Culture Isolation and Culture-Independent Clone Libraries Reveal New Marine Synechococcus Ecotypes with Distinctive Light and N Physiologies

Marine microbial communities often contain multiple closely related phylogenetic clades, but in many cases, it is still unclear what physiological traits differentiate these putative ecotypes. The numerically abundant marine cyanobacterium Synechococcus can be divided into at least 14 clades. In order to better understand ecotype differentiation in this genus, we assessed the diversity of a Synechococcus community from a well-mixed water column in the Sargasso Sea during March 2002, a time of year when this genus typically reaches its annual peak in abundance. Diversity was estimated from water sampled at three depths (approximately 5, 70, and 170 m) using both culture isolation and construction of cyanobacterial 16S-23S rRNA internal transcribed sequence clone libraries. Clonal isolates were obtained by enrichment with ammonium, nitrite, or nitrate as the sole N source, followed by pour plating. Each method sampled the in situ diversity differently. The combined methods revealed a total of seven Synechococcus phylotypes including two new putative ecotypes, labeled XV and XVI. Although most other isolates grow on nitrate, clade XV exhibited a reduced efficiency in nitrate utilization, and both clade XV and XVI are capable of chromatic adaptation, demonstrating that this trait is more widely distributed among Synechococcus strains than previously known. Thus, as in its sister genus Prochlorococcus, light and nitrogen utilization are important factors in ecotype differentiation in the marine Synechococcus lineage.     

Resolution of Prochlorococcus and Synechococcus ecotypes by using 16S-23S ribosomal DNA internal transcribed spacer sequences

Cultured isolates of the marine cyanobacteria Prochlorococcus and Synechococcus vary widely in their pigment compositions and growth responses to light and nutrients, yet show greater than 96% identity in their 16S ribosomal DNA (rDNA) sequences. In order to better define the genetic variation that accompanies their physiological diversity, sequences for the 16S-23S rDNA internal transcribed spacer (ITS) region were determined in 32 Prochlorococcus isolates and 25 Synechococcus isolates from around the globe. Each strain examined yielded one ITS sequence that contained two tRNA genes. Dramatic variations in the length and G+C content of the spacer were observed among the strains, particularly among Prochlorococcus strains. Secondary-structure models of the ITS were predicted in order to facilitate alignment of the sequences for phylogenetic analyses. The previously observed division of Prochlorococcus into two ecotypes (called high and low-B/A after their differences in chlorophyll content) were supported, as was the subdivision of the high-B/A ecotype into four genetically distinct clades. ITS-based phylogenies partitioned marine cluster A Synechococcus into six clades, three of which can be associated with a particular phenotype (motility, chromatic adaptation, and lack of phycourobilin). The pattern of sequence divergence within and between clades is suggestive of a mode of evolution driven by adaptive sweeps and implies that each clade represents an ecologically distinct population. Furthermore, many of the clades consist of strains isolated from disparate regions of the world's oceans, implying that they are geographically widely distributed. These results provide further evidence that natural populations of Prochlorococcus and Synechococcus consist of multiple coexisting ecotypes, genetically closely related but physiologically distinct, which may vary in relative abundance with changing environmental conditions.     

Clade-specific 16S ribosomal DNA oligonucleotides reveal the predominance of a single marine Synechococcus clade throughout a stratified water column in the Red Sea

Phylogenetic relationships among members of the marine Synechococcus genus were determined following sequencing of the 16S ribosomal DNA (rDNA) from 31 novel cultured isolates from the Red Sea and several other oceanic environments. This revealed a large genetic diversity within the marine Synechococcus cluster consistent with earlier work but also identified three novel clades not previously recognized. Phylogenetic analyses showed one clade, containing halotolerant isolates lacking phycoerythrin (PE) and including strains capable, or not, of utilizing nitrate as the sole N source, which clustered within the MC-A (Synechococcus subcluster 5.1) lineage. Two copies of the 16S rRNA gene are present in marine Synechococcus genomes, and cloning and sequencing of these copies from Synechococcus sp. strain WH 7803 and genomic information from Synechococcus sp. strain WH 8102 reveal these to be identical. Based on the 16S rDNA sequence information, clade-specific oligonucleotides for the marine Synechococcus genus were designed and their specificity was optimized. Using dot blot hybridization technology, these probes were used to determine the in situ community structure of marine Synechococcus populations in the Red Sea at the time of a Synechococcus maximum during April 1999. A predominance of genotypes representative of a single clade was found, and these genotypes were common among strains isolated into culture. Conversely, strains lacking PE, which were also relatively easily isolated into culture, represented only a minor component of the Synechococcus population. Genotypes corresponding to well-studied laboratory strains also appeared to be poorly represented in this stratified water column in the Red Sea.     

