Phenotypic and genomic diversification with isolation by environment along elevational gradients in a neotropical treefrog.

Understanding how geographic and environmental heterogeneity drive local patterns of genetic variation is a major goal of ecological genomics and a key question in evolutionary biology. The tropical Andes and inter-Andean valleys are shaped by markedly heterogeneous landscapes, where species experience strong selective processes. We examined genome-wide SNP data together with behavioural and ecological traits (mating calls and body size) known to contribute to genetic isolation in anurans in the banana tree-dwelling frog, Boana platanera, distributed across an environmental gradient in Central Colombia (northern South America). Here, we analysed the relationships between environmentally (temperature and precipitation) associated genetic and phenotypic differentiation and the potential drivers of isolation by environment along an elevation gradient. We identified candidate SNPs associated with temperature and body size, which follow a clinal pattern of genome-wide differentiation tightly coupled with phenotypic variation: as elevation increases, B. platanera exhibits larger body size and longer call duration with more pulses but lower pulse rate and frequency. Thus, the environmental landscape has rendered a scenario where isolation by environment and candidate loci show concordance with phenotypic divergence in this tropical frog along an elevation gradient in the Colombian Andes. Our study sets the basis for evaluating the role of temperature in the genetic structure and local adaptation in tropical treefrogs and its putative effect on life cycle (embryos, tadpoles, adults) along elevation gradients.

Non-adaptive evolutionary processes governed the diversification of a temperate conifer lineage after its migration into the tropics.

Constructing phylogenetic relationships among closely related species is a recurrent challenge in evolutionary biology, particularly for long-lived taxa with large effective population sizes and uncomplete reproductive isolation, like conifers. Conifers further have slow evolutionary rates, which raises the question of whether adaptive or non/adaptive processes were predominantly involved when they rapidly diversified after migrating from temperate regions into the tropical mountains. Indeed, fine-scale phylogenetic relationships within several conifer genus remain under debate. Here, we studied the phylogenetic relationships of endemic firs (Abies, Pinaceae) discontinuously distributed in the montane forests from the Southwestern United States to Guatemala, and addressed several hypotheses related to adaptive and non-adaptive radiations. We derived over 80 K SNPs from genotyping by sequencing (GBS) for 45 individuals of nine Mesoamerican species to perform phylogenetic analyses. Both Maximum Likelihood and quartets-inference phylogenies resulted in a well-resolved topology, showing a single fir lineage divided in four subgroups that coincided with the main mountain ranges of Mesoamerica; thus having important taxonomic implications. Such subdivision fitted a North-South isolation by distance framework, in which non-adaptive allopatric processes seemed the rule. Interestingly, several reticulations were observed within subgroups, especially in the central-south region, which may explain past difficulties for generating infrageneric phylogenies. Further evidence for non-adaptive processes was obtained from analyses of 21 candidate-gene regions, which exhibited diminishing values of πa/πs and Ka/Ks with latitude, thus indicating reduced efficiency of purifying selection towards the Equator. Our study indicates that non-adaptive allopatric processes may be key generators of species diversity and endemism in the tropics.

Evolutionary rate and genetic load in an emblematic Mediterranean tree following an ancient and prolonged population collapse.

Severe bottlenecks significantly diminish the amount of genetic diversity and the speed at which it accumulates (i.e., evolutionary rate). They further compromise the efficiency of natural selection to eliminate deleterious variants, which may reach fixation in the surviving populations. Consequently, expanding and adapting to new environments may pose a significant challenge when strong bottlenecks result in genetic pauperization. Herein, we surveyed the patterns of nucleotide diversity, molecular adaptation and genetic load across 177 gene-loci in a circum-Mediterranean conifer (Pinus pinea L.) that represents one of the most extreme cases of genetic pauperization in widespread outbreeding taxa. We found very little genetic variation in both hypervariable nuclear microsatellites (SSRs) and gene-loci, which translated into genetic diversity estimates one order of magnitude lower than those previously reported for pines. Such values were consistent with a strong population decline that began some ~1 Ma. Comparisons with the related and parapatric maritime pine (Pinus pinaster Ait.) revealed reduced rates of adaptive evolution (α and ωa ) and a significant accumulation of genetic load. It is unlikely that these are the result from differences in mutation rate or linkage disequilibrium between the two species; instead they are the presumable outcome of contrasting demographic histories affecting both the speed at which these taxa accumulate genetic diversity, and the global efficacy of selection. Future studies, and programs for conservation and management, should thus start testing for the effects of genetic load on fitness, and integrating such effects into predictive models.

