Correspondence between genetic clusters and Neocaridina species
A lag in taxonomic research is a major constraint on natural history research and ecosystem conservation. In Lake Biwa, the extinction of Neocaridina denticulata and the introduction of related alien species have been suspected. No studies have been conducted to support this, however, due to the difficulty of identification caused by taxonomic confusion. In this study, the analysis based on SNPs of Neocaridina suggested that there are three clusters in Lake Biwa. Of these, cluster 1 was identified as N. denticulata because of its long rostrum (RL/CL > 0.75; reaching distal end of 3rd segment of antennular peduncle; 78% individuals of cluster 1) and slightly excavated carpus of the 1st pereopod (Fig. 2a, e) (Klotz et al. 2013). Another diagnostic character, the shape of the propodus of the 3rd pereopod, was straight in many individuals in cluster 1 (angle less than 9°, 67% of all cluster 1 individuals), though the range of values was wide (2–19°). The populations identified as N. denticulata in this study were found to have elongated endopods of male 1st pleopod (1.4–2.1 times as long as broad), although the range of variation was wide. This feature is also shared by N. koreana (1.7–1.9; Kubo 1938; Liang 2004). Some populations of N. denticulata in Honshu, Japan, however, have been reported to have elongated endopods of male 1st pleopods (Kamita 1951, 1953). Cluster 2 and 3 showed a significant difference in the number of rostral teeth, but both were identified as N. davidi from the continent because of the short rostrum, the strong excavation of the carpus of the 1st pereopod, and the curvature of the propodus of the 3rd pereopod (Klotz et al. 2013, Mitsugi et al. 2017). We could not determine whether the two strains represent intraspecific polymorphism or cryptic species. Also, mtDNA suggested that clades 3 and 4 were included in cluster 2 as detected by SNP analysis. These two clades were treated as the same taxon for convenience because there was only one sample of clade 4 and they could not be distinguished by SNP analysis. The individuals identified as N. davidi were morphologically inconsistent with the 1915 record and specimen (see below) in many respects. Therefore, they were considered to belong to introduced populations that became established in the 2000s. The two alien clusters were found at 11 sites and were found to be established almost everywhere in and around Lake Biwa (Fig. 3a). In the absence of hybridization, N. denticulata and N. davidi can be distinguished by mtDNA (Fig. 3c); therefore, DNA barcoding may be helpful for invasive species surveys. Future taxonomic studies of the two lineages of N. davidi are needed to detect and manage invasive species.
Is Neocaridina denticulata in Lake Biwa native?
Determining whether a current population is native or alien is very challenging. Comparison with historical records and population genomic analysis are powerful approaches to this problem. Nelson Annandale first reported N. denticulata in 1915 from the northern part of Lake Biwa (near St. 07) and Seta River (near St. 16), a draining river (Annandale 1922; Kemp 1918). We confirmed another specimen of N. denticulata collected from northern Lake Biwa (near St. 07) in 1915. At this time, there is no record of transplantation of this species from other areas, and no aquatic organisms have been transplanted to Lake Biwa from western Japan, which is within the distribution range of this species, except for cultured eels Anguilla japonica (Furukawa and Awano 1969). Therefore, it is reasonable to assume that N. denticulata is a native species of Lake Biwa. The individuals identified as N. denticulata in this study were strongly suggested to belong to native populations because the rostrum length was consistent with Kemp (1918) and the past specimen, the proportion of endopod of the male 1st pleopod was consistent with the past specimen, and they were genetically in the same cluster as individuals in surrounding water systems (Fig. 3). Native populations that appeared to be pure strains were found in five sites in the rivers flowing into Lake Biwa. The possibility of hybridization was also suggested in St. 07, 14, and 16.
Evaluation of diagnostic characters between native and non-native species
Identification of Neocaridina species is difficult, but if we can predict their genetic characteristics from morphological traits, it will be possible to distinguish between native and non-native species easily. In the case of Neocaridina shrimps in Lake Biwa, among the traits considered diagnostic characters, RL/CL, AE, and FP showed significant differences between N. denticulata and N. davidi (Fig. 2a, d, e). However, all these traits had overlapping ranges of trait values among genetic clusters. Therefore, it is quite difficult to identify species based on a single trait alone. In the prediction of the N. denticulata ancestry proportion by GLMM, we found that using multiple traits as explanatory variables improved the prediction compared with using a single trait. The GLMM constructed in this study can be used for a simple survey of non-native Neocaridina shrimps using only morphological analysis in Lake Biwa. However, due to the overlap in the range of trait values, it is essentially difficult to completely discriminate between the genetic clusters of Neocaridina shrimps and their degree of admixture from morphological traits. Thus, genetic analysis is essential for a detailed survey.
