Research Article |
Corresponding author: Sun Hongying ( sunhongying@njnu.edu.cn ) Academic editor: Ingo S. Wehrtmann
© 2017 Xing Yuhui, Zhou Lijun, Hou Yue, Wang Xiaoqi, Zhang Chen, Zhang Huilun, Wang Ruoran, Pan Da, Sun Hongying.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Yuhui X, Lijun Z, Yue H, Xiaoqi W, Chen Z, Huilun Z, Ruoran W, Da P, Hongying S (2017) Complete mitochondrial genomes from two species of Chinese freshwater crabs of the genus Sinopotamon recovered using next-generation sequencing reveal a novel gene order (Brachyura, Potamidae). ZooKeys 705: 41-60. https://doi.org/10.3897/zookeys.705.11852
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Recent morphological and molecular evidence has challenged classical interpretations of eubrachyuran phylogeny and evolution. Complete mitochondrial genomes of two species of potamid freshwater crabs, Sinopotamon yaanense and Sinopotamon yangtsekiense were obtained using next-generation sequencing. The results revealed a novel gene order with translocations of a five-gene block and a tRNA gene in comparison to available brachyuran mitochondrial genomes. DNA sequence comparisons position the Potamidae, a primary freshwater crab family, outside of the clade for the traditional heterotreme families, and closer to the clade that includes the thoracotreme families of grapsoid and ocypodoid crabs. Mitogenomic comparisons using rapid next-generation sequencing and a much wider taxonomic sample are required for a high-resolution examination of the phylogenetic relationships within the Eubrachyura.
Eubrachyuran, gene rearrangement, mitochondrial genome, primary freshwater crab, Sinopotamon
Brachyuran crabs are one of the most species-rich and economically important groups in extant crustaceans with about 7200 species described (
Next-generation sequencing (NGS), combined with bioinformatic annotation, is becoming increasingly common for recovering animal mitogenome sequences and allows a rapid amplification-free sequencing (
Sinopotamon yaanense and S. yangtsekiense, as two representatives of the endemic genus Sinopotamon occur in China, are distributed in the middle and lower reaches of the Yangtze River Basin, respectively (
Specimens of S. yaanense and S. yangtsekiense were collected by hand from mountain streams of the Emei Mountain in Sichuan Province, China (29°36'3"N; 103°19'59"E) and Ningguo, Anhui Province, China (30°38'21"N; 118°59'27"E), respectively. These specimens were preserved in 95% ethanol and identified utilizing morphological information presented in
Next-generation transcriptome sequencing and next-generation total genomic DNA sequencing were used to obtain mitogenomes of the two Sinopotamon species. For transcriptome sequencing, total RNA of S. yangtsekiense was extracted from fresh tissue of one individual using the TRIzol (Takara). After determining the RNA quality, the sample was enriched by Oligo (dT) and broken into short RNA fragments. The cDNA library was then prepared and sequenced on the Illumina Hi-Seq 2000 platform (BGI). All raw data were processed to remove adaptors and clean data were assembled de novo using Trinity (
For total genomic DNA sequencing, total genomic DNA of S. yaanense was extracted using Cell and Tissue DNA Extraction Kit (Generay Biotech). The samples were sequenced on the Illumina HiSeq 4000 platform (BGI). The sequencing libraries with average insert sizes of approximately 300 bp were prepared, and then sequenced as 150 bp paired-end runs (about 2 Gb raw data each species). De novo assemblies were conducted with Geneious 9.1.4 using the Map to Reference program (
The assembled and identified mitochondrial DNA sequences were further annotated and analyzed. The locations of PCGs and rRNA genes were preliminarily annotated by DOGMA website (
In-group and out-group taxa are listed in Table
Taxon | Species | Length (bp) | GenBank Accession No. | Reference | |
Ingroup | |||||
Brachyura | Podotremata | ||||
Homoloidea | Homologenus malayensis | 15793 | KJ612407 | Unpublished | |
Moloha majora | 15903 | KT182069 |
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Raninoidea | |||||
Umalia orientalis | 15466 | NC_026688 |
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Ranina ranina | 15557 | NC_023474 | unpublished | ||
Lyreidus brevifrons | 16112 | NC_026721 |
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Eubrachyura | |||||
Heterotremata | |||||
