The freshwater species on Taiwan Island have been documented to have originated from mainland China and the Japanese islands from multiple events and by multiple colonization routes. Moreover, the sequences from the mitochondrial DNA cytochrome c oxidase subunit I (COI) have been used for DNA barcoding to identify the species. This study used the COI sequences to identify Neocaridina species in Taiwan and to examine their geographical and temporal origins.
In total, 479 specimens were collected from 35 localities, which covered almost all rivers in Taiwan. In addition, some sequences were downloaded from GenBank. The maximum likelihood (ML) tree displayed that all sequences were sorted into 13 taxa (clades), and all sequences in Taiwan were sorted into four clades. The Bayesian skyline plots revealed that these four Neocaridina species have declined recently in Taiwan.
All results support that (1) there are four Neocaridina species in Taiwan, which are N. davidi, N. saccam, N. ketagalan and an undescribed Neocaridina species (N. sp.); (2) these four species colonized Taiwan Island in four colonization events; (3) N. sp. colonized Taiwan first; (4) after the island reached its shape, N. ketagalan and N. saccam colonized Taiwan from the Japanese islands and mainland China, respectively; (5) N. davidi colonized northern Taiwan last; and (6) the cyclic glacial and landform changes in East Asia shaped the colonization events and population structures of the Neocaridina species.
The genus Neocaridina Kubo, 1938, is a group of land-locked species of the family Atyidae that consists of 26 species and is distributed in East Asia [1, 2]. Based on the Taiwanese atyid shrimp fauna [3, 4], only one species, N. denticulate, is distributed throughout Taiwan Island. Shih and Cai  proposed two new species, N. saccam and N. ketegalan, in southern and northern Taiwan, respectively. However, Shin and Cai  only sampled the specimens of N. ketegalan in one population and that of N. saccam in the two populations. Thus, our study aimed to determine how many Neocaridina species are present in Taiwan as well as the distribution pattern of each species.
Taiwan Island is located off the southeastern coast of mainland China and is separated from China by the shallow Taiwan Strait. Taiwan was first isolated from the mainland by rising sea levels four to five million years ago (mya) and reached its present shape ca. 2 mya . Previous biogeographic studies support that the many freshwater species easily migrated from the mainland to the island during the Pliocene and Pleistocene glaciations as a result of the lowered sea level [7,8,9,10]. Geological evidence indicates that during the glaciations, the land bridges connected Taiwan Island to the Asian continent three to four times, initially during the Pliocene glaciation and potentially two to three times during the Pleistocene glaciation [11, 12]. In addition, during the early and late Pleistocene, the sea basins of East Asia were exposed and Korea, the Japanese islands, Taiwan Island and mainland China connected [13, 14]. During these ice ages, the migrations between the Asian mainland and Taiwan, Ryuku Archipelago, and Japan may have been possible across the land bridges [15, 16]. Previous phylogeographical studies [9, 17, 18] have suggested that freshwater species in Taiwan might have originated from mainland China and Japan through multiple events and by multiple colonization routes.
Previous phylogeographic studies [18,19,20] have suggested that many geological barriers, e.g., the Central Range, Miaoli Plateau, and Kaoping foreland basins, shaped the structures and distribution patterns of the fauna in Taiwan Island. Taiwanese orogeny (mountain building) uplifted the longitudinal Central Range to nearly 4000 m (Fig. 1). The distribution patterns and phylogeographic studies of freshwater fishes [10, 19] indicate that the Central Range may have acted as a barrier to dispersal between the western and eastern populations. Lin  proposed that the Miaoli Plateau emerged at 0.150 mya based on geological studies, and many studies suggest that the Miaoli Plateau isolated freshwater fishes, preventing dispersal [9, 19]. The Kaoping foreland basins located in the southeastern Taiwan Strait reached a depth of 200 m within 3 km of the shoreline . Previous studies [23,24,25] have proposed that this sea trench interrupted the extension of the Kaoping River towards the land bridge during the glaciations. In other words, even during the ice ages, the freshwater species south of the Kaoping River could not cross the Kaoping foreland basins to the north of the land bridge (Fig. 1).
