James U. Van Dyke

Ancestral state reconstructions require biological evidence to test evolutionary hypotheses: A case study examining the evolution of reproductive mode in squamate reptiles

To understand evolutionary transformations it is necessary to identify the character states of extinct ancestors. Ancestral character state reconstruction is inherently difficult because it requires an accurate phylogeny, character state data, and a statistical model of transition rates and is fundamentally constrained by missing data such as extinct taxa. We argue that model based ancestral character state reconstruction should be used to generate hypotheses but should not be considered an analytical endpoint. Using the evolution of viviparity and reversals to oviparity in squamates as a case study, we show how anatomical, physiological, and ecological data can be used to evaluate hypotheses about evolutionary transitions. The evolution of squamate viviparity requires changes to the timing of reproductive events and the successive loss of features responsible for building an eggshell. A reversal to oviparity requires that those lost traits re-evolve. We argue that the re-evolution of oviparity is inherently more difficult than the reverse. We outline how the inviability of intermediate phenotypes might present physiological barriers to reversals from viviparity to oviparity. Finally, we show that ecological data supports an oviparous ancestral state for squamates and multiple transitions to viviparity. In summary, we conclude that the first squamates were oviparous, that frequent transitions to viviparity have occurred, and that reversals to oviparity in viviparous lineages either have not occurred or are exceedingly rare. As this evidence supports conclusions that differ from previous ancestral state reconstructions, our paper highlights the importance of incorporating biological evidence to evaluate model-generated hypotheses.

The evolution of viviparity: molecular and genomic data from squamate reptiles advance understanding of live birth in amniotes

Squamate reptiles (lizards and snakes) are an ideal model system for testing hypotheses regarding the evolution of viviparity (live birth) in amniote vertebrates. Viviparity has evolved over 100 times in squamates, resulting in major changes in reproductive physiology. At a minimum, all viviparous squamates exhibit placentae formed by the appositions of maternal and embryonic tissues, which are homologous in origin with the tissues that form the placenta in therian mammals. These placentae facilitate adhesion of the conceptus to the uterus as well as exchange of oxygen, carbon dioxide, water, sodium, and calcium. However, most viviparous squamates continue to rely on yolk for nearly all of their organic nutrition. In contrast, some species, which rely on the placenta for at least a portion of organic nutrition, exhibit complex placental specializations associated with the transport of amino acids and fatty acids. Some viviparous squamates also exhibit reduced immunocompetence during pregnancy, which could be the result of immunosuppression to protect developing embryos. Recent molecular studies using both candidate-gene and next-generation sequencing approaches have suggested that at least some of the genes and gene families underlying these phenomena play similar roles in the uterus and placenta of viviparous mammals and squamates. Therefore, studies of the evolution of viviparity in squamates should inform hypotheses of the evolution of viviparity in all amniotes, including mammals.

Bioenergetic components of reproductive effort in viviparous snakes: Costs of vitellogenesis exceed costs of pregnancy

Reproductive effort has been defined as the proportion of an organisms energy budget that is allocated to reproduction over a biologically meaningful time period. Historically, studies of reproductive bioenergetics considered energy content of gametes, but not costs of gamete production. Although metabolic costs of vitellogenesis (MCV) fundamentally reflect the primary bioenergetic cost of reproductive allocation in female reptiles, the few investigations that have considered costs of reproductive allocation have focused on metabolic costs of pregnancy (MCP) in viviparous species. We define MCP as energetic costs incurred by pregnant females, including all costs of maintaining gestation conditions necessary for embryogenesis. MCP by our definition do not include fetal costs of embryogenesis. We measured metabolic rates in five species of viviparous snakes (Agkistrodon contortrix, Boa constrictor, Eryx colubrinus, Nerodia sipedon, and Thamnophis sirtalis) during vitellogenesis and pregnancy in order to estimate MCV and MCP. Across all species, MCV were responsible for 30% increases in maternal metabolism. Phylogenetically-independent contrasts showed that MCV were significantly greater in B. constrictor than in other species, likely because B. constrictor yolk energy content was greater than that of other species. Estimates of MCP were not significantly different from zero in any species. In viviparous snakes, MCV appear to represent significant bioenergetic expenditures, while MCP do not. We suggest that MCV, together with yolk energy content, represent the most significant component of reptilian reproductive effort, and therefore deserve greater attention than MCP in studies of reptilian reproductive bioenergetics.