Portunoidea | |||||
Thalamita crenata | 15787 | NC_024438 |
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Callinectes sapidus | 16263 | NC_006281 |
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Portunus trituberculatus | 16026 | NC_005037 |
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Charybdis japonica | 15738 | NC_013246 | Liu et al. 2010 | ||
Chaceon granulatus | 16135 | NC_023476 | unpublished | ||
Scylla olivacea | 15723 | NC_012569 | unpublished | ||
Majoidea | |||||
Chionoecetes japonicus | 15341 | AB_735678 | unpublished | ||
Calappoidea | |||||
Ashtoret lunaris | 15807 | NC_024435 |
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Eriphioidea | |||||
Myomenippe fornasinii | 15658 | NC_024437 |
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Xanthoidea | |||||
Pseudocarcinus gigas | 15515 | NC_006891 |
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Bythograeoidea | |||||
Gandalfus yunohana | 15567 | NC_013713 |
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Austinograea alayseae | 15620 | NC_020314 |
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Potamoidea | |||||
Geothelphusa dehaani | 18197 | NC_007379 | Segawa et al. 2005 | ||
Sinopotamon xiushuiense | 18460 | NC_029226 |
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S. yangtsekiense | 17885 | KY785879 | Present study | ||
S. yaanense | 17126 | KY785880 | Present study | ||
Thoracotremata | |||||
Ocypodoidea | |||||
Ilyoplax deschampsi | 15460 | NC_020040 |
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Ocypode ceratophthalmus | 15564 | NC_025324 |
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Mictyris longicarpus | 15548 | LN_611670 |
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Grapsoidea | |||||
Eriocheir japonica sinensis | 16354 | NC_006992 |
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Xenograpsus testudinatus | 15798 | NC_013480 |
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Pachygrapsus crassipes | 15652 | NC_021754 |
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Cyclograpsus granulosus | 16300 | NC_025571 |
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Chiromantes neglectum | 15920 | KX156954 |
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Parasesarma tripectinis | 15612 | KU343209 | Unpublished | ||
Sesarmops sinensis | 15905 | KR336554 | Unpublished | ||
Metopaulias depressus | 15765 | KX118277 | Unpublished | ||
Outgroup | |||||
Anomura | Paguroidea | ||||
Pagurus longicarpus | 15630 | NC_003058 | Hickerson et al. 2000 | ||
Paralithodes brevipes | 16303 | NC_021458 | Unpublished | ||
Lithodes nintokuae | 15731 | NC_024202 | Unpublished | ||
Galatheoidea | Neopetrolisthes maculatus | 15324 | NC_020024 |
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Shinkaia crosnieri | 15182 | NC_011013 | Ya |
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Thalassinidea | Gebiidea | ||||
Austinogebia edulis | 15761 | NC_019606 |
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Upogebia major | 16119 | JF793665 |
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Polychelidae | Eryonoidea | ||||
Polycheles typhlops | 16221 | NC_020026 |
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Astacidea | Astacoidea | ||||
Cambaroides similis | 16220 | NC_016925 |
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Nephropoidea | |||||
Homarus americanus | 16432 | NC_015607 |
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Parastacoidea | |||||
Cherax destructor | 15894 | AY383557 |
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The complete mitogenome sequences of S. yaanense and S. yangtsekiense were, respectively, 17,126 bp and 17,885 bp in length, between 15,612 bp and 18,460 bp typical in length for eubrachyurans (Table
Mitochondrial genome sequenced in the present study. Gene order and sizes are shown relative to one another, including non-coding regions. Protein-coding genes encoded on the light strand are underlined. Transfer RNA (tRNA) genes encoded on the light strand are underlined. Each tRNA gene is designated by a single-letter amino acid code, except L1 (trnLeu (CUN)), L2 (trnLeu (UUR)), S1 (trnSer (AGN)) and S2 (trnSer (UCN)). Numbers inside circles represent the size of the non-coding region separating two adjacent genes or the amount of shared nucleotides between two overlapping genes. The translocations of gene or gene block are shaded gray.
