Identification of long-preserved specimens reveals the historical geographic range of the Patagonian lamprey Geotria macrostoma (Burmeister, 1868) in southern South America.

The lamprey genus Geotria Gray, 1851 currently includes only two species: G. australis and G. macrostoma. However, taxonomic relationships within the genus have traditionally been ambiguous and difficult to establish due to the extreme changes in morphology, dentition, and coloration that lampreys undergo during their life cycles, particularly during upstream migration and sexual maturation. Consequently, several lamprey specimens held in museum collections have remained unidentified, especially those from Argentina. In this study, a series of morphometric characters were subjected to discriminant function analysis (DFA) to identify the lamprey species collected during 1867-2004 from the de la Plata River and Patagonia. These specimens are housed at the Museo Argentino de Ciencias Naturales "Bernardino Rivadavia" in Buenos Aires, the Museo de Historia Natural de Montevideo, and the Naturhistoriska riksmuseet in Stockholm. Based on the proportions of the length of the oral disc, prebranchial, and pre-caudal body regions, and the depth of the trunk, DFA provided conclusive evidence that the specimens corresponded to the recently revalidated G. macrostoma (Burmeister, 1868), which was originally incorrectly named as Petromyzon macrostomus Burmeister, 1868, Exomegas macrostomus (Berg, 1899), Geotria chilensis (Berg, 1895), and Geotria macrostoma f. gallegensis Smitt, 1901, as well as other nontype museum individuals of uncertain taxonomic status. The identifications of these long-preserved museum specimens provided key information on the historical geographic range of Argentinian lampreys and suggest that the disappearance of the species reported from northern localities (the Pampean Region) can be attributed to the degradation of their critical habitats, primarily caused by anthropogenic impact and climate change.

The oldest three-dimensionally preserved vertebrate neurocranium.

The neurocranium is an integral part of the vertebrate head, itself a major evolutionary innovation1,2. However, its early history remains poorly understood, with great dissimilarity in form between the two living vertebrate groups: gnathostomes (jawed vertebrates) and cyclostomes (hagfishes and lampreys)2,3. The 100 Myr gap separating the Cambrian appearance of vertebrates4-6 from the earliest three-dimensionally preserved vertebrate neurocrania7 further obscures the origins of modern states. Here we use computed tomography to describe the cranial anatomy of an Ordovician stem-group gnathostome: Eriptychius americanus from the Harding Sandstone of Colorado, USA8. A fossilized head of Eriptychius preserves a symmetrical set of cartilages that we interpret as the preorbital neurocranium, enclosing the fronts of laterally placed orbits, terminally located mouth, olfactory bulbs and pineal organ. This suggests that, in the earliest gnathostomes, the neurocranium filled out the space between the dermal skeleton and brain, like in galeaspids, osteostracans and placoderms and unlike in cyclostomes2. However, these cartilages are not fused into a single neurocranial unit, suggesting that this is a derived gnathostome trait. Eriptychius fills a major temporal and phylogenetic gap in our understanding of the evolution of the gnathostome head, revealing a neurocranium with an anatomy unlike that of any previously described vertebrate.

Organization of the body wall in lampreys informs the evolution of the vertebrate paired appendages.

Vertebrate paired appendages are one of the most important evolutionary novelties in vertebrates. During embryogenesis, the skeletal elements of paired appendages differentiate from the somatic mesoderm, which is a layer of lateral plate mesoderm. However, the presence of the somatic mesoderm in the common ancestor of vertebrates has been controversial. To address this problem, it is necessary but insufficient to understand the developmental process of lateral plate mesoderm formation in lamprey (jawless vertebrates) embryos. Here, I show the presence of the somatic mesoderm in lamprey (Lethenteron camtschaticum) embryos using plastic sectioning and transmission electron microscopy analysis. During the early pharyngeal stages, the somatic mesoderm transforms from the lateral plate mesoderm in the trunk region. Soon after, when the cardiac structures were morphologically distinct, the somatic mesoderm was recognized through the cardiac to more caudal regions. These findings indicated that the somatic mesoderm evolved before the emergence of paired appendages. I also discuss the developmental changes in the body wall organization in the common ancestor of vertebrates, which is likely related to the evolution of the paired appendages.

