Geoclimatic drivers of diversification in the largest arid and semi-arid environment of the Neotropics: Perspectives from phylogeography.

The South American Dry Diagonal, also called the Diagonal of Open Formations, is a large region of seasonally dry vegetation extending from northeastern Brazil to northern Argentina, comprising the Caatinga, Cerrado, and Chaco subregions. A growing body of phylogeography literature has determined that a complex history of climatic changes coupled with more ancient geological events has produced a diverse and endemic-rich Dry Diagonal biota. However, the exact drivers are still under investigation, and their relative strengths and effects are controversial. Pleistocene climatic fluctuations structured lineages via vegetation shifts, refugium formation, and corridors between the Amazon and Atlantic forests. In some taxa, older geological events, such as the reconfiguration of the São Francisco River, uplift of the Central Brazilian Plateau, or the Miocene inundation of the Chaco by marine incursions, were more important. Here, we review the Dry Diagonal phylogeography literature, discussing each hypothesized driver of diversification and assessing degree of support. Few studies statistically test these hypotheses, with most support drawn from associating encountered phylogeographic patterns such as population structure with the timing of ancient geoclimatic events. Across statistical studies, most hypotheses are well supported, with the exception of the Pleistocene Arc Hypothesis. However, taxonomic and regional biases persist, such as a proportional overabundance of herpetofauna studies, and the under-representation of Chaco studies. Overall, both Pleistocene climate change and Neogene geological events shaped the evolution of the Dry Diagonal biota, though the precise effects are regionally and taxonomically varied. We encourage further use of model-based analyses to test evolutionary scenarios, as well as interdisciplinary collaborations to progress the field beyond its current focus on the traditional set of geoclimatic hypotheses.

Comparative transcriptome analysis reveals lineage- and environment-specific adaptations in cacti from the Brazilian Atlantic Forest.

MAIN CONCLUSION: Natural selection influenced adaptive divergence between Cereus fernambucensis and Cereus insularis, revealing key genes governing abiotic stress responses and supporting neoteny in C. insularis. Uncovering the molecular mechanisms driving adaptive divergence in traits related to habitat adaptation remains a central challenge. In this study, we focused on the cactus clade, which includes Cereus sericifer F.Ritter, Cereus fernambucensis Lem., and Cereus insularis Hemsley. These allopatric species inhabit distinct relatively drier regions within the Brazilian Atlantic Forest, each facing unique abiotic conditions. We leveraged whole transcriptome data and abiotic variables datasets to explore lineage-specific and environment-specific adaptations in these species. Employing comparative phylogenetic methods, we identified genes under positive selection (PSG) and examined their association with non-synonymous genetic variants and abiotic attributes through a PhyloGWAS approach. Our analysis unveiled signatures of selection in all studied lineages, with C. fernambucensis northern populations and C. insularis showing the most PSGs. These PSGs predominantly govern abiotic stress regulation, encompassing heat tolerance, UV stress response, and soil salinity adaptation. Our exclusive observation of gene expression tied to early developmental stages in C. insularis supports the hypothesis of neoteny in this species. We also identified genes associated with abiotic variables in independent lineages, suggesting their role as environmental filters on genetic diversity. Overall, our findings suggest that natural selection played a pivotal role in the geographic range of these species in response to environmental and biogeographic transitions.

A historical stepping-stone path for an island-colonizing cactus across a submerged "bridge" archipelago.

Here we use population genomic data (ddRAD-Seq) and ecological niche modeling to test biogeographic hypotheses for the divergence of the island-endemic cactus species Cereus insularis Hemsl. (Cereeae; Cactaceae) from its sister species C. fernambucensis Lem. The Cereus insularis grows in the Fernando de Noronha Islands (FNI), a Neotropical archipelago located 350 km off the Brazilian Atlantic Forest (BAF) coast. Phylogeographic reconstructions support a northward expansion by the common ancestor of C. insularis and C. fernambucensis along the mainland BAF coast, with C. insularis diverging from the widespread mainland taxon C. fernambucensis after colonizing FNI in the late Pleistocene. The morphologically distinct C. insularis is monophyletic and nested within C. fernambucensis, as expected from a progenitor-derivative speciation model. We tested alternative biogeographic and demographic hypotheses for the colonization of the FNI using Approximate Bayesian Computation. We found the greatest support for a stepping-stone path that emerged during periods of decreased sea level (the "bridge" hypothesis), in congruence with historical ecological niche modeling that shows highly suitable habitats on stepping-stone islands during glacial periods. The outlier analyses reveal signatures of selection in C. insularis, suggesting a putative role of adaptation driving rapid anagenic differentiation of this species in FNI.

Opsin diversity and evolution in the Elateroidea superfamily: Insights from transcriptome data.

