Volume 53

Investigation of molluscan phylogeny using nuclear protein-coding genes

Kevin M. Kocot and Kenneth M. Halanych,

Auburn University, Auburn, AL 36849

Mollusca constitutes the second most diverse lineage of animals and includes many economically, ecologically, and scientifically important species. Despite their diversity and importance, little is known about the phylogenetic relationships among the major lineages (‘classes’) of Mollusca which are Chaetodermomorpha (=Caudofoveata), Neomeniomorpha (=Solenogastres), Polyplacophora, Monoplacophora, Bivalvia, Cephalopoda, Scaphopoda, and Gastropoda (Haszprunar et al. 2008). Resolving mollusc class-level evolutionary relationships would have profound implications for our understanding of early animal evolution as molluscs are well represented in the early animal fossil record. Understanding mollusc relationships would also inform studies addressing the evolution of animal morphology, development, neurobiology, and physiology. Therefore, for a portion of my Ph.D. dissertation research, I am focusing on reconstructing a robust phylogenetic hypothesis for Mollusca using a molecular phylogenetic approach with sequence data from several carefully selected nuclear protein-coding genes.

The great disparity in morphology among the major lineages of Mollusca has prompted several diverse phylogenetic hypotheses (Table 1). Most previous molecular investigations of molluscan phylogeny have relied primarily on the nuclear small subunit (SSU or 18S) and large subunit (LSU or 28S) ribosomal genes (Giribet et al. 2006, Passamaneck et al. 2004, Rosenberg et al. 1997, Winnepennincks et al. 1996). Passamaneck et al. (2004) conducted a maximum likelihood analysis on complete 18S and partial 28S sequence data from 32 molluscs and recovered all classes except for Bivalvia monophyletic, but support values at higher-level nodes were generally weak. Giribet et al. (2006) analyzed a combined dataset of 18S, partial 28S, 16S, cytochrome oxidase I (COI), and histone H3 sequences for 101 molluscs. As in Passamaneck et al. (2004), bivalves were paraphyletic and support values at higher-level nodes were weak. Mitochondrial genome information has also provided little insight into mollusc class-level phylogeny (e.g. Dreyer and Steiner 2004).

Table 1 . Major hypotheses of molluscan phylogeny to be tested

Hypothesis name	Relationships proposed	Pertinent reference(s)
Aplacophora	Neomeniomorpha + Chaetodermomorpha	Scheltema 1993, 1996
Hepagastralia	Neomeniomorpha basal	Salvini-Plawen and Steiner 1996,
Adenopoda	Chaetodermomorpha basal	Salvini-Plawen 1972, 1980, 1985, 1991, 2003
Aculifera	Polyplacophora + Aplacophora	Ivanov 1996, Scheltema 1993, 1996
Conchifera	Monoplacophora, Gastropoda, Bivalvia, Cephalopoda, Scaphopoda	Wingstrand 1985
Testaria	Polyplacophora + Conchifera	Salvini-Plawen 1972, 1980, 1985, Salvini-Plawen and Steiner 1996, Haszprunar 2000
Cyrtosoma (=Visceroconcha)	Cephalopoda + Gastropoda	Runnegar and Pojeta 1974, Salvini-Plawen 1980
Diasoma (=Loboconcha)	Bivalvia + Scaphopoda	Runnegar and Pojeta 1974, Salvini-Plawen 1980
unnamed	Scaphopoda + Cephalopoda	Giribet and Wheeler 2002, Haszprunar 2000, Passamaneck et al. 2004, Steiner and Dreyer 2002, 2003, Waller 1998

Several studies have demonstrated that conserved nuclear protein-coding ‘housekeeping’ genes are very useful as molecular markers for higher-level phylogenetics (e.g., Anderson et al. 2004, Regier and Shultz 1997, Ruiz-Trillo et al. 2002). Results of these studies (and my own preliminary analyses) indicate that such markers have great potential to resolve long-standing questions in higher-level animal phylogeny. Specifically, preliminary analyses of sequence data available in public databases indicated that the genes elongation factor 1a (EF-1a), heat shock protein 90A (Hsp90A), sodium/potassium ATPase (Na ⁺/K ⁺ ATPase), and myosin II (My2) could be highly informative markers for mollusc higher-level phylogenetics. Therefore, I have chosen to utilize fragments of these genes as molecular markers in the study of mollusc class-level phylogeny.

