INTRODUCTION
Fiddler crabs of the genus Uca in the family Ocypodidae inhabit the intertidal to supratidal zone of tropical to temperate regions worldwide. Most are distributed in marginal seas, but species ranging from the continental coast to oceanic islands are limited (Uca tetragonon (Herbst) and U. crassipes (Adams & White)) (Crane, Reference Crane1975). Given their wide range, they are suspected to have greater dispersal during the larval stage and higher survival rates after settlement than other Uca spp. However, no studies have explored the cause of their wide distribution. Genetic structure analysis is useful for estimating larvae dispersal among populations based on gene flow. Several studies have examined the genetic structure of marine invertebrates with wide distributions (Benzie & Williams, Reference Benzie and Williams1995; Lavery et al., Reference Lavery, Moritz and Fielder1996; Palumbi, Reference Palumbi1996; Benzie, Reference Benzie1999; Gopurenko et al., Reference Gopurenko, Hughes and Keenan1999), some of which have suggested great larvae dispersal in the Pacific Ocean (Benzie & Williams, Reference Benzie and Williams1995; Lavery et al., Reference Lavery, Moritz and Fielder1996; Benzie, Reference Benzie1999; Gopurenko et al., Reference Gopurenko, Hughes and Keenan1999). Some researches have addressed the intraspecific genetic structure of marine invertebrates from the continental coast through the continental islands, and to oceanic islands (Williams & Benzie, Reference Williams and Benzie1998; Meyer et al., Reference Meyer, Geller and Paulay2005), but such research has not been conducted for intertidal brachyuran crabs.
Uca crassipes is distributed from the western Pacific Ocean to the Indian Ocean (Crane, Reference Crane1975). In Japan, U. crassipes is distributed only in the continental Ryukyu Islands and the oceanic Bonin Islands, which are separated by about 1500 km. Fukuda (Reference Fukuda1993, Reference Fukuda1994) compared the species composition of marine gastropods in the Bonin Islands with those in three other island groups: the Izu Islands, 700 km north; the Ryukyu Islands, 1500 km west; and the Northern Mariana Islands, 1000 km south of the Bonin Islands. These studies showed that most species from the Bonin Islands were common to the Ryukyu Islands. This implies long larvae dispersal along the oceanic current running from the Ryukyu to the Bonin Islands; however, marine invertebrate genetic relationships between the two island groups have not been studied.
Mitochondrial DNA sequencing, particularly of the most rapidly evolving and highly variable control region (CR), has proven useful in population genetic studies of many terrestrial and aquatic organisms (Avise, Reference Avise1994). Little is known regarding the structure and evolution of marine invertebrate mitochondrial control regions; however, in shrimp this region is divided into three polymorphic domains separated by two stretches with no intraspecific variability (Grabowski & Stuck, Reference Grabowski, Stuck, Schram and von Vaupel Klein1999). The CR has been used successfully to study decapod population genetics (Grabowski & Stuck, Reference Grabowski, Stuck, Schram and von Vaupel Klein1999; McMillen-Jackson & Bert, Reference McMillen-Jackson and Bert2003, Reference McMillen-Jackson and Bert2004; Diniz et al., Reference Diniz, Maclean, Ogawa, Cintra and Bentzen2005; Babbucci et al., Reference Babbucci, Buccoli, Cau, Cannas, Goñi, Díaz, Marcato, Zane and Patarnello2010). Furthermore, the hypervariable domain in the mtDNA CR could help elucidate the genetic structure of a population with high resolution.
This study aimed to clarify the genetic relationship between continental coast, continental island and oceanic island populations in the west Pacific region using 12Sr-RNA, 16Sr-RNA, and the control region of mitochondrial DNA in U. crassipes. Using these results, we examined the connectivity among these populations.
MATERIALS AND METHODS
Sample collection and DNA extraction
Uca crassipes specimens were collected from four localities in Japan (Chichi-jima and the Ryukyu Islands Okinawa-jima, Miyako-jima, and Iriomote-jima), one in Vietnam (Hoi An), and one in French Polynesia (Moorea Island) (Figure 1). For each specimen, muscle tissue from the third leg was fixed in 99% ethanol. Total DNA was extracted from the muscle tissue using proteinase K and sodium dodecyl sulphate (SDS)-phenol-chloroform extraction.
Locations of the six Uca crassipes sampling sites. The shaded region indicates the distribution of U. crassipes.