Deep divergence of Red-crowned Ant Tanager (Habia rubica: Cardinalidae), a multilocus phylogenetic analysis with emphasis in Mesoamerica.

Many neotropical species have a complex history of diversification as a result of the influence of geographical, ecological, climatic, and geological factors that determine the distribution of populations within a lineage. Phylogeography identifies such populations, determines their geographic distributions, and quantifies the degree of genetic divergence. In this work we explored the genetic structure of Habia rubica populations, a polytypic taxon with 17 subspecies described, in order to obtain hypotheses about their evolutionary history and processes of diversification. We undertook multilocus analyses using sequences of five molecular markers (ND2, ACOI-I9, MUSK, FGB-I5 and ODC), and sampling from across the species' distribution range, an area encompassing from Central Mexico throughout much of South America. With these data, we obtained a robust phylogenetic hypothesis, a species delimitation analysis, and estimates of divergence times for these lineages. The phylogenetic hypothesis of concatenated molecular markers shows that H. rubica can be divided in three main clades: the first includes Mexican Pacific coast populations, the second is formed by population from east of Mexico to Panama and the third comprises the South American populations. Within these clades we recognize seven principal phylogroups whose limits have a clear correspondence with important geographical discontinuities including the Isthmus of Tehuantepec in southern Mexico, the Talamanca Cordillera, and the Isthmus of Panama in North America. In South America, we observed a marked separation of two phylogroups that include the populations that inhabit mesic forests in western and central South America (Amazon Forest) and those inhabiting the seasonal forest from the eastern and northern regions of the South America (Atlantic Forest). These areas are separated by an intervening dry vegetation "diagonal" (Chaco, Cerrado and Caatinga). The geographic and genetic structure of these phylogroups describes a history of diversification more active and complex in the northern distribution of this species, producing at least seven well-supported lineages that could be considered species.

Genetic Resources in the "Calabaza Pipiana" Squash (Cucurbita argyrosperma) in Mexico: Genetic Diversity, Genetic Differentiation and Distribution Models.

Analyses of genetic variation allow understanding the origin, diversification and genetic resources of cultivated plants. Domesticated taxa and their wild relatives are ideal systems for studying genetic processes of plant domestication and their joint is important to evaluate the distribution of their genetic resources. Such is the case of the domesticated subspecies C. argyrosperma ssp. argyrosperma, known in Mexico as calabaza pipiana, and its wild relative C. argyrosperma ssp. sororia. The main aim of this study was to use molecular data (microsatellites) to assess the levels of genetic variation and genetic differentiation within and among populations of domesticated argyrosperma across its distribution in Mexico in comparison to its wild relative, sororia, and to identify environmental suitability in previously proposed centers of domestication. We analyzed nine unlinked nuclear microsatellite loci to assess levels of diversity and distribution of genetic variation within and among populations in 440 individuals from 19 populations of cultivated landraces of argyrosperma and from six wild populations of sororia, in order to conduct a first systematic analysis of their genetic resources. We also used species distribution models (SDMs) for sororia to identify changes in this wild subspecies' distribution from the Holocene (∼6,000 years ago) to the present, and to assess the presence of suitable environmental conditions in previously proposed domestication sites. Genetic variation was similar among subspecies (HE = 0.428 in sororia, and HE = 0.410 in argyrosperma). Nine argyrosperma populations showed significant levels of inbreeding. Both subspecies are well differentiated, and genetic differentiation (FST) among populations within each subspecies ranged from 0.152 to 0.652. Within argyrosperma we found three genetic groups (Northern Mexico, Yucatan Peninsula, including Michoacan and Veracruz, and Pacific coast plus Durango). We detected low levels of gene flow among populations at a regional scale (<0.01), except for the Yucatan Peninsula, and the northern portion of the Pacific Coast. Our analyses suggested that the Isthmus of Tehuantepec is an effective barrier isolating southern populations. Our SDM results indicate that environmental characteristics in the Balsas-Jalisco region, a potential center of domestication, were suitable for the presence of sororia during the Holocene.

