Revisiting phylogeny, systematics, and biogeography of a Pleistocene radiation.

PREMISE: Pilosocereus (Cactaceae) is an important dry forest element in all subregions and transitional zones of the neotropics, with the highest diversity in eastern Brazil. The genus is subdivided into informal taxonomic groups; however, most of these are not supported by recent molecular phylogenetic inferences. This lack of confidence is probably due to the use of an insufficient number of loci and the complexity of cactus diversification. Here, we explored the species relationships in Pilosocereus in more detail, integrating multilocus phylogenetic approaches with the assessment of the ancestral range and the effect of geography on diversification shifts. METHODS: We used 28 nuclear, plastid, and mitochondrial loci from 54 plant samples of 31 Pilosocereus species for phylogenetic analyses. We used concatenated and coalescent phylogenetic trees and Bayesian models to estimate the most likely ancestral range and diversification shifts. RESULTS: All Pilosocereus species were clustered in the same branch, except P. bohlei. The phylogenetic relationships were more associated with the geographic distribution than taxonomic affinities among taxa. The genus began diversifying during the Plio-Pleistocene transition in the Caatinga domain and experienced an increased diversification rate during the Calabrian age. CONCLUSIONS: We recovered a well-supported multispecies coalescent phylogeny. Our results refine the pattern of rapid diversification of Pilosocereus species across neotropical drylands during the Pleistocene and highlight the need for taxonomic rearrangements in the genus. We recovered a pulse of diversification during the Pleistocene that was likely driven by multiple dispersal and vicariance events within and among the Caatinga, Cerrado, and Atlantic Forest domains.

Exploring Phylogenetic Relationships and Divergence Times of Bioluminescent Species Using Genomic and Transcriptomic Data.

Next-generation sequencing (NGS) has dominated the scene of genomics and evolutionary biology as a great amount of genomic data have been accumulated for a diverse set of species. At the same time, phylogenetic approaches and programs are in development to allow better use of such large-size datasets. Phylogenomics appears as a promising field to accommodate and explore all the information of NGS data in phylogenetic methods, being an important approach to investigate the evolution of bioluminescence in different organisms. To guarantee accurate results in phylogenomic studies, it is mandatory to correctly identify orthologous genes in phylogenetic reconstruction. Here, we show a simplified step-by-step framework to perform phylogenetic analysis along with divergence time estimation, beginning with an orthologous search. As empirical data, we exemplify transcriptome sequences of six species of the Elateroidea superfamily (Coleoptera). We introduce several bioinformatics tools for handling genomic data, especially those available in the software OrthoFinder, IQTREE, BEAST2, and TreePL.

Localized Phylogenetic Discordance Among Nuclear Loci Due to Incomplete Lineage Sorting and Introgression in the Family of Cotton and Cacao (Malvaceae).

The economically important cotton and cacao family (Malvaceae sensu lato) have long been recognized as a monophyletic group. However, the relationships among some subfamilies are still unclear as discordant phylogenetic hypotheses keep arising when different sources of molecular data are analyzed. Phylogenetic discordance has previously been hypothesized to be the result of both introgression and incomplete lineage sorting (ILS), but the extent and source of discordance have not yet been evaluated in the context of loci derived from massive sequencing strategies and for a wide representation of the family. Furthermore, no formal methods have been applied to evaluate if the detected phylogenetic discordance among phylogenomic datasets influences phylogenetic dating estimates of the concordant relationships. The objective of this research was to generate a phylogenetic hypothesis of Malvaceae from nuclear genes, specifically we aimed to (1) investigate the presence of major discordance among hundreds of nuclear gene histories of Malvaceae; (2) evaluate the potential source of discordance; and (3) examine whether discordance and loci heterogeneity influence on time estimates of the origin and diversification of subfamilies. Our study is based on a comprehensive dataset representing 96 genera of the nine subfamilies and 268 nuclear loci. Both concatenated and coalescence-based approaches were followed for phylogenetic inference. Using branch lengths and topology, we located the placement of introgression events to directly evaluate whether discordance is due to introgression rather than ILS. To estimate divergence times, concordance and molecular rate were considered. We filtered loci based on congruence with the species tree and then obtained the molecular rate of each locus to distribute them into three different sets corresponding to shared molecular rate ranges. Bayesian dating was performed for each of the different sets of loci with the same parameters and calibrations. Phylogenomic discordance was detected between methods, as well as gene histories. At deep coalescent times, we found discordance in the position of five subclades probably due to ILS and a relatively small proportion of introgression. Divergence time estimation with each set of loci generated overlapping clade ages, indicating that, even with different molecular rate and gene histories, calibrations generally provide a strong prior.