Vision plays a vital biological role in organisms, which depends on the visual pigment molecules (opsin plus chromophore). The expansion or reduction of spectral channels in the organisms is determined by distinct opsin classes and copy numbers resulting from duplication or loss. Within Coleoptera, the superfamily Elateroidea exhibits a great diversity of morphological and physiological characteristics, such as bioluminescence, making this group an important model for opsin studies. While molecular and physiological studies have been conducted in Lampyridae and Elateridae, other families remain unexplored. Here, we reused transcriptome datasets from Elateroidea species, including members of Elateridae, Lampyridae, Phengodidae, Rhagophthalmidae, Cantharidae, and Lycidae, to detect the diversity of putative opsin genes in this superfamily. In addition, we tested the signature of sites under positive selection in both ultraviolet (UV)- and long-wavelength (LW)-opsin classes. Although the visual system in Elateroidea is considered simple, we observed events of duplication in LW- and UV-opsin, as well as the absence of UV-opsin in distinct families, such as larval Phengodidae individuals. We detected different copies of LW-opsins that were highly expressed in the eyes of distinct tribes of fireflies, indicating the possible selection of each copy during the evolution of the sexual mating to avoid spectrum overlapping. In Elateridae, we found that the bioluminescent species had a distinct LW-opsin copy compared with the non-bioluminescent species, suggesting events of duplication and loss. The signature of positive selection showed only one residue associated with the chromophore binding site in the Elateroidea, which may produce a bathochromic shift in the wavelength absorption spectra in this family. Overall, this study brings important content and fills gaps regarding opsin evolution in Elateroidea.

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.

Evolutionary Genetics of Cacti: Research Biases, Advances and Prospects.

Here, we present a review of the studies of evolutionary genetics (phylogenetics, population genetics, and phylogeography) using genetic data as well as genome scale assemblies in Cactaceae (Caryophyllales, Angiosperms), a major lineage of succulent plants with astonishing diversity on the American continent. To this end, we performed a literature survey (1992-2021) to obtain detailed information regarding key aspects of studies investigating cactus evolution. Specifically, we summarize the advances in the following aspects: molecular markers, species delimitation, phylogenetics, hybridization, biogeography, and genome assemblies. In brief, we observed substantial growth in the studies conducted with molecular markers in the past two decades. However, we found biases in taxonomic/geographic sampling and the use of traditional markers and statistical approaches. We discuss some methodological and social challenges for engaging the cactus community in genomic research. We also stressed the importance of integrative approaches, coalescent methods, and international collaboration to advance the understanding of cactus evolution.

Coalescent-based species delimitation meets deep learning: Insights from a highly fragmented cactus system.

Delimiting species boundaries is a major goal in evolutionary biology. An increasing volume of literature has focused on the challenges of investigating cryptic diversity within complex evolutionary scenarios of speciation, including gene flow and demographic fluctuations. New methods based on model selection, such as approximate Bayesian computation, approximate likelihoods, and machine learning are promising tools arising in this field. Here, we introduce a framework for species delimitation using the multispecies coalescent model coupled with a deep learning algorithm based on convolutional neural networks (CNNs). We compared this strategy with a similar ABC approach. We applied both methods to test species boundary hypotheses based on current and previous taxonomic delimitations as well as genetic data (sequences from 41 loci) in Pilosocereus aurisetus, a cactus species complex with a sky-island distribution and taxonomic uncertainty. To validate our method, we also applied the same strategy on data from widely accepted species from the genus Drosophila. The results show that our CNN approach has a high capacity to distinguish among the simulated species delimitation scenarios, with higher accuracy than ABC. For the cactus data set, a splitter hypothesis without gene flow showed the highest probability in both CNN and ABC approaches, a result agreeing with previous taxonomic classifications and in line with the sky-island distribution and low dispersal of P. aurisetus. Our results highlight the cryptic diversity within the P. aurisetus complex and show that CNNs are a promising approach for distinguishing complex evolutionary histories, even outperforming the accuracy of other model-based approaches such as ABC.

Comparative and predictive phylogeography in the South American diagonal of open formations: Unravelling the biological and environmental influences on multitaxon demography.

