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Systematic assessment of the Panopeidae and broader Eubrachyura (Decapoda: Brachyura) using mitochondrial genomics
expand article infoLucas A. Jennings, April M. H. Blakeslee§, Krista A. McCoy|, Donald C. Behringer, Jamie Bojko#
‡ University of Florida, Gainesville, United States of America
§ East Carolina University, Greenville, United States of America
| Florida Atlantic University, Fort Pierce, United States of America
¶ Teesside University, Middlesbrough, United Kingdom
# University of Leeds, Leeds, United Kingdom
Open Access

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.

Keywords

Xanthidae, Panopeus, Eurypanopeus, Rhithropanopeus, mud crab, marine, genomics

1. Introduction

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; Jia et al. 2018; Tan et al. 2018; Ma et al. 2019; Tan et al. 2019). Marine Brachyura boast a broad range of morphological and ecological diversity, leading to a complex taxonomy (Yong-kun et al. 2014). Historically, brachyurans were divided into two sections: the Eubrachyura and the Podotremata, with the Eubrachyura being further divided into the subsections: Heterotremata and Thoracotremata (Guinot 2013). The sub-sectioning of these Eubrachyura is based almost entirely on typological morphology (particularly the genital openings) and has been subject to debate with regards to monophyly. Due to the morphological complexity of this group, genetic tools and analytical methods are typically used to resolve systematic discrepancies (Basso et al. 2017; Bai et al. 2018; Jia et al. 2018; Tan et al. 2018; Wang et al. 2018).

High throughput sequencing (HTS) has proven effective in advancing and resolving taxonomies (Tan et al. 2018). Early studies on brachyuran mitogenomics relied on long PCR and primer walking techniques to read and assemble the mitogenome (Yamauchi 2003; Miller 2005). The rapid sequencing and assembly of the mitochondrial genome (mitogenome) using HTS has proven to be a powerful tool for conducting phylogenetic studies of eukaryotes (Gan et al. 2018). The small size of mitogenomes (~ 14–16 kb), potential for high mutation rate, and a simple closed structure, make them an ideal marker for inferring an organism’s mitogenetic phylogeny (Boore 1999). Brachyura has been classified into 93 families with over 7000 species (Ng 2008); for 111 species (representing 28 families) the complete mitogenomes are known (NCBI and Supplementary material 1). The Brachyuran genomes that have been sequenced thus far all are between 10-25 kb in length. Much of these data represent three brachyuran families [Portunidae (n = 15); Varunidae (n = 14); Sesarmidae (n = 10)]. The remaining 25 families have < 5 mitogenomes sequenced per family, many of them only having 1 (see Supplementary material). Recent publications using mitogenomics have challenged the validity of the morphologically founded Eubrachyuran subsections of Heterotremata and Thoracotremata. For example, freshwater crabs in the family Potamidae fall into the Heterotremata based on morphology but based on mitogenomics align with members of the Thoracotremata (Basso 2017; Bai et al. 2018; Tan et al. 2018).

Mitogenomes also offer insights into gene arrangement, which can have diagnostic properties at different systematic levels (Boore 1998; Boore 2000; Moret 2001; Perseke et al. 2008; Zhuang 2010; Babbucci 2014; Mindell 2016; Nakjima et al. 2016; Zhang 2020). Within the Malacostraca, mitogenome gene arrangements are conserved within certain groups (Shen 2011; Tan et al. 2019), which allows for simple comparisons at different taxonomic levels. Grouping the Crustacea with the Insecta to form the Pancrustacea has strong support based on the near-identical arrangement of the shared genes across taxa (Boore 1998). However, gene arrangement can vary greatly within crustacean orders. Specifically, in Brachyura many species share a gene arrangement that is thought to be ancestral to Brachyura (termed BraGO).However, recent research has shown that most groups deviate from this pattern forming new arrangements at the family and subfamily levels (Basso 2017; Tan et al. 2018; Wang et al. 2018; Wang 2020a; Wang 2020b). BraGO differs from the Pancrustacea genomic order (PanGO) by a transposition of the gene trnH from a location between the nad5 and nad4 genes to a location between the trnD and trnF genes (Basso 2017). To date, 20 different gene arrangements have been identified within the Brachyura (Basso 2017; Tan et al. 2018; Zhang 2020a), but many brachyuran groups remain un-sequenced and this syntenic diversity could be much higher.

