No link between population isolation and speciation rate in squamate reptiles.

Rates of species formation vary widely across the tree of life and contribute to massive disparities in species richness among clades. This variation can emerge from differences in metapopulation-level processes that affect the rates at which lineages diverge, persist, and evolve reproductive barriers and ecological differentiation. For example, populations that evolve reproductive barriers quickly should form new species at faster rates than populations that acquire reproductive barriers more slowly. This expectation implicitly links microevolutionary processes (the evolution of populations) and macroevolutionary patterns (the profound disparity in speciation rate across taxa). Here, leveraging extensive field sampling from the Neotropical Cerrado biome in a biogeographically controlled natural experiment, we test the role of an important microevolutionary process-the propensity for population isolation-as a control on speciation rate in lizards and snakes. By quantifying population genomic structure across a set of codistributed taxa with extensive and phylogenetically independent variation in speciation rate, we show that broad-scale patterns of species formation are decoupled from demographic and genetic processes that promote the formation of population isolates. Population isolation is likely a critical stage of speciation for many taxa, but our results suggest that interspecific variability in the propensity for isolation has little influence on speciation rates. These results suggest that other stages of speciation-including the rate at which reproductive barriers evolve and the extent to which newly formed populations persist-are likely to play a larger role than population isolation in controlling speciation rate variation in squamates.

Whole Snake Genomes from Eighteen Families of Snakes (Serpentes: Caenophidia) and Their Applications to Systematics.

We present genome assemblies for 18 snake species representing 18 families (Serpentes: Caenophidia): Acrochordus granulatus, Aparallactus werneri, Boaedon fuliginosus, Calamaria suluensis, Cerberus rynchops, Grayia smithii, Imantodes cenchoa, Mimophis mahfalensis, Oxyrhabdium leporinum, Pareas carinatus, Psammodynastes pulverulentus, Pseudoxenodon macrops, Pseudoxyrhopus heterurus, Sibynophis collaris, Stegonotus admiraltiensis, Toxicocalamus goodenoughensis, Trimeresurus albolabris, and Tropidonophis doriae. From these new genome assemblies, we extracted thousands of loci commonly used in systematic and phylogenomic studies on snakes, including target-capture datasets composed of UCEs and AHEs, as well as traditional Sanger loci. Phylogenies inferred from the two target-capture loci datasets were identical with each other, and strongly congruent with previously published snake phylogenies. To show additional utility of these non-model genomes for investigative evolutionary research, we mined the genome assemblies of two New Guinea island endemics in our dataset (Stegonotus admiraltiensis and Tropidonophis doriae) for the ATP1a3 gene, a thoroughly researched indicator of resistance to toad toxin ingestion by squamates. We find that both these snakes possess the genotype for toad toxin resistance despite their endemism to New Guinea, a region absent of any toads until the human-mediated introduction of Cane Toads in the 1930s. These species possess identical substitutions that suggest the same bufotoxin resistance as their Australian congenerics (Stegonotus cucullatus and Tropidonophis mairii) which forage on invasive Cane Toads. Herein, we show the utility of short-read high coverage genomes, as well as improving the deficit of available squamate genomes with associated voucher specimens.

Embryonic development of the skull in a parthenogenetic lizard, the mourning gecko (Lepidodactylus lugubris).

Gekkotans are one of the major clades of squamate reptiles. As one of the earliest-diverging lineages, they are crucial in studying deep-level squamate phylogeny and evolution. Developmental studies can shed light on the origin of many important morphological characters, yet our knowledge of cranial development in gekkotans is very incomplete. Here, we describe the embryonic development of the skull in a parthenogenetic gekkonid, the mourning gecko (Lepidodactylus lugubris), studied using non-acidic double staining and histological sectioning. Our analysis indicates that the pterygoid is the first ossifying bone in the skull, as in almost all other studied squamates, followed closely by the surangular and prearticular. The next to appear are the dentary, frontal, parietal and squamosal. The tooth-bearing upper jaw bones, the premaxilla and maxilla, develop relatively late. In contrast to previous reports, the premaxilla starts ossifying from two distinct centres, reminiscent of the condition observed in diplodactylids and eublepharids. Only a single ossification centre of the postorbitofrontal is observed. Some of the endochondral bones of the braincase (prootic, opisthotic, supraoccipital) and the dermal parasphenoid are the last bones to appear. The skull roof is relatively poorly ossified near the time of hatching, with a large frontoparietal fontanelle still present. Many bones begin ossifying relatively later in L. lugubris than in the phyllodactylid Tarentola annularis, which suggests that its ossification sequence is heterochronic with respect to T. annularis.

