Genetics, Vol. 170, 275-281, May 2005, Copyright © 2005
doi:10.1534/genetics.104.038273

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Evolution of Salamander Life Cycles

A Major-Effect Quantitative Trait Locus Contributes to Discrete and Continuous Variation for Metamorphic Timing

Department of Biology, University of Kentucky, Lexington, Kentucky 40506

¹ Corresponding author: Department of Biology, 101 T. H. Morgan Bldg., University of Kentucky, Lexington, KY 40506.
E-mail: srvoss{at}uky.edu

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS LITERATURE CITED

 
The evolution of alternate modes of development may occur through genetic changes in metamorphic timing. This hypothesis was examined by crossing salamanders that express alternate developmental modes: metamorphosis vs. paedomorphosis. Three strains were used in the crossing design: Ambystoma tigrinum tigrinum (Att; metamorph), wild-caught A. mexicanum (Am; paedomorph), and laboratory Am (paedomorph). Att/Am hybrids were created for each Am strain and then backcrossed to their respective Am line. Previous studies have shown that a dominant allele from Att (met^Att) and a recessive allele from lab Am (met^lab) results in metamorphosis in Att/Am hybrids, and met^Att/met^lab and met^lab/met^lab backcross genotypes are strongly associated with metamorphosis and paedomorphosis, respectively. We typed a molecular marker (contig325) linked to met and found that met^Att/met^lab and met^Att/met^wild were associated with metamorphosis in 99% of the cases examined. However, the frequency of paedomorphosis was 4.5 times higher for met^lab/met^lab than for met^wild/met^wild. We also found that met^Att/met^wild and met^wild/met^wild genotypes discriminated distributions of early and late metamorphosing individuals. Two forms of phenotypic variation are contributed by met: continuous variation of metamorphic age and expression of discrete, alternate morphs. We suggest that the evolution of paedomorphosis is associated with genetic changes that delay metamorphic timing in biphasic life cycles.

Like the majority of frogs and toads, many salamanders undergo an obligate metamorphosis that allows for the exploitation of both aquatic and terrestrial habitats during ontogeny. However, some salamander species express an alternate developmental mode in which they forego metamorphosis and remain in the aquatic habitat throughout their lifetimes (Figure 1). Nonmetamorphic forms are termed paedomorphic because they maintain juvenile features of the ancestral condition as they mature reproductively into large, larval forms (GOULD 1977). The exemplar of salamander paedomorphosis is the Mexican axolotl (Ambystoma mexicanum). Ambystoma mexicanum (Am) belongs to a group of several closely related species collectively known as the tiger salamander species complex (SHAFFER and MCKNIGHT 1996). Salamanders of this complex occupy a variety of North American breeding habitats ranging from temporary vernal pools to large permanent lakes. Among these habitats, populations are highly variable for metamorphic timing and expression of paedomorphosis. Some populations express metamorphosis (e.g., A. tigrinum tigrinum, Att) or paedomorphosis like Am, while in other populations both phenotypes are observed at varying frequencies. Presumably, the expression of paedomorphosis is an opportunistic strategy that allows individuals to more successfully colonize relatively permanent aquatic niches (WILBUR and COLLINS 1973; SPRULES 1974). Paedomorphic tiger salamanders are found in newly created habitats like cattle watering troughs and wastewater treatment ponds (ROSE and ARMENTROUT 1975; COLLINS 1981), as well as in stable, large lake systems (SHAFFER 1984).

View larger version (83K): [in this window] [in a new window] [Download PPT slide]   FIGURE 1.— Larval and adult phases of Ambystoma. (A) Larval A. mexicanum. (B) Adult A. t. tigrinum (metamorphic). (C) Adult A. mexicanum (paedomorphic).

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS LITERATURE CITED

The WILD2 backcrosses were created to obtain the largest-ever segregating population for genetic analysis of Ambystoma (SMITH 2002). WILD2 was created using Am individuals collected from Lake Xochimilco to make F₁ hybrids and first generation descendants of wild-caught Am to make backcrosses. The F₁ hybrids were generated from a single cross and backcross offspring were generated using three male Am and four female Att/Am hybrids. A total of nine backcross families compose WILD2 (Table 2). Artificial insemination was used in all crosses (ARMSTRONG and DUHON 1989; VOSS 1995).

