Sexual Dimorphism,Female Reproductive Characteristics and Egg Incubation in an Oviparous Forest Skink(Sphenomorphus incognitus) from South China

We studied sexual dimorphism and female reproduction in an oviparous forest skink(Sphenomorphus incognitus)from South China.We incubated eggs under five thermal regimes(22,25,28,25±3 and 27±5°C)to examine the effects of constant versus fluctuating temperatures on incubation length and hatchling morphology.In our sample the largest male and female were 110 mm and 108 mm snout-vent length(SVL),respectively.Adult males and females did not differ in mean SVL;adult males were larger in head size(both length and width),longer in foreand hind-limb lengths and shorter in abdomen length than females of the same SVL.Accordingly,we conclude that S.incognitus is a sexually monomorphic species in terms of SVL but shows sexual dimorphism in head size,abdomen length and appendage length.Females laid a single clutch of 3-10 eggs per breeding season from early May to midAugust,with larger females generally laying more(but not always larger)eggs per clutch than did smaller ones.Embryonic stages at laying ranged from Dufaure and Hubert's(1961)stage 31 to 32,with a mean stage of 31.3.The positive relationship between clutch mass and female SVL was not significant.The offspring size-number trade-off does not exist in S.incognitus,as revealed by the fact that egg mass was independent of relative fecundity.Incubation length decreased as temperature increased,and stable temperatures resulted in delayed hatching.Hatchlings incubated under the five thermal regimes did not differ from each other in any examined trait,suggesting that S.incognitus is among oviparous reptilian species where incubation temperature has no role in modifying hatchling morphology as long as eggs are not exposed to extreme temperatures for prolonged periods of time.     

Effects of Constant versus Fluctuating Incubation Temperatures on Hatching Success,Incubation Length,and Hatchling Morphology in the Chinese Skink(Plestiodon chinensis)

We incubated eggs of Plestiodon chinensis under five constant(24, 26, 28, 30, and 32 °C) and one fluctuating thermal regimes to examine the effects of constant versus fluctuating incubation temperatures on hatching success, incubation length, and hatchling morphology. The duration of incubation varied considerably among the six temperature treatments, whereas hatching success did not. The mean incubation length decreased as temperature increased in a nonlinear way, and increased as the thermal variance increased. Incubation temperature affected the body size(linear length and mass) and shape of hatchlings, with eggs incubated at 26, 28, and 30 °C producing larger and heavier hatchlings than did those incubated at 24 °C, 32 °C, or fluctuating temperatures. Our results showed that exposure of P. chinensis eggs to extreme temperatures for brief periods of time did not increase embryonic mortality and, in the fluctuating-temperature treatment, the thermal variance affected hatchling morphology more evidently than the thermal mean. Our results highlight the importance of the thermal variance in affecting embryonic development and hatchling morphology, and add further evidence that temperatures within the range of 26-30 °C are optimal for P. chinensis embryos.     

Embryonic Growth and Yolk Depletion during Incubation in the Chinese Skink,Plestiodon chinensis

We collected 24 gravid female Chinese skinks(Plestiodon chinensis) to study embryonic growth and yolk depletion during incubation. Females laid eggs between late May and mid-June. Eggs were incubated at 24(± 0.3) ℃. One egg from each clutch was dissected at 5-d intervals starting at laying. Embryonic stages at laying varied from Dufaure and Hubert's(1961) Stage 30-35, with a mean stage of 32.6. Incubation lengths at 24 ℃ varied from 35.1 to 48.3 d, with a mean of 41.5 d. Based on the derived functions describing instantaneous changes in embryo dry mass and yolk dry mass, we identified three phases of embryonic growth or yolk depletion in P. chinensis. Phase 1, from Day 0(at laying) to Day 15(～36% of the way through incubation), was one of minimal transfer of material from yolk to embryo. Phase 2, from Day 15 to Day 32-33(～77%-80% of the way through incubation), was characterized by increasingly rapid embryonic growth or yolk depletion. Phase 3, from Day 32-33 to hatching, was characterized by reduced embryonic growth or yolk depletion. The length of the last embryonic stage(Stage 40 = completely differentiated embryos) accounted for about 28% of incubation length, and the dry mass of the smallest embryos of Stage 40 accounted for only ～48% of the hatchling dry mass. Our study adds evidence to the idea that oviposition is not timed to coincide with the onset of rapid embryonic growth in oviparous reptiles, and is first to demonstrate that ～50% embryonic growth occurs in the last quarter of incubation in P. chinensis.     