According to geological history [11,12,13,14] and phylogeographic studies [9, 17, 18, 26], our study found that freshwater species colonized Taiwan Island from the Japanese islands or mainland China, once or multiple times, and through one or multiple routes. Moreover, the distribution patterns of the freshwater species were contributed by the origins of the colonization events and the colonization routes and times [9, 19]. Thus, our study also wanted to determine the distribution range of each Neocaridina species and their origins and phylogeographic patterns in Taiwan. To address the above problems, the mitochondrial DNA (mtDNA) cytochrome c oxidase subunit I (COI) was used to investigate the genetic diversity and structure of the Neocaridina species in Taiwan. The sequences of mtDNA are usually analysed in studies of animal phylogeography [9, 23, 27]. Among all the mtDNA genes, the COI gene is a widely accepted marker for resolving taxonomic identity and evaluating the levels of genetic diversity and differentiation in Decapoda species [28, 29]. The major questions in our study are (1) how many Neocaridina species are there in Taiwan, and (2) what is the colonization history of the Neocaridina species in Taiwan?
Based on all COI sequence data in our study (Table 1) and Shih et al.  from GenBank, the ML tree (Fig. 2) displayed that all sequences were assorted into 13 taxa (clades), and all sequences in Taiwan were assorted into four clades. The sequences of N. davidi in Taiwan, Kineme, Japan and Hawaii were grouped together and were close to those of N. denticulata in Japan. The sequences from the mainland (populations CJ and HJ) were assorted into three species [N. koreana, N. palmata, and one undescribed species (N. sp. in China)]. The range of the pairwise genetic distance between these 13 clades of Neocaridina (Fig. 2) was from 2.87% (between N. davidi and N. denticulata) to 15.23% (between N. sp. in Japan and N. spinose) (Table 2). The average pairwise genetic distance was 8.19%. These results suggested that there were four Neocaridina species in Taiwan: N. davidi (clade 1), N. saccam (clade 6), N. ketagalan (clade 12) and one undescribed species (N. sp. in Taiwan; clade 9). Neocaridina davidi was distributed widely; N. saccam was distributed in central and southern Taiwan; N. ketagalan was distributed in northern and southern Taiwan; and N. sp. was only distributed in eastern Taiwan (Fig. 2).Table 1 Samples used for mtDNA analysis, species, location, code and summary statistics. The number of the private haplotypes (P) and the distribution information of shared haplotypes (S). For sample site number see Fig. 1Full size table
Although the phylogeny of the Neocaridina species (Fig. 2) displayed that there were four species in Taiwan, this study attempted to understand the colonization routes of these four species. Thus, this study proposed seven population history scenarios with the program DIYABC to understand the colonization history of the Neocaridina species in Taiwan. In the first scenario (scenario A), which was based on the phylogenetic analysis (Fig. 2), these four species arrived in Taiwan via four different colonization events (Fig. 3a). Under scenarios B-D, according to a previous study , the freshwater fish in eastern Taiwan arrived from western Taiwan. Thus, we proposed that N. sp. might have originated from the other three species (Figs. 3b-d). Scenario E (Fig. 3e) showed that these four species colonized Taiwan by one colonization route and then diverged. In scenario F (Fig. 3f), N. ketagalan, N. saccam and N. sp. colonized Taiwan by the same colonization route and then diverged because these three species were allopatric. Finally, scenario G (Fig. 2g) showed that two species from northern Taiwan, N. davidi and N. ketagalan, colonized from one origin, and N. saccam and N. sp. colonized from another origin. The highest posterior probability was found for scenario A. Its posterior probability (D: 0.7560, 95% CI: 0.3795–1.0000; L: 0.9999, 95% CI: 0.9998–0.9999) was much higher than those of the other scenarios. The 95% CI of scenario A did not overlap with those for the other scenarios (Fig. 3). Thus, the Neocaridina species in Taiwan might have colonized the area through four different events.
The time to coalescence was estimated in the BEAST analyses using two substitution rates: 2.33% per million years [2, 5, 30] and 1.1% per million years . The TMRCA of these four species, N. davidi, N. ketagalan, N. saccam and N. sp., were 0.242–0.532, 0.37–0.774, 0.432–0.938 and 1.021–2.180, respectively (Table 3). However, the estimated Tajima’s D and Fu’s Fs values were largely consistent within each species; excluding N. sp. (Table 3), none of the calculated values supported population expansion. Although the statistical analysis of the species-specific mismatch distributions (the SSD and Rg indices) revealed that the observed distributions were not significantly different from those expected under a sudden expansion model for all species (Table 3), the Bayesian skyline plots revealed that these four Neocaridina species declined recently in Taiwan (Fig. 4).Table 3 Results of the dynamic tests and molecular clock analyses for the Neocaridina species in Taiwan.Full size table