Glucose Accumulation Can Account for the Initial Water Flux Triggered by Na+/Glucose Cotransport

Over the last decade, several cotransport studies have led to the proposal of secondary active transport of water, challenging the dogma that all water transport is passive. The major observation leading to this interpretation was that a Na⁺ influx failed to reproduce the large and rapid cell swelling induced by Na⁺/solute cotransport. We have investigated this phenomenon by comparing a Na⁺/glucose (hSGLT1) induced water flux to water fluxes triggered either by a cationic inward current (using ROMK2 K⁺ channels) or by a glucose influx (using GLUT2, a passive glucose transporter). These proteins were overexpressed in Xenopus oocytes and assayed through volumetric measurements combined with double-electrode electrophysiology or radioactive uptake measurements. The osmotic gradients driving the observed water fluxes were estimated by comparison with the swelling induced by osmotic shocks of known amplitude. We found that, for equivalent cation or glucose uptakes, the combination of substrate accumulations observed with ROMK2 and GLUT2 are sufficient to provide the osmotic gradient necessary to account for a passive water flux through SGLT1. Despite the fact that the Na⁺/glucose stoichiometry of SGLT1 is 2:1, glucose accumulation accounts for two-thirds of the osmotic gradient responsible for the water flux observed at t = 30 s. It is concluded that the different accumulation processes for neutral versus charged solutes can quantitatively account for the fast water flux associated with Na⁺/glucose cotransport activation without having to propose the presence of secondary active water transport.     

The sense of water in the blowfly Protophormia terraenovae

The gustatory system of the blowfly, Protophormia terraenovae, is a relatively simple biological model for studies on chemosensory input and behavioral output. It appears to have renewed interest as a model for studies on the role of water channels, namely aquaporins or aquaglyceroporins, in water detection. To this end, we investigated the presence of water channels, their role in “water” and “salt” cell responsiveness and the transduction mechanism involved. For the first time our electrophysiological results point to the presence of an aquaglyceroporin in the chemoreceptor membrane of the “water” cell in the blowfly taste chemosensilla whose transduction mechanism ultimately involves an intracellular calcium increase and consequently cell depolarization. This hypothesis is also supported by calcium imaging data following proper stimulation. This mechanism is triggered by “water” cell stimulation with hypotonic solutions and/or solutes such as glycerol which crosses the membrane by way of aquaglyceroporins. Behavioral output indicates that the “sense” of water in blowflies is definitely not dependent on the “water” cell only, but also on the “salt” cell sensitivity. These findings also hypothesize a new role for aquaglyceroporin in spiking cell excitability.     

Controversy regarding the secondary active water transport hypothesis.

Historically, water transport across biological membranes has always been considered a passive process, i.e., the net water transport is proportional to the gradients of hydrostatic and osmotic pressure. More recently, this dogma was challenged by the suggestion that secondary active transporters such as the Na/glucose cotransporter (SGLT1) could perform secondary active water transport with a fixed stoichiometry. In the case of SGLT1, the stoichiometry would consist of one glucose molecule to two Na+ ions to 220-400 water molecules. In the present minireview, we summarize and criticize the evidence supporting and opposing this water cotransport hypothesis. Published and unpublished observations from our own laboratory are also presented in support of the idea that transport-dependent osmotic gradients begin to build up immediately after cotransport commences and are fully responsible for the cell swelling observed.     

Water-Transporting Proteins

Transport through lipids and aquaporins is osmotic and entirely driven by the difference in osmotic pressure. Water transport in cotransporters and uniporters is different: Water can be cotransported, energized by coupling to the substrate flux by a mechanism closely associated with protein. In the K⁺/Cl⁻ and the Na⁺/K⁺/2Cl⁻ cotransporters, water is entirely cotransported, while water transport in glucose uniporters and Na⁺-coupled transporters of nutrients and neurotransmitters takes place by both osmosis and cotransport. The molecular mechanism behind cotransport of water is not clear. It is associated with the substrate movements in aqueous pathways within the protein; a conventional unstirred layer mechanism can be ruled out, due to high rates of diffusion in the cytoplasm. The physiological roles of the various modes of water transport are reviewed in relation to epithelial transport. Epithelial water transport is energized by the movements of ions, but how the coupling takes place is uncertain. All epithelia can transport water uphill against an osmotic gradient, which is hard to explain by simple osmosis. Furthermore, genetic removal of aquaporins has not given support to osmosis as the exclusive mode of transport. Water cotransport can explain the coupling between ion and water transport, a major fraction of transepithelial water transport and uphill water transport. Aquaporins enhance water transport by utilizing osmotic gradients and cause the osmolarity of the transportate to approach isotonicity.     

