Characterization of the core region and levels of genetic variation at 18 microsatellite loci from
1. Introduction
Mitten crabs (Brachyura, Varunidae, Varuninae) are native to east Asia and currently classified into eight species belonging to four genera:
Catadromous mitten crabs have the unusual life history of spawning in the sea and growing up in rivers. Fertilized eggs hatch into zoea, which leave the female and begin life in the sea as plankton. After passing through five ecdysis cycles over a period of several weeks, they metamorphose into megalopa (post-larval stage) that live in estuaries and migrate upstream to freshwaters where a second metamorphosis, into juvenile crabs, occurs [2]. The larval stage drifts passively with coastal currents, providing high potential for gene flow within coastal waters. Larvae mainly drift in proximity to the coastline rather than the open sea, so long distance dispersal across open seas is restricted [3]. Juvenile crabs move into rivers and dwell in their middle or upstream reaches where they grow until adulthood. They generally inhabit clear rushing waters as well as hiding in rock crevices by day and coming out at night to feed, their main food being periphyton growing on rocks and aquatic vegetation. They spend most of their lifetime (1–3 years) in freshwater and migrate downstream to coastal waters when mature to mate and spawn [3].
Of this group, only two genera and species,
This study attempted to distinguish
2. Experimental section
2.1. Sample collection
A total of 40
2.2. Genomic DNA isolation
Muscle tissues from all specimens were preserved in 95% ethanol until DNA extraction. Genomic DNA was isolated and purified from the muscle tissue of all individuals. Five hundred milligrams of tissue with 1 mL lysis buffer was digested with 55 µL proteinase K solution. Small amounts of DNA were extracted for polymerase chain reactions (PCR) using a Puregene core kit A (Qiagen, Valencia, CA, USA).
2.3. COI subcloning and analysis
The complete COI gene was amplified using the specific forward primer 5’-CTCTAACRGATTCCCCATCTTCTC-3’ and reverse primer 5’-ATCCTACACATCTGTCTGCC-3’ designed by the authors. A PCR consisted of approximately 50 ng genomic DNA, 50 pmol each of the forward and reverse primers, 25 mM dNTP, 0.05 ~ 0.1 mM MgCl2, 10× buffer, and 5 U
In total, 42 COI sequences were subcloned. All sequences were aligned using Clustal W [16] and then checked with the naked eye. Intraspecific and interspecific genetic distances and numbers of different nucleotides were calculated using MEGA software [17]. The interspecific variable site numbers and intraspecific nucleotide diversities were computed by DnaSP v5 [18]. The phylogenetic trees for COI sequences were constructed using neighbor-joining [19] and maximum parsimony methods [20]. Cluster confidence was assessed using a bootstrap analysis with 1000 replications [21]. The minimum spanning tree (MST) was computed from the matrix of pairwise distances between all pairs of haplotypes in each sample using a modification of the algorithm [22]. We evaluated whether sequences had evolved under strict neutrality. Fu’s
2.4. Genotyping and data analysis
All 18 microsatellite loci [30] were amplified in this study. A PCR was performed in a volume of 25 μL that included ~10 ng genomic DNA, 10 pmol reverse primer, 10 pmol forward primer, 25 mM dNTP, 0.05–0.1 mM MgCl2, 10× buffer, and 0.5 U
The total number of alleles (
3. Results and discussion
3.1. Interspecific diversity
Molecular systematics and historical population dynamics were also analyzed using mt COI gene sequences. The full-length of all 42 COI sequences in
The numbers of interspecific nucleotide differences ranged 196 - 221 with an average of 205.08 ± 4.14 between
The interspecific genetic distances ranged 0.142 - 0.163 with an average 0.150 ± 0.006 for
The complete COI gene can be translated into a 511 amino acid sequence. The number of different amino acids ranged 0 - 10 within
3.2. Intraspecific diversity and historical population dynamics
In total, 86 and 84 variable sites were respectively observed within intraspecific sequences of
A similar genetic pattern is observed in many marine species [34]. The most likely explanation is that the accumulation of mutations over time in a rapidly growing population leads to an increase in the number of haplotypes; even so, population sizes suffer seriously when there is low genetic diversity. Except for wild mitten crabs, which must counteract overharvesting, probable natural causes include climate oscillations that result in temperature and water quality changes, mudflows covering riverbeds that can block migratory pathways, and any other environmental factor that affects adult reproduction and larval survival in estuaries. A second explanation for low levels of genetic diversity in these species could be due to their high dispersal potential during the planktonic egg and larval stages, resulting in strong gene flow among populations. Intraspecific genetic diversity in
Four groups of marine fishes were defined based on the haplotype diversity (
The
Allele numbers and the effective allele numbers of all 18 microsatellite loci in
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PFO-4 | (CA)29 | 56 | 145-183 | 13 | 6.45 | 0.75/0.87 | JQ582816 |
PFO-5 | (TC)6 | 54 | 141-149 | 3 | 2.25 | 0.55/0.57 | JQ582817 |
PFO-7 | (GT)22 | 52 | 217-239 | 10 | 6.06 | 0.65/0.86 | JQ582818 |
PFO-9 | (CA)31 | 54 | 156-194 | 14 | 9.41 | 0.40*/0.92 | JQ582819 |
PFO-10 | (CA)10 | 50 | 94-128 | 8 | 7.27 | 0.65/0.88 | JQ582820 |
PFO-12 | (CA)32 | 56 | 186-216 | 14 | 7.84 | 0.45*/0.89 | JQ582821 |
PFO-15 | (GT)16 | 60 | 68-76 | 5 | 2.99 | 0.00*/0.68 | JQ582822 |
PFO-18 | (CA)33 | 60 | 134-170 | 13 | 6.84 | 0.35*/0.88 | JQ582823 |
PFO-19 | (CA)32 | 50 | 141-175 | 13 | 9.20 | 0.25*/0.91 | JQ582824 |
PFO-31 | (CA)17 | 52 | 79-93 | 6 | 2.74 | 0.35*/0.65 | JQ582825 |
PFO-34 | (CA)20 | 50 | 71-77 | 3 | 2.38 | 0.70*/0.59 | JQ582826 |
PFO-36 | (CA)35 | 54 | 101-125 | 10 | 6.11 | 0.50*/0.86 | JQ582827 |
PFO-37 | (CA)31 | 50 | 166-200 | 13 | 8.79 | 0.90/0.91 | JQ582828 |
PFO-51 | (CA)18 | 58 | 85-101 | 8 | 2.94 | 0.20*/0.68 | JQ582829 |
PFO-52 | (CA)12 | 50 | 97-109 | 6 | 4.10 | 0.95*/0.78 | JQ582830 |
PFO-54 | (GT)18 | 50 | 141-173 | 10 | 5.52 | 0.70*/0.84 | JQ582831 |
PFO-60 | (GT)17 | 50 | 118-146 | 11 | 7.62 | 0.60*/0.89 | JQ582832 |
PFO-79 | (GT)26 | 52 | 113-143 | 13 | 10.26 | 0.50*/0.93 | JQ582833 |
4. Conclusions
The interspecific and intraspecific genetic diversity of two native Taiwanese mitten crabs,
Acknowledgments
We are extremely grateful to YH Hung for her help with laboratory work.
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