Biodiversity of Costa Rican salamanders: Implications of high levels of genetic differentiation and phylogeographic structure for species formation

Although salamanders are characteristic amphibians in Holarctic temperate habitats, in tropical regions they have diversified evolutionarily only in tropical America. An adaptive radiation centered in Middle America occurred late in the history of a single clade, the supergenus Bolitoglossa (Plethodontidae), and large numbers of species now occur in diverse habitats. Sublineages within this clade decrease in number from the northern to southern parts of Middle America, and in Costa Rica, there are but three. Despite this phylogenetic constraint, Costa Rica has many species; the number of salamander species on one local elevational transect in the Cordillera de Talamanca may be the largest for any such transect in the world. Extraordinary variation in sequences of the mitochondrial gene cytochrome b within a clade of the genus Bolitoglossa in Costa Rica reveals strong phylogeographic structure within a single species, Bolitoglossa pesrubra. Allozymic variation in 19 proteins reveals a pattern largely concordant with the mitochondrial DNA phylogeography. More species exist than are currently recognized. Diversification occurs in restricted geographic areas and involves sharp geographic and elevational differentiation and zonation. In their degree of genetic differentiation at a local scale, these species of the deep tropics exceed the known variation of extratropical salamanders, which also differ in being less restricted in elevational range. Salamanders display “tropicality” in that although speciose, they are usually local in distribution and rare. They display strong ecological and physiological differentiation that may contribute importantly to morphological divergence and species formation.

Rapid, local adaptation of zooplankton behavior to changes in predation pressure in the absence of neutral genetic changes

Organisms producing resting stages provide unique opportunities for reconstructing the genetic history of natural populations. Diapausing seeds and eggs often are preserved in large numbers, representing entire populations captured in an evolutionary inert state for decades and even centuries. Starting from a natural resting egg bank of the waterflea Daphnia, we compare the evolutionary rates of change in an adaptive quantitative trait with those in selectively neutral DNA markers, thus effectively testing whether the observed genetic changes in the quantitative trait are driven by natural selection. The population studied experienced variable and well documented levels of fish predation over the past 30 years and shows correlated genetic changes in phototactic behavior, a predator-avoidance trait that is related to diel vertical migration. The changes mainly involve an increased plasticity response upon exposure to predator kairomone, the direction of the changes being in agreement with the hypothesis of adaptive evolution. Genetic differentiation through time was an order of magnitude higher for the studied behavioral trait than for neutral markers (DNA microsatellites), providing strong evidence that natural selection was the driving force behind the observed, rapid, evolutionary changes.

Genetic drift and within-host metapopulation dynamics of HIV-1 infection

Most HIV replication occurs in solid lymphoid tissue, which has prominent architecture at the histological level, which separates groups of productively infected CD4+ cells. Nevertheless, current population models of HIV assume panmixis within lymphoid tissue. We present a simple “metapopulation” model of HIV replication, where the population of infected cells is comprised of a large number of small populations, each of which is established by a few founder viruses and undergoes turnover. To test this model, we analyzed viral genetic variation of infected cell subpopulations within the spleen and demonstrated the action of founder effects as well as significant variation in the extent of genetic differentiation between subpopulations among patients. The combination of founder effects and subpopulation turnover can result in an effective population size much lower than the actual population size and may contribute to the importance of genetic drift in HIV evolution despite a large number of infected cells.

Nucleotide sequences provide evidence of genetic exchange among distantly related lineages of Trypanosoma cruzi

Simple phylogenetic tests were applied to a large data set of nucleotide sequences from two nuclear genes and a region of the mitochondrial genome of Trypanosoma cruzi, the agent of Chagas' disease. Incongruent gene genealogies manifest genetic exchange among distantly related lineages of T. cruzi. Two widely distributed isoenzyme types of T. cruzi are hybrids, their genetic composition being the likely result of genetic exchange between two distantly related lineages. The data show that the reference strain for the T. cruzi genome project (CL Brener) is a hybrid. Well-supported gene genealogies show that mitochondrial and nuclear gene sequences from T. cruzi cluster, respectively, in three or four distinct clades that do not fully correspond to the two previously defined major lineages of T. cruzi. There is clear genetic differentiation among the major groups of sequences, but genetic diversity within each major group is low. We estimate that the major extant lineages of T. cruzi have diverged during the Miocene or early Pliocene (3–16 million years ago).