Fish Invasions in the World's River Systems: When Natural Processes Are Blurred by Human Activities

Because species invasions are a principal driver of the human-induced biodiversity crisis, the identification of the major determinants of global invasions is a prerequisite for adopting sound conservation policies. Three major hypotheses, which are not necessarily mutually exclusive, have been proposed to explain the establishment of non-native species: the “human activity” hypothesis, which argues that human activities facilitate the establishment of non-native species by disturbing natural landscapes and by increasing propagule pressure; the “biotic resistance” hypothesis, predicting that species-rich communities will readily impede the establishment of non-native species; and the “biotic acceptance” hypothesis, predicting that environmentally suitable habitats for native species are also suitable for non-native species. We tested these hypotheses and report here a global map of fish invasions (i.e., the number of non-native fish species established per river basin) using an original worldwide dataset of freshwater fish occurrences, environmental variables, and human activity indicators for 1,055 river basins covering more than 80% of Earth's surface. First, we identified six major invasion hotspots where non-native species represent more than a quarter of the total number of species. According to the World Conservation Union, these areas are also characterised by the highest proportion of threatened fish species. Second, we show that the human activity indicators account for most of the global variation in non-native species richness, which is highly consistent with the “human activity” hypothesis. In contrast, our results do not provide support for either the “biotic acceptance” or the “biotic resistance” hypothesis. We show that the biogeography of fish invasions matches the geography of human impact at the global scale, which means that natural processes are blurred by human activities in driving fish invasions in the world's river systems. In view of our findings, we fear massive invasions in developing countries with a growing economy as already experienced in developed countries. Anticipating such potential biodiversity threats should therefore be a priority.     

Magnetic Navigation

Recent evidence suggests that some amphibians, reptiles and birds may be capable of homing using information about geographic position (“map” information) derived from subtle geographic gradients in the earth's magnetic field. The “magnetic map” hypothesis faces numerous theoretical difficulties, however, due to the extremely high level of sensitivity that would be necessary to detect natural magnetic gradients, and to the presence of spatial irregularities and temporal variation in the geomagnetic field that might make map coordinates derived from magnetic gradients unreliable. To date, the majority of studies carried out to test the magnetic map hypothesis have involved field observations of the effects on homing orientation of naturally occurring spatial or temporal variation in the geomagnetic field. While providing an important first step, these studies are subject to the criticism that the observed changes in homing orientation could result from effects on a magnetic compass, or some other unidentified component of the navigational system, rather than from effects on a magnetic map. The recent development of experimental systems in which navigational ability can be studied under controlled or semi-controlled laboratory conditions has opened up the possibility of using new experimental approaches to more rigorously test the magnetic map hypothesis. After briefly reviewing the available evidence of the geomagnetic field's involvement in the map component of homing, a simple graphical model is presented which describes how the home direction derived from a bicoordinate map varies as a function of the value of one of the map coordinates when the value of the second map coordinate is held constant. In studies of homing orientation in which the value of a specific magnetic field parameter (e.g., total intensity, inclination, etc.) can be varied independently of other putative map parameters, the graphical model can be used to generate qualitative predictions about the changes in the direction of homing orientation that should be observed if the magnetic field parameter being manipulated serves as one coordinate of a bicoordinate map. The relationship between the direction of homing orientation and the value of a putative magnetic map parameter can also be used to generate quantitative predictions about characteristics of the local gradient of that magnetic field parameter in the vicinity of the home site (i.e., the alignment and “home value” of the local gradient) which can then be compared with actual measured values. Together, the qualitative and quantitative predictions of the graphical model permit rigorous tests of whether one or both coordinates of a bicoordinate navigational map are derived from the geomagnetic field.     

