Dehydration during egg production alters egg composition and yolk immune function.

Parent-offspring conflicts occur when resources are limited for allocation, and, historically, energy has been the primary currency of focus when examining these trade-offs. Water is a fundamental resource that has received far less consideration for parent-offspring conflicts. Previous research suggests that, when water is limited, reproductive females are compromised in favor of developing embryos. However, these studies limited their assessments to standard metrics such as clutch size and mass. We tested the hypothesis that the mother-offspring conflict over limited water resources leads to finer scale morphological and physiological impacts on the eggs in Children's pythons (Antaresia childreni). We predicted that water deprivation during gravidity alters female investment into her eggs, impacting egg water content and shell development. Additionally, we predicted that the yolk in these dehydrated eggs would have enhanced immune performance metrics, as has been documented in dehydrated adults. We found that eggs from water-deprived females were dehydrated as indicated by reduced percent water and greater yolk osmolality compared to eggs from females that received ad libitum water. We also found that eggs from dehydrated mothers had thinner shells and higher water loss rates. The impacts were not entirely negative as dehydrated eggs had higher antimicrobial capabilities. Also, thinner and more permeability eggshells might allow for elevated rates of rehydration from nest substrate. Overall, by examining an array of egg traits, we demonstrated that dehydration of gravid females impacts the eggs, not just the females as previously reported. As a result, the mother-offspring conflicts are indeed two-sided.

Ex utero culture of viviparous embryos of the lizard, Zootoca vivipara, provides insights into calcium homeostasis during development.

The chorioallantoic membrane resides adjacent to either the inner surface of the egg shell or uterine epithelium in oviparous and viviparous reptiles, respectively. Chorionic cells face the shell or uterine epithelium and transport calcium to underlying embryonic capillaries. Calcium transport activity of the chorioallantois increases in the final stages of development coincident with rapid embryonic growth and skeletal ossification. We excised embryos from viviparous Zootoca vivipara females at a stage prior to significant calcium accumulation and incubated them ex utero with and without calcium to test the hypothesis that chorioallantois calcium transport activity depends on developmental stage and not calcium availability. We measured calcium uptake by monitoring incubation media calcium content and chorioallantois expression of calbindin-D28K, a marker for transcellular calcium transport. The pattern of calcium flux to the media differed by incubation condition. Eggs in 0mM calcium exhibited little variation in calcium gain or loss. For eggs in 2mM calcium, calcium flux to the media was highly variable and was directed inward during the last 3days of the experiment such that embryos gained calcium. Calbindin-D28K expression increased under both incubation conditions but was significantly higher in embryos incubated with 2mM calcium. We conclude that embryos respond to calcium availability, yet significant calcium accumulation is developmental stage dependent. These observations suggest the chorioallantois exhibits a degree of functional plasticity that facilitates response to metabolic or environmental fluctuations.

Influence of reproductive mode on metabolic costs of reproduction: insight from the bimodal lizard Zootoca vivipara.

Examination of the selective forces behind the transition from oviparity to viviparity in vertebrates must include an understanding of the relative energy costs of the two reproductive modes. However, interspecific comparisons of reproductive mode are confounded by numerous other inherent differences among the species. Therefore, we compared oxygen consumption, as a reflection of energy costs, during reproduction in oviparous and viviparous females of the reproductively bimodal lizard Zootoca vivipara (Jaquin 1787). Female oxygen consumption progressively increased over the course of reproduction, peaking just prior to parition when it was 46% (oviparous form) and 82% (viviparous form) higher than it was at the pre-reproductive stage. Total increase in oxygen consumption (TIOC) during the pre-ovulation period was not different between the reproductive modes. Conversely, post-ovulation TIOC was more than three times higher in viviparous females, reflecting a dramatic increase in embryonic metabolism as well as maternal metabolic costs of pregnancy (MCP). MCP accounted for 22% of total metabolism in viviparous females, whereas it was negligible in oviparous females. Our results demonstrate that egg retention through the first third of development, as is typical of most oviparous squamates, entails minimal maternal energy demand, while extending retention imposes much greater metabolic constraints. Selection for transition from oviparity to viviparity must therefore provide benefits that outweigh not only the added burden associated with prolonged embryonic retention, but also the substantial additional energy costs that are incurred.

Multilocus phylogeography of the common lizard Zootoca vivipara at the Ibero-Pyrenean suture zone reveals lowland barriers and high-elevation introgression.

