Genomic scale data shows that Parastacus nicoleti encompasses more than one species of burrowing continental crayfishes and that lineage divergence occurred with and without gene flow.

Delimiting species is a challenge, especially in scenarios of diversification with gene flow and when species are now allopatric where reproductive isolation cannot be directly tested. Continental burrowing crayfishes of the genus Parastacus present a disjoint distribution in southern South America. One of the species is P. nicoleti, which lives in underground waters in swampy and wooded areas of southern Chile. A previous assessment based on mitochondrial DNA sequences suggest that the taxon may represent a species complex. Here, using thousands of nuclear genomic single-nucleotide polymorphisms obtained via RADSeq from 81 specimens collected at 27 localities throughout the distributional range of the species, we apply an integrative species delimitation approach to test species boundaries and to investigate some aspects of the speciation process. Our analyses corroborate previous results; a scenario that we favor suggests that the P. nicoleti encompasses seven distinct species. Additionally, demographic analyses show that the distinct species have followed distinct trajectories in size change during the last 17.5 million years and that speciation in this group occurred both in strict isolation as well as in the presence of gene flow.

Geography and past climate changes have shaped the evolution of a widespread lizard from the Chilean hotspot.

The complex orogenic history and structure of Southern South America, coupled with Pleistocene glacial cycles, have generated paleoclimatic and environmental changes that influenced the spatial distribution and genetic composition of natural populations. Despite the increased number of phylogeographic studies in this region and given the frequent idiosyncratic phylogeographic patterns, there is still the need to focus research especially on species that are currently distributed within a wide range of bioclimatic regimes, and that historically have been subject to contrasting scenarios. Liolaemus tenuis is a widely distributed lizard species inhabiting latitudinally in almost 1000km through central and southern Chile. Here we describe the geographical patterns of genetic variation and lineage diversification within L. tenuis, and their association with geography and Pleistocene glaciations, using sequences from one mitochondrial and two nuclear genes, and five microsatellite loci, and covering most of the species distributional range. Our results revealed a high diversity both within and among populations, as well as two phylogeographic breaks, which are consistent with two of the larger rivers of central Chile, the Maipo and Biobío Rivers. Liolaemus tenuis is characterized by several allopatric lineages, especially in its north and central range, which suggest a history of multiple vicariance processes. Conversely, populations found in the southern range, south of the Biobío River, show signatures of recent decreases in effective population sizes, coupled with recent range expansions and secondary contact. Niche "envelope" data are consistent with patterns of genetic variation; both suggest a history of discontinuous areas of relatively stable populations throughout all of the distribution of L. tenuis. These data are also consistent with higher probabilities of habitat suitability north of the Maipo River (ca. 33°S), in both coastal areas and the "Intermediate Depression" between 34° and 37°S, as well as in the southern Coastal Cordillera between the Biobío and Araucanía regions. Interestingly, both molecular and niche envelope modeling data suggest that some populations may have persisted in fragmented refugia in Andean valleys, within the limits of the ice sheet. Finally, our results suggest that several populations of L. tenuis colonized glaciated regions from refugial areas in lowlands and coastal regions, and in the southern distribution, historic migration events would have occurred from refugial areas within the limits of the ice sheet.

Evolution and Conservation on Top of the World: Phylogeography of the Marbled Water Frog (Telmatobius marmoratus Species Complex; Anura, Telmatobiidae) in Protected Areas of Chile.

The Andean Altiplano has served as a complex setting throughout its history, driving dynamic processes of diversification in several taxa. We investigated phylogeographic processes in the Telmatobius marmoratus species complex occurring in this region by studying the geographic patterns of genetic variability, genealogies, and historical migration, using the cytochrome b (cyt-b) gene as a marker. DNA sequences from Telmatobius gigas and Telmatobius culeus, Bolivian species with an uncertain taxonomic status, were also included. Additionally, we evaluated the phylogenetic diversity (PD) represented within Chilean protected areas and the complementary contribution from unprotected populations. Phylogenetic reconstructions from 148 cyt-b sequences revealed 4 main clades, one of which corresponded to T. culeus. T. gigas was part of T. marmoratus clade indicating paraphyletic relationships. Haplotypes from Chilean and Bolivian sites were not reciprocally monophyletic. Geographic distribution of lineages, spatial Bayesian analysis, and migration patterns indicated that T. marmoratus displays a weaker geographic structure than expected based on habitat distribution and physiological requirements. Demographic and statistical phylogeography analyses pointed out to a scenario of recent population expansion and high connectivity events of a more recent age than the post Last Glacial Maximum, probably associated to more humid events in Altiplano. PD of T. marmoratus populations within protected areas represents 55.6% of the total estimated PD. The unprotected populations that would contribute the most to PD are Caquena and Quebe (21%). Recent evolutionary processes and paleoclimatic changes, potentially driving shifts in habitat connectivity levels and population sizes, could explain the phylogeographic patterns recovered herein.