Foraging mode constrains the evolution of cephalic horns in lizards and snakes.

A phylogenetically diverse minority of snake and lizard species exhibit rostral and ocular appendages that substantially modify the shape of their heads. These cephalic horns have evolved multiple times in diverse squamate lineages, enabling comparative tests of hypotheses on the benefits and costs of these distinctive traits. Here, we demonstrate correlated evolution between the occurrence of horns and foraging mode. We argue that although horns may be beneficial for various functions (e.g. camouflage, defence) in animals that move infrequently, they make active foragers more conspicuous to prey and predators, and hence are maladaptive. We therefore expected horns to be more common in species that ambush prey (entailing low movement rates) rather than in actively searching (frequently moving) species. Consistent with that hypothesis, our phylogenetic comparative analysis of published data on 1939 species reveals that cephalic horns occur almost exclusively in sit-and-wait predators. This finding underlines how foraging mode constrains the morphology of squamates and provides a compelling starting point for similar studies in other animal groups.

Genetic diversity and the origins of parthenogenesis in the teiid lizard Aspidoscelis laredoensis.

Unisexual vertebrates typically form through hybridization events between sexual species in which reproductive mode transitions occur in the hybrid offspring. This evolutionary history is thought to have important consequences for the ecology of unisexual lineages and their interactions with congeners in natural communities. However, these consequences have proven challenging to study owing to uncertainty about patterns of population genetic diversity in unisexual lineages. Of particular interest is resolving the contribution of historical hybridization events versus post formational mutation to patterns of genetic diversity in nature. Here we use restriction site associated DNA genotyping to evaluate genetic diversity and demographic history in Aspidoscelis laredoensis, a diploid unisexual lizard species from the vicinity of the Rio Grande River in southern Texas and northern Mexico. The sexual progenitor species from which one or more lineages are derived also occur in the Rio Grande Valley region, although patterns of distribution across individual sites are quite variable. Results from population genetic and phylogenetic analyses resolved the major axes of genetic variation in this species and highlight how these match predictions based on historical patterns of hybridization. We also found discordance between results of demographic modelling using different statistical approaches with the genomic data. We discuss these insights within the context of the ecological and evolutionary mechanisms that generate and maintain lineage diversity in unisexual species. As one of the most dynamic, intriguing, and geographically well investigated groups of whiptail lizards, these species hold substantial promise for future studies on the constraints of diversification in unisexual vertebrates.

Evolution of the albumin protein family in reptiles.

The albumin family of proteins consists of vitamin-D binding protein/group-specific component (GC), serum albumin (ALB), alpha-fetoprotein (AFP), and afamin (AFM), which are responsible for transporting many ligands throughout the body. The albumin family proteins are physiologically and medically important, but our understanding of their functions and applications is hindered by the dearth of information regarding these proteins' evolutionary relationships and functions in non-mammalian lineages. In this study we investigate the evolution of the albumin family proteins in reptiles, using bioinformatic methods to survey available reptile genomes and transcriptomes for albumin family proteins and phylogenetically characterize their relationships. We reinforce the established evolutionary relationships among the albumin protein family in reptiles, however, they are variable in their number of domains, overall genetic sequence, and synteny. We find a novel absence of the physiologically important ALB in squamates and identify two distinct lineages of AFP, one in mammals and another in reptiles. Our study provides a comparative genomic framework for further studies identifying lineage-specific gene expansions that may compensate for the lack of serum albumin in squamates.

The eggshell-matrix protein gene OC-17 is functionally lost in the viviparous Chinese crocodile lizard.