Methods

Trochozoan-specific degenerate oligonucleotide primers were designed for Hsp90A and Na ⁺/K ⁺ ATPase and primers previously reported in the literature were used for My2 (Ruiz-Trillo et al. 2002) and EF-1a (Regier and Shultz 1997). RNA was extracted from living, frozen, or RNAlater-preserved specimens using Trizol, purified using the Qiagen RNeasy kit, and reverse transcribed to first-strand cDNA. The genes of interest were amplified via touchdown PCR and/or nested PCR using the primers discussed above. Amplicons were then gel extracted, cloned, miniprepped, and bidirectionally sequenced using a Beckman Coulter CEQ 8000 in Dr. Halanych’s lab.

Sequences were then edited and assembled into contigs using Sequencher. Novel sequences generated in this project were combined with sequences already available in public databases and aligned using C lustalW. Amino acid sequences were analyzed on a 14-processor computing cluster using Bayesian inference in MrBayes using the amino acid substitution model suggested by MrModeltest.

Results and Discussion

To date, I have successfully amplified all four gene fragments from a diverse assemblage of molluscs indicating that, in general, my methods and primers are good. However, I have experienced difficulties amplifying all four genes from some taxa (i.e., I can amplify Na⁺/K ⁺ ATPase, EF-1a, and My2 from a particular species but not Hsp90). I am currently experimenting with new primers, cDNA library construction, and other methods in order to obtain recalcitrant sequences.

Figure 1. Preliminary Bayesian inference phylogram based on analysis of Hsp90A, Myosin II, Na +/K+ ATPase, and EF-1a amino acid sequence data (1687 positions) performed in Mr Bayes using the WAG + Γ model run for 20 6 generations. Polyplacophora (red), Aplacophora (green), Cephalopoda (brown), and Gastropoda (blue) are all strongly recovered monophyletic. Addition of data from more genes and increased taxon sampling is expected to increase the resolution at deeper nodes.

In the preliminary Bayesian inference analysis (Figure 1), Polyplacophora, Aplacophora, Cephalopoda, and Gastropoda are all recovered monophyletic with a posterior probability value of 1.0. Interestingly, Mollusca is not recovered monophyletic with respect to the outgroup taxon, Capitella sp. (Annelida). Instead, there is a 3-branch polytomy consisting of Capitella sp., Polyplacophora, and the remainder of Mollusca. Interestingly, a clade consisting of Scaphopoda and Aplacophora is strongly supported with a posterior probability of 1.0.

I feel that the preliminary results of this work are promising and intriguing. The monophyly of Polyplacophora, Cephalopoda, and Gastropoda have rarely been debated so recovering these taxa monophyletic with strong support was no surprise. However, the nature of the relationship between the two lineages of Aplacophoran molluscs has been intensely debated. The preliminary result presented here is strongly consistent with Aplacophoran monophyly as hypothesized by Scheltema (1993, 1996). However Aplacophora is recovered within Conchifera and is significantly separated from Polyplacophora in contrast to the Aculifera hypothesis (which predicts that Aplacophora and Polyplacophora form a monophyletic clade, Ivanov 1996, Scheltema 1993, 1996). I have successfully sequenced myosin II from several aplacophorans and chitons. A similar analysis conducted on these data also strongly supports aplacophoran monophyly but also weakly supports the monophyly of Aculifera (data not shown). Whether the strongly supported Aplacophora + Scaphopoda clade in the multi-gene analysis presented here is real or the result of long branch attraction requires further investigation.

Presently, the taxon sampling of this study is weak for Bivalvia, Scaphopoda, and Aplacophora. I am currently focusing on adding a complete dataset for additional members of each of these taxa. Funding generously provided by the Malacological Society of London was used to charter the R/VSusan Hudson from Duke University’s Beaufort Marine Laboratory in order to collect additional molluscs for use in this project. This collecting trip was successful and I am now working to collect sequence data from additional bivalves, scaphopods, and aplacophorans. Additionally, I am testing primers to amplify other gene fragments that performed well in preliminary in order to add additional molecular markers to this study.

References

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