Integrative Taxonomy Reveals Freshwater Shrimp Diversity (Decapoda: Atyidae: Neocaridina) from Kyushu and Southern Honshu of Japan, with a Discussion on Introduced Species

© 2024 Academia Sinica, Taiwan Open Access Integrative Taxonomy Reveals Freshwater Shrimp Diversity (Decapoda: Atyidae: Neocaridina) from Kyushu and Southern Honshu of Japan, with a Discussion on Introduced Species Hsi-Te Shih1,*,§ , Yixiong Cai2,§ , Nobuaki Niwa3,†, Hidenori Yoshigou4, and Yasuhiko Nakahara5 1Department of Life Science and Research Center for Global Change Biology, National Chung Hsing University, 250, Kuo Kuang Road, Taichung 402, Taiwan. *Correspondence: E-mail: [email protected] (Shih) 2National Biodiversity Centre, National Parks Board, 1 Cluny Road, Singapore 259569, Republic of Singapore. E-mail: [email protected].sg (Cai) 3Faculty of Science, Kyoto University, Kitashirakawa, Oiwake-cho, Sakyo-ku, Kyoto 606-8502, Japan 4Chugai Technos Co. LTD, 9-12, Yokogawa-shin-machi, Nishi-ku, Hiroshima City 733-0013, Japan. E-mail: [email protected] (Yoshigou) 5SEIBU Environmental Research Co., LTD, Mikawachishin-machi, Sasebo-City, Nagasaki 859-3153, Japan. E-mail: [email protected] (Nakahara) †Deceased. §HTS and YC contributed equally to this paper. Received 3 April 2023 / Accepted 14 March 2024 / Published 9 July 2024 Communicated by Benny K.K. Chan Correct identification of species is crucial for invasion ecology and management, particularly in aquatic systems. In this study, specimens of the freshwater shrimp genus Neocaridina from Kyushu and southern Honshu of Japan were identified by using an integrative approach that combined DNA barcoding of mitochondrial cytochrome oxidase subunit I (COI) and morphological examination. Among the eight species detected, two are native, viz. N. denticulata and N. ikiensis. Four are regarded as non-indigenous, viz. N. davidi, N. koreana, N. palmata, N. aff. palmata, which are believed to have been introduced from other East Asian countries either by the aquarium trade or as live fish bait. The remaining two species are likely cryptic native species, which have either been mistaken for known species, e.g., N. aff. denticulata, or species that have not been discovered before, e.g., N. aff. fukiensis. While the four alien species have spread widely in central Honshu, northern Kyushu and Tsushima Island, their impacts on the native species and the overall ecology remain mostly unexplored. Problems associated with using DNA barcoding for species identification are highlighted for further research. Key words: Japan, DNA barcoding, Cytochrome oxidase subunit I (COI), Morphology, Integrative taxonomy, Neocaridina denticulata, N. davidi, N. ikiensis, N. koreana, N. palmata BACKGROUND The impact of invasive species on ecology has been discussed extensively (Ehrenfeld 2010; Simberloff et al. 2013) and it has been suggested that the invasive pathways differ between terrestrial and aquatic ecosystems (Ehrenfeld 2010). Compared with terrestrial and marine systems, freshwater systems are more sensitive to introduced species due to the stronger impacts of introduced primary consumers on plant biomass, and a higher proportion of high-impact invaders. Additionally, freshwater systems exhibit similar degrees of extirpation of native species from either intraor inter-continental invaders (Ricciardi and MacIsaac 2011). Correct identification of species is important Citation: Shih HT, Cai Y, Niwa N, Yoshigou H, Nakahara Y. 2024. Integrative taxonomy reveals freshwater shrimp diversity (Decapoda: Atyidae: Neocaridina) from Kyushu and southern Honshu of Japan, with a discussion on introduced species. Zool Stud 63:18. doi:10.6620/ZS.2024.63-18. Zoological Studies 63:18 (2024) doi:10.6620/ZS.2024.63-18 1 © 2024 Academia Sinica, Taiwan for invasion ecology and biodiversity management (Darling and Blum 2007; Briski et al. 2016). In aquatic systems, species identification can be challenging due to difficult survey conditions, or because the life stages of organisms are poorly understood or not amenable to traditional identification methods. As a result, molecular detection methods using environmental samples often prove more successful (Darling and Mahon 2011; Darling 2015). DNA barcoding using the mitochondrial cytochrome c oxidase subunit I (COI) provides a reliable solution for species identification across all life stages (e.g., eggs, larvae, and juveniles), sexually dimorphic species, and species with large morphological variations within or between populations (Hebert et al. 2003a b; Radulovici et al. 2010). DNA barcoding has been recommended as an effective tool to identify species correctly as a basis for invasion ecology research (Darling and Blum 2007; Briski et al. 2016). The genus Neocaridina Kubo, 1938, native to freshwater habitats in East Asia, consists of over 30 recorded species (Liang 2004; De Grave and Fransen 2011; Shih et al. 2017 2019; Chen et al. 2018). While the species of both Caridina and Macrobrachium are either catadromous or land-locked, all known species of Neocaridina are land-locked with large eggs and abbreviated larval development (Shih and Cai 2007). Some species of atyid shrimps are popular in the aquarium trade due to their distinctive colors and land-locked habits. For example, Neocaridina davidi (Bouvier, 1904), with its varied coloration of red, yellow, blue, and black, is traded under names such as the Red Cherry Shrimp, Cherry Shrimp, and Fire Shrimp. Due to this trade, they have been introduced into regions like Hawaii, Japan, and Europe (Englund and Cai 1999; Klotz et al. 2013; Toyota et al. 2014; Hasegawa et al. 2015; Mitsugi et al. 2017; Shih et al. 2017; Jabłońska et al. 2018; Schoolmann and Arndt 2018). Species of Neocaridina are believed to have been imported into Japan from Korea since 1969 and from China since 1990 as live fish bait (Niwa 2010; Niwa and Ohtaka 2006; Shih et al. 2017). The first report of non-indigenous species of Neocaridina in Japan was in 2004, and they have since become widespread, ranging from Hokkaido to the Ryukyu Islands (e.g., Nishino and Niwa 2004; Niwa 2010; Fujita et al. 2011; Nishino 2017 2020; Fuke et al. 2021; Nagai and Imai 2021; Onuki 2021; Kakui and Komai 2022; Onuki and Fuke 2022). Niwa (2010 2017) suggested that more than one species of Neocaridina has been introduced from China and Korea to Japan. Morphological differences among Neocaridina species are subtle, with some species showing variations that complicate identification (Cai 1996; Liang 2004), especially among female specimens. Consequently, molecular evidence has been increasingly employed for reliable species identification (Shih and Cai 2007; Shih et al. 2017 2019). Recent findings suggest that the species diversity of Neocaridina in the main islands of Japan exceeds prior estimates, encompassing native N. denticulata (De Haan, 1844) and N. ikiensis Shih, Cai, Niwa & Nakahara, 2017, and probably introduced species (Shih et al. 2017). In this study, we examine the species of Neocaridina from Kyushu and southern Honshu of Japan using an integrative approach combining DNA barcoding and morphology (Dayrat 2005). We also discuss species-level taxonomy and highlight problems associated with DNA barcoding for species identification. MATERIALS AND METHODS Specimens of the genus Neocaridina examined in this study were collected from the Kyushu and southern Honshu of Japan (Tables 1, 3; Fig. 1), and were preserved in 70–95% ethanol after collection. Specimens with typical characters were selected for illustration, using a drawing tube attached to a Nikon stereo microscope (model SMZ 1000). All specimens were subsequently deposited in the Zoological Collections of the Department of Life Science, National Chung Hsing University, Taichung, Taiwan (NCHUZOOL); the Department of Environmental Biology and Fisheries Science, National Taiwan Ocean University, Keelung, Taiwan (NTOU); and the Zoological Reference Collection of the Lee Kong Chian Natural History Museum, National University of Singapore, Singapore (ZRC). The carapace length is abbreviated as “cl”. Following the recognition of several additional clades of Neocaridina from the main islands of Japan (Shih et al. 2017; Fuke et al. 2021), conflicting opinions have emerged regarding the valid names for these clades (e.g., Fuke et al. 2021; Nagai and Imai 2021; Kakui and Komai 2022). In this study, we follow most of the names used by Shih and Cai (2007) and Shih et al. (2017 2019). To confirm the species of Neocaridina collected from Japan, the main morphological characters of representative specimens from each species were compared with either other specimens or taxonomic references from China, Japan, and Taiwan, including Cai (1996), Liang (2004), Shih and Cai (2007), and Shih et al. (2017 2019). Genomic DNA was isolated from the muscle tissue of the abdomen by using the GeneMark tissue and cell genomic DNA purification kit (Taichung, Taiwan). A portion of the COI gene was amplified with PCR using the primers LCO1490 and HCO2198 page 2 of 30Zoological Studies 63:18 (2024) © 2024 Academia Sinica, Taiwan KOREA CHINA Honshu Kyushu 15 8,9 Lake Biwa 1 2 3 4 75 6 10 17,18 16 14 12,13 11 19 20 21 22 Gifu Hyogo Okayama Hiroshima Oita JAPAN (Folmer et al. 1994). The PCR conditions for these primers were denaturation for 50 s at 94°C, annealing for 70 s at 45–47°C, and extension for 60 s at 72°C (40 cycles), followed by extension for 10 min at 72°C. Sequences were obtained by automated sequencing (Applied Biosystems 3730), after verification with the complementary strand, with a 658-bp (base pair) segment of COI. Sequences of the different haplotypes have been deposited in the DNA Data Bank of Japan (DDBJ), along with other sequences published in Shih and Cai (2007) and Shih et al. (2017 2019) (Table 1). A neighbor-joining tree was constructed with the Fig. 1. Collection sites (nos. 1–18, black circles) for species of Neocaridina in the main islands of Japan (Honshu and Kyushu). For locality names and haplotypes, see table 1. Nos. 19–21 (gray circles) are the additional localities in Fuke et al. (2021). Table 1. New COI haplotypes of specimens of Neocaridina species collected from the main islands of Japan and other localities used in this study. Those from main islands of Japan in Shih et al. (2017; marked with “*”) are also included here. Numbers within brackets correspond to Japanese localities in figure 1. Accession numbers with identical haplotypes are parenthesized Species Locality Catalogue no. of NCHUZOOL (unless indicated) Sample size Japanese sample size Accession no. Haplotypes (for new sequences) N. davidi China: Qingdao, Shandong NTOU 20641 2 0 PP386817; PP386818 Ndv-1 China: Putian, Fujian 13342 6 0 AB563166* China: Jiangle, Fujian Japan: Himeji City, Hyogo [2] 13347; 14941; ZRC 2023.0214 4 3 AB563171*; PP386819– PP386821 Ndv-2 Japan: Awaji Island, Hyogo [3] 14943 1 1 PP386822 Ndv-3 Japan: Izumo City, Shimane [8] 14955 1 1 LC324766* page 3 of 30Zoological Studies 63:18 (2024) © 2024 Academia Sinica, Taiwan Species Locality Catalogue no. of NCHUZOOL (unless indicated) Sample size Japanese sample size Accession no. Haplotypes (for new sequences) Japan: Hiroshima City, Hiroshima [12] 14961 1 1 LC324767* Japan: Ube City, Yamaguchi [14] 14952 1 1 PP386823 Ndv-4 Japan: Ube City, Yamaguchi [14] 14952 1 1 PP386824 Ndv-5 N. denticulata Japan: Mizunami City, Gifu [1]; Awaji Island, Hyogo [3]; Higashihiroshima City, Hiroshima [11] 14944, 14942, 14958 3 3 LC324765* Akaiwa City, Okayama [4]; Ihara City, Okayama [6] 14948, 14949, 14968 3 3 LC324764* Japan: Ihara City, Okayama [6]; Fukuyama City, Hiroshima [7]; Ube City, Yamaguchi [14] 14946; 14953; 14968; 14969; ZRC 2023.0211 6 6 PP386825– PP386830 Ndt-1 Japan: Ihara City, Okayama [6]; Fukuyama City, Hiroshima [7]; Hiroshima City, Hiroshima [12]; Ube City, Yamaguchi [14] China: Pingyuan, Yunnan; Taihu Lake, Jiangsu 14947; 14953; 14961; 14969; 15168 7 4 PP386831– PP386837 Ndt-2 Japan: Izumo City, Shimane [9] 14956 1 1 PP386838 Ndt-3 Japan: Miyoshi City, Hiroshima [10] 14965 2 2 PP386839; PP386840 Ndt-4 Japan: Miyoshi City, Hiroshima [10] 14964 1 1 PP386841 Ndt-5 Japan: Higashihiroshima City, Hiroshima [11] 14945 1 1 PP386842 Ndt-6 Japan: Hiroshima City, Hiroshima [13] 14966 1 1 PP386843 Ndt-7 Japan: Hiroshima City, Hiroshima [12, 13] 14960; 14967 3 3 PP386844– PP386846 Ndt-8 Japan: Hiroshima City, Hiroshima [12] ZRC 2024.0055 2 2 PP386847 Ndt-9 China: Taihu Lake, Jiangsu NCHUZOOL 15264 1 0 PP386848 Ndt-10 N. ikiensis Japan: Iki Island, Nagasaki [16] 14935 2 2 LC324771* Japan: Iki Island, Nagasaki [16] 14933 (paratype); 14932 (paratype) 2 2 LC324772* Japan: Iki Island, Nagasaki [16] 14937; 14936 4 4 LC324773* Japan: Iki Island, Nagasaki [16] 14933 (paratype) 1 1 LC324774* Japan: Iki Island, Nagasaki [16] 14934; 14932 (paratype) 3 3 LC324775* N. koreana Japan: Miyoshi City, Hiroshima [10] 14964; 14965 2 2 LC324777* N. palmata China: Hanzhong, Shaanxi NCHUZOOL 15265 2 0 PP386849; PP386850 Npm-1 China: Nanping, Fujian NCHUZOOL 15266 1 0 PP386851 Npm-2 China: Changting, Fujian Japan: Hyogo [2] 15269; 14940 2 1 PP386852; PP386853 Npm-3 China: Leye, Guangxi Vietnam: Hoa An, Cao Bang; Thanh Thuy, Vi Xuyen, Hai Giang; Hai Gang Japan: Himeji City, Hyogo [2] 14975; 15174; 15175; 15176; 14940 5 1 LC324770*; PP386854– PP386857 Npm-4 N. aff. denticulata Japan: Mizunami City, Gifu [1] 14959 1 1 PP386858 Nad-1 Japan: Himeji City, Hyogo [2] 14940; ZRC 2023.0221 4 4 PP386859; PP386860– PP386862 Nad-2 Japan: Himeji City, Hyogo [2] ZRC 2023.0221 1 1 PP386863 Nad-3 Japan: Himeji City, Hyogo [2] 14940; 14941 2 2 PP386864; PP386865 Nad-4 Japan: Okayama City, Okayama [5] 14950; 14951 4 4 LC324778* Japan: Izumo City, Shimane [9]; Sasebo City, Nagasaki [15]; Tsushima Island, Nagasaki [17, 18] 14957; 