Evolving in islands of mud: old and structured hidden diversity in an endemic freshwater crayfish from the Chilean hotspot.

Parastacus is a genus of South American freshwater crayfishes disjunctively distributed in southern Chile, Northern Argentina, Uruguay and Southeastern Brazil. Parastacus pugnax is a Chilean endemic distributed along 700 km of latitude in central-southern Chile from the Pacific coast to the Andean piedmont, which is intensively captured for consumption for local communities. Considering the habitat (wet meadows) and natural history (primary burrower, non-migrant) of the species, we tested a hypothesis of highly structured genetic diversity using mtDNA of 465 specimens gathered at 56 localities across the species range. The crown age of P. pugnax was estimated at 38 Ma, predating the main Andean uplift. The genetic variation of P. pugnax is large and geographically structured. In some cases, genetic groups do not match basin limits, suggesting a previous to current dynamic of basin evolution. The uncovered intraspecific main lineages have different demographic histories. A latitudinal cline in past effective population size reduction suggests environmental singularities with a glacial effect in the southern populations. We suggest adding morphologic and more genetic data in order to assess species limits. Our results contribute to improve future conservation actions for this taxon, providing basic information to delimit conservation units.

Aerobic and anaerobic metabolic scaling in the burrowing freshwater crayfish Parastacus pugnax.

Metabolic scaling is a well-known biological pattern. Theoretical scaling exponents near 0.67 and 0.75 are the most widely accepted for aerobic metabolism, but little is known about the scaling of anaerobic metabolism. Furthermore, metabolic scaling has been mainly evaluated in organisms primarily relying on aerobic pathways. Here we evaluate both aerobic and anaerobic metabolic scaling in Parastacus pugnax, a burrowing freshwater crayfish endemic to Chile, which inhabits waters with low pO2 (~ 1 mg O2 L-1, measured in this study). We determined the metabolic rate, total oxidative capacity (Electron Transport System: ETS), critical oxygen tension (Pcrit) and muscular Lactate dehydrogenase (LHD) and Malate dehydrogenase (MDH) enzymatic activities (proxies of anaerobic metabolism) over a wide range of P. pugnax sizes (0.24-42.93 g wet mass). Aerobic metabolism scaled with crayfish size with an exponent of 0.78, remarkably similar to the 0.73 which scaled the ETS, the enzymatic complex behind respiration. Critical partial pressure of oxygen (Pcrit) was calculated as 15.6 ± 2.9 mmHg, showing that aerobic metabolism was efficiently maintained until ~ 10% air saturation. Below this threshold, P. pugnax switched to anaerobic metabolism, evidenced by a reduction in aerobic metabolism and ETS activity under chronic low oxygen conditions. None of the activities of MDH, LDH, their ratio (MDH/LDH), nor Pcrit scaled with crayfish size, indicating that these animals are equally adapted to hypoxic environments throughout their whole ontogeny. Given the particularities of its habitat, the information presented here is valuable for a proper management and successful conservation.

Assessing signals of selection and historical demography to develop conservation strategies in the Chilean emblematic Araucaria araucana.

Loss of genetic diversity reduces the ability of species to evolve and respond to environmental change. Araucaria araucana is an emblematic conifer species from southern South America, with important ethnic value for the Mapuche people (Pehuenche); the Chilean Government has catalogued its conservation status as vulnerable. Climatic fluctuations were potentially a major impact in the genetic variation within many tree species. In this context, the restricted geographic distribution of A. araucana in Chile appears to be a consequence of the Last Glacial Maximum (LGM). During the past two centuries, strong human intervention has also affected the geographical distribution and population sizes of A. araucana. Reduction of population size may cause loss of genetic diversity, which could affect frequency of adaptive loci. The aims of this study were to know the existence of potential loci under selection and populations with genetic, demographic disequilibrium in the Chilean distribution of A. araucana. Based on 268 polymorphic AFLP loci, we have investigated potential loci under selection and genetic, demographic disequilibrium within seven Chilean populations of Araucaria araucana. Correlation of 41 outlier loci with the environmental variables of precipitation and temperature reveals signatures of selection, whereas 227 neutral loci provide estimates of demographic equilibrium and genetic population structure. Three populations are recommended as priorities for conservation.

