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Anderson et al. BMC Evolutionary Biology (2017) 17:123 DOI 10.1186/s12862-017-0973-4 RESEARCHARTICLE Open Access Phylogenomic analyses of Crassiclitellata support major Northern and Southern Hemisphere clades and a Pangaean origin for earthworms Frank E. Anderson1* , Bronwyn W. Williams1,2, Kevin M. Horn1, Christer Erséus3, Kenneth M. Halanych4, Scott R. Santos4 and Samuel W. James5 Abstract Background: Earthworms (Crassiclitellata) are a diverse group of annelids of substantial ecological and economic importance. Earthworms are primarily terrestrial infaunal animals, and as such are probably rather poor natural dispersers. Therefore, the near global distribution of earthworms reflects an old and likely complex evolutionary history. Despite a long-standing interest in Crassiclitellata, relationships among and within major clades remain unresolved. Methods: In this study, we evaluate crassiclitellate phylogenetic relationships using 38 new transcriptomes in combination with publicly available transcriptome data. Our data include representatives of nearly all extant earthworm families and a representative of Moniligastridae, another terrestrial annelid group thought to be closely related to Crassiclitellata. We use a series of differentially filtered data matrices and analyses to examine the effects of data partitioning, missing data, compositional and branch-length heterogeneity, and outgroup inclusion. Results and discussion: We recover a consistent, strongly supported ingroup topology irrespective of differences in methodology. The topology supports two major earthworm clades, each of which consists of a Northern Hemisphere subclade and a Southern Hemisphere subclade. Divergence time analysis results are concordant with the hypothesis that these north-south splits are the result of the breakup of the supercontinent Pangaea. Conclusions: These results support several recently proposed revisions to the classical understanding of earthworm phylogeny, reveal two major clades that seem to reflect Pangaean distributions, and raise new questions about earthworm evolutionary relationships. Keywords: Clitellata, Crassiclitellata, Earthworm, Phylogenomics Background history of the world, as have these lowly organised creatures.” “The plough is one of the most ancient and most valuable of man's inventions; but long before he Charles Darwin, The formation of vegetable mould existed the land was in fact regularly ploughed, and through the actions of worms, with observations on still continues to be thus ploughed by earth-worms. It their habits, pg. 313 [1] may be doubted whether there are many other animals which have played so important a part in the Earthworms (Crassiclitellata) constitute a diverse group of primarily terrestrial, burrowing annelids com- prising 6000+ extant species in 18 families and found on * Correspondence: [email protected] all continents except Antarctica. Most earthworm 1Department of Zoology, Southern Illinois University, Carbondale, IL 62901, species live in soil, but some live in decaying logs, leaf USA litter, stream mud and riverbanks, as well as arboreal Full list of author information is available at the end of the article © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Anderson et al. BMC Evolutionary Biology (2017) 17:123 Page 2 of 18 (e.g., epiphytic root masses) and even marine littoral gene (16S, 18S and 28S ribosomal RNA genes) and taxon habitats. Charles Darwin famously extolled the import- sampling of Crassiclitellata and several outgroups and ance of earthworms as terrestrial ecosystem engineers, were able to reinterpret many morphological changes churning and aerating the soil with their burrows as well defining the families of crassiclitellates, proposing new as burying and processing large fragments of organic hypotheses of morphological evolution and rehabilitating matter and making their nutrients available to plants. older ones. Large-scale engineering by earthworms has recently Although James and Davidson [13] clarified many been documented in South America [2] and may occur aspects of earthworm phylogeny, relationships among elsewhere. Even apart from their direct agricultural several major groups remain poorly supported. Fortunately, importance as soil processors, earthworms have a sub- the advent of low-cost, high-throughput sequencing stantial economic impact—epigeic (leaf litter/compost- methods has revolutionized the study of higher-level rela- dwelling) species are