Co-occurring Synechococcus ecotypes occupy four major oceanic regimes defined by temperature,macronutrients and iron

Marine picocyanobacteria, comprised of the genera Synechococcus and Prochlorococcus, are the most abundant and widespread primary producers in the ocean. More than 20 genetically distinct clades of marine Synechococcus have been identified, but their physiology and biogeography are not as thoroughly characterized as those of Prochlorococcus. Using clade-specific qPCR primers, we measured the abundance of 10 Synechococcus clades at 92 locations in surface waters of the Atlantic and Pacific Oceans. We found that Synechococcus partition the ocean into four distinct regimes distinguished by temperature, macronutrients and iron availability. Clades I and IV were prevalent in colder, mesotrophic waters; clades II, III and X dominated in the warm, oligotrophic open ocean; clades CRD1 and CRD2 were restricted to sites with low iron availability; and clades XV and XVI were only found in transitional waters at the edges of the other biomes. Overall, clade II was the most ubiquitous clade investigated and was the dominant clade in the largest biome, the oligotrophic open ocean. Co-occurring clades that occupy the same regime belong to distinct evolutionary lineages within Synechococcus, indicating that multiple ecotypes have evolved independently to occupy similar niches and represent examples of parallel evolution. We speculate that parallel evolution of ecotypes may be a common feature of diverse marine microbial communities that contributes to functional redundancy and the potential for resiliency.     

Influence of molecular resolution on sequence-based discovery of ecological diversity among Synechococcus populations in an alkaline siliceous hot spring microbial mat

Previous research has shown that sequences of 16S rRNA genes and 16S-23S rRNA internal transcribed spacer regions may not have enough genetic resolution to define all ecologically distinct Synechococcus populations (ecotypes) inhabiting alkaline, siliceous hot spring microbial mats. To achieve higher molecular resolution, we studied sequence variation in three protein-encoding loci sampled by PCR from 60°C and 65°C sites in the Mushroom Spring mat (Yellowstone National Park, WY). Sequences were analyzed using the ecotype simulation (ES) and AdaptML algorithms to identify putative ecotypes. Between 4 and 14 times more putative ecotypes were predicted from variation in protein-encoding locus sequences than from variation in 16S rRNA and 16S-23S rRNA internal transcribed spacer sequences. The number of putative ecotypes predicted depended on the number of sequences sampled and the molecular resolution of the locus. Chao estimates of diversity indicated that few rare ecotypes were missed. Many ecotypes hypothesized by sequence analyses were different in their habitat specificities, suggesting different adaptations to temperature or other parameters that vary along the flow channel.     

Diversity of Synechococcus and Prochlorococcus populations determined from DNA sequences of the N-regulatory gene ntcA

The cyanobacteria Synechococcus and Prochlorococcus are abundant primary producers in the nitrogen-poor waters of the Gulf of Aqaba, northern Red Sea. Expression of the nitrogen regulatory gene ntcA is a useful indicator for determining the N-status of cyanobacteria, and preliminary work with this gene suggests that it may also serve as a useful biodiversity marker. Here we investigated the genotypic diversity of ntcA among the full spectrum of cultured Synechococcus and Prochlorococcus lineages and assessed cyanobacterial genotypic composition in environmental samples from the Gulf of Aqaba. The high level of ntcA diversification established this gene as an excellent biodiversity marker capable of distinguishing between numerous clades within each genus with high resolution. An unexpected large diversity was observed among Synechococcus populations, including the detection of four novel clades for which culture representatives have yet to be isolated. In addition, extensive microdiversity within a number of Synechococcus clades was revealed. Temporal differences in the detection of the various Synechococcus clades suggest seasonal fluctuations in the genotypic make-up of Synechococcus populations. In contrast, virtually all Prochlorococcus sequences fell within a single high-light adapted clade that was detected year round. We suggest that the limited genotypic diversity among Prochlorococcus in combination with a limited capacity for acclimation to environmental changes resulting from its small genome size led to the dramatic rise and demise of Prochlorococcus populations over the yearly cycle in the Gulf of Aqaba.     

