Research Article |
Corresponding author: Jamie Bojko ( j.bojko@tees.ac.uk ) Academic editor: Martin Schwentner
© 2021 Lucas A. Jennings, April M. H. Blakeslee, Krista A. McCoy, Donald C. Behringer, Jamie Bojko.
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.
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Abstract
This study provides a broad phylogenetic analysis for the Eubrachyura, with the inclusion of three new Panopeidae mitochondrial genomes: Eurypanopeus depressus (flatback mud crab) (15,854bp), Panopeus herbstii (Atlantic mud crab) (15,812bp) and Rhithropanopeus harrisii (Harris, or ‘white-fingered’ mud crab) (15,892bp). These new mitogenomes were analyzed alongside all available brachyuran mitochondrial genomes (n = 113), comprising 80 genera from 29 families, to provide an updated phylogenetic analysis of the infra-order Brachyura (“true crabs”). Our analyses support the subsection Potamoida within the Eubrachyura as the sister group to Thoracotremata. The family Panopeidae aligns with the family Xanthidae to form the Xanthoidea branch, which is supported by current morphological and genetic taxonomy. A unique gene arrangement termed ‘XanGO’ was identified for the panopeids and varies relative to other members of the subsection Heterotremata (within the Eubrachyura) via a transposition of the trnV gene. This gene arrangement is novel and is shared between several Xanthoidea species, including Etisus anaglyptus (hairy spooner crab), Atergatis floridus (brown egg crab), and Atergatis integerrimus (red egg crab), suggesting that it is a conserved gene arrangement within the Xanthoidea superfamily. Our study further reveals a need for taxonomic revision of some brachyuran groups, particularly the Sesarmidae. The inclusion of panopeid mitogenomes into the greater brachyuran phylogeny increases our understanding of crab evolution and higher level Eubrachyuran systematics.
Xanthidae, Panopeus, Eurypanopeus, Rhithropanopeus, mud crab, marine, genomics
Brachyura (“true” crabs) is the largest subgroup of the Decapoda (Crustacea). It is a ubiquitous group, whose members thrive in terrestrial and aquatic habitats but are particularly prevalent in marine environments (Tsang 2009;
High throughput sequencing (HTS) has proven effective in advancing and resolving taxonomies (
Mitogenomes also offer insights into gene arrangement, which can have diagnostic properties at different systematic levels (Boore 1998;
An example of an understudied brachyuran group is the superfamily Xanthoidea (Brachyura), which boasts high diversity across the world’s oceans (Karasawa 2006). Species within this superfamily share a high level of morphological similarity and are often poorly described both morphologically and genetically (Ng 2008; Thoma 2014). The number of families and subfamilies within the Xanthoidea has changed drastically in recent years (Lai 2011). Two common families, the Xanthidae and Panopeidae, share several morphological features that can lead to systematic confusion and difficulty in identifying them beyond the family level (Shih 2011). Both families are found in temperate and tropical shallow intertidal and subtidal zones, but xanthid crabs have a circumtropical distribution while panopeids are only found in the Americas, excluding global invasions (Thoma 2014). To date, there are only four mitogenomes available for the Xanthidae and none for the Panopeidae, whose systematics have primarily relied upon a select number of genes or morphological keys (
In this study, we enhance understanding of brachyuran systematics by adding three complete mitogenomes for the Panopeidae: Eurypanopeus depressus, Panopeus herbstii and Rhithropanopeus harrisii from their native range along the Atlantic coast of North America. The genetic composition, genetic similarity and gene arrangement of these three panopeid species are described relative to other brachyuran mitogenomes, allowing us to update the brachyuran mitogenomic phylogeny and explore brachyuran-wide classification. A new gene arrangement for the superfamily Xanthoidea is described as well as a renaming of previously reported gene arrangements suggested for other Brachyura.
Three species of panopeid mud crabs were collected for this study. First, an individual Eurypanopeus depressus was sampled on December 1, 2018 from Hoop Pole Creek, a polyhaline site located in Atlantic beach, North Carolina (NC), USA. The individual was hand-collected at low tide from an intertidal oyster reef and then brought back to the lab for dissection. Second, an individual Panopeus herbstii was sampled on August 12, 2019 from Middle Marsh (Beaufort, NC), another polyhaline site, using a passive sampler attached to a wooden stake that had been driven into the sediment. The sampler design is a small plastic milk crate (19×22×16 cm) filled with autoclaved oyster shell (Roche 2007). Third, an individual Rhithropanopeus harrisii was sampled on February 5, 2020 from Mallard Creek (Washington, NC), a mesohaline site, using the same passive sampling design as above, but this time attached to a small fishing dock. Crabs were brought back to the lab and anesthetized prior to dissection in a –20°C freezer. Dissections for all three species were carried out using a sterilized razor blade, and part of the hepatopancreas and gills were removed and placed into separate tubes for later DNA extraction.
