Elsevier

Pedobiologia

Volume 54, Supplement, 29 December 2011, Pages S3-S9
Pedobiologia

9th International Symposium on Earthworm Ecology
A critique of earthworm molecular phylogenetics

https://doi.org/10.1016/j.pedobi.2011.07.015Get rights and content

Abstract

2012 is the 10th anniversary of the publication of the first molecular phylogenetic study of earthworms by Jamieson and colleagues in 2002. Since then, a total of 29 papers on earthworm molecular phylogenetics have been published in peer-reviewed journals. However, only five of these focused on intergeneric or higher level systematics; moreover, no systematic revisions have been made. On the other hand, studies related to DNA barcoding and population genetics are flourishing, comprising more than half of published papers. In this review, we present a synthesis based on published papers of earthworm molecular phylogenetic studies, and discuss the evolutionary history and sequence characteristics that have caused the contrasting results found in the literature. We argue that multiple levels of rapid divergence in the evolutionary history of earthworms make phylogenetic reconstruction difficult. Many previous studies also suffered from using sequences that were too short and choosing genes that evolve too slowly. In response to this challenge, we recommend a strategy that involves analyzing long sequences (>2000 bp) by combining several genes with different evolutionary rates to provide enough information for different taxonomic levels, and we point out three genes (28S, 16S, and 12S rRNAs) suitable for intergeneric (within-family) analyses. Furthermore, the studies on closely related species demonstrate how molecular systematic and phylogeographic analyses have improved our understanding of earthworm speciation and intraspecific genetic variation. This knowledge about intra- and inter-specific genetic variations among closely related species provides information that can be further used for species delimitation in other earthworm groups. The numerous DNA barcoding studies have highlighted the usefulness of this technique in earthworm taxonomy, its possible applications in ecological studies, and an ongoing integrative taxonomic approach that combines morphology, DNA barcoding, and other independent data sources in species identification, discrimination and delimitation, plus new species description. Altogether, we believe that molecular phylogenetics is now on the verge of revolutionizing earthworm systematics.

Introduction

Excluding a few aquatic taxa (e.g., Almidae and Eiseniella tetraedra in freshwater habitats and Pontodrilus litoralis in marine littoral sediments), earthworms are terrestrial oligochaetes (Annelida: Clitellata) composed of about 5500 species (Blakemore et al. 2007). These clitellates, sometimes referred to as Crassiclitellata, a group of oligochaetes with multilayered cells on their clitellum, are considered by most researchers a natural group (Erseus 2005). Relationships among families have been debated extensively over the last 120 years, and the contents of suprafamilial taxa have been unstable (Michaelsen, 1900, Gates, 1972, Sims, 1980, Omodeo, 2000). Different authors recognize different numbers of families: 15 by Jamieson (1988), 18 by Blakemore, 2000, Blakemore, 2006 and 21 by Reynolds and Cook (1993). Even in the heavily investigated Lumbricidae, the numbers of genera vary between 6 and 20 genera in the traditional systems by Pop, Zicsi, and Omodeo (summarized in Csuzdi and Zicsi 2003) to 42 (Blakemore 2004) or 45 (Qiu and Bouché 1998).

Molecular phylogenetic analyses have been widely used to test taxonomic, phylogenetic and biogeographic hypotheses since the early 1990s and a total of 19 papers about clitellate phylogeny or systematics were published between 1996 and 2002 (see Erseus 2005 for review). The first molecular phylogenetic study that included earthworms was a study focusing on leeches and their relatives (Siddall et al. 2001), in which a sister group relationship between earthworms and the Enchytraeidae was weakly supported with limited sampling. This relationship was formally hypothesized by Erseus and Kallersjo (2004) using 18S rRNA gene sequences and later in a review of the phylogeny of oligochaetous Clitellata (Erseus 2005). Nevertheless, not until 2002 did the first phylogenetic study focusing on earthworms (Jamieson et al. 2002) get published. In that paper the hypothesis of the clade Crassiclitellata (Jamieson 1988) and the monophyly of the Megascolecidae were supported by 28S, 12S and 16S rRNAs data. Since then, a total of 29 papers have been published in peer-reviewed journals (Table 1). However, most of the papers focused on intraspecific genetic variations, DNA barcoding, and species complexes/groups; only five of the 29 papers focused on interfamilial or intergeneric systematics (Jamieson et al., 2002, Pop et al., 2003, Pop et al., 2007, Buckley et al., 2011, Novo et al., 2011). In addition, of nine papers on earthworm molecular phylogenetics published in the proceedings of the 1st–4th International Oligochaete Taxonomy Meetings, only six dealt with interfamilial or intergeneric systematics (Table 2). Regardless of all the above efforts and the seemingly promising idea that molecular phylogenetics could solve the long-lasting debates on the systematics of earthworms mentioned above, no higher level systematics has been proposed.