Phylogenomic systematics of the spotted skunks (Carnivora, Mephitidae, Spilogale): Additional species diversity and Pleistocene climate change as a major driver of diversification.

Four species of spotted skunks (Carnivora, Mephitidae, Spilogale) are currently recognized: Spilogale angustifrons, S. gracilis, S. putorius, and S. pygmaea. Understanding species boundaries within this group is critical for effective conservation given that regional populations or subspecies (e.g., S. p. interrupta) have experienced significant population declines. Further, there may be currently unrecognized diversity within this genus as some taxa (e.g., S. angustifrons) and geographic regions (e.g., Central America) never have been assessed using DNA sequence data. We analyzed species limits and diversification patterns in spotted skunks using multilocus nuclear (ultraconserved elements) and mitochondrial (whole mitogenomes and single gene analysis) data sets from broad geographic sampling representing all currently recognized species and subspecies. We found a high degree of genetic divergence among Spilogale that reflects seven distinct species and eight unique mitochondrial lineages. Initial divergence between S. pygmaea and all other Spilogale occurred in the Early Pliocene (~ 5.0 million years ago). Subsequent diversification of the remaining Spilogale into an "eastern" and a "western" lineage occurred during the Early Pleistocene (~1.5 million years ago). These two lineages experienced temporally coincident patterns of diversification at ~0.66 and ~0.35 million years ago into two and ultimately three distinct evolutionary units, respectively. Diversification was confined almost entirely within the Pleistocene during a timeframe characterized by alternating glacial-interglacial cycles, with the origin of this diversity occurring in northeastern Mexico and the southwestern United States of America. Mitochondrial-nuclear discordance was recovered across three lineages in geographic regions consistent with secondary contact, including a distinct mitochondrial lineage confined to the Sonoran Desert. Our results have direct consequences for conservation of threatened populations, or species, as well as for our understanding of the evolution of delayed implantation in this enigmatic group of small carnivores.

Paraphyly and evolutionary independent lineages in Gymnotus pantherinus (Gymnotiformes: Gymnotidae) in the Brazilian Atlantic Forest Coastal Streams.

In the Brazilian Atlantic Forest (AF), many terrestrial species with broad geographical distributions show high diversity and endemism of intraspecific lineages, as revealed by molecular genetic data. This pattern, however, is less explored in freshwater fishes. Gymnotus pantherinus is an electric fish endemic to the Brazilian coastal drainages that shows a wide distribution, ranging from the states of Bahia to Santa Catarina, an unusual pattern for AF fishes. It has been hypothesized that G. pantherinus is a species complex because distinct morphotypes were described for the species based on morphometric and meristic features. We used mitochondrial and nuclear data to test this hypothesis. Based on phylogenetic inference and multi-locus, multispecies coalescent methods, we identified six independent lineages, flagging them as candidate species. One such lineage is the recently described species G. refugio that is nested within G. pantherinus and renders it paraphyletic, showing it is a species complex. We named G. pantherinus stricto sensu the lineage that includes samples from the type locality (Santos, SP). Our results show that genetic lineages correspond only partially and far exceed the number of previously reported morphotypes. Genetic breaks in the group correspond to landscape features associated with the Serra do Mar mountain range and with riverine dynamics caused by sea level changes during the last glacial maximum. Moreover, we found evidence of river capture events affecting phylogeographic structure in the group. We uncovered an important dimension of diversity in the group and encourage further integration of genetic and phenotypic data. Such integration is a fruitful approach not only to reduce the gap between taxonomy and evolutionary history in Gymnotidae, but also to uncover the real AF biodiversity.

The number of species of degus (genus Octodon) is currently underestimated: An appraisal of species limits and their phylogenetic relationships (Rodentia: Hystricomorpha: Octodontidae).

As currently understood, the genus Octodon contains five species degus, lunatus, bridgesii, pacificus, and ricardojeda. Previous phylogenetic studies suggest that genus specific diversity is underestimated. In order to evaluate the taxonomic diversity of Octodon, we implemented unilocus (cytochrome-b) and multilocus (cytochrome-b + 4 nuclear genes) species delimitation methods. Octodon degus was recovered as a sister of the other species of the genus. The unilocus bGMYC and mPTP methods, based on cytochrome-b sequences, delimits 11 and 7 candidate species respectively, and both methods fail to recognize O. pacificus from O. ricardojeda. Results of the multilocus analysis (BPP) vary as a function of the dataset used. When the five genes are used 11 species are delimited, while eight species are delimited when only the nuclear genes are used. Octodon bridgesii is shown as comprising at least two species (one on the Pacific coast and the typical form found on the Andean slopes), while O. ricardojeda may comprise two species (one on the Chilean side of the Andes and the other in Argentina). Likewise, both multilocus matrices recover O. pacificus as a distinct species. This shows that species diversity of Octodon is underestimated. Remarkably, many of the delimited species based on genetic data are morphologically differentiated in cranio-dental characteristics. However, a pair of species has not achieved morphological differentiation, being cryptic species. Finally, the incongruence between mitochondrial and nuclear phylogenies suggests that processes such as incomplete lineage sorting and/or introgression have been present during the radiation of the genus.