Resolution of Prochlorococcus and Synechococcus Ecotypes by Using 16S-23S Ribosomal DNA Internal Transcribed Spacer Sequences

Cultured isolates of the marine cyanobacteria Prochlorococcus and Synechococcus vary widely in their pigment compositions and growth responses to light and nutrients, yet show greater than 96% identity in their 16S ribosomal DNA (rDNA) sequences. In order to better define the genetic variation that accompanies their physiological diversity, sequences for the 16S-23S rDNA internal transcribed spacer (ITS) region were determined in 32 Prochlorococcus isolates and 25 Synechococcus isolates from around the globe. Each strain examined yielded one ITS sequence that contained two tRNA genes. Dramatic variations in the length and G+C content of the spacer were observed among the strains, particularly among Prochlorococcus strains. Secondary-structure models of the ITS were predicted in order to facilitate alignment of the sequences for phylogenetic analyses. The previously observed division of Prochlorococcus into two ecotypes (called high and low-B/A after their differences in chlorophyll content) were supported, as was the subdivision of the high-B/A ecotype into four genetically distinct clades. ITS-based phylogenies partitioned marine cluster A Synechococcus into six clades, three of which can be associated with a particular phenotype (motility, chromatic adaptation, and lack of phycourobilin). The pattern of sequence divergence within and between clades is suggestive of a mode of evolution driven by adaptive sweeps and implies that each clade represents an ecologically distinct population. Furthermore, many of the clades consist of strains isolated from disparate regions of the world's oceans, implying that they are geographically widely distributed. These results provide further evidence that natural populations of Prochlorococcus and Synechococcus consist of multiple coexisting ecotypes, genetically closely related but physiologically distinct, which may vary in relative abundance with changing environmental conditions.     

Fine-scale distribution patterns of Synechococcus ecological diversity in microbial mats of Mushroom Spring, Yellowstone National Park

Past analyses of sequence diversity in high-resolution protein-encoding genes have identified putative ecological species of unicellular cyanobacteria in the genus Synechococcus, which are specialized to 60°C but not 65°C in Mushroom Spring microbial mats. Because these studies were limited to only two habitats, we studied the distribution of Synechococcus sequence variants at 1°C intervals along the effluent flow channel and at 80-μm vertical-depth intervals throughout the upper photic layer of the microbial mat. Diversity at the psaA locus, which encodes a photosynthetic reaction center protein (PsaA), was sampled by PCR amplification, cloning, and sequencing methods at 60, 63, and 65°C sites. The evolutionary simulation programs Ecotype Simulation and AdaptML were used to identify putative ecologically distinct populations (ecotypes). Ecotype Simulation predicted a higher number of putative ecotypes in cases where habitat variation was limited, while AdaptML predicted a higher number of ecologically distinct phylogenetic clades in cases where habitat variation was high. Denaturing gradient gel electrophoresis was used to track the distribution of dominant sequence variants of ecotype populations relative to temperature variation and to O₂, pH, and spectral irradiance variation, as measured using microsensors. Different distributions along effluent channel flow and vertical gradients, where temperature, light, and O₂ concentrations are known to vary, confirmed the ecological distinctness of putative ecotypes.     

Research note: Ecotype differentiation in qualitative content of water soluble organic compounds between marine and brackish Fucus vesiculosus L. (Phaeophyceae)

In the present study we compared the contents of water soluble organic compounds of the marine intertidal ecotype of Fucus vesiculosus (Phaeophyceae) from the Norwegian Sea (34 practical salinity units, psu) with the sublittoral ecotype of F. vesiculosus from the brackish Bothnian Sea (5 psu). Nuclear magnetic resonance spectra revealed that marine F. vesiculosus had additional types of water soluble organic compounds compared with brackish F. vesiculosus. The results suggested that glycine betaine in the marine ecotype could be the reason for this ecotype differentiation. Furthermore, the qualitative differences between the ecotypes were the same after one week's treatment of marine algae in brackish water and of brackish algae in marine water. These suggest that the additional types of water soluble organic compounds in marine F. vesiculosus are not caused by the salinity conditions at the growth sites. Further research concerning other environmental factors that may influence ecotype differentiation of water soluble organic compounds qualitative content and adaptation in F. vesiculosus is recommended.     