Maternal Thermal Effects on Female Reproduction and Hatchling Phenotype in the Chinese Skink(Plestiodon chinensis)

We maintained gravid Chinese skinks(Plestiodon chinensis) at three constant temperatures(25, 28 and 31 °C) during gestation, and randomly assigned eggs from each female to one of the same three temperatures for incubation to determine maternal thermal effects on female reproduction and hatchling phenotype. Maternal temperature affected egg-laying date, hatching success and hatchling linear size(snout-vent length, SVL) but not clutch size, egg size, egg component, and embryonic stage at laying. More specifically, females at higher temperatures laid eggs earlier than did those at low temperatures, eggs laid at 31 °C were less likely to hatch than those laid at 25 °C or 28 °C, and hatchlings from eggs laid at 31 °C were smaller in SVL. Our finding that maternal temperature(pre-ovipositional thermal condition) rather than incubation temperature(post-ovipositional thermal condition) affected hatching success indicated that embryos at early stages were more vulnerable to temperature than those at late stages. Our data provide an inference that moderate maternal temperatures enhance reproductive fitness in P. chinensis.     

Differences in Thermal Preference and Tolerance among Three Phrynocephalus Lizards （Agamidae） with Different Body Sizes and Habitat Use

We acclimated adults of two viviparous(Phrynocephalus guinanensis and P.vlangalii) and one oviparous(P.versicolor) species of toad-headed lizards(Agamidae) to 28 °C,33 °C and 38 °C to examine whether thermal preference(preferred body temperature,Tp) and thermal tolerance(critical thermal minimum,CTMin;critical thermal maximum,CTMax) were affected by acclimation temperature,and correlate with body size and habitat use.Both Tp and CTMax were highest in P.versicolor and lowest in P.vlangalii,with P.guinanensis in between.The two viviparous species did not differ in CTMin and thermal tolerance range,and they both were more resistant to low temperatures and had a wider range of thermal tolerance than the oviparous species.Both CTMin and CTMax shifted upward as acclimation temperature increased in all the three species.Tp was higher in the lizards acclimated to 33 °C than in those to 28 °C or 38 °C.The range of thermal tolerance was wider in the lizards acclimated to 28 °C than in those to 33 °C or 38 °C.The data showed that:1) thermal preference and tolerance were affected by acclimation temperature,and differed among the three species of Phrynocephalus lizards with different body sizes and habitat uses;2) both Tp and CTMax were higher in the species exchanging heat more rapidly with the environment,and CTMin was higher in the species using warmer habitats during the active season;and 3) thermal preference and tolerance might correlat with body size and habitat use in Phrynocephalus lizards.     

New Record of Lycodon liuchengchaoi in Anhui

One juvenile and one adult female wolf snake(Colubridae: Lycodon) were sampled at Yixian and Fuxi, Huangshan, Anhui, China in the summer of 2011 and 2012, respectively. The two specimens were identified as Lycodon liuchengchaoi based on external morphology and molecular data. This is a new reptile record in Anhui Province. In our laboratory, four eggs were laid and three neonates were hatched successfully. This is the first record of the laying and incubation of L. liuchengchaoi eggs. The five specimens were deposited at the Museum of Huangshan University(HUM20140001) and Guangdong Entomological Institute(HB-lcfsp12613, HB-lcfsp-ch1~3).     