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 (Williams 1984; Schubart 2000). Studies using conventional PCR to amplify and sequence mitochondrial and nuclear markers revealed that the genera Eurypanopeus and Panopeus are polyphyletic (Schubart 2000). Similarly, studies on the panopeid genus Hexapanopeus using 12S and 16S genes as markers have also suggested that this genus is polyphyletic (Thoma 2009). Later studies using three mitochondrial markers (COI, 12S and 16S) and three nuclear markers [18S, enolase (ENO) and Histone H3 (H3)] revealed that Xanthoidea is monophyletic, but its two families are not and are in need of taxonomic revision (Thoma 2014).

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.

2. Materials and methods

2.1. Specimen collection and dissection

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.

2.2. DNA extraction, sequencing and assembly

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 (Bankevich et al. 2012) with default parameters and k-mer lengths: 21, 33, 55, 77 and 99. The resulting datasets provided a series of contigs that were compared to the NCBI nr database using BLASTx. The mitochondrial genomes of E. depressus (574.088X coverage), P. herbstii (122.084X coverage) and R. harrisii (400.520X coverage) were each identified and circularized. Confirmation of their sequence coverage was conducted using CLC genomics workbench v.12.

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 13). The completed genomes were then annotated graphically using Circa (http://omgenomics.com/circa). The genomes are deposited in GenBank under accession numbers MN399962 (E. depressus), MT024989 (R. harrisii), and MT024990 (P. herbstii).

Table 1.

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
Table 2.

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
Table 3.

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

2.3. Phylogenetic and mitochondrial gene order assessment

There were 112 brachyuran mitogenomes (see Supplementary material 1) obtained from the GenBank database (NCBI) for phylogenetic comparison using the Brachyura taxonomic ID (txid6752) and filtering results to yield DNA sequences of 10,000–25,000 bp (search date: January 2020). The amino acid and nucleotide sequences were retrieved and annotated from these genomes (13 and 15 sequences, respectively) using the Mitophast pipeline (Tan et al. 2015) which downloads each gene in the mitochondrial genome as separate files. The amino acid and nucleotide sequences files were then aligned individually using MAFFT in Geneious (v10.0.2), trimmed to the smallest sequence and concatenated using Geneious. Phylogenetic analyses were conducted in IQtree (Trifinopoulos 2016), which computed the most appropriate evolutionary model (mtMet+F+I+G4) according to BIC for both the amino acid sequences and the nucleotide sequences. A maximum likelihood tree using the amino acid sequences was created using 1000 bootstrap replicates and an SH-aLRT branch test (Guindon 2010) over 3733 positions; the tree had a log score of –157295.9511. A maximum likelihood tree using the nucleotide sequences was created using 1000 bootstrap replicates and an SH-aLRT branch test over 13790 positions; the tree had a log score of –598390.2632. The resulting trees were annotated using FigTree (http://tree.bio.ed.ac.uk/software/figtree). Both trees were rooted with the mitogenomes of Coenobita brevimanus (KY352233), C. rugosus (KY352235) and C. perlatus (KY352234).

All previously reported gene orders for the Brachyura were annotated according to Basso et al. (2017) and Tan et al. (2018). Pairwise comparisons of the gene orders were performed using CREx software (Bernt et al. 2007) at common intervals. The nomenclature for the gene orders follows Basso et al. (2017). MITOS was used to determine the putative location of the control region (CoRe) for E. depressus, P. herbstii, R. harrisii, Etisus anaglyptus, Leptodius sanguineus, Atergatis floridus, and A. integerrimus through manual examination of the start and stop codons of the open reading frames to look for intergenic spacers. The CoRe for the crabs in the family Potamidae were obtained from Genbank. The CoRes for Echinoecus nipponicus and Pilumnus vespertilio were determined using MITOS following the same method as for the Panopeidae.