Phenotypic scores:
Individuals were scored as metamorphs upon complete resorption of all external gills (gills <1.0 mm in length). Age at metamorphosis was recorded as the number of days from fertilization to completion of metamorphosis. For WILD2, the experiment was terminated on day 350, at which point no individuals had completed metamorphosis within the previous 3 weeks. All remaining individuals showed no sign of having initiated metamorphosis (no apparent regression of the tail fin or external gills) and were scored as paedomorphs.

Genotyping:
A total of 98, 112, and 457 individuals from LAB, WILD1, and WILD2, respectively, were genotyped for contig325, a molecular marker that was isolated as a result of ongoing EST and genetic linkage mapping projects that generate genome resources for Ambystoma research (http://salamander.uky.edu). This marker was isolated from an Am tail regeneration blastema cDNA library (PUTTA et al. 2004). Additional coding sequence for this EST was obtained by 5'-RACE and assembled with existing EST sequences. The resulting 985-bp DNA sequence shows strong similarity to a human nerve growth factor receptor precursor (sequence data not shown; NP_002498, bit score = 164; BLASTX).

A 221-bp DNA fragment corresponding to contig325 was amplified from all individuals under standard PCR conditions (150 ng DNA, 50 ng each primer, 1.2 mM MgCl₂, 0.3 units Taq polymerase, 1x PCR buffer, 200 mM each of dATP, dCTP, dGTP, and dTTP; thermal cycling at 94° for 4 min; 33 cycles of 94° for 45 sec, 60° for 45 sec, 72° for 30 sec; and 72° for 7 min). DNA was isolated from all individuals using a previously described phenol extraction method (VOSS 1993). Primer sequences for amplifying contig325 are forward, 5'-GTGAAGTCAGTGATGAAAGTCCATGT-3', and reverse, 5'-CTAGGATACCAGTGGGAGAGTGTAAT-3'. Genotypes were assayed by restriction digestion of PCR products with a diagnostic AluI restriction enzyme (New England Biolabs, Beverly, MA) and agarose gel electrophoresis.

Linkage analysis:
Linkage and QTL mapping studies were performed using the software package MapMakerQTXb19 (http://www.mapmanager.org/mmQTX.html; MEER et al. 2004). Linkage distance and arrangement among contig325 and previously described amplified fragment length polymorphisms (AFLP) (VOSS and SHAFFER 1997) was estimated using the Kosambi mapping function at a linkage threshold of P = 0.001. The maximum-likelihood position of the met QTL was estimated using the interval mapping function. Significance thresholds for interval mapping were obtained through 10,000 permutations of trait values among backcross progeny. Associations between contig325 genotypes and phenotypic variation were measured using the marker regression function.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS LITERATURE CITED

 
Identification of a highly informative EST marker for met:
The met QTL was originally identified in LAB using AFLPs and interval mapping (VOSS and SHAFFER 1997). In LAB, met^Att/met^lab and met^lab/met^lab segregate as highly penetrant genotypes for metamorphosis and paedomorphosis, respectively; in fact, cosegregation of associated AFLPs and morph phenotypes was statistically consistent with simple Mendelian inheritance. However, given the anonymous nature of AFLPs and the nonspecific way in which these markers are generated, we identified a more informative and user-friendly marker for met: an expressed sequence tag that we refer to as contig325. To show correspondence of contig325 to met, we genotyped individuals from LAB. We observed a higher association between contig325 and met than was observed with the most closely linked AFLP marker (estimated proportion recombinants: AFLP32.17 = 0.15, N = 70; contig325 = 0.07, N = 91). Genotypes at contig325 explain 71% of discrete variation for segregation of metamorphosis vs. paedomorphosis in LAB. Interval mapping shows that contig325 is located near the maximum inflexion point of the previously determined AFLP LOD profile for met (Figure 2).

View larger version (12K): [in this window] [in a new window] [Download PPT slide]   FIGURE 2.— Likelihood-ratio statistic (LRS) plot for association of paedomorphosis with genetic factors in the met QTL region. The LRS for the contig325 marker is shown. Horizontal shaded lines represent LRS thresholds for suggestive (1.3), significant (6.6), and highly significant associations (15.6) as estimated using Map Manager QTXb19.