The DNA extractions were conducted on the hepatopancreas and gill tissue of E. depressus, P. herbstii, and R. harrisii using a Zymo DNA extraction kit, according to manufacturer’s protocols. The DNA samples were shipped on dry ice to Novogene, California, who conducted library preparation using the NEBNext Ultra DNA Library Prep Kit. The library was loaded on to a NovaSeq 6000 (Illumina) system using the 150 bp NovaSeq 600 SP reagent kit (300 cycles) for paired end metagenomic sequencing for each individual sample. The resulting data were delivered to the University of Florida for bioinformatic analysis. The data were quality checked and trimmed using Trimmomatic v.0.36 (Bolger 2014) using default parameters. The paired and unpaired reads were assembled using SPAdes v.3.13.0 (
The circularized mitogenomes were annotated using MITOS (Bernt 2013). Using the MITOS output, the location of the cox1 gene was determined and the sequences were re-annotated with the cox1 gene at the start of the genome. The putative amino acid and rRNA sequences determined by MITOS were checked using BLASTn and BLASTp (Tables
Nucleotide and protein similarity data for the protein-coding and non-coding genes of the Eurypanopeus depressus mitochondrial genome. The data represented were acquired from BLASTn and BLASTp outputs via comparison against the complete non-redundant database. The accession number of the specific nucleotide or amino acid sequence are provided in addition to the species, if known, belonging to the sequence isolate. The similarity (%), coverage comparison (%) and e-value are all provided. MCG = mitochondrion, complete genome.
Genome | Start | End | Gene | Strand | Gene hit | Gene similarity (%) | Gene Coverage (%) | Gene e-value | Gene accession | Protein hit | Protein similarity | Protein cover | Protein -value | Protein accession |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Eurypanopeus depressus mitochondrial genome | 1 | 1515 | cox1 | + | Rhithropanopeus harrisii MCG | 88.05 | 100 | 0.0 | Present study | cytochrome c oxidase subunit I [Rhithropanopeus harrisii] | 99.8 | 100 | 0.0 | Present study |
1535 | 1598 | trnL2(tta) | + | Panopeus herbstii MCG | 92.42 | 100 | 5e-23 | Present study | — | — | — | — | — | |
1606 | 2277 | cox2 | + | Rhithropanopeus harrisii MCG | 88.99 | 100 | 0.0 | Present study | cytochrome c oxidase subunit II [Panopeus herbstii] | 99.11 | 100 | 1e-170 | Present study | |
2291 | 2357 | trnK(aaa) | + | Panopeus herbstii MCG | 95.52 | 100 | 2e-27 | Present study | — | — | — | — | — | |
2358 | 2420 | trnD(gac) | + | Rhithropanopeus harrisii MCG | 100 | 100 | 3e-32 | Present study | — | — | — | — | — | |
2421 | 2573 | atp8 | + | - | — | — | — | — | ATP synthase F0 subunit 8 [Panopeus herbstii] | 90.20 | 100 | 6e-18 | Present study | |
2576 | 3238 | atp6 | + | Panopeus herbstii MCG | 87.80 | 100 | 0.0 | Present study | ATP synthase F0 subunit 6 [Panopeus herbstii] | 99.10 | 100 | 6e-158 | Present study | |
3256 | 4032 | cox3 | + | Rhithropanopeus harrisii MCG | 89.83 | 100 | 0.0 | Present study | cytochrome c oxidase subunit III [Rhithropanopeus harrisii] | 99.61 | 100 | 0.0 | Present study | |
4038 | 4100 | trnG(gga) | + | Rhithropanopeus harrisii MCG | 98.41 | 100 | 1e-30 | Present study | — | — | — | — | — | |
4107 | 4448 | nad3 | + | - | — | — | — | — | NADH dehydrogenase subunit 3 [Panopeus herbstii] | 96.49 | 99 | 1e-79 | Present study | |
4456 | 4518 | trnA(gca) | + | Panopeus herbstii MCG | 98.41 | 100 | 1e-28 | — | — | — | — | — | — | |
4519 | 4582 | trnR(cga) | + | Rhithropanopeus harrisii MCG | 98.44 | 100 | 4e-31 | Present study | — | — | — | — | — | |
4583 | 4649 | trnN(aac) | + | - | — | — | — | — | — | — | — | — | — | |
4652 | 4718 | trnS1(aga) | + | Rhithropanopeus harrisii MCG | 98.51 | 100 | 9e-31 | Present study | — | — | — | — | — | |
4721 | 4786 | trnE(gaa) | + | Rhithropanopeus harrisii MCG | 95.45 | 100 | 6e-29 | Present study | — | — | — | — | — | |
4808 | 4871 | trnH(cac) | — | Rhithropanopeus harrisii MCG | 95.31 | 100 | 9e-26 | Present study | — | — | — | — | — | |
4872 | 4935 | trnF(ttc) | — | Rhithropanopeus harrisii MCG | 95.31 | 100 | 9e-26 | Present study | — | — | — | — | — | |
4943 | 6574 | nad5 | — | Rhithropanopeus harrisii MCG | 88.79 | 98 | 0.0 | Present study | NADH dehydrogenase subunit 5 [Rhithropanopeus harrisii] | 92.75 | 98 | 0.0 | Present study | |
