Species tree analyses and speciation-based species delimitation support new species in the relict catfish family Diplomystidae and provide insights on recent glacial history in Patagonia.

Diplomystidae is an early-diverged family of freshwater catfish endemic to southern South America. We have recently collected five juvenile specimens belonging to this family from the Bueno River Basin, a basin which the only previous record was a single juvenile specimen collected in 1996. This finding confirms the distribution of the family further South in northern Patagonia, but poses new questions about the origin of this population in an area with a strong glacial history. We used phylogenetic analyses to evaluate three different hypotheses that could explain the origin of this population in the basin. First, the population could have originated in Atlantic basins (East of the Andes) and dispersed to the Bueno Basin after the Last Glacial Maximum (LGM) via river reversals, as it has been proposed for other population of Diplomystes as well as for other freshwater species from Patagonia. Second, the population could have originated in the geographically close Valdivia Basin (West of the Andes) and dispersed south to its current location in the Bueno Basin. Third, regardless of its geographic origin (West or East of the Andes), the Bueno Basin population could have a longer history in the basin, surviving in situ through the LGM. In addition, we conducted species delimitation analyses using a recently developed method that uses a protracted model of speciation. Our goal was to test the species status of the Bueno Basin population along with another controversial population in Central Chile (Biobío Basin), which appeared highly divergent in previous studies with mtDNA. The phylogenetic analyses showed that the population from the Bueno Basin is more related to Atlantic than to Pacific lineages, although with a deep divergence that predated the LGM, supporting in situ survival rather than postglacial dispersal. In addition, these analyses also showed that the species D. nahuelbutaensis is polyphyletic, supporting the need for a taxonomic reevaluation. The species delimitation analyses supported two new species which are described using molecular diagnostic characters: Diplomystes arratiae sp. nov. from the Biobío, Carampangue, and Laraquete basins, maintaining D. nahuelbutaensis valid only for the Imperial Basin, and Diplomystes habitae sp. nov. from the Bueno Basin. This study greatly increases the number of species within both the family Diplomystidae and Patagonia, and contributes substantially to the knowledge of the evolution of southern South American freshwater biodiversity during its glacial history. Given the important contribution to the phylogenetic diversity of the family, we recommend a high conservation priority for both new species. Finally, this study highlights an exemplary scenario where species descriptions based only on DNA data are particularly valuable, bringing additional elements to the ongoing debate on DNA-based taxonomy.

High genetic diversity and low-population differentiation in the Patagonian sprat (Sprattus fuegensis) based on mitochondrial DNA.

The Patagonian sprat, Sprattus fuegensis, is a small pelagic marine fish that inhabits the continental shelf along the coasts of Chilean Patagonian and Argentina, a distribution that was highly impacted during the Last Glacial Maximum (LGM). In order to identify how the LGM played a role on the current observed genetic diversity and population structure of S. fuegensis, we analyzed 1438 nucleotide positions from the control region of 335 individuals collected at 12 sites across its distribution. Genetic diversity and differentiation indices were calculated to identify population structure, and a Bayesian skyride plot (BSRP) reconstruction was carried out to infer the historic population dynamics. Extremely high genetic diversity was found at all locations analyzed, non-population structure was found across its distribution, and the BSRP showed two increases in effective population size over time. Our outcomes suggest that the current genetic diversity, population structure and population expansion may have occurred during the medium and late Pleistocene.

Tabula rasa in the Patagonian Channels? The phylogeography of Oreobolus obtusangulus (Cyperaceae).

The extent of the Pleistocene glaciations in the Patagonian Channel region (southwesternmost South America) and their impact on the vegetation there are largely unknown. Whether the regional flora was wiped out completely (tabula rasa) or survived in ice-free pockets (in situ survival) is still an open question. The molecular imprint of either scenario should still be visible in extant populations. Therefore, DNA sequence data of Oreobolus obtusangulus Gaudich. (Cyperaceae) were analysed. This species is an abundant constituent of Patagonian cushion peat bogs, one of the Patagonian Channel region's major vegetation types. Three hundred and eighty-four individuals from 48 populations were sequenced for two chloroplast (ycf3-psaA and trnQUUG -psbK intergenic spacers) and 14 nuclear loci containing simple sequence repeats (SSRs; microsatellites). Phylogenetic reconstructions and the geographic distribution of genetic diversity revealed that the species was split into three main lineages whose general distributions comprise three separate major regions, that is, south-central Chile, Fuego-Patagonia and the East Patagonian Andes, which probably constitute glacial refugia. Postglacial migration fronts formed a suture zone with high levels of genetic diversity in the Northwest Patagonian Andes, where remnants of a supposedly ancestral lineage were also found to be locally restricted to a single population (Huinay). The heavily glaciated Patagonian Channels were likely recolonized from the northwest, and partly from the south. Although the westernmost Patagonian Channel population (Estero Bachem) harboured private SSR alleles (singletons) and showed slightly elevated genetic diversity, it remained unclear whether this population actually survived in situ. This study helps fill a major gap in reconstructing the Pleistocene vegetation history of West and Andean Patagonia.