Thickness reduction or loss of the calcareous eggshell is one of major phenotypic changes in the transition from oviparity to viviparity. Whether the reduction of eggshells in viviparous squamates is associated with specific gene losses is unknown. Taking advantage of a newly generated high-quality genome of the viviparous Chinese crocodile lizard (Shinisaurus crocodilurus), we found that ovocleidin-17 gene (OC-17), which encodes an eggshell matrix protein that is essential for calcium deposition in eggshells, is not intact in the crocodile lizard genome. Only OC-17 transcript fragments were found in the oviduct transcriptome, and no OC-17 peptides were identified in the eggshell proteome of crocodile lizards. In contrast, OC-17 was present in the eggshells of the oviparous Mongolia racerunner (Eremias argus). Although the loss of OC-17 is not common in viviparous species, viviparous squamates show fewer intact eggshell-specific proteins than oviparous squamates. Our study implies that functional loss of eggshell-matrix protein genes may be involved in the reduction of eggshells during the transition from oviparity to viviparity in the crocodile lizard.

Scaling and relations of morphology with locomotor kinematics in the sidewinder rattlesnake Crotalus cerastes.

The movement of limbless terrestrial animals differs fundamentally from that of limbed animals, yet few scaling studies of their locomotor kinematics and morphology are available. We examined scaling and relations of morphology and locomotion in sidewinder rattlesnakes (Crotalus cerastes). During sidewinding locomotion, a snake lifts sections of its body up and forward while other sections maintain static ground contact. We used high-speed video to quantify whole-animal speed and acceleration; the height to which body sections are lifted; and the frequency, wavelength, amplitude and skew angle (degree of tilting) of the body wave. Kinematic variables were not sexually dimorphic, and most did not deviate from isometry, except wave amplitude. Larger sidewinders were not faster, contrary to many results from limbed terrestrial animals. Free from the need to maintain dynamic similarity (because their locomotion is dominated by friction rather than inertia), limbless species may have greater freedom to modulate speed independently of body size. Path analysis supported: (1) a hypothesized relationship between body width and wavelength, indicating that stouter sidewinders form looser curves; (2) a strong relationship between cycle frequency and whole-animal speed; and (3) weaker effects of wavelength (positive) and amplitude (negative) on speed. We suggest that sidewinding snakes may face a limit on stride length (to which amplitude and wavelength both contribute), beyond which they sacrifice stability. Thus, increasing frequency may be the best way to increase speed. Finally, frequency and skew angle were correlated, a result that deserves future study from the standpoint of both kinematics and physiology.

Investment in chemical signalling glands facilitates the evolution of sociality in lizards.

The evolution of sociality and traits that correlate with, or predict, sociality, have been the focus of considerable recent study. In order to reduce the social conflict that ultimately comes with group living, and foster social tolerance, individuals need reliable information about group members and potential rivals. Chemical signals are one such source of information and are widely used in many animal taxa, including lizards. Here, we take a phylogenetic comparative approach to test the hypothesis that social grouping correlates with investment in chemical signalling. We used the presence of epidermal glands as a proxy of chemical investment and considered social grouping as the occurrence of social groups containing both adults and juveniles. Based on a dataset of 911 lizard species, our models strongly supported correlated evolution between social grouping and chemical signalling glands. The rate of transition towards social grouping from a background of 'epidermal glands present' was an order of a magnitude higher than from a background of 'no epidermal glands'. Our results highlight the potential importance of chemical signalling during the evolution of sociality and the need for more focused studies on the role of chemical communication in facilitating information transfer about individual and group identity, and ameliorating social conflict.

Parallel evolution of placental calcium transfer in the lizard Mabuya and eutherian mammals.

An exceptional case of parallel evolution between lizards and eutherian mammals occurs in the evolution of viviparity. In the lizard genus Mabuya, viviparity provided the environment for the evolution of yolk-reduced eggs and obligate placentotrophy. One major event that favored the evolution of placentation was the reduction of the eggshell. As with all oviparous reptiles, lizard embryos obtain calcium from both the eggshell and egg yolk. Therefore, the loss of the eggshell likely imposes a constraint for the conservation of the egg yolk, which can only be obviated by the evolution of alternative mechanisms for the transport of calcium directly from the mother. The molecular and cellular mechanisms employed to solve these constraints, in a lizard with only a rudimentary eggshell such as Mabuya, are poorly understood. Here, we used RT-qPCR on placental and uterine samples during different stages of gestation in Mabuya, and demonstrate that transcripts of the calcium transporters trpv6, cabp28k, cabp9k and pmca are expressed and gradually increase in abundance through pregnancy stages, reaching their maximum expression when bone mineralization occurs. Furthermore, CABP28K/9K proteins were studied by immunofluorescence, demonstrating expression in specific regions of the mature placenta. Our results indicate that the machinery for calcium transportation in the Mabuya placenta was co-opted from other tissues elsewhere in the vertebrate bodyplan. Thus, the calcium transportation machinery in the placenta of Mabuya evolved in parallel with the mammalian placenta by redeploying the expression of similar calcium transporter genes.

