Evolutionary genetics of metamorphic failure using wild-caught vs. laboratory axolotls (Ambystoma mexicanum)

In many organisms metamorphosis allows for an ecologically important habitat-shift from water to land. However, in some salamanders an adaptive life cycle mode has evolved that is characterized by metamorphic failure (paedomorphosis); these species remain in the aquatic habitat throughout the life cycle. Perhaps the most famous example of metamorphic failure is the Mexican axolotl (Ambystoma mexicanum), which has become a focal species for developmental biology since it was introduced into laboratory culture in the 1800s. Our previous genetic linkage mapping analysis, using an interspecific crossing design, demonstrated that a major gene effect underlies the expression of metamorphic failure in laboratory stocks of the Mexican axolotl. Here, we repeated this experiment using A. mexicanum that were sampled directly from their natural habitat at Lake Xochimilco, Mexico. We found no significant association between the major gene and metamorphic failure when wild-caught axolotls were used in the experimental design, although there is evidence of a smaller genetic effect. Thus, there appears to be genetic variation among Mexican axolotls (and possibly A. tigrinum tigrinum) at loci that contribute to metamorphic failure. This result suggests a role for more than one mutation and possibly artificial selection in the evolution of the major gene effect in the laboratory Mexican axolotl.     

Larval growth in polyphenic salamanders: making the best of a bad lot

Polyphenisms are excellent models for studying phenotypic variation, yet few studies have focused on natural populations. Facultative paedomorphosis is a polyphenism in which salamanders either metamorphose or retain their larval morphology and eventually become paedomorphic. Paedomorphosis can result from selection for capitalizing on favorable aquatic habitats (paedomorph advantage), but could also be a default strategy under poor aquatic conditions (best of a bad lot). We tested these alternatives by quantifying how the developmental environment influences the ontogeny of wild Arizona tiger salamanders (Ambystoma tigrinum nebulosum). Most paedomorphs in our study population arose from slow-growing larvae that developed under high density and size-structured conditions (best of a bad lot), although a few faster-growing larvae also became paedomorphic (paedomorph advantage). Males were more likely to become paedomorphs than females and did so under a greater range of body sizes than females, signifying a critical role for gender in this polyphenism. Our results emphasize that the same phenotype can be adaptive under different environmental and genetic contexts and that studies of phenotypic variation should consider multiple mechanisms of morph production.     

EVOLUTION OF PAEDOMORPHOSIS IN PLETHODONTID SALAMANDERS: ECOLOGICAL CORRELATES AND RE‐EVOLUTION OF METAMORPHOSIS

Life‐history modes can profoundly impact the biology of a species, and a classic example is the dichotomy between metamorphic (biphasic) and paedomorphic (permanently aquatic) life‐history strategies in salamanders. However, despite centuries of research on this system, several basic questions about the evolution of paedomorphosis in salamanders have not been addressed. Here, we use a nearly comprehensive, time‐calibrated phylogeny of spelerpine plethodontids to reconstruct the evolution of paedomorphosis and to test if paedomorphosis is (1) reversible; (2) associated with living in caves; (3) associated with relatively dry climatic conditions on the surface; and (4) correlated with limited range size and geographic dispersal. We find that paedomorphosis arose multiple times in spelerpines. We also find evidence for re‐evolution of metamorphosis after several million years of paedomorphosis in a lineage of Eurycea from the Edwards Plateau region of Texas. We also show for the first time using phylogenetic comparative methods that paedomorphosis is highly correlated with cave‐dwelling, arid surface environments, and small geographic range sizes, providing insights into both the causes and consequences of this major life history transition.     

Thyroid hormone responsive QTL and the evolution of paedomorphic salamanders

The transformation of ancestral phenotypes into novel traits is poorly understood for many examples of evolutionary novelty. Ancestrally, salamanders have a biphasic life cycle with an aquatic larval stage, a brief and pronounced metamorphosis, followed by a terrestrial adult stage. Repeatedly during evolution, metamorphic timing has been delayed to exploit growth-permissive environments, resulting in paedomorphic salamanders that retain larval traits as adults. We used thyroid hormone (TH) to rescue metamorphic phenotypes in paedomorphic salamanders and then identified quantitative trait loci (QTL) for life history traits that are associated with amphibian life cycle evolution: metamorphic timing and adult body size. We demonstrate that paedomorphic tiger salamanders (Ambystoma tigrinum complex) carry alleles at three moderate effect QTL (met1–3) that vary in responsiveness to TH and additively affect metamorphic timing. Salamanders that delay metamorphosis attain significantly larger body sizes as adults and met2 explains a significant portion of this variation. Thus, substitution of alleles at TH-responsive loci suggests an adaptive pleiotropic basis for two key life-history traits in amphibians: body size and metamorphic timing. Our study demonstrates a likely pathway for the evolution of novel paedomorphic species from metamorphic ancestors via selection of TH-response alleles that delay metamorphic timing and increase adult body size.     