14962, 14963; 14971; 14970 11 11 LC324779*; PP386866– PP386875 Nad-5 N. aff. fukiensis Japan: Fukuyama City, Hiroshima [7] 14947; 14946; ZRC 2023.0211 5 5 LC324776* N. aff. palmata Japan: Himeji City, Hyogo [2] 14941 1 1 PP386876 Nap-1 China: Yunnan Japan: Himeji City, Hyogo [2] 15170; 15267; 14941; 14940 5 2 PP386877– PP386881 Nap-2 China: Fujian: Jiangle; Nanping 15169; 15172 2 0 PP386882; PP386883 Nap-3 Japan: Izumo City, Shimane [8] 14954 1 1 PP386884 Nap-4 Japan: Miyoshi City, Hiroshima [10] 14965 1 1 PP386885 Nap-5 Total 128 85 Table 1. (Continued) page 4 of 30Zoological Studies 63:18 (2024) © 2024 Academia Sinica, Taiwan program MEGA (vers. 11, Tamura et al. 2021), with Kimura 2-parameter (K2P) distance (Kimura 1980) and 2000 bootstrap reiterations. Other analyses, including the nucleotide composition, variable and parsimony informative positions, and K2P distance between haplotypes were also calculated using MEGA. COI sequences of Neocaridina, obtained from various studies conducted in Japan (Fuke et al. 2021; Nagai and Imai 2021; Kakui and Komai 2022), Taiwan (Han et al. 2019), China (Zhou et al. 2021) and Israel (Levitt-Barmats et al. 2019), as well as unpublished sequences in GenBank (from Canada and Hungary), were incorporated into the analysis if their sequence length was ≥ 495 bp. This inclusion aimed to elucidate the phylogenetic relationships among species within the East Asian Arc and to identify introduced species (Table 2). The best-fitting model for sequence evolution was determined by PartitionFinder (ver. 2.1.1, Lanfear et al. 2017) and selected based on the Bayesian information criterion (BIC). The obtained best model (HKY+I+G) was subsequently employed for Bayesian inference (BI) and maximum likelihood (ML) analyses. BI analysis was performed with MrBayes (ver. 3.2.3, Ronquist et al. 2012), running four chains for 10 million generations across four independent runs, with trees sampled every 1,000 generations. The convergence of chains was determined using the average standard deviation of split frequency values, which remained below the recommended threshold of 0.01 (Ronquist et al. 2020). The first 1200 trees were accordingly discarded as “burn-in”. ML analysis was performed using IQ-TREE (vers. 2.2.0, Minh et al. 2020) with the best model, and 20,000 ultrafast bootstrap replicates were generated (Hoang et al. 2017). As the phylogeny of Neocaridina is still not clear, due to sequences of most species not being available in GenBank, a midpoint rooting method was employed (calculating tip to tip distances and then placing the root midway between the two longest tips) (Kinene et al. 2016). RESULTS Distribution of Japanese Neocaridina species Specimens of eight species of Neocaridina (see “TAXONOMY”) collected from the 18 collection sites in Kyushu and southern Honshu of Japan (Fig. 1), as well as other related species, are shown in table 3. Among these species, N. denticulata was found at 11 out of 18 localities, all in Honshu, sometimes sympatric with other species, i.e., N. davidi, N. koreana, N. aff. fukiensis, N. aff. palmata, and N. aff. denticulata. The second most widespread species was N. aff. denticulata, found at 7 localities in Honshu (4), Kyushu (1), and Tsushima (2). N. davidi, the next most widespread, was found at four localities in Honshu and one on Awaji Island, adjacent to Shikoku. Neocaridina palmata was collected from three localities in Honshu. Neocaridina ikiensis was only found at three localities on Iki Island. Neocaridina koreana, N. palmata, and N. aff. fukiensis, were only collected from one locality each in Honshu. Half of the studied localities supported two or more species living sympatrically (Table 3). For example, Himeji City, Hyogo (Site 2) had four species occurring together, viz. N. davidi, N. palmata, N. aff. palmata, and N. aff. denticulata; Miyoshi City, Hiroshima (Site 10) had three species collected, viz. N. denticulata, N. aff. palmata and N. koreana. While Awaji Island (Site 3) had two species (N. davidi and N. denticulata), only a single species was found on the other two islands under study, viz. Iki Island (Site 16) with N. ikiensis and Tsushima Island (Sites 17, 18) with N. aff. denticulata. Molecular analyses A 658-bp (base pair) segment of COI from 115 specimens representing 12 Neocaridina species (with 84 specimens from Japan) was amplified, resulting in 53 different haplotypes (Table 1). The studied COI segment was AT rich (59.4%) (T, 32.5%; A, 26.8%; G, 19.8%; C, 20.9%). Within this gene fragment, 170 positions were variable and 143 were parsimoniously informative. The neighbor-joining tree, based on 658 bp of COI (Fig. 2), reveals that the Japanese specimens collected in this study belong to eight clades, each corresponding to a specific species: N. davidi, N. denticulata, N. ikiensis, N. aff. palmata, N. palmata, N. aff. fukiensis, N. koreana, and N. aff. denticulata. These species were identified through morphological examination (see “TAXONOMY”). All clades are highly supported, except the N. davidi clade, which is moderately supported. In the COI tree (Fig. 2), N. denticulata and N. davidi appear as sister species, and they, along with N. palmata and N. aff. palmata, form a highly-supported large clade. Neocaridina ikiensis is closely related to N. aff. fukiensis, while N. koreana is sister to N. aff. denticulata. The pairwise nucleotide divergences for COI with K2P distance (and differences in the total bp numbers) are shown in table 4. Most species are well separated from others by a minimum divergence of 3.77% (24 bp difference), except for a smaller divergence of 1.7% (11 bp difference) between N. davidi and N. denticulata. By incorporating sequences from other studies, including those available in GenBank, the phylogeny of Neocaridina from East Asia is shown in figure 3, page 5 of 30Zoological Studies 63:18 (2024) © 2024 Academia Sinica, Taiwan with respective support values from BI and ML. The tree was rooted using midpoint rooting (see “Molecular analyses”), positioning N. spinosa and N. hofendopoda at the basal positions, followed by N. iriomotensis and N. ishigakiensis from the southern Ryukyus. The remaining species form a major group with two large clades. One large clade with high support is composed of four species: two sister species, N. davidi and N. denticulata, and another species pair, N. aff. palmata and N. palmata. Another large clade with moderate support consists of 11 species. Neocaridina sp. 1, N. sp. 2, N. sp. 3 (see Remarks under N. ikiensis), N. aff. fukiensis and N. ikiensis are more closely related, and form two groups, one of which includes N. sp. 1, N. Table 2. Other COI haplotypes of Neocaridina species from GenBank used in figure 3. Accession numbers with identical haplotypes are parenthesized Species Original name (if different) Locality Accession no. Reference N. davidi Taiwan: Sinshe, Taichung; Dounan, Yunlin AB300183 (AB300184) Shih and Cai 2007 Taiwan: Kinmen AB300187 Hawaii: Oahu AB300185; AB300186 N. davidi “N. denticulata” Canada: Big Al's Fish Store MG319788 unpublished “N. denticulata” Hungary MH780819 (MH780820, MH780821) unpublished “N. denticulata” Israel MN336479 Levitt-Barmats et al. 2019 MN336445 (MN336446, MN336449, MN336450, MN336451, MN336452, MN336453, MN336461, MN336473, MN336482, MN336483); MN336447 (MN336448, MN336454, MN336455, MN336456, MN336457, MN336459, MN336460, MN336462, MN336463, MN336465, MN336475, MN336480, MN336481, MN336484) N. denticulata Japan: Biwa Lake AB300191 Shih and Cai 2007 “N. davidi Type II” Japan: Okinawa: Nanjo: Kakinohana spring LC659919 Nagai and Imai 2021 “N. denticulata” Israel: HaHula MN336458 Levitt-Barmats et al. 2019 N. fonticulata Taiwan: Kenting, Pingtung LC427866 Shih et al. 2019 N. hofendopoda China: Hubei: Yichang: Sanxia MN701609 (MN701610) Chen et al. 2020 N. ketagalan Taiwan: Sijhih, New Taipei City AB300180 (AB300181); AB300182 Shih and Cai 2007 N. koreana “N. heteropoda koreana” Korea: Gyeongsangnam: Geojedo MK907783 Park et al. 2019 N. palmata China: Meizhou, Guangdong; Jiangle, Fujian LC324769 Shih et al. 2017 China: Leye, Guangxi LC324770 N. saccam Taiwan: Longci, Tainan; Houjha, Tainan AB300177 (AB300179) Shih and Cai 2007 Taiwan: Longci, Tainan AB300178 N. spinosa China: Tong-an, Fujian AB300188 N. aff. fukiensis “N. denticulata” Japan: Oita: Machida R. LC612372 Fuke et al. 2021 Japan: Oita: Miya R. LC612374 (LC612376, LC612378); LC612379 N. aff. palmata “N. koreana” aquarium in South Korea LC324768 Shih et al. 2017 “N. sp. aff. davidi” Japan: Hokkaido: Sapporo LC664097 (LC664098, LC664099) Kakui and Komai 2022 Japan: Chiba LC664096 N. sp. 1 “N. denticulata” Japan: Kumatoto: Kuro R. LC612358 (LC612359, LC612360, LC612361, LC612362, LC612363); LC612364; LC612357 Fuke et al. 2021 Japan: Nagasaki: Ishiki R. LC612349; LC612352; LC612356 N. sp. 2 “N. denticulata” Japan: Nagasaki: Goto Islands: Fukue-jima LC612339 (LC612340, LC612342, LC612343, LC612344, LC612345, LC612346, LC612348); LC612341 (LC612347) N. sp. 3 “N. denticulata” Japan: Oita: Machida R. LC612365; LC612368; LC612366; LC612371; LC612370 N. sp. 4 “N. sp.” Taiwan: Hualien MG734216; MG734217; MG734218; MG734219; MG734220; MG734221 Han et al. 2019 page 6 of 30Zoological Studies 63:18 (2024) © 2024 Academia Sinica, Taiwan sp. 2 and N. aff. fukiensis, and the other consists of N. ikiensis and N. sp. 3. The remaining six species do not exhibit clear groupings, except for N. aff. denticulata, which is closely related to N. koreana. The sequences of the species introduced to Hawaii, Canada, Hungary and Israel cluster within the same clade as N. davidi. Another species found in common between Okinawa and Israel appears to belong to N. denticulata (but see Remarks under N. denticulata). Some specimens collected from Japan share identical haplotypes with those from China and Vietnam, including N. davidi (NCHUZOOL 14941 Table 3. Geographic positions of the collection sites in the main islands of Japan, with the species of Neocaridina collected in this study Species collected No. on Fig. 1 Prefecture City Latitude Longitude N. davidi N. denticulata N. koreana N. ikiensis 1 Gifu Mizunami City 35°22'03.9"N 137°14'07.6"E * 2 Hyogo Himeji City 34°56'31.9"N 134°38'19.0''E * 3 Hyogo Awaji City (Awaji Island) 34°26'09.8"N 134°53'22.8"E * * 4 Okayama Akaiwa City 34°53'48"N 134°01'05"E * 5 Okayama Okayama City 34°45'47"N 133°55'05"E 6 Okayama Ihara City 34°33'57.4"N 133°28'50.4"E * 7 Hiroshima Fukuyama City 34°36'43"N 133°15'33''E * 8 Shimane Izumo City 35°23'33.3"N 132°43'37.1"E * 9 Shimane Izumo City 35°22'42.9"N 132°50'35.4"E * 10 Hiroshima Miyoshi City 34°46'51"N 132°54'39"E * * 11 Hiroshima Higashihiroshima City 34°23'14"N 132°43'50''E * 12 Hiroshima Hiroshima City 34°29'11"N 132°31'51"E * * 13 Hiroshima Hiroshima City 34°22'53"N 132°38'17"E * 14 Yamaguchi Ube City N34°01'08" 131°14'46"E * * 15 Nagasaki Sasebo City 33°09'45.5"N 129°50'32.9''E 16 Nagasaki Iki City (Iki Island) 33°47'26.7"N 129°42'49.4"E * 17 Nagasaki Tsushima City 34°14'1.9''N 129°17'45.7''E 18 Nagasaki Tsushima City 34°15'32.4''N 129°18' 47.5''E Species collected No. on Fig. 1 Prefecture City Latitude Longitude N. palmata N. aff. denticulata N. aff. fukiensis N. aff. palmata 1 Gifu Mizunami City 35°22'03.9"N 137°14'07.6"E * 2 Hyogo Himeji City 34°56'31.9"N 134°38'19.0''E * * * 3 Hyogo Awaji City (Awaji Island) 34°26'09.8"N 134°53'22.8"E 4 Okayama Akaiwa City 34°53'48"N 134°01'05"E 5 Okayama Okayama City 34°45'47"N 133°55'05"E * 6 Okayama Ihara City 34°33'57.4"N 133°28'50.4"E 7 Hiroshima Fukuyama City 34°36'43"N 133°15'33''E * 8 Shimane Izumo City 35°23'33.3"N 132°43'37.1"E * 9 Shimane Izumo City 35°22'42.9"N 132°50'35.4"E * 10 Hiroshima Miyoshi City 34°46'51"N 132°54'39"E * 11 Hiroshima Higashihiroshima City 34°23'14"N 132°43'50''E 12 Hiroshima Hiroshima City 34°29'11"N 132°31'51"E 13 Hiroshima Hiroshima City 34°22'53"N 132°38'17"E 14 Yamaguchi Ube City 34°01'08"N 131°14'46"E 15 Nagasaki Sasebo City 33°09'45.5"N 129°50'32.9''E * 16 Nagasaki Iki City (Iki Island) 33°47'26.7"N 129°42'49.4"E 17 Nagasaki Tsushima City 34°14'1.9''N 129°17'45.7''E * 18 Nagasaki Tsushima City 34°15'32.4''N 129°18' 47.5''E * page 7 of 30Zoological Studies 63:18 (2024) © 2024 Academia Sinica, Taiwan from Hyogo, Japan; NCHUZOOL 13347 from Fujian, China), N. palmata (NCHUZOOL 14940 from Hyogo, Japan; NCHUZOOL 14975 from Guangxi, China; NCHUZOOL 15174, 15175, 15176 from northern Vietnam) and N. aff. palmata (NCHUZOOL 14940, 14941 from Hyogo, Japan; NCHUZOOL 15170 from Yunnan, China) (Fig. 3). Morphological identification Based on the taxonomic literature for Neocaridina from mainland China, Japan and Taiwan (see “MATERIALS AND METHODS”), five of the eight clades from Japan are identified with precise published names (Fig. 3), including the native N. denticulata and N. ikiensis, as well as the introduced N. davidi, N. palmata, and N. koreana. The remaining three species are temporarily referred to as N. aff. fukiensis, N. aff. palmata, and N. aff. denticulata. The identity of these three species will be further compared morphologically, in conjunction with known species in the region, and the results will be published separately. TAXONOMY Family Atyidae De Haan, 1849 Neocaridina Kubo, 1938 Neocaridina denticulata (De Haan, 1844) (Figs. 4, 5) Specimens examined: Japan: 1 male, cl 6.5 mm, 1 male, cl 5.2 mm, 6 males, cl 4.2–4.7 mm, ZRC 2023.0211, Kaya R., Ashida River system, Fukuyama City, Hiroshima Prefecture, N34°36'43", E133°15'33", coll. H. Yoshigou, 17 Oct. 2015; 1 female, cl 6.2 mm, Fig. 2. A neighbor-joining COI tree, based on 53 haplotypes (115 specimens) of Neocaridina species from the main islands of Japan and the related taxa from East Asia. Only bootstrap values > 50% are shown on the nodes. *, species collected from Japan in this study. 