Skull diversity and evolution in miniaturized amphibians, genus Brachycephalus (Anura: Brachycephalidae).

Miniaturized amphibians of the genus Brachycephalus are phenotypically diverse. The species of Brachycephalus have bufoniform or leptodactyliform Baupläne and any of three skeletal states: nonhyperossified, hyperossified without dorsal shield, and hyperossified with dorsal shield. We integrate high-resolution microcomputed tomography, geometric morphometrics, and an estimate of molecular phylogenetic relationships to investigate skull diversity in shape and size-shape space in selected species of Brachycephalus. Skull diversity amongst species of Brachycephalus can be partitioned into shape and size-shape space according to the four conditions of skeletal states-Baupläne, namely, nonhyperossified leptodactyliform, nonhyperossified bufoniform, hyperossified bufoniform without dorsal shield, and hyperossified bufoniform with dorsal shield. Skull diversity in shape and size-shape space in nonhyperossified leptodactyliform species of Brachycephalus is markedly larger, when compared to skull diversity in species of the three other conditions of skeletal states-Baupläne. Variation in skull shape scales with size across Brachycephalus and, therefore, can be explained by allometry. Skull diversity, Baupläne, and skeletal states covary to a large extent with monophyletic lineages of Brachycephalus, as revealed by a mitochondrial DNA species tree. Nonhyperossified bufoniform species and hyperossified bufoniform species with or without dorsal shield are monophyletic lineages, as inferred from a mitochondrial DNA species tree. Nonhyperossified leptodactyliform species of Brachycephalus do not share, however, a most recent common ancestor. The nonhyperossified leptodactyliform species of Brachycephalus, due to their marked skull diversity and lack of monophyly, emerge as evolutionarily complex. Therefore, further sampling of the nonhyperossified leptodactyliform condition of skeletal states-Baupläne will be necessary to further understand the evolutionary history of Brachycephalus.

Os anfíbios miniaturizados do gênero Brachycephalus são fenotipicamente diversos. As espécies de Brachycephalus têm o plano corporal bufoniforme ou leptodactyliforme e três estados esqueléticos: não-hiperossificado, hiperossificado sem placa dorsal e hiperossificado com placa dorsal. Neste trabalho nós integramos tomografia micro-computadorizada de alta resolução, morfometria geométrica e uma estimativa de relações filogenéticas moleculares para investigar diversidade craniana nos espaços de forma e tamanho-forma em determinadas espécies de Brachycephalus. A diversidade craniana entre espécies de Brachycephalus pode ser dividida no espaço de forma e tamanho-forma segundo as quatro condições de plano corporal-esqueleto, a saber, leptodactiliforme não-hiperossificado, bufoniforme não-hiperossificado, bufoniforme hiperossificado sem placa dorsal e bufoniforme hiperossificado com placa dorsal. A diversidade craniana nos espaços de forma e tamanho-forma nas espécies de Brachycephalus leptodactiliformes não-hiperossificadas é pronunciadamente maior quando comparada àquela das espécies nas outras trcs condições de plano corporal-esqueleto. A variação na forma craniana aumenta com o tamanho craniano em Brachycephalus e, portanto, pode ser explicada por alometria. Diversidade craniana, plano corporal e estados esqueléticos covariam consideravelmente com as linhagens monofiléticas de Brachycephalus, como estimado pela filogenia mitocondrial. As espécies de Brachycephalus leptodactiliformes não-hiperossificadas e bufoniformes hiperossificadas com ou sem placa dorsal são linhagens monofiléticas, como estimado pela filogenia mitocondrial. As espécies leptodactiliformes não-hiperossificadas não compartilham, todavia, um ancestral comum mais recente. As espécies de Brachycephalus leptodactiliformes não-hiperossificadas devido a sua pronunciada diversidade e não-monofilia emergem claramente como entidades evolutivamente complexas. Por conseguinte, a amostragem adicional de populações leptodactiliformes não-hiperossificadas será necessária para uma melhor compreensão da história evolutiva do gênero Brachycephalus.

Geographic restriction, genetic divergence, and morphological disparity in the Brazilian Atlantic Forests: Insights from Leposoma lizards (Gymnophthalmidae, Squamata).

