Rediscovery of a native freshwater shrimp, Neocaridina denticulata, and expansion of an invasive species in Lake Biwa, Japan: Genetic and morphological approach

doi:10.21203/rs.3.rs-1448326/v1

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Rediscovery of a native freshwater shrimp, Neocaridina denticulata, and expansion of an invasive species in Lake Biwa, Japan: Genetic and morphological approach

https://doi.org/10.21203/rs.3.rs-1448326/v1

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published 12 Aug, 2022

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Invasive species prevention involves the prevention of two aspects: introduction and secondary spread. The latter is important in places that can become hubs for the spread of invasive species. Lake Biwa, the largest lake in Japan, is an important area for biodiversity and fisheries. However, several invasive species of fish and crustaceans became established in the lake during the last century. One of the conservation problems in Lake Biwa is the unresolved suspicion that the native freshwater shrimp Neocaridina denticulata have been replaced with alien Neocaridina species. To verify whether the native Neocaridina population in Lake Biwa has been replaced by alien species, we estimated the population structure of Neocaridina spp., collected from 19 sites in and around Lake Biwa, based on genome-wide SNPs and mitochondrial DNA. The three detected genetic clusters were characterized by quantitative analysis of multiple morphological traits. Two clusters were identified as non-native N. davidi and the other as native N. denticulata. However, species discrimination based solely on morphological analysis was difficult, highlighting the importance of genetic analysis. We rediscovered the native populations in the region for the first time in a century; however, in 11 sites the invasive species were dominant. These findings suggest that the native populations are in a critical situation. Furthermore, stocking in Lake Biwa as a source for fishery resources throughout Japan can cause the secondary spread of the invasive shrimps from the lake, acting as a hub, to other parts of the country.

Invasive species

Lake Biwa

MIG-seq

mitochondrial DNA

Neocaridina

secondary spread

Since the impact of invasive species, which is critical in biodiversity conservation, on native species is often irreversible, the prevention of invasion is generally the most effective countermeasure against this issue (Pyšek and Richardson 2010). However, once an invasive species is allowed to invade and become established, it is important to prevent its secondary spread. Prevention of secondary spread refers to stopping or slowing down the spread of invasive species that have become already established in an area (Vander Zanden and Olden 2008). It is especially important in areas that can potentially become hubs for the spread of invasive species through anthropogenic or natural movement (Jaspers et al. 2018; Seebens et al. 2016; Vander Zanden and Olden 2008).

Lake Biwa, the largest lake in Japan, is home to more than 2,000 species of aquatic and semi-aquatic animals, including endemic fishes and mollusks (Nishino 2020a). Since the 1930s, 22 species of invasive fish and six species of invasive crustaceans have been introduced in Lake Biwa through release of fishery resources, including ornamental pets and fish for angling (Nakai and Kaneko 2020; Maehata 2020). These invasive species, along with environmental degradation, have become a major threat to the native species of Lake Biwa (Nishino et al. 2020). With the natural population of fish and crustaceans and an active fishery, Lake Biwa is important for the fisheries industry and act as a source for release in other parts of Japan (e.g., the ayu Plecoglossus altivelis and the lake prawn Palaemon paucidens) (Aino et al. 2015; Fujioka 2012; Fujioka and Maehata 2012; Nishino 1980). The presence of various freshwater fish originating from Lake Biwa have been confirmed in various parts of the country (Kitanishi et al. 2016; Watanabe 2012) and are thought to have been introduced by these releases through mixing.

Neocaridina is a genus of land-locked freshwater shrimp mainly distributed in East Asia. In Japan, four native species, N. denticulata (De Haan 1844), N. ishigakiensis (Fujino and Shokita 1975), N. iriomotensis Naruse et al. 2006, and N. ikiensis Shih et al. 2017, as well as one invasive species from China, N. davidi (Bouvier 2004), have been reported (Cai 1996; Cai and Shokita 2006; Kamita 1970; Mitsugi et al. 2017; Shih et al. 2017). Although N. denticulata, which is widely distributed in western Japan, used to inhabit Lake Biwa, it has been considered extinct since there has been no record of its presence since Annandale’s survey in 1915 (Annandale 1922; Kemp 1918; Nishino 2020b). Since the 2000s, Neocaridina shrimps have been found in various parts of the lake, and they are suspected to be an invasive species (Nishino 2017). However, identification based on genetic and quantitative morphological surveys and comparisons with native Neocaridina populations have not yet been carried out in Lake Biwa. If the native population has been replaced by invasive species, then the secondary spread of invasive shrimp via Lake Biwa may occur by mixing with fish and crustacean stocks. Clarifying the actual state of the Neocaridina population in the lake is, therefore, important for understanding and conserving the ecosystem in the region, as well as for assessing the risk of secondary spread of invasive species using Lake Biwa as a hub.

