Further evidence of the existence of oviparous populations of Lacerta (Zootoca) vivipara in the NW of the Balkan Peninsula

The lizard Lacerta (Zootoca) vivipara, which is viviparous in the greatest part of its distribution range, has however some oviparous populations on the southern margin of its range. The present study aimed at determining the reproductive mode and the ATA (aspartate transaminase) enzyme characteristics of four populations in Slovenia and one population in Croatia. The Slovenian females studied here presented an oviparous reproductive mode which strongly resembled those observed in the oviparous populations of south-western France and north-western Spain. Our electrophoresis analyses revealed the existence of two distinct alleles, ATA-150 and ATA-200, in the oviparous populations of Slovenia. These alleles were identical to those observed in the French and Spanish oviparous group and were distinct from the allele ATA-100 characterizing the viviparous populations that we had previously studied. Although we did not study the reproductive mode of Croatian females, the allele ATA-200 observed in one population of Croatia strongly suggested that this population might also be oviparous.     

Experimental evidence of early costs of reproduction in conspecific viviparous and oviparous lizards

Reproduction entails costs, and disentangling the relative importance of each stage of the reproductive cycle may be important to assess the costs and benefits of different reproductive strategies. We studied the early costs of reproduction in oviparous and viviparous lizard females of the bimodal reproductive species Zootoca vivipara. Egg retention time in oviparous females is approximately one-third of the time in viviparous females. We compared the vitellogenesis and egg retention stages that are common to both reproductive modes. Precisely, we monitored the thermoregulatory behaviour, the weight gain and the immunocompetence of the females. Moreover, we injected an antigen in half of the females (immune challenge) to study the trade-offs between reproductive mode and immune performance and between different components of the immune system. Finally, we experimentally induced parturition in viviparous females at the time of egg laying in oviparous females. Oviparous and viviparous females did not show strong differences in response to the immune challenge. However, viviparous females spent more time thermoregulating while partially hidden and gained more weight than oviparous females. The greater weight gain indicates that the initial period of egg retention is less costly for viviparous than for oviparous females or that viviparous females are able to save and accumulate energy at this period. This energy may be used by viviparous females to cope with the subsequent costs of the last two-third of the gestation. Such an ability to compensate the higher costs of a longer egg retention period may account for the frequent evolution of viviparity in squamate reptiles.     

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.     

Intraspecific phylogeography of Lacerta vivipara and the evolution of viviparity

The lacertid lizard Lacerta vivipara is one of the few squamate species with two reproductive modes. We present the intraspecific phylogeny obtained from neighbor-joining and maximum-parsimony analyses of the mtDNA cytochrome b sequences for 15 individuals from Slovenian oviparous populations, 34 individuals from western oviparous populations of southern France and northern Spain, 92 specimens from European and Russian viviparous populations, and 3 specimens of the viviparous subspecies L. v. pannonica. The phylogeny indicates that the evolutionary transition from oviparity to viviparity probably occurred once in L. vivipara. The western oviparous group from Spain and southern France is phylogenetically most closely related to the viviparous clade. However, the biarmed W chromosome characterizing the western viviparous populations is an apomorphic character, whereas the uniarmed W chromosome, existing both in the western oviparous populations and in the geographically distant eastern viviparous populations, is a plesiomorphic character. This suggests an eastern origin of viviparity. Various estimates suggest that the oviparous and viviparous clades of L. vivipara split during the Pleistocene. Our results are discussed in the framework of general evolutionary models: the concept of an oviparity-viviparity continuum in squamates, the cold climate model of selection for viviparity in squamates, and the contraction-expansion of ranges in the Pleistocene resulting in allopatric differentiation.     

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.     

Is the Evolution of Viviparity Accompanied by a Relative Increase in Maternal Abdomen Size in Lizards?

Female reptiles with viviparous reproduction should leave space for their eggs that reach the maximum mass and volume in the oviducts. Is the evolution of viviparity accompanied by a relative increase in maternal abdomen size, thus allowing viviparous females to increase the amount of space for eggs? To answer this question, we compared morphology and reproductive output between oviparous and viviparous species using three pairs of lizards, which included two Eremias, two Eutropis and two Phrynocephalus species with different reproductive modes. The two lizards in each pair differed morphologically, but were similar in the patterns of sexual dimorphism in abdomen and head sizes and the rates at which reproductive output increased with maternal body and abdomen sizes. Postpartum females were heavier in viviparous species, suggesting that the strategy adopted by females to allocate energy towards competing demands differs between oviparous and viviparous species. Reproductive output was increased in one viviparous species, but decreased in the other two, as compared with congeneric oviparous species. The space requirement for eggs did not differ between oviparous and viviparous females in one species pair, but was greater in viviparous females in the other two pairs greater in relative clutch mass and relative litter mass. In the two Phrynocephalus species, viviparous females produced heavier clutches than did oviparous females not by increasing the relative size of the abdomen, but by being more full of eggs. In none of the three species pairs was the maternal abdomen size greater in the viviparous species after accounting for body size. Our data show that the evolution of viviparity is not accompanied by a relative increase in maternal abdomen size in lizards. Future work could usefully investigate other lineages of lizards to determine whether our results are generalisable to all lizards.     

