The mitogenome of the black yabby, Geocharax gracilis, was sequenced using the MiSeq Personal Sequencer. It has 15,924 base pairs consisting of 13 protein-coding genes, 2 ribosomal subunit genes, 23 transfer RNAs, and a non-coding AT-rich region. The base composition of G. gracilis mitogenome is 32.18% for T, 22.32% for C, 34.83% for A, and 10.68% for G, with an AT bias of 67.01%. The mitogenome gene order is typical for that of parastacid crayfish with the exception of some minor rearrangements involving tRNA genes.
Although it is possible to recover the complete mitogenome directly from shotgun sequencing data, currently reported methods and pipelines are still relatively time consuming and costly. Using a sample of the Australian freshwater crayfish Engaeus lengana, we demonstrate that it is possible to achieve three-day turnaround time (four hours hands-on time) from tissue sample to NCBI-ready submission file through the integration of MiSeq sequencing platform, Nextera sample preparation protocol, MITObim assembly algorithm and MITOS annotation pipeline.
The infraorder Anomura consists of a morphologically and ecologically heterogeneous group of decapod crustaceans, and has attracted interest from taxonomists for decades attempting to find some order out of the seemingly chaotic diversity within the group. Species-level diversity within the Anomura runs the gamut from the "hairy" spindly-legged yeti crab found in deep-sea hydrothermal vent environments to the largest known terrestrial invertebrate, the robust coconut or robber crab. Owing to a well-developed capacity for parallel evolution, as evidenced by the occurrence of multiple independent carcinization events, Anomura has long tested the patience and skill of both taxonomists attempting to find order, and phylogeneticists trying to establish stable hypotheses of evolutionary inter-relationships. In this study, we performed genome skimming to recover the mitogenome sequences of 12 anomuran species including the world's largest extant invertebrate, the robber crab (Birgus latro), thereby over doubling these resources for this group, together with 8 new brachyuran mitogenomes. Maximum-likelihood (ML) and Bayesian-inferred (BI) phylogenetic reconstructions based on amino acid sequences from mitogenome protein-coding genes provided strong support for the monophyly of the Anomura and Brachyura and their sister relationship, consistent with previous studies. The majority of relationships within families were supported and were largely consistent with current taxonomic classifications, whereas many relationships at higher taxonomic levels were unresolved. Nevertheless, we have strong support for a polyphyletic Paguroidea and recovered a well-supported clade of a subset of paguroids (Diogenidae + Coenobitidae) basal to all other anomurans, though this requires further testing with greater taxonomic sampling. We also introduce a new feature to the MitoPhAST bioinformatics pipeline (https://github.com/mht85/MitoPhAST) that enables the extraction of mitochondrial gene order (MGO) information directly from GenBank files and clusters groups based on common MGOs. Using this tool, we compared MGOs across the Anomura and Brachyura, identifying Anomura as a taxonomic "hot spot" with high variability in MGOs among congeneric species from multiple families while noting the broad association of highly-rearranged MGOs with several anomuran lineages inhabiting extreme niches. We also demonstrate the value of MGOs as a source of novel synapomorphies for independently reinforcing tree-based relationships and for shedding light on relationships among challenging groups such as the Aegloidea and Lomisoidea that were unresolved in phylogenetic reconstructions. Overall, this study contributes a substantial amount of new genetic material for Anomura and attempts to further resolve anomuran evolutionary relationships where possible based on a combination of sequence and MGO information. The new feature in MitoPhAST adds to the relatively limited number of bioinformatics tools available for MGO analyses, which can be utilized widely across animal groups.
To further understand the evolutionary history and mitogenomic features of Australia's highly distinctive freshwater crayfish fauna, we utilized a recently described rapid mitogenome sequencing pipeline to generate 24 new crayfish mitogenomes including a diversity of burrowing crayfish species and the first for Astacopsis gouldi, the world's largest freshwater invertebrate. Whole mitogenome-based phylogeny estimates using both Bayesian and Maximum Likelihood methods substantially strengthen existing hypotheses for systematic relationships among Australian freshwater crayfish with evidence of pervasive diversifying selection and accelerated mitochondrial substitution rate among the members of the clade representing strongly burrowing crayfish that may reflect selection pressures for increased energy requirement for adaptation to terrestrial environment and a burrowing lifestyle. Further, gene rearrangements are prevalent in the burrowing crayfish mitogenomes involving both tRNA and protein coding genes. In addition, duplicated control regions were observed in two closely related Engaeus species, together with evidence for concerted evolution. This study significantly adds to the understanding of Australian freshwater crayfish evolutionary relationships and suggests a link between mitogenome evolution and adaptation to terrestrial environments and a burrowing lifestyle in freshwater crayfish.