Probing the perils of dichotomous binning: how categorizing female dogs as spayed or intact can misinform our assumptions about the lifelong health consequences of ovariohysterectomy

In 2009, we reported findings from the first study evaluating the relationship between canine longevity and number of years of lifetime ovary exposure. All previous studies examining gonadal influences on canine longevity relied upon categorizing females as “intact” or “spayed” based on gonadal status at the time of death. Our study of Rottweilers generated a novel result: Keeping ovaries longer was associated with living longer. This result challenged previous assumptions that spayed females live longer. In the present investigation, we explored a methodological explanation for the apparent contradiction between our results and those of others, so we might better understand the impact that timing of spaying has on longevity. We hypothesized that naming female dogs as “spayed” or “intact” based upon gonadal status at time of death – a method we refer to as dichotomous binning – inadequately represents important biological differences in lifetime ovary exposure among bitches spayed at different ages. This hypothesis predicts that a strong relationship between years of lifetime ovary exposure and longevity in a population could be obscured by categorizing females as spayed or intact. Herein, we provide support for this hypothesis by reanalyzing longevity data from 183 female Rottweilers. In this study population, there was a three-fold increased likelihood of exceptional longevity (living ≥13 yr) associated with the longest duration of ovary exposure. However, categorizing females in this population as spayed or intact yielded the spurious, contradictory assertion that spayed females (presumed to have the least ovary exposure) are more likely to reach exceptional longevity than those that are intact. Thus, by ignoring the timing of spaying in each bitch, the inference from these data was distorted. It follows from this new understanding that dichotomous binning—naming females as spayed or intact—is inadequate for representing lifetime ovary exposure, introducing misclassification bias that can generate misleading assumptions regarding the lifelong health consequences of ovariohysterectomy.     

Intracellular hypertonicity is responsible for water flux associated with Na+/glucose cotransport

Detection of a significant transmembrane water flux immediately after cotransporter stimulation is the experimental basis for the controversial hypothesis of secondary active water transport involving a proposed stoichiometry for the human Na(+)/glucose cotransporter (SGLT1) of two Na(+), one glucose, and 264 water molecules. Volumetric measurements of Xenopus laevis oocytes coexpressing human SGLT1 and aquaporin can be used to detect osmotic gradients with high sensitivity. Adding 2 mM of the substrate alpha-methyl-glucose (alphaMG) created mild extracellular hypertonicity and generated a large cotransport current with minimal cell volume changes. After 20, 40, and 60 s of cotransport, the return to sugar-free, isotonic conditions was accompanied by measurable cell swelling averaging 0.051, 0.061, and 0.077 nl/s, respectively. These water fluxes are consistent with internal hypertonicities of 1.5, 1.7, and 2.2 mOsm for these cotransport periods. In the absence of aquaporin, the measured hypertonicites were 4.6, 5.0, and 5.3 mOsm for the same cotransport periods Cotransport-dependent water fluxes, previously assumed to be water cotransport, could be largely explained by hypertonicities of such amplitudes. Using intracellular Na(+) injection and Na(+)-selective electrode, the intracellular diffusion coefficient for Na(+) was estimated at 0.29 +/- 0.03 x 10(-5) cm(2) s(-1). Using the effect of intracellular alphaMG injection on the SGLT1-mediated outward current, the intracellular diffusion coefficient of alphaMG was estimated at 0.15 +/- 0.01 x 10(-5) cm(2) s(-1). Although these intracellular diffusion coefficients are much lower than in free aqueous solution, a diffusion model for a single solute in an oocyte would require a diffusion coefficient three times lower than estimated to explain the local osmolyte accumulation that was experimentally detected. This suggests that either the diffusion coefficients were overestimated, possibly due to the presence of convection, or the diffusion in cytosol of an oocyte is more complex than depicted by a simple model.     

Beyond dual-lattice models: Incorporating plant strategies when modeling the interplay between facilitation and competition along environmental severity gradients

We introduce a spatially explicit model that evaluates how the trade-offs between the life strategies of two interacting plant species affect the outcome of their interaction along environmental severity gradients. In our model, we represent the landscape as a two-dimensional lattice, with environmental severity increasing from left to right. Two species with different strategies, a competitor and a stress-tolerant, interact in the lattice. We find that facilitation expands the realized niche of the competitor into harsh environments by suppressing the stress-tolerant species. Most of their coexisting range is dominated by a positive effect of one species on another, with a reciprocal negative effect from the species receiving the benefits on its benefactor (“+, −”), whereas mutualistic (“+, +”) interactions are only found in the harshest part of the environmental gradient. Contrarily as assumed by models commonly used in facilitation research (e.g. dual-lattice models), our results indicate that “+, +” interactions are not dominant, and that their differences with “+, −” interactions along environmental severity gradients depend on the strategies of the interacting species. By integrating the trade-off between competitive ability and stress tolerance, our model provides a new framework to investigate the interplay of facilitative and competitive interactions along environmental gradients and their impacts on processes such as population dynamics and community organization.     