In this study, we conducted the following to verify whether the native Neocaridina population in and around Lake Biwa has been replaced by non-native species: 1) population structure estimation based on genome-wide SNPs using region-wide samples, 2) quantitative morphological comparison between clusters detected by genetic analysis, and 3) species identification and native/non-native population determination based on the genetic and morphological analysis. This study provides necessary insights for conserving native populations and managing invasive species. Furthermore, our statistical evaluation of multiple morphological traits for genetic cluster discrimination and the provision of reference information for genetic analysis will contribute to solve alien species problems of this taxon.

Sampling shrimps

Sampling was conducted from July 2019 to November 2021 at 19 sites in and around Lake Biwa, Japan (Fig. 1; Table 1; Online Resource, Table S1). Two sites, St. 05 and St. 06, were located on the east shore of Lake Biwa, 13 sites, St. 01 − 04 and St. 07 − 15, were located in rivers flowing into the lake, and the remaining four sites, St. 16 − 19, were located in the Yodo River system downstream of the lake (Fig. 1). The specimens were collected using a hand-net. All specimens were anesthetized on ice and stored in 70–99% ethanol. All specimens used in this study were deposited in the Zoological Collection of Kyoto University (KUZ) (Table 1). Since some diagnostic characters of Neocaridina are male traits, 111 adult males (five to nine per site) were used for analysis (Table 1; Online Resource, Table S1). Sex was determined by the shape of the endopod of the 1st pleopod and the appendix masculina of the 2nd pleopod (Kubo 1938).

Table 1

List of *Neocaridina* samples used in this study
Site	Species	Locality	Latitude, longitude	Date	N	Specimen ID	mtDNA accession number	mtDNA haplotype
St. 01	N. denticulata	Toru River	35.133452 N, 135.885919 E	Jan-17-2021	5	KUZ Z3821	LC699728–LC699732	dt05, 06, 07
St. 02	N. davidi	Wadauchi River	35.298455 N, 136.020433 E	Jan-17-2021	5	KUZ Z3822	LC699770–LC699774	dvA01, 02, 05, dvB04
St. 03	N. denticulata	Chinai River	35.494733 N, 136.055203 E	Apr-15-2021	9	KUZ Z4010	LC699682–LC699690	dt03
St. 04	N. davidi	Yogo River	35.481708 N, 136.209194 E	Apr-10-2021	5	KUZ Z4008	LC699788–LC699792	dvB01, 05
St. 05	N. davidi	Lake Biwa	35.444170 N, 136.190366 E	Apr-10-2021	5	KUZ Z4009	LC699723–LC699727	dvA01, 05, dvB04
St. 06	N. davidi	Lake Biwa	35.348536 N, 136.277314 E	Jul-24-2019	5	KUZ Z3816	LC699718–LC699722	dvA01, 02, 05, 06
St. 07	N. spp.	Seri River	35.254116 N, 136.261098 E	Oct-28-2021	7	KUZ Z4014	LC699738–LC699744	dt04, dvA01, 03, 05, dvB01
St. 08	N. denticulata	Uso River	35.167673 N, 136.234434 E	Oct-28-2021	6	KUZ Z4015	LC699764–LC699769	dt03
St. 09	N. davidi	Sakura River	35.057908 N, 136.187638 E	Jun-21-2020	5	KUZ Z3819	LC699701–LC699705	dvB01, 02, 08, 09
St. 10	N. davidi	Shiratori River	35.127494 N, 136.083379 E	Nov-6-2021	5	KUZ Z4020	LC699745–LC699749	dvB02, 06
St. 11	N. davidi	Doji River	35.091781 N, 136.028706 E	Nov-6-2021	5	KUZ Z4019	LC699696–LC699700	dvB01, 03, 10
St. 12	N. denticulata	Yasu River	34.945510 N, 136.228728 E	Nov-5-2021	8	KUZ Z4016	LC699780–LC699787	dt03, 05, 06, 09, 10
St. 13	N. davidi	Oyama River	35.039339 N, 136.034966 E	Jul-25-2019	5	KUZ Z3817	LC699775–LC699779	dvB03
St. 14	N. spp.	Kusatsu River	35.005933 N, 135.985086 E	Nov-6-2021	7	KUZ Z4018	LC699711–LC699717	dt04, dvA04, dvB06, 11, 12, dvC01
St. 15	N. denticulata	Kusatsu River	35.012549 N, 135.947124 E	Apr-19-2021	5	KUZ Z4011	LC699706–LC699710	dt01, 04, 05, 08
St. 16	N. spp.	Senjo River	34.948170 N, 135.886881 E	Nov-6-2021	5	KUZ Z4017	LC699733–LC699737	dt04, dvB07
St. 17	N. denticulata	Umakado River	34.922611 N, 136.074531 E	Mar-9-2020	5	KUZ Z4006	LC699691–LC699695	dt05
St. 18	N. denticulata	Tawara River	34.854037 N, 135.862465 E	Oct-16-2021	7	KUZ Z4013	LC699757–LC699763	dt02
St. 19	N. denticulata	Takano River	35.108909 N, 135.820908 E	Feb-11-2021	7	KUZ Z4007	LC699750–LC699756	dt01
KUZ	N. denticulata	Hikone, Shiga	—	Apr-12-1915	1	KUZ Ar 081 − 2	—	—

