By Kevin E. Noonan --
The
coelacanth, an aquatic animal described as a "living fossil" when discovered in 1938, was thought to have gone
extinct during late Cretaceous period, ~70 million years ago. Only about 300 specimens of the African
coelacanth, Latimeria chalumnae, are known to exist and a second species, Latimeria menadoensis, was discovered
in 1997. These animals are morphologically
primitive, resembling fossils dating ~300 million years ago, which has
suggested that these are "slowly evolving" species (although this is
more a descriptive than an informed characterization). There have been sporadic reports of
coelacanth gene sequencing over the past decade, but last week Nature
published the first report on whole genome sequencing (Ameniya et al., "The
African coelacanth genome provides insighted into tetrapod evolution," Nature
496: 311-16, April 18, 2013).
The article reports that the coelacanth genome
comprises 2.68 gigabases, on 48 chromosomes, with 19,033 protein-coding genes
comprising 21,817 transcripts, 2,894 "short" non-coding RNAs, and 1,214 "long"
non-coding RNAs. 336 of protein-encoding
genes were found to have undergone gene duplication. The authors examined transcripts from 251
genes from coelacanth and compared these genes with Protopterus annectens
(the African lungfish) expressed in brain, gonad, and kidney and with 15
terrestrial vertebrate lineages (dog, human, mouse, elephant, armadillo, Tammar
wallaby, opossum, platypus, chicken, turkey, zebra finch, lizard, Western
clawed frog, Chinese brown frog) and five modern fish species (tilapia,
pufferfish, zebra fish, spotted catshark, little skate, and elephant shark). The results of these analysis, comprising
100,583 concatenated amino acid positions, was consistent with the lungfish
being the earliest relative of modern tetrapods (four-limbed animals),
answering a previously unresolved uncertainty as to the role of the coelacanth
in vertebrate evolution.
Turning to the question of the status of the
coelacanth as a "living fossil," the authors confirmed earlier
conclusions (based on Hox genes and protocadherins) that this species
evolves slowly, the authors assessed the data from the 251 genes used in their
phylogenetic analyses. This was done as
follows:
Pair-wise
distances between taxa were calculated from the branch lengths of the tree
using the two-cluster test proposed previously to test for equality of average
substitution rates. Then, for each of the following species and species clusters
(coelacanth, lungfish, chicken and mammals), we ascertained their respective
mean distance to an outgroup consisting of three cartilaginous fishes (elephant
shark, little skate and spotted catshark). Finally, we tested whether there was
any significant difference in the distance to the outgroup of cartilaginous
fish for every pair of species and species clusters, using a Z
statistic.
The coelacanth genes showed 0.89 substitutions per
site, compared with 1.05 substitutions/site in the lungfish, 1.09 substitutions/site
in chicken, and 1.21 substitutions/site in mammals.
The authors also ascertained the "abundance"
of transposable elements in the coelacanth genome, because these elements are
believed to provide "templates for exaptation," i.e., to facilitate
formation of novel protein exons and regulatory elements, as well as providing
targets for genomic rearrangement. Transposable element content was "high" (~25%, which the
authors consider an underestimate) and also showed a "wide variety of
transposable-element superfamilies," with 14 such families being "currently
active." The authors acknowledge
that these results "contrast[] with the slow protein evolution observed."
"[E]xtensive conservation of synteny" was
observed in a comparison of chromosomal breakpoints in coelacanth and tetrapod
genomes, and "indicate that large-scale rearrangements have occurred at a
generally low rate in the coelacanth lineage." Interchromosomal rearrangements indicated
that "karyotypic evolution in the coelacanth lineage has occurred at a
relatively slow rate, similar to that of non-mammalian tetrapods" (31 in
coelacanth, 20 in salamander and 21 in Xenopus species). Comparison of the two coelacanth species, L.
chalumnae (Africa) and L. menadoensis (Indonesia), showed divergence
rates similar to those found between humans and chimpanzees, and the authors
estimated that these species diverged "slightly more than" 6-8 million
years ago, based on the slower substitution rates found in coelacanth species.