Candidate gene analysis of metamorphic timing in ambystomatid salamanders

Although much is known about the ecological significance of metamorphosis and metamorphic timing, few studies have examined the underlying genetic architecture of these traits, and no study has attempted to associate phenotypic variation to molecular variation in specific genes. Here we report on a candidate gene approach (CGA) to test specific loci for a statistical contribution to variation in metamorphic timing. Three segregating populations (SP1, SP2 and SP3) were constructed utilizing three species of paedomorphic Mexican ambystomatid salamander, including the axolotl, Ambystoma mexicanum. We used these replicated species to test the hypothesis that inheritance of alternate genotypes at two thyroid hormone receptor loci (TRalpha, TRbeta) affects metamorphic timing in ambystomatid salamanders. A significant TRalpha*SP effect indicated that variation in metamorphic timing may be influenced by TRalpha genotype, however, the effect was not a simple one, as both the magnitude and direction of the phenotypic effect depended upon the genetic background. These are the first data to implicate a specific gene in contributing to variation in metamorphic timing. In general, candidate gene approaches can be extended to any number of loci and to any organism where simple genetic crosses can be performed to create segregating populations. The approach is thus of particular value in ecological studies where target genes have been identified but the study organism is not one of the few well-characterized model systems that dominate genetic research.     

Adenohypophysial-thyroid activity of the tiger salamander, Ambystoma tigrinum, as a function of metamorphosis and captivity

The aim of this study was to determine the timing of adenohypophysial activation during metamorphosis of the tiger salamander, Ambystoma tigrinum. It consisted of two parts: 1) determination of plasma thyroid hormone concentrations and analysis of thyroid gland histology as a function of metamorphic stage and 2) analysis of the time-course of uptake of 125I by the thyroids during metamorphosis as an indicator of endogenous thyrotropin (TSH) levels. Significant increases in both triiodothyronine (T3) and thyroxine (T4) first were evident at the onset of metamorphic climax (stage II). Maximum levels of both hormones were not observed, however, until the completion of gill resorption (stage VII). No changes in thyroid histology were observed that could be unambiguously related to metamorphic transformation. The thyroids accumulated 125I in a slow but linear fashion in premetamorphic larvae (stage I). However, uptake exhibited a rapid peak during early climax (stage II), before maximum concentrations of thyroid hormones were observed. In addition, uptake was maintained above premetamorphic levels at stage VII, in conjunction with maximum levels of T4 and T3. Captivity alone produced a small but significant increase in plasma concentrations of T3. It produced no significant effect on either thyroid histology or uptake of 125I. These results indicate that adenohypophysial activation occurs rapidly and is maximal at the onset of metamorphic climax.     

Considering alternative life history modes and genetic divergence in conservation: a case study of the Oklahoma salamander

Alternative life history strategies can provide important variation for the long-term persistence of a lineage. However, conservation of such lineages can be complicated because each life history mode may have different habitat requirements and may be vulnerable to different environmental perturbations. The Oklahoma salamander (Eurycea tynerensis) is endemic to the Ozark Plateau of North America, and has two discrete life history modes, biphasic (metamorphic) and aquatic (paedomorphic). Until recently, these modes were considered separate species and conservation attention focused only on paedomorphic populations. We perform phylogenetic analyses of the mitochondrial gene cytochrome b (Cytb) and nuclear gene proopiomelanocortin (POMC) to assess patterns of historical isolation in E. tynerensis, and test whether life history mode is randomly distributed with respect to the phylogeny and geography. We find three divergent Cytb lineages and significant shifts in POMC allele frequencies between the eastern, western, and southwestern portions of the distribution. Life history mode varies extensively, but paedomorphosis is largely restricted to the widespread western clade. Therefore, the two most divergent and narrowly distributed clades (southwestern and eastern) were previously overlooked due to their metamorphic life history. Paedomorphosis has allowed E. tynerensis to drastically increase its niche breadth and distribution size. Nevertheless, metamorphosis is also an important attribute, and metamorphic populations are the ultimate source for paedomorphic evolution. Preservation of divergent genetic lineages, and regions that include adjacent habitat for both life history modes, may be the most effective way to maintain historical and adaptive variation and provide gateways for ongoing life history evolution.     

