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Lingulid Brachiopod Genome Sequence Gives Peek Into Evolution of 'Living Fossil'

NEW YORK (GenomeWeb) – A team of Japanese researchers has sequenced the genome of a lingulid brachiopod to find that this so-called 'living fossil' is still evolving and to get a glimpse into how it forms its shell.

The earliest brachiopods appeared during the Cambrian period, some 520 million years ago, and became dominant in the oceans during the Paleozoic era. As they have barely changed in appearance since the Silurian period about 443 million to 419 million years ago, brachiopods have been dubbed 'living fossils.' They are also one of the earliest known examples of animal biomineralization.

Researchers led by Noriyuki Satoh, the head of the Okinawa Institute of Science and Technology Graduate University Marine Genomics Unit, sequenced the brachiopod Lingula anatine. Based on their analysis of its genome, transcriptome, and proteome, they reported in Nature Communications that the L. anatina genome has been evolving rapidly. They also noted that brachiopods, based on the L. anatina genome, appear to be close phylogenetic relatives of mollusks, and that the biomineralization mechanism L. anatina uses to form its shell differs from the processes employed by mollusks to create shells and by vertebrates to build bones.

"This is one step toward untangling the mysteries of animal evolution," Satoh said in a statement. "The study highlights the fact that various animals have taken evolutionary paths independently from one another."

The researchers collected L. anatina samples from Amami Island in Japan, and sequenced its genome to some 226-fold coverage using four next-generation sequencing platforms. The Lingula genome, they reported, shows high levels of heterozygosity and low levels of repetitive sequences.

Using transcriptomic data obtained from adult and embryonic tissues as a guide, they estimated that the Lingula genome includes 34,105 protein-coding gene models.

A Blast search indicated that a little more than a quarter of Lingula genes are most similar to mollusk genes, and about a quarter of its genes appeared to be unique to brachiopods.

Further phylogenetic analyses based on one-to-one orthologs, lineage-specific domain losses, microsyntenic blocks, and intron structures confirmed that Lingula is more closely related to Mollusca than to Annelida, the researchers reported.

Despite Lingula's reputation as a living fossil, Satoh and his colleagues found a high degree of change within its genome structure and gene families. They particularly noted that it includes a disorganized Hox cluster, which is divided in two and lacks Lox2 and Lox4.

Lingula also exhibits the highest rate of gene family turnover in bilaterians, the researchers said. It has 3,525 unique gene families, and it has had 7,263 gains and 8,441 losses of gene families.

Further, the gene duplication rate in Lingula is some two times to four times faster than in other lophotrochozoans, they noted. Though a large number of young duplicated genes appear to be under negative selection, they found that genes associated with the extracellular matrix seem to be under positive selection.

A sizeable portion of the protein domain and gene families that have expanded in Lingula have been linked to shell formation. For instance, Lingula contains 31 copies of chitin synthase (CHS) genes and 30 copies of carbohydrate sulfotransferase genes. Interestingly, the researchers found that the Lingula CHS genes include a myosin head domain (MHD), which appears to be a feature unique to lophotrochozoans.

Transcriptomic analysis indicated that Lingula CHS genes are expressed in all adult tissues as well as in larvae, and the MHD-containing CHS genes are highly expressed in larvae as well as in the mantle.

This expansion and expression profiles suggested to Satoh and his colleagues that chitins — which are a characteristic component of arthropod and mollusk shells — are also important in brachiopod biomineralization.

In contrast to mollusks' calcium carbonate shells, Lingula shells are made of calcium phosphate and collagen, as are vertebrate bones, though the molecular mechanism of biomineralization isn't clear.

Using a comparative genomics approach, Satoh and his colleagues examined genes linked to biomineralization in Lingula, vertebrates, and mollusks. Lingula and vertebrates, they found, don't have any specific similarities; however, Lingula and mollusks shared shell formation genes like calcium-dependent protein kinase and chitin synthase.

Through a proteomic analysis of Lingula shells, Satoh and his colleagues uncovered 65 shell matrix proteins, which they found to be similar to those of mollusks and amphioxus. Lingula proteins, they noted, were more likely to contain higher levels of glycine and alanine and have an abundance of cadherin and collagen domains. Of these, 26 were ubiquitously expressed in adult tissues and 20 were specifically expressed in the mantle, including collagen, chitinase, and glutathione peroxidase, among others.

Lingula calcium phosphate production relies on a different gene set than that used by vertebrates, the researchers added. They said that the Lingula shell and mantel contains a number of calcium-binding and extracellular matrix proteins, indicating that metazoan biomineralization likely originated from a calcium-regulated extracellular matrix system.

Because of this, Satoh and his colleagues argued that calcification might be a derived feature in both mollusks and brachiopods, with chitin localized to epithelial cells as the primitive character.

They then proposed a possible mechanism for how Lingula form their shells using a chintinous scaffold. In their model, myosin-head containing chitin synthases and actin filaments turn the cytoskeleton organization into an extracellular chitin scaffold. Then, chitinases in the shell matrix remodel the scaffold to enable the interaction of chitin and chitin-binding proteins. Meanwhile, calcium-binding proteins regulate calcium levels in the shell matrix and initiate the deposition of calcium phosphate with other structural proteins.

"Taken together, our genomic, transcriptomic and proteomic analyses of Lingula biomineralization show similar patterns to those in mollusks and corals, where co-option, domain shuffling and novel genes are the fundamental mechanisms for metazoan biomineralization," Satoh and his colleagues wrote in their paper.