6721 | 8046 | nad4 | — | Rhithropanopeus harrisii MCG | 87.30 | 99 | 0.0 | Present study | NADH dehydrogenase subunit 4 [Panopeus herbstii] | 96.15 | 99 | 0.0 | Present study | |
8043 | 8318 | nad4L | — | Rhithropanopeus harrisii MCG | 91.21 | 98 | 1e-107 | Present study | NADH dehydrogenase subunit 4L [Panopeus herbstii] | 100.00 | 100 | 8e-66 | Present study | |
8345 | 8408 | trnT(aca) | + | Rhithropanopeus harrisii MCG | 98.39 | 95 | 2e-29 | Present study | — | — | — | — | — | |
8409 | 8473 | trnP(cca) | — | Rhithropanopeus harrisii MCG | 98.46 | 100 | 1e-31 | Present study | — | — | — | — | — | |
8476 | 8970 | nad6 | + | Rhithropanopeus harrisii MCG | 85.51 | 98 | 4e-147 | Present study | NADH dehydrogenase subunit 6 [Rhithropanopeus harrisii] | 92.73 | 100 | 6e-90 | Present study | |
8982 | 10118 | cob | + | Rhithropanopeus harrisii MCG | 86.77 | 99 | 0.0 | Present study | cytochrome b [Panopeus herbstii] | 98.68 | 100 | 0.0 | Present study | |
Eurypanopeus depressus mitochondrial genome | 10117 | 10183 | trnS2(tca) | + | Echinoecus nipponicus voucher MABIK CR00241788 MCG | 95.85 | 97 | 2e-15 | NC_039618.1 | — | — | — | — | — |
10235 | 11134 | nad1 | — | Rhithropanopeus harrisii MCG | 88.95 | 99 | 0.0 | Present study | NADH dehydrogenase subunit 1 [Rhithropanopeus harrisii] | 98.67 | 100 | 0.0 | Present study | |
11171 | 11242 | trnL1(cta) | — | — | — | — | — | — | — | — | — | — | — | |
11217 | 12610 | rrnL | — | Eurypanopeus depressus voucher USNM 16S RNA gene | 91.03 | 98 | 0.0 | KT959469.1 | — | — | — | — | — | |
12705 | 13522 | rrnS | — | Eurypanopeus depressus voucher ULLZ 3976 12S ribosomal RNA gene, partial sequence; mitochondrial | 99.73 | 44 | 0.0 | EU863325.2 | — | — | — | — | — | |
14140 | 14204 | trnV(gta) | — | Rhithropanopeus harrisii MCG | 95.45 | 100 | 3e-26 | Present study | — | — | — | — | — | |
14422 | 14489 | trnI(atc) | + | Panopeus herbstii MCG | 97.06 | 100 | 4e-31 | Present study | — | — | — | — | — | |
14487 | 14555 | trnQ(caa) | — | Rhithropanopeus harrisii MCG | 95.65 | 100 | 2e-30 | Present study | — | — | — | — | — | |
14579 | 14646 | trnM(atg) | + | Atergatis floridus MCG | 98.53 | 100 | 1e-22 | NC_037201.1 | — | — | — | — | — | |
14659 | 15621 | nad2 | + | Rhithropanopeus harrisii MCG | 82.18 | 100 | 0.0 | Present study | NADH dehydrogenase subunit 2 [Panopeus herbstii] | 90.62 | 99 | 0.0 | Present study | |
15656 | 15723 | trnW(tga) | + | — | — | — | — | — | — | — | — | — | — | |
15724 | 15787 | trnC(tgc) | — | Panopeus herbstii MCG | 96.88 | 100 | 2e-29 | NC_037201.1 | — | — | — | — | — | |
15788 | 15852 | trnY(tac) | — | Etisus anaglyptus MCG | 90.77 | 100 | 4e-12 | NC_042208.1 | — | — | — | — | — |
Nucleotide and protein similarity data for the protein-coding and non-coding genes of the Panopeus herbstii mitochondrial genome. The data represented were acquired from BLASTn and BLASTp outputs via comparison against the complete non-redundant database. The accession number of the specific nucleotide or amino acid sequence are provided in addition to the species, if known, belonging to the sequence isolate. The similarity (%), coverage comparison (%) and e-value are all provided. MCG = mitochondrion, complete genome.
Genome | Start | End | Gene | Strand | Gene hit | Gene similarity (%) | Gene Coverage (%) | Gene e-value | Gene accession | Protein hit | Protein similarity | Protein cover | Protein e-value | Protein accession |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Panopeus herbstii Mitochondrial Genome | 1 | 1515 | cox1 | + | Rhithropanopeus harrisii MCG | 87.52 | 100 | 0.0 | Present study | cytochrome c oxidase subunit I [Rhithropanopeus harrisii] | 100.00 | 100 | 0.0 | Present study |
1535 | 1600 | trnL2(tta) | + | Eurypanopeus depressus MCG | 92.42 | 100 | 6e-23 | Present study | — | — | — | — | — | |
1607 | 2278 | cox2 | + | Rhithropanopeus harrisii MCG | 88.24 | 100 | 0.0 | Present study | cytochrome c oxidase subunit II [Eurypanopeus depressus] | 99.11 | 100 | 1e-170 | Present study | |
2292 | 2358 | trnK(aaa) | + | Eurypanopeus depressus MCG | 95.52 | 100 | 2e-29 | Present study | — | — | — | — | — | |
2359 | 2421 | trnD(gac) | + | Rhithropanopeus harrisii MCG | 95.24 | 100 | 3e-27 | Present study | — | — | — | — | — | |
2422 | 2574 | atp8 | + | — | — | — | — | — | ATP synthase F0 subunit 8 [Eurypanopeus depressus] | 90.20 | 100 | 6-e18 | Present study | |
2577 | 3239 | atp6 | + | Eurypanopeus depressus MCG | 87.80 | 100 | 0.0 | Present study | ATP synthase F0 subunit 6 [Eurypanopeus depressus] | 99.10 | 100 | 6e-158 | Present study | |