A non-coding region between rrnS and trnI was identified as the main non-coding region (mNCR, or AT-rich region) for the two Sinopotamon crab species. The mNCR consisted of 1231 bp in S. yaanense (with 78.9% A + T content), whereas it consisted of 1194 bp in S. yangtsekiense (79.3%). The alignment of the entire sequences of mNCR revealed a high similarity between the two Sinopotamon species in two dormains (Suppl. material
The overall A + T content of 13 PCGs, calculated from the coding strand of each gene, was 70.7% in S. yaanense and 71.8% in S. yangtsekiense (Table
Nucleptide composition in 13 PCGs of the Sinopotamon yaanense and S. yangtsekiense mitogenomes.
Species | Base composition(%) | A+T (%) | Length (nt) | Start codons | Stop codons | ||||
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A | T | G | C | ||||||
cox1 | S. yaanense | 27.60 | 37.20 | 15.70 | 19.50 | 64.80 | 1539 | ATG | TAA |
S. yangtsekiense | 27.70 | 37.90 | 16.00 | 18.30 | 65.60 | 1539 | ATG | TAA | |
cox2 | S. yaanense | 31.88 | 37.26 | 11.79 | 19.07 | 69.14 | 688 | ATG | T |
S. yangtsekiense | 31.43 | 39.62 | 12.13 | 16.81 | 71.05 | 685 | ATG | T | |
atp8 | S. yaanense | 26.14 | 47.71 | 7.19 | 18.95 | 73.86 | 159 | ATG | TAG |
S. yangtsekiense | 27.45 | 49.02 | 4.58 | 18.95 | 76.47 | 159 | ATG | TAG | |
atp6 | S. yaanense | 30.06 | 40.63 | 10.42 | 18.90 | 70.68 | 675 | ATT | TAA |
S. yangtsekiense | 29.76 | 41.52 | 10.71 | 18.01 | 71.28 | 675 | ATT | TAA | |
cox3 | S. yaanense | 26.62 | 39.67 | 14.45 | 19.26 | 66.29 | 792 | ATG | TAA |
S. yangtsekiense | 27.25 | 40.43 | 13.81 | 18.50 | 67.68 | 792 | ATG | TAA | |
nad3 | S. yaanense | 27.64 | 44.73 | 11.68 | 15.95 | 72.36 | 354 | ATC | TAG |
S. yangtsekiense | 27.64 | 44.44 | 11.11 | 16.81 | 72.08 | 354 | ATC | TAG | |
nad5 | S. yaanense | 30.84 | 41.61 | 18.75 | 8.80 | 72.45 | 1729 | ATG | T |
S. yangtsekiense | 30.84 | 41.61 | 18.75 | 8.80 | 72.45 | 1729 | ATG | T | |
nad4 | S. yaanense | 32.35 | 42.19 | 17.36 | 8.10 | 74.54 | 1335 | ATG | TAG |
S. yangtsekiense | 30.94 | 42.92 | 18.35 | 7.79 | 73.86 | 1338 | ATG | TAA | |
nad4L | S. yaanense | 26.00 | 46.33 | 22.00 | 5.67 | 72.33 | 303 | ATG | TAA |
S. yangtsekiense | 27.00 | 47.33 | 20.33 | 5.33 | 74.33 | 303 | ATG | TAA | |
nad6 | S. yaanense | 26.91 | 47.39 | 6.63 | 19.08 | 74.30 | 504 | ATT | TAA |
S. yangtsekiense | 27.38 | 49.60 | 6.55 | 16.47 | 76.98 | 507 | ATT | TAA | |
cob | S. yaanense | 27.43 | 40.39 | 12.35 | 19.84 | 67.81 | 1135 | ATG | T |
S. yangtsekiense | 27.43 | 41.53 | 12.70 | 18.34 | 68.96 | 1135 | ATG | T | |
nad1 | S. yaanense | 26.82 | 45.09 | 19.23 | 8.87 | 71.90 | 939 | ATA | TAA |
S. yangtsekiense | 27.67 | 45.09 | 18.48 | 8.76 | 72.76 | 939 | ATA | TAA | |
nad2 | S. yaanense | 28.44 | 45.41 | 7.49 | 18.66 | 73.85 | 1005 | ATG | TAA |
S. yangtsekiense | 29.24 | 46.11 | 7.58 | 17.07 | 75.35 | 1005 | ATG | TAA | |
total | S. yaanense | 28.36 | 42.74 | 13.46 | 15.43 | 70.67 | 11157 | ||
S. yangtsekiense | 28.59 | 43.63 | 13.16 | 14.61 | 71.81 | 11160 |
Gene orders of S. yaanense and S. yangtsekiense mitogenomes were identical between both species (Fig.