Forebrain Architecture and Development in Cyclostomes, with Reference to the Early Morphology and Evolution of the Vertebrate Head.

The vertebrate head and brain are characterized by highly complex morphological patterns. The forebrain, the most anterior division of the brain, is subdivided into the diencephalon, hypothalamus, and telencephalon from the neuromeric subdivision into prosomeres. Importantly, the telencephalon contains the cerebral cortex, which plays a key role in higher order cognitive functions in humans. To elucidate the evolution of the forebrain regionalization, comparative analyses of the brain development between extant jawed and jawless vertebrates are crucial. Cyclostomes - lampreys and hagfishes - are the only extant jawless vertebrates, and diverged from jawed vertebrates (gnathostomes) over 500 million years ago. Previous developmental studies on the cyclostome brain were conducted mainly in lampreys because hagfish embryos were rarely available. Although still scarce, the recent availability of hagfish embryos has propelled comparative studies of brain development and gene expression. By integrating findings with those of cyclostomes and fossil jawless vertebrates, we can depict the morphology, developmental mechanism, and even the evolutionary path of the brain of the last common ancestor of vertebrates. In this review, we summarize the development of the forebrain in cyclostomes and suggest what evolutionary changes each cyclostome lineage underwent during brain evolution. In addition, together with recent advances in the head morphology in fossil vertebrates revealed by CT scanning technology, we discuss how the evolution of craniofacial morphology and the changes of the developmental mechanism of the forebrain towards crown gnathostomes are causally related.

Morphometric and physical characteristics distinguishing adult Patagonian lamprey, Geotria macrostoma from the pouched lamprey, Geotria australis.

The pouched lamprey, Geotria australis Gray, 1851, has long been considered monotypic in the Geotriidae family with a wide southern temperate distribution across Australasia and South America. Recent studies have provided molecular and morphological evidence for a second Geotria species in South America; Geotria macrostoma (Burmeister, 1868). The aim of this study was to determine morphometric and physical characteristics of adult G. macrostoma that further differentiate this re-instated species of Geotriidae from G. australis. The diagnostic features discriminating immature adult G. macrostoma from G. australis when entering fresh water, are distinct differences in dentition, oral papillae and fimbriae counts and differences in coloration. In addition, G. macrostoma display greater growth of the prebranchial region and oral disc and has a deeper body depth and higher condition factor. All current ecological knowledge of the genus Geotria is based on Australasian populations, which may not be applicable to G. macrostoma. To ensure the conservation and protection of the Patagonian lamprey as a re-identified species, further investigations are needed to understand its life history, biology and ecology throughout its range.

Non-ammocoete larvae of Palaeozoic stem lampreys.

Ammocoetes-the filter-feeding larvae of modern lampreys-have long influenced hypotheses of vertebrate ancestry1-7. The life history of modern lampreys, which develop from a superficially amphioxus-like ammocoete to a specialized predatory adult, appears to recapitulate widely accepted scenarios of vertebrate origin. However, no direct evidence has validated the evolutionary antiquity of ammocoetes, and their status as models of primitive vertebrate anatomy is uncertain. Here we report larval and juvenile forms of four stem lampreys from the Palaeozoic era (Hardistiella, Mayomyzon, Pipiscius, and Priscomyzon), including a hatchling-to-adult growth series of the genus Priscomyzon from Late Devonian Gondwana. Larvae of all four genera lack the defining traits of ammocoetes. They instead display features that are otherwise unique to adult modern lampreys, including prominent eyes, a cusped feeding apparatus, and posteriorly united branchial baskets. Notably, phylogenetic analyses find that these non-ammocoete larvae occur in at least three independent lineages of stem lamprey. This distribution strongly implies that ammocoetes are specializations of modern-lamprey life history rather than relics of vertebrate ancestry. These phylogenetic insights also suggest that the last common ancestor of hagfishes and lampreys was a macrophagous predator that did not have a filter-feeding larval phase. Thus, the armoured 'ostracoderms' that populate the cyclostome and gnathostome stems might serve as better proxies than living cyclostomes for the last common ancestor of all living vertebrates.

Cephalochordates: A window into vertebrate origins.

