Cephalochordata

Cephalochordata (from the Greek kephalē, "head," and chordē, "cord," commonly known as lancelets or amphioxus) is the most basal living subphylum of Chordata, comprising approximately 35 extant marine species across three genera: Branchiostoma (~25 species, including the European B. lanceolatum, Floridian B. floridae, and East Asian B. belcheri), Epigonichthys (~3 species), and the basal genus Asymmetron (A. lucayanum, ~7 species). Slender, laterally compressed, translucent fish-like animals measuring 3 to 8 cm in length, lancelets spend their adult lives burrowed tail-first in shallow coastal sands with only their oral hood protruding to filter phytoplankton from seawater, while swimming via rapid lateral undulations when disturbed.

Although 19th- and 20th-century zoologists placed Cephalochordata as the immediate sister group to Vertebrata based on their striking V-shaped segmented muscle blocks (myomeres) and fish-like swimming posture, modern phylogenomics (Delsuc et al., 2006; Putnam et al., 2008) conclusively established that Tunicata (urochordates) is the true sister clade to Vertebrata (forming Olfactores), while Cephalochordata represents the basal sister lineage to all other living chordates. Because tunicates underwent rapid genomic rearrangement and morphological simplification into sessile filter-feeders, whereas amphioxus evolved remarkably slowly at both the morphological and chromosomal levels, Branchiostoma serves as the premier living proxy for reconstructing the anatomy, embryology, and pre-duplication genome of the last common ancestor of all chordates (~540–520 Ma, mirrored in Cambrian fossils such as Pikaia gracilens and Cathaymyrus).

Unlike tunicates (which lose the notochord and dorsal nerve cord during metamorphosis) and vertebrates (which replace most of the embryonic notochord with bony or cartilaginous vertebral centra), amphioxus retains all five canonical chordate synapomorphies in a functional, textbook configuration throughout adult life:

1. A Muscular Notochord Extending into the Anterior Rostrum: The notochord of amphioxus runs not only from the tip of the tail to the hindbrain (as in vertebrates and tunicates), but projects all the way forward to the very tip of the anterior snout (rostrum) ahead of the cerebral vesicle—giving the subphylum its name (Cephalochordata, "head-chord") and stiffening the snout for burrowing into coarse sand. Uniquely among chordates, the coin-stacked notochordal lamellae of amphioxus are actual striated muscle cells containing actin and paramyosin myofilaments innervated directly from the dorsal nerve cord via upward rootlets ("horns"): contracting these notochordal muscle plates increases internal hydrostatic turgor and dynamically tunes the flexural stiffness of the axial rod during fast swimming vs. burrowing!
2. Dorsal Hollow Nerve Cord and the Cerebral Vesicle: Dorsal to the notochord runs a hollow ectodermal neural tube (formed by rolling and overgrowth of the embryonic neural plate). Although amphioxus lacks a tripartite cranial box or paired camera eyes, molecular gene-expression mapping (Otx, Pax2/5/8, Gbx, Hox1–4) by Linda Z. Holland, Nicholas D. Holland, and Thurston Lacalli proved that the slightly dilated anterior cerebral vesicle of amphioxus is homologous region-for-region with the vertebrate forebrain (diencephalon/hypothalamus) and midbrain, followed posteriorly by a hindbrain homolog and spinal cord, complete with four distinct photoreceptive systems:
   • An anterior unpaired ciliary photoreceptor "frontal eye" (homologous to the vertebrate paired lateral eyes / retina);
   • A dorsal lamellar body of ciliary photoreceptors (homologous to the vertebrate pineal gland); and
   • Midbrain Joseph cells and segmental dorsal rhabdomeric photoreceptors (Organs of Hesse) wrapped by cup-shaped melanocytes along the length of the neural tube (homologous to vertebrate melanopsin-expressing ipRGCs) that detect whether the burrowed body is fully covered by sand!
3. Pharyngeal Gill Slits (Up to 180 Pairs) and the Atrium: Behind the oral hood (ringed by sensory buccal cirri and a ciliated wheel organ), the spacious pharynx is perforated by 50 to 180 pairs of oblique pharyngeal gill slits supported by acellular collagenous gill bars (branchial basket). Cilia lining the gill bars pump water through the slits into a protective surrounding cavity (atrium) that vents through a single ventral atriopore, while food particles are trapped on a mucous net.
4. Endostyle (Thyroid Homolog) and Midgut Hepatic Caecum: Running along the ventral floor of the pharynx is the endostyle—a ciliated glandular groove that secretes iodinated mucoproteins and expresses the transcription factors Nkx2.1 (TTF-1), Pax8, and FoxE4 plus thyroid peroxidase (TPO), synthesizing thyroid hormones (triiodothyronine T₃ and thyroacetic acid TRIAC, which trigger amphioxus larval metamorphosis via the thyroid hormone receptor!). Behind the pharynx, a blind-ending hepatic diverticulum (caecum) buds off the midgut and synthesizes vertebrate-liver-homologous proteins (vitellogenin, plasminogen, antithrombin, and insulin-like peptide).
5. Segmented V-Shaped Myomeres and Post-Anal Tail: Along both sides of the body lie 50 to 85 chevron-shaped muscle blocks (myomeres / myotomes) separated by connective-tissue myosepta. Unlike vertebrates (where motor nerves grow out from the spinal cord to innervate muscle fibers), amphioxus muscle cells send long cytoplasmic tails (muscle tails) inward to synapse directly on the surface of the dorsal nerve cord!