The objective of the present study is to review published studies to reveal the evolutionary history and sequence characteristics that present difficulties in earthworm molecular phylogeny. We further discuss the recent progress in different disciplines within earthworm molecular phylogenetics, and highlight challenges and promise.

Section snippets

Multiple levels of rapid divergence

One of the most daunting challenges of earthworm molecular phylogenetics is that it is usually hard to achieve reasonable phylogenetic resolution. In fact, it is a challenge not only for earthworms, but also for the whole Annelida. Martin et al. (2000) argued that the poor resolution of higher level phylogenetic relationships among Clitellata was due to rapid divergence, or radiation at the root of Clitellata phylogeny. McHugh, 2000, McHugh, 2001 proposed the same reason to explain the poorly

Sequence divergences and choice of genes

Different genes evolve at different rates and therefore are suitable for studying different taxonomic levels. At the same time, the same gene may evolve at different rates in different animal taxa. Therefore, for studying earthworm phylogeny, it is important to understand the sequence divergences both among different genes within a taxon and within the same gene across different taxonomic levels.

Here we present a comparison of interspecific sequence divergences within a species group/complex,

Interfamilial and intergeneric systematics

Molecular phylogenetic studies focusing on the whole Crassiclitellata support the monophyly of the Megascolecidae (Jamieson et al. 2002), Ocnerodrilidae, Eudrilidae, Lumbricidae, and Glossoscolecidae (Pop et al. 2005a), but the support for all the above families except the Megascolecidae is weak due to insufficient and biased taxon sampling. Furthermore, many families were not included in the two studies and the interfamilial relationships remained unresolved. Similarly, although some studies

Speciation, molecular clock, and biogeography

Compared to higher level systematics, the studies regarding closely related species have been more fruitful. The systematics of the M. formosae species group of the Megascolecidae and the A. caliginosa species complex of the Lumbricidae were revised or re-evaluated (Chang et al., 2008, Pérez-Losada et al., 2009), and cryptic speciation within Hormogaster elisae s. l. of the Hormogastridae was discovered (Novo et al., 2009, Novo et al., 2010). Among these studies, two different modes of

DNA barcoding and integrative taxonomy

Many genes have been used in earthworms to evaluate cryptic diversity and to help species identification, including 16S rRNA (Boyer and Wratten, 2010) and COII (Heethoff et al. 2004). A 658-bp fragment of the mitochondrial gene COI has been proposed as the standard DNA barcode for most animal taxa (Hebert et al., 2003a, Hebert et al., 2003b), and it has been widely used in earthworms (Chang and Chen, 2005, Pérez-Losada et al., 2005, Chang et al., 2007, Chang et al., 2008, Chang et al., 2009,

Pitfalls and promise

Molecular phylogenetics is now at the point of revolutionizing earthworm systematics. The semi-standardized procedure of data management and sharing through GenBank and the Barcode of Life Data Systems (BOLD, http://www.boldsystems.org/) provides enormous resources for studies that need a large body of data from broad taxonomic and/or geographic sampling. With a large quantity of data, however, good taxonomy is required to ensure quality. In GenBank many specific names that refer to earthworm

Acknowledgements

C.-H. Chang is grateful to Katalin Szlavecz for supporting this work and Jiun-Hong Chen for supporting Chang's earlier work. We are also thankful for the helpful comments made by Carlos Fragoso, Stefan Scheu, and two anonymous reviewers on an earlier version of this manuscript. This study was partially funded by the United States National Science Foundation awards EAR-0748442 and DEB-0516439, the Canadian Centre for DNA Barcoding, and a Marie Curie France Regions fellowship award to the

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    Current address: Department of Biology, University of Iowa, Iowa City, Iowa 52242, USA.

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