Phylogeny of the bristle worm family Eunicidae (Eunicida, Annelida) and the phylogenetic utility of noncongruent 16S, COI and 18S in combined analyses
Introduction
Eunicidae comprises benthic marine annelids inhabiting soft and hard marine substrates; many burrow into hard corals and calcareous algae or live in their crevices (Hutchings, 1986). They are distributed worldwide, but are most common in shallow tropical waters and play important roles in coral reef communities (Fauchald, 1992a). Many eunicid1 species are economically important as bait for leisure and commercial fishing in diverse regions including the Mediterranean Coast, Australia, Japan and the United States of America (Gambi et al., 1994, Olive, 1994). Additionally, the reproductive swimming phase of members of Palola is a delicacy for natives from several of South Pacific Islands (Schulze, 2006).
The current generally accepted composition of Eunicidae has been stable since proposed by Hartman (1944, modified from Kinberg, 1865). This classification proposes that Onuphidae is the closest lineage to Eunicidae, a hypothesis consistently recovered in both morphological and molecular phylogenies (Rouse and Fauchald, 1997, Struck et al., 2002, Struck et al., 2006, Struck et al., 2008, Rousset et al., 2007, Zanol et al., 2007). However, rather than using unique diagnostic features (apomorphies), Hartman’s classification employs plesiomorphies or homoplasies, such as asymmetric labidognath jaws, one to five prostomial appendages, peristomium double ringed and wide bilobed prostomium (Orensanz, 1990). Previous molecular and morphological phylogenetic analyses focusing, respectively, on the order Eunicida (Struck et al., 2002, Struck et al., 2006) or on the genus Eunice (Zanol et al., 2007) did not always recover the current Eunicidae as monophyletic. In both, morphological analysis of Eunice using 59 characters (Zanol et al., 2007) and molecular analysis of Eunicida using just 18S rDNA data (Struck et al., 2002, Struck et al., 2006), Onuphidae is nested within a paraphyletic Eunicidae. Molecular analyses of Eunicida using more genes (COI, 16S rDNA, 18S rDNA and 28S rDNA) have lacked sufficient taxonomic representation to evaluate monophyly of Eunicidae (Struck et al., 2006). Therefore, under a phylogenetic framework, the current composition for Eunicidae is dubious, and further analysis is warranted.
Species of Eunicidae (∼326 spp.) are grouped in nine currently accepted genera (Table 1). Proposed diagnostic characters of most genera also appear to be plesiomorphic (e.g., five prostomial appendages, a pair of peristomial cirri and labidognath jaws in Eunice; Zanol et al., 2007) or ontogenetically variable (number of prostomial appendages in Lysidice and Nematonereis, and absence of peristomial cirri in Marphysa; Åkesson, 1967, Giangrande, 1989). Despite these inconsistencies, this generic classification has remained unmodified for many years (Orensanz, 1990). Just three of the nine currently accepted genera have been revised and had their monophyletic status phylogenetically tested, with these results: Eunice is polyphyletic (Fauchald, 1992a, Zanol et al., 2007), and Palola (Fauchald, 1992b, Schulze, 2006) and Euniphysa (Lu and Fauchald, 2000) are monophyletic. Eunice can be divided in at least two monophyletic groups Eunice sensu stricto and Leodice (junior synonym of Eunice, see below), however, composition and diagnostic features of such groups are still unclear (Zanol et al., 2007).
In addition to the nine currently recognized genera, 18 additional genera have been described for eunicid species (see Fauchald, 1992a for a revision). Most of these have been synonymized with other genera and a few are indeterminable (Fauchald, 1992a). Of genera later synonymized to the genus Eunice, Leodice Lamarck, 1818 and Nicidion Kinberg, 1865 had the most widespread use by several authors. Leodice named for Leodice antennata Lamarck, 1818, was not clearly differentiated from Eunice, and both names were used interchangeably through the first third of the 20th century (Hartman, 1944). Nicidion was described for abranchiate species, a characteristic currently not considered valid at the generic level (Fauchald, 1992a). The original description of Nicidion includes three species, none of which was identified as the type species. Two of these species, N. gualapaguensis and N. longicirrata, were placed in the genus Palola and are indeterminable beyond the genus level because of the incomplete original description and poorly preserved type specimens (Fauchald, 1992b). The third species, N. cincta, is the only determinable species and has been referred to Eunice (Hartman, 1948, Fauchald, 1992a).
