BACKGROUND: The deep (> 200 m) ocean floor is often considered to be a refugium of biodiversity; many benthic marine animals appear to share ancient common ancestry with nearshore and terrestrial relatives. Whether this pattern holds for vertebrates is obscured by a poor understanding of the evolutionary history of the oldest marine vertebrate clades. Hagfishes are jawless vertebrates that are either the living sister to all vertebrates or form a clade with lampreys, the only other surviving jawless fishes.
RESULTS: We use the hagfish fossil record and molecular data for all recognized genera to construct a novel hypothesis for hagfish relationships and diversification. We find that crown hagfishes persisted through three mass extinctions after appearing in the Permian ~ 275 Ma, making them one of the oldest living vertebrate lineages. In contrast to most other deep marine vertebrates, we consistently infer a deep origin of continental slope occupation by hagfishes that dates to the Paleozoic. Yet, we show that hagfishes have experienced marked body size diversification over the last hundred million years, contrasting with a view of this clade as morphologically stagnant.
CONCLUSIONS: Our results establish hagfishes as ancient members of demersal continental slope faunas and suggest a prolonged accumulation of deep sea jawless vertebrate biodiversity.

Jawless vertebrates possess an alternative adaptive immune system in which antigens are recognized by variable lymphocyte receptors (VLRs) generated by combinatorial assembly of leucine-rich repeat (LRR) cassettes. Three types of receptors, VLRA, VLRB, and VLRC, have been previously identified. VLRA- and VLRC-expressing cells are T cell-like, whereas VLRB-expressing cells are B cell-like. Here, we report two types of VLRs in lampreys, VLRD and VLRE, phylogenetically related to VLRA and VLRC. The germline VLRD and VLRE genes are flanked by 39 LRR cassettes used in the assembly of mature VLRD and VLRE, with cassettes from chromosomes containing the VLRA and VLRC genes also contributing to VLRD and VLRE assemblies. VLRD and VLRE transcription is highest in the triple-negative (VLRA-/VLRB-/VLRC-) population of lymphocytes, albeit also detectable in VLRA+ and VLRC+ populations. Tissue distribution studies suggest that lamprey VLRD+ and VLRE+ lymphocytes comprise T-like sublineages of cells.

Retinoic acid (RA) is involved in antero-posterior patterning of the chordate body axis and, in jawed vertebrates, has been shown to play a major role at multiple levels of the gene regulatory network (GRN) regulating hindbrain segmentation. Knowing when and how RA became coupled to the core hindbrain GRN is important for understanding how ancient signaling pathways and patterning genes can evolve and generate diversity. Hence, we investigated the link between RA signaling and hindbrain segmentation in the sea lamprey Petromyzon marinus, an important jawless vertebrate model providing clues to decipher ancestral vertebrate features. Combining genomics, gene expression, and functional analyses of major components involved in RA synthesis (Aldh1as) and degradation (Cyp26s), we demonstrate that RA signaling is coupled to hindbrain segmentation in lamprey. Thus, the link between RA signaling and hindbrain segmentation is a pan vertebrate feature of the hindbrain and likely evolved at the base of vertebrates.

On occasion, retroviruses infect the genome of germline cell, forming endogenous retroviruses (ERVs), which provide molecular fossils for studying the deep evolution of retroviruses. While ERVs have been extensively characterized in the genomes of jawed vertebrates, much remains contentious and unexplored about the diversity and evolution of ERVs within jawless vertebrates. Here, we report the discovery of a novel ERV lineage, designated EbuERVs, in the genome of a hagfish Eptatretus burgeri. Phylogenetic analyses show that EbuERVs pertain to epsilon-retroviruses and might have derived from cross-species transmission from jawed vertebrates. EbuERVs are estimated to have invaded in the hagfish genome at least tens of millions of years ago. Evolutionary dynamics analyses indicate that EbuERVs might have experienced one proliferation peak and have been not active in transposition anymore. However, some EbuERVs can transcribe in embryo and might serve as lncRNA. Overall, these findings expand the distribution of retroviruses from jawed vertebrates to jawless vertebrates.

Three types of variable lymphocyte receptor (VLR) genes, VLRA, VLRB, and VLRC, encode antigen recognition receptors in the extant jawless vertebrates, lampreys and hagfish. The somatically diversified repertoires of these VLRs are generated by serial stepwise copying of leucine-rich repeat (LRR) sequences into an incomplete germline VLR gene. Lymphocytes that express VLRA or VLRC are T cell-like, while VLRB-expressing cells are B cell-like. Here, we analyze the composition of the VLRB locus in different jawless vertebrates to elucidate its configuration and evolutionary modification. The incomplete germline VLRB genes of two hagfish species contain short noncoding intervening sequences, whereas germline VLRB genes in six representative lamprey species have much longer intervening sequences that exhibit notable genomic variation. Genomic clusters of potential LRR cassette donors, fragments of which are copied to complete VLRB gene assembly, are identified in Japanese lamprey and sea lamprey. In the sea lamprey, 428 LRR cassettes are located in five clusters spread over a total of 1.7 Mbp of chromosomal DNA. Preferential usage of the different donor cassettes for VLRB assemblage is characterized in our analysis, which reveals evolutionary modifications of the lamprey VLRB genes, elucidates the organization of the complex VLRB locus, and provides a comprehensive catalog of donor VLRB cassettes in sea lamprey and Japanese lamprey.