Phylogenetic Analysis of Brine Shrimp （Artemia） in China Using DNA Barcodinge

DNA barcoding is a powerful approach for characterizing species of organisms, especially those with almost identical morphological features, thereby helping to to establish phylogenetic relationships and reveal evolutionary histories. In this study, we chose a 648-bp segment of the mitochondrial gene, cytochrome c oxidase subunit 1 （COI）, as a standard barcode region to establish phylogenetic relationships among brine shrimp （Artemia） species from major habitats around the world and further focused on the biodiversity of Artemia species in China, especially in the Tibetan Plateau. Samples from five major salt lakes of the Tibetan Plateau located at altitudes over 4,000 m showed clear differences from other Artemia populations in China. We also observed two consistent amino acid changes, 153A/V and 183L/F, in the COI gene between the high and low altitude species in China. Moreover, indels in the COI sequence were identified in cyst and adult samples unique to the Co Qen population from the Tibetan Plateau, demonstrating the need for additional investigations of the mitochondrial genome among Tibetan Artemia populations.     

Molecular mechanism of diapause in insect (I) --Expression of diapause hormone gene and incubation temperature in embryonic stage of Bombyx mori

The embryonic diapause of the silkworm, Bombyx mori, is induced by the diapause hormone (DH) which is secreted from the suboesophageal ganglion of pupae. The diapause nature of bivoltine strains uses environmental stimuli as the initial signal to determine the diapause nature. The experiments showed that DH gene expression is a direct response to the environmental stimulus, such as high incubation temperature. The cDNA from the embryonic stage was cloned and sequence analysis showed the cDNA encoding DH. Expression patterns of the DH gene in embryonic stage are different at incubation temperatures 15℃ and 25℃, suggesting that the incubation temperature as an environmental signal is kept within the body to control the DH gene expression at the pupal stage, so that the embryonic diapause of next generation can be determined.     

Effect of Incubation Temperature on the Somatic Development of the Snail-Eating Turtle Malayemys macrocephala

The rising average global temperature can lead to changes both in the physical and biological environments and affect the survival of organisms. Freshwater turtles are considered to be susceptible species since their development is dependent on incubation temperature. In Thailand, although several species of freshwater turtle are present, the extent of their susceptibility to temperature change is unknown due to the lack of information on their developmental patterns. This research, therefore, is aimed at examining the effects of temperature on somatic development in Malayemys macrocephala, a native species and the most common freshwater turtle in Thailand. Turtle eggs were collected from rice fields in the central part of Thailand during December 2011 to February 2012 inclusive. Eggs （237-238 per condition） were incubated in microprocessor-controlled incubators at three different temperatures （26 ℃, 29℃ and 32℃） with a relative humidity in excess of 80%. Each week, until the remaining eggs hatched, eggs were randomly selected, removed and dissected to reveal the developing embryo to screen for developmental stage and any abnormalities. The incubation period （lay to hatch） was not significantly different among the three temperatures （115 ±11.3 d, 115 ±20.3 d and 109±17.8 d, respectively）, but the growth patterns, as indicated by the stages of development and carapace lengths, were significantly different. At a high temperature （32℃）, turtle embryos showed a significantly faster growth rate than at the intermediate and low temperatures （29 ℃and 26 ℃）, but had a significantly （over 3.3-fold） higher incidence of developmental abnormalities （especially deformed bodies） than at the lower temperatures. Overall, the results indicate that incubation temperature is an important variable affecting the somatic development of this tropical freshwater turtle species, whilst abnormalities in the embryonic body may be a sensitive indicator of extreme thermal stress.     

