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Acknowledgments: We thank R. Axel, E. Morrow, and
members of the Barnea laboratory for critical reading of the
manuscript. We thank R. Y. Korsak and M. Talay for artwork
for the figures. This work was supported by NIH grants
T32GM007601 (L. T.) and 5R01MH086920 (G.B.), as well
as by funds from the Pew Scholar in the Biomedical
Materials and Methods
Figs. S1 to S4
21 November 2013; accepted 28 February 2014
Acquisition of Germ Plasm
Accelerates Vertebrate Evolution
Teri Evans,1 Christopher M. Wade,1 Frank A. Chapman,2 Andrew D. Johnson,1 Matthew Loose1*
Primordial germ cell (PGC) specification occurs either by induction from pluripotent cells
(epigenesis) or by a cell-autonomous mechanism mediated by germ plasm (preformation). Among
vertebrates, epigenesis is basal, whereas germ plasm has evolved convergently across lineages
and is associated with greater speciation. We compared protein-coding sequences of vertebrate
species that employ preformation with their sister taxa that use epigenesis and demonstrate
that genes evolve more rapidly in species containing germ plasm. Furthermore, differences in
rates of evolution appear to cause phylogenetic incongruence in protein-coding sequence
comparisons between vertebrate taxa. Our results support the hypothesis that germ plasm liberates
constraints on somatic development and that enhanced evolvability drives the evolution of
The germ line of metazoans is established early in development with the specifica- tionofprimordialgermcells(PGCs). Among
vertebrates, the conserved mechanism for PGC
specification involves their induction from pluripotent cells by extracellular signals, a process referred to as epigenesis (1, 2). However, in several
lineages of vertebrates, an alternative mechanism
evolved, termed preformation. Here, PGCs are
determined by inheritance of germ plasm. Preformation evolved by convergence, which suggests that it may confer a selective advantage.
Accordingly, the evolution of germ plasm is associated with morphological innovations and enhanced numbers of species within individual
clades (1, 3, 4). Why this derived mode of PGC
specification evolved repeatedly in vertebrates is
The best-studied contrast of epigenesis and
preformation is within amphibians. The PGCs of
urodele amphibians (salamanders) are specified
by epigenesis, whereas in its sister lineage, anu-
rans (frogs), PGCs contain germ plasm (5). Using
the axolotl (Ambystoma mexicanum) as a model
urodele, the ancestral gene regulatory networks
(GRNs) for pluripotency and mesoderm specifi-
cation in vertebrates were identified (6, 7). These
GRNs were conserved through the evolution of
mammals (6, 7), which also employ epigenesis
(8). In contrast, in frogs the master regulators of
pluripotency as employed in mammals have been
deleted (6, 9, 10), and the GRN for mesoderm
underwent expansions of key regulatory mole-
cules (7, 11). Similar genetic innovations evolved
in the GRNs for zebrafish development (12), which
also uses preformation (13). The correlation of
germ plasm with genetic change has been pro-
posed to result from the relaxation of constraints
on somatic development imposed by maintain-
ing the PGC induction pathway (1, 3, 4). To
investigate this possibility, we compiled available
expressed sequence tag, mRNA and cDNA se-
quences from vertebrates (fig. S1A and table S1)
identifying ortholog pairs shared between sister
taxa with different modes of PGC specification
and an appropriate mammal and outgroup sequence
(14) (fig. S1B). To increase sequence numbers from
organisms using epigenesis, we generated tran-
scriptomes from the axolotl and an Acipenseriforme,
Acipenser ruthenus (the sterlet) (14), identifying
82,954 sequence clusters across all vertebrates.
All analyses were performed with protein coding
DNA sequence, excluding the saturated third po-
sition (14) (figs. S2 and S3).
Of the 56 published gene trees involving an
anuran and a urodele, 29 do not recapitulate the
known species phylogeny (table S2). The majority of the incongruent gene trees group urodele
1School of Life Sciences, University of Nottingham, Nottingham,
NG7 2UH, UK. 2Program of Fisheries and Aquatic Sciences,
University of Florida, Gainesville, FL 32653–3071, USA.
*Corresponding author. E-mail: firstname.lastname@example.org
(M.L.); email@example.com (A.D.J.)
Fig. 1. Amphibian four-taxon tree topologies. (A) Number of significant trees by bootstrapping (>70%)
and SH test (P < 0.05) for each topology rooted with a Teleostei sequence. (B and C) The proportions of
species phylogeny (black), mammal-urodele (gray), and mammal-anuran (white) topologies per species.
(D and E) The likelihood of each species grouping with mammals when the tree is incongruent; species
using preformation are shown in red, those using epigenesis in blue. Dashed lines indicate equal
probability of species grouping with mammal or outgroup. [(B) to (E)] Only species with >20 significant
trees are shown. The results excluding the transcriptome are shown in fig. S4.