Interspecific differences in egg production affect egg trace element concentrations after a coal fly ash spill.

In oviparous vertebrates, trace elements transfer from mother to offspring during egg production. For animals that produce eggs slowly, like turtles, the trace element concentration of each egg reflects an integration of dietary and stored accumulation over the duration of vitellogenesis. Because turtles also produce eggs synchronously, all eggs within a clutch should exhibit uniform trace element concentrations. In contrast, for animals that produce eggs in sequence and primarily from current dietary resources, like many birds, the trace element concentrations of eggs should be less uniform within a clutch, and likely reflect short-term changes in dietary exposure. We tested the hypothesis that stinkpot turtle (Sternotherus odoratus) clutches exhibit lower variability and higher repeatability in barium, selenium, strontium, and thallium concentrations than those of tree swallows (Tachycineta bicolor) from a site impacted by a recent coal ash spill. All four trace elements exhibited significantly lower variability and significantly higher repeatability in stinkpot clutches than in swallow clutches. Mean trace element concentrations of stinkpot eggs were also significantly higher than those of swallow eggs although both species feed primarily on aquatic invertebrates. Variability in swallow egg trace element concentrations was partially due to significant laying order effects. Our results support the hypothesis that interspecific variation in the source of resources and in the synchronicity and rate of egg production can lead to interspecific differences in the variability of egg trace element concentrations.

Stable isotope tracer reveals that viviparous snakes transport amino acids to offspring during gestation

SUMMARY Viviparity and placentation have evolved from oviparity over 100 times in squamate reptiles (lizards and snakes). The independent origins of placentation have resulted in a variety of placental morphologies in different taxa, ranging from simple apposition of fetal and maternal tissues to endotheliochorial implantation that is homoplasious with mammalian placentation. Because the eggs of oviparous squamates transport gases and water from the environment and calcium from the eggshell, the placentae of viviparous squamates are thought to have initially evolved to accomplish these functions from within the maternal oviduct. Species with complex placentae have also been shown to rely substantially, or even primarily, on placental transport of organic nutrients for embryonic nutrition. However, it is unclear whether species with only simple placentae are also capable of transporting organic nutrients to offspring. Among viviparous squamates, all of the snakes that have been studied thus far have been shown to have simple placentae. However, most studies of snake placentation are limited to a single lineage, the North American Natricinae. We tested the abilities of four species of viviparous snakes – Agkistrodon contortrix (Viperidae), Boa constrictor (Boidae), Nerodia sipedon (Colubridae: Natricinae) and Thamnophis sirtalis (Colubridae: Natricinae) – to transport diet-derived amino acids to offspring during gestation. We fed [15N]leucine to pregnant snakes, and compared offspring 15N content with that of unlabeled controls. Labeled females allocated significantly more 15N to offspring than did controls, but 15N allocation did not differ among species. Our results indicate that viviparous snakes are capable of transporting diet-derived amino acids to their offspring during gestation, possibly via placentation.

High Food Abundance Permits the Evolution of Placentotrophy: Evidence from a Placental Lizard, Pseudemoia entrecasteauxii

Mechanisms of reproductive allocation are major determinants of fitness because embryos cannot complete development without receiving sufficient nutrition from their parents. The nourishment of offspring via placentas (placentotrophy) has evolved repeatedly in vertebrates, including multiple times in squamate reptiles (lizards and snakes). Placentotrophy has been suggested to evolve only if food is sufficiently abundant throughout gestation to allow successful embryogenesis. If scarcity of food prevents successful embryogenesis, females should recoup nutrients allocated to embryos via abortion, reabsorption, and/or cannibalism. We tested these hypotheses in the placentotrophic southern grass skink Pseudemoia entrecasteauxii. We fed females one of four diets (high constant, high variable, low constant, and low variable) during gestation and tested the effects of both food amount and schedule of feeding on developmental success, cannibalism rate, placental nutrient transport, offspring size, and maternal growth and body condition. Low food availability reduced developmental success, placental nutrient transport, offspring size, and maternal growth and body condition. Cannibalism of offspring also increased when food was scarce. Schedule of feeding did not affect offspring or mothers. We suggest that high food abundance and ability to abort and cannibalize poor-quality offspring are permissive factors necessary for placentotrophy to be a viable strategy of reproductive allocation.