Phylogenetic diversity of Synechococcus strains isolated from the East China Sea and the East Sea

Phylogenetic relationships among 33 Synechococcus strains isolated from the East China Sea (ECS) and the East Sea (ES) were studied based on 16S rRNA gene sequences and 16S–23S rRNA gene internal transcribed spacer (ITS) sequences. Pigment patterns of the culture strains were also examined. Based on 16S rRNA gene and ITS sequence phylogenies, the Synechococcus isolates were clustered into 10 clades, among which eight were previously identified and two were novel. Half of the culture strains belonged to clade V or VI. All strains that clustered into novel clades exhibited both phycoerythrobilin and phycourobilin. Interestingly, the pigment compositions of isolates belonging to clades V and VI differed from those reported for other oceanic regions. None of the isolates in clade V showed phycourobilin, whereas strains in clade VI exhibited both phycourobilin and phycoerythrobilin, which is in contrast to previous studies. The presence of novel lineages and the different pigment patterns in the ECS and the ES suggests the possibility that some Synechococcus lineages are distributed only in geographically restricted areas and have evolved in these regions. Therefore, further elucidation of the physiological, ecological, and genetic characteristics of the diverse Synechococcus strains is required to understand their spatial and geographical distribution.     

The quorum sensing diversity within and between ecotypes of Bacillus subtilis

Ecological sociobiology is an emerging field that aims to frame social evolution in terms of ecological adaptation. Here we explore the ecological context for evolution of quorum sensing diversity in bacteria, where social communication is limited to members of the same quorum sensing type (pherotype). We sampled isolates of Bacillus subtilis from soil on a microgeographical scale and identified three ecologically distinct phylogenetic groups (ecotypes) and three pherotypes. Each pherotype was strongly associated with a different ecotype, suggesting that it is usually not adaptive for one ecotype to ‘listen’ to the signalling of another. Each ecotype, however, contained one or more minority pherotypes shared with the other B. subtilis ecotypes and with more distantly related species taxa. The pherotype diversity within ecotypes is consistent with two models: first, a pherotype cycling model, whereby minority pherotypes enter a population through horizontal genetic transfer and increase in frequency through cheating the social interaction; and second, an occasional advantage model, such that when two ecotypes are each below their quorum densities, they may benefit from listening to one another. This is the first survey of pherotype diversity in relation to ecotypes and it will be interesting to further test the hypotheses raised and supported here, and to explore other bacterial systems for the role of ecological divergence in fostering pherotype diversity.     

Clade-Specific 16S Ribosomal DNA Oligonucleotides Reveal the Predominance of a Single Marine Synechococcus Clade throughout a Stratified Water Column in the Red Sea

Phylogenetic relationships among members of the marine Synechococcus genus were determined following sequencing of the 16S ribosomal DNA (rDNA) from 31 novel cultured isolates from the Red Sea and several other oceanic environments. This revealed a large genetic diversity within the marine Synechococcus cluster consistent with earlier work but also identified three novel clades not previously recognized. Phylogenetic analyses showed one clade, containing halotolerant isolates lacking phycoerythrin (PE) and including strains capable, or not, of utilizing nitrate as the sole N source, which clustered within the MC-A (Synechococcus subcluster 5.1) lineage. Two copies of the 16S rRNA gene are present in marine Synechococcus genomes, and cloning and sequencing of these copies from Synechococcus sp. strain WH 7803 and genomic information from Synechococcus sp. strain WH 8102 reveal these to be identical. Based on the 16S rDNA sequence information, clade-specific oligonucleotides for the marine Synechococcus genus were designed and their specificity was optimized. Using dot blot hybridization technology, these probes were used to determine the in situ community structure of marine Synechococcus populations in the Red Sea at the time of a Synechococcus maximum during April 1999. A predominance of genotypes representative of a single clade was found, and these genotypes were common among strains isolated into culture. Conversely, strains lacking PE, which were also relatively easily isolated into culture, represented only a minor component of the Synechococcus population. Genotypes corresponding to well-studied laboratory strains also appeared to be poorly represented in this stratified water column in the Red Sea.     