Comparative phylogeography of codistributed species of Chilean Liolaemus (Squamata: Tropiduridae) from the central-southern Andean range.

In this study, we used a recently developed supertrees method to test for shared phylogeographical signal in partially overlapping geographical ranges of lizards of the genus Liolaemus from the Andean Range in south-central Chile. We reconstruct mtDNA gene trees for three partially codistributed species (Liolaemus tenuis, L. lemniscatus and L. pictus), and our sampling effort is sufficient to allow statistical tests of shared signal between the combinations L. tenuis-L. pictus, and L. tenuis-L. lemniscatus. For both combinations, standardized maximum agreement subtrees scores showed statistically significant signal for shared pattern in regions of overlap, as evaluated by randomization tests (P < 0.001 and < 0.05, respectively). The matrix representation with parsimony tree obtained from the combination of the three different gene trees revealed concordant phylogeographical associations of all species, and was consistent with the geographical association of intraspecific haploclades with three Chilean bioclimatic zones. A multidimensional scaling analysis of several climate variables showed highly significant differences among these zones, which further suggests that they may have contributed to similar patterns of intraspecific divergence across all three species. In the mesomorphic zone in Central Chile, the species L. tenuis and L. lemniscatus may have codiverged in response to shared orogenic vicariant events, which likely predominated over climatic events associated with cycles of glacial advance and retreat. In the hygromorphic zone in southern Chile, however, glacial cycles likely predominated in structuring the phylogeographical histories of L. tenuis and L. pictus, although important ecological differences between these two caution against broad generalizations at this point.

First steps towards assessing the evolutionary history and phylogeography of a widely distributed Neotropical grassland bird (Motacillidae: Anthus correndera).

Grasslands in southern South America are extensive ecosystems which harbor a unique biodiversity; however, studies on the evolution of their taxa are scarce. Here we studied the phylogeography and population history of the Correndera Pipit (Anthus correndera), a grassland specialist bird with a large breeding distribution in southern South America, with the goals of investigating its phylogeographic history and relate it to the historical development of South American grasslands. The mitochondrial NADH dehydrogenase subunit II gene (ND2) was sequenced in 66 individuals from 19 localities and the intron 9 of the sex-linked gene for aconitase (ACOI9) was sequenced from a subset of those individuals, including all five subspecies of A. correndera, as well as the closely related A. antarcticus. Phylogenetic analysis revealed two distinct lineages within the complex: the first (A) corresponding to Andean subspecies A. c. calcaratus and A. c. catamarcae and the second (B) including birds traditionally assigned to A. c. correndera, A. c. chilensis, A. c. grayi and some individuals of A. c. catamarcae. A. antarcticus is nested within this second lineage. These results were also supported by evidence of niche divergence for variables associated with precipitation. The oldest split between clade A and B was estimated at c. 0.37 Mya, during the middle Pleistocene. Species distribution models for the present and the Last Glacial Maximum (LGM) suggest that grassland areas in southern South America remained relatively stable, in contrast to the general view of a reduction in grassland cover in South America since the LGM. Recent divergences and low phylogeographic structure (for lowland vs. highland geographic groups, intra-population genetic variance was greater than inter-groups; e.g., for ACOI9: 95.47% and ND2: 51.51% respectively), suggest widespread gene flow between lowland populations.

Phylogenomic analysis of the Chilean clade of Liolaemus lizards (Squamata: Liolaemidae) based on sequence capture data.