Lineage differentiation, long-term persistence, and range limitation promote high levels of phylogenetic and phylogeographic endemisms and likely underlie the abundant morphologically cryptic diversity observed in the Brazilian Atlantic Forests (AF). We explore lineage differentiation and range restriction in the AF and ask if genetic divergence and morphological disparity are correlated by integrating coalescent-based species delimitation, molecular phylogenetic, and morphological analyses in the lizard genus Leposoma. We present the first species tree for Leposoma and of their tribe, the Ecpleopodini. The analyses are based on the largest dataset ever assembled for Leposoma in terms of number of species (all represented), genetic markers (12 loci), and geographic coverage (~2,500 km). The exercise allows us to robustly delimit species within the genus and phylogeographic lineages within all species. We find support for the monophyly of the genus and for the recognition of a yet undescribed species around the Baía de Todos-os-Santos, in the state of Bahia; this form is distinct from all other congeners, both genetically and morphologically. We find that L. baturitensis, from the northeastern state of Ceará, is basal to the genus - and sister to a clade of six species restricted to the AF across the eastern coast of Brazil. Relationships within this coastal clade are ((((L. annectans, Leposoma sp.), L. scincoides), L. puk) (L. nanodactylus, L. sinepollex)). Phylogenetic and phylogeographic analyses, together with precise distribution data, allowed us to update the ranges of species and phylogeographic lineages. We reveal pervasive geographic restriction of divergent lineages in Leposoma at and below species level and discuss how forest refuges and rivers might have contributed to it. We find that morphological disparity lags behind genetic divergence in the genus because although they are correlated, the first accumulates at a much slower rate than the latter. We hope to encourage new studies in the area of AF north of the Doce river; phylogeographic sampling in that region has been much less common relative to southern sites, yet it may hold the key to several important processes defining biodiversity patterns in eastern Brazil. This appears to specially apply to processes underlying geographic restriction of morphologically cryptic, yet genetic divergent lineages, as the case of Leposoma.

A phylogenomic study of Steganinae fruit flies (Diptera: Drosophilidae): strong gene tree heterogeneity and evidence for monophyly.

BACKGROUND: The Drosophilidae family is traditionally divided into two subfamilies: Drosophilinae and Steganinae. This division is based on morphological characters, and the two subfamilies have been treated as monophyletic in most of the literature, but some molecular phylogenies have suggested Steganinae to be paraphyletic. To test the paraphyletic-Steganinae hypothesis, here, we used genomic sequences of eight Drosophilidae (three Steganinae and five Drosophilinae) and two Ephydridae (outgroup) species and inferred the phylogeny for the group based on a dataset of 1,028 orthologous genes present in all species (> 1,000,000 bp). This dataset includes three genera that broke the monophyly of the subfamilies in previous works. To investigate possible biases introduced by small sample sizes and automatic gene annotation, we used the same methods to infer species trees from a set of 10 manually annotated genes that are commonly used in phylogenetics. RESULTS: Most of the 1,028 gene trees depicted Steganinae as paraphyletic with distinct topologies, but the most common topology depicted it as monophyletic (43.7% of the gene trees). Despite the high levels of gene tree heterogeneity observed, species tree inference in ASTRAL, in PhyloNet, and with the concatenation approach strongly supported the monophyly of both subfamilies for the 1,028-gene dataset. However, when using the concatenation approach to infer a species tree from the smaller set of 10 genes, we recovered Steganinae as a paraphyletic group. The pattern of gene tree heterogeneity was asymmetrical and thus could not be explained solely by incomplete lineage sorting (ILS). CONCLUSIONS: Steganinae was clearly a monophyletic group in the dataset that we analyzed. In addition to ILS, gene tree discordance was possibly the result of introgression, suggesting complex branching processes during the early evolution of Drosophilidae with short speciation intervals and gene flow. Our study highlights the importance of genomic data in elucidating contentious phylogenetic relationships and suggests that phylogenetic inference for drosophilids based on small molecular datasets should be performed cautiously. Finally, we suggest an approach for the correction and cleaning of BUSCO-derived genomic datasets that will be useful to other researchers planning to use this tool for phylogenomic studies.

Genetic diversity and species delimitation in Rhamdia (Siluriformes: Heptapteridae) in South America, with a redescription of R. quelen (Quoy amp; Gaimard, 1824).