To confirm the morphological characteristics of the native population in Lake Biwa, we examined one male specimen among the N. denticulata specimens collected in Hikone (east shore of the lake; near St. 06) in 1915 and housed in the KUZ (KUZ Ar 081 − 2) prior to the invasion of non-native Neocaridina species. These specimens were found by us mixed in a specimen jar of Palaemon paucidens (KUZ Ar 081 − 1). In addition, we refer to the description of Kemp (1918), who reported N. denticulata from the Hikone and Seta Rivers (the outflow river of Lake Biwa; near St. 16).

DNA extraction and MIG-seq library sequencing

Total DNA was extracted from the 5th pereiopod and/or the 4th and/or 5th pleopod of 111 male specimens using the Genomic DNA Purification Kit (Promega, Madison, WI, USA) or the Monarch Genomic DNA Purification Kit (New England BioLabs, Ipswich, MA, USA). To obtain genome-wide SNPs, we used the multiplexed ISSR genotyping method (MIG-seq). We followed Watanabe et al. (2020) and Suyama et al. (2021) for MIG-seq library preparation but changed the sequencing platform from MiSeq to NovaSeq and used a newly designed primer set for the forward side to improve data volume and cost performance (Online Resource, Table S2). Pooling libraries were prepared, and 150 bp paired-end sequencing by NovaSeq was outsourced to Novogene. The raw MIG-seq data were deposited in DDBJ Sequence Read Archive (DRA) (accession number: DRA013666).

MIG-seq analysis

The raw-data obtained were demultiplexed using the process_shortread in Stacks 2.49 (Rochette et al. 2019). The adapter sequences and low-quality bases (< Q30) were removed using fastp 0.20.1 (Chen et al. 2018). Since the read lengths of Read 1 and Read 2 were different in our method, the read length was unified to 109 bp to match the shorter Read 1 in the downstream analysis. SNP calling was performed on these reads using the Denovo_map.pl pipeline of Stacks with paired-end mode. The minimum depth of coverage (m) was set to five, and the maximum allowable number of substitutions between stacks (M) was set to three. When calling SNPs by populations in Stacks, only one SNP from a locus (--write-single-snp), common to more than 50% of all samples (R = 0.5), was output. We excluded SNPs with heterozygosity greater than 75% (--max-obs-het = 0.75) and minor alleles found only in two or fewer individuals (--min-mac = 2). All other parameters were set to the default setting.

To estimate population structure based on SNPs data, we conducted principal component analysis (PCA) using GenoDive 3.0.4 (Meirmans 2020). To estimate individual admixture proportions, we used the likelihood model-based program ADMIXTURE 1.3.0 (Alexander et al. 2009). We performed unsupervised clustering at each number of genetic populations (K) from 1 to 10 with a convergence criterion set to 10^− 4 (C = 0.0001). These analyses were repeated 100 times, and the optimal K value was estimated based on the lowest cross-validation error value calculated by ADMIXTURE for each K. Summary of cross-validation error values and visualization of the output data were performed in R 4.1.1 (R Core Team 2021).