Phylogeographic structure of Canthon cyanellus (Coleoptera: Scarabaeidae), a Neotropical dung beetle in the Mexican Transition Zone: Insights on its origin and the impacts of Pleistocene climatic fluctuations on population dynamics.

Canthon cyanellus is a roller dung beetle with a wide distribution range in the tropical forests of the New World. In Mexico, it inhabits the Pacific and the Gulf coasts, the Yucatan Peninsula and the south mainly in the State of Chiapas. This species shows a wide geographical variation in cuticle color, which has been used as defining trait for subspecies. In this study we analyzed the phylogeographic and demographic history of the Mexican populations of C. cyanellus using DNA sequences of the nuclear ITS2, and the mitochondrial COI and 16S genes. We found that not all the current valid subspecies are supported by the molecular analysis. The populations are genetically and geographically structured in five lineages. The diversification events that gave origin to the main lineages within this species complex occurred during the Pleistocine in a time range of 1.63-0.91Myr. The demographic history of these lineages suggests post-glacial expansions toward the middle and the end of the Pleistocene. The combined data of mitochondrial and nuclear DNA suggest that the phylogeographic structure and demographic history of the C. cyanellus populations are the result of: the geological and volcanic activity that occurred from the end of the Pliocene to the Pleistocene; and the contraction and expansion of tropical forests due to the glacial and inter-glacial cycles during the Pleistocene. Landscape changes derived from historical events have affected the demographic history of the populations of this species. The results presented here point to the need to review the taxonomic status and delimitation of the lineages encompassed in the Canthon cyanellus complex.

Drift effects on the multivariate floral phenotype of Calceolaria polyrhiza during a post-glacial expansion in Patagonia.

Quaternary environmental changes substantially impacted the landscape and promoted rapid evolutionary changes in many species; however, analyses of adaptive phenotypic variation in plants have usually neglected the underlying historical context. Here, we associate phylogeography and phenotypic evolution by analysing the divergence of Calceolaria polyrhiza multivariate floral phenotype after a Pleistocene post-glacial expansion in Patagonia. Phenotypic matrix (P) properties (size, shape, orientation and phenotypic integration) of six refugium and six recent populations from two different phylogroups were compared following different approaches. We found that P-matrix shape and orientation remained stable despite the strong phylogeographic footprint of post-glacial expansion. However, average proportional reductions in matrix size supported the expectation that drift had a significant effect on the floral phenotype in the northern phylogroup. When phylogeographic history was not included in the analyses, the results overestimated phenotypic differences, whereas under explicit phylogeographic control, drift appeared as the best explanation for matrix differences. In general, recent populations showed a larger phenotypic divergence among them, but a lower overall phenotypic variation than refugium populations. Random Skewers analyses indicated a lower potential response to selection in recently colonized populations than in refugium populations. We discuss that the combination of phylogeographic analyses with geographical distribution of functional phenotypic (genotypic) variation is critical not only to understand how historical effects influence adaptive evolution, but also to improve field comparisons in evolutionary ecology studies.

Genetic divergence in the common bush-tanager Chlorospingus ophthalmicus (Aves: Emberizidae) throughout Mexican cloud forests: The role of geography, ecology and Pleistocene climatic fluctuations.

By integrating mitochondrial DNA (mtDNA), microsatellites and ecological niche modelling (ENM), we investigated the phylogeography of Mexican populations of the common bush-tanager Chlorospingus ophthalmicus to examine the relative role of geographical and ecological features, as well as Pleistocene climatic oscillations in driving the diversification. We sequenced mtDNA of individuals collected throughout the species range in Mexico and genotyped them at seven microsatellite loci. Phylogeographic, population genetics and coalescent methods were used to assess patterns of genetic structure, gene flow and demographic history. ENM was used to infer contractions and expansions at different time periods as well as differences in climatic conditions among lineages. The retrieved mitochondrial and microsatellite groups correspond with the fragmented cloud forest distribution in mountain ranges and morphotectonic provinces. Differing climatic conditions between mountain ranges were detected, and palaeodistribution modelling as well as demographic history analyses, indicated recent population expansions throughout the Sierra Madre Oriental (SMO). The marked genetic structure of C. ophthalmicus was promoted by the presence of ecological and geographical barriers that restricted the movement of individuals among mountain ranges. The SMO was mainly affected by Pleistocene climatic oscillations, with the moist forests model best fitting the displayed genetic patterns of populations in this mountain range.