More than 100 species have been referred to Rhamdia since its description, but in the last revision of the genus more than two decades ago, only eleven species have been considered as valid. Rhamdia quelen was then redefined to include 47 junior synonyms, resulting in a species with a large distribution in the Neotropics, from Mexico to Argentina. Populations of Rhamdia from west of the Andes have been recently removed from the synonymy of R. quelen, and currently there are 27 species recognized in the genus. Most cis-Andean populations, however, remain referred to the R. quelen species complex. In this paper, we use species delimitation methods and multilocus phylogenetic analyses to delimit the species contained in the R. quelen species complex in cis-Andean river drainages. Eighteen MOTUs in the gene tree were recovered with high support. We further demonstrate that the previous designation of a neotype for R. quelen is invalid, and has to be replaced by the proper designation of a neotype from the type locality of the species. The neotype is designated herein and R. quelen is diagnosed and redescribed.

Multilocus phylogeny of Paratelmatobiinae (Anura: Leptodactylidae) reveals strong spatial structure and previously unknown diversity in the Atlantic Forest hotspot.

The Brazilian Atlantic Forest harbors high levels of anuran diversity and endemism, including several taxa restricted to small geographic ranges. Here, we provide a multilocus phylogeny for Paratelmatobiinae, a leptodactylid subfamily composed of small-ranged species distributed in the Brazilian Atlantic Forest and in the campo rupestre ecosystem. We performed Bayesian inference and maximum likelihood analyses using three mitochondrial and five nuclear markers, and a matrix comprising a broad taxonomic sampling. We then delimitated independently evolving lineages within the group. We recovered Paratelmatobiinae and each of its four genera as monophyletic and robustly supported. Five putatively new species included in our analyses were unambiguously supported in the phylogenetic trees and delimitation analyses. We also recovered other deeply divergent and geographically structured lineages within the four genera of Paratelmatobiinae. Our estimation of divergence times indicates that diversification in the subfamily began in the Eocene and continued until the Pleistocene. We discuss possible scenarios of diversification for the four genera of Paratelmatobiinae, and outline the implications of our findings for taxonomy and conservation.

Cryptic molecular diversity in the morphologically variable rotiferan Brachionus quadridentatus (Rotifera: Monogononta)

Brachionus quadridentatus es una especie morfológicamente variable, distribuida por todo el mundo. Su taxonomía es confusa debido a las numerosas variantes infrasubespecíficas descritas en este taxón. Con la taxonomía basada en la morfología, B. quadridentatus tiene tres variantes reconocidas: B. quadridentatus quadridentatus, B. quadridentatus f. brevispinus and B. quadridentatus f. cluniorbicularis. En este estudio, exploramos la diversidad genética entre algunas poblaciones de B. quadridentatus, usando secuencias de los genes COI ADNmt y 18S ARNr. El análisis de delimitación de especies coalescente usando el gen 18S apoya la presencia de al menos tres especies putativas dentro del complejo B. quadridentatus. Estos resultados estuvieron en concordancia con los análisis filogenético y GMYC usando el gen 18S. Sin embargo, se encontró variación en morfología y secuencias del gen COI dentro de cada una de las tres especies putativas. Se encontraron siete linajes delimitados por las secuencias del gen COI usando el método de delimitación ABGD, que además están morfologicamente diferenciadas. Se encontró discordancia mitonuclear entre la filogenia del gen COI y la del gen 18S. La incongruencia entre el marcador mitocondrial y el nuclear puede ser explicada por sorteo incompleto de linaje.

Brachionus quadridentatus is a morphologically variable species of rotifer distributed worldwide. The taxonomy of this species is confused, with numerous infrasubspecific variants described in the taxon: B. quadridentatus quadridentatus, B. quadridentatus f. brevispinus and B. quadridentatus f. cluniorbicularis. In this study, we explored genetic diversity among some populations of B. quadridentatus, using sequences of mitochondrial COI and nuclear 18S genes. The coalescent species delimitation analysis with the 18S gene highly supports the presence of at least three putative species within the B. quadridentatus complex. These results were in agreement with the phylogenetic and GMYC analysis using the 18S gene. However, we also found variation within each of these three putative species in morphology and COI gene sequences. There were seven morphologically differentiated lineages that were recovered as distinct based on COI gene sequences using the ABGD delimitation method. As such, mitonuclear discordance between COI and 18S phylogenies was found. The incongruence between mitochondrial and nuclear markers could be explained by incomplete lineage sorting.

Phylogenetic relationships and historical biogeography of Oligosarcus (Teleostei: Characidae): Examining riverine landscape evolution in southeastern South America.