How vertebrates evolved from their invertebrate ancestors has long been a central topic of discussion in biology. Evolutionary developmental biology (evodevo) has provided a new tool-using gene expression patterns as phenotypic characters to infer homologies between body parts in distantly related organisms-to address this question. Combined with micro-anatomy and genomics, evodevo has provided convincing evidence that vertebrates evolved from an ancestral invertebrate chordate, in many respects resembling a modern amphioxus. The present review focuses on the role of evodevo in addressing two major questions of chordate evolution: (1) how the vertebrate brain evolved from the much simpler central nervous system (CNS) in of this ancestral chordate and (2) whether or not the head mesoderm of this ancestor was segmented.

Evo-devo studies of cyclostomes and the origin and evolution of jawed vertebrates.

Modern vertebrates consist of two sister groups: cyclostomes and gnathostomes. Cyclostomes are a monophyletic jawless group that can be further divided into hagfishes and lampreys, which show conspicuously different developmental and morphological patterns. However, during early pharyngula development, there appears to be a stage when the embryos of hagfishes and lampreys resemble each other by showing an "ancestral" craniofacial pattern; this pattern enables morphological comparison of hagfish and lamprey craniofacial development at late stages. This cyclostome developmental pattern, or more accurately, this developmental pattern of the jawless grade of vertebrates in early pharyngula was very likely shared by the gnathostome stem before the division of the nasohypophyseal placode led to the jaw and paired nostrils. The craniofacial pattern of the modern jawed vertebrates seems to have begun in fossil ostracoderms (including galeaspids), and was completed by the early placoderm lineages. The transition from jawless to jawed vertebrates was thus driven by heterotopy of development, mainly caused by separation and shift of ectodermal placodes and resultant ectomesenchymal distribution, and shifts of the epithelial-mesenchymal interactions that underlie craniofacial differentiation. Thus, the evolution of the jaw was not a simple modification of the mandibular arch, but a heterotopic shift of the developmental interactions involving not only the mandibular arch, but also the premandibular region rostral to the mandibular arch.

Predicting larval metamorphosis of Pacific lamprey Entosphenus tridentatus through measurements of length and mass.

The utility of length and mass measurements to predict the larval metamorphosis of Pacific lamprey Entosphenus tridentatus was evaluated. During 2004-2008, larval E. tridentatus were collected from Cedar Creek (Washington, USA) in either the spring or autumn, measured for total length and total mass, reared in captivity and monitored for metamorphosis. The minimum total length, total mass and condition factor of larvae that were observed to go through metamorphosis were 102 mm, 2.0 g and 1.52, respectively. Logistic models indicated that total length and condition factor in both spring and autumn were the most significant variables for predicting metamorphosis of Pacific lamprey during the subsequent summer. Mass in the autumn also appeared important to predict whether metamorphosis occurred in the subsequent summer. Collectively, all models using specific minimums of total length, total mass or condition factor of larvae as criteria for them to metamorphose were sometimes (5 of 14 cases) able to predict the percentage of larvae that would metamorphose but rarely (1 of 12 cases) able to predict which individual larvae would metamorphose. Similar to other anadromous species of lampreys, the size and condition of larval E. tridentatus have utility for predicting metamorphic fate.

Revalidation of the Argentinian pouched lamprey Geotria macrostoma (Burmeister, 1868) with molecular and morphological evidence.