Metamorphosis of the feeding mechanism in tiger salamanders (Ambystoma tigrinum): the ontogeny of cranial muscle mass

Most previous research on metamorphosis of the musculoskeletal system in vertebrates has focused on the transformation of the skeleton. In this paper we focus on the transformation of the muscles of the head during metamorphosis in tiger salamanders ( Ambystoma tigrinum ) in order (1) to provide new data on changes in myology during ontogeny, and (2) to aid in interpreting previous data on the metamorphosis of function in the head of salamanders.
The physiological cross-sectional area of nine head muscles was calculated by measuring fibre angles, fibre lengths, and muscle mass in two samples of tiger salamanders obtained just before and just after metamorphosis. The major mouth-opening muscles (rectus cervicis and depressor mandibulae) exhibit a significant decrease in estimated maximum tetanic tension (MTT) across metamorphosis of about 36%. The jaw-closing muscles (adductor mandibulae internus and externus) and the head-lifting muscles (epaxials) also decrease in MTT but not significantly. The muscles associated with tongue projection during feeding on land (the subarcualis rectus I, geniohyoideus, interhyoideus and intermandibularis) all show a slight increase in MTT at metamorphosis.
Metamorphic transformation of feeding behaviour in Ambystoma tigrinum involves changes in performance, the design of skeletal elements, changes in muscle force-generating capability, and changes in hydrodynamic design from unidirectional flow in larvae to bidirectional flow during aquatic feeding after metamorphosis. Although muscle activity patterns during aquatic feeding do not change across metamorphosis, tongue-based terrestrial feeding involves a suite of novel muscle activity patterns, morphological characters acquired at metamorphosis, and a metamorphic increase in the masses of muscles important in tongue projection.     

Morph switching in a dimorphic population of <Emphasis Type="Italic">Triturus alpestris</Emphasis> (Amphibia,Caudata)

The usual life cycle of Alpine newts comprises an aquatic larval stage and a terrestrial juvenile and adult stage. However, some populations differ from this pattern in exhibiting facultative paedomorphosis where some individuals reach sexual maturity while retaining larval traits such as gills and gill slits. While paedomorphic newts can, in some circumstances, initiate metamorphosis, once a newt has commenced metamorphosis, the state is irreversible. Because the frequency of this switching from one morph to the other has never been quantified in the wild, we attempted to estimate switching rate and survival by carrying out a 3-year monitoring survey of a population inhabiting an alpine lake. While morph switching did occur in this population, it involved a relatively low proportion of the paedomorphs (approx. 12%), suggesting that metamorphosis is not favoured in the study population. The hypothesis of paedomorphic advantage was not supported since neither survival nor body condition differed between morphs. The ontogenetic pathway of wild Alpine newts is thus characterised by two forks in the developmental pathway. The first occurs during the larval stage (metamorphosis vs. paedomorphosis), and the second occurs in paedomorphic adults (switching for metamorphosis vs. continuation of the paedomorphic lifestyle). Such a two-level decision process may allow individuals to cope with environmental uncertainty.     

Carry‐over Effects of Size at Metamorphosis in Red‐eyed Treefrogs: Higher Survival but Slower Growth of Larger Metamorphs

Most animals have complex life histories, composed of a series of ecologically distinct stages, and the transitions between stages are often plastic. Anurans are models for research on complex life cycles. Many species exhibit plastic timing of and size at metamorphosis, due to both environmental constraints on larval growth and development and adaptive plastic responses to environmental variation. Models predicting optimal timing of metamorphosis balance cost/benefit ratios across stages, assuming that size affects growth and mortality rates in each stage. Much research has documented such effects in the larval period, but we lack an equal understanding of juvenile growth and mortality. Here, we examine how variation in size at metamorphosis in the Neotropical red‐eyed treefrog, Agalychnis callidryas, affects post‐metamorphic growth, foraging, and behavior in the lab as well as growth and survival in the field. Surprisingly, many individuals lost mass for weeks after metamorphosis. In the lab, larger metamorphs lost more mass following metamorphosis, ate similar amounts, had lower food conversion efficiencies, and grew more slowly after mass loss ceased than did smaller ones. In field cages larger metamorphs were more likely to survive than smaller ones; just one froglet died in the lab. Our data suggest that size‐specific differences in physiology and behavior influence these trends. Comparing across species and studies, large size at metamorphosis generally confers higher survival; size effects on growth rates vary substantially among species, in both magnitude and direction, but may be stronger in the tropics.     