The pike-characin Oligosarcus is a group of Characidae composed of 22 species, which have mostly allopatric distributed species in southeastern South America and sympatric occurrence of few species. Oligosarcus shares a similar distribution pattern with other fish genera and therefore, can help us to understand biogeographic events that influenced freshwater fish distribution in the southeastern South America. Our paper presents the most extensive taxonomic coverage for molecular analysis of Oligosarcus and uses various methods to examine the evolutionary history of the genus. Phylogenetic relationships among species of Oligosarcus were examined using a multilocus dataset by Maximum Likelihood and Bayesian methods. A relaxed molecular clock was used to estimate lineage divergence times, which provide a framework to examine the biogeographic history of this clade across the drainage basins of southeastern South America. Oligosarcus was resolved as monophyletic with strong support, and related to lineages currently assigned to the genus Astyanax. Within Oligosarcus, two groups of approximately equal species richness were resolved as monophyletic, mainly restricted to continental and coastal drainages of southeastern South America. Oligosarcus radiation is estimated to the late Neogene, with its origin in the Pliocene and most speciation events occurring in the Pleistocene. Some apomorphic characteristics associated with piscivory (e.g. large caniniform teeth) in Oligosarcus likely have evolved once, and are convergent to similar phenotypes observed in a distantly related clade of Astyanax (formerly Bramocharax). In addition, the presence of morphological convergence within the genus Oligosarcus (e.g. trophic morphology) seems to explain the difference between the present molecular hypothesis and some previous morphological studies. Ancestral geographical range estimation using analytical methods (e.g. DIVALIKE and DEC) demonstrated the effects of different Landscape Evolution Models (LEMs) on diversification of Oligosarcus. The results suggest that the two main Oligosarcus clades evolved in allopatry in continental and coastal drainages, with subsequent range extension and vicariance events that established the modern distributions. LEM analyses indicate the importance of formation of riverine barriers across the watershed of the La Plata basin and the effects of sea-level changes during the Pleistocene for delineating lineage distributions of Oligosarcus.

Coalescent-based species delimitation in the sand lizards of the Liolaemus wiegmannii complex (Squamata: Liolaemidae).

Coalescent-based algorithms coupled with the access to genome-wide data have become powerful tools for assessing questions on recent or rapid diversification, as well as delineating species boundaries in the absence of reciprocal monophyly. In southern South America, the diversification of Liolaemus lizards during the Pleistocene is well documented and has been attributed to the climatic changes that characterized this recent period of time. Past climatic changes had harsh effects at extreme latitudes, including Patagonia, but habitat changes at intermediate latitudes of South America have also been recorded, including expansion of sand fields over northern Patagonia and Pampas). In this work, we apply a coalescent-based approach to study the diversification of the Liolaemus wiegmannii species complex, a morphologically conservative clade that inhabits sandy soils across northwest and south-central Argentina, and the south shores of Uruguay. Using four standard sequence markers (mitochondrial DNA and three nuclear loci) along with ddRADseq data we inferred species limits and a time-calibrated species tree for the L. wiegmannii complex in order to evaluate the influence of Quaternary sand expansion/retraction cycles on diversification. We also evaluated the evolutionary independence of the recently described L. gardeli and inferred its phylogenetic position relative to L. wiegmannii. We find strong evidence for six allopatric candidate species within L. wiegmannii, which diversified during the Pleistocene. The Great Patagonian Glaciation (∼1 million years before present) likely split the species complex into two main groups: one composed of lineages associated with sub-Andean sedimentary formations, and the other mostly related to sand fields in the Pampas and northern Patagonia. We hypothesize that early speciation within L. wiegmannii was influenced by the expansion of sand dunes throughout central Argentina and Pampas. Finally, L. gardeli is supported as a distinct lineage nested within the L. wiegmannii complex.

Target sequence capture in the Brazil nut family (Lecythidaceae): Marker selection and in silico capture from genome skimming data.

Reconstructing species trees from multi-loci datasets is becoming a standard practice in phylogenetics. Nevertheless, access to high-throughput sequencing may be costly, especially with studies of many samples. The potential high cost makes a priori assessments desirable in order to make informed decisions about sequencing. We generated twelve transcriptomes for ten species of the Brazil nut family (Lecythidaceae), identified a set of putatively orthologous nuclear loci and evaluated, in silico, their phylogenetic utility using genome skimming data of 24 species. We designed the markers using MarkerMiner, and developed a script, GoldFinder, to efficiently sub-select the best makers for sequencing. We captured, in silico, all designed 354 nuclear loci and performed a maximum likelihood phylogenetic analysis on the concatenated sequence matrix. We also calculated individual gene trees with maximum likelihood and used them for a coalescent-based species tree inference. Both analyses resulted in almost identical topologies. However, our nuclear-loci phylogenies were strongly incongruent with a published plastome phylogeny, suggesting that plastome data alone is not sufficient for species tree estimation. Our results suggest that using hundreds of nuclear markers (i.e. 354) will significantly improve the Lecythidaceae species tree. The framework described here will be useful, generally, for developing markers for species tree inference.