Molluscan mobile elements similar to the vertebrate Recombination-Activating Genes

Animal genomes contain ∼20,000 genes. Additionally millions of genes for antigen receptors are generated in cells of the immune system from the sets of separate gene segments by a mechanism known as the V(D)J somatic recombination. The components of the V(D)J recombination system, Recombination-Activating Gene proteins (RAG1 and RAG2) and recombination signal sequence (RSS), are thought to have “entered” the vertebrate genome as a hypothetical “RAG transposon”. Recently discovered mobile elements have terminal inverted repeats (TIRs) similar to RSS and may encode proteins with a different degree of similarity to RAG1. We describe a novel N-RAG-TP transposon identified from the sea slug Aplysia californica that encodes a protein similar to the N-terminal part of RAG1 in vertebrates. This refines the “RAG transposon” hypothesis and allows us to propose a scenario for V(D)J recombination machinery evolution from a relic transposon related to the existing mobile elements N-RAG-TP, Chapaev, and Transib.     

Osmotic water transport with glucose in GLUT2 and SGLT

Carrier-mediated water cotransport is currently a favored explanation for water movement against an osmotic gradient. The vestibule within the central pore of Na⁺-dependent cotransporters or GLUT2 provides the necessary precondition for an osmotic mechanism, explaining this phenomenon without carriers. Simulating equilibrative glucose inflow via the narrow external orifice of GLUT2 raises vestibular tonicity relative to the external solution. Vestibular hypertonicity causes osmotic water inflow, which raises vestibular hydrostatic pressure and forces water, salt, and glucose into the outer cytosolic layer via its wide endofacial exit. Glucose uptake via GLUT2 also raises oocyte tonicity. Glucose exit from preloaded cells depletes the vestibule of glucose, making it hypotonic and thereby inducing water efflux. Inhibiting glucose exit with phloretin reestablishes vestibular hypertonicity, as it reequilibrates with the cytosolic glucose and net water inflow recommences. Simulated Na⁺-glucose cotransport demonstrates that active glucose accumulation within the vestibule generates water flows simultaneously with the onset of glucose flow and before any flow external to the transporter caused by hypertonicity in the outer cytosolic layers. The molar ratio of water/glucose flow is seen now to relate to the ratio of hydraulic and glucose permeability rather than to water storage capacity of putative water carriers.     

Characterisation of physiological and immunological differences between Pacific oysters (Crassostrea gigas) genetically selected for high or low survival to summer mortalities and fed different rations under controlled conditions

Within the framework of a national scientific program named “MORtalités ESTivales de l'huître creuse Crassostrea gigas” (MOREST), a family-based experiment was developed to study the genetic basis of resistance to summer mortality in the Pacific oyster, Crassostrea gigas. As part of the MOREST project, the second generation of three resistant families and two susceptible families were chosen and pooled into two respective groups: “R” and “S”. These two groups of oysters were conditioned for 6 months on two food levels (4% and 12% of oyster soft-tissue dry weight in algal dry weight per day) with a temperature gradient that mimicked the Marennes-Oléron natural cycle during the oyster reproductive period. Oyster mortality remained low for the first two months, but then rapidly increased in July when seawater temperature reached 19 °C and above. Mortality was higher in “S” oysters than in “R” oysters, and also higher in oysters fed the 12% diet than those fed 4%, resulting in a decreasing, relative order in cumulative mortality as follows; 12% “S” > 12% “R” > 4% “S” > 4% “R”. Although the observed mortality rates were lower than those previously observed in the field, the mortality differential between “R” and “S” oysters was similar. Gonadal development, estimated by tissue lipid content, followed a relative order yielding a direct, positive relationship between reproductive effort and mortality as we reported precedently by quantitative histology. Regarding hemocyte parameters, one of the most striking observations was that reactive oxygen species (ROS) production was significantly higher in “S” oysters than in “R” oysters in May and June, regardless of food level. The absence of known environmental stress under these experimental conditions suggests that the ROS increase in “S” oyster could be related to their higher reproductive activity. Finally, a higher increase in hyalinocyte counts was observed for”S” oysters, compared to “R” oysters, in July, just before mortality. Taken together, our results suggest an association of genetically based resistance to summer mortality, reproductive strategy and hemocyte parameters.     