The location of Z- and W-linked marker genes and sequence on the homomorphic sex chromosomes of the ostrich and the emu

Perhaps the most striking fact about early Cenozoic avian history some 70 million years ago was the rapid radiation of large, flightless, ground-living birds. It has been suggested that, for a time, there was active competition between these large terrestrial birds and the early mammals. Probably reflecting the above noted early start of Ratitae of the infraclass Eoaves, the presumptive sex chromosomes of their present day survivors, such as the emu and the ostrich, largely remained homomorphic. The signs of genetic differentiation between their still-homomorphic Z and W chromosomes were tested by using two marker genes (Z-linked ZOV3 and the gene for the iron-responsive element-binding protein) and one marker sequence of a part of a presumptive pseudogene (W-linked EE0.6 of the chicken). Their homologues, maintaining 71–92% identities to the chicken counterparts, were found in both the emu (Dromaius novaehollandiae) and the ostrich (Struthio camelus). Their locations were visualized on chromosome preparations by fluorescence in situ hybridization. In the case of the emu, these three marker sequences were localized on both members of the fifth pair of a female, thus revealing no sign yet of genetic differentiation between the Z and the W. The finding was the same with regard to both members of the fourth pair of male ostriches. In the female ostrich, however, the sequence of the gene for the iron-responsive element-binding protein was missing from one of the pairs, thus revealing the differentiation by a small deletion of the W from the Z.

Disrupting evolutionary processes: The effect of habitat fragmentation on collared lizards in the Missouri Ozarks

Humans affect biodiversity at the genetic, species, community, and ecosystem levels. This impact on genetic diversity is critical, because genetic diversity is the raw material of evolutionary change, including adaptation and speciation. Two forces affecting genetic variation are genetic drift (which decreases genetic variation within but increases genetic differentiation among local populations) and gene flow (which increases variation within but decreases differentiation among local populations). Humans activities often augment drift and diminish gene flow for many species, which reduces genetic variation in local populations and prevents the spread of adaptive complexes outside their population of origin, thereby disrupting adaptive processes both locally and globally within a species. These impacts are illustrated with collared lizards (Crotaphytus collaris) in the Missouri Ozarks. Forest fire suppression has reduced habitat and disrupted gene flow in this lizard, thereby altering the balance toward drift and away from gene flow. This balance can be restored by managed landscape burns. Some have argued that, although human-induced fragmentation disrupts adaptation, it will also ultimately produce new species through founder effects. However, population genetic theory and experiments predict that most fragmentation events caused by human activities will facilitate not speciation, but local extinction. Founder events have played an important role in the macroevolution of certain groups, but only when ecological opportunities are expanding rather than contracting. The general impact of human activities on genetic diversity disrupts or diminishes the capacity for adaptation, speciation, and macroevolutionary change. This impact will ultimately diminish biodiversity at all levels.

Genes, peoples, and languages

The genetic history of a group of populations is usually analyzed by reconstructing a tree of their origins. Reliability of the reconstruction depends on the validity of the hypothesis that genetic differentiation of the populations is mostly due to population fissions followed by independent evolution. If necessary, adjustment for major population admixtures can be made. Dating the fissions requires comparisons with paleoanthropological and paleontological dates, which are few and uncertain. A method of absolute genetic dating recently introduced uses mutation rates as molecular clocks; it was applied to human evolution using microsatellites, which have a sufficiently high mutation rate. Results are comparable with those of other methods and agree with a recent expansion of modern humans from Africa. An alternative method of analysis, useful when there is adequate geographic coverage of regions, is the geographic study of frequencies of alleles or haplotypes. As in the case of trees, it is necessary to summarize data from many loci for conclusions to be acceptable. Results must be independent from the loci used. Multivariate analyses like principal components or multidimensional scaling reveal a number of hidden patterns and evaluate their relative importance. Most patterns found in the analysis of human living populations are likely to be consequences of demographic expansions, determined by technological developments affecting food availability, transportation, or military power. During such expansions, both genes and languages are spread to potentially vast areas. In principle, this tends to create a correlation between the respective evolutionary trees. The correlation is usually positive and often remarkably high. It can be decreased or hidden by phenomena of language replacement and also of gene replacement, usually partial, due to gene flow.