BACKGROUND: The Argentinian pouched lamprey, classified as Petromyzon macrostomus Burmeister, 1868 was first described in 1867 in De La Plata River, in Buenos Aires, Argentina, and subsequently recorded in several rivers from Patagonia. Since its original description, the validity of P. macrostomus was questioned by several ichthyologists and 36 years after its original discovery it was considered a junior synonym of Geotria australis Gray, 1851. For a long time, the taxonomic status of G. australis has been uncertain, largely due to the misinterpretations of the morphological alterations that occur during sexual maturation, including the arrangement of teeth, size and position of fins and cloaca, and the development of an exceptionally large gular pouch in males. In this study, the taxonomic status of Geotria from across the "species" range was evaluated using both molecular analysis and examination of morphological characteristics. METHODOLOGY/PRINCIPAL FINDINGS: Phylogenetic and species delimitation analyses based on mitochondrial DNA sequences of Cytochrome b (Cyt b) and Cytochrome C Oxidase Subunit 1 (COI) genes, along with morphological analysis of diagnostic characters reported in the original descriptions of the species were used to assess genetic and morphological variation within Geotria and to determine the specific status of the Argentinian lamprey. These analyses revealed that Geotria from Argentina constitutes a well differentiated lineage from Chilean and Australasian populations. The position of the cloaca and the distance between the second dorsal and caudal fins in sub-adult individuals, and at previous life stages, can be used to distinguish between the two species. In addition, the genetic distance between G. macrostoma and G. australis for the COI and Cyt b mitochondrial genes is higher than both intra- and inter-specific distances reported for other Petromyzontiformes. CONCLUSIONS/SIGNIFICANCE: Our results indicate that the Argentinian pouched lamprey, found along a broad latitudinal gradient on the south-west Atlantic coast of South America, should be named as Geotria macrostoma (Burmeister, 1868) and not as G. australis Gray 1851, returning to its earliest valid designation in Argentina. Geotria macrostoma can now be considered as the single lamprey species inhabiting Argentinian Patagonia, with distinct local adaptations and evolutionary potential. It is essential that this distinctiveness is recognized in order to guide future conservation and management actions against imminent threats posed by human actions in the major basins of Patagonia.

The role of the optic tectum for visually evoked orienting and evasive movements.

As animals forage for food and water or evade predators, they must rapidly decide what visual features in the environment deserve attention. In vertebrates, this visuomotor computation is implemented within the neural circuits of the optic tectum (superior colliculus in mammals). However, the mechanisms by which tectum decides whether to approach or evade remain unclear, and also which neural mechanisms underlie this behavioral choice. To address this problem, we used an eye-brain-spinal cord preparation to evaluate how the lamprey responds to visual inputs with distinct stimulus-dependent motor patterns. Using ventral root activity as a behavioral readout, we classified 2 main types of fictive motor responses: (i) a unilateral burst response corresponding to orientation of the head toward slowly expanding or moving stimuli, particularly within the anterior visual field, and (ii) a unilateral or bilateral burst response triggering fictive avoidance in response to rapidly expanding looming stimuli or moving bars. A selective pharmacological blockade revealed that the brainstem-projecting neurons in the deep layer of the tectum in interaction with local inhibitory interneurons are responsible for selecting between these 2 visually triggered motor actions conveyed through downstream reticulospinal circuits. We suggest that these visual decision-making circuits had evolved in the common ancestor of vertebrates and have been conserved throughout vertebrate phylogeny.

Development and Functional Organization of the Cranial Nerves in Lampreys.

Lampreys, together with hagfishes, are the only extant representatives of the oldest branch of vertebrates, the agnathans, which are the sister group of gnathostomes; therefore, studies on these animals are of great evolutionary significance. Lampreys exhibit a particular life cycle with remarkable changes in their behavior, concomitant, in part, with important modifications in the head and its musculature, which might influence the development of the cranial nerves. In this context, some cranial nerves such as the optic nerve and the ocular motor nerves, which develop slowly during an extremely long larval period lasting more than five years, have been more thoroughly investigated; however, much less experimental information is available about others, such as the facial or the hypoglossal nerves. In addition, the possible existence of a "true" accessory nerve in these animals is still a matter of conjecture. Although growing in last decades, investigations on the physiology of the lamprey cranial nerves is scanty. This review focuses on past and recent findings that have contributed to characterize the anatomical organization of the cranial nerves in lampreys, including their components and nuclei, and their relations in the brain; in addition, comments on their development and functional role are also included. Anat Rec, 302:512-539, 2019. © 2018 Wiley Periodicals, Inc.

Inner ear development in cyclostomes and evolution of the vertebrate semicircular canals.