Mitochondrial DNA analysis

The partial COI gene was amplified by PCR using the primer pairs LCO1-1490 (5′-GGT CAA CAA ATC ATA AAG ATA TTG G-3′) and HCO1-2198 (5′-TAA ACT TCA GGG TGA CCA AAA AAT CA-3′) (Folmer et al. 1994) or LCO-nc (5′-TTG TCA ACA AAC CAC AAA GAY ATT GG-3′) and HCO-nc (5′-ATA CYT CAG GGT GYC CAA AGA ATC A-3′) (designed for Neocaridina species; present study) under the following conditions: initial denaturation (94°C, 2 min); 30 cycles of denaturation (94°C, 30 s), annealing (52°C, 30 s), extension (72°C, 60 s), and a single final extension (72°C, 7 min). The PCR products were purified using Illustra ExoStar (GE Healthcare Japan, Tokyo, Japan) and sequenced on an ABI 3130xl Genetic Analyzer (Applied Biosystems, Waltham, USA) using amplification primers and the BigDye Terminator Cycle Sequencing FS Ready Reaction Kit v. 3.1 (Applied Biosystems). The DNA sequences were deposited in DDBJ/EMBL/GenBank (accession numbers: LC699682–LC699792).

The sequences obtained were checked for quality and edited using the mapping function of Unipro UGENE 40.1 (Okonechnikov et al. 2012). Multiple alignments of the sequences were performed using MAFFT 7.475 (Katoh and Standley 2013) on Unipro UGENE. COI haplotypes were detected using POPART 1.7 (Leigh and Bryant 2015). To estimate phylogenetic relationships of detected haplotypes, a maximum likelihood tree was estimated using IQ-TREE 2.1.3 (Minh et al. 2020) based on the HKY + F + G4 model selected by ModelFinder (Kalyaanamoorthy et al. 2017) with 1,000 times ultrafast-bootstrapping (Hoang et al. 2018) and SH-like approximate likelihood ratio test (Guindon et al. 2010), and an approximate Bayes test (Anisimova et al. 2011).

Morphometric analysis

The 111 males used in the genetic analysis and one male collected in 1915 were measured for postorbital carapace length (CL) and the following five diagnostic characters of Neocaridina: 1) rostrum length (RL), 2) number of dorsal rostrum teeth (RT), 3) curvature of propodus of 3rd pereopod (TP), 4) aspect ratio of the endopod of the 1st pleopod (AE), and 5) excavation of carpus of 1st pereopod (FP) (Fig. 2, left column; Kemp 1918; Kubo 1938; Liang 2004; Klotz et al. 2013; Mitsugi et al. 2017). The CL measurements were performed under an optical microscope using a digital caliper. The RL, RT, TP, AE, and FP were confirmed by taking and observing micrographs using ImageJ (Schneider et al. 2012). Following Klotz et al. (2013), the FP was classified into three categories: strongly excavated, excavated, and slightly excavated.

Photographs of the 111 male specimens are registered in the ffish.asia (https://ffish.asia), biodiversity database of freshwater fish and freshwater organisms in Asia (Watanabe et al. 2010; Kano et al. 2013) and can be referred to using the ID of NeocaridinaBIWA (https://ffish.asia/NeocaridinaBIWA).

Quantitative morphological comparison between genetic clusters

The five diagnostic characters (ratio of RL to CL [RL/CL], FP, RT, TP, AE) were compared among genetic clusters. All statistical analyses were performed using R 4.1.1 (R Core Team 2021). The comparisons were based on the results of ADMIXTURE analysis with K = 3, the best K. Individuals with any one of the ancestry proportions ≥ 95% were used. For FP, Fisher’s exact probability test was performed on the ratio of the above three categories of excavation. The p-values were adjusted using the Holm method. For all the other characters, data were tested for normality and homogeneity of variances using Shapiro-Wilk test and Levene’s test implemented in the R package “car” (Fox and Weisberg 2019), respectively. Significant differences between populations were estimated using the Kruskal-Wallis test, followed by the Steel-Dwass test implemented in the R package “NSM3” (pSDCFlig function; method = "Monte Carlo"; seed was set to 0) (Schneider et al. 2021). Statistical significance was set at p < 0.05. Based on the results of the above analysis and the trait values of the sample collected in 1915, the species of each genetic cluster were identified and the native and non-native populations were distinguished.

