The Inference of the Evolution of Immune Traits as Constrained by Phylogeny: Insight into the Immune System of the Basal Diapsid.

Among vertebrates, some of the most vulnerable taxa to emergent fungal pathogens are members of Reptilia. In light of the growing threat of emergent fungal pathogens affecting wildlife, it is important to broaden the current understanding of immune system function, development, and evolution. The homologous condition of a trait is necessary in order to study its evolution, as such, homology is necessary in the study of immunological evolution. Here, we explore the evolution of toll-like receptors (TLRs), a series of homologous receptors crucial to the initial immune response. The homologous condition of TLR genes provides a unique system in which to explore the evolution of the TLR; using a Reptilian phylogeny, we elucidate the immune condition of the basal diapsid. Our analysis revealed that the basal diapsid may have had an immune system that lacked two receptors: TLR 15, a receptor uniquely present in Reptilia, and TLR 13, a receptor important in the recognition of nucleic acid motifs. Additionally, our analysis showed multiple losses and convergences for various TLRs, likely attributed to redundancies in receptor function. Further exploration into the immune condition of extinct taxa may shed light on the evolution of the reptilian immune system.

Empirical and philosophical problems with the subspecies rank.

Species-level taxonomy derives from empirical sources (data and techniques) that assess the existence of spatiotemporal evolutionary lineages via various species "concepts." These concepts determine if observed lineages are independent given a particular methodology and ontology, which relates the metaphysical species concept to what "kind" of thing a species is in reality. Often, species concepts fail to link epistemology back to ontology. This lack of coherence is in part responsible for the persistence of the subspecies rank, which in modern usage often functions as a placeholder between the evolutionary events of divergence or collapse of incipient species. Thus, prospective events like lineages merging or diverging require information from unknowable future information. This is also conditioned on evidence that the lineage already has a detectably distinct evolutionary history. Ranking these lineages as subspecies can seem attractive given that many lineages do not exhibit intrinsic reproductive isolation. We argue that using subspecies is indefensible on philosophical and empirical grounds. Ontologically, the rank of subspecies is either identical to that of species or undefined in the context of evolutionary lineages representing spatiotemporally defined individuals. Some species concepts more inclined to consider subspecies, like the Biological Species Concept, are disconnected from evolutionary ontology and do not consider genealogy. Even if ontology is ignored, methods addressing reproductive isolation are often indirect and fail to capture the range of scenarios linking gene flow to species identity over space and time. The use of subspecies and reliance on reproductive isolation as a basis for an operational species concept can also conflict with ethical issues governing the protection of species. We provide a way forward for recognizing and naming species that links theoretical and operational species concepts regardless of the magnitude of reproductive isolation.

The evolution of crocodilian nesting ecology and behavior.

Crocodilians comprise an ancient and successful lineage of archosaurs that repeatedly raises questions on how they survived a mass extinction and remained relatively unchanged for ~100 million years. Was their success due to the change-resistant retention of a specific set of traits over time (phylogenetic conservatism) or due to flexible, generalist capabilities (e.g., catholic diets, phenotypic plasticity in behavior), or some combination of these? We examined the evolution of reproductive ecology and behavior of crocodilians within a phylogenetic perspective, using 14 traits for all 24 species to determine whether these traits were phylogenetically constrained versus (ecologically) convergent. Our analysis revealed that the ancestral crocodilian was a mound nester that exhibited both nest attendance and defense. Nesting mode exhibited 4-5 transformations from mound to hole nesting, a convergence of which habitat may have been a driving factor. Hole nesters were more likely to nest communally, but this association may be biased by scale. Although there were exceptions, mound nesters typically nested during the wet season and hole nesters during the dry season; this trait was relatively conserved, however. About two-thirds of species timed their nesting with the wet season, while the other third timed their hatching with the onset of the wet season. Nest attendance and defense were nearly ubiquitous and thus exhibited phylogenetic conservatism, but attendance lodging was diverse among species, showing multiple reversals between water and burrows. Collectively, our analysis reveals that reproductive trait evolution in crocodilians reflects phylogenetic constraint (nest attendance, nest defense), ecological convergence (seasonal timing of nesting, nest attendance lodging), or both (mode of nesting). Some traits (e.g., communal nesting and mode of nesting) were autocorrelated. Our analysis provides a framework for addressing hypotheses raised for why there has been trait convergence in reproductive ecology and behavior in crocodilians and why some traits remained phylogenetically conserved.