Jawed vertebrates have inner ears with three semicircular canals, the presence of which has been used as a key to understanding evolutionary relationships. Ostracoderms, the jawless stem gnathostomes, had only two canals and lacked the lateral canal1-3. Lampreys, which are modern cyclostomes, are generally thought to possess two semicircular canals whereas the hagfishes-which are also cyclostomes-have only a single canal, which used to be regarded as a more primitive trait1,4. However, recent molecular and developmental analyses have strongly supported the monophyly of cyclostomes5-7, which has left the evolutionary trajectory of the vertebrate inner ear unclear8. Here we show the differentiation of the otic vesicle of the lamprey Lethenteron camtschaticum and inshore hagfish Eptatretus burgeri. This is the first time, to our knowledge, that the development of the hagfish inner ear is reported. We found that canal development in the lamprey starts with two depressions-which is reminiscent of the early developmental pattern of the inner ear in modern gnathostomes. These cyclostome otic vesicles show a pattern of expression of regulatory genes, including OTX genes, that is comparable to that of gnathosomes. Although two depressions appear in the lamprey vesicle, they subsequently fuse to form a single canal that is similar to that of hagfishes. Complete separation of the depressions results in anterior and posterior canals in gnathostomes. The single depression of the vesicle in hagfishes thus appears to be a secondarily derived trait. Furthermore, the lateral canal in crown gnathostomes was acquired secondarily-not by de novo acquisition of an OTX expression domain, but by the evolution of a developmental program downstream of the OTX genes.

GABAergic modulation of olfactomotor transmission in lampreys.

Odor-guided behaviors, including homing, predator avoidance, or food and mate searching, are ubiquitous in animals. It is only recently that the neural substrate underlying olfactomotor behaviors in vertebrates was uncovered in lampreys. It consists of a neural pathway extending from the medial part of the olfactory bulb (medOB) to locomotor control centers in the brainstem via a single relay in the caudal diencephalon. This hardwired olfactomotor pathway is present throughout life and may be responsible for the olfactory-induced motor behaviors seen at all life stages. We investigated modulatory mechanisms acting on this pathway by conducting anatomical (tract tracing and immunohistochemistry) and physiological (intracellular recordings and calcium imaging) experiments on lamprey brain preparations. We show that the GABAergic circuitry of the olfactory bulb (OB) acts as a gatekeeper of this hardwired sensorimotor pathway. We also demonstrate the presence of a novel olfactomotor pathway that originates in the non-medOB and consists of a projection to the lateral pallium (LPal) that, in turn, projects to the caudal diencephalon and to the mesencephalic locomotor region (MLR). Our results indicate that olfactory inputs can induce behavioral responses by activating brain locomotor centers via two distinct pathways that are strongly modulated by GABA in the OB. The existence of segregated olfactory subsystems in lampreys suggests that the organization of the olfactory system in functional clusters may be a common ancestral trait of vertebrates.

The blueprint of the vertebrate forebrain - With special reference to the habenulae.

The medial and lateral habenulae are conserved throughout vertebrate evolution, and form an integrated part in the forebrain control of behavior together with the basal ganglia, the dopamine and serotonin systems and cortex. The lateral habenula plays a role in the control of dopamine activity in the context of aversive behavior and the converse, a reward situation. These circuits are important for a value-based evaluation of the success of prior actions. The medial habenula is involved in mediating escape and freezing behavior. These structures are reviewed with a focus on the lamprey, belonging to the oldest group of now living vertebrate, showing that most aspects of the habenular structure and function have been conserved throughout vertebrate phylogeny.

The Lamprey Pallium Provides a Blueprint of the Mammalian Layered Cortex.

The basic architecture of the mammalian neocortex is remarkably similar across species. Pallial structures in the reptilian brain are considered amniote precursors of mammalian neocortex, whereas pallia of anamniotes ("lower" vertebrates) have been deemed largely insignificant with respect to homology. Here, we examine the cytoarchitecture of the lateral pallium in the lamprey, the phylogenetically oldest group of extant vertebrates. We reveal a three-layered structure with similar excitatory cell types as in the mammalian cortex and GABAergic interneurons. The ventral parts are sensory areas receiving monosynaptic thalamic input that can be activated from the optic nerve, whereas the dorsal parts contain motor areas with efferent projections to the brainstem, receiving oligosynaptic thalamic input. Both regions receive monosynaptic olfactory input. This three-layered "primordial" lamprey lateral pallium has evolved most features of the three-layered reptilian cortices and is thereby a precursor of the six-layered "neo" cortex with a long-standing evolutionary precedent (some 500 million years ago).

Characterization of the encoding properties of intraspinal mechanosensory neurons in the lamprey.

