ORDER within the chaos: Insights into phylogenetic relationships within the Anomura (Crustacea: Decapoda) from mitochondrial sequences and gene order rearrangements

https://doi.org/10.1016/j.ympev.2018.05.015Get rights and content

Highlights

  • This study more than doubles complete mitogenome resources for the decapod infraorder Anomura.

  • New feature in MitoPhAST version 2.0 extracts and clusters mitochondrial gene orders (MGO) information.

  • Anomura is a MGO “hot spot”, with major events associated with lineages in extreme niches.

  • Highly variable MGO patterns are found in brachyuran freshwater crabs.

  • A putative ancestral anomuran MGO is predicted based on current available data.

Abstract

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.

Introduction

The crustacean decapod infraorder Anomura (meaning “varied tailed”), formerly referred to as the Anomala (meaning “irregular”), lives up to its name and has challenged many taxonomists and phylogeneticists who have struggled to resolve the evolutionary relationships among the extraordinary diversity of species. While the anomurans are highly heterogeneous morphologically, they do appear to form a distinct monophyletic lineage that is consistently placed as the sister group to the true crabs, the Brachyura (Ahyong and O’Meally, 2004, Dixon et al., 2003, Scholtz and Richter, 1995, Shen et al., 2013, Tan et al., 2015). Confusingly, a number of anomuran groups are also referred to as “crabs” due to their resemblance to brachyurans, but it is now well established that a crab-like body form has independently evolved several times, in a process referred to as carcinization (Cunningham et al., 1992).

Anomura consists of approximately 2450 extant species (De Grave et al., 2009) with a broad range of ecological specializations and with various lineages having successfully adapted to freshwater, terrestrial and diverse marine environments, including the abyssal depths such as deep-sea hydrothermal vents. With an extraordinary array of body forms, functions and sizes, this group includes the asymmetrical and symmetrical hermit crabs, squat lobsters with elongated folded pleons (e.g. aeglids, yeti crabs) and crab-like species (e.g. king crabs, porcelain crabs, hairy stone crabs) including the largest terrestrial invertebrate, the infamous robber or coconut crab. Crab-like morphologies (Cunningham et al., 1992) within this group have been well studied, and it is hypothesized to have evolved from multiple independent carcinization events, i.e. the tendency of a non-crab ancestor to morph into the crab-like form of generally broadened, fully-calcified carapace and reduced pleon fully tucked under the body (Ahyong et al., 2009, Bracken-Grissom et al., 2013, Keiler et al., 2015, Keiler et al., 2017, Morrison et al., 2002, Noever and Glenner, 2017, Tsang et al., 2011). The tendency for anomuran lineages to undergo this form of parallel evolution, unrelated to the exploitation of specific habitats or lifestyles, has made it difficult to establish robust hypotheses of phylogenetic relationships among them based on morphological characters alone. Thus, while morphological inferences may have been confused by the problems of correctly interpreting plesiomorphies and traits influenced by parallel and convergent evolution, alternative perspectives on anomuran relationships are emerging from molecular information (Ahyong et al., 2009, Bracken-Grissom et al., 2013, Hamasaki et al., 2017, Morrison et al., 2002, Pérez-Losada et al., 2002, Tsang et al., 2011).

Molecular studies have reshaped our understanding of evolutionary relationships within Anomura, leading to the reclassifications of major taxonomic groups over the past decade (Ahyong et al., 2010, McLaughlin et al., 2007, Schnabel et al., 2011, Schnabel and Ahyong, 2010). Previously considered to consist of only three major lineages Hippoidea, Paguroidea and Galatheoidea (Ahyong et al., 2009), this infraorder has since been reclassified into 7 superfamilies, which includes the elevation of Aeglidae and Lithodidae to superfamily status (the Aegloidea and Lithodoidea) (McLaughlin et al., 2007), the restriction of the superfamily Galatheoidea to include just the families Galatheidae and Porcellanidae (Ahyong et al., 2010, Schnabel et al., 2011), and the transfer of the Chirostylidae and the Kiwaidae to a new superfamily, the Chirostyloidea (Schnabel et al., 2011, Schnabel and Ahyong, 2010). While the monophyly of Anomura is now well accepted (Ahyong and O’Meally, 2004, Porter et al., 2005, Scholtz and Richter, 1995, Schram and Dixon, 2004, Shen et al., 2013, Tan et al., 2015, Toon et al., 2009, Tsang et al., 2011), many of the inter-relationships within and among the major anomuran lineages are still largely unresolved (Ahyong et al., 2010, Bracken-Grissom et al., 2013, Lemaitre and McLaughlin, 2009, McLaughlin et al., 2007, Schnabel and Ahyong, 2010, Tsang et al., 2011).

Given the level of interest in anomuran evolution and classification, it is surprising that only ten complete anomuran mitogenomes are currently available on the NCBI database, making it one of the most poorly represented of the major crustacean groups for these resources (Tan et al., 2018). At the time of their study, Tan et al. (2018) showed that only 0.4% of the diversity of this taxon have mitogenome representation on the NCBI RefSeq database (in contrast with other groups with much better representation, e.g. 6.9% for Astacidea and 7.9% for Achelata). The mitogenome has been demonstrated to effectively resolve relationships in studies on various animal groups (Kayal et al., 2015, Yuan et al., 2016), not only for phylogenetic reconstruction but also on the basis of novel gene orders, which provide an additional source of phylogenetic information (Basso et al., 2017, Morrison et al., 2002, Weigert et al., 2016). As a group that lacks genomic resources due to large reported genome sizes ranging from 1 Gbp to 40 Gbp (http://www.genomesize.com), decapod crustaceans benefit from the relative ease of recovering the mitogenome (as opposed to nuclear genes), further adding to the usefulness of the mitogenome as a source of molecular and phylogenetic information.