Refuge areas and suture zones in the Pyrenean and Cantabrian regions: geographic variation of the female MPI sex-linked alleles among oviparous populations of the lizard Lacerta (Zootoca) vivipara

The viviparous lizard Lacerta (Zootoca) vivipara exhibits several alleles of the mannose-6-phosphate isomerase (MPI) enzyme that are carried exclusively on the female W sex-chromosome. Previous studies showed that both the oviparous and viviparous forms of L. (Zootoca) vivipara have these female sex-linked alleles. We document the existence of geographic variation of these alleles among the oviparous populations of southwestern France and northwestern Spain. Two oviparous subgroups were identified: all females from the eastern and central Pyrenees and most females from Aquitaine and from the northern slope of the western Pyrenees exhibited the fast migrating alleles MPI¹¹⁰ or MPI¹²⁰, whereas all females from the Cantabric mountains, Spanish Basque country, and from the south slope of the western Pyrenees exhibited the slow migrating allele MPI⁹⁰. Populations with both fast and slow migrating alleles occurred at some stations in the upper Ossau valley (western Pyrenees) and also at a lowland station of south Aquitaine. The hypothesis that several oviparous forms could have retreated to different places of the Pyreneo-Iberian refugia during the Quaternary glaciations could explain the conservation or the evolution of the polymorphism of the MPI alleles, and that is consistent with the phylogeographic scenario previously proposed to account for the reproductive and cytogenetical variation observed in this species.     

Is increased maternal basking an adaptation or a pre-adaptation to viviparity in lizards?

Pregnant females modify their thermoregulatory behaviour in many species of viviparous (live-bearing) reptiles, typically maintaining higher and more stable body temperatures at this time. Such modifications often have been interpreted as adaptations to viviparity, functioning to accelerate embryonic development and/or modify phenotypic traits of hatchlings. An alternative possibility is that similar maternal thermophily may be widespread also in oviparous species and if so, would be a pre-adaptation (rather than an adaptation) to viviparity. Because eggs are retained in utero for a significant proportion of development even in oviparous reptiles, maternal thermophily might confer similar advantages in oviparous as in viviparous taxa. Experimental trials on montane oviparous scincid lizards (Bassiana duperreyi) support the pre-adaptation hypothesis. First, captive females (both reproductive and non-reproductive) selected higher temperatures than males. Second, experimentally imposing thermal regimes on pregnant females significantly affected their oviposition dates and the phenotypic traits (body shape, running speed) of their hatchlings. Thus, as for many other behavioural correlates of pregnancy in viviparous reptiles, maternal thermophily likely may have already been present in the ancestral oviparous taxa that gave rise to present-day viviparous forms.     

Biogeography of Lacerta (Zootoca) vivipara: reproductive mode and enzyme phenotypes in Bulgaria

Lacerta (Zootoca) vivipara , has allopatric oviparous and viviparous populations viviparity is observed from central France and the British Isles to Scandinavia and Russia, while oviparity is restricted to northern Spain and southwestern France, i e the extreme southwestern part of the range Recent observations in the Rila, Balkan, Vitocha, Pirin and Rhodopes mountains indicate that Bulgarian populations of Lacerta (Zootoca) vivipara are indeed viviparous The electrophoretic study of allozymes and the estimation of genetic distances indicate that viviparous lizards from northwest and central France are more closely related to those of Bulgaria, than to the oviparous lizards of southwest France and northwest Spain Variations in reproductive mode and allozymes are not directly related to geographic distances between populations, nor to their latitude populations located at the southwest limit of distribution are oviparous and exhibit alleles ATA^-150 or ATA^-200, whereas, at a comparable latitude, the Bulgarian populations are viviparous and exhibit allele ATA^-100 characteristic of other distant viviparous populations These findings underline the orginality of the oviparous southwestern populations They do not contradict our previous biogeographic scenario     