Large ancestral effective population size explains the difficult phylogenetic placement of owl monkeys.

The phylogenetic position of owl monkeys, grouped in the genus Aotus, has been a controversial issue for understanding Neotropical primate evolution. Explanations of the difficult phylogenetic assignment of owl monkeys have been elusive, frequently relying on insufficient data (stochastic error) or scenarios of rapid speciation (adaptive radiation) events. Using a coalescent-based approach, we explored the population-level mechanisms likely explaining these topological discrepancies. We examined the topological variance of 2,192 orthologous genes shared between representatives of the three major Cebidae lineages and the outgroup. By employing a methodological framework that allows for reticulated tree topologies, our analysis explicitly tested for non-dichotomous evolutionary processes impacting the finding of the position of owl monkeys in the cebid phylogeny. Our findings indicated that Aotus is a sister lineage of the callitrichines. Most gene trees (>50%) failed to recover the species tree topology, although the distribution of gene trees mismatching the true species topology followed the standard expectation of the multispecies coalescent without reticulation. We showed that the large effective population size of the common ancestor of Aotus and callitrichines was the most likely factor responsible for generating phylogenetic uncertainty. On the other hand, fast speciation scenarios or introgression played minor roles. We propose that the difficult phylogenetic placement of Aotus is explained by population-level processes associated with the large ancestral effective size. These results shed light on the biogeography of the early cebid diversification in the Miocene, highlighting the relevance of evaluating phylogenetic relationships employing population-aware approaches.

Appropriate Assignment of Fossil Calibration Information Minimizes the Difference between Phylogenetic and Pedigree Mutation Rates in Humans.

Studies that measured mutation rates in human populations using pedigrees have reported values that differ significantly from rates estimated from the phylogenetic comparison of humans and chimpanzees. Consequently, exchanges between mutation rate values across different timescales lead to conflicting divergence time estimates. It has been argued that this variation of mutation rate estimates across hominoid evolution is in part caused by incorrect assignment of calibration information to the mean coalescent time among loci, instead of the true genetic isolation (speciation) time between humans and chimpanzees. In this study, we investigated the feasibility of estimating the human pedigree mutation rate using phylogenetic data from the genomes of great apes. We found that, when calibration information was correctly assigned to the human⁻chimpanzee speciation time (and not to the coalescent time), estimates of phylogenetic mutation rates were statistically equivalent to the estimates previously reported using studies of human pedigrees. We conclude that, within the range of biologically realistic ancestral generation times, part of the difference between whole-genome phylogenetic and pedigree mutation rates is due to inappropriate assignment of fossil calibration information to the mean coalescent time instead of the speciation time. Although our results focus on the human⁻chimpanzee divergence, our findings are general, and relevant to the inference of the timescale of the tree of life.

Reconstructing the origins and the biogeography of species' genomes in the highly reticulate allopolyploid-rich model grass genus Brachypodium using minimum evolution, coalescence and maximum likelihood approaches.

The identification of homeologous genomes and the biogeographical analyses of highly reticulate allopolyploid-rich groups face the challenge of incorrectly inferring the genomic origins and the biogeographical patterns of the polyploids from unreliable strictly bifurcating trees. Here we reconstruct a plausible evolutionary scenario of the diverging and merging genomes inherited by the diploid and allopolyploid species and cytotypes of the model grass genus Brachypodium. We have identified the ancestral Brachypodium genomes and inferred the paleogeographical ranges for potential hybridization events that originated its allopolyploid taxa. We also constructed a comprehensive phylogeny of Brachypodium from five nuclear and plastid genes using Species Tree Minimum Evolution allele grafting and Species Network analysis. The divergence ages of the lineages were estimated from a consensus maximum clade credibility tree using fossil calibrations, whereas ages of origin of the diploid and allopolyploid species were inferred from coalescence Bayesian methods. The biogeographical events of the genomes were reconstructed using a stratified Dispersal-Extinction-Colonization model with three temporal windows. Our combined Minimum Evolution-coalescence-Bayesian approach allowed us to infer the origins and the identities of the homeologous genomes of the Brachypodium allopolyploids, matching the expected ploidy levels of the hybrids. To date, the current extant progenitor genomes (species) are only known for B. hybridum. Putative ancestral homeologous genome have been inherited by B. mexicanum, ancestral and recent genomes by B. boissieri, and only recently evolved genomes by B. retusum and the core perennial clade allopolyploids (B. phoenicoides, B. pinnatum 4x, B. rupestre 4x). We dissected the complex spatio-temporal evolution of ancestral and recent genomes and have detected successive splitting, dispersal and merging events for dysploid homeologous genomes in diverse geographical scenarios that have led to the current extant taxa. Our data support Mid-Miocene splits of the Holarctic ancestral genomes that preceded the Late Miocene origins of Brachypodium ancestors of the modern diploid species. Successive divergences of the annual B. stacei and B. distachyon diploid genomes were implied to have occurred in the Mediterranean region during the Late Miocene-Pliocene. By contrast, a profusion of splits, range expansions and different genome mergings were inferred for the perennial diploid genomes in the Mediterranean and Eurasian regions, with sporadic colonizations and further mergings in other continents during the Quaternary. A reliable biogeographical scenario was obtained for the Brachypodium genomes and allopolyploids where homeologous genomes split from their respective diploid counterpart lineages in the same ancestral areas, showing similar or distinct dispersals. By contrast, the allopolyploid taxa remained in the same ancestral ranges after hybridization and genome doubling events. Our approach should have utility in deciphering the genomic composition and the historical biogeography of other allopolyploid-rich organismal groups, which are predominant in eukaryotes.