A comprehensive expressed sequence tag linkage map for tiger salamander and Mexican axolotl: enabling gene mapping and comparative genomics in Ambystoma

Expressed sequence tag (EST) markers were developed for Ambystoma tigrinum tigrinum (Eastern tiger salamander) and for A. mexicanum (Mexican axolotl) to generate the first comprehensive linkage map for these model amphibians. We identified 14 large linkage groups (125.5-836.7 cM) that presumably correspond to the 14 haploid chromosomes in the Ambystoma genome. The extent of genome coverage for these linkage groups is apparently high because the total map size (5251 cM) falls within the range of theoretical estimates and is consistent with independent empirical estimates. Unlike most vertebrate species, linkage map size in Ambystoma is not strongly correlated with chromosome arm number. Presumably, the large physical genome size ( approximately 30 Gbp) is a major determinant of map size in Ambystoma. To demonstrate the utility of this resource, we mapped the position of two historically significant A. mexicanum mutants, white and melanoid, and also met, a quantitative trait locus (QTL) that contributes to variation in metamorphic timing. This new collection of EST-based PCR markers will better enable the Ambystoma system by facilitating development of new molecular probes, and the linkage map will allow comparative studies of this important vertebrate group.     

The evolution of sex-independent transmission ratio distortion involving multiple allelic interactions at a single locus in rice

Transmission ratio distortion (TRD) is frequently observed in inter- and intraspecific hybrids of plants, leading to a violation of Mendelian inheritance. Sex-independent TRD (siTRD) was detected in a hybrid between Asian cultivated rice and its wild ancestor. Here we examined how siTRD caused by an allelic interaction at a specific locus arose in Asian rice species. The siTRD is controlled by the S(6) locus via a mechanism in which the S(6) allele acts as a gamete eliminator, and both the male and female gametes possessing the opposite allele (S(6)(a)) are aborted only in heterozygotes (S(6)/S(6)(a)). Fine mapping revealed that the S(6) locus is located near the centromere of chromosome 6. Testcross experiments using near-isogenic lines (NILs) carrying either the S(6) or S(6)(a) alleles revealed that Asian rice strains frequently harbor an additional allele (S(6)(n)) the presence of which, in heterozygotic states (S(6)/S(6)(n) and S(6)(a)/S(6)(n)), does not result in siTRD. A prominent reduction in the nucleotide diversity of S(6) or S(6)(a) carriers relative to that of S(6)(n) carriers was detected in the chromosomal region. These results suggest that the two incompatible alleles (S(6) and S(6)(a)) arose independently from S(6)(n) and established genetically discontinuous relationships between limited constituents of the Asian rice population.     

Are direct density cues,not resource competition,driving life history trajectories in a polyphenic salamander?

Polyphenisms, where multiple, discrete, environmentally-cued phenotypes can arise from a single genotype, are extreme forms of phenotypic plasticity. Cue acquisition and interpretation are vital for matching phenotypes to varying environments, but can be difficult if cues are unreliable indicators or if multiple cues are present simultaneously. Facultative paedomorphosis, where juvenile traits are retained at sexual maturity, is a density-dependent polyphenism exhibited by many salamanders. Favorable conditions such as low larval densities and stable hydroperiod delay metamorphosis and promote a paedomorphic strategy. We investigated proximate cues affecting facultative paedomorphosis in order to understand how larval newts (Notophthalmus viridescens louisianensis) assess conspecific density. To isolate the effects of density cues from the effects of resources and agonistic behavior, we caged larval newts in mesocosms in a 2?×?2 factorial design that manipulated both background larval newt densities (high or low) and food levels (ambient or supplemented). We found strong effects of both food and density on caged individuals. Under high densities, caged larvae were more likely to become efts, a long-lasting juvenile terrestrial stage, across both food levels, while paedomorphs were more common under low densities. Though food levels increased growth rates, density had strong independent effects on metamorphic timing and phenotype. Competition for food and space are classical density-dependent processes, but density cues themselves may be a mediator of density-dependent effects on polyphenisms and life history responses.     

