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.

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.

Unravelling phylogenetic relationships of the tribe Cereeae using target enrichment sequencing.

BACKGROUND AND AIMS: Cactaceae are succulent plants, quasi-endemic to the American continent, and one of the most endangered plant groups in the world. Molecular phylogenies have been key to unravelling phylogenetic relationships among major cactus groups, previously hampered by high levels of morphological convergence. Phylogenetic studies using plastid markers have not provided adequate resolution for determining generic relationships within cactus groups. This is the case for the tribe Cereeae s.l., a highly diverse group from tropical America. Here we aimed to reconstruct a well-resolved phylogenetic tree of tribe Cereeae and update the circumscription of suprageneric and generic groups in this tribe. METHODS: We integrated sequence data from public gene and genomic databases with new target sequences (generated using the customized Cactaceae591 probe set) across representatives of this tribe, with a denser taxon sampling of the subtribe Cereinae. We inferred concatenated and coalescent phylogenetic trees and compared the performance of both approaches. KEY RESULTS: Six well-supported suprageneric clades were identified using different datasets. However, only genomic datasets, especially the Cactaceae591, were able to resolve the contentious relationships within the subtribe Cereinae. CONCLUSIONS: We propose a new taxonomic classification within Cereeae based on well-resolved clades, including new subtribes (Aylosterinae subtr. nov., Uebelmanniinae subtr. nov. and Gymnocalyciinae subtr. nov.) and revised subtribes (Trichocereinae, Rebutiinae and Cereinae). We emphasize the importance of using genomic datasets allied with coalescent inference to investigate evolutionary patterns within the tribe Cereeae.

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.

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.

Maintaining genetic integrity with high promiscuity: Frequent hybridization with low introgression in multiple hybrid zones of Melocactus (Cactaceae).

Hybridization and introgression between species in contact/hybrid zones provide important insight into the genetic and ecological mechanisms of speciation. Cactaceae represents the most important radiation of true succulent angiosperms in the New World. This diversification continues to date, with species experiencing few intrinsic barriers to gene flow and the frequent occurrence of natural hybridization. Here, we used RAD-Seq single-nucleotide polymorphism (SNP) data to investigate the genetic architecture of hybridization in four hybrid zones hosting Melocactus concinnus and four congeneric species (M. ernestii, M. glaucescens, M. paucispinus, and M. zehntneri). Our results revealed that M. concinnus is highly promiscuous in sympatric areas and hybridizes with various species distributed in Morro do Chapéu (Diamantina Plateau, Bahia), eastern Brazil. However, the contemporary genomic introgression among the investigated species is very low (c. 2-5%), confirming that even in the face of hybridization, Melocactus species maintain their genetic integrity. The genomic cline approach showed a large fraction of loci deviating from a model of neutral introgression, where most of the loci are consistent with selection favoring parental genotypes. Our results suggest the occurrence of weak premating but strong postmating reproductive isolation in the analyzed cactus species. Furthermore, as most of the Melocactus species are restricted in distribution, hybridization might negatively affect their integrity if hybrids replace the parental species.

The potential of genome-wide RAD sequences for resolving rapid radiations: a case study in Cactaceae.

The reconstruction of relationships within recently radiated groups is challenging even when massive amounts of sequencing data are available. The use of restriction site-associated DNA sequencing (RAD-Seq) to this end is promising. Here, we assessed the performance of RAD-Seq to infer the species-level phylogeny of the rapidly radiating genus Cereus (Cactaceae). To examine how the amount of genomic data affects resolution in this group, we used datasets and implemented different analyses. We sampled 52 individuals of Cereus, representing 18 of the 25 species currently recognized, plus members of the closely allied genera Cipocereus and Praecereus, and other 11 Cactaceae genera as outgroups. Three scenarios of permissiveness to missing data were carried out in iPyRAD, assembling datasets with 30% (333 loci), 45% (1440 loci), and 70% (6141 loci) of missing data. For each dataset, Maximum Likelihood (ML) trees were generated using two supermatrices, i.e., only SNPs and SNPs plus invariant sites. Accuracy and resolution were improved when the dataset with the highest number of loci was used (6141 loci), despite the high percentage of missing data included (70%). Coalescent trees estimated using SVDQuartets and ASTRAL are similar to those obtained by the ML reconstructions. Overall, we reconstruct a well-supported phylogeny of Cereus, which is resolved as monophyletic and composed of four main clades with high support in their internal relationships. Our findings also provide insights into the impact of missing data for phylogeny reconstruction using RAD loci.

Anonymous nuclear markers reveal taxonomic incongruence and long-term disjunction in a cactus species complex with continental-island distribution in South America.

The Pilosocereus aurisetus complex consists of eight cactus species with a fragmented distribution associated to xeric enclaves within the Cerrado biome in eastern South America. The phylogeny of these species is incompletely resolved, and this instability complicates evolutionary analyses. Previous analyses based on both plastid and microsatellite markers suggested that this complex contained species with inherent phylogeographic structure, which was attributed to recent diversification and recurring range shifts. However, limitations of the molecular markers used in these analyses prevented some questions from being properly addressed. In order to better understand the relationship among these species and make a preliminary assessment of the genetic structure within them, we developed anonymous nuclear loci from pyrosequencing data of 40 individuals from four species in the P. aurisetus complex. The data obtained from these loci were used to identify genetic clusters within species, and to investigate the phylogenetic relationship among these inferred clusters using a species tree methodology. Coupled with a palaeodistributional modelling, our results reveal a deep phylogenetic and climatic disjunction between two geographic lineages. Our results highlight the importance of sampling more regions from the genome to gain better insights on the evolution of species with an intricate evolutionary history. The methodology used here provides a feasible approach to develop numerous genealogical molecular markers throughout the genome for non-model species. These data provide a more robust hypothesis for the relationship among the lineages of the P. aurisetus complex.