99 99 98 82 92 98 99 99 99 99 99 77 99 99 99 N. denticulata* N. spinosa N. fonticulata N. palmata* N. aff. palmata* N. davidi* N. ikiensis* N. aff.fukiensis* N. koreana* N. aff. denticulata* N. ketagalan N. saccam page 8 of 30Zoological Studies 63:18 (2024) © 2024 Academia Sinica, Taiwan Table 4. Matrix of percentage pairwise nucleotide divergences with K2P distance and mean number of differences based on 658 bp of COI within and between species of Neocaridina used in this study. In the interspecific (right) part, lower-left values are K2P distances and upper-right ones are bp differences. Range of values are given in parentheses Intraspecific Interspecific Nucleotide divergence Mean nucleotide difference N. davidi N. denticulata N. ikiensis N. aff. fukiensis N. koreana N. davidi 0.89 (0–2.17) 5.8 (0–14) 18.42 (11–25) 44.68 (39–50) 51.87 (50–54) 46.96 (45–49) N. denticulata 1.17 (0–2.01) 7.59 (0–13) 2.88 (1.7–3.93) 45.2 (41–51) 54.77 (52–59) 52.85 (50–56) N. ikiensis 1.22 (0–2.49) 7.86 (0–16) 7.21 (6.25–8.13) 7.31 (6.58–8.31) 36.92 (35–40) 41.92 (41–43) N. aff. fukiensis 0 0 8.48 (8.14–8.85) 9 (8.52–9.76) 5.91 (5.59–6.43) 47 (47–47) N. koreana 0 0 7.6 (7.26–7.95) 8.64 (8.12–9.21) 6.74 (6.59–6.92) 7.63 (7.63–7.63) N. aff. denticulata 0.77 (0–2.01) 5.02 (0–13) 9.32 (8.62–10.08) 9.71 (8.8–10.27) 7.17 (6.39–7.77) 8.64 (8.12–9.01) 3.97 (3.78–4.43) N. palmata 0.41 (0–0.61) 2.68 (0–4) 4.77 (4.24–5.56) 5.46 (4.9–5.89) 8.39 (7.56–8.95) 9.47 (9.31–9.49) 9.12 (8.79–9.5) N. aff. palmata 0.7 (0.15–1.23) 4.6 (1–8) 4.51 (3.77–5.58) 4.99 (4.26–5.59) 8.63 (8.12–9.18) 9.88 (9.72–10.1) 8.84 (8.63–9.15) N. fonticulata 0 0 5.69 (5.4–6.24) 6.14 (5.57–6.59) 6.76 (6.43–6.94) 8.36 (8.36–8.36) 6.08 (6.08–6.08) N. saccam 0.31 (0–0.46) 2 (0–3) 7.13 (6.74–8.14) 8.02 (7.43–8.68) 7.85 (7.11–8.33) 8.88 (8.52–9.06) 6.53 (6.42–6.59) N. ketagalan 0.1 (0–0.15) 0.67 (0–1) 7.2 (6.57–7.78) 8.06 (7.44–8.5) 6.58 (6.08–7.11) 7.7 (7.64–7.81) 6.32 (6.26–6.43) N. spinosa — — 14.41 (13.94–14.7) 15.06 (14.51–15.3) 15.56 (14.73–16.12) 16.27 (16.27–16.27) 15.68 (15.68–15.68) Interspecific N. aff. denticulata N. palmata N. aff. palmata N. fonticulata N. saccam N. ketagalan N. spinosa N. davidi 56.78 (53–61) 30.21 (27–35) 28.53 (24–35) 35.74 (34–39) 44.17 (42–50) 44.59 (41–48) 84.52 (82–86) N. denticulata 58.98 (54–62) 34.39 (31–37) 31.4 (27–35) 38.38 (35–41) 49.33 (46–53) 49.56 (46–52) 87.85 (85–89) N. ikiensis 44.5 (40–48) 51.83 (47–55) 52.88 (50–56) 41.92 (40–43) 48.25 (44–51) 40.92 (38–44) 90.17 (86–93) N. aff. fukiensis 52.87 (50–55) 57.88 (57–58) 59.8 (59–61) 51 (51–51) 54 (52–55) 47.33 (47–48) 94 (94–94) N. koreana 25.22 (24–28) 55.88 (54–58) 54.2 (53–56) 38 (38–38) 40.67 (40–41) 39.33 (39–40) 91 (91–91) N. aff. denticulata 65.04 (61–68) 63.6 (60–67) 47 (44–48) 50.86 (47–52) 50.12 (47–52) 97.22 (95–101) N. palmata 10.76 (10.04–11.3) 36.28 (34–40) 46.63 (45–47) 52.46 (51–53) 48.21 (47–49) 91.25 (90–92) N. aff. palmata 10.51 (9.84–11.13) 5.77 (5.39–6.4) 44.4 (43–46) 51.93 (50–54) 47.33 (46–49) 89.4 (89–90) N. fonticulata 7.61 (7.1–7.77) 7.5 (7.22–7.56) 7.14 (6.9–7.41) 34.33 (34–35) 34.67 (34–35) 83 (83–83) N. saccam 8.29 (7.61–8.49) 8.52 (8.27–8.62) 8.45 (8.11–8.81) 5.49 (5.43–5.6) 38 (37–39) 83 (82–85) N. ketagalan 8.17 (7.61–8.51) 7.79 (7.58–7.93) 7.66 (7.43–7.95) 5.53 (5.42–5.58) 6.08 (5.91–6.25) 85.67 (85–86) N. spinosa 16.94 (16.49–17.72) 15.75 (15.5–15.9) 15.35 (15.26–15.46) 14.13 (14.13–14.13) 14.15 (13.95–14.54) 14.67 (14.54–14.73) page 9 of 30Zoological Studies 63:18 (2024) © 2024 Academia Sinica, Taiwan Fig. 7. Neocaridina davidi (Bouvier, 1904). (A) 1st pereiopod; (B) 2nd pereiopod; (C) 3rd pereiopod; (D) the same, dactylus; (E) 5th pereiopod; (F) the same, dactylus. Scale bars: A, B, C, E = 0.5 mm; D, F = 0.2 mm (male, cl 5.6 mm, ZRC 2023.0214, Sugo R., Yumesaki River system, Himeji City, Hyogo Prefecture, N34°56'31.9", E134°38'19.0'', coll. N. Niwa, 14 Aug. 2015). page 16 of 30Zoological Studies 63:18 (2024) © 2024 Academia Sinica, Taiwan that roughly correspond to local populations of N. denticulata, but one clade was confirmed as N. davidi, and it is sympatric with N. denticulata in three rivers in western Japan. Neocaridina davidi has also been found in eastern Japan (Chiba) where no populations of N. denticulata were found (Toyota et al. 2014; Mitsugi et al. 2017; Mitsugi and Suzuki 2018). Mitsugi et al. (2017) reported the occurrence of N. davidi at Tomoe R., in Boso Peninsula, Chiba, eastern Japan. They found that their specimens are conspecific genetically with samples collected from Gono R. in Shimane Prefecture, Kako R. in Hyogo Prefecture, and Saba R. in Yamaguchi Prefecture. Mitsugi and Suzuki (2018) studied the life history of that population. Onuki (2021) investigated the distribution of N. davidi in relation to environmental parameters at Mama-shita Springs Park at Kunitachi, Tokyo. Neocaridina palmata (Shen, 1948) (Figs. 8, 9) Specimens examined: Japan: 1 male, cl 4.6 mm, 1 male, cl 4.5 mm, NCHUZOOL 14940, 7 males, cl 3.6– 5.5 mm, ZRC 2023.0215, 1 male, cl 4.2 mm, 1 male, cl 5.0 mm, ZRC 2023.0216, Sugo R., Yumesaki River system, Himeji City, Hyogo Prefecture, N34°56'31.9", E134°38'19.0'', coll. N. Niwa, 14 Aug. 2015. Native distribution: Mainland China and northern Vietnam (Cai 1996; Liang 2004). Remarks: Shen (1948) described Caridina palmata based on specimens from “Sha-Ping-Pa”, Chungking (= Chongqing), southwestern China, with no mention of specific comparison with other congeners. It had been totally ignored in the Chinese fauna until Dai et al. (1993) redescribed and illustrated it in detail and transferred it to Neocaridina. Morphologically, N. palmata is similar to N. denticulata and N. davidi. However, it can be distinguished from N. denticulata and N. davidi by its distinct palm-shaped endopod of the male first pleopods (vs. pear-shaped in the latter two species); and the much longer and stouter appendix interna of the male second pleopods, as well as the stouter spinules surrounding the appendix masculina (Figs. 8, 9). It can be further distinguished from N. denticulata by the sexual dimorphism in the third pereiopods, and from N. davidi by the distinctly longer appendix interna of the male second pleopods. Two specimens (1 male, cl 4.2 mm; 1 male, cl 5.0 mm; ZRC 2023.0216) from Himeji City, Hyogo are morphologically typical of N. palmata, but the COI data show them to be clustered within N. davidi (Fig. 3). We suspect that interspecific hybridization might have occurred between the two species living in the same waterbody (see “DISCUSSION”). In their natural range in China, the two species have not been found from the same collection sites (Cai 1996; Liang 2004), while in Japan they were found to be sympatric in the Sugo River. Neocaridina palmata is mainly distributed in central and southern China, with the northern limit in the Hubei and Anhui provinces and a western limit in the Yunnan