3. Results

3.1. The mitochondrial genomes of panopeid crabs

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. 1). The E. depressus mitogenome was 15,854 bp in length. The P. herbstii mitogenome was 15,812 bp in length. The R. harrisii mitogenome was 15,967 bp in length. As with most brachyurans, the rrnL (16S) and rrnS (12S) genes were located on the negative strand, as are the nad5, nad4, nad4L, nad1 and 8 tRNA genes (Table 1).

Figure 1. 

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 1 layout: Portrait. Associated with section 3.1

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 (nad1nad6 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 1).

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 2).

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 3).

3.2. Phylogenetics

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. 2). Two sequences that are publicly available for brachyurans were not included in our analysis due to inconsistences with the sequences. (1) The protein sequences for Gecarcoidea natalis contained ambiguous amino acid identifications, resulting in poor alignment with other members within the superfamily Grapsoidea. (2) The protein sequences for Pyrhila pisum aligned poorly with other members of the Brachyura; however, there were no missing protein codes. When tested in BLASTp, the proteins for P. pisum yielded low identity with other brachyurans; < 60% identity in most cases.

Four distinct clades were identified (Fig. 2). One clade belongs to crabs in the subsection Heterotremata (n=40), a second belongs to crabs in the subsection Thoracotremata (n=44) and a third belongs to crabs in the section Podotremata (n=7). The fourth clade belongs to the ‘Old World’ freshwater crabs in the superfamilies Potamoidea and Gecarcinucoidea (n=20). This fourth clade forms a subsection termed Potamoida, a sister group to Thoracotremata. The split between the Heterotremata and the Potamoida/Thoracotremata clades is well supported using both amino acid sequences (Sh-aLRT/UFBoot: 100/100) as well as nucleotide sequences (Sh-aLRT/UFBoot:100/100). The Potamoida and Thoracotremata split is also well supported using both sequence types (amino acids- Sh-aLRT/UFBoot: 98.9/99; nucleotides- Sh-aLRT/UFBoot: 92.9/98).

The panopeid crab species E. depressus, P. herbstii and R. harrisii formed a branch for the family Panopeidae (Fig. 2 and Fig. 3; “β”) aligned alongside the xanthid branch to form the superfamily Xanthoidea (amino acids- Sh-aLRT/UFBoot: 100/100; nucleotides- Sh-aLRT/UFBoot: 100/100). The xanthid branch contains members of the Xanthidae family: E. anaglyptus, A. floridus and A. integerrimus (Fig. 2 and Fig. 3; “α”). When considering its amino acid sequences, the crab species Epixanthus frontalis from the family Oziidae aligns with the Xanthoidea superfamily with moderate support (Sh-aLRT/UFBoot: 89.4/94) (Fig. 2). The nucleotide sequences for E. frontalis show a similar pattern; however, Leptodius sanguineus is part of the branch with middling support (Sh-aLRT/UFBoot: 66.7/93) (Fig. 3). Based on amino acid comparison, L. sanguineus (considered a member of the Xanthidae) aligns between E. frontalis and members of the Pilumnidae, on a branch separate from other xanthid crabs (Sh-aLRT/UFBoot: 16/66) (Fig. 2). The amino acid phylogeny suggests that the hydrothermal vent crabs in the family Xenograpsidae align with the terrestrial crabs in the family Ocypodidae (Sh-aLRT/UFBoot: 83.2/72), yet the nucleotide sequences suggest that the xenograpids form their own branch alongside of the sesarmid crabs (Sh-aLRT/UFBoot: 100/99).

Figure 2. 

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 1 for a list of the species used and their accession numbers involved in this analysis.

Figure 3. 

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 1 for a list of the species used and their accession numbers involved in this analysis.

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. 2 and Fig. 3).