3257 | 4033 | cox3 | + | Rhithropanopeus harrisii MCG | 89.32 | 100 | 0.0 | Present study | cytochrome c oxidase subunit III [Eurypanopeus depressus] | 98.46 | 100 | 0.0 | Present study | |
4039 | 4102 | trnG(gga) | + | Rhithropanopeus harrisii MCG | 96.88 | 100 | 6e-29 | Present study | — | — | — | — | — | |
4109 | 4450 | nad3 | + | — | — | — | — | — | NADH dehydrogenase subunit 3 [Eurypanopeus depressus] | 96.49 | 99 | 1e-79 | Present study | |
4458 | 4520 | trnA(gca) | + | Eurypanopeus depressus MCG | 98.41 | 100 | 1e-28 | Present study | — | — | — | — | — | |
4521 | 4583 | trnR(cga) | + | Rhithropanopeus harrisii MCG | 96.88 | 100 | 6e-29 | Present study | — | — | — | — | — | |
4584 | 4651 | trnN(aac) | + | — | — | — | — | — | — | — | — | — | — | |
4653 | 4719 | trnS1(aga) | + | Eurypanopeus depressus MCG | 97.01 | 100 | 4e-29 | Present study | — | — | — | — | — | |
4722 | 4785 | trnE(gaa) | + | Eurypanopeus depressus MCG | 95.45 | 100 | 2e-28 | Present study | — | — | — | — | — | |
4805 | 4868 | trnH(cac) | — | Rhithropanopeus harrisii MCG | 96.88 | 100 | 2e-27 | Present study | — | — | — | — | — | |
4869 | 4935 | trnF(ttc) | — | — | — | — | — | — | — | — | — | — | — | |
4943 | 6550 | nad5 | — | Rhithropanopeus harrisii MCG | 87.93 | 99 | 0.0 | Present study | NADH dehydrogenase subunit 5 [Eurypanopeus depressus] | 93.64 | 99 | 0.0 | Present study | |
6725 | 8047 | nad4 | — | Rhithropanopeus harrisii MCG | 85.54 | 99 | 0.0 | Present study | NADH dehydrogenase subunit 4 [Eurypanopeus depressus] | 96.15 | 99 | 0.0 | Present study | |
8044 | 8319 | nad4L | — | Rhithropanopeus harrisii MCG | 90.84 | 98 | 6e-106 | Present study | NADH dehydrogenase subunit 4L [Eurypanopeus depressus] | 100.00 | 100 | 8e-66 | Present study | |
8346 | 8409 | trnT(aca) | + | — | — | — | — | — | — | — | — | — | — | |
8410 | 8474 | trnP(cca) | — | Rhithropanopeus harrisii MCG | 98.46 | 100 | 1e-31 | Present study | — | — | — | — | — | |
8477 | 8974 | nad6 | + | Eurypanopeus depressus MCG | 83.54 | 97 | 2e-130 | Present study | NADH dehydrogenase subunit 6 [Rhithropanopeus harrisii] | 91.52 | 99 | 7e-89 | Present study | |
Panopeus herbstii Mitochondrial Genome | 8983 | 10119 | cob | + | Rhithropanopeus harrisii MCG | 87.13 | 99 | 0.0 | Present study | cytochrome b [Eurypanopeus depressus] | 98.68 | 100 | 0.0 | Present study |
10118 | 10184 | trnS2(tca) | + | Rhithropanopeus harrisii MCG | 92.65 | 100 | 4e-26 | Present study | — | — | — | — | — | |
10230 | 11135 | nad1 | — | Rhithropanopeus harrisii MCG | 89.40 | 98 | 0.0 | Present study | NADH dehydrogenase subunit 1 [Eurypanopeus depressus] | 97.00 | 99 | 0.0 | Present study | |
11171 | 11239 | trnL1(cta) | — | — | — | — | — | — | — | — | — | — | — | |
11194 | 12584 | rrnL | — | Panopeus herbstii voucher USNM: 16S RNA gene, mitochondrial | 100.00 | 37 | 0.0 | KT959516.1 | — | — | — | — | — | |
12683 | 13502 | rrnS | — | Panopeus herbstii voucher ULLZ 8457 12S ribosomal RNA gene, partial sequence; mitochondrial | 99.46 | 44 | 0.0 | EU863296 | — | — | — | — | — | |
14124 | 14190 | trnV(gta) | — | — | — | — | — | — | — | — | — | — | — | |
14357 | 14423 | trnI(atc) | + | Eurypanopeus depressus MCG | 97.06 | 100 | 4e-31 | Present study | — | — | — | — | — | |
14421 | 14489 | trnQ(caa) | — | Eurypanopeus depressus MCG | 95.65 | 100 | 2e-30 | Present study | — | — | — | — | — | |
14541 | 14609 | trnM(atg) | + | Etisus anaglyptus MCG | 98.55 | 100 | 9e-25 | NC_042208 | — | — | — | — | — | |
14622 | 15581 | nad2 | + | — | — | — | — | — | NADH dehydrogenase subunit 2 [Eurypanopeus depressus] | 90.62 | 100 | 0.0 | Present study | |
15619 | 15685 | trnW(tga) | + | — | — | — | — | — | — | — | — | — | — | |
15685 | 15748 | trnC(tgc) | — | Rhithropanopeus harrisii MCG | 98.44 | 100 | 4e-31 | Present study | — | — | — | — | — | |
15749 | 15812 | trnY(tac) | — | Rhithropanopeus harrisii MCG | 92.31 | 100 | 2e-24 | Present study | — | — | — | — | — |
Nucleotide and protein similarity data for the protein-coding and non-coding genes of the Rhithropanopeus harrisii mitochondrial genome. The data represented were acquired from BLASTn and BLASTp outputs via comparison against the complete non-redundant database. The accession number of the specific nucleotide or amino acid sequence are provided in addition to the species, if known, belonging to the sequence isolate. The similarity (%), coverage comparison (%) and e-value are all provided. MCG = mitochondrion, complete genome.
