Materials and methods
Experimental design and samplingnext section
Two great advantages of using newts for experimental analysis are that European newt females, including crested newts, deposit their eggs one by one and embryonic development occurs in transparent mucoid capsules, making the tracing of developmental stages easy and accurate (Nieuwkoop, 1996).
We collected large, gravid females with inflated abdomens and swollen cloacae from natural populations of four species prior to the oviposition period. Females of T. macedonicus (N = 7) were collected in March 2008: six originated from Ceklin (42º21´N; 18º59´E, 290 m above sea level (a.s.l.)) and one female from nearby Donji Ljubotinj (42º23´N, 19º07´E, 225 m a.s.l.), Montenegro. Females of T. arntzeni (N = 8) were collected in April 2008 from Borovsko polje, Serbia (42º58´N, 22º43´E, 890 m a.s.l.). Females of T. dobrogicus (N = 4) were collected near Kikinda town, Serbia (March 2006, 45º40´N, 20º29´E, 75 m a.s.l.). Females of T. cristatus females (N = 6) were collected in April 2006: five females were taken from Miroč, Serbia (44º29´N, 22º20´E, 440 m a.s.l.), and one female from Vršački breg, Korkana (45º06´N, 21º27´E, 180 m a.s.l.). The females were transferred to a laboratory at the Institute of Biological Research, University of Belgrade, within 24 h. All females were returned to their original population sites after the oviposition period ended.
Since crested newt species prefer different temperatures (Litvinchuk et al., 2007), we provided two different experimental temperature conditions when examining interspecific differences in embryonic development. Triturus dobrogicus and T. cristatus were kept at 16-17ºC, while the more thermophylic species T. macedonicus and T. arntzeni were kept at 18-19ºC. Since the duration of European newt embryonic development correlates with temperature (Griffiths and Wijer, 1994; Bonacci et al., 2005; D’Amen et al., 2007), temperatures were kept constant to provide similar ecological settings for all individuals during embryonic development. In the laboratory, females were housed individually in 12-litre aquaria containing six litres of dechlorinated tap water. The captive females were fed every other day with worms and Tubifex. Plastic strips were provided for egg deposition and eggs were collected daily. Eggs were allowed to develop under laboratory conditions in Petri dishes (5 cm in diameter) containing a maximum of 10 eggs per Petri dish and filled with enough dechlorinated tap water to cover the eggs. The water was changed every second day.
The eggs deposited by one female over a 24 h period were used as cohorts, and the mean values of analysed traits were calculated separately for each cohort. Eggs were photographed immediately after removal from the plastic strips for measurements. The morphologically defined developmental events (stages of embryonic development) were established according to a description of embryonic stages for M. alpestris (Knight, 1938; see also Epperlein and Junginger, 1982). To estimate the variation in timing of developmental events within and between species, the developing embryos were examined under a binocular microscope by M. C. and N. T. K. at three different time points: at 7, 11 and 15 days following egg laying. The embryonic developmental stage was recorded at each particular checkpoint (at day 7, 11, and 15 of embryonic development), hereafter designated as S7, S11 and S15, respectively. Such experimental design provided a basis for analysing the variation in developmental events and developmental rates within and between species. Undeveloped eggs and dead embryos were removed regularly. Due to the possible effect of signalling between embryos on embryonic induction (Hall, 1999; 2003), median values for recorded stages of embryos per Petri dish (up to 10 embryos) were calculated; these data were used in further analyses. Hatched larvae were photographed with a digital camera (Nikon Coolpix 4500) and a 10-mm scale bar to measure the total length of larvae.
The following eight life-history traits were recorded: time of egg deposition relative to the oviposition period (DO), number of eggs laid per cohort (NE), mean vitellus diameter per cohort (RV), volume of galerta calculated as a difference among egg’s volume and vitellus volume (VG), number of hatched larvae per cohort (NH), total length of hatched larvae per cohort (TL), duration of embryonic period of embryos per cohort (EP) and total hatchling survival rate of embryos per cohort (SR). Measurements of egg and vitellus diameter and TL of hatched larvae were taken by M. C. using UTHSCSA IMAGETOOL version 3.0 (http://ddsdx.uthscsa.edu/dig/itdesc.html).
To investigate the pattern of mortality during the embryonic period, we estimated survival rate (SR) as a proportion of live embryos recorded between two checkpoints. Survival rate confidence intervals were calculated according to the following formula:
where q = 1-SR, SR is the survival rate and n is number of eggs at the beginning of the experiment. Two survival rates were considered significantly different (at 0.95 level) when respective confidence intervals did not overlap (Geller, 1983; Miaud, 1994).
To investigate the relationship between recorded life-history traits for each cohort (NE, RV, VG, SR, TL, EP), as well as between recorded developmental stages of embryos at each particular checkpoint (S7, S11, S15), we calculated Pearson correlation coefficients between these traits separately for each species. The pattern of correlation between recorded traits was analysed with matrix correlations. The similarity of these correlations between species was tested using Quadratic Assignment Procedures (Mantel’s test) with 10,000 iterations. The significance test is based on the null hypothesis of no similarity in correlation patterns between compared matrices. A significant correlation between matrices would suggest a non-negligible concordance in correlation pattern between compared matrices.
Matrix correlations were used to estimate the degree of correspondence between interspecies observed correlation patterns and to estimate the robustness of observed correlations. We estimated the robustness and repeatability of observed correlation matrices using the resampling with replacement, or “bootstrapping”, method (Cheverud et al., 1989; Marroig and Cheverud, 2001). For each species of sample size n, n samples were randomly resampled with replacements from the original dataset. This procedure was repeated 500 times (Efron and Tibshirani, 1993), and 500 bootstrap datasets were generated separately for each species using Poptools 2.62 (Hood, 2004). For each generated dataset, correlation matrices were calculated and compared with the correlation matrix obtained from the original data using a matrix correlation. The frequency distributions of matrix self-correlations were used to estimate the robustness of correlation matrices. The repeatability of matrix self-correlations were used to estimate the theoretical maximum matrix correlation (R max ) and to obtain an adjusted matrix correlation (R adj ) between two observed matrices (Marroig and Cheverud, 2001). The value of R max was calculated as (tA tB ) 0.5, where tA and tB denoted the mean of the matrix self-correlations of matrices A and B, respectively. The adjusted matrix correlation (R adj ) was calculated as the observed correlation between two matrices (R obs ) divided by the maximum matrix correlation (R max ).
Since temperature is one of the most important variables affecting amphibian embryonic development, direct comparisons of embryonic developmental timing between different species must be assayed at the animals’ preferred developmental temperatures (Bonacci et al., 2005; D’Amen et al., 2007). Taking these limitations into account, we compared the rate and timing of developmental events of crested newt species at two temperatures. To analyse the differences in the embryonic developmental stages between species at each particular checkpoint (S7, S11, S15), we performed Friedman and Kruskal-Wallis tests. We then used a Mann-Whitney analysis to test for statistical significance in pair-wise comparisons.