To examine which of the five diagnostic traits, or combination of traits, better distinguishes between native and non-native populations, a generalized linear mixed model (GLMM; distribution: binomial, link function: logit) was constructed using the R package “lme4” (Bates et al. 2015). The response variable was the native ancestry proportion. The explanatory variables were the trait value of the five characters. Sampling sites were included as random effects. All the subsets of the models based on the global model were produced and ranked based on Akaike’s information criterion (AIC). The best performing models with ΔAIC < 2 were selected.

Genetic population structure

The results of PCA based on 183 SNPs showed that the 111 male Neocaridina shrimps formed three clusters (Fig. 3b). Exceptionally, the individuals collected from St. 14 were located in the middle of the three clusters. The ADMIXTURE analysis evaluated the optimal number of clusters to be three (Fig. 3a; henceforth referred to as cluster 1–3). At most sites, all individuals were estimated to have originated from almost a single cluster, although at St. 07, 14, and 16, some individuals were estimated to have originated from multiple clusters (Fig. 3a). The three PCA clusters corresponded well with clusters 1, 2, and 3 in the ADMIXTURE results (Fig. 3b).

A total of 29 haplotypes were detected in the 596 bp mtDNA COI region of 111 individuals (Table 1; Fig. 3c). Phylogenetic analysis showed that the Neocaridina shrimps in Lake Biwa consist of four clades (Fig. 3c). The most divergent clade 1 consisted of all individuals from St. 01, 03, 08, 12, 15, 17–19 and some from St. 07, 14, 16, which corresponded to SNP cluster 1 (Fig. 3a). Clade 3, consisting of all individuals from St. 06 and some from St. 02, 05, and 07, was sister to clade 4, consisting of only one individual from St. 14, and generally corresponded to SNP clusters 2 and 3, respectively. Clade 2, the sister clade to these two clades, consisted of all individuals from St. 04, 09–11, and 13 and some individuals from St. 02, 05, 14, and 16, and generally corresponded to SNP cluster 3. In St. 07, clades 1 and 2 were present; in St. 16, clades 1 and 3 were present; in St. 02 and 05, clades 2 and 3 were present; and in St. 14, all clades were present.

Morphological comparison between genetic clusters

The trait values of the five diagnostic characters differed generally corresponding to the three clusters of PCA using SNPs (Fig. 2, middle column). The upper-left cluster was characterized by large RL/CL, slightly excavated FP, small RT, and large AE; the lower-middle cluster by small RL/CL, strongly excavated FP, small RT, and small AE; and the upper-right cluster by small RL/CL, strongly excavated FP, large RT, and small AE. TP was not clearly different among the clusters (Fig. 2).

The same trend was observed in the comparisons using individuals with ancestry proportions of ≥ 95% for any genetic clusters in the ADMIXTURE analysis (Fig. 2, right column). No homogeneity of variances was found for the TP (Levene’s test, p = 0.041), and no normality was found for the TP of cluster 1 (Shapiro-Wilk test, p = 0.002). The proportions of the three categories of FP were significantly different between cluster 1 and the other two clusters (Fisher’s exact probability test, 1 vs. 2: p = 0.001, 1 vs. 3: p = 0.02, respectively; Fig. 2e). In cluster 1, there were more slightly excavated individuals than in clusters 2 and 3. For the other four diagnostic characters, significant differences were detected in RL/CL, RT, and AE among clusters (Kruskal-Wallis test, p < 0.001, = 0.002, = 0.001, respectively); no significant difference was found for TP (Fig. 2c). Compared to individuals in the cluster 2 and 3, individuals in the cluster 1 had larger RL/CL (Steel-Dwass test, 1 vs. 2: p < 0.001, 1 vs. 3: p < 0.001; Fig. 2a) and larger AE (Steel-Dwass test, 1 vs. 2: p = 0.003, 1 vs. 3: p = 0.027; Fig. 2d). Compared to individuals in the clusters 1 and 2, individuals in cluster 3 had more RT (Steel-Dwass test, 1 vs. 3: p = 0.002, 2 vs. 3: p = 0.015; Fig. 2b). The diagnostic characters of male specimens from the KUZ collection were all within the range of trait values of the three clusters, although RL/CL was particularly close to cluster 2 (0.65), RT to clusters 1 and 2 (14), TP to cluster 1 (5.55), and AE to cluster 1 (1.83) (Fig. 2).