used to process food waste tionships across the tree of life, allowing researchers to (vermiculture), larger species are sold as bait for fish, bring dozens to thousands of genes to bear on previously and some earthworm species are considered delicacies intractable questions. To test previous hypotheses of rela- and are sold for human consumption. Earthworms are tionships among earthworms and provide a robust frame- prey items for many other species, including planarians, work for historical biogeographic inference and studies of leeches, mollusks, insects, amphibians, lizards, snakes, character evolution, we generated transcriptomic data from birds and mammals, and thus serve as a crucial link in representatives of nearly all major extant lineages of Crassi- numerous terrestrial food webs. Many earthworms are clitellata and performed a series of analyses to infer considered invasive; approximately one-third of all relationships among the major lineages of earthworms. earthworm species in North America are introduced from Europe and Asia [3, 4]. As invasive earthworms spread in recently glaciated and otherwise earthworm- Methods free forests in North America, they affect many micro- Taxon sampling bial, plant and invertebrate species that have come to A total of forty taxa (33 crassiclitellates, one moniligastrid rely on large amounts of undisturbed leaf material [5]. and six outgroup taxa) were sampled for this study Widespread distribution and limited dispersal abilities (Table 1). James and Davidson [13] used representa- make earthworms a promising model of historical tives of several clitellate taxa as outgroups for their biogeographic patterns at a global scale. Indeed, specula- analysis of crassiclitellate phylogeny based on 18S tion about earthworm biogeography has a long history, data, but only used an enchytraeid for most other perhaps unusually attractive to history-of-science enthu- analyses (including combined analyses of multiple loci). siasts. Early ideas about earthworm distributions relied Their 18S Bayesian phylogeny (Fig. 1 in [13]) suggested on dubious land bridge hypotheses (review in [6]). Apoc- that Haplotaxidae s. str. (represented by Haplotaxis ryphal lore has it that J.W. Michaelsen (a great Clitellata gordioides) was sister to Metagynophora (Crassiclitellata + taxonomist of the late 19th and early 20th centuries; Moniligastridae), and our preliminary analyses of a e.g., [7]) and Alfred Wegener were office neighbors in broader sample of clitellate transcriptomes also suggested Hamburg, Germany for a time. Michaelsen [8] cited that members of Haplotaxidae are the closest extant Wegener’s hypothesis of continental drift [9] as providing relatives of Metagynophora (not shown). Haplotaxidae, considerable explanatory power for the distributions of with its currently recognized eight genera, is no longer earthworms, and named an amphi-Atlantic genus after considered to be monophyletic and has long been him (Wegeneriella Michaelsen 1933). Despite Michaelsen’s regarded as a “dustbin” for slender, primitive-looking cli- contribution, speculation about land bridges continued to tellates [10, 14–17]. We chose representatives of four hap- pervade the earthworm biogeographic literature. lotaxid species, Lumbriculidae (Lumbriculus variegatus) Earthworms have a very poor fossil record, and spe- and Propappidae (Propappus volki) as outgroups; P. volki cialists have long disagreed about directions of character was used to root the phylogeny. No leeches or branchiob- evolution within the group. Early earthworm phylogenies dellidans were used in this study, for two reasons. First, were highly intuitive (cf. [10]) and shed little light on previous work [13, 18] and preliminary analyses including earthworm historical biogeography. Earthworm phylo- several leech and branchiobdellidan transcriptomes sup- genetic understanding has progressed slowly since these ported a clade comprising Lumbriculidae, Branchiobdellida initial attempts. The few applications of cladistic analysis, and Hirudinea. Second, all available leech and bran- such as Jamieson’s (1988) morphological study [11], chiobdellidan transcriptomes showed appreciably lon- yielded mixed conclusions, and the first use of molecular ger branch lengths on preliminary ML trees than did all data [12] overturned many of the morphology-based other clitellates. Sampling only the relatively short- hypotheses. James and Davidson [13] included a broader branch Lumbriculus variegatus allows this outgroup Anderson et al.