Evandro M. Moraes

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.

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.

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.

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.

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.

Isolation and characterization of ten new microsatellite markers in Machaerium villosum Vogel (Fabaceae)

Machaerium villosum is an important tree species from Southeastern Brazil. We report hereby 10 new microsatellite markers to investigate the structure and genetic diversity of this species. Ninety-seven alleles were detected in 60 specimens from a natural population in Minas Gerais State. High genetic diversity has been found. The mean observed and expected heterozygosities were 0.771 and 0.802, respectively. One locus showed significant Hardy-Weinberg departure and five loci combinations showed significant linkage disequilibrium. These 10 new microsatellite loci will be used to evaluate the genetic diversity of this species in order to understand the fragmentation effects of the Brazilian Atlantic Rain Forest.

Polymorphic microsatellite markers for the rare and endangered cactus Uebelmannia pectinifera (Cactaceae) and its congeneric species

The cactus genus Uebelmannia includes 3 narrow endemic species associated with rocky savanna habitats in eastern South America. Because of their rarity and illegal over-collection, all of these species are endangered. Taxonomic uncertainties resulting from dramatic local variation in morphology within Uebelmannia species preclude effective conservation efforts, such as the reintroduction or translocation of plants, to restore declining populations. In this study, we developed and characterized 18 perfect, dinucleotide simple-sequence repeat markers for U. pectinifera, the most widely distributed species in the genus, and tested the cross-amplification of these markers in the remaining congeneric species and subspecies. All markers were polymorphic in a sample from 2 U. pectinifera populations. The effective number of alleles ranged from 1.6 to 8.7, with an average per population of 3.3 (SE ± 0.30) and 4.5 (SE ± 0.50). Expected heterozygosity ranged from 0.375 to 0.847 and 8-10 loci showed departures from Hardy- Weinberg equilibrium in the analyzed populations. Based on the observed polymorphism level of each marker, as well as the analysis of null allele presence and evidence of amplification of duplicate loci, a subset of 12 loci can be used as reliable markers to investigate the genetic structure, diversity, and species limits of the Uebelmannia genus.

Extreme population subdivision or cryptic speciation in the cactus Pilosocereus jauruensis? A taxonomic challenge posed by a naturally fragmented system

In allopatric systems, where gene flow is highly constrained, the distinction between intraspecific differentiation and speciation is a challenging task. Pilosocereus jauruensis is a columnar cactus restricted to patchy dry habitats of Pantanal, Cerrado, and Chaco biomes in south-western Brazil and neighbouring countries. This species was recently reinstated after having been synonymized with P. machriisi, now including P. paraguayensis, P. saudadensis, and P. densivillosus as its synonyms. These taxa have been placed within the informal Aurisetus group. The present study aimed to investigate the genetic structure of P. jauruensis in order to identify possible distinct evolutionary lineages, and to support further refinement of the taxonomy of this species. For this purpose, 10 microsatellite loci were genotyped for 156 individuals of P. jauruensis. To provide a proxy for the expected interspecific differentiation level within this allopatric system, two populations (49 individuals) of P. vilaboensis (a species closely related to P. jauruensis) were also included. Additionally, two cpDNA markers (trnSGCU-trnGUCC and trnT-trnL) for a subsample of 44 individuals of both species were sequenced. The results obtained showed that even though P. jauruensis populations are relatively small and isolated, they still maintain a high level of genetic diversity (e.g., HE = 0.557 to 0.725; HO = 0.253 to 0.409). The study suggests the existence of two distinct taxa/evolutionary lineages through their entire distribution. Two populations found in the same geographic region of the now synonymized P. densivillosus showed significant genetic divergence when compared with other populations of P. jauruensis. In contrast, our results did not support an evolutionary independence hypothesis for populations that occur in the same geographic region of the synonymous P. saudadensis.

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.