Genome | Start | End | Gene | Strand | Gene hit | Gene similarity (%) | Gene Coverage (%) | Gene e-value | Gene accession | Protein hit | Protein similarity | Protein cover | Protein e-value | Protein accession |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Rhithropanopeus harrisii Complete Mitochondrial Genome | 1 | 1515 | cox1 | + | Rhithropanopeus harrisii mitochondrial partial COI gene for cytochrome oxidase subunit 1, isolate R617-8 | 99.39 | 65 | 0.0 | LN810615 | cytochrome c oxidase subunit I [Panopeus herbstii] | 100.00 | 100 | 0.0 | Present study |
1535 | 1599 | trnL2(tta) | + | - | — | — | — | — | — | — | — | — | — | |
1607 | 2278 | cox2 | + | Eurypanopeus depressus MCG | 88.99 | 100 | 0.0 | Present study | cytochrome c oxidase subunit II [Panopeus herbstii] | 99.11 | 100 | 8-e170 | Present study | |
2292 | 2357 | trnK(aaa) | + | Panopeus herbstii MCG | 95.52 | 100 | 7e-29 | Present study | — | — | — | — | — | |
2358 | 2420 | trnD(gac) | + | Eurypanopeus depressus MCG | 100.00 | 100 | 3e-32 | Present study | — | — | — | — | — | |
2421 | 2573 | atp8 | + | - | — | — | — | — | ATP synthase F0 subunit 8 [Panopeus herbstii] | 84.31 | 100 | 2e-16 | Present study | |
2576 | 3238 | atp6 | + | Eurypanopeus depressus MCG | 88.54 | 100 | 0.0 | Present study | ATP synthase F0 subunit 6 [Eurypanopeus depressus] | 97.74 | 100 | 1e-156 | Present study | |
3256 | 4032 | cox3 | + | Eurypanopeus depressus MCG | 89.86 | 100 | 0.0 | Present study | cytochrome c oxidase subunit III [Eurypanopeus depressus] | 99.61 | 100 | 0.0 | Present study | |
4038 | 4100 | trnG(gga) | + | Eurypanopeus depressus MCG | 98.41 | 100 | 1e-30 | Present study | — | — | — | — | — | |
4107 | 4448 | nad3 | + | - | — | — | — | — | NADH dehydrogenase subunit 3 [Eurypanopeus depressus] | 94.74 | 100 | 9e-73 | Present study | |
4455 | 4517 | trnA(gca) | + | Eurypanopeus depressus MCG | 98.41 | 100 | 1e-28 | Present study | — | — | — | — | — | |
4518 | 4581 | trnR(cga) | + | Eurypanopeus depressus MCG | 98.44 | 100 | 4e-31 | Present study | — | — | — | — | — | |
4582 | 4648 | trnN(aac) | + | - | — | — | — | — | — | — | — | — | — | |
4651 | 4717 | trnS1(aga) | + | Eurypanopeus depressus MCG | 98.51 | 100 | 9e-31 | Present study | — | — | — | — | — | |
4721 | 4786 | trnE(gaa) | + | Eurypanopeus depressus MCG | 95.45 | 100 | 6e-29 | Present study | — | — | — | — | — | |
4803 | 4866 | trnH(cac) | — | Panopeus herbstii MCG | 96.88 | 100 | 2e-27 | Present study | — | — | — | — | — | |
4867 | 4930 | trnF(ttc) | — | Eurypanopeus depressus MCG | 95.31 | 100 | 8e-28 | Present study | — | — | — | — | — | |
4941 | 6554 | nad5 | — | Eurypanopeus depressus MCG | 88.79 | 99 | 0.0 | Present study | NADH dehydrogenase subunit 5 [Eurpanopeus depressus] | 92.75 | 98 | 0.0 | Present study | |
6712 | 8037 | nad4 | — | Eurypanopeus depressus MCG | 87.30 | 99 | 0.0 | Present study | NADH dehydrogenase subunit 4 [Eurypanopeus depressus] | 94.80 | 100 | 0.0 | Present study | |
8034 | 8309 | nad4L | — | Eurypanopeus depressus MCG | 91.21 | 98 | 1e-107 | Present study | NADH dehydrogenase subunit 4L [Eurypanopeus depressus] | 96.74 | 100 | 2e-64 | Present study | |
8336 | 8400 | trnT(aca) | + | Eurypanopeus depressus MCG | 98.39 | 95 | 2e-29 | Present study | — | — | — | — | — | |
8401 | 8465 | trnP(cca) | — | Panopeus herbstii MCG | 98.46 | 100 | 1e-31 | Present study | — | — | — | — | — | |
Rhithropanopeus harrisii Complete Mitochondrial Genome | 8468 | 8962 | nad6 | + | Eurypanopeus depressus MCG | 85.45 | 98 | 3e-148 | Present study | NADH dehydrogenase subunit 6 [Eurypanopeus depressus] | 92.73 | 100 | 6e-90 | Present study |
8974 | 10107 | cob | + | Panopeus herbstii MCG | 87.13 | 100 | 0.0 | Present study | cytochrome b [Panopeus herbstii] | 98.68 | 100 | 0.0 | Present study | |
10109 | 10175 | trnS2(tca) | + | Panopeus herbstii MCG | 92.65 | 100 | 4e-24 | Present study | — | — | — | — | — | |
10224 | 11123 | nad1 | — | Panopeus herbstii MCG | 89.40 | 99 | 0.0 | Present study | NADH dehydrogenase subunit 1 [Eurypanopeus depressus] | 98.67 | 100 | 0.0 | Present study | |
11160 | 11228 | trnL1(cta) | — | - | — | — | — | — | — | — | — | — | — | |
11184 | 12583 | rrnL | — | Rhithropanopeus harrisii voucher USNM 12S ribosomal RNA gene, partial sequence; mitochondrial | 100.00 | 37 | 0.0 | KT959486.1 | — | — | — | — | — | |
12683 | 13499 | rrnS | — | Rhithropanopeus harrisii voucher ULLZ 3995 12S ribosomal RNA gene, partial sequence; mitochondrial | 98.90 | 44 | 0.0 | EU863280 | — | — | — | — | — | |
14143 | 14208 | trnV(gta) | — | Eurypanopeus depressus MCG | 95.45 | 100 | 3e-26 | Present study | — | — | — | — | — | |