Phylogenetic analysis of macroecological patterns of home range area in snakes.

A home range is the area animals use to carry out routine activities such as mating, foraging, and caring for young. Thus, the area of a home range is an important indicator of an animal's behavioural and energetic requirements. While several studies have identified the factors that influence home range area (HRA), none of them has investigated global patterns of HRA among and within snake species. Here, we used a phylogenetic mixed model to determine which factors influence HRA in 51 snake species. We analysed 200 HRA estimates to test the influence of body mass, sex, age, diet, precipitation, latitude, winter and summer temperature, while controlling for the duration of the study and sample size. We found that males had larger HRA than females, that adults had larger HRA than juveniles, and that snake species with fish-based diets had smaller HRA than snake species with terrestrial vertebrate-based and invertebrate-based diets. We also found that HRA tended to increase as mean winter temperature decreases and tended to decrease with precipitation. After accounting for these factors, the phylogenetic heritability of HRA in snakes was low (0.21 ± 0.14). Determining the factors that dictate macroecological patterns of space use has important management implications in an era of rapid climate change.

Squamate egg tooth development revisited using three-dimensional reconstructions of brown anole (Anolis sagrei, Squamata, Dactyloidae) dentition.

The egg tooth is a hatching adaptation, characteristic of all squamates. In brown anole embryos, the first tooth that starts differentiating is the egg tooth. It develops from a single tooth germ and, similar to the regular dentition of all the other vertebrates, the differentiating egg tooth of the brown anole passes through classic morphological and developmental stages named according to the shape of the dental epithelium: epithelial thickening, dental lamina, tooth bud, cap and bell stages. The differentiating egg tooth consists of three parts: the enamel organ, hard tissues and dental pulp. Shortly before hatching, the egg tooth connects with the premaxilla. Attachment tissue of the egg tooth does not undergo mineralization, which makes it different from the other teeth of most squamates. After hatching, odontoclasts are involved in resorption of the egg tooth's remains. This study shows that the brown anole egg tooth does not completely conform to previous reports describing iguanomorph egg teeth and reveals a need to investigate its development in the context of squamate phylogeny.

Surprising simplicities and syntheses in limbless self-propulsion in sand.

Animals moving on and in fluids and solids move their bodies in diverse ways to generate propulsion and lift forces. In fluids, animals can wiggle, stroke, paddle or slap, whereas on hard frictional terrain, animals largely engage their appendages with the substrate to avoid slip. Granular substrates, such as desert sand, can display complex responses to animal interactions. This complexity has led to locomotor strategies that make use of fluid-like or solid-like features of this substrate, or combinations of the two. Here, we use examples from our work to demonstrate the diverse array of methods used and insights gained in the study of both surface and subsurface limbless locomotion in these habitats. Counterintuitively, these seemingly complex granular environments offer certain experimental, theoretical, robotic and computational advantages for studying terrestrial movement, with the potential for providing broad insights into morphology and locomotor control in fluids and solids, including neuromechanical control templates and morphological and behavioral evolution. In particular, granular media provide an excellent testbed for a locomotion framework called geometric mechanics, which was introduced by particle physicists and control engineers in the last century, and which allows quantitative analysis of alternative locomotor patterns and morphology to test for control templates, optimality and evolutionary alternatives. Thus, we posit that insights gained from movement in granular environments can be translated into principles that have broader applications across taxa, habitats and movement patterns, including those at microscopic scales.

Molecular structure of sauropsid ß-keratins from tuatara (Sphenodon punctatus).

