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Facultative oviposition of Eastern Newt (Notophthalmus viridescens) in response to water reduction of aquatic habitats.


A single genotype can express a range of different phenotypes in response to various environmental stimuli (phenotypic plasticity; Newman, 1992; Stearns, 1992; Pigliucci, 2001; Roff, 2002). Although there are costs and constraints (DeWitt et al., 1998), phenotypic plasticity is often adaptive, providing organisms greater chances of survival and reproduction under heterogeneous environments (Via et al, 1995). Amphibians have served as excellent model species to study adaptive significance of phenotypic plasticity. For example developmental plasticity of larval amphibians in response to different pond hydroperiods, which varies from year to year depending on the balance between precipitation and evaporation, has received much empirical and theoretical attention (Wilbur and Collins, 1973; Smith-Gill and Berven, 1979; Wilbur, 1980; Semlitsch and Wilbur, 1988; Newman, 1992; Babbitt et al., 2003). Previous studies showed that when reared under short hydroperiods, amphibian larvae typically undergo metamorphosis more quickly into smaller-bodied juveniles in order to escape from the deteriorating aquatic habitats (Newman, 1988; Semlitsch and Wilbur, 1988; Denver et al., 1998; Ryan and Winne, 2001; Takahashi and Parris, 2008).

In contrast to the notable attention paid to larval plasticity, few studies have examined maternal plasticity in oviposition in response to varied hydroperiods. Evolution of maternal plasticity in oviposition would allow mothers to achieve greater overall fitness. Timing of oviposition is one example of maternal plasticity. It varies among individuals within populations and also varies between years (Forchhammer et al., 1998; Paton and Crouch, 2002). A transplant experiment of Lithobates sylvaticus (Wood Frog; Berven, 1982) also revealed that timing of reproduction and opposition is highly plastic and determined largely by environmental factors. Thus, it is possible that females of those species that breed in ephemeral ponds (e.g., vernal pools in North America) deposit eggs earlier in response to gradual reduction of aquatic breeding habitats in order for a greater number of offspring to be able to complete metamorphosis before the ponds dry out. In particular, in species whose breeding seasons last a few to several months (e.g., many species in the family Salamandiridae, many North American species in family Hylidae, some species in family Ranidae such as Lithobates catesbianus and L. clamitans), females may deposit proportionally more eggs earlier in their breeding season.

Alternatively, reproductive females of iteroparous amphibians may skip current opposition and invest more effort into future reproduction when the quality of aquatic habitats is unsuitable for offspring surPval (i.e., facultative breeding; Church et al., 2007). Under uncertain enPronments, selection should favor iteroparity and the ability to allocate reproductive efforts among multiple reproductive events in order for indiPduals to avoid risk of failed recruitment and preclude cost of current reproduction which can reduce chances of future surPval and reproduction (Murphy, 1968; Steams, 1992; Harshman and Zera, 2007). Based on this tradeoff between cost and benefit of current and future reproduction, reproductive females of iteroparous pond-breeding amphibians may plastically alter timing of opposition or forgo opposition in response to water reduction of aquatic habitats.

Similarly, reproductive female may have evolved to appear as though making decisions regarding size and number of eggs. Kaplan (1987) demonstrated that female Bombina orientalis possess plasticity and can produce eggs of different sizes and numbers when exposed during their Ptellogenesis to different temperature and food availability conditions. Using California newt (Taricha tarosa) as a model, Kaplan (1985) also showed that larvae from larger eggs completed metamorphosis earlier at a larger body size under abundant food conditions. There is a general inverse relationship between egg size and clutch size across taxa (Steams, 1992). Therefore, amphibian females experiencing water reduction during Ptellogenic phase may plastically change yolk allocation and deposit a smaller number of larger eggs so that an increased number of offspring are able to surPve to metamorphosis under unfavorable drying conditions.

