Friday, September 14, 2012

Nuclear Genes Result in a Resolved Squirrelfish-Soldierfish Species Tree

Fifteen years ago Wayne Maddison articulated a vision for phylogenetics, which was realized with the introduction of methods that incorporate coalescent theory into molecular phylogenetic inferences (1, 2). These “species tree” methods attempt to estimate the optimal tree that contains the set of sampled gene trees, using simple coalescent models to account for ancestral polymorphism (3).

There is a growing frequency of fish phylogenetic studies that are using multi-locus DNA sequence datasets analyzed with species tree methods (e.g., 4). Most molecular phylogenetic studies of closely related animals have relied on mitochondrial DNA because there is no recombination in the mitochondrial genome and the rate of nucleotide substitution is substantially higher than nuclear DNA. Also, universal primers to amplify specific regions of the mtDNA genome have been around since the late 1980s (5). This historical reliance on mtDNA means that we do not really know if we can expect phylogenetic resolution for clades of closely related species when sampling DNA sequences from a modest number of nuclear genes.

A print of Holocentrus ascensionis sampled
from Wikipedia
My graduate student Alex Dornburg is working on the phylogenetic relationships and patterns of diversification in the teleost clade Beryciformes. The first paper from his work on these fishes was published this week in MPE (6). Alex’s paper focuses on the phylogenetic relationships of the beryciform lineage Holocentridae (Squirrelfishes and Soldierfishes), a clade of 84 large eyed and primarily nocturnal species. The most recent common ancestor of living holocentrid species is minimally dated at 50 million years ago based on the phylogenetic relationships of two fossil holocentrid species from the Pesciara beds at Bolca, Italy (7-10). The Eocene age of the clade indicates that many holocentrid species likely share a fairly recent common ancestry.

Alex worked with a closely with a Yale College student, Rachel Webster, to compile a nice dataset of DNA sequences of six protein coding genes sampled from 39 holocentrid species. A single mtDNA gene was sequenced as well because data for this gene was available on Genbank for Corniger spinosus, which we did not have access to specimens for DNA sequencing. The phylogenetic analyses of the concatenated nuclear genes, as well as the species tree inference, were well resolved with very impressive bootstrap and Bayesian posterior clade support. We included the mtDNA gene and the 6 nuclear genes in a subsequent set of phylogenetic analyses, including a species tree inference. Inclusion of the mtDNA tree did not appear to alter the phylogenetic inferences, nor did it increase the posterior clade support in the *BEAST inferred species tree. These results indicate that DNA sequences sampled from a set of nuclear genes result in a strong inference of phylogenetic relationships among the closely related species of Holocentridae.
Phylogenetic relationships of Holocentridae inferred from six nuclear genes. A. Optimal tree from RAxML anlaysis, black and open circles are strong bootstrap values. B. Maximum clade credibility tree from a MrBayes analysis, black and open circles denote nodes with a Bayesian posterior equal to or greater than 0.95. C. *BEAST inferred maximum clade credibility species tree, black and open circles denote nodes with a Bayesian posterior equal to or greater than 0.95.

*BEAST inferred maximum clade credibility species tree inferred from 
six nuclear genes and a single mtDNA gene, black and open circles
denote nodes with a Bayesian posterior equal to or greater than 0.95.
The inferred phylogenies are orthodox in the resolution of the two major holocentrid taxonomic groups, Holocentrinae (Squirrelfishes) and Myripristinae (Soldierfishes), as monophyletic; however, two of the three genera of Holocentrinae (Sargocentron and Neoniphon) are dramatically non-monophyletic in these phylogenies. We use the phylogenetic trees to show that many of the characters used to diagnose and delimit these genera have a complex evolutionary history. A new taxonomy for Holocentrinae is proposed where several species previously classified as Sargocentron are treated as species of Neoniphon, Sargocentron is restricted to the least inclusive clade containing S. spiniferum and S. rubrum, and the subgenus Flammeo is elevated to contain F. marianus. Holocentrus is monophyletic in the phylogenies.

These results are exciting, as they indicate that molecular datasets being collected by ichthyologists in the early 21st Century provide well-resolved phylogenies for closely related lineages, as well as the deepest branches in the Actinopterygian Tree of Life (e.g., 11). Also, these well resolved phylogenies reveal substantial incongruence between the formal classifications of species and inferred phylogenetic relationships. Perhaps most exciting are the numerous evolutionary studies, aimed at understanding the mechanisms that have generated the abundant species diversity of fishes, which will be guided by these well resolved species level phylogenies.


1.  Maddison, W.P. 1997. Gene trees in species trees. Systematic Biology. 46:523-536.
2.  Edwards, S.V., L. Liu, and D.K. Pearl. 2007. High-resolution species trees without concatenation. Proceedings of the National Academy of Sciences of the United States of America. 104:5936-5941.
3.  Edwards, S.V. 2009. Is a new and general theory of molecular systematics emerging? Evolution. 63:1-19.
4.  Hollingsworth, P.R. and C.D. Hulsey. 2011. Reconciling gene trees of eastern North American minnows. Molecular Phylogenetics and Evolution. 61:149-156.
5.  Kocher, T.D., W.K. Thomas, A. Meyer, S.V. Edwards, S. Paabo, F.X. Villablanca, and A.C. Wilson. 1989. Dynamics of mitochondrial DNA evolution in animals:  amplification and sequencing with conserved primers. Proceedings of the National Academy of Sciences of the United States of America. 86:6196-6200.
6.  Dornburg, A., J.A. Moore, R. Webster, D.L. Warren, M.C. Brandley, T.L. Iglesias, P.C. Wainwright, and T.J. Near. 2012. Molecular phylogenetics of squirrelfishes and soldierfishes (Teleostei: Beryciformes: Holocentridae): reconciling more than 100 years of taxonomic confusion. Molecular Phylogenetics and Evolution. 65:727-738.
7.  Sorbini, L. 1975. Gli Holocentridae di Monte Bolca. II: Tenuicentrum pattersoni nov. gen. nov. sp.  Nuovi dati a favoure dell’origine monofiletica dei beryciformi (Pisces). Studi e Ricerche sui Giaciamenti Terziari di Bolca. 2:456-472.
8.  Sorbini, L. 1979. Gli Holocentridae di Monte Bolca. III. Berybolcensis leptacanthus (Agassiz). Studi e Ricerche sui Giaciamenti Terziari di Bolca. 4:19-35.
9.  Sorbini, L. 1975. Gli Holocentridae di Monte Bolca.  I: Eoholocentrum, nov. gen., Eoholocentrum macrocephalum (de Blainville) (Pisces-Actinopterygii). Studi e Ricerche sui Giaciamenti Terziari di Bolca. 2:205-228.
10.  Stewart, J.D., Taxonomy, paleoecology, and stratigraphy of the halecostome-inoceramid associations of the North American Upper Cretaceous epicontinental seaway. 1984, University of Kansas: Lawrence. p. 201.
11.  Near, T.J., R.I. Eytan, A. Dornburg, K.L. Kuhn, J.A. Moore, M.P. Davis, P.C. Wainwright, M. Friedman, and W.L. Smith. 2012. Resolution of ray-finned fish phylogeny and timing of diversification. Proceedings of the National Academy of Sciences of the United States of America. 109:13698-13703.

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