The birds and reptiles, collectively known as the sauropsids, can be subdivided phylogenetically into the archosaurs (birds, crocodiles), the testudines (turtles), the squamates (lizards, snakes) and the rhynchocephalia (tuatara). The structural framework of the epidermal appendages from the sauropsids, which include feathers, claws and scales, has previously been characterised by electron microscopy, infrared spectroscopy and X-ray diffraction analyses, as well as by studies of the amino acid sequences of the constituent ß-keratin proteins (also referred to as the corneous ß-proteins). An important omission in this work, however, was the lack of sequence and structural data relating to the epidermal appendages of the rhynchocephalia (tuatara), one of the two branches of the lepidosaurs. Considerable effort has gone into sequencing the tuatara genome and while this is not yet complete, there are now sufficient sequence data for conclusions to be drawn on the similarity of the ß-keratins from the tuatara to those of other members of the sauropsids. These results, together with a comparison of the X-ray diffraction pattern of tuatara claw with those from seagull feather and goanna claw, confirm that there is a common structural plan in the ß-keratins of all of the sauropsids, and not just those that comprise the archosaurs (birds and crocodiles), the testudines (turtles) and the squamates (lizards and snakes).

5-Methylcytosine-Rich Heterochromatin in Reptiles.

An experimental approach using monoclonal anti-5-methylcytosine antibodies and indirect immunofluorescence was elaborated for detecting 5-methylcytosine-rich chromosome regions in reptilian chromosomes. This technique was applied to conventionally prepared mitotic metaphases of 2 turtle species and 12 squamate species from 8 families. The hypermethylation patterns were compared with C-banding patterns obtained by conventional banding techniques. The hypermethylated DNA sequences are species-specific and are located in constitutive heterochromatin. They are highly reproducible and often found in centromeric, pericentromeric, and interstitial positions of the chromosomes. Heterochromatic regions in differentiated sex chromosomes are particularly hypermethylated.

Is genomic diversity a useful proxy for census population size? Evidence from a species-rich community of desert lizards.

Species abundance data are critical for testing ecological theory, but obtaining accurate empirical estimates for many taxa is challenging. Proxies for species abundance can help researchers circumvent time and cost constraints that are prohibitive for long-term sampling. Under simple demographic models, genetic diversity is expected to correlate with census size, such that genome-wide heterozygosity may provide a surrogate measure of species abundance. We tested whether nucleotide diversity is correlated with long-term estimates of abundance, occupancy and degree of ecological specialization in a diverse lizard community from arid Australia. Using targeted sequence capture, we obtained estimates of genomic diversity from 30 species of lizards, recovering an average of 5,066 loci covering 3.6 Mb of DNA sequence per individual. We compared measures of individual heterozygosity to a metric of habitat specialization to investigate whether ecological preference exerts a measurable effect on genetic diversity. We find that heterozygosity is significantly correlated with species abundance and occupancy, but not habitat specialization. Demonstrating the power of genomic sampling, the correlation between heterozygosity and abundance/occupancy emerged from considering just one or two individuals per species. However, genetic diversity does no better at predicting abundance than a single day of traditional sampling in this community. We conclude that genetic diversity is a useful proxy for regional-scale species abundance and occupancy, but a large amount of unexplained variation in heterozygosity suggests additional constraints or a failure of ecological sampling to adequately capture variation in true population size.

Evolutionary transitions in body plan and reproductive mode alter maintenance metabolism in squamates.

BACKGROUND: Energy (resources) acquired by animals should be allocated towards competing demands, maintenance, growth, reproduction and fat storage. Reproduction has the second lowest priority in energy allocation and only is allowed after meeting the energetic demands for maintenance and growth. This hierarchical allocation of energy suggests the hypothesis that species or taxa with high maintenance costs would be less likely to invest more energy in reproduction or to evolve an energetically more expensive mode of reproduction. Here, we used data on standard metabolic rate so far reported for 196 species of squamates to test this hypothesis. RESULTS: We found that maintenance costs were lower in snakes than in lizards, and that the costs were lower in viviparous species than in oviparous species. As snakes generally invest more energy per reproductive episode than lizards, and viviparity is an energetically more expensive mode of reproduction than oviparity, our results are consistent with the hypothesis tested. CONCLUSION: The transition from lizard-like to snake-like body form and the transition from oviparity to viviparity are major evolutionary transitions in vertebrates, which likely alter many aspects of biology of the organisms involved. Our study is the first to demonstrate that evolutionary transitions in body plan and reproductive mode alter maintenance metabolism in squamates.

Does Population Structure Predict the Rate of Speciation? A Comparative Test across Australia's Most Diverse Vertebrate Radiation.