Maternal body-volume as a constraint on reproductive output in lizards: evidence from the evolution of viviparity

A few species of squamate reptiles contain both oviparous (egg-laying) and viviparous (live-bearing) populations, and thus offer exceptional opportunities to test adaptationist hypotheses on the determinants of reproductive output. We focus on the hypothesis that maternal body-volume constrains reproductive output in squamate reptiles. If females are “full” of eggs, what happens when viviparity evolves within a lineage? Eggs increase in volume and mass during development, primarily due to the uptake of water, so how can they be accommodated within the mother's abdomen? We predict that the resultant increase in relative clutch mass (RCM) will be lessened by (1) a decrease in reproductive output (by reducing the number or size of offspring), and/or (2) an increase in maternal body-volume (via modifications of size or shape of adult females). Our comparisons of conspecific oviparous and viviparous lizards (Lerista bougainvillii) confirm that live-bearers carry heavier clutches (in both absolute and relative terms) and show the predicted shifts in body size and shape of reproductive females. However, offspring size and number were unaffected by the evolution of viviparity, and the shifts in maternal morphology were too small to fully offset the increase in clutch mass. Thus, RCMs increased by 50%, indicating that viviparous females produced clutches which more completely filled the space available in the abdominal cavity. We conclude that maternal body-volume does play a role in determining reproductive output, but that the observed clutch masses may be optimized, rather than maximized, with respect to the abdominal space available.     

Karyological investigations of populations of the lizard Zootoca vivipara (Juaquin, 1787) from the Baltic Sea basin (western region of Russia)

Populations of the lizard Zootoca vivipara (Juaquin, 1787) (Lacertidae, Sauria) from Western and Central Europe are poorly differentiated in morphology. However, they differ from each other by several karyotype markers and their reproductive mode (oviparous and viviparous types). Here we report for the first time the results of combined investigations of Z. vivipara from Eastern Europe (the Baltic Sea basin of Western Russia). The karyotype and reproductive mode of the specimens from four previously not examined populations in Kaliningrad region have been studied. It has been shown that these lizards have viviparous mode of reproduction and their karyotypes vary on morphology of W-sex chromosomes. Females possess 2n = 35 : 32 A + Z1Z2W, with W--SV and 2n = 35 : 32 A + Z1Z2W, with W--A/ST. We have identified these populations karyologically and have first revealed that they belong to two different viviparous forms of nominative subspecies Z. v. vivipara, namely to its "western" and "Russian (eastern)" forms. These data have confirmed that the Baltic Sea basin in Eastern Europe is a zone of secondary contact of these chromosomal forms of Z. v. vivipara. The results obtained do not now allow us to speak about their sympatry, parapatry and (or) hybridization events. The results suggest that during postglasial time "western" form might colonize this region from western Europe whereas "Russian (eastern)" form might do it from Southern-Eastern Europe. It has been recommended to save the biodiversity of Z. vivipara in Kaliningrad region, namely the peripheral and very rare on vast territory of Russia "western" form and unique for western region of Russia "Russian (eastern)" form.     

Mating does not influence reproductive investment, in a viviparous lizard

Mating is crucial for females that reproduce exclusively sexually and should influence their investment into reproduction. Although reproductive adjustments in response to mate quality have been tested in a wide range of species, the effect of exposure to males and mating per se has seldom been studied. Compensatory mechanisms against the absence of mating may evolve more frequently in viviparous females, which pay higher direct costs of reproduction, due to gestation, than oviparous females. To test the existence of such mechanisms in a viviparous species, we experimentally manipulated the mating opportunity of viviparous female lizard, Lacerta (Zootoca) vivipara. We assessed the effect of mating on ovulation, postpartum body condition and parturition date, as well as on changes in locomotor performances and body temperatures during the breeding cycle. Female lizards ovulated spontaneously and mating had no influence on litter size, locomotor impairment or on selected body temperature. However, offspring production induced a more pronounced locomotor impairment and physical burden than the production of undeveloped eggs. Postpartum body condition and parturition dates were not different among females. This result suggests that gestation length is not determined by an embryonic signal. In the common lizard, viviparity is not associated with facultative ovulation and a control of litter size after ovulation, in response to the absence of mating.     