Genomic footprints of adaptation in a cooperatively breeding tropical bird across a vegetation gradient.

Identifying the genetic basis of phenotypic variation and its relationship with the environment is key to understanding how local adaptations evolve. Such patterns are especially interesting among populations distributed across habitat gradients, where genetic structure can be driven by isolation by distance (IBD) and/or isolation by environment (IBE). Here, we used variation in ~1,600 high-quality SNPs derived from paired-end sequencing of double-digest restriction site-associated DNA (ddRAD-Seq) to test hypotheses related to IBD and IBE in the Yucatan jay (Cyanocorax yucatanicus), a tropical bird endemic to the Yucatán Peninsula. This peninsula is characterized by a precipitation and vegetation gradient-from dry to evergreen tropical forests-that is associated with morphological variation in this species. We found a moderate level of nucleotide diversity (π = .008) and little evidence for genetic differentiation among vegetation types. Analyses of neutral and putatively adaptive SNPs (identified by complementary genome-scan approaches) indicate that IBD is the most reliable explanation to account for frequency distribution of the former, while IBE has to be invoked to explain those of the later. These results suggest that selective factors acting along a vegetation gradient can promote local adaptation in the presence of gene flow in a vagile, nonmigratory and geographically restricted species. The putative candidate SNPs identified here are located within or linked to a variety of genes that represent ideal targets for future genomic surveys.

Evolution of an ancient microsatellite hotspot in the conifer mitochondrial genome and comparison with other plants.

In plants, mitochondrial sequence tandem repeats (STRs) have been associated with intragenomic recombination, a process held responsible for evolutionary outcomes such as gene regulation or cytoplasmic male-sterility. However, no link has been established between the recurrent accumulation of STRs and increased mutation rates in specific regions of the plant mtDNA genome. Herein, we surveyed this possibility by comparing, in a phylogenetic context, the variation of a STR-rich mitochondrial intron (nad5-4) with eleven mtDNA genes devoid of STRs within Abies (Pinaceae) and its related genera. This intron has been accumulating repeated stretches, generated by at least three-independent insertions, before the split of the two Pinaceae subfamilies, Abietoideae and Pinoideae. The last of these insertions occurred before the divergence of Abies and produced, exclusively within this genus, a tenfold increase of both the indel and substitution rates in the STR hotspot of the intron. The regions flanking the STRs harbored mutation rates as low as those estimated in mitochondrial genes devoid of repeated stretches. Further searches in complete plant mtDNA genomes, and previous studies reporting polymorphic mtSTRs, revealed that repeated stretches are common in all sorts of plants, but their accumulation in STR hotspots appears to be taxa specific. Our study suggests a new mutagenic role for repeated sequences in the plant mtDNA.

Phylogeny, diversification rates and species boundaries of Mesoamerican firs (Abies, Pinaceae) in a genus-wide context.

The genus Abies is distributed discontinuously in the temperate and subtropical montane forests of the northern hemisphere. In Mesoamerica (Mexico and northern Central America), modern firs originated from the divergence of isolated mountain populations of migrating North American taxa. However, the number of ancestral species, migratory waves and diversification speed of these taxa is unknown. Here, variation in repetitive (Pt30204, Pt63718, and Pt71936) and non-repetitive (rbcL, rps18-rpl20 and trnL-trnF) regions of the chloroplast genome was used to reconstruct the phylogenetic relationships of the Mesoamerican Abies in a genus-wide context. These phylogenies and two fossil-calibrated scenarios were further employed to estimate divergence dates and diversification rates within the genus, and to test the hypothesis that, as in many angiosperms, conifers may exhibit accelerated speciation rates in the subtropics. All phylogenies showed five main clusters that mostly agreed with the currently recognized sections of Abies and with the geographic distribution of species. The Mesoamerican taxa formed a single group with species from southwestern North America of sections Oiamel and Grandis. However, populations of the same species were not monophyletic within this group. Divergence of this whole group dated back to the late Paleocene and the early Miocene depending on the calibration used, which translated in very low diversification rates (r(0.0)=0.026-0.054, r(0.9)=0.009-0.019 sp/Ma). Such low rates were a constant along the entire genus, including both the subtropical and temperate taxa. An extended phylogeographic analysis on the Mesoamerican clade indicated that Abies flinckii and A. concolor were the most divergent taxa, while the remaining species (A. durangensis, A. guatemalensis, A. hickelii, A. religiosa and A. vejari) formed a single group. Altogether, these results show that divergence of Mesoamerican firs coincides with a model of environmental stasis and decreased extinction rate, being probably prompted by a series of range expansions and isolation-by-distance.