Incipient species formation in salamanders of the Ensatina complex

The Ensatina eschscholtzii complex of plethodontid salamanders, a well-known “ring species,” is thought to illustrate stages in the speciation process. Early research, based on morphology and coloration, has been extended by the incorporation of studies of protein variation and mitochondrial DNA sequences. The new data show that the complex includes a number of geographically and genetically distinct components that are at or near the species level. The complex is old and apparently has undergone instances of range contraction, isolation, differentiation, and then expansion and secondary contact. While the hypothesis that speciation is retarded by gene flow around the ring is not supported by molecular data, the general biogeographical hypothesis is supported. There is evidence of a north to south range expansion along two axes, with secondary contact and completion of the ring in southern California. Current research targets regions once thought to show primary intergradation, but which molecular markers reveal to be zones of secondary contact. Here emphasis is on the subspecies E. e. xanthoptica, which is involved in four distinct secondary contacts in central California. There is evidence of renewed genetic interactions upon recontact, with greater genetic differentiation within xanthoptica than between it and some of the interacting populations. The complex presents a full array of intermediate conditions between well-marked species and geographically variable populations. Geographically differentiated segments represent a diversity of depths of time of isolation and admixture, reflecting the complicated geomorphological history of California. Ensatina illustrates the continuing difficulty in making taxonomic assignments in complexes studied during species formation.

Gene genealogies and population variation in plants

Early in the development of plant evolutionary biology, genetic drift, fluctuations in population size, and isolation were identified as critical processes that affect the course of evolution in plant species. Attempts to assess these processes in natural populations became possible only with the development of neutral genetic markers in the 1960s. More recently, the application of historically ordered neutral molecular variation (within the conceptual framework of coalescent theory) has allowed a reevaluation of these microevolutionary processes. Gene genealogies trace the evolutionary relationships among haplotypes (alleles) with populations. Processes such as selection, fluctuation in population size, and population substructuring affect the geographical and genealogical relationships among these alleles. Therefore, examination of these genealogical data can provide insights into the evolutionary history of a species. For example, studies of Arabidopsis thaliana have suggested that this species underwent rapid expansion, with populations showing little genetic differentiation. The new discipline of phylogeography examines the distribution of allele genealogies in an explicit geographical context. Phylogeographic studies of plants have documented the recolonization of European tree species from refugia subsequent to Pleistocene glaciation, and such studies have been instructive in understanding the origin and domestication of the crop cassava. Currently, several technical limitations hinder the widespread application of a genealogical approach to plant evolutionary studies. However, as these technical issues are solved, a genealogical approach holds great promise for understanding these previously elusive processes in plant evolution.

An intrinsic genetic program for autonomous differentiation of muscle cells in the ascidian embryo.

The B-line presumptive muscle cells of ascidian embryos have extensive potential for self-differentiation dependent on determinants prelocalized in the myoplasm of fertilized eggs. Ascidian larval muscle cells therefore provide an experimental system with which to explore an intrinsic genetic program for autonomous specification of embryonic cells. Experiments with egg fragments suggested that maternal mRNAs are one of the components of muscle determinants. Expression of larval muscle actin genes begins as early as the 32-cell stage, prior to the developmental fate restriction of the cells. The timing of initiation of the actin gene expression proceeds the expression of an ascidian homologue of vertebrate MyoD by a few hours. Mutations in the proximal E-box of the 5' flanking region of the actin genes did not alter the promoter activity for muscle-specific expression of reporter gene. These results, together with results of deletion constructs of fusion genes, suggest that muscle determinants regulate directly, or indirectly via regulatory factors other than MyoD, the transcription of muscle-specific structural genes leading to the terminal differentiation.

Lens complementation system for the genetic analysis of growth, differentiation, and apoptosis in vivo.

A genetic approach has been established that combines the advantages of blastocyst complementation with the experimental attributes of the developing lens for the functional analysis of genes governing cellular proliferation, terminal differentiation, and apoptosis. This lens complementation system (LCS) makes use of a mutant mouse strain, aphakia (ak), homozygotes of which fail to develop an ocular lens. We demonstrate that microinjection of wild-type embryonic stem (ES) cells into ak/ak blastocysts produces chimeras with normal ES-cell-derived lenses and that microinjection of Rb-/- ES cells generates an aberrant lens phenotype identical to that obtained through conventional gene targeting methodology. Our determination that a cell autonomous defect underlies the aphakia condition assures that lenses generated through LCS are necessarily ES-cell-derived. LCS provides for the rapid phenotypic analysis of loss-of-function mutations, circumvents the need for germ-line transmission of null alleles, and, most significantly, facilitates the study of essential genes whose inactivation is associated with early lethal phenotypes.