Differential performance among LDH-B genotypes in Rana lessonae tadpoles

The European pool frog, Rana lessonae, is widely polymorphic for two common alleles (b,e) at the lactate dehydrogenase-B (LDH-B) locus. We compared fitness-related larval life-history traits among LDH-B genotypes, which originated from segregation in heterozygous parents, in an artificial pond experiment where tadpoles of R. lessonae from a Swiss population were raised together with tadpoles of the hemiclonal hybrid R. esculenta at two densities. In R. lessonae, LDH-B e/e homozygotes at each density had a higher proportion of metamorphs among survivors, reached metamorphosis earlier, and were heavier at metamorphosis than b/b homozygotes; b/e heterozygotes had intermediate values. That e/e individuals were superior to b/b in both time to and mass at metamorphosis is surprising because these two life-history traits are thought to reflect a performance trade-off; e/e genotypes apparently compensated for shorter time to metamorphosis by a higher growth rate. The two alleles showed the same performance ranking when combined in hybrids with a R. ridibunda allele: When R. esculenta from Swiss populations reared in the same ponds had received the e allele rather than the b allele from their R. lessonae parent, they reached metamorphosis earlier, but did not differ in mass at metamorphosis. The degree of linkage disequilibrium in the source population of the eight R. lessonae used as parents of the R. lessonae tadpoles is unknown, so we cannot exclude the possibility that the performance differences are caused by some anonymous tightly linked gene, rather than the LDH-B locus, that constitutes the genomically localized target of natural selection. A causal involvement of LDH-B is plausible, nevertheless, because this enzyme takes part in the central energy-metabolizing processes and has been reported to underlie fitness differences in other animals; also, differential performance of LDH-B genotypes has been observed in R. lessonae larvae from another population. The present results suggest strong directional selection for allele e; the sum of available data, including an independent laboratory experiment, suggests that partial environment-dependent overdominance combined with balancing selection favoring e/e homozygotes under some and b/b homozygotes under other conditions may be partially responsible for the broad maintenance of the LDH-B polymorphism in R. lessonae.     

Tissue-specific profile of DNA replication in the swimming larvae of Ciona intestinalis

The cell cycle is strictly regulated during development and its regulation is essential for organ formation and developmental timing. Here we observed the pattern of DNA replication in swimming larvae of an ascidian, Ciona intestinalis. Usually, Ciona swimming larvae obtain competence for metamorphosis at about 4-5 h after hatching, and these competent larvae initiate metamorphosis soon after they adhere to substrate with their papillae. In these larvae, three major tissues (epidermis, endoderm and mesenchyme) showed extensive DNA replication with distinct pattern and timing, suggesting tissue-specific cell cycle regulation. However, DNA replication did not continue in aged larvae which kept swimming for several days, suggesting that the cell cycle is arrested in these larvae at a certain time to prevent further growth of adult organ rudiments until the initiation of metamorphosis. Inhibition of the cell cycle by aphidicolin during the larval stage affects only the speed of metamorphosis, and not the formation of adult organ rudiments or the timing of the initiation of metamorphosis. However, after the completion of tail resorption, DNA replication is necessary for further metamorphic events. Our data showed that DNA synthesis in the larval trunk is not directly associated with the organization of adult organs, but it contributes to the speed of metamorphosis after settlement.     

Why grow up? A perspective on insect strategies to avoid metamorphosis

1. Insects with complete metamorphosis (holometaboly) are extremely successful, constituting over 60% of all described animal species. Complete metamorphosis confers significant advantages because it enables organisms to optimise life‐history components through temporal partitioning, and thereby to exploit multiple ecological niches. Yet holometaboly can also impose costs, and several lineages have evolved life cycle modifications to avoid complete metamorphosis. 2. In this review, we discuss different strategies that have evolved that result in the loss of complete metamorphosis (type I and type II paedomorphosis). In addition, the ecological pressures and developmental modifications that facilitate this avoidance are considered, as well as the importance of life cycle complexity in life‐history evolution. 3. Interestingly, only female holometabolous insects have entirely avoided complete metamorphosis, and it is always the ancestrally juvenile morphology that is retained. These findings point to a strong sex‐biased trade‐off between investment in reproduction and development. While the loss of complete metamorphosis in females has occurred independently on several occasions across holometabolous insects, only a small number of species possessing this ability have been described. 4. Thus, complete metamorphosis, which originated only once in insects, appears to have been almost fully retained. This indicates that significant modifications to the holometabolan metamorphic ground plan are highly constrained, and suggests that the transition to complete metamorphosis is evolutionarily irreversible.     