A mini-review of advances in the study of the evolution of Perdix species

The Northern Eurasia origin hypothesis is one key point in the evolutionary model. This indicates that species riginated in Northern Eurasia. Perdix, belonging to Phasianidae of Galliformes, is a valuable type of wildfowl widely distributed Northern Eurasia and contains three species. This mini-review discusses the taxonomic status, phylogeny, and origin of Perdix. Morphological and molecular evidence suggest that the three species, P. hodgsoniae, P. dauuricae and P. perdix, form a monophyletic group and they should be classified as "pheasant" rather than "quail." The current review also shows that results were affected by the methods selected for data analysis. A taxonomic category should contain as many species as possible, have a clear system history, remain evolutionarily stable, and lack adaptive radiation. Appropriate type, number and length of genes are important as molecular markers and out-group affects the bootstrap support value. P. hodgsoniae is located at the root of the phylogenetic tree and has the phenotype of ancestral Perdix. The genus Perdix may originate in the Qinghai-Tibet Plateau and expanded into high latitude areas, towards the northern plateau. This phylogenetic and evolutionary statement differs from the Eurasian origin hypothesis.     

Unhatched and Hatched Eggshells of the Chinese Cobra Naja atra

Changes in structure and composition of the eggshell resulting from embryonic mobilization of minerals from the eggshell are found in all oviparous reptiles studied thus far. In this study, we measured samples of unhatched and hatched eggshells of the Chinese cobra Naja atra to determine the percentage of ash and the phase composition of calcium carbonate. The mean percentage of ash was significantly higher in unhatched eggshells （24.6%） than in hatched eggshells （22.3%）. The dominant phase in unhatched eggshells was the calcite form of calcium carbonate. In addition to the peaks of calcite, a few small peaks were found to be caused by the aragonite and vaterite phases of calcium carbonate, implying that there are small amounts of aragonite and vaterite in the eggshell. The concentration of the various phases calculated from the intensity of the X-ray diffraction spectra allowed the estimation that percentages of calcite, aragonite and vaterite were about 92%, 4% and 4%, respectively. Hatched eggshells produced similar spectral characteristics as unhatched eggshells, with one exception. The dominant phase composition in the hatched eggshell was also calcite, but the amount of the aragonite phase had a marked increase. Our study adds evidence that embryonic mobilization of minerals from the eggshell may result in changes in structure of the eggshell.     

Incubation temperature and phenotypic traits of Sceloporus undulatus: implications for the northern limits of distribution

Cold environmental temperature is detrimental to reproduction by oviparous squamate reptiles by prolonging incubation period, negatively affecting embryonic developmental processes, and by killing embryos in eggs directly. Because low soil temperature may prevent successful development of embryos in eggs in nests, the geographic distributions of oviparous species may be influenced by the thermal requirements of embryos. In the present study, we tested the hypothesis that low incubation temperature determines the northern distributional limit of the oviparous lizard Sceloporus undulatus. To compare the effects of incubation temperature on incubation length, egg and hatchling survival, and hatchling phenotypic traits, we incubated eggs of S. undulatus under temperature treatments that simulated the thermal environment that eggs would experience if located in nests within their geographic range at 37°N and north of the species’ present geographic range at latitudes of 44 and 42°N. After hatching, snout–vent length (SVL), mass, tail length, body condition (SVL relative to mass), locomotor performance, and growth rate were measured for each hatchling. Hatchlings were released at a field site to evaluate growth and survival under natural conditions. Incubation at temperatures simulating those of nests at 44°N prolonged incubation and resulted in hatchlings with shorter SVL relative to mass, shorter tails, shorter hind limb span, slower growth, and lower survival than hatchlings from eggs incubated at temperatures simulating those of nests at 37 and 42°N. We also evaluated the association between environmental temperature and the northern distribution of S. undulatus. We predicted that the northernmost distributional limit of S. undulatus would be associated with locations that provide the minimum heat sum (∼495 degree-days) required to complete embryonic development. Based on air and soil temperatures, the predicted northern latitudinal limit of S. undulatus would lie at ∼40.5–41.5°N. Our predicted value closely corresponds to the observed latitudinal limit in the eastern United States of ∼40°N. Our results suggest that soil temperatures at northern latitudes are not warm enough for a sufficient length of time to permit successful incubation of S. undulatus embryos. These results are consistent with the hypothesis that incubation temperature is an important factor limiting the geographic distributions of oviparous reptile species at high latitudes and elevations.     