Early usage and meaning of evolvability.

Evolvability has become an enormously popular concept in evolutionary biology and in machine learning software architecture. While it is claimed that the term was coined in 1988 by Richard Dawkins, it was used as early as 1931 as a characteristic of life by John A. Thomson. We quote and review the earliest uses and definitions of evolvability in biological frameworks up until 1989, which are remarkably few. The meaning changed from simply the "ability to evolve" as a characteristic of life to various versions of including necessary variation to predict whether or not something could evolve to the rate and quality of that evolution. Or, meaning changed from the ability to evolve to the "quality" of the ability to evolve. Since then, evolvability has taken on many definitions as it has exploded in usage.

Squamate areas of endemism in Cuba as determined by parsimony analysis of endemicity.

Cuba, the largest island in the Greater Antilles, hosts a high diversity of native squamate reptiles and is characterized by a complex geological history. The island has undergone repeated submergence and emergence, positioned on the dynamic border between the Caribbean and North American tectonic plates. Here, we infer current areas of endemism on Cuba based on squamate distributions using standard parsimony analysis of endemism under the "areas of endemism as individuals" hypothesis. We diagnose 29 areas of endemism, 14 of which are nested within other areas of endemism, from 52 squamate taxa. We suspect the current biotic pattern is a composite view of layered histories, and we summarize the geological history of the island to contemplate historical periods that left stronger marks on squamate distributions than others.

Translating a clade based classification into one that is valid under the international code of zoological nomenclature: the case of the lizards of the family Dactyloidae (Order Squamata).

In a tour-de-force for anole biology, Poe et al. (2017) provide the most complete phylogenetic analysis of members of the family Dactyloidae yet attempted. The contribution is remarkable in the completeness of sampled taxa and breath of included characters. It is equally remarkable in the concordance of their consensus tree with the topology of previous phylogenetic inferences. Thus, the creation of a near-complete data matrix of extant taxa demonstrates that an asymptote in tree topological stability likely was reached in previous studies with more limited sampling (e.g. Alfoldi 2011, Jackman et al. 1999, Nicholson et al. 2012). Such a result provides hope that major lineages within the anole radiation can be examined consistently by scientists interested in parsing evolutionary patterns emerging within and among them.

Skin glands of an aquatic salamander vary in size and distribution and release antimicrobial secretions effective against chytrid fungal pathogens.

Amphibian skin is unique among vertebrate classes, containing a large number of multicellular exocrine glands that vary among species and have diverse functions. The secretions of skin glands contain a rich array of bioactive compounds including antimicrobial peptides (AMPs). Such compounds are important for amphibian innate immune responses and may protect some species from chytridiomycosis, a lethal skin disease caused by the fungal pathogens Batrachochytrium dendrobatidis (Bd) and Batrachochytrium salamandrivorans (Bsal). While the bioactivity of skin secretions against Bd has been assessed for many amphibian taxa, similar studies are lacking for Bsal, a chytrid fungus that is especially pathogenic for salamanders. We studied the skin glands and their potential functions in an aquatic salamander, the three-toed amphiuma (Amphiuma tridactylum). Skin secretions of captive adult salamanders were analyzed by RP-HPLC and tested against the growth of Bd and Bsal using in vitro assays. We found that compounds within collected skin secretions were similar between male and female salamanders and inhibited the growth of Bd and Bsal. Thus, skin secretions that protect against Bd may also provide protection against Bsal. Histological examination of the skin glands of preserved salamanders revealed the presence of enlarged granular glands concentrated within caudal body regions. A site of potential gland specialization was identified at the tail base and may indicate specialized granular glands related to courtship and communication.

