Population diversity of N. saccam and N. species in eastern Taiwan
A total of eight N. saccam COI haplotypes from 47 sequences were defined by 17 variable sites and 15 phylogenetically informative sites. The nucleotide sequences were A + T rich (58.7%). The mean COI haplotype diversity in each population was 0.17 (range: 0.00 to 0.68) (Table 1). The haplotype and nucleotide diversities in most populations were 0.00. This result revealed very high levels of differentiation among the populations. A comparison of the fixation indices NST (0.95) and GST (0.58) revealed a weak relationship between phylogeny and geography (Table 3). Among the eight haplotypes, three haplotypes (S1-S3) were shared between two adjacent populations (Table 1). The haplotype trees reconstructed with different methods (ML and BI) were identical. In the BI tree (Fig. 5c), all haplotypes fell into two lineages (NS1 and NS2). Lineage NS1 included four populations south of the Formosa Bank, and lineage NS2 contained two populations north of the Formosa Bank (Figs. 1 and 5c). The results of the S-DIVA analysis showed that the ancestral populations of N. saccam were distributed in north- (C1) and south (C2) of Formosa Bank (Figs. 1 and 5c).
The six haplotypes from the 43 sequences of N. sp. in eastern Taiwan were defined by 30 variable sites and 6 phylogenetically informative sites. The nucleotide sequences were A + T rich (59.1%). The mean haplotype diversity in each population was 0.30 (range: 0.00 to 0.53) (Table 1). A comparison of the fixation indices NST (0.28) and GST (0.51) displayed that the most related haplotypes were found in different populations (Table 3). Compared with those of other species, the NST of N. sp. was the smallest (Table 3). These results suggested that the level of population differentiation of N. sp. was much lower than that of the other three species. The phylogenetic analyses also revealed mixed populations (Fig. 5d). The results of the S-DIVA analysis indicated that the ancestral population was distributed in the population SK and then to the north.
Systematics of the genus Neocaridina
Many studies suggest that species should fulfil two criteria, monophyly and distinctness [33,34,35]. In the present study, the freshwater shrimp Neocaridina in Taiwan formed four monophyletic clades (clades 1, 6, 9 and 12; Fig. 2), and the mean genetic distance among these four clades was 6.64% (ranging 5.74 to 7.50%; Table 2). 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), and the average pairwise distance was 8.19% (Table 2). Robe et al.  evaluated the utility of mtDNA COI in the identification of species of Palaemonidae (Crustacea, Decapoda) and found that the mean genetic distances between the species within the genus Macrobrachium ranged from 0.000 to 0.312 (mean = 0.198). Hebert et al.  suggested that the best threshold for distinguishing intra- from interspecific divergence was approximately 3% sequence divergence, although this value was later modified approximately ten times by many studies [37,38,39,40]. Thus, the present study suggested that the four clades in Taiwan corresponded to four species: N. davidi, N. saccam, N. ketagalan and one undescribed species (N. sp. in Taiwan) (Fig. 2; Table 2). Our study provides a table comparing the morphologic characteristics among these four Neocaridina species in Taiwan and their identification keys (Table 4).Table 4 Comparison of the morphological characters among four Neocaridina species in Taiwan and their identification keysFull size table
Recently, Shih et al.  proposed the third endemic species of Neocaridina known from Taiwan. This new species of land-locked freshwater shrimp, N. fonticulata, is described from Kenting, Hengchun Peninsula, and southern Taiwan . However, although our study sampled specimens from 35 localities in Taiwan, which covers almost all rivers within the island, we did not sample any specimens in the Hengchun Peninsula (Fig. 1). After comparing the morphological characteristics (Table 4 and Shih et al. ), our study found that N. sp. in Taiwan may be synonymous with N. fonticulata. However, the molecular data of N. fonticulata have not been released. Therefore, this question needs to be confirmed in the future.
Moreover, our study found that the systematics of N. davidi were unclear. Neocaridina davidi, once named N. denticulate sinensis , and Shih et al.  suggested that it was synonymous with N. davidi. However, our study found that the genetic distance between N. davidi and N. denticulata was the smallest (2.87%). Thus, we could not suggest that “N. davidi in Taiwan” was a species, subspecies or population. The systematics and distribution area of N. denticulata were also unclear. Moreover, our study also found that there were many questions about the systematics of the genus Neocaridina in East Asia. For example, clade 11 (name: Nak; accession no. LC324777) was named N. aff. Koreana , but it is not close to N. koreana (clade 3; Fig. 2) (name: Nkr; accession no. LC324768 in Shih et al. ). Furthermore, undescribed Neocaridina species were found not only in our study (Fig. 2; clade 4, N. sp. in China, and clade 9, N. sp. in Taiwan) but also in the study of Shih et al.  (clade 10, N. sp. in Japan). Our study suggests that the systematics and species diversity of the genus Neocaridina in East Asia need revisions in future studies.