14430 | 14496 | trnI(atc) | + | Eurypanopeus depressus MCG | 97.06 | 100 | 4e-31 | Present study | — | — | — | — | — | |
14494 | 14562 | trnQ(caa) | — | Panopeus herbstii MCG | 92.75 | 100 | 3e-27 | Present study | — | — | — | — | — | |
14620 | 14687 | trnM(atg) | + | Panopeus herbstii MCG | 97.10 | 100 | 1e-29 | Present study | — | — | — | — | — | |
14700 | 15680 | nad2 | + | Eurypanopeus depressus MCG | 82.16 | 98 | 0.0 | Present study | NADH dehydrogenase subunit 2 [Eurpanopeus depressus] | 91.28 | 98 | 2e-168 | Present study | |
15697 | 15764 | trnW(tga) | + | - | — | — | — | — | — | — | — | — | — | |
15764 | 15827 | trnC(tgc) | — | Panopeus herbstii MCG | 98.44 | 100 | 4e-31 | Present study | — | — | — | — | — | |
15828 | 15892 | trnY(tac) | — | Panopeus herbstii MCG | 92.31 | 100 | 2e-24 | Present study | — | — | — | — | — |
There were 112 brachyuran mitogenomes (see Supplementary material
All previously reported gene orders for the Brachyura were annotated according to
The mitochondrial genomes of the panopeid crabs used in this study were closed circular molecules containing 13 protein coding genes, 22 tRNA genes, 2 rRNA genes, and a single control region (CoRe) (Fig.
Annotated Circa plots for the circular mitochondrial genomes of Eurypanopeus depressus, Panopeus herbstii and Rithropanopeus harrisii. Each mitogenome is represented by a thick circular black line near the centre of the plot. Protein coding genes are on the outside of this line (negative = dark violet, positive = maroon). Non-coding RNA genes are on the inside of this line (negative = light violet, positive = light maroon). The genome sizes are written in the centre of each plot. The protein coding gene names are represented in the outer most circle (dark grey). The ncRNA gene names are listed in the second internal circle (light grey). The green rectangle labelled “CoRe” indicates the putative control region of the mitochondrial genomes. Figure
The nucleotide composition of the complete E. depressus mitochondrial genome was as follows: A=5442 (34.32%), T=5509 (34.75%), G=1652 (10.42%), C=3251 (20.51%). The A+T and G+C contents were 69.07% and 30.93%, respectively. The protein coding regions include 7 NADH dehydrogenases (nad1–nad6 and nad4L), three cytochrome c oxidases (cox1–cox3), 2 ATPases (atp6 and atp8) and 1 cytochrome b (cob) and account for 10,838 bp of the mitogenome. The 22 rRNA genes present in the mitogenome range in size from 62 (trnD)–71 (trnL1) bp in length, and the ribosomal RNA genes rrnL (16S) and rrnS (12S) have a length of 1393 bp and 817 bp, respectively. The 13 protein coding genes and majority of the ncRNA sequences showed similarity among the panopeid crabs used in this study (Table
The nucleotide composition of the complete P. herbstii mitochondrial genome was as follows: A=5520 (34.91%), T=5687 (35.97%), G=1627 (10.29%), C=2980 (18.85%). The A+T and the G+C contents were 70.87% and 29.13%, respectively. The protein coding region contains 7 NADH dehydrogenases (nad1–nad6 and nad4L), three cytochrome c oxidases (cox1–cox3), 2 ATPases (atp6 and atp8) and 1 cytochrome b (cob) and accounts for 10947 bp of the mitogenome of P. herbstii. The 22 rRNAs present in the mitogenome range in size from 63 (trnD, trnA, trnR) – 69 (trnL1, trnQ, trnM) bp in length, and the ribosomal RNA genes rrnL (16S) and rrnS (12S) have a length of 1392 bp and 820 bp, respectively. All 13 protein coding genes showed high similarity to the panopeid crabs used in this study. The ncRNAs all showed similarity to decapod crustaceans with the majority having high similarity with the Panopeidae (Table
The nucleotide composition of the complete R. harrisii mitochondrial genome was as follows: A=5595 (34.21%), T=5873 (37.00%), G=1556 (9.82%), C=2866 (17.99%). The A+T and the G+C contents were 72.20% and 27.080%, respectively. The protein coding region contains 7 NADH dehydrogenases (nad1–nad6 and nad4L), three cytochrome c oxidases (cox1–cox3), 2 ATPases (atp6 and atp8) and 1 cytochrome b (cob) and account for 10,848 bp of the mitogenome. The 22 rRNAs present in the mitogenome range in size from 63 (trnD, trnG, trnA) – 69 (trnL1, trnQ) bp in length, and the ribosomal RNA genes rrnL (16S) and rrnS (12S) have a length of 1400 bp and 817 bp, respectively. The 13 protein coding genes showed high similarity with the panopeid crabs used in this study (Table
To establish where the panopeid crabs align within the Eubrachyrua, amino acid and nucleotide sequences from 112 mitogenomes comprising 77 genera from 28 families of brachyuran crabs were used along with the three new mitogenomes (Fig.