Phylogeography investigates historical drivers of the geographical distribution of intraspecific lineages. Particular attention has been given to ecological, climatic and geological processes in the diversification of the Neotropical biota. Several species sampled across the South American diagonal of open formations (DOF), comprising the Caatinga, Cerrado and Chaco biomes, experienced range shifts coincident with Quaternary climatic changes. However, comparative studies across different spatial, temporal and biological scales on DOF species are still meagre. Here, we combine phylogeographical model selection and machine learning predictive frameworks to investigate the influence of Pleistocene climatic changes on several plant and animal species from the DOF. We assembled mitochondrial/chloroplastic DNA sequences in public repositories and inferred the demographic responses of 44 species, comprising 70 intraspecific lineages of plants, lizards, frogs, spiders and insects. We then built a random forest model using biotic and abiotic information to identify the best predictors of demographic responses in the Pleistocene. Finally, we assessed the temporal synchrony of species demographic responses with hierarchical approximate Bayesian computation. Biotic variables related to population connectivity, gene flow and habitat preferences largely predicted how species responded to Pleistocene climatic changes, and demographic changes were synchronous primarily during the Middle Pleistocene. Although 22 (~31%) lineages underwent demographic expansion, presumably associated with the spread of aridity during the glacial Pleistocene periods, our findings suggest that nine lineages (~13%) exhibited the opposite response due to taxon-specific attributes.

Phylogenomic analyses and divergence time estimation of Elateroidea (Coleoptera) based on RNA-Seq data.

Bioluminescence, the emission of visible light in a living organism, is an intriguing phenomenon observed in different species and environments. In terrestrial organisms, the bioluminescence is observed mainly in beetles of the Elateroidea superfamily (Coleoptera). Several phylogenetic studies have been used different strategies to propose a scenario for the origin and evolution of the bioluminescence within this group, however some of them showed incongruences, mainly about the relationship of the bioluminescent families. In order to increase the number of molecular markers available for Elateroidea species and to propose a more accurate phylogeny, with high supported topology, we employed the Next-Generation Sequencing (NGS) methodology to perform the RNA-Seq analysis of luminescent (Elateridae, Phengodidae, Rhagophthalmidae, and Lampyridae) and non-luminescent (Cantharidae) species of Neotropical beetles. We used the RNA-Seq data to construct a calibrated phylogeny of Elateroidea superfamily using a large number of nuclear molecular markers. The results indicate Lampyridae and Phengodidae/Rhagophthalmidae as sister-groups, suggesting that the bioluminescence evolved later in Elateridae than other families (Lampyridae, Phengodidae, and Rhagophthalmidae), and indicating the Upper Cretaceous as the period for the main diversification of Elateroidea bioluminescent species.

Weak population structure and no genetic erosion in Pilosocereus aureispinus: A microendemic and threatened cactus species from eastern Brazil.

Succulent cacti (Cactaceae) are among the most threatened taxonomic groups assessed to date. Here we evaluated the genetic diversity and population structure of a narrow endemic columnar cactus Pilosocereus aureispinus. This species is only found in a small area of c. 300 km2 of rocky savanna from eastern Brazil and it is currently classified as vulnerable (VU) on the International Union for Conservation of Nature (IUCN) red list. Eight microsatellite loci were genotyped for 91 individuals from four localities of the known P. aureispinus range. In contrast with expectations for narrow endemic species, we found relatively high levels of genetic diversity (e.g., HE = 0.390 to 0.525; HO = 0.394 to 0.572) and very low population structure based on the variation of six loci. All the analyzed individuals were clustered in one unique genetic group in assignment tests. We also generated the sequences of two plastid markers (trnT-trnL and psbD-trnT) and found no variation on a subsample of 39 individuals. We used Landsat 8 images and Normalized Difference Vegetation Index to estimate a potential extent of occurrence of c. 750 km2 for this species. Our results showed that P. aureispinus is not suffering from erosion of nuclear genetic variability due to its narrow distribution. However, we advocate that because of the extremely limited extent of occurrence, the ongoing anthropogenic disturbances in its habitat, and phylogenetic distinctiveness of P. aureispinus, this species should be classified as endangered (EN) on the IUCN Red List.

Using Next Generation RAD Sequencing to Isolate Multispecies Microsatellites for Pilosocereus (Cactaceae).

Microsatellite markers (also known as SSRs, Simple Sequence Repeats) are widely used in plant science and are among the most informative molecular markers for population genetic investigations, but the development of such markers presents substantial challenges. In this report, we discuss how next generation sequencing can replace the cloning, Sanger sequencing, identification of polymorphic loci, and testing cross-amplification that were previously required to develop microsatellites. We report the development of a large set of microsatellite markers for five species of the Neotropical cactus genus Pilosocereus using a restriction-site-associated DNA sequencing (RAD-seq) on a Roche 454 platform. We identified an average of 165 microsatellites per individual, with the absolute numbers across individuals proportional to the sequence reads obtained per individual. Frequency distribution of the repeat units was similar in the five species, with shorter motifs such as di- and trinucleotide being the most abundant repeats. In addition, we provide 72 microsatellites that could be potentially amplified in the sampled species and 22 polymorphic microsatellites validated in two populations of the species Pilosocereus machrisii. Although low coverage sequencing among individuals was observed for most of the loci, which we suggest to be more related to the nature of the microsatellite markers and the possible bias inserted by the restriction enzymes than to the genome size, our work demonstrates that an NGS approach is an efficient method to isolate multispecies microsatellites even in non-model organisms.