BACKGROUND: The geographic distribution of evolutionary lineages and the patterns of gene flow upon secondary contact provide insight into the process of divergence and speciation. We explore the evolutionary history of the common lizard Zootoca vivipara (= Lacerta vivipara) in the Iberian Peninsula and test the role of the Pyrenees and the Cantabrian Mountains in restricting gene flow and driving lineage isolation and divergence. We also assess patterns of introgression among lineages upon secondary contact, and test for the role of high-elevation trans-mountain colonisations in explaining spatial patterns of genetic diversity. We use mtDNA sequence data and genome-wide AFLP loci to reconstruct phylogenetic relationships among lineages, and measure genetic structure. RESULTS: The main genetic split in mtDNA corresponds generally to the French and Spanish sides of the Pyrenees as previously reported, in contrast to genome-wide AFLP data, which show a major division between NW Spain and the rest. Both types of markers support the existence of four distinct and geographically congruent genetic groups, which are consistent with major topographic barriers. Both datasets reveal the presence of three independent contact zones between lineages in the Pyrenean region, one in the Basque lowlands, one in the low-elevation mountains of the western Pyrenees, and one in the French side of the central Pyrenees. The latter shows genetic evidence of a recent, high-altitude trans-Pyrenean incursion from Spain into France. CONCLUSIONS: The distribution and age of major lineages is consistent with a Pleistocene origin and a role for both the Pyrenees and the Cantabrian Mountains in driving isolation and differentiation of Z. vivipara lineages at large geographic scales. However, mountain ranges are not always effective barriers to dispersal, and have not prevented a recent high-elevation trans-Pyrenean incursion that has led to asymmetrical introgression among divergent lineages. Cytonuclear discordance in patterns of genetic structure and introgression at contact zones suggests selection may be involved at various scales. Suture zones are important areas for the study of lineage formation and speciation, and our results show that biogeographic barriers can yield markedly different phylogeographic patterns in different vertebrate and invertebrate taxa.

Developmental expression of calcium transport proteins in extraembryonic membranes of oviparous and viviparous Zootoca vivipara (Lacertilia, Lacertidae).

The eggshell of oviparous lizards is a significant source of calcium for embryos, whereas the eggshell of viviparous lizards, when present, contains little calcium. In view of the potential cost to embryonic nutrition occasioned by the loss of eggshell calcium, the large number of independent origins of viviparity among lizards is surprising. Concomitant evolution of viviparity and calcium placentotrophy would ameliorate the loss of eggshell calcium, but a mechanism linking these events has yet to be discovered. Zootoca vivipara, a lizard with geographic variation in its mode of parity, is an excellent model for studying mechanisms of calcium transport to oviparous and viviparous embryos because each is highly dependent on calcium secreted by the uterus (eggshell or placenta) and ontogenetic patterns of embryonic calcium mobilization are similar. We compared developmental expression of the calcium transport protein calbindin-D(28K) in yolk splanchnopleure and chorioallantoic membranes of oviparous and viviparous embryos to test the hypothesis that the mechanism of calcium transport does not differ between modes of parity. We found that the ontogenetic pattern of protein expression is similar between reproductive modes and is correlated with calcium uptake from yolk and either eggshell or placenta. Calbindin-D(28K) is localized in the chorionic epithelium of embryos of both reproductive modes. These findings suggest that the embryonic calcium transport machinery is conserved in the transition between reproductive modes and that an adaptation of oviparous embryos for calcium uptake from eggshells functions similarly to transport calcium directly from uterine secretions.

Models of density-dependent genic selection and a new rock-paper-scissors social system.

We describe new ESS models of density regulation driven by genic selection to explain the cyclical dynamics of a social system that exhibits a rock-paper-scissors (RPS) set of three alternative strategies. We tracked changes in morph frequency and fitness of Lacerta vivipara and found conspicuous RPS cycles. Morphs of Uta and Lacerta exhibited parallel survival-performance trade-offs. Frequency cycles in both species of lizards are driven by genic selection. In Lacerta, frequency of each allele in adult cohorts had significant impacts on juvenile recruitment, similar to mutualistic, altruistic, and antagonistic relations of RPS alleles in Uta. We constructed evolutionarily stable strategy (ESS) models in which adults impact juvenile recruitment as a function of self versus nonself color recognition. ESS models suggest that the rapid 4-year RPS cycles exhibited by Lacerta are not possible unless three factors are present: behaviors evolve that discriminate self versus nonself morphs at higher rates than random, self- versus non-self-recognition contributes to density regulation, and context-dependent mate choice evolves in females, which choose sire genotypes to enhance progeny survival. We suggest genic selection coupled to density regulation is widespread and thus fundamental to theories of social system evolution as well as theories of population regulation in diverse animal taxa.