Four distinct clades were identified (Fig.
The panopeid crab species E. depressus, P. herbstii and R. harrisii formed a branch for the family Panopeidae (Fig.
Maximum-likelihood phylogenetic relationships derived from 112 species of brachyuran crabs, using 13 concatenated amino acid sequences (cox1–cox3, cob, atp6, atp8, nad1, nad5, nad4, nad4-L). Some families have been collapsed for increased clarity (triangles). Black circles on nodes represent an SH-aLRT and bootstrap support of greater than 90/90. Stars (*) indicate areas on the tree with taxonomic conflicts related to previous literature. The symbol α indicates the family Xanthidae; β indicates the family Panopeidae. See Supplementary material
Maximum-likelihood phylogenetic relationships derived from 112 species of brachyuran crabs, using 15 concatenated nucleotide sequences (cox1–cox3, cob, atp6, atp8, nad1, nad5, nad4, nad4-L, rrnL, rrnS). Some families have been collapsed for increased clarity (triangles). Black circles on nodes represent an SH-aLRT and bootstrap support of greater than 90/90. Stars (*) indicate areas on the tree with taxonomic conflicts related to previous literature. The symbol α indicates the family Xanthidae; β indicates the family Panopeidae. See Supplementary material
The family Sesarmidae (10 mitogenomes) appears to be polyphyletic. Rather than grouping together, the genus Chiromantes is split, where C. dehaani aligns with Sesarma neglectum (amino acids- Sh-aLRT/UFBoot: 99.5/100; nucleotides- Sh-aLRT/UFBoot: 100/100), and C. haematocheir aligns with Sesarmops sinensis (amino acids- Sh-aLRT/UFBoot: 99.7/100; nucleotides- Sh-aLRT/UFBoot: 100/100) (Fig.
The gene arrangements for the panopeid crabs E. depressus, P. herbstii and R. harrisii (Fig.
The mitogenomes of the crabs in the family Panopeidae all shared a ~600 bp long intergenic spacer between the rrnS and trnV ncRNA genes (E. depressus, 618 bp; P. herbstii, 622 bp; R. harrisii, 644 bp) representing the control region (CoRe) (Fig.
Gene orders (-GO) found among brachyuran crabs. Red boxes indicate protein coding genes. Blue boxes indicate tRNA’s. Green boxes indicate rRNAs. Purple boxes indicate the control region (CoRe). The red lines along the bottom of the gene orders represents areas within the gene order that are located on the negative strand. Not shown are the 9 unique gene orders for the freshwater crabs (see
This study provides the first mitochondrial genomes for three members of the Panopeidae and an updated concatenated mito-phylogenetic analysis for the Eubrachyura (excluding nuclear genetic data), informing upon the systematics of multiple families and higher taxonomic rankings. In addition, the mitochondrial genomes for members of the Panopeidae are identified with a consensus gene arrangement shared with other Xanthoidea (XanGO). These results advance our systematic understanding of the brachyurans through the exploration of mitochondrial genomics and gene synteny rearrangement events.
The mitogenomes of the panopeid crabs E. depressus, P. herbstii and R. harrisii support the position of the Panopeidae within the Heterotremata, helping to build/support the branch belonging to the superfamily Xanthoidea (Ng 2008). Along this branch, the Xanthidae and Panopeidae form sister groups, additionally supported by previous genetic data using five or less mitochondrial and nuclear genes (Thoma 2009; Lai 2011; Thoma 2014). The genera within these families have been historically identified as polyphyletic (Thoma 2009) and the limited number of mitogenomes available makes it difficult to determine their validity. We acknowledge that the families Xanthidae and Panopeidae both occur in two forms: sensu stricto and sensu lato (Ng 2008). There are 4 publicly available mitogenomes for the Xanthidae (GenBank) and we provide 3 additional mitogenomes for the Panopeidae. We have treated these families in their simple form due to the lack of genetic information to split them further. As more mitogenomes become available, the validity of the two forms should be revisited.