The genus Liolaemus is one of the most ecologically diverse and species-rich genera of lizards worldwide. It currently includes more than 250 recognized species, which have been subject to many ecological and evolutionary studies. Nevertheless, Liolaemus lizards have a complex taxonomic history, mainly due to the incongruence between morphological and genetic data, incomplete taxon sampling, incomplete lineage sorting and hybridization. In addition, as many species have restricted and remote distributions, this has hampered their examination and inclusion in molecular systematic studies. The aims of this study are to infer a robust phylogeny for a subsample of lizards representing the Chilean clade (subgenus Liolaemus sensu stricto), and to test the monophyly of several of the major species groups. We use a phylogenomic approach, targeting 541 ultra-conserved elements (UCEs) and 44 protein-coding genes for 16 taxa. We conduct a comparison of phylogenetic analyses using maximum-likelihood and several species tree inference methods. The UCEs provide stronger support for phylogenetic relationships compared to the protein-coding genes; however, the UCEs outnumber the protein-coding genes by 10-fold. On average, the protein-coding genes contain over twice the number of informative sites. Based on our phylogenomic analyses, all the groups sampled are polyphyletic. Liolaemus tenuis tenuis is difficult to place in the phylogeny, because only a few loci (nine) were recovered for this species. Topologies or support values did not change dramatically upon exclusion of L. t. tenuis from analyses, suggesting that missing data did not had a significant impact on phylogenetic inference in this data set. The phylogenomic analyses provide strong support for sister group relationships between L. fuscus, L. monticola, L. nigroviridis and L. nitidus, and L. platei and L. velosoi. Despite our limited taxon sampling, we have provided a reliable starting hypothesis for the relationships among many major groups of the Chilean clade of Liolaemus that will help future work aimed at resolving the Liolaemus phylogeny.

Low Genetic Diversity in Diplomystes camposensis, an Endemic and Endangered Catfish from South Chile.

Carlos P. Muñoz-Ramírez, Evelyn Habit, Peter J. Unmack, Jerald B. Johnson, and Pedro F. Victoriano (2016) Despite the fundamental importance of the family Diplomystidae for understanding catfish evolution, its species are poorly known and most of them endangered. Diplomystes camposensis, restricted to a single river basin in southern Chile, is perhaps the most vulnerable species due to its small geographic range and imminent habitat alterations by dam constructions. Using mitochondrial DNA sequences, we describe the genetic diversity across its entire distribution in the Valdivia basin and test hypotheses related to the impact of glacial cycles on the genetic diversity and structure. We found that Diplomystes camposensis has low genetic diversity and structure across the entire Valdivia basin along with a pattern of decreasing nucleotide and haplotype diversity from West to East. Demographic analyses showed evidence of population expansion in agreement with the glaciated history of the basin. Analyses of population structure showed no evidence of population subdivision. However, coalescent analyses indicated that very recent subdivision (in the last 50 years) cannot be ruled out. Low genetic diversity and genetic structure across the entire basin suggest that the species might be highly vulnerable to habitat fragmentation. Thus, the imminent construction of hydropower dams represents a serious threat to its conservation. Our results suggest that the low genetic diversity can be the product of the glaciated history of the basin, although the influence of species-specific biological traits may also add to this condition. Despite the overall low genetic diversity, higher diversity was found in the central portion of the basin suggesting high priority of conservation for this area as it might be used as a source population in case translocations are required among potential management plans.

Lizards on ice: evidence for multiple refugia in Liolaemus pictus (Liolaemidae) during the last glacial maximum in the Southern Andean beech forests.

Historical climate changes and orogenesis are two important factors that have shaped intraspecific biodiversity patterns worldwide. Although southern South America has experienced such complex events, there is a paucity of studies examining the effects on intraspecific diversification in this part of the world. Liolaemus pictus is the southernmost distributed lizard in the Chilean temperate forest, whose genetic structure has likely been influenced by Pleistocene glaciations. We conducted a phylogeographic study of L. pictus in Chile and Argentina based on one mitochondrial and two nuclear genes recovering two strongly divergent groups, Northern and Southern clades. The first group is distributed from the northernmost limit of the species to the Araucanía region while the second group is distributed throughout the Andes and the Chiloé archipelago in Southern Chile. Our results suggest that L. pictus originated 751 Kya, with divergence between the two clades occurring in the late Pleistocene. Demographic reconstructions for the Northern and Southern clades indicate a decrease in effective population sizes likely associated with Pleistocene glaciations. Surprisingly, patterns of genetic variation, clades age and historical gene flow in populations distributed within the limits of the Last Glacial Maximum (LGM) are not explained by recent colonization. We propose an "intra-Andean multiple refuge" hypothesis, along with the classical refuge hypothesis previously proposed for the biota of the Chilean Coastal range and Eastern Andean Cordillera. Our hypothesis is supported by niche modelling analysis suggesting the persistence of fragments of suitable habitat for the species within the limits of the LGM ice shield. This type of refuge hypothesis is proposed for the first time for an ectothermic species.