and Sichuan provinces, and its range extends to southern China (Liang 2004) and northern Vietnam (Li and Liang 2004). The sequences of specimens from northern Vietnam have been included in the phylogenetic tree (Fig. 3). The occurrence of N. palmata at Sugo River, Himeji City, Hyogo Prefecture, represents the first confirmed record of the species (Figs. 2, 3) being introduced outside its natural range in China and Vietnam. According to Cai (1996) and Liang (2004), Neocaridina palmata is a hardy, well-adapted species that can be found in various freshwater habitats, e.g., small streams, large rivers, and open water like ponds, reservoirs, and lakes in temperate and subtropical areas, and always occurs in abundance. The species may pose threats to native Japanese atyid species. Close monitoring of the species is highly recommended for follow-up action. Neocaridina aff. palmata (Fig. 10) Specimens examined: Japan: 1 male, cl 4.2 mm, NCHUZOOL 14940, 1 female, cl 5.5 mm, 1 ovig. female, cl 5.1 mm, non-eyed eggs 1.0 × 0.7 mm, NCHUZOOL 14941, Sugo R., Yumesaki River system, Himeji City, Hyogo Prefecture, N34°56'31.9", E134°38'19.0'', coll. N. Niwa, 14 Aug. 2015; 1 female, cl 4.1 mm, NCHUZOOL 14954, Takahamagawa R., Takahamagawa system, Izumo City, Shimane Prefecture, N35°23'33.3", E132°43'37.1", coll. Y. Nakahara, 10 Mar. 2015; 1 female, cl 6.1 mm, NCHUZOOL 14965; Basen-gawa R., Go-no-gawa system, Miyoshi City, Hiroshima Prefecture, N34°46'51", E132°54'39", coll. H. Yoshigou, 28 Feb. 2016. 1 female, cl 4.7 mm, ZRC 2023.0217; Basen-gawa R., Go-no-gawa system, Miyoshi City, Hiroshima Prefecture, N34°46'51", E132°54'39", coll. H. Yoshigou, 28 Feb. 2016. Korea: 1 female, cl 5.0 mm, NCHUZOOL 14972, aquarium, coll. Jul. 2005. Mainland China: 1 female, cl 3.9 mm, NCHUZOOL 15169, Jiangle, Fujian, coll. H.-T. Shih, 5 Jul. 2004; 1 female, cl 4.3 mm, NCHUZOOL 15172, Nanping, Fujian, coll. H.-T. Shih, 5 Jul. 2004; 1 male, cl 4.1 mm, NCHUZOOL 15170, Yunnan, coll. H.-T. Shih, 5 Nov. 2002; 1 ovig. female, cl 6.2 mm, non-eyed eggs 0.9 × 0.7 mm, ZRC 2023.0218, Shilin, Yunnan, coll. Y. Cai, 11 Apr. 2005; 1 female, cl 6.0 mm, NCHUZOOL page 17 of 30Zoological Studies 63:18 (2024) © 2024 Academia Sinica, Taiwan Fig. 8. Neocaridina palmata (Shen, 1948). (A) cephalothorax and cephalic appendages, lateral view; (B) telson; (C) distal portion of telson; (D) scaphocerite; (E) male 1st pleopod; (F) male 2nd pleopod; (G) uropodal diaeresis. Scale bars: A = 2 mm; B, D, E, F = 0.5 mm; C, G = 0.2 mm (A, D–G, male, cl 4.6 mm, NCHUZOOL 14940, Sugo R., Yumesaki River system, Himeji City, Hyogo Prefecture, N34°56'31.9", E134°38'19.0'', coll. N. Niwa, 14 Aug. 2015; B, C, male, cl 4.3 mm, ZRC 2023.0215, Sugo R., Yumesaki River system, Himeji City, Hyogo Prefecture, N34°56'31.9", E134°38'19.0'', coll. N. Niwa, 14 Aug. 2015). page 18 of 30Zoological Studies 63:18 (2024) © 2024 Academia Sinica, Taiwan Fig. 9. Neocaridina palmata (Shen, 1948). (A) 1st pereiopod; (B) 2nd pereiopod; (C) 3rd pereiopod; (D) the same, dactylus; (E) 5th pereiopod; (F) the same, dactylus. Scale bars: A, B, C, E = 0.5 mm; D, F = 0.2 mm (male, cl 4.6 mm NCHUZOOL 14940, Sugo R., Yumesaki River system, Himeji City, Hyogo Prefecture, N34°56'31.9", E134°38'19.0'', coll. N. Niwa, 14 Aug. 2015). page 19 of 30Zoological Studies 63:18 (2024) © 2024 Academia Sinica, Taiwan Fig. 10. Neocaridina aff. palmata. (A) cephalothorax and cephalic appendages, lateral view; (B) 1st pereiopod; (C) 2nd pereiopod; (D) 3rd pereiopod; (E) the same, dactylus; (F) 5th pereiopod; (G) the same, dactylus; (H) male 1st pleopod; (I) male 2nd pleopod. Scale bars: A–D, F = 0.5 mm; E, G, H, I = 0.2 mm (male, cl 4.2 mm, NCHUZOOL 14940, Sugo R., Yumesaki River system, Himeji City, Hyogo Prefecture, N34°56'31.9", E134°38'19.0'', coll. N. Niwa, 14 Aug. 2015). page 20 of 30Zoological Studies 63:18 (2024) © 2024 Academia Sinica, Taiwan 15171, Kunming, Yunnan, coll. H.-T. Shih, 5 Nov. 2002. Native distribution: Korea and mainland China. Remarks: This species is morphologically very close to N. palmata, and currently we can only separate them based on subtle differences displayed by the available specimens (Fig. 10), e.g., the position of the appendix interna located at 0.35 times the length of the endopod (vs. 0.30 in N. palmata); the telson terminating in a prominent projection (vs. hardly discernible in N. palmata) and a broader scaphocerite (2.8 times as long as wide vs. 3.4 in N. palmata) (cf. Figs. 8, 9). Shih et al. (2017) assigned a specimen that they obtained from South Korea (aquarium dealer) to “N. koreana”. The specimen (NCHUZOOL 14972) was reexamined morphologically in the current study, but we are unable to confirm its identity morphologically as it is a female specimen. However, COI sequence data (Figs. 2, 3) firmly indicated that it should be re-assigned to N. aff. palmata instead. Kakui and Komai (2022) reported the first occurrence of the freshwater ectoparasitic platyhelminth Scutariella japonica from Yasuharu River, Sapporo, Hokkaido, Japan and discussed the identity of the host shrimps, a species of Neocaridina. Phylogenetic analyses using COI (Kakui and Komai 2022: fig. 5) showed that the host species is clustered with “N. koreana” identified by Shih et al. (2017) (see above), sister to N. palmata, and also close to two clades, “N. davidi” (types I and II, after Nagai and Imai 2021). The authors thus tentatively referred their shrimps to “N. sp. aff. davidi”. The Hokkaido COI sequences (LC664097, LC664098, LC664099) were re-analyzed in our study and the results show that genetically they are conspecific with our material of “N. aff. palmata” (Fig. 3). The specimens from the same clade in Kakui and Komai (2022: fig. 5) would also be assigned to the same species, including specimens from Hyogo (AB524970), Shimane (AB524966, AB524968), and Chiba (LC664096), and Chinese material from Henan Province (MW069628, MW069631, MW069644, MW069650, MW069652, MW069653, MW069657, MW069661, MW069670). Notably, based on one of the specimens, the drawings of the endopod of the male first pleopods, the appendix masculina of the male second pleopods, and the form of the dactylus and propodus of the male third pereiopods provided by Kakui and Komai (2022: fig. 3E, F, H) clearly exhibit morphological characteristics consistent with N. davidi, despite the authors initially believing this specimen to be a young male. It is worth noting that our current study observed both N. aff. palmata and N. davidi living sympatrically in the streams at Hyogo, Himeji City and Shimane, Izumo City. The conflict between genetic and morphological findings suggests the possibility of interspecific hybridization (see “DISCUSSION”). Further investigation with both morphology and DNA testing would facilitate a taxonomic decision. The species is found in Fujian (Jiangle and Nanping) and Yunnan (Shilin and Kunming) in southern China, as well