Pleistocene niche stability and lineage diversification in the subtropical spider Araneus omnicolor (Araneidae).

The influence of Quaternary climate oscillations on the diversification of the South American fauna is being increasingly explored. However, most of these studies have focused on taxa that are endemic to tropical environments, and relatively few have treated organisms restricted to subtropical biomes. Here we used an integrative phylogeographical framework to investigate the effects of these climate events on the ecological niche and genetic patterns of the subtropical orb-weaver spider Araneus omnicolor (Araneidae). We analyzed the mitochondrial (Cytochrome Oxidase I, COI) and nuclear (Internal Transcribed Subunit II, ITS2) DNA of 130 individuals throughout the species' range, and generated distribution models in three different climate scenarios [present, Last Glacial Maximum (LGM), and Last Interglacial Maximum (LIG)]. Additionally, we used an Approximate Bayesian Computation (ABC) approach to compare possible demographic scenarios and select the hypothesis that better explains the genetic patterns of A. omnicolor. We obtained high haplotype diversity but low nucleotide variation among sequences. The population structure and demographic analyses showed discrepancies between markers, suggesting male-biased dispersal in the species. The time-calibrated COI phylogenetic inference showed a recent diversification of lineages (Middle/Late Pleistocene), while the paleoclimate modeling indicated niche stability since ~120 Kya. The ABC results agreed with the niche models, supporting a panmictic population as the most likely historical scenario for the species. These results indicate that A. omnicolor experienced no niche or population reductions during the Late Pleistocene, despite the intense landscape modifications that occurred in the subtropical region, and that other factors beside LGM and LIG climate oscillations might have contributed to the demographic history of this species. This pattern may be related to the high dispersal ability and wide environmental tolerance of A. omnicolor, highlighting the need for more phylogeographical studies with invertebrates and other generalist taxa, in order to understand the effects of Quaternary climate changes on Neotropical biodiversity.

The evolution of phylogeographic data sets.

Empirical phylogeographic studies have progressively sampled greater numbers of loci over time, in part motivated by theoretical papers showing that estimates of key demographic parameters improve as the number of loci increases. Recently, next-generation sequencing has been applied to questions about organismal history, with the promise of revolutionizing the field. However, no systematic assessment of how phylogeographic data sets have changed over time with respect to overall size and information content has been performed. Here, we quantify the changing nature of these genetic data sets over the past 20 years, focusing on papers published in Molecular Ecology. We found that the number of independent loci, the total number of alleles sampled and the total number of single nucleotide polymorphisms (SNPs) per data set has improved over time, with particularly dramatic increases within the past 5 years. Interestingly, uniparentally inherited organellar markers (e.g. animal mitochondrial and plant chloroplast DNA) continue to represent an important component of phylogeographic data. Single-species studies (cf. comparative studies) that focus on vertebrates (particularly fish and to some extent, birds) represent the gold standard of phylogeographic data collection. Based on the current trajectory seen in our survey data, forecast modelling indicates that the median number of SNPs per data set for studies published by the end of the year 2016 may approach ~20,000. This survey provides baseline information for understanding the evolution of phylogeographic data sets and underscores the fact that development of analytical methods for handling very large genetic data sets will be critical for facilitating growth of the field.

Interglacial microrefugia and diversification of a cactus species complex: phylogeography and palaeodistributional reconstructions for Pilosocereus aurisetus and allies.

The role of Pleistocene climate changes in promoting evolutionary diversification in global biota is well documented, but the great majority of data regarding this subject come from North America and Europe, which were greatly affected by glaciation. The effects of Pleistocene changes on cold- and/or dry-adapted species in tropical areas where glaciers were not present remain sparsely investigated. Many such species are restricted to small areas surrounded by unfavourable habitats, which may represent potential interglacial microrefugia. Here, we analysed the phylogeographic structure and diversification history of seven cactus species in the Pilosocereus aurisetus complex that are restricted to rocky areas with high diversity and endemism within the Neotropical savannas of eastern South America. We combined palaeodistributional estimates with standard phylogeographic approaches based on two chloroplast DNA regions (trnT-trnL and trnS-trnG), exon 1 of the nuclear gene PhyC and 10 nuclear microsatellite loci. Our analyses revealed a phylogeographic history marked by multiple levels of distributional fragmentation, isolation leading to allopatric differentiation and secondary contact among divergent lineages within the complex. Diversification and demographic events appear to have been affected by the Quaternary climatic cycles as a result of isolation in multiple patches of xerophytic vegetation. These small patches presently harbouring P. aurisetus populations seem to operate as microrefugia, both at present and during Pleistocene interglacial periods; the role of such microrefugia should be explored and analysed in greater detail.