Segregation of discrete developmental modes and contig325 in WILD2 and WILD1:
We observed the segregation of metamorphs and paedomorphs in all nine WILD2 crosses (Table 2). Segregation ratios were not significantly heterogeneous (G = 13.32, d.f. = 8, P = 0.10) among crosses; therefore segregation ratios were pooled for hypothesis testing. The majority of offspring generated (453 of 497) metamorphosed before day 350. In total, only 44 (9%) of the offspring exhibited paedomorphosis and ratios were significantly different from the simple Mendelian expectation of 1:1 (G = 392, d.f. = 1, P = 4 x 10^–87, N = 497). Significantly lower-than-expected numbers of paedomorphs (19%) were also observed in WILD1 (VOSS and SHAFFER 2000). Thus, results from WILD1 and WILD2 indicate that the proportion of paedomorphs is significantly lower in backcrosses using wild-caught Am, relative to laboratory Am.

To determine if met contributed to the segregation of discrete developmental modes in WILD2, we genotyped all individuals for contig325 (325) (Table 3). Inheritance of 325^Att/325^Am, and thus presumably of met^Att/met^wild2 (Table 1), yielded the expected metamorphic phenotype in >99% of the cases. The 325^Am/325^Am genotype (presumably marking met^wild2/met^wild2 ) was not as penetrant for the paedomorphic phenotype as only 17% of individuals in this genotypic class were paedomorphs. However, inheritance of met^wild2/met^wild2 is apparently necessary for expression of paedomorphosis as only one paedomorph inherited a met^Att/met^wild2 genotype. To investigate linkage results between WILD2 and WILD1, which had previously been examined using only the informative AFLP makers (VOSS and SHAFFER 2000), we genotyped individuals from WILD1 for contig325. The pattern of segregation for contig325 in WILD1 was the same as that observed for WILD2. All but one individual that inherited 325^Att/325^Am was metamorphic and 325^Am/325^Am yielded incomplete penetrance for paedomorphosis (only 16 of 51 325^Am/325^Am genotypes were paedomorphic). Observation of the same pattern of segregation between WILD1 and WILD2 suggests no sex linkage or maternal effect on the segregation of genotypes and phenotypes because the crossing designs were reversed to create WILD1 and WILD2 backcrosses (i.e., F₁ hybrids were male in creating WILD1 but female in creating WILD2). Overall, our results show that 325^Att/325^Am is strongly associated with the metamorphic phenotype; this association did not vary across LAB, WILD1, or WILD2. However, the proportion of 325^Am/325^Am genotypes that were associated with paedomorphosis was 4.5 times higher in LAB than in WILD1 and WILD2. This indicates a genetic difference in the basis of paedomorphosis between the natural and domestic strains of Am.

View larger version (24K): [in this window] [in a new window] [Download PPT slide]   FIGURE 3.— Distribution of ages at metamorphosis for WILD2 plotted separately for contig325 genotypes.

View larger version (17K): [in this window] [in a new window] [Download PPT slide]   FIGURE 4.— Distribution of ages at metamorphosis for WILD1 plotted separately for contig325 genotypes.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS LITERATURE CITED

Genetic basis of discrete variation: expression of metamorphosis vs. paedomorphosis:
A conceptual framework for understanding how polygenes give rise to discrete phenotypic variation is the threshold model (FALCONER 1989). Under this model, the expression of alternate phenotypes depends upon an individual's liability value relative to a threshold value, with liability values above and below the threshold yielding alternate phenotypes. We suggest that contig325 makes a major contribution to the liability or threshold underlying the expression of metamorphosis vs. paedomorphosis. Within LAB, both 325^Att/325^Am and 325^Am/325^Am were highly predictive of their expected phenotypes, indicating highly significant linkage to a single locus (met) (² = 84.97, d.f. = 1, N = 98, P < 0.001; Table 3). Thus, in the LAB genetic background, the threshold for expressing metamorphosis vs. paedomorphosis is traversed by the segregation of alternate met genotypes at a single locus. Apparently, 325^Att/325^Am is not sensitive to genetic background because this genotype was also highly predictive of metamorphosis in WILD1 and WILD2. Thus, in both LAB and WILD genetic backgrounds, substitution of a single Am met allele with a dominant Att met allele rescued the metamorphic phenotype in essentially all cases.

In contrast to 325^Att/325^Am, the penetrance of 325^Am/325^Am for paedomorphosis varied between LAB and the WILD backcrosses. This suggests that met^lab and met^wild1,2 contribute differently to the underlying genetic architecture or that LAB and WILD genetic backgrounds influence the probability of paedomorphosis differently. Although we cannot differentiate between these two possibilities, the genetic basis of paedomorphosis clearly differs between the natural population and a recently derived laboratory strain of Am, thus indicating the potential for rapid evolution of genetic architecture. This supports the idea that the simple Mendelian basis of paedomorphosis in LAB evolved recently during the domestication of Am (VOSS and SHAFFER 2000; see also MALACINSKI 1978). Although paedomorphosis is expressed by both the wild strain and the laboratory strain, our results indicate that selection has canalized expression of paedomorphosis to a greater degree in the laboratory strain, as assayed by our interspecific crossing design. Thus, although paedomorphosis has been cited as a classic example of heterochrony by a major gene effect, our study shows that factors beyond a single major gene are important in discrete trait expression in Am.