We used the Eastern Newts Notophthalamus viridescens to test maternal plasticity in opposition in response to water reduction of aquatic habitats. The Eastern Newt is an ideal model species because it has a prolonged breeding (up to 6 mo) and opposition season (2-3 mo) in aquatic habitats (Petranka, 1998), which may proPde sufficient time for reproductive females to directly assess the quality of the aquatic habitat and alter maternal investment into opposition. Newts and other amphibians in temperate regions begin Ptellogenesis shortly after the end of the prePous opposition season (i.e., summer), whereas prePtellogenic phase overlaps with the late Ptellogenetic phase of the prePous oogenic cycle (Verrell et al., 1986; Gilbert, 2000). This pattern of oogenic cycle suggests that the mid to late Ptellogenic phase of the Eastern Newts occurs while females are in aquatic habitats for mating and opposition. Therefore females may alter egg size and clutch size by adjusting yolk investment among ova in response to the quality of aquatic habitats. It is unlikely that females are able to change the number of maturing ova once they enter breeding season. However, they appear not to deposit all mature ova during a single breeding season (Petranka, 1998), which may provide means for females to adjust clutch size. In addition the Eastern Newts can live up to 13 y or more in nature and reproduce multiple times (Caetano and Leclaii Jr., 1996). Therefore this species is also a good model to test the possibility of facultative breeding. We predicted that reproductive females under water reduction conditions would deposit a smaller number of larger eggs earlier than those under constant water conditions. Alternatively, females under water reduction conditions may forgo opposition in order to avoid risk of failed recruitment, which would allow females to save the cost of current reproduction and invest more into future reproduction. In such cases we predicted that those that do not deposit eggs would show greater body-mass gain than those that deposit eggs.



We collected 20 female and 40 male adult newts from local ephemeral ponds in Union County, Pennsylvania, U.S.A., (40.9600[degrees] N, 77.0600[degrees] W) between April 5 and April 14, 2014, during which time we observed spring breeding migration to the ponds. A sole purpose of this spring migration is to breed. Their readiness for reproduction was evident by secondary sexual characteristics in males (i.eenlarged cloacae, black excrescences on the medial surfaces of thighs and toes, and broaden tail height) and noticeable swelling of abdominal regions of females. We recorded body mass and snout-vent length (SVL hereafter) of each adult before the experiment. On April 15 (Day 0), we started the experiment by placing one female and two males in each of 20 plastic lOgal (38 liter, 46.5 x 45.7 x 71.2 cm) tubs filled with 36.5 cm of aged tap water, which were located in the Amphibian Research Greenhouse at Bucknell University. To simulate natural fluctuation of temperature and photoperiod that is important to facilitate newt reproduction (Takahashi, pers. obs.), all greenhouse widows were left open and the fan was run continuously until the termination of the experiment. Female newts lay eggs singly by wrapping them with leaves and aquatic vegetation. We added five white oak (Quercus alba) leaves collected from the ephemeral ponds to each tub to provide materials for oviposition and also hiding. As soon as we established the breeding units, we observed frequent mating activities such as amplexus and hula (the behavior a male displays before spermatophore deposition), which lasted until we separated males from females on Day 26. We fed newts with 0 to 200 mg of live aquatic blackworms (Lumbriculus variegatus) per tub once a week. We changed the amount of feeding over the course of the experiment depending on the rate of consumption but kept it consistent across all tubs for any given week.

We randomly assigned 10 females to water reduction and the other 10 to constant water treatment and started the water reduction treatment on April 17 (Day 2). There was no significant difference in initial female body mass between the treatments (constant water treatment = 2.96 [+ or -] 0.19 g (se), water reduction treatment = 2.79 [+ or -] 0.18 g; t = 0.624, P = 0.541). We reduced water depth by 2.25 cm twice a week (4.5 cm per week) by draining water through premade small holes, which were sealed with corks until the assigned date of water reduction. We checked leaves for eggs on a daily basis. When we found eggs, we collected them, recorded the number of eggs, and also took two measurements by a caliper, the longest and shortest diameters of each egg including its elliptical envelope. We did not take these measurements when eggs were tightly wrapped with leaves, which made it difficult to unwrap them without destroying the eggs. We found the first eggs on April 22 (Day 7). We removed males from each tub on May 11 (Day 26) because the presence of males often suppresses oviposition (Takahashi, pers. obs.). We also judged, based on the frequent mating activities observed, that the 26 d period was sufficient for females to pick up necessary spermatophores for fertilization.

We completed the water reduction treatment on May 22 (Day 37) at the water depth of 11.75 cm. Based on the observation that captive newts that have lived in an aquarium with water depth of 8 to 12 cm for several years without showing obvious behavioral signs of stress (Takahashi, pers. obs.), we determined this ending depth to ensure that oviposition decision would be based on predicted quality of offspring habitat, rather than on mortality risk of mothers. We terminated the experiment on May 23 (Day 38) when three females from the water reduction treatment became terrestrial morphs, which were characterized by water repelling coarse skins and narrower tail fins, and started climbing up the walls of the tubs. We measured the final body mass of each female and released all animals back to the natal ponds.