Hybridization between crops and wild relatives: the contribution of cultivated lettuce to the vigour of crop–wild hybrids under drought, salinity and nutrient deficiency conditions

With the development of transgenic crop varieties, crop-wild hybridization has received considerable consideration with regard to the potential of transgenes to be transferred to wild species. Although many studies have shown that crops can hybridize with their wild relatives and that the resulting hybrids may show improved fitness over the wild parents, little is still known on the genetic contribution of the crop parent to the performance of the hybrids. In this study, we investigated the vigour of lettuce hybrids using 98 F(2:3) families from a cross between cultivated lettuce and its wild relative Lactuca serriola under non-stress conditions and under drought, salinity and nutrient deficiency. Using single nucleotide polymorphism markers, we mapped quantitative trait loci associated with plant vigour in the F(2:3) families and determined the allelic contribution of the two parents. Seventeen QTLs (quantitative trait loci) associated with vigour and six QTLs associated with the accumulation of ions (Na(+), Cl(-) and K(+)) were mapped on the nine linkage groups of lettuce. Seven of the vigour QTLs had a positive effect from the crop allele and six had a positive effect from the wild allele across treatments, and four QTLs had a positive effect from the crop allele in one treatment and from the wild allele in another treatment. Based on the allelic effect of the QTLs and their location on the genetic map, we could suggest genomic locations where transgene integration should be avoided when aiming at the mitigation of its persistence once crop-wild hybridization takes place.     

ARTIFICIAL SELECTION FOR PAEDOMORPHOSIS IN THE SALAMANDER AMBYSTOMA TALPOIDEUM

Heterochronic ontogenetic mechanisms such as paedomorphosis are potentially important mechanisms of both microevolutionary and macroevolutionary change. The salamander Ambystoma talpoideum is facultatively paedomorphic. Expression of paedomorphosis in this species varies among local natural populations. Two breeding lines, one from a population associated with a temporary pond where metamorphosis to a terrestrial adult always occurs, another from a population associated with a nearly permanent pond where paedomorphosis is common, were selected artificially for paedomorphosis over four generations. The F₅ generation of each breeding line was reared in a “common garden” field experiment under two drying regimes to simulate the larval environment in a temporary and in a permanent pond. There was a significantly different response to the drying regimes and to the artificial selection in the two lines. A significant population × selection interaction indicated that the two populations responded differently to artificial selection for paedomorphosis. The presence of heritable genetic variation suggests that evolution and divergence among populations of salamanders is possible with intense natural selection over short periods of ecological time.     

Ontogeny discombobulates phylogeny: paedomorphosis and higher-level salamander relationships

Evolutionary developmental biology ("evo-devo") has revolutionized evolutionary biology but has had relatively little impact on systematics. We show that similar large-scale developmental changes in distantly related lineages can dramatically mislead phylogenetic analyses based on morphological data. Salamanders are important model systems in many fields of biology and are of special interest in that many species are paedomorphic and thus never complete metamorphosis. A recent study of higher-level salamander phylogeny placed most paedomorphic families in a single clade based on morphological data. Here, we use new molecular and morphological data to show that this result most likely was caused by the misleading effects of paedomorphosis. We also provide a well-supported estimate of higher-level salamander relationships based on combined molecular and morphological data. Many authors have suggested that paedomorphosis may be problematic in studies of salamander phylogeny, but this hypothesis has never been tested with a rigorous phylogenetic analysis. We find that the misleading effects of paedomorphosis on phylogenetic analysis go beyond the sharing of homoplastic larval traits by paedomorphic adults, and the problem therefore is not solved by simply excluding suspected paedomorphic characters. Instead, two additional factors are critically important in causing paedomorphic species to be phylogenetically "misplaced": (1) the absence of clade-specific synapomorphies that develop during metamorphosis in nonpaedomorphic taxa and allow their "correct" placement and (2) parallel adaptive changes associated with the aquatic habitat of the larval stage. Our results suggest that the effects of paedomorphosis on phylogenetic analyses may be complex, difficult to detect, and can lead to results that are both wrong and statistically well supported by parsimony and Bayesian analyses.