Genetic basis of continuous variation: variation in metamorphic age:
Because the WILD backcrosses yielded a large number of metamorphosing offspring reared under identical conditions, we were able to estimate the contribution of met to variation in metamorphic age. We found that metamorphic age varied significantly between 325^Am/325^Am and 325^Att/325^Am genotypic classes. This indicates that met^wild/met^wild delays timing of metamorphosis relative to met^Att/met^wild. Because met^wild/met^wild was associated with paedomorphosis in WILD1,2 (all but two paedomorphs were met^wild/met^wild), our results show that both delayed metamorphosis and expression of paedomorphosis are associated with this genotype; we note that these associations were observed in the same genetic background. Conversely, an earlier metamorphosis was associated with the alternate met^Att/met^wild genotype, again within the same WILD genetic backgrounds. This indicates that met alleles deriving from paedomorphic Am delay metamorphosis while met alleles from the metamorphic Att decrease the time to metamorphosis. We suggest that metamorphic age is a continuous variable that is closely associated with the underlying liability or threshold that determines the expression of alternate developmental modes. It is possible that met influences metamorphic timing via changes in the timing of the sensitive period for hormonal initiation of metamorphosis, as has been suggested for dung beetles (Onthophagus taurus) that express alternate male morphs (MOCZEK and NIJHOUT 2002). A comparative mapping project is underway to identify likely candidate genes in the vicinity of contig325 (http://salamander.uky.edu).

Evolutionary maintenance of the biphasic life cycle and evolution of paedomorphosis:
Our results suggest that two distinct evolutionary processes—(1) adaptation of biphasic life cycles through selection of metamorphic timing (VOSS et al. 2003) and (2) evolution of novel paedomorphic developmental modes that isolate lineages and promote speciation (SHAFFER 1984)—are apparently linked by a common genetic architecture. Selection for met alleles that increase or decrease age at metamorphosis is expected to allow the evolution of a continuum of metamorphic timing phenotypes. Because met did not account for all of the variation in metamorphic timing in WILD2, it is likely that other loci make a contribution to continuous variation (VOSS et al. 2003). The average difference in metamorphic age that we observed between met genotypic classes was 36 days. This amount of variation may significantly affect larval survivorship in natural populations that use unpredictable, ephemeral ponds (WILBUR and COLLINS 1973). In more predictable ephemeral ponds, selection is expected to favor alleles that delay metamorphic timing because larvae that attain larger body sizes have increased survival probabilities after metamorphosis (SEMLITSCH et al. 1988). In our study, inheritance of the same met genotype was associated with delayed metamorphosis and expression of paedomorphosis. Because both of these life history strategies would be favored in a stable aquatic habitat, we propose that the evolution of paedomorphosis in Am occurred gradually via selection for delayed metamorphic timing. Overall, our results provide a framework for understanding how metamorphic timing and paedomorphic phenotypes can evolve to be fixed or variable within and between species and thus how microevolutionary processes lead to macroevolutionary patterns.

	ACKNOWLEDGEMENTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS LITERATURE CITED

 
We thank the Axolotl Colony for animal resources and historical information and two anonymous reviewers for their comments. The work was supported by the U.S. National Science Foundation EPSCoR's (Experimental Program to Stimulate Competitive Research) support of a functional genomics initiative at the University of Kentucky and grants to S.R.V. by the U.S. National Science Foundation (IBN-0242833; IBN-0080112) and the National Institutes of Health (5 R24 RR16344-03).

	FOOTNOTES

 
We dedicate our work to the memory of Virginia Graue, without whose efforts in conserving natural axolotls we would have not gained the evolutionary insights reported in this article.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS LITERATURE CITED

 

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This Article Abstract Full Text (PDF) All Versions of this Article: genetics.104.038273v1 170/1/275    most recent Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Voss, S. R. Articles by Smith, J. J. Search for Related Content PubMed PubMed Citation Articles by Voss, S. R. Articles by Smith, J. J.