In order to compare timing of oviposition between the water reduction treatment and control, we calculated the average elapsed time (by day) of oviposidon per female since the first oviposition event observed on Day 7. Because only one female from the water reduction treatment deposited eggs, we could not statistically compare the elapsed time between the treatments to test the prediction regarding the timing of oviposidon. For the same reason, we could not statistically compare clutch and egg size between the treatments. For the presentation of egg size data, we used an average value of two measurements (the longest and shortest diameter). We used one-tailed Fisher's exact test to examine the facultative reproduction prediction that more females from the water reduction treatment would skip oviposition. We used a t-test to compare the average number of eggs per female (oviposition investment per female) between the treatments. Analysis of covariance (ANCOVA) was used to test the effect of the water reduction on body-mass change before and after the experiment by having SVL as a covariate. Another ANCOVA was also conducted to compare body-mass change before and after the experiment between oviposited females and females that did not deposit any eggs regardless of the treatment designation by controlling for SVL as a covariate.


In total six out of ten control females laid 265 eggs (treatment mean [+ or -] SE = 26.5 [+ or -] 9.2) whereas one out often females from the water reduction treatment laid 17 eggs (1.7 [+ or -] 1.7, Table 1). Oviposition investment per female was significantly lower in the water reduction treatment (t = 2.659, P = 0.025). The proportion of females that laid eggs during the experiment was significantly lower in the water reduction treatment than in the control (Fisher's Exact Test, P = 0.029). The average egg size for the six control females was 3.1 [+ or -] 0.26 mm (N = 92). The average egg size for the single female from the water reduction treatment was 2.7 [+ or -] 0.28 mm (N = 6). The average elapsed time for the control females was 15.1 [+ or -] 2.7 d whereas the average elapsed time for the single treatment female that laid eggs was 21.3 d. There was no significant difference in body-mass change between the treatments ([F.sub.1,17] = 1.77, P = 0.202, Fig. 1A). However, we found oviposited females, regardless of the treatment, gained significantly more body mass than the females that did not deposit any eggs ([F.sub.1,17]= 6.18, P = 0.024, Fig. IB). ANCOVA revealed no effect of SVL on body-mass change (treatment comparison: [F.sub.1,17] = 0.706, P = 0.412; oviposition status comparison: [F.sub.1,17] = 0.239, P = 0.631).


Few amphibian studies have examined maternal plasticity in multiple traits associated with oviposition in response to variable pond hydrology. The present study is among the first to document experimentally that female amphibians facultatively forgo oviposition in response to water reduction of aquatic habitats. In particular those three females in the water reduction treatment that transformed to terrestrial adults and attempted to leave the aquatic environment provide assured cases of forgone oviposition. In addition the smaller body-mass gain of the females without oviposition most likely suggests that those females stopped vitellogenesis during the experiment. This result was unexpected as we predicted otherwise because females that do not oviposit invest no resources into opposition. There was a combination of two conditions that was likely responsible for this unexpected outcome. First, we fed newts sufficiently throughout the experiment. Although we adjusted the amount of feeding depending on the rate of consumption, we consistently observed the presence of live blackworms at the bottom of all tubs, suggesting that the females had unlimited accesses food throughout the experiment. Therefore, those females that deposited eggs were able to constantly replenish their resource reserve. Second, it is likely that we terminated the experiment before the oviposited females completed opposition. Those females deposited 6 to 74 eggs (Table 1). In contrast Bishop (1941) dissected five adult females and found they contained 232-376 mature ova. Moreover, eight females housed in outdoor mesocosms deposited on average 152.8 [+ or -] 10.4 se eggs during a single breeding season (Takahashi, pers. obs.). Therefore, the females that deposited eggs in our experiment likely contained more mature ova with full yolk at the end of the experiment, resulting in greater body-mass gain. Given the significandy lower body-mass gain, it is likely that those females that did not deposit any eggs during the experiment would have skipped opposition if we continued the experiment until the end of the opposition season {i.e., June).