Multi-locus sequence analysis, taxonomic resolution and biogeography of marine Synechococcus

Conserved markers such as the 16S rRNA gene do not provide sufficient molecular resolution to identify spatially structured populations of marine Synechococcus, or 'ecotypes' adapted to distinct ecological niches. Multi-locus sequence analysis targeting seven 'core' genes was employed to taxonomically resolve Synechococcus isolates and correlate previous phylogenetic analyses encompassing a range of markers. Despite the recognized importance of lateral gene transfer in shaping the genomes of marine cyanobacteria, multi-locus sequence analysis of more than 120 isolates reflects a clonal population structure of major lineages and subgroups. A single core genome locus, petB, encoding the cytochrome b(6) subunit of the cytochrome b(6) f complex, was selected to expand our understanding of the diversity and ecology of marine Synechococcus populations. Environmental petB sequences cloned from contrasting sites highlight numerous genetically and ecologically distinct clusters, some of which represent novel, environmentally abundant clades without cultured representatives. With a view to scaling ecological analyses, the short sequence, taxonomic resolution and accurate automated alignment of petB is ideally suited to high-throughput and high-resolution sequencing projects to explore links between the ecology, evolution and biology of marine Synechococcus.     

Convergent evolution across the Australian continent: ecotype diversification drives morphological convergence in two distantly related clades of Australian frogs

Animals from different clades but subject to similar environments often evolve similar body shapes and physiological adaptations due to convergent evolution, but this has been rarely tested at the transcontinental level and across entire classes of animal. Australia's biome diversity, isolation and aridification history provide excellent opportunities for comparative analyses on broad‐scale macroevolutionary patterns. We collected morphological and environmental data on eighty‐four (98%) Australian hylid frog species and categorized them into ecotypes. Using a phylogenetic framework, we tested the hypothesis that frogs from the same ecotype display similar body shape patterns: (i) across all the Australian hylids, and (ii) through comparison with a similar previous study on 127 (97%) Australian myobatrachid species. Body size and shape variation did not follow a strong phylogenetic pattern and was not tightly correlated with environment, but there was a stronger association between morphotype and ecotype. Both arboreal and aquatic frogs had long limbs, whereas limbs of fossorial species were shorter. Other terrestrial species were convergent on the more typical frog body shape. We quantified the strength of morphological convergence at two levels: (i) between fossorial myobatrachid and hylid frogs, and (ii) in each ecomorph within the hylids. We found strong convergence within ecotypes, especially in fossorial species. Ecotypes were also reflected in physiological adaptations: both arboreal and cocooned fossorial frogs tend to have higher rates of evaporative water loss. Our results illustrate how adaptation to different ecological niches plays a crucial role in morphological evolution, boosting phenotypic diversity within a clade. Despite phylogenetic conservatism, morphological adaptation to repeatedly emerging new environments can erase the signature of ancestral morphotypes, resulting in phenotypic diversification and convergence both within and between diverse clades.     

Differentiation in parasitism among ecotypes of whitefish segregating along depth gradients

Parasitism is a potential mechanism initiating or facilitating ecotypic differentiation and speciation in freshwater fish. While recent studies have begun to explore this question, there are no empirical studies of parasitism in evolutionary replicates of ecotype‐pairs at variable stages of speciation. Such comparative studies of parasitism along continuums of host differentiation are needed as a first step towards testing the role of parasites in ecological speciation. We explored parasitism of whitefish Coregonus lavaretus in four pre‐alpine lakes in Switzerland that hold replicate species radiations of whitefish. We sampled shallow and deep‐spawning ecotypes on their breeding grounds. We found significant and consistent differences in infection between the ecotypes so that the shallow‐spawning fish had more trematode infections, whereas the deepspawning fish had more cestodes. The magnitude of these differences correlated positively with the degree of the genetic differentiation among the ecotypes and negatively with the extent of eutrophication of the lakes. Although the overall diversity of infections was low, some parasite species with potential effects on fish showed marked differences in infection between the ecotypes, suggesting that parasitism may have a role in maintaining ecotype differentiation in this system. Our results also indicate previously unknown habitat segregation of the better differentiated ecotypes, i.e. species, along the depth gradient outside the breeding season. Moreover, oligotrophic lakes tended to have higher parasite species richness and higher abundances of infection, than mesotrophic and eutrophic lakes, suggesting that the history of eutrophication affects parasite diversity.     

Genetic variation for shell traits in a direct-developing marine snail involved in a putative sympatric ecological speciation process

Populations of the marine gastropod Littorina saxatilis from exposed rocky shores of NW Spain provide one of the few putative cases of sympatric ecological speciation. Two ecotypes with large differences in shell morphology and strong assortative mating are living at different vertical levels of the shore separated by a few meters. It has been hypothesized that shell size is the main determinant for the reproductive isolation observed between the ecotypes, and that several shell shape traits are subject to divergent natural selection and are responsible for the adaptation of each ecotype to its respective habitat. Using embryos extracted from wild females we obtain estimates of genetic variation for shell size and shape and compare them with those from neutral molecular markers. Estimates of heritability are significantly larger for the ecotype found in the upper shore than for that in the lower shore, in concordance with a similar result observed for heterozygosity of neutral markers. The large genetic differentiation between ecotypes for the shell traits, contrasting the smaller close to neutral differentiation between populations of the same ecotype, supports the implication of the traits in adaptation.     