Changing distribution of cadherins during gestation in the uterine epithelium of lizards

The uterine epithelium provides the interface between an embryo and its mother during pregnancy. Calcium-dependent cadherins are adherens junction proteins that undergo major shifts in the uterine epithelium to facilitate the communication between maternal cells and the embryonic milieu during implantation in mammals. They are, therefore, important in trophoblast invasion and the maintenance of pregnancy. We investigated spatiotemporal changes of cadherins throughout pregnancy in the uterine epithelium of two viviparous skinks and one oviparous population, which all exhibit a noninvasive (epitheliochorial) placenta. Cadherins were identified for the first time in squamate reptiles. In all species, cadherins are reduced in the uterine epithelium as gestation progresses, which would lessen the attachment between uterine epithelial cells and allow them to stretch to accommodate embryonic growth. Interestingly, cadherins were reduced sooner after ovulation in the oviparous species than in the viviparous species. In viviparous species, the different expression of cadherins between barren and pregnant uteri from the same mother indicates that expression of cadherins may not be driven solely by maternal hormones, but also by the presence of an embryo. The redistribution of cadherins in squamates is comparable to that of mammals, reflecting establishment of feto-maternal communication during the peri-implantation period. As there is no breaching of maternal tissue in lizards, the change in adherens junctional properties are thus not exclusive to mammals with invasive placentae, which suggests that similar molecular mechanisms regulate changes to uterine epithelia during pregnancy across placental types.     

Evolutionary and biogeographical implications of the karyological variations in the oviparous and viviparous forms of the lizard Lacerta (Zootoca) vivipara

The lizard Lacerta ( Zootoca ) vivipara has two modes of reproduction and is variable karyologically. We describe its karyological variation from literature data and from new data on two viviparous populations from France, on two oviparous populations from the Pyrenees in south-western France and on three oviparous populations recently discovered in Slovenia. Males have 36 chromosomes, whereas females have only 35 chromosomes in all viviparous populations and in the Pyrenean oviparous populations. This karyotype has been interpreted to result from a fusion of an ancestral sexual W chromosome with an autosome from the Zl or from the Z2 pair. The karyotype formula is 32 autosomes + ZIZ2W for the female and 32 autosomes + Z1Z1Z2Z2 for the male. The karyotype of the Slovenian oviparous populations, 34 autosomes + ZW in the male and 34 autosomes +ZW in the female, represents an evolutionary stage that preceded the chromosomal fusion. There is minor karyological variation, mainly concerning the W and Z2 chromosomes, within the Pyrenean oviparous populations. This parallels the geographic variation of the W-linked alleles of the MPI enzyme and suggests that allopatric differentiation of these oviparous populations might have occurred in the vicinity of the Pyrenees during the Pleistocene.
The viviparous populations from western Europe carry a metacentric W chromosome, whereas oviparous populations from south-western Europe and eastern viviparous populations both show an acrocentrie, or a subtelocentrie. W chromosome. This suggests that the acrocentric-subtelocentric W is a primitive character and that viviparity probably arose in an eastern lineage of the species.     

From oviparity to viviparity: a preliminary note on the morphometric differentiation between oviparous and viviparous species assigned to the genus Liolaemus (Reptilia, Squamata, Liolaemidae)

Embryonic growth requires a considerable internal space in viviparous female lizards and this need for space should be reflected in their external morphometry. External morphological differences associated with the reproductive mode in 12 viviparous and 18 oviparous species of Liolaemus lizards were identified. Size differences between viviparous and oviparous species were elucidated by axilla-groin/snout-vent relationship. Axilla-groin distance, considered a size estimator of visceral cavity, surpassed 50% of snout-vent length in viviparous females, while it is always less than 50% in oviparous females. This difference between the two reproductive modes is statistically significant.     

Ovarian steroid synthesis and the hormonal control of the elasmobranch reproductive tract

Synopsis The reproductive biology of the chondricthyan fishes is remarkably sophisticated. Using both oviparous and viviparous reproductive modes, the group has generally adapted the style of bringing forth relatively few young at one time, each representing the investment of a great deal of maternal energy. The oviparous species foreshadow the situation common in oviparous reptiles and universal in birds. On the other hand, viviparous species range from simple internal incubators, in which large yolked eggs are retained, to other species in which the complexity of placentation and yolk reduction approach the eutherian condition. Further, in certain viviparous elasmobranchs the phenomenon of histotrophic nutrition attains an importance and complexity not seen in any other vertebrate group including mammals. Internal fertilization and amniote patterns of reproductive tract development also operate in virtually all elasmobranchs. The summary of work presented here suggests that these female reproductive styles are associated with a reproductive endocrinology which is the archetype for amniote vertebrates.     