As described above, the diversity of the genus Neocaridina is outside of our understanding. The distribution of the shared haplotypes (Table 1) and NST (Table 3) within each species showed high population differentiation. These results suggested weak migrating potential in the Neocaridina species. Thus, their distribution patterns were restricted (Fig. 2), and the ancestral populations were easily isolated. These results were also supported by the DIYABC analysis (Fig. 3). The four species did not colonize Taiwan by one colonization route (scenario E, Fig. 3e) because the ancestral populations could not disperse over the entire island. Thus, these four Neocaridina species in Taiwan may have colonized the islands through four different groups of ancestral populations.
Multiple origins of the genus Neocaridina in Taiwan
Our study found four Neocaridina species in Taiwan. The distribution ranges of the three species, N. saccam, N. ketagalan and N. sp., were restricted, and only N. davidi was widely distributed (Fig. 2). The phylogenetic analysis of Neocaridina species in the world revealed that these four species in Taiwan were polytomous (Fig. 2). The TMRCA of these four Taiwanese species were different (Table 3). Moreover, the results of the DIYABC analysis demonstrated that these four Neocaridina species colonized Taiwan during four colonization events (Fig. 3). Chang et al.  also found that two endemic Microphysogobio species colonized Taiwan from two origins and through two colonization centres. Previous studies [9, 19, 42] propose that due to the geological history of Taiwan Island, the different colonization times shaped the different distribution patterns. These present results of the genus Neocaridina in Taiwan agreed with those of previous studies [9, 19, 42]. The four Neocaridina species in Taiwan displayed different distribution patterns, and they may have colonized the islands at different times.
Many studies [9, 18, 19, 42] have suggested that when the freshwater species colonized Taiwan after the island reached its present shape, their distribution range was restricted. Among the four species in Taiwan, N. sp. was restricted to eastern Taiwan. However, the distribution patterns of the freshwater fishes and the phylogeographic studies [10, 19] indicate that the Central Range has acted as a barrier to dispersal between the western and eastern populations of species. Thus, many freshwater species were not distributed in eastern Taiwan, and some species were distributed in eastern Taiwan by human activities . Thus, the freshwater species in eastern Taiwan colonized before those in western Taiwan or originated from populations in western Taiwan through human activities. The results of TMRCA estimated that N. sp. colonized before other species (Table 3). Based on the substitution rate of 1.1% per million years , the TMRCA of N. sp. was 2.180 mya, which was before the Central Range in Taiwan formed (ca. 2 mya). In addition, the results of the DIYABC analysis also supported that the genus Neocaridina colonized Taiwan through four colonization events. Thus, this study suggested that N. sp. colonized Taiwan before the island reached its current shape, and the TMRCA based on the substitution rate of 1.1% per million years was likely an appropriate estimate.
According to previous studies [9, 17, 18, 20, 26], the freshwater species colonized Taiwan through five colonization centres: two to the south of the Formosa Bank, two to the north of the Formosa Bank and the south of the Miaoli Plateau, and one to the north of the Miaoli Plateau. In the phylogeny of the genus Neocaridina (Fig. 2), N. ketagalan was grouped with N. aff. Koreana and N. sp. in Japan as monophyletic. The pairwise p-distance between the clades of Neocaridina suggested that N. ketagalan was close to N. aff. Koreana in Japan (Table 2). Moreover, the S-DIVA analyses showed that the ancestral populations of N. ketagalan were distributed north of the Taoyuan Plateau (Fig. 5b). Our study found that M. brevirostris and N. ketagalan had the same distribution area, but Chang et al.  proposed that M. brevirostris might have originated from mainland China. However, in a study by Chang et al. , we found that M. brevirostris was close to M. koreensis from South Korea. Moreover, Chiu et al.  found that the freshwater snail in northern Taiwan originated from Japan. Thus, this study considered that the freshwater species in northern Taiwan might not have colonized from mainland China and suggested that N. ketagalan originated from Japan (Fig. 1).
Neocaridina saccam was only distributed south of the Miaoli Plateau, and the results of the S-DIVA analysis demonstrated that the ancestral populations of N. saccam were distributed south and north of the Formosa Bank (Figs. 1 and 5c). Based on these results, N. saccam did not colonize Japan. According to the geographic locations, our study suggested that N. saccam originated from mainland China (Fig. 1). Actually, this colonization route was the most common to Taiwan Island [17, 23, 32, 43]. In addition, our study found that N. sp. in eastern Taiwan may be synonymous with N. fonticulata in the Hengchun Peninsula (Fig. 1). If this hypothesis is supported, N. sp. was also distributed in southern Taiwan. This distribution pattern was similar to that of two freshwater fishes, Spinibarbus hollandi and Onychostoma alticorpus. Chiang et al.  proposed that these two fishes colonized the island in the southern region of the Kaoping foreland basins, followed by eastern and northward dispersal. Thus, our study considered that N. sp. in Taiwan (N. fonticulata) may have colonized mainland China (Fig. 1).
In addition, although the S-DIVA analyses showed that the ancestral populations of N. davidi were distributed in northern Taiwan, this species is widely distributed around the world. Moreover, the phylogenetic analysis showed that N. davidi was close to N. denticulata in Japan. Neocaridina davidi may have colonized Japan (Fig. 1). However, our study could not suggest a geographical origin because the systematic status of this species was unidentified. Accordingly, this study suggested that the Neocaridina species in Taiwan colonized the area from multiple geographical and temporal origins, but the deterministic geographical sources need further study.