Development of the uterine shell glands during the preovulatory and early gestation periods in oviparous and viviparous Lacerta vivipara.

The evolutionary process leading to the emergence of viviparity in Squamata consists of lengthening the period of egg retention in utero coupled with marked reduction in the thickness of the eggshell. We used light microscopy and scanning electron microscopy to study uterine structure during the reproductive cycle of oviparous and viviparous females of the reproductively bimodal Lacerta vivipara. We compared the structure of the uterine shell glands, which secrete components of the eggshell, during preovulatory and early gestation phases of the reproductive cycle and also compared histochemistry of the eggshells. The uterine glands of both reproductive forms undergo considerable growth within a period of a few weeks during folliculogenesis and vitellogenesis preceding ovulation. The majority of the proteinaceous fibers of the shell membrane are secreted early in embryonic development and the uterine glands regress shortly thereafter. This supports previous observations indicating that, in Squamata, secretion of the shell membrane occurs very rapidly after ovulation. The most striking differences between reproductive modes were larger uterine glands at late vitellogenesis in oviparous females, 101 microm compared to 60 microm in viviparous females, and greater thickness of the shell membrane during early gestation in oviparous females (52-73 microm) compared to viviparous females (4-8 microm). Our intraspecific comparison supports the conclusions of previous studies that, prior to ovulation, the uterine glandular layer is less developed in viviparous than in oviparous species, and that this is the main factor accounting for differences in the thickness of the shell membrane of the two reproductive forms of squamates.

Variation in eggshell characteristics and in intrauterine egg retention between two oviparous clades of the lizard Lacerta vivipara: insight into the oviparity-viviparity continuum in squamates.

The concept of the oviparity-viviparity continuum refers to the wide range in the length of intrauterine egg retention and, hence, in the stage of embryonic development at oviposition existing in squamates. The evolutionary process underlying this continuum may involve not only a lengthening of egg retention in utero, but also a marked reduction in the thickness of the eggshell. The idea that there may exist a negative correlation between the developmental stage reached by the embryo at oviposition and the eggshell thickness within squamates, although supported by the comparison of oviparous vs. viviparous species, has seldom been evaluated by comparing eggshell thickness of oviparous forms with different lengths of intrauterine egg retention. Eggs of two distinct oviparous clades of the lizard Lacerta vivipara were compared. The eggs laid by females from Slovenian and Italian populations have thicker eggshells, contain embryos on average less developed at the time of oviposition, and require a longer incubation period before hatching than the eggs laid by females from French oviparous populations. Our data and several other examples available from the literature support the idea that the lengthening of intrauterine retention of eggs and the shortening of the subsequent external incubation of eggs are associated with reduction in the thickness of the eggshell, at least in some lineages of oviparous squamates. The current hypotheses that may account for this correlation are presented and a few restrictions and refinements to those hypotheses are discussed. In particular, other changes, such as increased vascularization of the oviduct and of the extraembryonic membranes, may play the same role as the decrease of eggshell thickness in facilitating prolonged intrauterine egg retention in squamates. Future studies should also consider the hypothesis that the length of intrauterine retention might directly depend on the extent of maternal-fetal chemical communication through the eggshell barrier.

Extraembryonic membrane development in a reproductively bimodal lizard, Lacerta (Zootoca) vivipara.

Reproductive mode has been remarkably labile among squamate reptiles and the evolutionary transition from oviparity to viviparity commonly has been accompanied by a shift in the pattern of embryonic nutrition. Structural specializations for placental transfer of nutrients during intrauterine gestation are highly diverse and many features of the extraembryonic membranes of viviparous species differ markedly from those of oviparous species. However, because of a high degree of evolutionary divergence between the species used for comparisons it is likely that the observed differences arose secondarily to the evolution of viviparity. We studied development of the extraembryonic membranes and placentation in the reproductively bimodal lizard Lacerta vivipara because the influence of reproductive mode on the structural/functional relationship between mothers and embryos can best be understood by studying the most recent evolutionary events. Lecithotrophic viviparity has evolved recently within this species and, although populations with different reproductive modes are allopatric, oviparous and viviparous forms interbreed in the laboratory and share many life history characteristics. In contrast to prior comparisons between oviparous and viviparous species, we found no differences in ontogeny or structure of the extraembryonic membranes between populations with different reproductive modes within L. vivipara. However, we did confirm conclusions from previous studies that the tertiary envelope of the egg, the eggshell, is much reduced in the viviparous population. These conclusions support a widely accepted model for the evolution of squamate placentation. We also found support for work published nearly 80 years ago that the pattern of development of the yolk sac of L. vivipara is unusual and that a function of a unique structure of squamate development, the yolk cleft, is hematopoiesis. The structure of the yolk sac splanchnopleure of L. vivipara is inconsistent with a commonly accepted model for amniote yolk sac function and we suggest that a long standing hypothesis that cells from the yolk cleft participate in yolk digestion requires further study.