Among the GLMMs that explained ancestry proportion in cluster 1, which was considered to be the native population (see below), with four diagnostic characters, three models were selected with ΔAIC < 2 (Table 2). All selected models included multiple diagnostic characters as explanatory variables; all models included RL/CL and RT, the best and 3rd best models included AE, and the 3rd best model included TP. In the best model, the coefficient of RL/CL was significantly positive (18.84; Pr(>|z|) = 0.007), the coefficient of RT was significantly negative (− 0.51; Pr(>|z|) = 0.025), and the coefficient of AE was positive, though not significant (4.92; Pr(>|z|) = 0.056).

Table 2

Parameter coefficients for the *Neocaridina denticulata* (cluster 1) ancestry proportion estimated with the three GLMM models selected by ΔAIC < 2
	Models
Explanatory variables	Best			2nd			3rd
Explanatory variables	Estimate	Pr(>\|z\|)		Estimate	Pr(>\|z\|)		Estimate	Pr(>\|z\|)
RL/CL	18.84	0.007	**	16.64	0.020	*	18.07	0.010	*
RT	−0.51	0.025	*	−0.42	0.057		−0.50	0.029	*
TP							−0.06	0.663
AE	4.92	0.056					4.44	0.108
FP
(Intercept)	−14.03			−5.82			−12.26
AIC	69.1			70.7			71.0
ΔAIC	0.00			1.54			1.88
RL/CL: proportion of rostrum length to carapace length, RT: number of dorsal rostrum teeth, TP: curvature of propodus of 3rd pereopod, AE: aspect ratio of the endopod of the 1st pleopod, FP: excavation of carpus of 1st pereopod. * p-value < 0.05, ** p-value < 0.01.

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.

Data availability

Raw MIG-seq data are available at DDBJ Sequence Read Archive (DRA) with Accession Number DRA013666. The mtDNA COI sequences are available at DDBJ/EMBL/GenBank with Accession Numbers LC699682–LC699792.

Acknowledgments

We are grateful to Mr. S. Kunimatsu and Mr. M. Asano for providing valuable specimens; Dr. T. Nakano for registering the specimens and supporting the research in KUZ; Dr. S. Matsui for supporting the MIG-seq primer selection process; Dr. Y. Kano for registering the photographs to the database; Dr. K. Kakui and Dr. T. Komai for letting us use their beautiful illustrations; Dr. K. Watanabe for giving helpful comments to our manuscript.

Funding

This work was partly supported by Grant-in-Aid for JSPS Research Fellow Grant Number JP19J23130 to Yusuke Fuke.

Competing Interests

There are no conflicts of interest to declare.

Author Contributions

All authors contributed to the study conception and design, sampling, morphometry, and manuscript preparation. All statistical analyses were performed by Keisuke Onuki. Primer development and genetic analysis were performed by Yusuke Fuke. All authors read and approved the final manuscript.

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No competing interests reported.

TableS1.xlsx
Supplemental Table 1 Measurements of morphological characters of Neocaridina shrimps used in the study
TableS2.xlsx
Supplemental Table 2 List of primer sequences used to prepare the MIG-seq library. Bold and underlined bases in the primer sequences represent the index sequences

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Rediscovery of a native freshwater shrimp, Neocaridina denticulata, and expansion of an invasive species in Lake Biwa, Japan: Genetic and morphological approach

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Abstract

Figures

Introduction

Materials And Methods

Sampling shrimps

DNA extraction and MIG-seq library sequencing

MIG-seq analysis

Mitochondrial DNA analysis

Morphometric analysis

Quantitative morphological comparison between genetic clusters

Results

Genetic population structure

Morphological comparison between genetic clusters

Discussion

Evaluation of diagnostic characters between native and non-native species

Importance of conservation of native population and preventing secondary spread of invasive species

Declarations

References

Additional Declarations

Supplementary Files

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