The role of thermal contrast in infrared-based defensive targeting by the copperhead, Agkistrodon contortrix

The facial pit organs of the copperhead are the end organs of a complex infrared-imaging system that allows accurate and precise strikes on potential prey. Anecdotal and recent experimental observations show that pit vipers can use their infrared-imaging systems to discriminate between cool and warm features in the environment. We tested the hypothesis that the infrared-imaging system is a thermal contrast detector and determined whether behavioural responses of copperheads differ with contrast type. We found that blindfolded copperheads responded behaviourally towards both warm objects moving against cool backgrounds and cool objects moving against warm backgrounds. Responses towards the former were more robust than those towards the latter. Furthermore, oscillating warm targets generated rhythmic following behaviour in-phase with target motion, while oscillating cool targets generated rhythmic behavioural movement in antiphase to target motion. The results show that the infrared-imaging system of pit vipers operates on the basis of thermal contrast, but the differences in behavioural responses with respect to contrast type indicate that pit vipers preferentially target the warm aspect of thermal differentials. Our results also demonstrate that the infrared-imaging system allows defensive targeting of potential endothermic predators.

Prevalence of ingested fish hooks in freshwater turtles from five rivers in the southeastern United States.

Freshwater turtles may ingest baited fish hooks because many are opportunistic scavengers. Although the ingestion of fish hooks is known to be a source of mortality in multiple vertebrate groups, the prevalence of hook ingestion by freshwater turtles has not been well studied. We trapped turtles from five rivers in the southeastern United States and used radiographs to examine over 600 individuals of four species. Depending on the species, sex, and age class, 0–33% of turtles contained ingested fish hooks. For some species, larger turtles were more likely to contain a fish hook than smaller individuals. Freshwater turtle demography suggests that even small increases in adult mortality may lead to population declines. If our study areas are representative of other aquatic systems that receive fishing pressure, this work likely identifies a potential conflict between a widespread, common recreational activity (i.e., fishing) and an imperiled taxonomic group.

Residual yolk energetics and postnatal shell growth in Smooth Softshell Turtles, Apalone mutica

We examined functions of residual yolk (RY) in hatchling Smooth Softshell Turtles (Apalone mutica). Removal of RY did not affect survival, shell growth, or resting metabolic rates of turtles for 40 d after hatching. Our estimates of metabolic rate suggest that RY can fuel maintenance and activity metabolism for approximately 25 days. A. mutica absorb more than 1g of water in the first 2 weeks of life, which appears to be the basis of post-hatch shell expansion rather than yolk-provisioned growth. Post-hatch growth may be limited by the magnitude of RY remaining at hatching, but RY protein and lipid proportions do not differ from those of freshly-laid eggs. In addition, A. mutica did not use RY to fuel nest emergence. Our results suggest that RY does not fulfill several hypothetical functions in A. mutica, including postnatal growth, catabolic fuel for nest emergence, and long-term nutritional sustenance for maintenance, activity, or hibernation. Instead, A. mutica appear to absorb most yolk prior to hatching, and are left with a minimum of RY. Variation in RY mass with incubation regime in other species suggests that mothers may overprovision their eggs to ensure successful development across a diversity of possible incubation conditions.

Influence of relative trophic position and carbon source on selenium bioaccumulation in turtles from a coal fly-ash spill site

Selenium (Se) is a bioaccumulative constituent of coal fly-ash that can disrupt reproduction of oviparous wildlife. In food webs, the greatest enrichment of Se occurs at the lowest trophic levels, making it readily bioavailable to higher consumers. However, subsequent enrichment at higher trophic levels is less pronounced, leading to mixed tendencies for Se to biomagnify. We used stable isotopes ((1)(5)N and (13)C) in claws to infer relative trophic positions and relative carbon sources, respectively, of seven turtle species near the site of a recently-remediated coal fly-ash spill. We then tested whether Se concentrations differed with relative trophic position or relative carbon source. We did not observe a strong relationship between δ(15)N and Se concentration. Instead, selenium concentrations decreased with increasing δ(13)C among species. Therefore, in an assemblage of closely-related aquatic vertebrates, relative carbon source was a better predictor of Se bioaccumulation than was relative trophic position.