Speciation is a continuous process. Although it is known that differential adaptation can initiate divergence even in the face of gene flow, we know relatively little about the mechanisms driving complete reproductive isolation and the genomic patterns of divergence and introgression at the later stages of speciation. Sticklebacks contain many pairs of sympatric species differing in levels of reproductive isolation and divergence history. Nevertheless, most previous studies have focused on young species pairs. Here, we investigated two sympatric stickleback species, Pungitius pungitius and P. sinensis , whose habitats overlap in eastern Hokkaido; these species show hybrid male sterility, suggesting that they may be at a late stage of speciation. Our demographic analysis using whole-genome sequence data showed that these species split 1.73 Ma and came into secondary contact 37 200 years ago after a period of allopatry. This long period of allopatry might have promoted the evolution of intrinsic incompatibility. Although we detected on-going gene flow and signatures of introgression, overall genomic divergence was high, with considerable heterogeneity across the genome. The heterogeneity was significantly associated with variation in recombination rate. This sympatric pair provides new avenues to investigate the late stages of the stickleback speciation continuum. This article is part of the theme issue ‘Towards the completion of speciation: the evolution of reproductive isolation beyond the first barriers’.

The invasive alien crayfish Pacifastacus leniusculus is considered harmful to freshwater pearl mussels Margaritifera laevis and M. togakushiensis. It also often colonises mussel habitats in Japan. In order to test the negative effects of alien crayfish on mussels, we evaluated the predation impact of signal crayfish on freshwater pearl mussels in vitro. We tested the relationship between the survival/injury rates of mussels and crayfish predation with respect to different sizes of mussels (four classes based on shell length: 10, 30, 50 and 70 mm). Crayfish selectively fed on the flesh of the 10-mm size class mussels after breaking their shells. The shell margins of mussels in all size classes were injured by crayfish. Results also showed that crayfish particularly injured the 50-mm size class of mussels. This observation could be attributed to this mussel size being the most suitable shell size (29.56-37.73 mm in carapace length) that the crayfish can effectively hold. This study shows that the presence of invasive crayfish reduces freshwater pearl mussel populations by damaging the shell margins and/or killing the mussels. This negative impact of invasive crayfish not only decreases the mussel population but could also limit mussel recruitment, growth and reproduction.

The nest site characteristics and distribution patterns of nests of three sympatric ninespine sticklebacks, the brackish-and fresh-water types and Pungitius tymensis, were investigated in the Shiomi River, eastern Hokkaido, Japan. In this river, most of the males of the brackish-water type were found to build their nests in the lower reach with higher salinity (mean 13.5%) and water temperature (mean 13.3°C), whereas the males of the freshwater type and P. tymensis built their nests in the upper reach with lower salinity (mean 0.6 and 0.8%, respectively) and water temperature (both mean 11.3°C). The ranges of their nesting areas, however, overlapped within the river, because the nests of the brackish-water type were widely distributed in the river. A comparison of the nest site characteristics in the area where nest overlapped indicated that there were no significant differences for all of five characteristics (the distance from the nest to the shore, the distance from the nest to the bottom, the distance to the nearest nest, the nest density and the amount of cover around the nest). Using canonical discriminant analysis, the nests of the three sticklebacks could not be distinguished using the five nest characteristics. These results suggest that habitat isolation through physical factors functions as an important mechanism of reproductive isolation between the brackish-water type and the other two sticklebacks.

Species identification is a fundamental process for ecological studies and conservation practices, and simple nonlethal identification criteria are important for endangered species. This study developed species identification criteria for two endangered freshwater pearl mussels (Margaritifera laevis and Margaritifera togakushiensis) based on linear discriminant functions (LDFs) that were established considering intraspecific regional morphological differences from sites at Hokkaido, northern Japan. We collected a total of 1,110 mussels from 52 rivers across 32 watersheds in two geographical regions (east and west). Shell morphologies (length, height, and width) of the collected mussels were measured, followed by species identification with gel electrophoresis banding patterns of 16S rRNA polymerase chain reaction products. LDFs were constructed for two size classes (≥40 mm and < 40 mm) using forward stepwise procedures to determine key morphological differences between the two species and consideration of whether regional morphological differences improved identification accuracies. The LDFs revealed that the morphologies of the two species were clearly distinguished by the relationship between height and length for both size classes. Region‐specific LDFs produced better identification accuracies. Regardless of region, the maximum length of M. laevis exceeded 100 mm, whereas all M. togakushiensis were < 100 mm in length. Identification accuracies of the established LDFs were high for each of the five length classes (0–20 mm, 20–40 mm, 40–60 mm, 60–80 mm, and 80–100 mm) with 85–96% (mean: 92%) accuracy in the east and 67–96% (mean: 80%) in the west. These criteria for species identification will progress future ecological studies and conservation practices for freshwater mussels.

Egg hatching rates of F1 hybrids among three fishes, Japanese dace Tribolodon hakonensis, Pacific redfin T. brandtii, and rosyface dace T. sachalinensis were compared with pure crosses. They are deeply diverged (10-20 million years ago) but naturally hybridizing. Although crosses involving female rosyface dace were not available, hatching success was high among all combinations of hybrids (76 ± 23 %) and comparable to pure crosses (86 ± 8 %), implying ecological or genetic isolation mechanisms maintaining each species in later life stages.