Previous methods for obtaining complete mitogenome sequences have primarily relied on PCR and Sanger sequencing techniques, while new genome skimming methods utilizing high-throughput short-read sequencing (Gan et al., 2014, Grandjean et al., 2017) allow rapid and cost-effective acquisition of complete mitogenome sequences. In addition, these new methods allow recovery of mitogenome sequences from older museum specimens that have previously been considered problematic due to DNA degradation (Besnard et al., 2016, Grandjean et al., 2017, Tan et al., 2018). Not only can the complete mitogenome of a species be efficiently recovered using these methods, additional information on mitochondrial gene rearrangement patterns is also made available in the process, which can be highly informative for resolving deeper phylogenetic relationships (Boore et al., 1998, Dowton and Austin, 1999, Dowton et al., 2002), and sometimes even at more shallow taxonomic levels (Gan et al., 2017, Tan et al., 2017). Relative to Brachyura, for which the majority of the studied species contain a conserved mitochondrial gene order with a few exceptions (Basso et al., 2017), the Anomura appears to be a “hot spot” for gene rearrangements, with different gene orders being reported for almost every published mitogenome (Gan et al., 2016, Lee et al., 2016, Zhang et al., 2017). Diversity in gene order patterns provides a rich source of molecular markers, with limited homoplasy, that have the potential to act as synapomorphies for specific lineages and taxonomic groups, and thus providing support for existing or new hypotheses for phylogenetic relationships (Hickerson and Cunningham, 2000, Morrison et al., 2002, Tan et al., 2017).

In this study, we recover and report the mitogenome sequences for 12 anomuran and 8 brachyuran species using whole genome skimming methods. We then generated a dataset consisting of amino acid characters of 13 protein-coding gene sequences from all complete mitogenome sequences currently available for the Anomura and Brachyura, and assessed the evolutionary relationships within and between these diverse decapod groups via Bayesian and Maximum-likelihood phylogenetic reconstruction methods. Additionally, we introduce a new feature to our MitoPhAST pipeline (Tan et al., 2015) that enables the comparison of mitogenome gene orders (MGOs) from GenBank files (Benson et al., 2018). Using this pipeline, in combination with other existing bioinformatics tools, we compare MGOs across taxonomic groups and confirm that MGOs are highly variable within the Anomura, and also for some groups of the Brachyura. We further demonstrated the utility of MGO patterns in reinforcing tree-supported relationships, or resolving evolutionary relationships where tree-based relationships are weak. In the latter context, we hypothesize that the lomisoids and chirostyloids share a common ancestor to the exclusion of the aegloids on the basis of a shared MGO arrangement that is present in lomisoids and chirostyloids but not in the aegloids.

Section snippets

Sampling, sequencing, mitogenome assemblies and annotation

Tissue samples from a total of 20 species (12 anomuran, 8 brachyuran) were acquired from various geographic locations as well as from museums, including National Museum Victoria (NMV) and the Museum and Art Gallery of the Northern Territory (MAGNT) (Table 1). Total genomic DNA was extracted from the tissue samples (Sokolov, 2000) and subsequently sequenced at low coverage on the Illumina MiSeq platform as previously described (Gan et al., 2014). Sequence reads were pre-processed with

An expanded and updated Meiura phylogeny based on whole mitogenome

Mitogenomes of 12 anomuran species (genera: Aegla, Birgus, Coenobita, Gastroptychus, Lomis, Munida, Pylocheles, Stemonopa) and 8 brachyuran species (genera: Cardisoma, Cranuca, Epixanthus, Macrophthalmus, Pachygrapsus, Pilumnus, Tubuca) were successfully recovered. Of these, the mitogenomes of Aegla longirostri sensu lato, Pylocheles mortensenii and Macrophthalmus darwinensis were partially recovered with gaps in the 12S rRNA gene in the former and putative control regions in the latter two

Discussion

This study increases the number of available complete mitogenomes for Anomura by 120% from 10 to 22, contributing the first mitogenome-based resources for the families Coenobitidae, Albuneidae, Pylochelidae, Aeglidae, Lomisidae and Chirostylidae. We also generate sequences for multiple brachyuran lineages, including some that are under-represented or that have not previously been subject to mitogenome-based phylogenetic analysis (Cardisoma, Cranuca, Tubuca, Epixanthus, Pilumnus), resulting in a

Conclusion

In this study, we show that not only can mitogenome gene rearrangements be used to support relationships recovered from phylogenetic reconstruction (i.e. closely-related members of a same taxonomic clade often share identical MGOs, with the exception of Munida species), it can also provide insights into relationships where tree-based relationships are poorly resolved (i.e. the Aegla-Lomis relationship) or in conflict (i.e. suggesting a basal Hippoidea as opposed to paguroids in the tree). In

Acknowledgements

This work was supported by the Malaysia Tropical Medicine and Biology Platform. We are grateful to Joanne Taylor (Museum Victoria), Gavin Dally (Museum and Art Gallery of the Northern Territory) and Sara Fratini (University of Florence) for making available samples used in this study. We would also like to thank Tin-Yam Chan (National Taiwan Ocean University), Sandro Santos (Universidade Federal de Santa Maria) and Olga Zimina (Arctic Megabenthos, http://megabenthos.info) for the provision of

Conflict of interest

The authors declare that they have no conflict of interest.

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