Proprioceptive sensory inputs are an integral part of the closed-loop system of locomotion. In the lamprey, a model organism for vertebrate locomotion, such sensory inputs come from intraspinal mechanosensory cells called "edge cells". These edge cells synapse directly onto interneurons in the spinal central pattern generator (CPG) circuit and allow the CPG to adjust the motor output according to how the body is bending. However, the encoding properties of the edge cells have never been fully characterized. To identify these properties and better understand edge cells' role in locomotion, we isolated spinal cords of silver lampreys (Ichthyomyzon unicuspis) and recorded extracellularly from the lateral tracts where edge cell axons are located. We identified cells that responded to mechanical stimuli and used standard spike sorting algorithms to identify separate units, then examined how the cells respond to bending rate and bending angle. Although some cells respond to the bending angle, as was previously known, the strongest and most common responses were to bending velocity. These encoding properties will help us better understand how lampreys and other basal vertebrates adapt their locomotor rhythms to different water flow patterns, perturbations, or other unexpected changes in their environments.

The Central Nervous System of Jawless Vertebrates: Encephalization in Lampreys and Hagfishes.

Lampreys and hagfishes are the sole surviving representatives of the early agnathan (jawless) stage in vertebrate evolution, which has previously been regarded as the least encephalized group of all vertebrates. Very little is known, however, about the extent of interspecific variation in relative brain size in these fishes, as previous studies have focused on only a few species, even though lampreys exhibit a variety of life history traits. While some species are parasitic as adults, with varying feeding behaviors, others (nonparasitic species) do not feed after completing their macrophagous freshwater larval phase. In addition, some parasitic species remain in freshwater, while others undergo an anadromous migration. On the basis of data for postmetamorphic individuals representing approximately 40% of all lamprey species, with representatives from each of the three families, the aforementioned differences in life history traits are reflected in variations in relative brain size. Across all lampreys, brain mass increases with body mass with a scaling factor or slope (α) of 0.35, which is less than those calculated for different groups of gnathostomatous (jawed) vertebrates (α = 0.43-0.62). When parasitic and nonparasitic species are analyzed separately, with phylogeny taken into account, the scaling factors of both groups (parasitic α = 0.43, nonparasitic α = 0.45) approach those of gnathostomes. The relative brain size in fully grown adults of parasitic species is, however, less than that of the adults of nonparasitic species, paralleling differences between fully grown adults and recently metamorphosed individuals of anadromous species. The average degree of encephalization is found in anadromous parasitic lampreys and might thus represent the ancestral condition for extant lampreys. These results suggest that the degree of encephalization in lampreys varies according to both life history traits and phylogenetic relationships.

The neural control of respiration in lampreys.

This review focuses on past and recent findings that have contributed to characterize the neural networks controlling respiration in the lamprey, a basal vertebrate. As in other vertebrates, respiration in lampreys is generated centrally in the brainstem. It is characterized by the presence of a fast and a slow respiratory rhythm. The anatomical and the basic physiological properties of the neural networks underlying the generation of the fast rhythm have been more thoroughly investigated; less is known about the generation of the slow respiratory rhythm. Comparative aspects with respiratory generators in other vertebrates as well as the mechanisms of modulation of respiration in association with locomotion are discussed.

Regulatory evolution of Tbx5 and the origin of paired appendages.

The diversification of paired appendages has been a major factor in the evolutionary radiation of vertebrates. Despite its importance, an understanding of the origin of paired appendages has remained elusive. To address this problem, we focused on T-box transcription factor 5 (Tbx5), a gene indispensable for pectoral appendage initiation and development. Comparison of gene expression in jawless and jawed vertebrates reveals that the Tbx5 expression in jawed vertebrates is derived in having an expression domain that extends caudal to the heart and gills. Chromatin profiling, phylogenetic footprinting, and functional assays enabled the identification of a Tbx5 fin enhancer associated with this apomorphic pattern of expression. Comparative functional analysis of reporter constructs reveals that this enhancer activity is evolutionarily conserved among jawed vertebrates and is able to rescue the finless phenotype of tbx5a mutant zebrafish. Taking paleontological evidence of early vertebrates into account, our results suggest that the gain of apomorphic patterns of Tbx5 expression and regulation likely contributed to the morphological transition from a finless to finned condition at the base of the vertebrate lineage.