Energy consumption by embryos of a viviparous lizard, Eulamprus tympanum, during development

Energy consumption during development has been measured in many oviparous lizards, but not in viviparous lizards in utero. It has always been assumed that energy consumption by embryos of viviparous lizards during development is similar to that of oviparous species. Estimation of energy consumption of viviparous lizards in vivo are confounded by the possible influence of pregnancy on maternal metabolism. Here we separated maternal and embryonic metabolism in measurements of pregnant Eulamprus tympanum throughout pregnancy. Our data support the hypothesis that the energetic cost of development in viviparous lizards (19.8 kJ g(-1)) is similar to that in oviparous lizards (mean 16.2 kJ g(-1)), at least for a species with a simple placenta. An increase in maternal metabolism of 29% above that for non-pregnant E. tympanum goes to maintain pregnancy, and represents an important component of the reproductive effort in E. tympanum.     

Fundamentals of viviparity: comparison of seasonal changes in the uterine epithelium of oviparous and viviparous Lerista bougainvillii (Squamata: Scincidae)

Distinct differences in epithelial response between oviparous and viviparous species of skinks led us to investigate morphological differences in the uterus of a species that exhibits bi-modal reproduction and that may indicate specialities for the different requirements of viviparity and oviparity. The uteri of females from oviparous and viviparous populations of the Australian scincid lizard, Lerista bougainvillii, are described in detail to determine whether the occurrence of uterodomes and the plasma membrane transformation, found in other viviparous species but not oviparous species, are indeed features characteristic of viviparity. Oviductal tissue was dissected at three different stages of reproduction from lizards from both populations: 1) vitellogenic, 2) gravid or pregnant, and 3) non-reproductive or quiescent. Tissue was observed using both scanning and transmission electron microscopy. Lerista bougainvillii has a simple placental morphology with simple squamous epithelium. In contrast to mammals and other viviparous skinks, L. bougainvillii does not undergo a plasma membrane transformation, but early signs of placentation in viviparous individuals are indicated by changes in the uterine surface that occur largely after embryonic stage 30. There are no obvious cellular differences between the uteri of oviparous and viviparous L. bougainvillii at the non-reproductive and vitellogenic phase of the reproductive cycle but throughout gestation/gravidity, the cellular differences that could be related to the changing functional requirements with the retention of the viviparous embryo, became apparent. A plasma membrane transformation with ensuing uterodome formation does not occur, which suggests that these more sophisticated changes are a feature of advanced placental development in reptiles.     

Intraspecific Phylogeography of Lacerta vivipara and the Evolution of Viviparity

The lacertid lizard Lacerta vivipara is one of the few squamate species with two reproductive modes. We present the intraspecific phylogeny obtained from neighbor-joining and maximum-parsimony analyses of the mtDNA cytochrome b sequences for 15 individuals from Slovenian oviparous populations, 34 individuals from western oviparous populations of southern France and northern Spain, 92 specimens from European and Russian viviparous populations, and 3 specimens of the viviparous subspecies L. v. pannonica. The phylogeny indicates that the evolutionary transition from oviparity to viviparity probably occurred once in L. vivipara. The western oviparous group from Spain and southern France is phylogenetically most closely related to the viviparous clade. However, the biarmed W chromosome characterizing the western viviparous populations is an apomorphic character, whereas the uniarmed W chromosome, existing both in the western oviparous populations and in the geographically distant eastern viviparous populations, is a plesiomorphic character. This suggests an eastern origin of viviparity. Various estimates suggest that the oviparous and viviparous clades of L. vivipara split during the Pleistocene. Our results are discussed in the framework of general evolutionary models: the concept of an oviparity–viviparity continuum in squamates, the cold climate model of selection for viviparity in squamates, and the contraction–expansion of ranges in the Pleistocene resulting in allopatric differentiation.     

Subsociality and female reproductive success in a mycophagous thrips: An observational and experimental analysis

Oviparous females of the haplodiploid, facultatively viviparous thrips Elaphrothrips tuberculatus(Thysanoptera: Phlaeothripidae) guard their eggs against female conspecifics and other egg predators. The intensity of maternal defense increases with clutch size. Field and laboratory observations indicate that cannibalism by females is an important selective pressure favoring maternal care. Experimental removals of guarding females showed that egg guarding substantially increases egg survivorship and that the survivorship of undefended eggs is higher in the absence of nonguarding female conspecifics than in their presence. The fecundity of viviparous females increases with the number of eggs cannibalized. The reproductive success of oviparous females increases with body size and local food density and decreases with local density of breeding females. Social behavior may not have advanced beyond maternal care in Elaphrothrips tuberculatusbecause, relative to Hymenoptera, capabilities for helping relatives are few or nonexistent, and the causes of variation in female reproductive success are not influenced easily by cooperation among females.