Mitochondrial and nuclear DNA survey of Zootoca vivipara across the eastern Italian Alps: evolutionary relationships, historical demography and conservation implications.

The European common lizard Zootoca vivipara exhibits reproductive bimodality, with populations being either viviparous or oviparous. In the central-eastern Italian Alps oviparous populations (Z. v. carniolica) and viviparous populations (Z. v. vivipara) partly overlap geographically. Studying the evolutionary relationship between these taxa presents an interesting opportunity to gain insight into the evolution of this trait. We aim to: i) test whether Z. v. carniolica, which is endangered, constitutes an ESU (Evolutionary Significant Unity); ii) infer mtDNA divergence time between the Z. v. carniolica clade and all the other Z. vivipara subspecies with the aid of an external calibration point; and iii) describe the phylogeographical and demographic scenarios in the area. To do so we sequenced about 200 individuals for mitochondrial variation; 64 of them were also analysed for three nuclear genes. Furthermore, we analysed the same nuclear markers in 17 individuals from the other oviparous subspecies Z. v. louislantzi and 11 individuals of Z. v. vivipara from widespread geographical origins. The mtDNA and nDNA loci that we examined supported the monophyly of Z. v. carniolica. The mtDNA-based estimate of divergence time between Z. v. carniolica and all the other subspecies indicated a separation at 4.5 Mya (95% CI 6.1-2.6), with about 5% of sequence divergence. Considering that Z. v. carniolica harbours higher genetic diversity, while Z. v. vivipara from central-eastern Alps shows a signature of recent population and spatial expansion, we argue that Z. v. carniolica represents a distinct evolutionary unit, with a presumably long-term evolutionary history of separation. Z. v. carniolica populations, occurring at higher latitudes and altitudes than insofar supposed, live in peat bogs, a seriously threatened habitat: taking into account also its evolutionary distinctness, specific conservation measures should be considered.

The role of python eggshell permeability dynamics in a respiration-hydration trade-off.

Parental care is taxonomically widespread because it improves developmental conditions and thus fitness of offspring. Although relatively simplistic compared with parental behaviors of other taxa, python egg-brooding behavior exemplifies parental care because it mediates a trade-off between embryonic respiration and hydration. However, because egg brooding increases gas-exchange resistance between embryonic and nest environments and because female pythons do not adjust their brooding behavior in response to the increasing metabolic requirements of developing offspring, python egg brooding imposes hypoxic costs on embryos during the late stages of incubation. We conducted a series of experiments to determine whether eggshells coadapted with brooding behavior to minimize the negative effects of developmental hypoxia. We tested the hypotheses that python eggshells (1) increase permeability over time to accommodate increasing embryonic respiration and (2) exhibit permeability plasticity in response to chronic hypoxia. Over incubation, we serially measured the atomic and structural components of Children's python (Antaresia childreni) eggshells as well as in vivo and in vitro gas exchange across eggshells. In support of our first hypothesis, A. childreni eggshells exhibited a reduced fibrous layer, became more permeable, and facilitated greater gas exchange as incubation progressed. Our second hypothesis was not supported, as incubation O(2) concentration did not affect the shells' permeabilities to O(2) and H(2)O vapor. Our results suggest that python eggshell permeability changes during incubation but that the alterations over time are fixed and independent of environmental conditions. These findings are of broad evolutionary interest because they demonstrate that, even in relatively simple parental-care models, successful parent-offspring relationships depend on adjustments made by both the parent (i.e., egg-brooding behavioral shifts) and the offspring (i.e., changes in eggshell permeability).

Erosion of lizard diversity by climate change and altered thermal niches.

It is predicted that climate change will cause species extinctions and distributional shifts in coming decades, but data to validate these predictions are relatively scarce. Here, we compare recent and historical surveys for 48 Mexican lizard species at 200 sites. Since 1975, 12% of local populations have gone extinct. We verified physiological models of extinction risk with observed local extinctions and extended projections worldwide. Since 1975, we estimate that 4% of local populations have gone extinct worldwide, but by 2080 local extinctions are projected to reach 39% worldwide, and species extinctions may reach 20%. Global extinction projections were validated with local extinctions observed from 1975 to 2009 for regional biotas on four other continents, suggesting that lizards have already crossed a threshold for extinctions caused by climate change.