Configural processing of different topologically structured figures: an ERP Study

According to Chen’s theory, topological differences are perceived faster than feature differences in early visual perception. We hypothesized that topological perception is caused by the sensitivity in discriminating figures with and without “holes”. An ERP experiment was conducted utilizing a passive paradigm to investigate the differences in perceiving figures with “hole” and with “no-hole”. The results showed differences in N170 components between figures with “holes” and with “no-holes”. The inversion of the “hole” could influence the latency of N170, but the inversion of the “no-hole” could not, which indicated that global features are processed first in the “hole” perception whilst local features are given priority to the “no-hole” perception. This result was similar to studies concerning face and non-face objects, suggesting a configural processing of the “hole”.     

Two applications of the thermogram of the alcohol/water binary system with compositions of cryobiological interests

Quantitative analyses of the bound water content in the alcohol aqueous solution and its osmotic behavior should be cryobiologically significant. This paper has presented two applications of the thermogram of the alcohol/water system recorded by differential scanning calorimeter (DSC). Both applications are: (1) generating the quantitative relationship between the bound water content and the solution composition; (2) calculating the osmotic virial coefficients for alcohols. Five alcohols including methanol, ethanol, ethylene glycol, propylene glycol and glycerol are investigated. In the present study, partial binary phase diagrams of these five alcohol solutions are determined in the first place. The bound water contents in these solutions are quantitatively evaluated by three criteria afterwards. In the end, the osmotic virial coefficients for these alcohols are calculated according to the osmotic virial equation. It is turned out that the bound water fraction out of the total water content increases with a rising molality. The ability of the solute to restrict water molecules can be weakened when the solution becomes more concentrated. The results also indicate that propylene glycol should be the strongest “water-blocker” while methanol the weakest one. These findings can deepen our understanding of the cryoprotective properties of the alcohols from the perspectives of their roles in binding free water and promoting the osmotic efflux of cell water.     

Dynamics of the glutamic acid 242 side chain in cytochrome c oxidase

In many cytochrome c oxidases glutamic acid 242 is required for proton transfer to the binuclear heme a₃/Cu_B site, and for proton pumping. When present, the side chain of Glu-242 is orientated “down” towards the proton-transferring D-pathway in all available crystal structures. A nonpolar cavity “above” Glu-242 is empty in these structures. Yet, proton transfer from Glu-242 to the binuclear site, and for proton-pumping, is well established, and the cavity has been proposed to at least transiently contain water molecules that would mediate proton transfer. Such proton transfer has been proposed to require isomerisation of the Glu-242 side chain into an “up” position pointing towards the cavity. Here, we have explored the molecular dynamics of the protonated Glu-242 side chain. We find that the “up” position is preferred energetically when the cavity contains four water molecules, but the “down” position is favoured with less water. We conclude that the cavity might be deficient in water in the crystal structures, possibly reflecting the “resting” state of the enzyme, and that the “up/down” equilibrium of Glu-242 may be coupled to the presence of active-site water molecules produced by O₂ reduction.     

The relative cost of bent-hip bent-knee walking is reduced in water

The debate about how early hominids walked may be characterised as two competing hypotheses: They moved with a fully upright (FU) gait, like modern humans, or with a bent-hip, bent-knee (BK) gait, like apes. Both have assumed that this bipedalism was almost exclusively on land, in trees or a combination of the two. Recent findings favoured the FU hypothesis by showing that the BK gait is 50–60% more energetically costly than a FU human gait on land. We confirm these findings but show that in water this cost differential is markedly reduced, especially in deeper water, at slower speeds and with greater knee flexion. These data suggest that the controversy about australopithecine locomotion may be eased if it is assumed that wading was a component of their locomotor repertoire and supports the idea that shallow water might have been an environment favourable to the evolution of early forms of “non-optimal” hominid bipedalism.     

