The distributions of two bitterling fish (subfamily: Acheilognathinae), Tanakia lanceolata and T. limbata, overlap in western Japan. Acheilognathinae fish lay their eggs in the gills of freshwater bivalves, and the early juvenile stage develops in the gills. Populations of freshwater bivalves are declining worldwide, which has limited the number of spawning substrate for bitterlings. T. limbata has been artificially introduced to some rivers in Ehime, Japan, where it coexists with native T. lanceolata, and some hybrids have been observed. We collected both species from several sites in western Japan, and from the Kunichi River system in Ehime, and analyzed genetic population structure based on six microsatellite loci and sequences of the mitochondrial cytochrome b gene. Structure analysis identified three genetically distinct populations: T. lanceolata, T. limbata “West Kyushu”, and T. limbata “Setouchi”. Two clades of T. limbata were also supported by molecular phylogenetic analyses based on cytochrome b. Hybrids in Ehime originated mostly from interbreeding between male T. lanceolata and female T. limbata “West Kyushu”, and made up 10.2% of all collected fish, suggesting that hybrids occurred frequently between females of colonizing species and males of native species. On the other hand, interspecific hybrids were detected at rates of 40.0%, 20.0%, and 17.6% in the Ima River (Fukuoka), Midori River (Kumamoto), and Kase River (Saga), respectively, which are naturally sympatric regions. We found a few T. limbata “Setouchi” in the Midori and Kase Rivers, which were supposed to be introduced from other regions, coexisting with native T. limbata “West Kyushu”, and this cryptic invasion may have triggered the interspecific hybridization. These results suggest that artificial introduction of a fish species, a decline in the unionid population, and degradation of habitat have caused broad hybridization of bitterlings in western Japan.

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.

1. Bitterling fishes (Subfamily: Acheilognathinae) spawn in the gills of living freshwater mussels and obligately depend on the mussels for reproduction. On the Matsuyama Plain, Japan, populations of unionid mussels-Pronodularia japanensis, Nodularia douglasiae, and Sinanodonta lauta-have decreased rapidly over the past 30 years. Simultaneously, the population of a native bitterling fish, Tanakia lanceolata, which depends on the three unionids as a breeding substrate, has decreased. Furthermore, a congeneric bitterling, Tanakia limbata, has been artificially introduced, and hybridisation and genetic introgression occur between them. Here, we hypothesised that decline of the unionids has enhanced this invasive hybridisation through competition for the breeding substrate. 2. Three study sites were set in three streams on the Matsuyama Plain. We collected adult bitterling fishes (native T. lanceolata, introduced T. limbata, and foreign Rhodeus ocellatus ocellatus) once a week from April to October 2013 to measure their densities in streams and to examine seasonal differences in female ovipositor length, which elongates in the breeding season. Simultaneously, we set quadrats and captured unionids and measured environmental conditions. Each unionid individual was kept separately in its own aquarium to collect ejected bitterling eggs/ larvae. Tanakia eggs and larvae were genotyped using six microsatellite markers and the mitochondrial cytochrome b gene. 3. Introduced T. limbata was more abundant, had a longer breeding period, and produced more juveniles than native T. lanceolata. Hybrids between the two species occurred at all sites, and in total 101 of the 837 juveniles genotyped were hybrids. The density of P. japanensis was low, at most 0.42 individuals/m 2. Nodularia douglasiae and S. lauta have nearly or totally disappeared from these sites. Hybrid clutches of Tanakia species occurred more frequently where the local density of P. japanensis was low. Mussels were apparently overused and used simultaneously by three species of bitterlings. 4. Decline of freshwater unionid populations has enhanced hybridisation of native and invasive bitterling fishes through increasing competition for breeding substrate. We showed that rapid decline of host mussel species and introduction of

Although parasites reduce host health, parasite infections also occur as a consequence of compromised host health. Both causalities could induce positive feedback, in which infected hosts with poor body conditions may suffer further infection, but it has rarely been demonstrated in the wild, possibly due to methodological difficulties. We used a mark-recapture survey combined with structural equation modelling (SEM) to examine whether both causalities and positive feedback occurred in stream salmonid and parasitic copepod systems. We found that parasitic copepods reduced host conditions and hosts with poor conditions were likely to be infected, suggesting that positive feedback can occur in the wild. Importantly, heavily infected hosts with poor body conditions showed lower apparent survival rates. Our findings provide robust evidence showing host condition–parasite infection dynamics, offering novel insights into how positive feedback could strongly undermine the wild host population via reduction of host survival.