3.3. Gene arrangement among the Brachyura, incorporating the Panopeidae

The gene arrangements for the panopeid crabs E. depressus, P. herbstii and R. harrisii (Fig. 3) correspond in synteny to other sequenced xanthid species: E. anaglyptus, A. floridus and A. integerrimus. This gene arrangement differs from both the PanGO and BraGO, where the rrnL and rrnS are adjacent to each other and the trnV is transposed past the CoRe (Table 1). The gene order for the xanthid crab species L. sanguineus reported by Tan et al. (2018) is different from the other xanthids presented in this study, following the basic BraGO pattern rather than the shared pattern of the superfamily Xanthoidea. Based on the CREx test, the new gene arrangement XanGO shares 870 common intervals with PanGO and 988 common intervals with BraGO (Fig. 3), suggesting it to be a low-level rearrangement relative to the common gene arrangements. The new XanGO is most different to the MaVaGO, sharing only 80 common intervals.

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. 1). The CoRe in the panopeid mitogenomes are A + T skewed (78.40–80.22%) and contain the repeated motifs TA (125–107), AT (112–104), TAA (47–39), TTA (30–40), ATA (43–35) and TAT (41–37). The mitogenomes of the xanthid crabs used in this study also have similar sized intergenic spacers in this region, suggesting that this is the putative location of the CoRe for members of the superfamily Xanthoidea. The CoRe nucleotide sequence for all species within Xanthoidea were isolated and run through BLASTn, resulting in a lack of any significant similarity, suggesting high mutability.

Figure 4. 

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 Zhang et al. 2020). The CREx results are listed for the different gene orders. In the associated table, gene orders with high similarity (> 1000) have red boxes while those with low similarity (< 200) have blue boxes. Intermediate similarity remains white.

4. Discussion

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.

4.1. Xanthid systematics considering panopeid mitogenomic data

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.

4.2. Mitogenomic gene arrangements across the Brachyura

Gene arrangement changes were once thought to be rare (Boore 2000) but with greater availability of mitogenome sequencing, it appears that changes in gene arrangements can be common across groups. For example, gene order is conserved within Osteichthyes and some subgroups of Mammalia, while it varies strongly in e.g. Ctenophora (Arafat 2018), Mollusca (Guerra 2018), Hymenoptera (Dowton 1999) and Anomura (Tan et al. 2018). For Crustacea, some species within the Stomatopoda, Amphipoda and Dendrobranchiata still carry the PanGO ground pattern of Pancrustacea (Shen 2011), while no sequenced species within the Brachyura have retained this gene order. Studies on gene order rearrangement are ongoing with some hypothesizing that the evolution to living within harsh environments, such as the deep sea or hydrothermal vents, can lead to new gene synteny (Nakajima 2016; Gan 2018; Tan et al. 2019).

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 Tan et al. (2019) who found little evidence for linking gene order rearrangements with adaptations to extreme environments, concluding that these cues are poorly understood and merit a more detailed approach.

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 Tan et al. (2018) and differ from BraGO in having the trnL gene transposed from its location between the cox1 and cox2 genes to a location between the second trnL and rrnL genes. This gene arrangement was reported by Tan et al. (2018) as number 12, but we propose Pilumnidae gene order (PilGO) to follow the original gene nomenclature determined by Basso et al. (2017). Similarly, the gene arrangement reported as number 5 by Tan et al. (2018) we rename to the Somanniathelphusa gene order (SomGO). Basso et al. (2017) report the gene arrangement GeoGO as having the trnL gene between the cox1 and cox2 genes, but based on the gene arrangement listed in Genbank, this is nonconcurrent. The correct gene arrangement was reported by Tan et al. (2019) and is supported here with the addition of the mitogenome for Geothelphusa sp. (MG674171), where the trnL gene is located between nad1 and the second trnL gene. This corrected nomenclature should be incorporated into further taxonomic assessments.

4.3. Conclusions

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.

5. Author contributions

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.

6. Competing interests

The authors declare no competing interests.

7. Acknowledgements

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).

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