Ancestry and divergence of subtropical montane forest isolates: molecular biogeography of the genus Abies (Pinaceae) in southern México and Guatemala.

The genus Abies has a complex history in southern México and Guatemala. In this region, four closely related species, Abies flinckii, A. guatemalensis, A. hickelii, and A. religiosa, are distributed in fragmented and isolated montane populations. Range-wide genetic variation was investigated across species using cytoplasmic DNA markers with contrasted inheritance. Variation at two maternally inherited mitochondrial DNA markers was low. All species shared two of the nine mitotypes detected, while the remaining seven mitochondrial DNA types were restricted to a few isolated stands. Mitochondrial genetic differentiation across taxa was high (G(ST) = 0.933), it was not related to the taxonomic identity (amova; P > 0.05) of the populations, and it was not phylogeographically structured (G(ST) approximately N(ST)). In contrast, variation at three paternally inherited chloroplast DNA microsatellites was high. Chloroplast genetic differentiation was lower (G(ST) = 0.402; R(ST) = 0.547) than for mitochondrial DNA, but it was significantly related to taxonomy (amova; P < 0.001), and exhibited a significant phylogeographical structure (G(ST) < R(ST)). Different analyses of population structure indicated that A. flinckii was the most divergent taxon, while the remaining three species formed a relatively homogeneous group. However, a small number of the populations of these three taxa, all located at the limits of their respective ranges or in the Transverse Volcanic Belt, diverged from this main cluster. These trends suggest that the Mesoamerican Abies share a recent common ancestor and that their divergence and speciation is mainly driven by genetic drift and isolation during the warm interglacial periods.

Decoupled mitochondrial and chloroplast DNA population structure reveals Holocene collapse and population isolation in a threatened Mexican-endemic conifer.

Chihuahua spruce (Picea chihuahuana Martínez) is a montane subtropical conifer endemic to the Sierra Madre Occidental in northwestern México. Range-wide variation was investigated using maternally inherited mitochondrial (mtDNA) and paternally inherited chloroplast (cpDNA) DNA markers. Among the 16 mtDNA regions analysed, only two mitotypes were detected, while the study of six cpDNA microsatellite markers revealed eight different chlorotypes. The average cpDNA diversity (H = 0.415) was low but much higher than that for mtDNA (H = 0). The distribution of mitotypes revealed two clear nonoverlapping areas (G(ST) = N(ST) = 1), one including northern populations and the second one including the southern and central stands, suggesting that these two regions may represent different ancestral populations. The cpDNA markers showed lower population differentiation (G(ST) = 0.362; R(ST) = 0.230), implying that the two ancestral populations continued to exchange pollen after their initial geographic separation. A lack of a phylogeographic structure was revealed by different spatial analyses of cpDNA (G(ST) > R(ST); and samova), and reduced cpDNA gene flow was noted among populations (Nm = 0.873). Some stands deviated significantly from the mutation-drift equilibrium, suggesting recent bottlenecks. Altogether, these various trends are consistent with the hypothesis of a population collapse during the Holocene warming and suggest that most of the modern P. chihuahuana populations are now effectively isolated with their genetic diversity essentially modelled by genetic drift. The conservation efforts should focus on most southern populations and on the northern and central stands exhibiting high levels of genetic diversity. Additional mtDNA sequence analysis confirmed that P. martinezii (Patterson) is not conspecific with P. chihuahuana, and thus deserves separate conservation efforts.