as the Korean Peninsula, so we presume that China and Korea are likely its native range. We have recorded its presence in several Japanese localities, including Hyogo, Shimane, Hiroshima, Chiba, and Hokkaido (Fig. 3). These localities are considered to represent one or more introductions, especially since the sites (Sites 2, 8 and 10 in Fig. 1) are also associated with other introduced species (Table 3). Neocaridina ikiensis Shih, Cai, Niwa & Nakahara, 2017 Specimens examined: See Shih et al. (2017). Native distribution: This species is only distributed in Iki Island, Nagasaki Prefecture, western Japan (Shih et al. 2017). Remarks: This species was the second native species of Neocaridina discovered in the main islands of Japan; and its distribution is restricted to Iki Island, an offshore island in northern Kyushu (Shih et al. 2017). Fuke et al. (2021) questioned the validity of N. ikiensis, commenting that Shih et al. (2017) may not have adequately considered the genetic and morphological variations between their new species and the populations of N. denticulata in nearby regions, including what they referred to as “N. denticulata” from Fukue-jima Island, Goto, Nagasaki; Ishiki River, Kawatana, Nagasaki; Kuro River, Aso, Kumamota; and Machida R., Kokoneo, Oita. Their COI tree showed that N. ikiensis clustered together with several populations of their “N. denticulata”. By re-analyzing their sequences using longer lengths (Table 3), along with ours, the results (Fig. 3) indicate that N. ikiensis forms a sister clade with the Machida population. The samples collected from Kuro River and Ishiki River form one clade, which is sister to a clade composed of samples from the Goto Islands. The supported values of these two clades suggest that each clade may represent a separate species not previously recognized, here assigned as N. sp. 1 and N. sp. 2, from the Kuro/Ishiki River and from the Goto Islands, respectively. These two clades are distinctly differentiated from the taxa N. denticulata and N. aff. denticulata that our study recognizes based on both genetic and morphological data (cf. Fig. 3). The group formed by N. sp. 1, N. sp. 2, and N. aff. fukiensis is sister to another group comprising N. ikiensis and the previously mentioned Machida population (Fig. page 21 of 30Zoological Studies 63:18 (2024) © 2024 Academia Sinica, Taiwan 3). Neocaridina aff. fukiensis is morphologically very distinct from N. denticulata (see Remarks under N. aff. fukiensis), and the Machida specimens may represent another separate species, named here as N. sp. 3, that is genetically similar to N. ikiensis. Since Fuke et al. (2021) did not provide morphological characters for the species they identified, we cannot compare these three potential unknown species to any of the known ones. Neocaridina aff. fukiensis (Fig. 11) Specimens examined: Japan: 1 male, cl 6.6 mm, 1 male, cl 5.7 mm, 1 male, cl 6.0 mm, 10 males, cl 3.7–5.8 mm, NCHUZOOL 14946, 1 female, cl 7.7 mm, 1 female, cl 4.5 mm, NCHUZOOL 14947, Kaya R., Ashida River system, Fukuyama City, Hiroshima Prefecture, N34°36'43", E133°15'33'', coll. H. Yoshigou, 17 Oct. 2015; 1 male, cl 4.5 mm, 1 male, cl 4.2 mm, 14 males, cl 3.8–5.7 mm, ZRC 2023.0219, Kaya R., Ashida River system, Fukuyama City, Hiroshima Prefecture, N34°36'43", E133°15'33'', coll. H. Yoshigou, 1 Dec. 2016. Remarks: This species morphologically resembles Neocaridina fukiensis (Liang & Yan, 1978) including the form of its rostrum, which is elongated, reaching beyond the end of the antennular peduncle and with a slight upward curve distally; the telson terminates in a prominent projection; no sexual dimorphism in the third pereiopods; and the form of the appendix interna and the appendix masculina of the male second pleopods, with the appendix interna projecting out distinctly from the appendix masculina (Fig. 11). However, the endopod of the male first pleopods does not possess a distinct projection at its base (vs. with a distinct projection in N. fukiensis) and the fewer spines on the flexor margin of dactylus of the third pereiopods (6–7 vs. 9–10 in N. fukiensis) can be used to separate the two species easily. Fuke et al. (2021) assigned specimens collected from Miya River, Yufuin, Oita to “Neocaridina sp.” based on both morphological and molecular analyses. Upon re-analysis of their COI sequence data (LC612372, LC612374, LC612376, LC612378, LC612379) in conjunction with our own data, the findings revealed that the Miya material clusters in a clade with our Fukuyama specimens, suggesting they are conspecific (Fig. 3). Despite these findings, a pending detailed morphological examination is necessary to confirm the identity of this clade, as it may represent an undescribed native species endemic to Japan. Additional geographic data are requisite to accurately establish its distributional range. Neocaridina koreana Kubo, 1938 (Fig. 12) Specimens examined: Japan: 1 male, cl 4.1 mm, 3 males, cl 4.1–5.3 mm, NCHUZOOL 14964, 1 female, cl 4.1 mm, NCHUZOOL 14965, Go-no-gawa River system, Basen-gawa R., Miyoshi City, Hiroshima Prefecture, N34°46'51", E132°54'39", coll. H. Yoshigou, 28 Feb. 2016; 2 males, cl 5.0–6.5 mm, ZRC 2023.0220, Basen-gawa R., Go-no-gawa system, Miyoshi City, Hiroshima Prefecture, N34°46'51", E132°54'39", coll. H. Yoshigou, 28 Feb. 2016. Native distribution: Korea and probably China (Cai 1996; Liang 2004). Remarks: The original description of Neocaridina denticulata koreana Kubo, 1938 mentioned that “first cheliped rather short, with proportions against finger: palm 0.9, carpus 1.2, merus 1.4…” and “Second cheliped much longer than first, with proportions against finger: palm 0.9, merus 2.0.” “Endopodite of first pleopod pear-shaped, length about 1.7 times as long as breadth in male…” (Kubo 1938: 81). Our specimens agree well with this description (Fig. 12), but with a slightly more elongated endopod of the male first pleopods (1.85 times as long as wide) in adult males, which could be within the range of individual variation if a larger sample size were available. Liang (2004) redescribed the species as having characteristic sexual dimorphism of the third pereiopods and transferred the taxon to the subspecies status under Neocaridina heteropoda (= N. davidi), based on specimens from Zhejiang Province (Jinhua, Quxian). However, this reassignment could not be confirmed, as Kubo’s (1938) original description did not mention this character, and no topo-type specimens are available for comparison. Notably, our specimens do not exhibit any sexual dimorphism in the third pereiopods (Fig. 12). Park et al. (2019) reported the complete mitochondrial genome of Neocaridina koreana (as Neocaridina heteropoda koreana) based on specimens collected from Geojedo, which is close to the type locality Huzan (Busan). Comparison of the available sequences shows that our specimens are conspecific with those from