The authors then looked at estimates of how
vertebrates adapted to the terrestrial environment. They identified 50 genes found in coelacanth
but not terrestrial tetrapods, presumably because these genes were not needed
when the animal left water for land. These genes included "components of fibroblast growth factor (FGF)
signalling, TGF-β and bone morphogenic protein (BMP) signalling, and WNT
signalling pathways, as well as many transcription factor genes," and
specifically that 4 genes (And1, And2, Fgf24 and Asip2)
not present in tetrapod genomes were indeed present in the coelacanth genome. These genes also included 13 genes involved
in fin development, 8 genes in otolith and ear development, 7 genes for kidney
development, 13 genes for eye development, and 23 genes for brain
development. In contrast, there were
only slight differences in homeobox genes. There were also changes in gene regulation, wherein 6% of "conserved
non-coding elements (CNEs)" ("promoters, enhancers, repressors and
insulators") had originated after divergence of the coelacanth from the
ancestral lineage. Further analysis
showed that tetrapod-specific CNEs were most closely (genetically) linked to
genes involved in smell perception (consistent with the recognized expansion of
olfactory receptor genes in the evolution of tetrapods from teleost fishes) and
"morphogenesis (radial pattern formation, hind limb morphogenesis, kidney
morphogenesis) and cell differentiation (endothelial cell fate commitment,
epithelial cell fate commitment)" and immunoglobulin VDJ
recombination.
A particular set of genes compared in the study are
genes for digits, "[a] major innovation in tetrapod evolution," and
specifically Hox genes for regulating limb development in ray-finned
fish, coelacanths and tetrapods (mouse). Three of the six "cis-regulatory elements" showed
sequence conservation limited to tetrapods, with one element being shared by
tetrapods and coelacanth but not the ray-finned fish; this latter element could
function in transgenic mouse assays to "drive reporter gene expression in
a limb-specific pattern." Another
particular set of genes compared between tetrapod and coelacanth lineages were
genes for the urea cycle, because "[e]xcretion of nitrogen is a major
physiological challenge for terrestrial vertebrates." Urea cycle genes involved in producing urea
for nitrogenous waste disposal (such as carbamoyl phosphate synthase I) showed
strong evidence of selection whereas genes (such as mitochondrial arginase)
involved in arginine metabolism but not excretion did not show such selection. The authors conclude that this is evidence of adaptive evolution in the
transition from water to land. Hox
gene studies also indicated changes from the coelacanth and tetrapod lineages
implicated in placental and other reproductive structures not found in animals
living in an aquatic environment. Finally,
the coelacanth genome lacks genes for immunoglobuins of the M class but did
possess two IgW genes previously found only in lungfish and certain
cartilaginous fish.
In addition to establishing that lungfish not the
coelacanth was the common ancestor to all terrestrial vertebrate, the authors also
established that the coelacanth also showed a slow rate of evolutionary change,
which they speculate might be due to "a static habitat and lack of
predation" and promising that "[f]urther study of these changes
between tetrapods and the coelacanth may provide important insights into how a
complex organism like a vertebrate can markedly change its way of life."
The
authors were affiliated with the following institutions: The Broad Institute at
MIT; Benaroya Research Institute and University of Washington; University of Konstanz, Germany; Universite
de Montreal; University of Oregon; Institute of Molecular and Cell Biology,
Singapore; Universidade Federal do Para, Brazil; Harvard University; University
of Utah; Ecole Normale Superieure de Lyon; University of Kentucky; Rhodes
University, South Africa; Wellcome Trust Sanger Institute; University of
Trieste; University of Liege; Victoria University, Australia; University of
Tuscia; University of Hamburg; Polytechnic University of Marche, Italy;
University of South Florida; University of Western Cape, South Africa; Woods
Hole Oceanographic Institution; Oxford University; Universitat Leipzig; Keio
University, Japan; The Graduate University
for Advanced Studies, Japan; European Molecular Biology Laboratory; University
of Wuerzburg, Germany; University of Illinois at Chicago; National Institute of
Genetics, Japan; and Uppsala University.
Image of Latimeria chalumnae (above) by Alberto Fernandez Fernandez, from the Wikipedia Commons under the Creative Commons Attribution 3.0 Unported license.