Phylogenetic patterns of trait and trait plasticity evolution: Insights from amphibian embryos.

Environmental variation favors the evolution of phenotypic plasticity. For many species, we understand the costs and benefits of different phenotypes, but we lack a broad understanding of how plastic traits evolve across large clades. Using identical experiments conducted across North America, we examined prey responses to predator cues. We quantified five life-history traits and the magnitude of their plasticity for 23 amphibian species/populations (spanning three families and five genera) when exposed to no cues, crushed-egg cues, and predatory crayfish cues. Embryonic responses varied considerably among species and phylogenetic signal was common among the traits, whereas phylogenetic signal was rare for trait plasticities. Among trait-evolution models, the Ornstein-Uhlenbeck (OU) model provided the best fit or was essentially tied with Brownian motion. Using the best fitting model, evolutionary rates for plasticities were higher than traits for three life-history traits and lower for two. These data suggest that the evolution of life-history traits in amphibian embryos is more constrained by a species' position in the phylogeny than is the evolution of life history plasticities. The fact that an OU model of trait evolution was often a good fit to patterns of trait variation may indicate adaptive optima for traits and their plasticities.

Entities on a Temporal Scale.

Ontological understanding of biological units (i.e. what kinds of things are they) is crucial to their use in experimental design, analysis, and interpretation. Conceptualizing fundamental units in biology as individuals or classes is important for subsequent development of discovery operations. While the criteria for diagnosing individuals are acknowledged, temporal boundedness is often misinterpreted and temporal minima are applied to units in question. This results in misdiagnosis or abandonment of ontological interpretation altogether. Biological units such as areas of endemism in biogeography and species in evolutionary biology fall victim to such problems. Our goal here is to address the misconception that biological individuals such as species and areas of endemism have a temporal minimum. Areas of endemism can persist within small temporal boundaries in the context of metapopulation dynamics, island biogeography, and range expansion and contraction. Similarly, lineage reticulation illustrates examples of short-lived species. Here, examples of known entities are provided to illustrate their persistence on short time scales in attempt to rescue future interpretation of biological units from ontological misdiagnosis, elucidate the philosophical individuality of areas of endemism and species with short lifespans, and provide justification for the "snapshot in time" diagnostic approach.

Bi-modal strategy of gastrulation in reptiles.

BACKGROUND: Amniote gastrulation is often described with respect to human, mouse and chick development by the presence of the primitive streak, a posterior-to-anterior midline morphological cell ingression feature that has come to define Amniote gastrulation. How this midline, ingression-based strategy of gastrulation evolved from the ancestral blastopore, a circumferential involution event in Anamniotes, is unknown. However, within the Amniote clade there exists a more diverse range of gastrulation strategies than just the primitive streak. Investigating gastrulation in a wider range of Amniotes provides a way to understand evolutionary transition from blastopore to the primitive streak. RESULTS: We analysed early to late gastrulation stages of Chamaeleo calyptratus, showing their unique morphology through confocal imaging of F-actin and laminin-stained embryos to visualise cell morphology and assess basal lamina integrity. We analysed the expression pattern of core mesodermal markers Brachyury and Fgf8 and complimented this analysis with that of the turtle, Trachemys scripta. CONCLUSIONS: Our analysis suggests that reptile gastrulation is bi-modal; primary internalization occurs anteriorly by means of an incomplete blastopore-like opening, while posteriorly the cells undergo ingression in the Brachyury-expressing blastoporal plate. This strategy stands mid-way between Anamniotes and Avians/Mammals, suggesting that blastoporal plate is a precursor of the avian primitive streak. Developmental Dynamics 244:1144-1157, 2015. © 2015 Wiley Periodicals, Inc.