According to Hartman (1944), eunicid phylogeny has a basal split separating Palola from a grade containing the remaining genera recognized by her, Nematonereis, Lysidice, Marphysa + Paramarphysa, Eunice + subgenus Nicidion. Genera are disposed in the grade according to the increasing number of prostomial and peristomial appendages, implying that such appendages evolved in the grade by stepwise addition as observed in the ontogeny of Eunice species, median antennae (Nematonereis) – lateral antennae (Lysidice) – palps (Marphysa) – pair of peristomial cirri (Eunice). Hartman’s (1944) scenario results from her knowledge and ideas about the evolution of the group, not on an objective and repeatable methodology, thus the justification of why certain taxa are grouped together is not clear.
In an 18S rDNA phylogenetic hypothesis for Eunicida, Eunicidae was recovered as paraphyletic and grouped in three clades (Lysidice, Nematonereis), (Eunice species with yellow subacicular hook) and (Marphysa, Eunice torquata) (Struck et al., 2006). However, such hypothesis is based on a small fraction of eunicid diversity and just one molecular marker, which can be biased by biological processes (e.g., natural selection, horizontal gene transfer).
In the present study, we provide a phylogenetic hypothesis for Eunicidae which supports a poorly supported monophyletic family and Palola as the only monophyletic genus among the ones for which we tested the monophyly (Eunice, Marphysa, Palola, Lysidice and Nematonereis). Moreover, we examined evolution of prostomial and peristomial appendages in Eunicidae, which may be explained by sequential heterochrony, and the phylogenetic information content of the three genes (16S rDNA, COI and 18S rDNA) at different levels on the trees.
Section snippets
Taxon sampling
We sampled 50 ingroup species representing six (Eunice, Marphysa, Palola, Lysidice, Euniphysa and Nematonereis) of the nine currently valid genera of Eunicidae. These genera are the most species rich and best studied in the family (Table 1). No sample was available for the remaining three genera (Aciculomarphysa, Fauchaldius, Nauphanta), which have only one or two species. Choice of species took into account specimen availability, diversity of characters within the different genera and
Sequence characteristics and incongruence
After removal of COI nucleotide insertions, combined data sets had 4966 aligned positions in the Mafft alignment and 4911 in the Clustal alignment. Of these, 2165 and 2145 sites, respectively, were variable and 1680 and 1685 sites were parsimony informative. Alignments are available online at TreeBASE (www.TreeBASE.org). Table 3 summarizes characteristics of all single gene sequences.
SH tests results showed that trees obtained from a single gene data set are rejected by all remaining single
Congruence and phylogenetic utility of different genes
The additive phylogenetic signal contributed by each gene to clades in different regions of the trees likely explains the improved resolution and support of combined analyses, as it has been observed in previous studies (e.g., Creer et al., 2003). Even though most single gene trees are incongruent with other data sets in the SH tests, WSRT results and SH tests for trees obtained from combined analyses show that single gene data sets are generally congruent with the combined data set. This
Conclusion
The combination of 16S, COI and 18S sequences was essential for resolution and recovery of stable clades at different depths of the trees. Moreover, combining three genes, despite their incongruence, allowed for phylogenetic signal present in data sets of different genes, but hidden in single gene analyses, to emerge. Further supporting the notion that congruence is not a requirement for combining different partitions.
Eunicidae is monophyletic and sister to Onuphidae, supporting the presence of
Acknowledgments
We are greatly thankful to Diana L. Lipscomb, Allen G. Collins, Sheri A. Church, James M. Clark, Gustavo Hormiga, Fernando Alvarez, M. Rosario Castañeda, Vinita Gowda, Lara Lopardo, Dimitar Dimitrov, Christoph Bleidorn, Tiago B. Quental, Omar Torres-Carvajal and Kevin de Queiroz for their contribution at different phases of this study. Many thanks to all the staff in Invertebrate Zoology Department (NMNH, Smithsonian Institution), Heather Blasczyk, all other members of Halanych’s lab in the
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