Calcium provision to oviparous and viviparous embryos of the reproductively bimodal lizard Lacerta (Zootoca) vivipara.

Embryos of oviparous squamate reptiles typically obtain calcium from both yolk and eggshell but differ from other oviparous amniotes (turtles, birds and crocodilians) because they are heavily dependent on calcium-rich yolk. Eggs of viviparous squamates lack calcareous eggshells, and embryos receive calcium solely from yolk or from both yolk and placenta. The pattern of calcium mobilization by amniote embryos has been predicted to influence the evolution of viviparity if embryos are dependent on calcium from the eggshell and calcium placentotrophy evolves subsequent to viviparity. We studied the pattern of maternal provision and embryonic utilization of calcium of an oviparous and a viviparous population of the reproductively bimodal lizard Lacerta vivipara to test the hypotheses: (1) oviparous embryos are not dependent on eggshell calcium and (2) calcium content of viviparous hatchlings does not differ from oviparous hatchlings. Our findings do not support either of these hypotheses because oviparous females oviposited eggs with heavily calcified shells and calcium-poor yolk, and embryonic mobilization of shell calcium was greater than for other oviparous squamates. The calcium content of yolk from viviparous females did not differ from oviparous yolk, but viviparous eggs lacked calcareous eggshells. Uterine secretion by viviparous females compensated for the low calcium content of yolk, and placental calcium transfer was among the highest recorded for squamates. The pattern of calcium provision in these two populations suggests that dependence on uterine calcium, either stored temporarily in an eggshell or transferred directly across a placenta, did not constrain the evolution of reproductive mode in this lineage.

Cytokines in the oviparity/viviparity transition: evidence of the interleukin-1 system in a species with reproductive bimodality, the lizard Lacerta vivipara.

Placental viviparity is a reproductive strategy usually attributed to mammals. However, it is also present in other vertebrate species, e.g. in Squamate reptiles. Although the immunological mechanisms that allow the survival of the semi-allogenic embryo in maternal tissues are still largely unknown, cytokines seem to play an important role in mammalian reproduction. Previous studies in our laboratory showed that interleukin-1 (IL-1), a cytokine associated with implantation in mice, is also expressed at the materno-fetal interface of placental viviparous Squamates. In this study, we used the model of Lacerta vivipara, which exhibits reproductive bimodality, that is, the coexistence of oviparous and viviparous populations. By means of immunohistochemistry and anti-human antibodies, we showed that uterine tissues of L. vivipara (seven oviparous and six viviparous animals) expressed the two IL-1 isoforms, IL-1alpha and IL-1beta, and the type I IL-1 receptor (IL-1R tI) both at the pre-ovulatory stage and during gestation, with no significant difference between oviparous and viviparous females. In L. vivipara, as in most oviparous Squamates, an important phase of embryonic development takes place in the mother's oviduct, before egg-laying. Moreover, although thinner than in oviparous females, an eggshell membrane persists throughout gestation in viviparous females also, which develop a very simple type of placenta. The data suggest that immunological mechanisms that allow the survival of the semi-allogenic embryo in maternal tissues are independent of the timing or intimacy of contact between maternal and fetal tissues.

Comparison of the cold hardiness capacities of the oviparous and viviparous forms of Lacerta vivipara.

The lizard Lacerta vivipara has allopatric oviparous and viviparous populations. The cold hardiness strategy of L. vivipara has previously been studied in viviparous populations, but never in oviparous ones. The present study reveals that both the oviparous and viviparous individuals of this species are able to survive in a supercooled state at -3 degrees C for at least one week when kept on dry substrates. The mean crystallisation temperatures of the body, around -4 degrees C on dry substrata and -2 degrees C on wet substrata, do not differ between oviparous and viviparous individuals. All the individuals are able to tolerate up to 48-50% of their body fluid converted into ice, but only viviparous individuals were able to stabilize their body ice content at 48%, and hence were able to survive even when frozen at -3 degrees C for times of up 24 hours. Ice contents higher than 51% have been constantly found lethal for oviparous individuals. This suggests that, in L. vivipara, the evolution towards a higher degree of freezing tolerance could parallel the evolution of the viviparous reproductive mode, a feature believed to be strongly selected under cold climatic conditions. This is the first report, among reptiles, of an intraspecific variation regarding the freeze tolerance capacities.