Genetic influence on neurogenesis in the dentate gyrus of adult mice

To address genetic influences on hippocampal neurogenesis in adult mice, we compared C57BL/6, BALB/c, CD1(ICR), and 129Sv/J mice to examine proliferation, survival, and differentiation of newborn cells in the dentate gyrus. Proliferation was highest in C57BL/6; the survival rate of newborn cells was highest in CD1. In all strains ≈60% of surviving newborn cells had a neuronal phenotype, but 129/SvJ produced more astrocytes. Over 6 days C57BL/6 produced 0.36% of their total granule cell number of 239,000 as new neurons, BALB/c 0.30% of 242,000, CD1 (ICR) 0.32% of 351,000, and 129/SvJ 0.16% of 280,000. These results show that different aspects of adult hippocampal neurogenesis are differentially influenced by the genetic background.

Differentiation of pancreatic epithelial progenitor cells into hepatocytes following transplantation into rat liver

The ability to identify, isolate, and transplant progenitor cells from solid tissues would greatly facilitate the treatment of diseases currently requiring whole organ transplantation. In this study, cell fractions enriched in candidate epithelial progenitor cells from the rat pancreas were isolated and transplanted into the liver of an inbred strain of Fischer rats. Using a dipeptidyl dipeptidase IV genetic marker system to follow the fate of transplanted cells in conjunction with albumin gene expression, we provide conclusive evidence that, after transplantation to the liver, epithelial progenitor cells from the pancreas differentiate into hepatocytes, express liver-specific proteins, and become fully integrated into the liver parenchymal structure. These studies demonstrate the presence of multipotent progenitor cells in the adult pancreas and establish a role for the liver microenvironment in the terminal differentiation of epithelial cells of foregut origin. They further suggest that such progenitor cells might be useful in studies of organ repopulation following acute or chronic liver injury.

A novel bacterial tyrosine kinase essential for cell division and differentiation

Protein kinases play central roles in the regulation of eukaryotic and prokaryotic cell growth, division, and differentiation. The Caulobacter crescentus divL gene encodes a novel bacterial tyrosine kinase essential for cell viability and division. Although the DivL protein is homologous to the ubiquitous bacterial histidine protein kinases (HPKs), it differs from previously studied members of this protein kinase family in that it contains a tyrosine residue (Tyr-550) in the conserved H-box instead of a histidine residue, which is the expected site of autophosphorylation. DivL is autophosphorylated on Tyr-550 in vitro, and this tyrosine residue is essential for cell viability and regulation of the cell division cycle. Purified DivL also catalyzes phosphorylation of CtrA and activates transcription in vitro of the cell cycle-regulated fliF promoter. Suppressor mutations in ctrA bypass the conditional cell division phenotype of cold-sensitive divL mutants, providing genetic evidence that DivL function in cell cycle and developmental regulation is mediated, at least in part, by the global response regulator CtrA. DivL is the only reported HPK homologue whose function has been shown to require autophosphorylation on a tyrosine, and, thus, it represents a new class of kinases within this superfamily of protein kinases.

Developmental signal transduction pathways uncovered by genetic suppressors

We have found conditions for saturation mutagenesis by restriction enzyme mediated integration that result in plasmid tagging of disrupted genes. Using this method we selected for mutations in genes that act at checkpoints downstream of the intercellular signaling system that controls encapsulation in Dictyostelium discoideum. One of these genes, mkcA, is a member of the mitogen-activating protein kinase cascade family while the other, regA, is a novel bipartite gene homologous to response regulators in one part and to cyclic nucleotide phosphodiesterases in the other part. Disruption of either of these genes results in partial suppression of the block to spore formation resulting from the loss of the prestalk genes, tagB and tagC. The products of the tag genes have conserved domains of serine proteases attached to ATP-driven transporters, suggesting that they process and export peptide signals. Together, these genes outline an intercellular communication system that coordinates organismal shape with cellular differentiation during development.