Hybridization between an endangered freshwater fish and an introduced congeneric species and consequent genetic introgression

Abstract

Artificial transplantation of organisms and consequent invasive hybridization can lead to the extinction of native species. In Matsuyama, Japan, a native bitterling fish, Tanakia lanceolata, is known to form hybrids with another bitterling species, T. limbata, which was recently introduced from western Kyushu, Japan. These bitterlings spawn in the gills of two freshwater unionid species, Pronodularia japanensis and Nodularia douglasiae nipponensis, which have rapidly declined on the Matsuyama Plain in the past 30 years. To gauge the effect of invasive hybridization, we determined the genetic introgression between T. lanceolata and T. limbata and analyzed the morphology of these species and their hybrids to infer their niche overlap. We collected adult individuals of Tanakia spp. and genotyped them based on six microsatellite loci and mitochondrial cytochrome b sequences. We analyzed their meristic characters and body shapes by geometric morphometrics. We found that 10.9% of all individuals collected were hybrids. Whereas T. lanceolata were more densely distributed downstream and T. limbata were distributed upstream, their hybrids were widely distributed, covering the entire range of native T. lanceolata. The body height and anal fin length of T. limbata were greater than those of T. lanceolata, but their hybrids were highly morphologically variable, covering both parental morphs, and were widely distributed in the habitats of both parental species. Hybridization has occurred in both directions, but introduced T. limbata females and native T. lanceolata males are more likely to have crossed. This study shows that invasive hybridization with the introduced T. limbata is a potential threat to the native population of T. lanceolata via genetic introgression and replacement of its niche in streams.

Fig 1. Study species and maps of the study sites on the Matsuyama Plain, Ehime prefecture, Japan, and distribution of native Tanakia lanceolata, introduced T. limbata, and their hybrids.

(a) Tanakia lanceolata and (b) T. limbata with the scale bar of 10 mm. (c) Location of the study areas in Japan. (d) Distribution of T. lanceolata (red), T. limbata (green), and hybrids of these two species (gray) classified based on six microsatellite markers. Circle size indicates the number of individuals. We have drawn these maps ourselves based on maps provided under CC BY 4.0 by the Geospatial Information Authority of Japan (https://maps.gsi.go.jp).

Fig 2. Body shape landmarks for geometric morphometrics.

(1) Anterior tip of snout, (2 and 3) anterior and posterior insertion of the dorsal fin, (4 and 5) upper and lower insertion of the caudal fin, (6 and 7) posterior and anterior insertion of the anal fin, (8) insertion of the pelvic fin, (9 and 10) ventral and dorsal insertion of the operculum on the profile, and (11) dorsal insertion of the pectoral fin. Number of lateral line scales and number of anal fin soft rays are observed as meristic characters.

Fig 3. Results of population genetic analysis of Tanakia lanceolata, T. limbata, and their hybrids in streams in Ehime, Japan, based on six microsatellite loci.

Squares above bars indicate mitochondrial cytochrome b genotypes. Asterisks above squares indicate hybrid individuals. Numbers indicate the locality as shown in Fig 1.

Fig 4. Longitudinal distribution of Tanakia lanceolata, T. limbata, and their hybrids in study streams.

(a) Tanakia lanceolata, (b) T. limbata, and (c) their hybrids. Solid curves indicate estimated values based on the result of a GLM without any variance among streams, and dotted curves indicate estimated values based on a GLMM with variance among streams.

Fig 5. Meristic characters and genotypes of Tanakia lanceolata, T. limbata, and their hybrids.

Circle size indicates the number of individuals.

Fig 6. Differences in the body shapes of Tanakia lanceolata, T. limbata, and their hybrids.

(a) Variations in body shape based on geometric morphometrics. Genotyping was conducted in NewHybrids based on six microsatellite loci. Circles indicate individuals with T. lanceolata mitochondrial genotype; squares indicate T. limbata mitochondrial genotypes. (b) Variations in shape along the PC1 axis. (c) Variations in shape along the PC2 axis. The lines in (b and c) indicate shape changes following the plot shifts in (a) with 0.1 unit in the positive PC direction. (d) Comparison between individuals with the maximum (T. lanceolata morph) and minimum (T. limbata morph) PC1 scores.

Fig 7. Relationships between body shape and longitudinal distribution in rivers of T. lanceolata, T. limbata, and their hybrids.

(a) Relationship between distance from the river mouth and PC1 values of geometric morphometrics of body shape; (b) relationship between distance from the river mouth and PC2 values.