrid status and can help determine the extent and direction of hybridization (e.g., Avise and Saunders, 1984; Dowling et al., 1989; Avise et al., 1990; Albert et al., 2006; Chelomina et al., 2008). In practice, the combined application of nuclear DNA (nDNA) and mitochondrial DNA (mtDNA) sequences has proven useful in determination of hybridization in fish species (e.g., Sonnenberg et al., 2007; Lee et al., 2009; Yoon et al., 2009). These techniques are simple and easy to implement as the result of recent advances in molecular techniques, such as polymerase chain reaction (PCR), DNA sequencing, etc.
The mitochondrial genome is haploid, located in the cytoplasm and is maternally inherited without recombination. Given these distinct properties, analyses of mtDNA sequences provide an opportunity to determine the direction of gene flow in a hybrid cross (Avise and Saunders, 1984; Moritz et al., 1987; DeMarais et al., 1992; Rhymer and Simberloff, 1996). However, the information provided by mtDNA alone is limited due to its strict maternal mode of inheritance. Thus, to determine the directionality of species hybridization, mtDNA analysis is often used in conjunction with analysis of a nuclear gene, which is inherited biparentally or in a simple Mendelian fashion. Hybrid state is reliably identifiable in monomorphic (homozygous) versus polymorphic (heterozygous) states; i.e., each pure parent species possesses a clear DNA sequence with monomorphic base positions, whereas their hybrids exhibit polymorphisms different to the parent (Scribner et al., 2001; Sonnenberg et al., 2007).
The striped shiner
A single natural hybrid (
[Fig. 1.] Specimens of a natural hybrid and its supposed parent species, Pungtungia herzi and Pseudorasbora parva (left) and fragments of their electropherograms showing the recombination activating gene 1 gene (rag1) (right). A) Pu. herzi (♀), B) the natural hybrid and C) Ps. parva (♂). The fragments of the electropherograms show differences in base positions for the two parent species and double peaks at the respective positions for the natural hybrid (indicated by inverted arrowheads).
A total of 25 morphological characters were used in the calculation of the hybrid index (Hindex) to measure the degree of phenotypic intermediacy of the hybrid in comparison to its parent species. The number of fin rays and vertebrae were counted using a soft X-ray photographer (Hitex HA-80; Hitex, Osaka, Japan). Total of six meristic and 19 morphometric characters were counted or measured as described by Hubbs and Lagler (1964) (Table 2). The mean of
where H is the numerical value of a character of the hybrid, and M1 and M2 are those of the same character in
[Table 2.] Comparison of six meristic and 19 morphometric characters of a natural hybrid and its supposed parent species, Pungtungia herzi and Pseudorasbora parva, captured in the Geumho River, a tributary of the Nakdong River basin of Korea. Hybrid index (Hindex) values were calculated for each character state based on the mean values of the two parent species
A section of the pectoral or anal fin was excised from each specimen to extract genomic DNA (gDNA). The tissue was incubated with TNES-Urea buffer (10 mM Tris-HCl, pH 8.0; 125 mM NaCl; 10 mM EDTA, pH 8.0; 1% SDS; 8 M urea) (Asahida et al., 1996) containing 100-μg proteinase K (Sigma-Aldrich, St. Louis, MO, USA) at 37℃ for one day, followed by separation of the aqueous phase with a phenol : chloroform : isoamyl alcohol (25 : 24 : 1) solution, and ethanol precipitation according to Sambrook and Russell (2001). The extracted gDNA was resuspended in 1× TE buffer (10 mM Tris-HCl, pH 8.0; 1 mM EDTA, pH 8.0). Its quantity and quality were checked using a spectrophotometer, NanoDrop 1000 (Thermo Fisher Scientific, Wilmington, DE, USA) and by electrophoresis in a 0.7% agarose gel after staining with GelRed™ Nucleic Acid Gel Stain (Biotium, Hayward, CA, USA).
To amplify a complete fragment of the mitochondrial cytochrome
The purified PCR product of the
After cycle sequencing using the ABI PRISM® BigDye™ Terminator v3.1 Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Foster City, CA, USA), the purified PCR products and plasmid DNAs were sequenced by Macrogen (Seoul, Korea), a commercial company, on an ABI 3730xl DNA Analyzer (Applied Biosystems) using both sets of PCR primers. The sequences analyzed in this study were deposited in GenBank under accession numbers KP053615–KP053620.
DNA sequences of the
Phylogenetic analyses were carried out in PAUP* 4.0b10 (Swofford, 2002). A neighbour-joining (NJ) tree was reconstructed with the Kimura 2-parameter model, and a maximum-parsimony (MP) tree with the heuristic search option with the random addition of sequences (10 replicates) and tree-bisection-reconnection (TBR) branch swapping. The robustness of the tree topologies was evaluated by bootstrap analyses for NJ and MP analyses with 1,000 pseudoreplicates.
The morphological data for six meristic and 19 morphometric characters along with their Hindex values are presented in Table 2. A Hindex of 50 indicates an exact phenotypic intermediacy between the two parent species. The hybrid was considered to resemble
Of the meristic characters, the number of dorsal and ventral fin rays did not differ among the hybrid and parent species, but the number of anal fin rays of the hybrid and
Mouth shape is an important morphological character that distinguishes two parent species (Mori, 1935).
Partial DNA sequences of the single-copy nuclear
[Fig. 3.] Nucleotide sequence alignment of the nuclear recombination activating gene 1 gene (rag1) of a natural hybrid and its supposed parent species, Pungtungia herzi (♀) and Pseudorasbora parva (♂). The polymorphic base positions are indicated according to IUPAC codes.
Twelve randomly chosen PCR clones of the
[Fig. 4.] Phylogenetic trees inferred from the nuclear recombination activating gene 1 gene (rag1) (A) and mitochondrial cytochrome b gene (mt-cyb) (B) of a natural hybrid and its supposed parent species, Pungtungia herzi (♀) and Pseudorasbora parva (♂). Bootstrap values above 50% for the neighbour-joining and maximum-parsimony trees, respectively, are shown at each branch node. Species analyzed in this study are in bold faced font. Two haplotypes of rag1 of the hybrid were deduced from twelve randomly chosen PCR clones.
Analyses of the complete nucleotide sequences of the mitochondrial
A natural hybrid of a probable intergeneric mating between