Mitochondrial DNA amplification and sequencing
The target DNA fragments of the small subunit of ribosomal RNA (12Sr-RNA) and the large subunit of ribosomal RNA (16Sr-RNA) were amplified via polymerase chain reaction (PCR) using the primers H2492i (5′CAG ACA TGT TTT TAA TAA ACA GGC-3′; modified from Palumbi et al., Reference Palumbi, Martin, Romano, McMillan, Stice and Grabowski1991), L1496i (5′-GTA CAT ATC GCC CGT CGC TT-3′; Kitaura et al., Reference Kitaura, Wada and Nishida1998), H3059i (5′-CCG GTC TGA ACT CAG ATC ATG T-3′), H3062 (5′-CCG GTC TGA ACT CAG ATC A-3′), and L2510C (5′-CGC CTG TTT AAC AAA GAC AT-3′; modified from Palumbi et al., Reference Palumbi, Martin, Romano, McMillan, Stice and Grabowski1991). The PCR was performed in a total reaction volume of 25 µl containing 0.2 µl of TaKaRa Ex Taq (5 units/μl), 2.5 µl of 10× Ex Taq buffer, 2.0 µl of dNTP mixture (2.5 mM each), 0.4 mM of each primer, and 1.0 µl of template. The PCR consisted of 25–30 cycles of 94°C for 30 seconds, 46–50°C for 30 seconds, and 72°C for 60 seconds using a GeneAmp PCR System 2400 (Applied Biosystems) or TaKaRa PCR Thermal Cycler Dice Version 3 Model TP600 (TaKaRa).
The CR portion of the mitochondrial DNA gene, located between the 12Sr-RNA and the isoleucine transfer RNA (tRNA-Ile) gene, was amplified by PCR using primers that Kitamura et al. (Reference Kitamura, Nitta, Wakao and Oda2005) designed for the mtDNA genome of the Japanese spiny lobster Panulirus japonicus (Von Siebold) (GenBank Accession No. NC004251; Yamauchi et al., Reference Yamauchi, Miya and Nishida2002) Pjapo13350 (CCT TTA AGT TTA ACC GCA GAT GC) and the swimming crab Portunus trituberculatus (Miers) (GenBank Accession No. NC005037; Yamauchi et al., Reference Yamauchi, Miya and Nishida2003) Pjapo14471 (ACG GGG TAT GAG CCC ATT AGC). The PCR consisted of 30 cycles of 94°C for 30 seconds, 65°C for 30 seconds and 72°C for 60 seconds. The amplification products were purified using ExSAP-IT (USB) and sequenced using the BigDye Terminator v.3.1 Cycle Sequencing Kit (Applied Biosystems) with an automated 3500/3500xL Genetic Analyzer (Applied Biosystems) DNA sequencer. The sequencing reactions followed the manufacturer's protocol. The partial mitochondrial 12Sr-RNA and 16Sr-RNA genes were sequenced to determine the phylogenetic relationships among haplotypes. In addition, the partial mitochondrial CR gene was sequenced and used to analyse genetic diversity, genetic structure and gene flow.
The DNA sequences were aligned using the program ClustalW (Thompson et al., Reference Thompson, Higgins and Gibson1994) and inspected visually with a chromatogram viewer editor. Polymorphic sites were assessed visually on the original chromatogram and alignments were refined manually when necessary.
Data analysis
PHYLOGENETIC RELATIONSHIPS, GENE DIVERSITY AND GENETIC STRUCTURE
Some consensus sequences of the 611-bp 12Sr-RNA–16Sr-RNA and 428-bp 16Sr-RNA were obtained for 18 individuals, which represented all of the sample populations. The sequences were first analysed with the software Modeltest (Posada & Crandall, Reference Posada and Crandall1998) to identify the evolutionary model that best fit the data. The 12Sr-RNA–16Sr-RNA and 16Sr-RNA sequence data were combined because Modeltest selected the same evolutionary model, HKY, for each sequence.
The combined sequences were analysed with the maximum likelihood (ML) and neighbour-joining (NJ) methods using PAUP version 4.0b10 (Swofford, Reference Swofford2001). The ML analysis was performed using a heuristic search algorithm, setting the parameters to the values calculated by Modeltest. For both tests, bootstrap analyses as heuristic searches were applied, with 100 replications for ML and 1000 replications for NJ. The tree was visualized and drawn using Fig Tree Version 1.3.1 (Rambaut, Reference Rambaut2009).
Gene diversity at the mitochondrial CR was described as haplotype (h) and nucleotide (π) diversities (Nei, Reference Nei1987) using ARLEQUIN 3.1 (Excoffier et al., Reference Excoffier, Laval and Schneider2005). To test for regional subdivisions in the sequences, we used an analysis of molecular variance (AMOVA), and pairwise F ST values between populations were calculated in ARLEQUIN ver. 3.1. All F-statistics were computed using the method of Weir & Cockerham (Reference Weir and Cockerham1984), and F values significantly different from zero were identified by comparison with the results of 10,000 data permutations (Raymond & Rousset, Reference Raymond and Rousset1995). We adjusted the critical P values for these tests using Bonferroni corrections. Gene flow (Nm) was estimated as Nm = (1 – F ST )/2F ST (Hudson et al., Reference Hudson, Slatkin and Maddison1992). The haplotype network using statistical parsimony (Templeton et al., Reference Templeton, Crandall and Sing1992) was built with TCS ver.1.21 (Clement et al., Reference Clement, Posada and Crandall2000). However, it was difficult to estimate the connections between all of the haplotypes from Vietnam and several from Iriomote-jima (H75, H76 and H77) and the other haplotypes by TCS because these haplotypes differed greatly from those from the Ryukyu Islands and Moorea Island (Supplementary Appendix 2). Therefore, we drew the haplotype network with 80 haplotypes, excluding these haplotypes.