Cl- absorption in European eel intestine and its regulation

The intestinal epithelium of the euryhaline teleost fish, Anguilla anguilla, absorbs Cl(-) transepithelially. This gives rise to a negative transepithelial potential at the basolateral side of the epithelium and to a measured short circuit current. Cl(-) absorption occurs via bumetanide-sensitive Na(+)-K(+)-2Cl(-) cotransport, localized on the luminal membrane. The cotransport operates in parallel with a luminal K(+) conductance that recycles the ion into the lumen. Cl(-) leaves the cell across the basolateral membrane by way of Cl(-) conductance and presumably via a KCl cotransport. The driving force for this process is provided by the electrochemical sodium gradient across the plasma membrane, generated and maintained by the basolateral Na(+)-K(+)-ATPase. The resulting NaCl absorption process is active and enables marine fish to take up water, thereby compensating for water that was lost passively from the body. Fresh water acclimatized eel also absorb Cl(-) actively, although in smaller quantities, utilizing the same ion transport mechanisms as marine eels. This mechanism is basically the same as the model proposed for the thick ascending limb (cTAL). Cl(-) absorption is regulated by a number of cellular factors, such as HCO(3) (-), pH, Ca(2+), cyclic nucleotides, and cytoskeletal elements. It is sensitive to osmotic stress, and therefore is a good physiological model to study ion transport mechanisms that are activated when osmotic stress induces cell volume regulation. The activation of these various ion transport pathways is dependent on cellular transduction mechanisms in which phosphorylation events (mainly by PKC and MLCK for the hypertonic response) and cytoskeletal elements, either microfilaments or microtubules, seem to play key roles.     

L’art Paléolithique est-il un « art » ?: Réflexions autour d’une question d’actualité

During the last years, an international debate about the concept of “Palaeolithic art” has taken place. On one hand, several specialists have critiqued the use of the concept of “art” in naming the images created by Homo sapiens during the Palaeolithic era. They claim that the use of this term implies the projection of a modern category to a world - that of prehistoric humans - which is completely different from our own. On the other hand, some archaeologists consider that the term “Palaeolithic art” does not imply an anachronistic interpretation of prehistoric representations. In presenting the historical context, which has made such a discussion possible, we consider the causes and effects of this controversy. Firstly, we analyze the traditional interpretation, which considered Palaeolithic images as “works of art”. Secondly, we examine the connections, which can be found between the debate about “Palaeolithic art” and certain polemics, which have arisen from the history of art and anthropological frameworks. Thirdly, we consider the arguments utilized by those who are for and those who are against the term “Palaeolithic art”. Finally, we emphasize the importance of this debate to better understand the categories and the concepts, which determine the scientific practice.     

Are Victoria West cores “proto-Levallois”? A phylogenetic assessment

Cores from South Africa assigned to the “Victoria West” industry have long been purported as a “proto-Levallois” core form, and thus regarded as ancestral to the Levallois prepared core technologies of the Middle Paleolithic and African Middle Stone Age. Similarities in form between Victoria West cores, in terms of surface morphology and the removal of large flakes from a prepared surface, led to hypothesized schemes of technological evolution from Victoria West cores through to fully developed Levallois cores. However, the phylogenetic basis of this Victoria West “proto-Levallois” hypothesis, and the assumptions of phylogenetic homology upon which it rests, have never been tested formally. In recent years, archaeologists have begun to use phylogenetic methods drawn from biology to test hypotheses of technological and cultural evolution. Here, the phylogenetic assumptions of the Victoria West “proto-Levallois” hypothesis are tested directly using a cladistic (maximum parsimony) protocol. The cladistic analyses indicate that Victoria West cores are not the basal sister taxon of a Levallois clade, as predicted by the proto-Levallois hypothesis. Moreover, character analyses demonstrate that several characters relating to core surfaces and flake scar morphology are not phylogenetically homologous, but result from convergent technological evolution within the Acheulean techno-complex. Post hoc analyses further determine that these results are not confounded by choice of outgroup or raw material factors. The results were also shown to be robust on the basis of the ensemble retention index statistic, bootstrap analyses, and permutation tests. Hence, it is concluded that Victoria West cores do not represent a “proto-Levallois” core form, and that the term “para-Levallois” should more correctly be applied on phylogenetic grounds. It is further argued that even in cases where different technologies are found to share phylogenetically homologous features, use of the term “proto” is questionable on theoretical grounds.