Both incubation temperature and posthatching temperature affect swimming performance and morphology of wood frog tadpoles (Rana sylvatica)

In many oviparous vertebrates, hatchling phenotypes are influenced by egg incubation temperature. Many of those phenotypic traits can also acclimate to long-term thermal conditions of juveniles and adults, yet the interactive effects of prehatching and posthatching temperatures on phenotypes have not been studied. To address such interaction, we incubated eggs of wood frogs (Rana sylvatica) at two temperatures and subsequently reared larvae at three temperatures in a fully factorial design. We measured body size, size-independent morphology, and burst swimming speed at one developmental stage. Body size was independent of egg temperature but decreased significantly with increasing larval temperature. Size-independent morphology depended in complex ways on both temperature treatments directly and on their interaction. Burst speed was not influenced directly by egg temperature but was influenced by larval temperature and by the interactions among egg temperature, larval temperature, and test temperature. Our results indicate pervasive effects of egg temperature even late in the larval period and show that prehatching and posthatching temperatures can interact to affect various phenotypic traits. Tadpoles may be able to alter the long-term effects of incubation temperature by choosing particular larval developmental temperatures. Thus, the importance of incubation temperature in oviparous vertebrates should be evaluated by considering the effects of posthatching temperatures.     

Living at the edge: lower success of eggs and hatchlings at lower elevation may shape range limits in an alpine lizard

Studies on range limits clarify the factors involved in the extent of species occurrence and shed light on the limits to adaptation. We studied the effects of elevational variation on the thermal dependence of fitness‐related traits (incubation time, hatching rate, and survivorship, size, and condition of hatchlings) to assess the role of incubation requirements in distribution range limits of the alpine endemic Iberolacerta cyreni. We captured gravid females from two core (summit) and two marginal (low‐elevation edge) populations, we incubated their eggs at three temperatures (22, 26, and 30 °C), and we monitored phenotypic effects. Viability of eggs and hatchlings decreased, independently of elevation, as incubation temperature increased. Hatching success and embryo survivorship were lower for clutches from low‐elevation areas than for those from mountain summits, showing that lizards face difficulties thriving at the low‐elevation edge of their range. Such difficulties were partly counterbalanced by faster postnatal growth at lower elevations, leading to increased adult size and higher fecundity. High incubation temperature had detrimental effects also at low‐elevation areas, and no elevational variation in the thermal dependence of hatchling traits was detected. We suggest that temperature effects on egg development and the lack of selective pressures strong enough to foster local adaptation at marginal areas, combined with extended egg retention, may contribute to shape the range limits of these alpine oviparous reptiles.     

Embryonic growth and mobilization of energy and material during incubation in the checkered keelback snake, Xenochrophis piscator

We collected 20 checkered keelback snakes (Xenochrophis piscator) to study embryonic growth and mobilization of energy and material during incubation. Females laid eggs between late May and late June. The eggs were incubated at 27 degrees C (+/-0.3). One egg from each clutch was dissected at five-day intervals starting at oviposition. The mean incubation length at 27 degrees C was 48.9 days. We identified three phases of embryonic growth or yolk depletion in X. piscator. Phase 1, between oviposition and Day 20, was one of minimal transfer of energy and material from yolk to embryo. Phase 2, between Day 20 and Day 39-40, was characterized by increasingly rapid embryonic growth or yolk depletion. Phase 3, between Day 39-40 and hatching, was characterized by reduced embryonic growth or yolk depletion. Approximately 71% of dry mass, 53% of non-polar lipids and 66% of energy were transferred from the egg contents to the hatchling during incubation. Our data confirm that oviposition is not timed to coincide with the onset of rapid embryonic growth in oviparous squamate reptiles. The greater conversion efficiencies of energy and material from egg to hatchling in snakes can be attributed to their lower energetic costs of embryonic development and greater residual yolk sizes.     