When the 520-megabase genome of Branchiostoma floridae was sequenced by Nicholas H. Putnam, Daniel S. Rokhsar, Linda Z. Holland, and colleagues (2008) (followed by functional epigenomic comparisons across Branchiostoma lanceolatum by Marlétaz et al., 2018), it provided the definitive genomic proof of Susumu Ohno's (1970) 2R Whole-Genome Duplication Hypothesis:

1. 1-to-4 Macrosynteny with Vertebrate Chromosomes: Across the genome, every single ancestral chordate linkage group preserved in amphioxus maps onto four paralogous chromosomal segments (ohnologons) in jawed vertebrates (such as the four human Hox clusters: HOXA, HOXB, HOXC, HOXD), confirming that two rounds of whole-genome duplication (1R and 2R) occurred at the base of Vertebrata after the divergence of Cephalochordata and Tunicata.
2. An Intact 14-GeneSingle Hox Cluster (AmphiHox1–14): Whereas tunicates (Ciona) shattered their Hox cluster across multiple chromosomes and lost central Hox genes, and mammals duplicated theirs into 39 genes across four clusters, amphioxus retains a single, unbroken, collinear cluster of 15 Hox genes (AmphiHox1 through AmphiHox15) spanning ~470 kb—preserving the ancestral spatial and temporal collinearity of the early Cambrian chordate ancestor.
3. Hyper-Polymorphic Innate Immunity and Proto-RAG Transposons: Although amphioxus lacks the rearranging immunoglobulin/TCR adaptive immune system of jawed vertebrates and the LRR-based VLR system of cyclostomes, its genome expanded innate immune receptor families by an order of magnitude (>70 Toll-like receptors [TLRs], >90 NLR inflammasome receptors, and >1,200 scavenger receptors), alongside variable region-containing chitin-binding proteins (VCBPs) in the gut and an active ProtoRAG transposon (Huang et al., 2016)—the living evolutionary ancestor of the vertebrate RAG1/RAG2 V(D)J recombinase!

What was the ProtoRAG transposon discovered in amphioxus, and how did it solve the origin of the vertebrate adaptive immune system?

For decades, immunologists debated how jawed vertebrates suddenly acquired the RAG1 and RAG2 recombinase enzymes that cut and paste V, D, and J gene segments at recombination signal sequences (RSSs) to generate billions of antibodies and T-cell receptors. In 2016, Shengfeng Huang, Anlong Xu, David G. Schatz, and colleagues discovered an active Transib-superfamily DNA transposon in the lancelet Branchiostoma belcheri—named ProtoRAG—that encodes both adjacent intronless RAG1-like and RAG2-like proteins flanked by terminal inverted repeats (TIRs) whose sequence and cleavage mechanism match vertebrate RSSs! Cryo-EM structures confirmed that an ancient ProtoRAG transposon infected a stem-chordate genome, lost its ability to transpose in jawed vertebrates, and was domesticated into the RAG1/2 V(D)J recombinase.

How does the basal lancelet genus Asymmetron differ from Branchiostoma?

Phylogenomic and mitogenomic analyses (Kon et al., 2007; Yue et al., 2014) show that although living lancelets look nearly identical externally, the genus Asymmetron (which possesses a single row of gonads on the right side only and a long urostyloid tail process, compared to paired left and right gonads in Branchiostoma) diverged from Branchiostoma roughly 120 to 160 million years ago during the Mesozoic—demonstrating remarkable morphological stasis across >100 million years of independent evolution.

1. Delsuc, F., Brinkmann, H., Chourrout, D., & Philippe, H. (2006). Tunicates and not cephalochordates are the closest living relatives of vertebrates. Nature, 439(7079), 965–968. doi:10.1038/nature04336
2. Putnam, N. H., Butts, T., Ferrier, D. E. K., Furlong, R. F., Hellsten, U., Kawashima, T., ... & Rokhsar, D. S. (2008). The amphioxus genome and the evolution of the chordate karyotype. Nature, 453(7198), 1064–1071. doi:10.1038/nature06967
3. Holland, L. Z., Carvalho, J. E., Escriva, H., Laudet, V., Schubert, M., Shimeld, S. M., & Yu, J.-K. (2013). Evolution of bilaterian central nervous systems: A single origin? EvoDevo, 4, 27. doi:10.1186/2041-9139-4-27
4. Huang, S., Tao, X., Yuan, S., Zhang, Y., Li, P., Beilinson, H. A., ... & Xu, A. (2016). Discovery of an active RAG transposon illuminates the origins of V(D)J recombination. Cell, 166(1), 102–114. doi:10.1016/j.cell.2016.05.032
5. Marlétaz, F., Firbas, P. N., Maeso, I., Tena, J. J., Bogdanovic, O., Perry, M., ... & Irimia, M. (2018). Amphioxus functional genomics and the origins of vertebrate gene regulation. Nature, 564(7734), 64–70. doi:10.1038/s41586-018-0734-6