RESULTS
The combined 1039-bp 12Sr-RNA–16Sr-RNA sequences were obtained for 18 individuals in six populations. We identified eight haplotypes using the 46 variable sites, including 12 gaps, in the combined sequences (Supplementary Appendix 1). The forty of 46 variation sites were found in the Vietnamese samples. The HKY model was selected for DNA substitution by hLRT. The parameter values under the selected model were as follows: base frequencies A = 0.3801, C = 0.1573, G = 0.0702, T = 0.3924; substitution model, Ti/Tv ratio = 7.9768; proportion of invariable sites = 0.0000; and variable sites (G) = equal rates for all sites. The NJ analysis produced the same topology as ML. Two major clusters were supported strongly: the Vietnamese population and the other populations (Figure 2). In addition, in the non-Vietnamese population cluster, the haplotypes from Chichi-jima (H2 and H3) were local haplotypes, whereas haplotype H1 was shared with the Ryukyu Islands, Okinawa-jima, Miyako-jima, Iriomote-jima and the Moorea Island populations.
Maximum-likelihood (ML) tree for the combined sequence of mtDNA 12Sr-RNA and 16Sr-RNA (1039 base pairs) haplotypes using the HKY model. Eight haplotypes are shown in 6 sampling sites. The numbers are bootstrap values from 100 replications with ML (the prior value) and 1000 replications with neighbour-joining (the posterior value). Bootstrap values higher than 50% are shown in the tree.
A 504-bp portion of the CR sequence was obtained from 133 individuals in six Uca crassipes populations. We identified 94 haplotypes using the 166 variable sites, including seven gaps, with 107 informative sites (Supplementary Appendix 2).
The h and π diversities were lowest in the Okinawa-jima population (h = 0.8719, π = 0.0034), whereas those of the Vietnamese population (h = 1.0000, π = 0.0603) had the highest values (Table 1). The AMOVA revealed significant overall population differentiation (Φ ST = 0.5807, P < 0.001). In the pairwise F ST values (Table 2), all combinations of populations, excluding two combinations, Miyako-jima and Okinawa-jima and Miyako-jima and Iriomote-jima, showed significant genetic differentiation (F ST = 0.0929–0.8527; P < 0.0033; Table 2).
Haplotype and nucleotide diversities of six populations of Uca crassipes.
Pairwise F ST values between populations (below diaonal) and Nm (above the diagonal) for the mtDNA control region among six populations of Uca crassipes, as by Arlequin version 3.1.
Significance was tested at the 5% level with a Bonfeferroni-corrected P for multiple test (*P < 0.05,**P < 0.01).
The haplotype network by TCS, excluding the Vietnamese haplotypes and some from Iriomote-jima, indicated that the three Ryukyu Islands populations shared four haplotypes: H1, H2, H3 and H12 (Figure 3). A common haplotype, H1, occurred in the three populations and was designated ancestral by TCS. Private haplotypes that were unique to one locality and were connected to the shared haplotypes by a few steps did not form a cluster for each locality. All of the Moorea Island haplotypes were private (Figure 3) and were connected with the Miyako-jima haplotype H70 within ten steps. Haplotypes from Chichi-jima formed two clusters many steps apart and connected with the Moorea Island haplotypes by >19 steps (Figure 3). All of the Chichi-jima haplotypes were private and separated by many steps from each other and the Ryukyu and Moorea Islands haplotypes.
Statistical parsimony cladogram for the mtDNA control region haplotypes using TCS. The individual oval sizes in the diagram are proportional to the number of individuals possessing each haplotype. Branches correspond to single mutations and additional circles on the branches represent additional inferred mutations. Haplotype 1 is the ancestral haplotype inferred by TCS.