Geographic variation in hatchling size in an oviparous skink: effects of maternal investment and incubation thermal environment

Geographic variation in offspring size can be viewed as an adaptive response to local environmental conditions, but the causes of such variation remain unclear. Here, we compared the size and composition of eggs laid by female Chinese skinks (Plestiodon chinensis) from six geographically distinct populations in southeastern China to evaluate geographic variation in hatchling size. We also incubated eggs from these six populations at three constant temperatures (24, 28 and 32 °C) to evaluate the combined effects of incubation temperature and population source on hatchling size. Egg mass and composition varied among populations, and interpopulation differences in yolk dry mass and energy content were still evident after accounting for egg mass. Population mean egg mass and thus hatchling mass were greater in the colder localities. Females from three northern populations increased offspring size by laying larger eggs relative to their own size. Females from an inland population in Rongjiang could increase offspring size by investing relatively more dry materials and thus more energy into individual eggs without enlarging the size of their eggs. The degree of embryonic development at oviposition was almost the same across the six populations, so was the rate of embryonic development and thus incubation length at any given temperature. Both incubation temperature and population source affected hatchling traits examined, but the relative importance of these two factors varied between traits. Our data show that in P. chinensis hatchling traits reflecting overall body size (body mass, snout‐vent length and tail length) are more profoundly affected by population source. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 113 , 283–296.     

Effects of incubation temperature on the energetics of embryonic development and hatchling morphology in the Brisbane river turtle Emydura signata

Incubation temperature and the amount of water taken up by eggs from the substrate during incubation affects hatchling size and morphology in many oviparous reptiles. The Brisbane river turtle Emydura signata lays hard-shelled eggs and hatchling mass was unaffected by the amount of water gained or lost during incubation. Constant temperature incubation of eggs at 24 °C, 26 °C, 28 °C and 31 °C had no effect on hatchling mass, yolk-free hatchling mass, residual yolk mass, carapace length, carapace width, plastron length or plastron width. However, hatchlings incubated at 26 °C and 28 °C had wider heads than hatchlings incubated at 24 °C and 31 °C. Incubation period varied inversely with incubation temperature, while the rate of increase in oxygen consumption during the first part of incubation and the peak rate of oxygen consumption varied directly with incubation temperature. The total amount of oxygen consumed during development and hatchling production cost was significantly greater at 24 °C than at 26 °C, 28 °C and 31 °C. Hatchling mass and dimensions and total embryonic energy expenditure was directly proportional to initial egg mass. Accepted: 18 March 1998     

孵化温度对王锦蛇胚胎代谢和幼体适合度的影响

孵化温度所驱动的爬行动物的表型变异是生理生态学研究的热点。本研究以王锦蛇(Elaphe carinata)为实验动物,检验了24℃和28℃孵化温度对王锦蛇胚胎代谢速率、孵化过程中的卵重量、孵出幼体代谢和行为的影响。研究结果显示:卵重和胚胎的呼吸代谢均与孵化时间呈正相关;28℃下胚胎代谢速率大于24℃;幼蛇孵出15 d内体重随着生长时间的延长而减小,24℃孵出幼体的代谢速率大于28℃孵出幼体,两温度下孵出幼体的呼吸代谢速率和生长时间无显著关系;28℃孵出幼体的疾游速和吐信频次均大于24℃;两孵化温度孵出幼体的选择体温无显著差异,但在消耗完体内的剩余卵黄后28℃孵出幼体有60%的个体摄食,而24℃孵出幼体无摄食个体。总体而言,王锦蛇28℃孵出幼体适合度优于24℃孵出幼体。