Several taxonomic conflicts appear when considering mitogenetics surrounding the Xanthoidea. First, based on morphology and limited mitogenome availability, the genus Leptodius is considered a member of the family Xanthidae. However, despite this genus having 12 separate species, only one mitogenome (the species L. sanguineus) is available for analysis. Previous studies showed that L. sanguineus aligns closely with other members of the Xanthidae (Sung 2016; Karagozlu 2018; Xie 2018; Ma 2019), but these studies use fewer brachyuran mitogenomes in their analysis prior to our study. When considering all mitogenomes available for the Brachyura in our investigation, L. sanguineus aligns more closely with the members of the family Oziidae, rather than the Xanthidae. This interesting observation merits further exploration.
Gene arrangement changes were once thought to be rare (
The brachyurans include several families found in the deep sea. Two of them are represented herein: Bythograeidae and Xenograpsidae. Bythograeidae possess the BraGO arrangement plesiomorphic for Brachyura, while Xenograpsidae have their own gene arrangement (XenGO). In contrast, the freshwater crab family Potamidae has 9 different gene arrangements (Zhang 2020). Brachyuran crabs represent both cases: the adaptation from a marine to a freshwater environment was likely harsh and may have resulted in several new gene arrangements, while in contrast, the evolution of crabs to the deep-sea benthos resulted in some retaining the ancestral gene order in the face of a new environmental extreme. Therefore, when considering crabs, living within harsh environments does not seem to be the only answer to gene arrangement plasticity, but perhaps requires consideration at the finer scale of environmental adaption. Similar findings have been reported by
A comparison of the eubrachyuran subsections shows that Heterotremata has a higher diversity of gene arrangements than Thoracotremata. Both subsections share species whose gene arrangement follows the basic BraGO pattern. Aside from the BraGO, Thoracotremata only has 3 unique gene arrangements while Heterotremata has 8 unique gene arrangements (including the herein newly established XanGO). This does not include the gene arrangements for the freshwater crabs in the superfamilies Potamoidea and Gecarcinucoidea. The freshwater crabs have more unique gene arrangements than the known Heterotremata.
The panopeid crabs E. depressus, P. herbstii and R. harrisii all have the trnV gene transposed from between the rrnL and rrnS genes to a location past the CoRe. This differs from the PanGO, BraGO, SesGO, XenGO, DamGO, MajGO and DynGO, which all have the trnV gene located between the rrnL and rrnS genes, with the CoRe following the rrnS gene. The xanthid crabs E. anaglyptus, L. sanguineus, A. floridus and A. integerrimusi all share the latter gene arrangement, suggesting that it might be a conserved arrangement within Xanthoidea and thus support our interpretation of the new Xanthoidea gene arrangement (XanGO). The intergenic spacer found between the rrnS and trnV genes in panopeids appears to be the putative location of the CoRe for these species and is shared with xanthid species, E. anaglyptus, A. floridus and A. integerrimus. All have similarly sized intergenic spacers (600–750 bp long) at this location, suggesting that this may be the location of the CoRe across Xanthoidea. Apart from L. sanguineus, the Xanthidae all follow the new gene arrangement XanGO. Leptodius sanguineus follows the plesiomorphic brachyuran gene arrangement BraGO and based on its amino acid sequences, it groups more closely with the family Pilumnidae than the members of the Xanthidae or the panopeids presented here; however, nodal support is low, meriting further study and sequencing of closer relatives. Higher nodal support is offered with the nucleotide tree, where L. sanguineus groups with Epixanthus frontalis from Oziidae rather than with the xanthids. Based on the molecular taxonomy and its gene arrangement, the placement of L. sanguineus within Xanthidae appears to be invalid and in need of revision, adding to our explanation above.
The mitogenome analysis we performed also supports the renaming of two gene arrangements and confirms the correct gene sequence for another. Two mitogenomes were available for the pilumnid crabs, Echinoecus nipponicus and Pilumnus vespertilio. They follow the gene arrangement reported by
This study provides an updated mitophylogeny for the Brachyura, utilizing all available mitogenomes, along with the first mitogenomes for the Panopeidae, a highly abundant group of ecologically important estuarine crabs with a limited phylogenetic understanding. Our data support the subsection, Potamoida, within the Eubrachyura. The addition of E. depressus¸ P. herbstii and R. harrisii mitogenomes provides a greater phylogenetic understanding of a group that has been taxonomically challenging in the past. Moreover, the addition of mitogenomes from the Panopeidae further supports the split of the Xanthoidea into multiple families. The novel gene arrangement we describe within the Heterotremata, increases the total number of unique gene arrangements within this subsection to eight. Whilst our results clarify some phylogenetic relationships, they also highlight the need for further study of the genus Leptodius which appears to be incorrectly placed within the subfamily Xanthoidea. Greater sequencing efforts will provide more comparative data for these underrepresented crab groups, and should include the incorporation of nuclear genetic data where possible.
AMHB collected the crabs used in the study. JB performed the extraction and bioinformatic processing/assembly of the mitogenomes. LAJ and JB performed the phylogenetics and gene similarity assessments. Gene order analysis and annotation was performed by LAJ and JB. LAJ, AMHB, KAM, DCB and JB contributed to the writing of the manuscript.
The authors declare no competing interests.
Thanks to Mr. Christopher Moore, who helped to collect the crabs used in the study. Funding for the research and staff time was attained from East Carolina University (AMHB and KAM), the University of Florida (LAJ and DCB) and National Horizon Centre, Teesside University (JB).
Table S1
Data type: .docx
Explanation note: NCBI accession numbers for species used to conduct phylogenetic analysis.