DISCUSSION
Vietnamese population
The pairwise F ST and relationship of some sequences from the regions of 12Sr-RNA to 16Sr-RNA and the mtDNA CR revealed that the Vietnamese population exhibited high genetic differentiation from the other Pacific Ocean populations (Table 2; Figure 2). In addition, the Vietnamese population contained higher gene and nucleotide diversity than the other populations, suggesting that the Vietnamese population has maintained its large population longer than the other populations. Hoi An is located in Vietnam on the South China Sea, which, with an average depth of about 1500 m, is known to have been reduced in size as recently as the last glacial maximum (Li et al., Reference Li, Zhong, Tian, Wang and Li2009). Over 2 Mya, the connections between the South China Sea and Pacific Ocean were limited by low sea levels (Tjia, Reference Tjia1980). Therefore, the limited connection of the South China Sea with the Pacific Ocean could have caused the remarkable differentiation of the Vietnamese Uca crassipes population. Similar genetic differentiation of South China Sea populations has been detected in the Kuruma shrimp Penaeus japonicus (Bate) (Tsoi et al., Reference Tsoi, Wang and Chu2005) and the oval squid Sepioteuthis cf. lessoniana (Lesson) (Aoki et al., Reference Aoki, Imai, Naruse and Ikeda2008).
The differentiation of the Vietnamese population may be observed at an intraspecific level, as the haplotypes H75, H76 and H77 found on Iriomote-jima were similar to haplotypes from the Vietnamese population. However, more taxonomic studies (morphological feature, breeding feature and nucleus marker) will be needed to resolve this issue.
Connections between the Ryukyu Islands and Moorea Island populations
The private haplotypes from the Ryukyu Islands were connected with the shared haplotypes by several steps (Figure 3). In addition, the pairwise F ST showed no significant difference between Miyako-jima and Okinawa-jima or Iriomote-jima (Table 2), indicating that gene flow has occurred within the Ryukyu Islands. However, two lineages were found in the Iriomote-jima population, one connected to the populations of the Ryukyu Islands and the other with the Vietnamese population, suggesting that the Iriomote-jima population was formed by the ingression of different gene pools.
The Moorea Island population is located in the Society Islands and exhibited significant differentiation from all other populations, although the haplotypes showed a close relationship with the Ryukyu Islands haplotypes. The tree topology created from the combined 12Sr-RNA–16Sr-RNA sequences also showed a close genetic relationship between the Ryukyu Islands and Moorea Island populations. Therefore, the Moorea and Ryukyu Islands populations were likely either derived from the same historical gene pool or there was great gene flow that spanned almost 10,000 km between the Ryukyu Islands and Moorea Island via oceanic currents.
Chichi-jima population
Chichi-jima is an oceanic island of the Bonin Islands, 1000 km north of the Northern Mariana Islands. The Bonin Island biota has been influenced greatly by oceanic currents. Fukuda (Reference Fukuda1993, Reference Fukuda1994) compared the composition of marine gastropod species of the Bonin Islands with the three surrounding island groups: the Izu Islands, Ryukyu Islands, and Northern Mariana Islands; 70% of the species were common to the Ryukyu Islands, 45% to the Izu Islands and 20% to the Northern Mariana Islands. Furthermore, there is a sister relationship between the Bonin Islands and Ryukyu Islands in the intertidal gastropod Lunella coronata (Gmelin) group (Williams et al., Reference Williams, Apte, Ozawa, Kaligis and Nakano2011). However, our results did not identify shared haplotypes between the Ryukyu and Bonin Islands, suggesting little recent transport of larvae between the two island groups.
If a population was isolated from other populations during a long period, the isolated population could show low genetic diversity by the effect of inbreeding. However, our results are in contrast with this; the oceanic island haplotypes of the Chichi-jima population had higher variability, with two different lineages (Figure 3), than the continental Ryukyu Islands populations. This implies that larvae have been transported from Chichi-jima from a large gene pool, possibly the Mariana Islands, as both groups of islands share the same geological history (Seno & Maruyama, Reference Seno and Maruyama1984). Further genetic study of U. crassipes, including the Mariana Island population, will determine the origin of the Chichi-jima population.
The U. crassipes population in the Bonin Islands occurs only in Chichi-jima. The habitat area, which is limited to a few sites, and the population size (<50 individuals) are extremely small, suggesting the possibility of extirpation. Considering the genetic lineage of the Chichi-jima population, their conservation status should be amended.
ACKNOWLEDGEMENTS
We thank Dr M. Tsuchiya, Dr M. Izawa, and Dr T. Naruse (University of the Ryukyus) and Mr T. Tannai (Ogasawara Subprefecture, Tokyo) for their cooperation with specimen sampling. We thank Dr H. Imai (University of the Ryukyus), Professor Y. Yusa, and the members of the Laboratory of Population and Community Ecology, Nara Women's University, for their invaluable input and support. This study was partly supported by the Mikimoto Fund for Marine Ecology in Japan, and by Nara Women's University Intramural Grant for Young Women Researchers to M.A.
Supplementary materials and methods
The supplementary material refered to in this paper can be found online at journals.cambridge.org/mbi.
