Eumalacostracan phylogeny and total evidence: limitations of the usual suspects

Background The phylogeny of Eumalacostraca (Crustacea) remains elusive, despite over a century of interest. Recent morphological and molecular phylogenies appear highly incongruent, but this has not been assessed quantitatively. Moreover, 18S rRNA trees show striking branch length differences between species, accompanied by a conspicuous clustering of taxa with similar branch lengths. Surprisingly, previous research found no rate heterogeneity. Hitherto, no phylogenetic analysis of all major eumalacostracan taxa (orders) has either combined evidence from multiple loci, or combined molecular and morphological evidence. Results We combined evidence from four nuclear ribosomal and mitochondrial loci (18S rRNA, 28S rRNA, 16S rRNA, and cytochrome c oxidase subunit I) with a newly synthesized morphological dataset. We tested the homogeneity of data partitions, both in terms of character congruence and the topological congruence of inferred trees. We also performed Bayesian and parsimony analyses on separate and combined partitions, and tested the contribution of each partition. We tested for potential long-branch attraction (LBA) using taxon deletion experiments, and with relative rate tests. Additionally we searched for molecular polytomies (spurious clades). Lastly, we investigated the phylogenetic stability of taxa, and assessed their impact on inferred relationships over the whole tree. We detected significant conflict between data partitions, especially between morphology and molecules. We found significant rate heterogeneity between species for both the 18S rRNA and combined datasets, introducing the possibility of LBA. As a test case, we showed that LBA probably affected the position of Spelaeogriphacea in the combined molecular evidence analysis. We also demonstrated that several clades, including the previously reported and surprising clade of Amphipoda plus Spelaeogriphacea, are 'supported' by zero length branches. Furthermore we showed that different sets of taxa have the greatest impact upon the relationships within molecular versus morphological trees. Conclusion Rate heterogeneity and conflict between data partitions mean that existing molecular and morphological evidence is unable to resolve a well-supported eumalacostracan phylogeny. We believe that it will be necessary to look beyond the most commonly utilized sources of data (nuclear ribosomal and mitochondrial sequences) to obtain a robust tree in the future.

19. Ocular lobe: Absent (0). Present (1). An ocular lobe can be present in the absence of compound eyes, and vice versa.
21. Ultrastructure of ommatidia: Crystalline cone completely round in transverse section, cone without any extensions (0). Cone with two lateral extensions (in transverse section button-like), formed by one cone cell each (1).
22. Ultrastructure of ommatidia: Crystalline cones with four cone cell processes (0). Only the two accessory cone cell processes are present; the processes of the main cone cells are missing (1). All cone cell processes missing (2).

Posteriormost appendages
119. Last pleopods: Small, far from the telson, and not forming a tail fan (0). Modified to broad uropods, forming a tail fan with the telson (1). Oriented posteriorly and close to the telson, but not forming a tail fan with it (2).
122. Uropod numbers: None (0). One set (1). Three sets (2). Tagmosis 125. Number of maxillipeds: None (0). One (1). Two (2). Three or more (3). Richter and Scholtz (2001) prefer to break the single character for the number of maxillipeds used by Schram and Hof (1998) and Wills (1997) down into two. The first codes for the first maxilliped (presence/absence) since this is regarded as a robust homology. The second codes for the transformation of additional and subsequent thoracopods (none, second and third, or five), where homologies between groups are less certain. We assume that this second character is unordered, recognizing only that different, homologous conditions exist, but not associating these in an ordered sequence according to actual number. If so, the treatment here is different, and the entire character is treated as ordered.
129. Reduction or absence of pleon appendages: All pleon appendages present (0). All pleon appendages absent, or up to three highly-reduced and vestigial appendages clearly demarcated from those of the thorax (1). 138. Atrium between the inferomediana connecting the cardiac primary filter grooves with the pyloric filter grooves: Absent (0). Present (1).
144. Position of the heart: In whole thorax and pleon (0). In thorax (1). Only in posterior part of the thorax and pleon (2).