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.

Phylogeography and palaeodistribution modelling of Nassauvia subgenus Strongyloma (Asteraceae): exploring phylogeographical scenarios in the Patagonian steppe.

The Patagonian steppe is an immense, cold, arid region, yet phylogeographically understudied. Nassauvia subgen. Strongyloma is a characteristic element of the steppe, exhibiting a continuum of morphological variation. This taxon provides a relevant phylogeographical model not only to understand how past environmental changes shaped the genetic structure of its populations, but also to explore phylogeographical scenarios at the large geographical scale of the Patagonian steppe. Here, we (1) assess demographic processes and historical events that shaped current geographic patterns of haplotypic diversity; (2) analyze hypotheses of isolation in refugia, fragmentation of populations, and/or colonization of available areas during Pleistocene glaciations; and (3) model extant and palaeoclimatic distributions to support inferred phylogeographical patterns. Chloroplast intergenic spacers, rpl32-trnL and trnQ-5'rps16, were sequenced for 372 individuals from 63 populations. Nested clade analysis, analyses of molecular variance, and neutrality tests were performed to assess genetic structure and range expansion. The present potential distribution was modelled and projected onto a last glacial maximum (LGM) model. Of 41 haplotypes observed, ten were shared among populations associated with different morphological variants. Populations with highest haplotype diversity and private haplotypes were found in central-western and south-eastern Patagonia, consistent with long-term persistence in refugia during Pleistocene. Palaeomodelling suggested a shift toward the palaeoseashore during LGM; new available areas over the exposed Atlantic submarine platform were colonized during glaciations with postglacial retraction of populations. A scenario of fragmentation and posterior range expansion may explain the observed patterns in the center of the steppe, which is supported by palaeomodelling. Northern Patagonian populations were isolated from southern populations by the Chubut and the Deseado river basins during glaciations. Pleistocene glaciations indirectly impacted the distribution, demography, and diversification of subgen. Strongyloma through decreased winter temperatures and water availability in different areas of its range.

Phylogeography of the ancient catfish family Diplomystidae: biogeographic, systematic, and conservation implications.

The catfish family Diplomystidae is one of the earliest branching lineages within the diverse order Siluriformes and shows a deep phylogenetic split from all other extant and extinct major catfish groups. Despite its relevance in the evolution of siluriforms, phylogenetic relationships within the Diplomystidae are poorly understood, and prior to this study, no phylogenetic hypotheses using molecular data had been published. By conducting a phylogeographic study across the entire distribution of the family, that encompasses river systems from Central-South Chile and Argentina, we provide the first molecular phylogenetic hypothesis among all known species of Diplomystidae, and in addition, investigate how their evolutionary history relates to major historical events that took place in southern South America. Our phylogenetic analyses show four main lineages and nine sub-lineages strongly structured geographically. All Pacific basin populations, with one exception (those found in the Baker basin) clustered within three of the four main lineages (clades I-III), while all populations from Atlantic basins and those from the Baker basin clustered in a single main clade (clade IV). There was a tendency for genetic diversity to decrease from north to south for Pacific basins consistent with an increasing north-south ice coverage during the last glacial maximum. However, we did not find a statistically significant correlation between genetic diversity and latitude. Analysis of molecular variance (AMOVA) showed that river basins and the barrier created by the Andes Mountains explained a high percentage of the genetic variation. Interestingly, most of the genetic variation among drainages was explained among Pacific basins. Molecular phylogenetic analyses agree only partially with current systematics. The geographical distribution of main lineages did not match species distribution and suggests a new taxonomic hypothesis with support for four species of Diplomystes, three species distributed allopatrically from the Rapel to the Valdivia basin, and only one species distributed in Baker and Atlantic basins. High genetic differentiation among river basins suggests that conservation efforts should focus on protecting populations in each basin in order to preserve the genetic diversity of one of the oldest groups of catfishes on the earth today.