Ostracoderms (fossil armoured jawless fishes) shed light on early vertebrate evolution by revealing the step-wise acquisition of jawed vertebrate characters, and were important constituents of Middle Palaeozoic vertebrate faunas. A wide variety of head shield shapes are observed within and between the ostracoderm groups, but the timing of these diversifications and the consistency between different measures of their morphospace are unclear. Here, we present the first disparity (explored morphospace) versus diversity (number of taxa) analysis of Pteraspidiformes heterostracans using continuous and discrete characters. Patterns of taxic diversity and morphological disparity are in accordance: they both show a rise to a peak in the Lochkovian followed by a gradual decline in the Middle-Late Devonian. Patterns are largely consistent for disparity measures using sum of ranges or total variance, and when using continuous or discrete characters. Pteraspidiformes heterostracans can be classified as a \"bottom-heavy clade\", i.e., a group where a high initial disparity decreasing over time is detected. In fact, the group explored morphospace early in its evolutionary history, with much of the subsequent variation in dermal armour occurring as variation in the proportions of already evolved anatomical features. This Early Devonian radiation is also in agreement with the paleobiogeographic distribution of the group, with a maximum of dispersal and explored morphospace during the Lochkovian and Pragian time bins.

Stem-group gnathostomes reveal the sequence of character acquisition in the origin of modern jawed vertebrates. The petalichthyids are placoderm-grade stem-group gnathostomes known from both isolated skeletal material and rarer articulated specimens of one genus. They are of particular interest because of anatomical resemblances with osteostracans, the jawless sister group of jawed vertebrates. Because of this, they have become central to debates on the relationships of placoderms and the primitive cranial architecture of gnathostomes. However, among petalichthyids, only the braincase of Macropetalichthys has been studied in detail, and the diversity of neurocranial morphology in this group remains poorly documented. Using X-ray computed microtomography, we investigated the endocranial morphology of Shearsbyaspis oepiki Young, a three-dimensionally preserved petalichthyid from the Early Devonian of Taemas-Wee Jasper, Australia. We generated virtual reconstructions of the external endocranial surfaces, orbital walls and cranial endocavity, including canals for major nerves and blood vessels. The neurocranium of Shearsbyaspis resembles that of Macropetalichthys, particularly in the morphology of the brain cavity, nerves and blood vessels. Many characters, including the morphology of the pituitary vein canal and the course of the trigeminal nerve, recall the morphology of osteostracans. Additionally, the presence of a parasphenoid in Shearsbyaspis (previously not known with confidence outside of arthrodires and osteichthyans) raises some questions about current proposals of placoderm paraphyly. Our detailed description of this specimen adds to the known morphological diversity of petalichthyids, and invites critical reappraisal of the phylogenetic relationships of placoderms.

The brain, comprising billions of neurons and intricate neural networks, is arguably the most complex organ in vertebrates. The diversity of individual neurons is fundamental to the neuronal network complexity and the overall function of the vertebrate brain. In jawed vertebrates, clustered protocadherins provide the molecular basis for this neuronal diversity, through stochastic and combinatorial expression of their various isoforms in individual neurons. Based on analyses of transcriptomes from the Japanese lamprey brain and sea lamprey embryos, genome assemblies of the two lampreys, and brain expressed sequence tags of the inshore hagfish, we show that extant jawless vertebrates (cyclostomes) lack the clustered protocadherins. Our findings indicate that the clustered protocadherins originated from a nonclustered protocadherin in the jawed vertebrate ancestor, after the two rounds of whole-genome duplication. In the absence of clustered protocadherins, cyclostomes might have evolved novel molecules or mechanisms for generating neuronal diversity which remains to be discovered.

Jawless vertebrates, which occupy a unique position in chordate phylogeny, employ leucine-rich repeat (LRR)-based variable lymphocyte receptors (VLR) for antigen recognition. During the assembly of the VLR genes (VLRA, VLRB and VLRC), donor LRR-encoding sequences are copied in a step-wise manner into the incomplete germ-line genes. The assembled VLR genes are differentially expressed by discrete lymphocyte lineages: VLRA- and VLRC-producing cells are T-cell like, whereas VLRB-producing cells are B-cell like. VLRA(+) and VLRC(+) lymphocytes resemble the two principal T-cell lineages of jawed vertebrates that express the αβ or γδ T-cell receptors (TCR). Reminiscent of the interspersed nature of the TCRα/TCRδ locus in jawed vertebrates, the close proximity of the VLRA and VLRC loci facilitates sharing of donor LRR sequences during VLRA and VLRC assembly. Here we discuss the insight these findings provide into vertebrate T- and B-cell evolution, and the alternative types of anticipatory receptors they use for adaptive immunity.

In jawed (gnathostome) vertebrates, the inner ears have three semicircular canals arranged orthogonally in the three Cartesian planes: one horizontal (lateral) and two vertical canals. They function as detectors for angular acceleration in their respective planes. Living jawless craniates, cyclostomes (hagfish and lamprey) and their fossil records seemingly lack a lateral horizontal canal. The jawless vertebrate hagfish inner ear is described as a torus or doughnut, having one vertical canal, and the jawless vertebrate lamprey having two. These observations on the anatomy of the cyclostome (jawless vertebrate) inner ear have been unchallenged for over a century, and the question of how these jawless vertebrates perceive angular acceleration in the yaw (horizontal) planes has remained open. To provide an answer to this open question we reevaluated the anatomy of the inner ear in the lamprey, using stereoscopic dissection and scanning electron microscopy. The present study reveals a novel observation: the lamprey has two horizontal semicircular ducts in each labyrinth. Furthermore, the horizontal ducts in the lamprey, in contrast to those of jawed vertebrates, are located on the medial surface in the labyrinth rather than on the lateral surface. Our data on the lamprey horizontal duct suggest that the appearance of the horizontal canal characteristic of gnathostomes (lateral) and lampreys (medial) are mutually exclusive and indicate a parallel evolution of both systems, one in cyclostomes and one in gnathostome ancestors.