Testable but not falsifiable?

We are puzzled by a recent comment that suggested that historical hypotheses can be tested but are unfalsifiable. We argue that phylogenetic hypotheses are falsifiable without the aid of a time machine and that they are like any other hypothesis: they are tentative knowledge propositions capable of falsification with character evidence.

Evolution of the germ line-soma relationship in vertebrate embryos.

The germ line and soma together maintain genetic lineages from generation to generation: the germ line passes genetic information between generations; the soma is the vehicle for germ line transmission, and is shaped by natural selection. The germ line and somatic lineages arise simultaneously in early embryos, but how their development is related depends on how primordial germ cells (PGC) are specified. PGCs are specified by one of two means. Epigenesis describes the induction of PGCs from pluripotent cells by signals from surrounding somatic tissues. In contrast, PGCs in many species are specified cell-autonomously by maternally derived molecules, known as germ plasm, and this is called preformation. Germ plasm inhibits signaling to PGCs; thus, they are specified cell-autonomously. Germ plasm evolved independently in many animal lineages, suggesting convergent evolution, and therefore it would be expected to convey a selective advantage. But, what this is remains unknown. We propose that the selective advantage that drives the emergence of germ plasm in vertebrates is the disengagement of germ line specification from somatic influences. This liberates the evolution of gene regulatory networks (GRNs) that govern somatic development, and thereby enhances species evolvability, a well-recognized selective advantage. We cite recent evidence showing that frog embryos, which contain germ plasm, have modified GRNs that are not conserved in axolotls, which represent more basal amphibians and employ epigenesis. We also present the correlation of preformation with enhanced species radiations, and we discuss the mutually exclusive trajectories influenced by germ plasm or pluripotency, which shaped chordate evolution.

Expression of Dazl and Vasa in turtle embryos and ovaries: evidence for inductive specification of germ cells.

In bilaterian animals, germ cells are specified by the inductive/regulative mode or the predetermined (germ plasm) mode. Among tetrapods, mammals and urodeles use the inductive mode, whereas birds and anurans use the predetermined mode. From histological data it has been predicted that some reptiles including turtles use the inductive mode. Examining turtle oocytes, we find that Dazl RNA, Vasa RNA, and Vasa protein are not localized, suggesting that germ plasm is not present. In turtle embryos at somite stages, primordial germ cells (PGCs) expressing Dazl lie on a path from the lateral posterior extraembryonic endoderm through the gut to the gonad as previously described. In gastrulating embryos, cells expressing Dazl are found in the blastoporal plate and subsequently below the blastoporal plate, indicating that PGCs are generated at the equivalent of the early posterior primitive streak of mammals. Vasa RNA is expressed in somatic cells of gastrula to early somite stages, and Vasa RNA and protein are expressed in PGCs of later embryos. Taken together the evidence strongly suggests that turtles, and other reptiles (lacertoid lizards) with the same location of PGCs in embryos, use the inductive mode of germ cell specification. Phylogenetic analysis of the available evidence supports the following hypotheses: (1) the inductive mode is basal among reptiles, indicating that this mode was maintained as basal tetrapods evolved to amniotes, (2) the predetermined mode arose twice within reptiles, and (3) the induced mode may be used in several lepidosaurs whose PGCs are located in an unusual pattern distributed around the embryo.

Evolution of germ cell development in tetrapods: comparison of urodeles and amniotes.