A suppression subtractive hybridization approach reveals niche-specific genes that may be involved in predator avoidance in marine Synechococcus isolates

Picocyanobacteria of the genus Synechococcus are important contributors to marine primary production and are ubiquitous in the world's oceans. This genus is genetically diverse, and at least 10 discrete lineages or clades have been identified phylogenetically. However, little if anything is known about the genetic attributes which characterize particular lineages or are unique to specific strains. Here, we used a suppression subtractive hybridization (SSH) approach to identify strain- and clade-specific genes in two well-characterized laboratory strains, Synechococcus sp. strain WH8103 (clade III) and Synechococcus sp. strain WH7803 (clade V). Among the genes that were identified as potentially unique to each strain were genes encoding proteins that may be involved in specific predator avoidance, including a glycosyltransferase in strain WH8103 and a permease component of an ABC-type polysaccharide/polyol phosphate export system in WH7803. During this work the genome of one of these strains, WH7803, became available. This allowed assessment of the number of false-positive sequences (i.e., sequences present in the tester genome) present among the SSH-enriched sequences. We found that approximately 9% of the WH8103 sequences were potential false-positive sequences, which demonstrated that caution should be used when this technology is used to assess genomic differences in genetically similar bacterial strains.     

Population genetic structure of Tomicus piniperda L. (Curculionidae: Scolytinae) on different pine species and validation of T. destruens (Woll.)

Genetic diversity and population structure of Tomicus piniperda was assessed using mitochondrial sequences on 16 populations sampled on 6 pine species in France. Amplifications of Internal transcribed space 1 (ITS1) were also performed. Our goals were to determine the taxonomic status of the Mediterranean ecotype T. piniperda destruens, and to test for host plant or geographical isolation effect on population genetic structure. We showed that T. piniperda clusters in two mtDNA haplotypic groups. Clade A corresponds to insects sampled in continental France on Pinus sylvestris, P. pinaster and P. uncinata, whereas clade B gathers the individuals sampled in Corsica on P. pinaster and P. radiata and in continental France on P. pinea and P. halepensis. Insects belonging to clade A and clade B also consistently differ in the length of ITS1. Individuals belonging to both clades were found once in sympatry on P. pinaster. Genetic distances between clades are similar to those measured between distinct species of Tomicus. We concluded that clade B actually corresponds to the destruens ecotype and forms a good species, T. destruens. Analyses of molecular variance ( amova ) were conducted separately on T. destruens and T. piniperda to test for an effect of either geographical isolation or host species. Interestingly, the effect of host plant was significant for T. piniperda only, while the effect of geographical isolation was not. Pine species therefore seems to act as a significant barrier to gene flow, even if host race formation is not observed. These results still need to be confirmed by nuclear markers.     

AN ADAPTIVE RADIATION OF FROGS IN A SOUTHEAST ASIAN ISLAND ARCHIPELAGO

Living amphibians exhibit a diversity of ecologies, life histories, and species‐rich lineages that offers opportunities for studies of adaptive radiation. We characterize a diverse clade of frogs (Kaloula, Microhylidae) in the Philippine island archipelago as an example of an adaptive radiation into three primary habitat specialists or ecotypes. We use a novel phylogenetic estimate for this clade to evaluate the tempo of lineage accumulation and morphological diversification. Because species‐level phylogenetic estimates for Philippine Kaloula are lacking, we employ dense population sampling to determine the appropriate evolutionary lineages for diversification analyses. We explicitly take phylogenetic uncertainty into account when calculating diversification and disparification statistics and fitting models of diversification. Following dispersal to the Philippines from Southeast Asia, Kaloula radiated rapidly into several well‐supported clades. Morphological variation within Kaloula is partly explained by ecotype and accumulated at high levels during this radiation, including within ecotypes. We pinpoint an axis of morphospace related directly to climbing and digging behaviors and find patterns of phenotypic evolution suggestive of ecological opportunity with partitioning into distinct habitat specialists. We conclude by discussing the components of phenotypic diversity that are likely important in amphibian adaptive radiations.