Species replacement and its causes in the Neocaridina population of Lake Biwa
Based on preliminary observations, alien Neocaridina spp. were suspected to have been established in Lake Biwa (Nishino 2017; Nishino 2020b). However, these populations were not identified, and it was unclear whether replacement of the native species by alien species occurred. Our results showed that N. davidi was established at 11 of the 19 sites in and around Lake Biwa, suggesting that the native species has been replaced by an invasive species at most sites in this region.
The invasive species of the genus Neocaridina have been imported from China and Korea since the 1970s as fishing bait and ornamental species. Some of them are believed to have been introduced and became established (Niwa 2010). One of the factors involved in the successful establishment of invasive species is the structure of the ecosystem and the richness of biodiversity in the introduced area (Gallien and Carboni 2017). A decrease in the population size of a native species increases the niche opportunity for non-native species to use similar niches, facilitating the establishment of non-native species (Shea and Chesson 2002; Dlugosch et al. 2015). No specimen-based records of N. denticulata in Lake Biwa have been found since 1915 until this study, suggesting that the population has declined significantly, but the timing and factors behind the decline are unclear. Among the fishes in Lake Biwa, the kissing loach Parabotia curtus, golden venus chub Hemigrammocypris neglectus, Itasenpara bitterling Acheilognathus longipinnis, and Japanese rosy bitterling Rhodeus ocellatus kurumeus are believed to have become extinct from within Lake Biwa due to the development of coastal areas, habitat modifications such as land reclamation of the inner lake in the 1940s and 1950s, and hybridization with closely related invasive species (Nakanishi and Sekino 1996; Fujita et al. 2008; Kanao and Matsuda 2012). In Lake Biwa, mass die-offs of fish and shellfish caused by pesticide use in rice paddies (pentachlorophenol, PCP) were reported several times in the 1960s (Kawabe 1965; Matida 1968). Neocaridina denticulata, which inhabits coastal areas and shallow rivers, may have been strongly affected by development and pesticide use, leading to its extinction in some populations in and around Lake Biwa. Neocaridina davidi in Lake Biwa may have taken advantage of the vacant niche created by the drastic decrease in or extinction of the native population and became established. Elucidation of the factors involved in the establishment of N. davidi will contribute to the prevention of the further spread of this species.
Importance of conservation of native population and preventing secondary spread of invasive species
The native population of Lake Biwa, rediscovered after a century, is a valuable local population characterizing the biodiversity of this region. Furthermore, it can become a good model for studying the effects of habitat degradation and disturbance caused by introduced species on native organisms. Based on the population structure of Neocaridina in this region, the conservation unit of this regional population should be determined in the future, and further distribution and ecological surveys should be promoted. The development of molecular markers that can distinguish non-native and native species may promote these surveys.
Once an invasive species is established, approaches to prevent the secondary spread from potential hubs are necessary (Vander Zanden and Olden 2008). In Japan, the signal crayfish Pacifastacus leniusculus, introduced from North America into Lake Mashu, Hokkaido, has been secondarily spread and has become established through illegal release and mixing with fish stock from the lake to water bodies throughout Hokkaido (Usio et al. 2016). Since the ayu Plecoglossus altivelis and the prawn Palaemon paucidens from Lake Biwa, which are important fishery resources, have been released throughout Japan (Aino et al. 2015; Nishino 1980), the invasive Neocaridina species, reported from various parts of Japan, may have also been introduced by mixing with these releases from Lake Biwa (Hasegawa et al. 2015; Fuke et al. 2021; Niwa et al. 2014; Katayama et al. 2017; Kakui and Komai 2022; Mitsugi et al. 2017; Mitsui et al. 2019; Nagai and Imai 2021; Nishida 2016). In the future, it will be necessary to conduct a comprehensive and detailed population analysis using molecular markers of Neocaridina populations in various regions to verify the migration route and to understand the actual status of the introduction. It has been suggested that N. davidi might negatively affect native N. denticulata and other native shrimp species through competition (Katayama et al. 2017; Mitsugi and Suzuki 2018). Therefore, preventing further spread of N. davidi via Lake Biwa as a hub is important for the conservation of biodiversity in other regions.