The embryonic development of germ cells in tetrapods is described, focusing on groups with the inductive mode of germ cell specification. In mammals PGCs are induced early in the gastrulation process, they are internalized with future extraembryonic mesoderm in the early posterior primitive streak, and specified soon thereafter. Strong evidence indicates that a similar process occurs in turtles and some other reptiles. In amniotes, the PGCs appear well before formation of the gonad in the posterior trunk, resulting in a period in which they are located outside the embryo before their migration to the gonad. In contrast, in urodeles the PGCs appear relatively late, and throughout development maintain a position close to precursors of the somatic cells of the gonad so that migration is not required. In lampreys early development of germ cells is strikingly similar to that in urodeles, suggesting this is the primitive process. As amniotes evolved large yolky eggs and better access to nutrition, development of the posterior half of the trunk became more dependent on cell proliferation; this was followed or accompanied by a shift of early germ cell development to the equivalent of the early primitive streak. A similar process may have occurred as some basal vertebrates developed large yolky eggs.

Inferring developmental constraint and constraint release: primordial germ cell determination mechanisms as examples.

Developmental constraint and its converse constraint release are significant concepts in understanding pattern and process in macroevolution. The purpose of this paper is to propose a two-step method for identifying constraints and constraint release. The first step is a phylogenetic optimization procedure to identify which trait/process is primitive and which is derived. The primitive trait is inferred to be the constraint and the convergently derived trait the release. The second criterion uses sister-clade asymmetry. Clades diagnosed by the constraint will have fewer taxa than clades diagnosed by the release. As an example, we use the process of germ cell specification, in which there are three modes of specification. Our results corroborate previous conclusions that the induced mode is the constraint and the predetermined mode is the release and we speculate on the importance of these two processes in terms of robustness and evolvability.

Regulative germ cell specification in axolotl embryos: a primitive trait conserved in the mammalian lineage.

How germ cells are specified in the embryos of animals has been a mystery for decades. Unlike most developmental processes, which are highly conserved, embryos specify germ cells in very different ways. Curiously, in mouse embryos germ cells are specified by extracellular signals; they are not autonomously specified by maternal germ cell determinants (germ plasm), as are the germ cells in most animal model systems. We have developed the axolotl (Ambystoma mexicanum), a salamander, as an experimental system, because classic experiments have shown that the germ cells in this species are induced by extracellular signals in the absence of germ plasm. Here, we provide evidence that the germ cells in axolotls arise from naive mesoderm in response to simple inducing agents. In addition, by analysing the sequences of axolotl germ-cell-specific genes, we provide evidence that mice and urodele amphibians share a common mechanism of germ cell development that is ancestral to tetrapods. Our results imply that germ plasm, as found in species such as frogs and teleosts, is the result of convergent evolution. We discuss the evolutionary implications of our findings.

Evolution of predetermined germ cells in vertebrate embryos: implications for macroevolution.

The germ line is established in animal embryos with the formation of primordial germ cells (PGCs), which give rise to gametes. Therefore, the need to form PGCs can act as a developmental constraint by inhibiting the evolution of embryonic patterning mechanisms that compromise their development. Conversely, events that stabilize the PGCs may liberate these constraints. Two modes of germ cell determination exist in animal embryos: (a) either PGCs are predetermined by the inheritance of germ cell determinants (germ plasm) or (b) PGCs are formed by inducing signals secreted by embryonic tissues (i.e., regulative determination). Surprisingly, among the major extant amphibian lineages, one mechanism is found in urodeles and the other in anurans. In anuran amphibians PGCs are predetermined by germ plasm; in urodele amphibians PGCs are formed by inducing signals. To determine which mechanism is ancestral to the tetrapod lineage and to understand the pattern of inheritance in higher vertebrates, we used a phylogenetic approach to analyze basic morphological processes in both groups and correlated these with mechanisms of germ cell determination. Our results indicate that regulative germ cell determination is a property of embryos retaining ancestral embryological processes, whereas predetermined germ cells are found in embryos with derived morphological traits. These correlations suggest that regulative germ cell formation is an important developmental constraint in vertebrate embryos, acting before the highly conserved pharyngula stage. Moreover, our analysis suggests that germ plasm has evolved independently in several lineages of vertebrate embryos.