Population divergence is the first step in allopatric speciation, as has long been recognized in both theoretical models of speciation and empirical explorations of natural systems. All else being equal, lineages with substantial population differentiation should form new species more quickly than lineages that maintain range-wide genetic cohesion through high levels of gene flow. However, there have been few direct tests of the extent to which population differentiation predicts speciation rates as measured on phylogenetic trees. Here, we explicitly test the links between organismal traits, population-level processes, and phylogenetic speciation rates across a diverse clade of Australian lizards that shows remarkable variation in speciation rate. Using genome-wide double digest restriction site-associated DNA data from 892 individuals, we generated a comparative data set on isolation by distance and population differentiation across 104 putative species-level lineages (operational taxonomic units). We find that species show substantial variation in the extent of population differentiation, and this variation is predicted by organismal traits that are thought to be proxies for dispersal and deme size. However, variation in population structure does not predict variation in speciation rate. Our results suggest that population differentiation is not the rate-limiting step in species formation and that other ecological and historical factors are primary determinants of speciation rates at macroevolutionary scales.

Human land use promotes the abundance and diversity of exotic species on Caribbean islands.

Human land use causes major changes in species abundance and composition, yet native and exotic species can exhibit different responses to land use change. Native populations generally decline in human-impacted habitats while exotic species often benefit. In this study, we assessed the effects of human land use on exotic and native reptile diversity, including functional diversity, which relates to the range of habitat use strategies in biotic communities. We surveyed 114 reptile communities from localities that varied in habitat structure and human impact level on two Caribbean islands, and calculated species richness, overall abundance, and evenness for every plot. Functional diversity indices were calculated using published trait data, which enabled us to detect signs of trait filtering associated with impacted habitats. Our results show that environmental variation among sampling plots was explained by two Principal Component Analysis (PCA) ordination axes related to habitat structure (i.e., forest or nonforest) and human impact level (i.e., addition of man-made constructions such as roads and buildings). Several diversity indices were significantly correlated with the two PCA axes, but exotic and native species showed opposing responses. Native species reached the highest abundance in forests, while exotic species were absent in this habitat. Human impact was associated with an increase in exotic abundance and species richness, while native species showed no significant associations. Functional diversity was highest in nonforested environments on both islands, and further increased on St. Martin with the establishment of functionally unique exotic species in nonforested habitat. Habitat structure, rather than human impact, proved to be an important agent for environmental filtering of traits, causing divergent functional trait values across forested and nonforested environments. Our results illustrate the importance of considering various elements of land use when studying its impact on species diversity and the establishment and spread of exotic species.

Immunohistochemical localization of 3ß-Hydroxysteroid dehydrogenase and progesterone receptors in the ovary and placenta during gestation of the placentotrophic lizard Mabuya sp (Squamata: Scincidae).

In squamates, progesterone (P) plays a key role in the inhibition of uterine mobility during egg retention in oviparous species, and during gestation in viviparous species. The corpus luteum (CL) is the main organ responsible for the production of P; however, in some species, the CL degenerates early and the P needed for gestation maintenance should be produced in other tissues. Mabuya sp (Scincidae) is a viviparous lizard with a prolonged gestation, it produces microlecithal eggs and, consequently, has an obligate placentotrophy related with a highly complex placenta. Its CL degenerates at early stages of gestation and therefore, other sources of P should exist. The aim of this study was to determine and localize by immunohistochemistry the production of P by detection of the enzyme 3ß-Hydroxysteroid dehydrogenase (3ß-HSD) and P receptors (PR) during gestation in the ovary and placenta of Mabuya sp. Positive and negative control sections were used. The ovary of this species localizes 3ß-HSD and PR in the same tissues. The CL of the ovaries of females at early stages of gestation were positive for both molecules, whereas they did not localize from mid gestation to the end of pregnancy. Previtellogenic and vitellogenic follicles labelled for both molecules in the follicular epithelium and thecae. The placenta of Mabuya sp. demonstrated the potential for P production from mid gestation to the end of gestation in the uterine and chorionic tissues. PR were located in the uterine tissues throughout gestation, with a decrease towards its completion. Western blot analysis confirmed the presence of 3ß-HSD mainly in the ovary of early pregnant females and in the placental tissues at mid gestation stages. Therefore, the chorioallantoic placenta of Mabuya sp. has an endocrine function producing the P needed for gestation and replacing the CL from mid gestation to the end of pregnancy.