We collected newts as they arrived at the ephemeral ponds for spring breeding. Despite the fact that the females were prepared to reproduce, four control females did not deposit any eggs. It is unlikely that the lack of opposition by those control females was caused by the lack of mating opportunities, as we observed active mating behaPors (i.e., amplexus) of all of those four females. Rather, this and the other reports (Bishop, 1941; Verrell, 1986) suggest the difficulty of facilitating opposition of captive newts. The possible issues in our experiment include the daily egg checking (although this was not avoidable), the tubs being too small, and the lack of sufficient plant materials for opposition. Also, water temperature might have become too warm as the greenhouse trapped heat during daytime despite the continuous air exchange via fan and windows (the greenhouse air temperature went up to ~30 C a few times). Using the Alpine Newt (Triturus alpestris) that has a similar life history to the Eastern Newt, Dvorak and Gvozdlk (2009) tested relative importance of thermal condition and availability of egg-wrapping vegetation in oviposition behavior. Most females, when aquatic vegetation was removed, did not oviposit even in the optimal thermal condition (15-20 C). Females also did not oviposit below 12.5 C or above 22.5 C.

Although our results supported the facultative reproduction prediction, we do not know whether females that did not oviposit gained increased chance of survival and future reproduction. One interesting question yet to be explored is whether amphibians are able to transfer vitellogenic ova from one reproductive season to the following. The discrepancy between number of mature ovarian eggs (232-376; Bishop, 1941) and actual clutch size (19 to 181; Bishop, 1941; Verrell, 1986; Takahashi, pers. obs.) suggests female newts do not always deposit all mature ova during a single reproductive season. This discrepancy was also found in Slimy Salamander (Plethodon glutinosus) (Petranka, 1998) and may be common among other iteroparous amphibians. Whereas female Plethodontid salamanders reproduce annually in southern regions, those in northern regions typically reproduce biennially, presumably because it takes multiple years for females to develop ova due to the shorter growing season in northern regions (Highton, 1962; Sayler, 1966; Takahashi and Pauley, 2010). A long-term monitoring of female Four-toed Salamanders (Hemidactylium scutatum) revealed that females that skipped reproduction gained more body mass, which can result in higher fecundity during their next reproduction (Harris and Ludwig, 2004). Harris and Ludwig (2004) also experimentally demonstrated the causality between food availability and reproductive frequency of Fourtoed Salamanders. These lines of evidence suggest female newts that forgo oviposition or deposit fewer eggs may gain reproductive advantage in the following reproduction, especially under resource-limited conditions, by depositing transferred ova from the previous reason.

While demonstrating facultative oviposition in response to water reduction, our data did not allow us to evaluate the predictions regarding timing of oviposition, egg size, and clutch size, which can affect offspring fitness (Kaplan, 1980, 1985; Kaplan, 1989). In temperate amphibians with complex life cycles that have long breeding and oviposition season in aquatic habitats, vitellogenesis occurs in the aquatic habitats (Verrell et al., 1986; Gilbert, 2000). In such cases females may plastically adjust yolk allocation among ova and alter the life history traits associated with oviposition by direcdy assessing the quality of aquatic habitats. Even in the pond-breeding species whose vitellogenesis occur mostly on land prior to breeding migration, females may able to indirectly assess the quality of aquatic habitats through the amount of precipitation and soil moisture content. Church et al. (2007) found that more females of Ambystoma tigrinum skipped reproduction during dry years. In order to define dry vs. wet years, Church et al. (2007) used Standardized Precipitation Index which reflected the hydrology of the breeding ponds that they studied. The authors attributed the cause of forgone reproduction of A. tigrinum to avoidance of breeding in shallower pond water which causes high adult mortality. In the meantime, shallow pond water and low precipitation certainly impose high mortality risk on their larvae as well. Therefore, the study by Church et al. (2007) implies an important possibility that females may be able to alter reproductive output through indirect assessment of the aquatic habitat prior to breeding migration. Maternal plasticity of amphibians in oviposition behavior has been largely unexplored. The present study showed that the Eastern Newt is a potentially useful model for future studies in this field.

Acknowledgments.--We thank Brian Case, Trevor Reitz, and Mayu Uemura for field assistance, Sabrina Kirby, Brian Sullivan, two anonymous reviewers for helpful comments on our early draft, and Pennsylvania Department of Conservation for Natural Resources for Scientific Collection Permit (#624-1). Animal use was approved by Bucknell University LACUC (#MT-04).


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Submitted 1 June 2015

Accepted 5 October 2015


Department of Biology, Bucknell University, Lewisburg, Pennsylvania 17837

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Author:Takahashi, Mizuki K.; McPhee, Carolyn
Publication:The American Midland Naturalist
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Date:Jan 1, 2016
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