The phylogenetic trees were reconstructed based on the two different datasets A (13 PCGs) and B (13 PCGs + two rRNAs). Both ML and Bayesian analyses resulted in congruent tree topologies with the exception of some minor difference within “Grapsoidea + Ocypodoidea”. Branch lengths and topologies came from ML analysis. Bootstrap value (BP) and Bayesian posterior probability (BPP) of nodes are shown like BP/BPP in Fig.
Phylogenetic analyses derived for brachyurans using the maximum likelihood (ML) analyses and Bayesian inferences (BI) using dataset A (13 PCGs) and dataset B (13 PCGs + two rRNAs). Branch lengths and topologies came from ML analysis. Values at the branches represent BP (Bootstrap value)/BPP (Bayesian posterior probability). 100/1.00 is denoted by an asterisk. The horizontal line stands for BP under 50 or BPP under 0.9 ML analyses. The gene rearrangement is denoted by the block on (A): (I) the translocation of trnH shared by the Brachyura taxa sampled; (II) the transposition of trnQ shared by potamid species; (III) the five-gene block, (trnM-nad2-trnW-trnC-trnY), translocation shared by three Sinopotamon crabs sampled.
Within the Eubrachyura, strong support was obtained for the non-monophyly of Heterotremata, with potamid crabs nesting outside the heterotreme clade ((((Portunoidea + Calappoidea) + (Xanthoidea + Eriphioidea)) + Bythograeoidea) + Majoidea); instead, four potamid crabs appeared to constitute a close affinity to Thoracotremata, and form a robust clade ((Grapsoidea + Ocypodoidea)+Potamidae). All potamids, S. yaanense, S. yangtsekiense and S. xiushuiense constituted a robust clade with G. dehaani being sister to this clade in all analyses, with relatively high nodal support (BP = 100 and 100; BPP = 1.00 and 1.00).
The mitogenomic organizations found in the two Sinopotamon species are identical to those of the typical decapods (
The largest non-coding region between rrnS and trnI was predicted to be the putative mNCR in the two Sinopotamon crab species ranging from 1194 bp (S. yangtsekiense) to 1231 bp (S. yaanense), where the position is similar to that found in Geothelphusa dehaani and other brachyurans (see
Gene arrangements and primary sequences in mitogenomes embrace useful signals in reconstruction of phylogenetic relationships (
Striking species diversification has challenged the phylogeny of brachyurans and led to a high number of hypotheses about affinities within the Eubrechyura (reviewed in
The authors declare no competing interests. We thank Qiang Zhao, Yongkun Ji (College of Life Sciences, Nanjing Normal University) for assistance with sample collection, Dr. Ingo Wehrtmann and one anonymous reviewer for insightful comments. This work was supported by the National Natural Science Foundation of China (No. 31471972 and No. 31071902) to SHY.
Figure S1
Data type: TIF image file
Explanation note: Comparisons of the consensus sequence and variable sites in the entire mNCR for Sinopotamon yaanense, S. yangtsekiense and S. xiushuiense. The conserved central domain is grey shaded, and the extended termination associated sequences (ETAS) is underlined.