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.

Cross-species amplification of microsatellites reveals incongruence in the molecular variation and taxonomic limits of the Pilosocereus aurisetus group (Cactaceae).

The Pilosocereus aurisetus group contains eight cactus species restricted to xeric habitats in eastern and central Brazil that have an archipelago-like distribution. In this study, 5-11 microsatellite markers previously designed for Pilosocereus machrisii were evaluated for cross-amplification and polymorphisms in ten populations from six species of the P. aurisetus group. The genotypic information was subsequently used to investigate the genetic relationships between the individuals, populations, and species analyzed. Only the Pmac101 locus failed to amplify in all of the six analyzed species, resulting in an 88 % success rate. The number of alleles per polymorphic locus ranged from 2 to 12, and the most successfully amplified loci showed at least one population with a larger number of alleles than were reported in the source species. The population relationships revealed clear genetic clustering in a neighbor-joining tree that was partially incongruent with the taxonomic limits between the P. aurisetus and P. machrisii species, a fact which parallels the problematic taxonomy of the P. aurisetus group. A Bayesian clustering analysis of the individual genotypes confirmed the observed taxonomic incongruence. These microsatellite markers provide a valuable resource for facilitating large-scale genetic studies on population structures, systematics and evolutionary history in this group.

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.

Isolation, characterization, and cross-species amplification of polymorphic microsatellite markers for Pilosocereus machrisii (Cactaceae).

PREMISE OF THE STUDY: Microsatellite primers were developed in Pilosocereus machrisii, a columnar cactus with a patchy distribution in eastern tropical South America, to assess its genetic diversity and population structure. METHODS AND RESULTS: Eleven microsatellite markers were developed, of which one was monomorphic among 51 individuals from two populations. The number of alleles per polymorphic locus ranged from two to eight, and the total number of alleles was 57. From the 11 isolated loci, nine were successfully amplified in the other four Pilosocereus species. CONCLUSIONS: The results showed that these markers will be useful for studies of genetic diversity, patterns of gene flow, and population genetic structure in P. machrisii, as well as across other congeneric 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.

Anonymous nuclear markers data supporting species tree phylogeny and divergence time estimates in a cactus species complex in South America.

Supportive data related to the article "Anonymous nuclear markers reveal taxonomic incongruence and long-term disjunction in a cactus species complex with continental-island distribution in South America" (Perez et al., 2016) [1]. Here, we present pyrosequencing results, primer sequences, a cpDNA phylogeny, and a species tree phylogeny.

Lineage-specific evolutionary rate in plants: Contributions of a screening for Cereus (Cactaceae).

PREMISE OF THE STUDY: Predictable chloroplast DNA (cpDNA) sequences have been listed for the shallowest taxonomic studies in plants. We investigated whether plastid regions that vary between closely allied species could be applied for intraspecific studies and compared the variation of these plastid segments with two nuclear regions. METHODS: We screened 16 plastid and two nuclear intronic regions for species of the genus Cereus (Cactaceae) at three hierarchical levels (species from different clades, species of the same clade, and allopatric populations). RESULTS: Ten plastid regions presented interspecific variation, and six of them showed variation at the intraspecific level. The two nuclear regions showed both inter- and intraspecific variation, and in general they showed higher levels of variability in almost all hierarchical levels than the plastid segments. DISCUSSION: Our data suggest no correspondence between variation of plastid regions at the interspecific and intraspecific level, probably due to lineage-specific variation in cpDNA, which appears to have less effect in nuclear data. Despite the heterogeneity in evolutionary rates of cpDNA, we highlight three plastid segments that may be considered in initial screenings in plant phylogeographic studies.

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.

Population genetic structure of two columnar cacti with a patchy distribution in eastern Brazil.

The genetic variability and population genetic structure of six populations of Praecereus euchlorus and Pilosocereus machrisii were investigated. The genetic variability in single populations of Pilosocereus vilaboensis, Pilosocereus aureispinus, and Facheiroa squamosa was also examined. All of these cacti species have a patchy geographic distribution in which they are restricted to small areas of xeric habitats in eastern Brazil. An analysis of genetic structure was used to gain insights into the historical mechanisms responsible for the patchy distribution of P. euchlorus and P. machrisii. High genetic variability was found at the populational level in all species (P=58.9-92.8%, A(p)=2.34-3.33, H(e)=0.266-0.401), and did not support our expectations of low variability based on the small population size. Substantial inbreeding was detected within populations (F(IS)=0.370-0.623). In agreement with their insular distribution patterns, P. euchlorus and P. machrisii had a high genetic differentiation (F(ST)=0.484 and F(ST)=0.281, respectively), with no evidence of isolation by distance. Accordingly, estimates of gene flow (N(m)) calculated from F(ST) and private alleles were below the level of N(m)=1 in P. machrisii and P. euchlorus. These results favored historical fragmentation as the mechanism responsible for the patchy distribution of these two species. The genetic distance between P. machrisii and P. vilaboensis was not compatible with their taxonomic distinction, indicating a possible local speciation event in this genus, or the occurrence of introgression events.