GET_PHYLOMARKERS, a Software Package to Select Optimal Orthologous Clusters for Phylogenomics and Inferring Pan-Genome Phylogenies, Used for a Critical Geno-Taxonomic Revision of the Genus Stenotrophomonas.

The massive accumulation of genome-sequences in public databases promoted the proliferation of genome-level phylogenetic analyses in many areas of biological research. However, due to diverse evolutionary and genetic processes, many loci have undesirable properties for phylogenetic reconstruction. These, if undetected, can result in erroneous or biased estimates, particularly when estimating species trees from concatenated datasets. To deal with these problems, we developed GET_PHYLOMARKERS, a pipeline designed to identify high-quality markers to estimate robust genome phylogenies from the orthologous clusters, or the pan-genome matrix (PGM), computed by GET_HOMOLOGUES. In the first context, a set of sequential filters are applied to exclude recombinant alignments and those producing anomalous or poorly resolved trees. Multiple sequence alignments and maximum likelihood (ML) phylogenies are computed in parallel on multi-core computers. A ML species tree is estimated from the concatenated set of top-ranking alignments at the DNA or protein levels, using either FastTree or IQ-TREE (IQT). The latter is used by default due to its superior performance revealed in an extensive benchmark analysis. In addition, parsimony and ML phylogenies can be estimated from the PGM. We demonstrate the practical utility of the software by analyzing 170 Stenotrophomonas genome sequences available in RefSeq and 10 new complete genomes of Mexican environmental S. maltophilia complex (Smc) isolates reported herein. A combination of core-genome and PGM analyses was used to revise the molecular systematics of the genus. An unsupervised learning approach that uses a goodness of clustering statistic identified 20 groups within the Smc at a core-genome average nucleotide identity (cgANIb) of 95.9% that are perfectly consistent with strongly supported clades on the core- and pan-genome trees. In addition, we identified 16 misclassified RefSeq genome sequences, 14 of them labeled as S. maltophilia, demonstrating the broad utility of the software for phylogenomics and geno-taxonomic studies. The code, a detailed manual and tutorials are freely available for Linux/UNIX servers under the GNU GPLv3 license at https://github.com/vinuesa/get_phylomarkers. A docker image bundling GET_PHYLOMARKERS with GET_HOMOLOGUES is available at https://hub.docker.com/r/csicunam/get_homologues/, which can be easily run on any platform.

Taxonomic changes and description of two new species for the Phyllodactylus lanei complex (Gekkota: Phyllodactylidae) in Mexico.

An integrative taxonomy approach was implemented based on analysis of genetic, phylogenetic, morphological and ecological data to identify the cryptic diversity within the Phyllodactylus lanei complex. At least six species can be identified, of which four are currently considered subspecies: Phyllodactylus lanei, Phyllodactylus rupinus, Phyllodactylus isabelae, Phyllodactylus lupitae and two corresponding to undescribed taxa, which are identified and described in this contribution. These differ from other Mexican geckos in several characters: genetic distance (DNAmt), position in molecular phylogeny (concatened data DNAmt+DNAnu), species tree, morphological characters such as snout-vent length, longitudinal scales, tubercles from head to tail, interorbital scales, scales across venter, third labial-snout scales and rows of tubercles across dorsum; there are also differences in their bioclimatic profiles (temperature and precipitation) and geographical distribution. The most recent studies on taxonomy and evolution of Mexican geckos (Phyllodactylus) show that the diversity of this group of reptiles is currently underestimated, suggesting that more research and conservation efforts are should be addressed at these lizards.