Geojedo (Fig. 3). The Japanese record in our study represents the first confirmed record of the species to be introduced outside its natural range, Korea, and probably China (Cai 1996; Liang 2004). Neocaridina aff. denticulata (Fig. 13) Specimens examined: Japan: 3 males, cl 3.5–4.6 mm, ZRC 2023.0221, Sugo R., Yumesaki River system, Himeji City, Hyogo Prefecture, page 22 of 30Zoological Studies 63:18 (2024) © 2024 Academia Sinica, Taiwan Fig. 11. Neocaridina aff. fukiensis. (A) cephalothorax and cephalic appendages, lateral view; (B, C) 1st pereiopod; (D) 2nd pereiopod; (E) same, basis; (F) male 1st pleopod; (G, H) male 2nd pleopod. Scale bars: A–D = 0.5 mm; E–H = 0.2 mm (male, cl 5.7 mm, NCHUZOOL 14946, Kaya R., Ashida River system, Fukuyama City, Hiroshima Prefecture, N34°36'43", E133°15'33'', coll. H. Yoshigou, 17 Oct. 2015). page 23 of 30Zoological Studies 63:18 (2024) © 2024 Academia Sinica, Taiwan Fig. 12. Neocaridina koreana Kubo, 1938. (A) cephalothorax and cephalic appendages, lateral view; (B) 1st pereiopod; (C) 2nd pereiopod; (D) 3rd pereiopod; (E) the same, dactylus; (F) 5th pereiopod; (G) the same, dactylus; (H, I) male 1st pleopod. Scale bars: A = 2 mm; B–D, F = 0.5 mm; E, G–I = 0.2 mm (male, cl 4.1 mm, NCHUZOOL 14964, Basen-gawa R., Go-no-gawa system, Miyoshi City, Hiroshima Prefecture, N34°46'51", E132°54'39", coll. H. Yoshigou, 28 Feb. 2016). page 24 of 30Zoological Studies 63:18 (2024) © 2024 Academia Sinica, Taiwan N34°56'31.9", E134°38'19.0'', coll. N. Niwa, 14 Aug. 2015; 8 males, cl 4.6–5.5 mm, 15 females, cl 5.5–5.9 mm, ZRC 2023.0222, Oura upstream way (on Oura Dam), Izuharamachi-koura, Tsushima City, Nagasaki Prefecture, N34°14'1.9'', E129°17'45.7'', coll. H. Yoshigou, 15 Dec. 2016; 7 males, cl 5.2–6.0 mm, 10 females, cl 4.5–7.4 mm, ZRC 2023.0223, Takahama R., Mitsushimamachi-kechikou, Tsushima-shi, Tsushima City, Nagasaki Prefecture, N34°15'32.4'', E129°18'47.5'', coll. H. Yoshigou, 16 Dec. 2016. Remarks: Neocaridina aff. denticulata is similar to N. denticulata in the general appearance of the rostrum, the absence of sexual dimorphism in the third pereiopods, and the appendix masculina of the male second pleopods. However, it can be distinguished by its relatively shorter rostrum which reaches to or near to the end of the antennular peduncle (vs. mostly reaching beyond the antennular peduncle in N. denticulata); the exopod of the male first pleopods is 1.5 times as long as wide (vs. 1.2 times in N. denticulata); and the carpus of the first pereiopods is stouter (Fig. 13) (vs. slender in N. denticulata). Its distribution is confined to western Japan, encompassing western Honshu and Kyushu. The phylogenetic relationship with other species (Fig. 3) suggests that it is most probably a cryptic native species, which may have long been mistaken for N. denticulata. DISCUSSION In Japan, several introduced species of Neocaridina, including N. davidi, were found in western Japan in the early 2000s (Niwa et al. 2005; Niwa 2010; Fujita et al. 2011; Toyota et al. 2014). These species have recently spread to eastern Japan (Nishida 2016; Katayama et al. 2017; Mitsugi et al. 2017). Nishino (2009) reported that among the Neocaridina species in Japan tested with mtDNA sequencing of COI and 16S, there are two major haplotype clades, one composed of the haplotypes detected only from Japan, while the other is composed of those observed from both Japan and China, including haplotypes that are identical or show only a few nucleotide differences between the two countries. They considered the former to be indigenous to Japan (N. denticulata denticulata) and the latter to be exotic (i.e., N. davidi, see Nishino 2020). “In four localities of Japan, the two clades were observed sympatrically, implying the genetic pollution through hybridization or introgression between indigenous and exotic populations” (Nishino 2009). Several studies have successfully used DNA barcoding to distinguish closely related species of freshwater shrimps (e.g., Klotz and von Rintelen 2014; Shih et al. 2017 2019; Chen et al. 2020; Xu et al. 2020; Feng et al. 2021; Zhou et al. 2021). Genetic distances of COI among related species can serve as important references for molecular thresholds in certain groups of crustacean taxa (Lefébure et al. 2006; Costa et al. 2007; Chu et al. 2015). Most of the minimum interspecific COI distances (either uncorrected p-distances or K2P) of atyid shrimps are larger than 3.5%, e.g., 3.5% of K2P between Caridina serrata and C. tetrazona (Chen et al. 2020); 8.7% of p-distance between C. pacho and C. pseudoserrata (Do et al. 2020) and 5.57% of K2P between N. fonticulata and N. davidi (Shih et al. 2019). In our study, eight species of Neocaridina from Kyushu and southern Honshu of Japan were detected using the COI marker, with the K2P distances ≥ 3.77% among most species (Table 4), which is larger than the 3.5% threshold found in most studies and could treated as separate species. However, the distance between N. davidi and N. denticulata is small (1.7%; Table 4) and similar to values found in previous studies (e.g., Shih et al. 2017 2019; Han et al. 2019) (Table 4) although they are morphologically very distinct. Additionally, mitochondrial COI barcoding may encounter problems in species delimitation and identification, including asymmetrical introgression (genes introgressed to another species) and incomplete lineage sorting (where some mitochondrial and/or nuclear genes of descendants inherited from a common ancestor do not sort neatly). Furthermore, the coalescence times in mitochondrial markers are 3–4 times faster than those in nuclear markers. Therefore, using additional evidence from nuclear markers is recommended to strengthen the morphology-based taxonomy (Chu et al. 2015; Eberle et al. 2019 2020; Ahrens et al. 2021). Among the introduced species of Neocaridina in Japan, three species (N. davidi, N. koreana and N. aff. palmata) are difficult to identify. This is mainly due to the lack of distinct and consistent morphological characters, and the unavailability of COI sequences for most Chinese species. Based on Japanese literature (Niwa 2010; Toyota et al. 2014; Hasegawa et al. 2015; Mitsugi et al. 2017), these alien species are believed to have been introduced from China and Korea, either for the aquarium trade or as live fish bait. The extent of environmental impact from some invasive freshwater decapod crustaceans has been well documented. Examples include, the Chinese mitten crab (Eriocheir sinensis; Dittel and Epifanio 2009), the red swamp crayfish (Procambarus clarkii; Gherardi 2006; Gherardi and Acquistapace 2007), the giant river prawn (Macrobrachium rosenbergii; Iketani et al. 2016), and land-locked freshwater crabs (Sayamia germaini; Shih et al. 2011). Regarding atyid shrimps, Weber and Traunspurger (2016) reported that introduced N. davidi page 25 of 30Zoological Studies 63:18 (2024)