YABBY transcription factor family in the octoploid Fragaria × ananassa and five diploid Fragaria species.

YABBY genes encode specific TFs of seed plants involved in development and formation of leaves, flowers, and fruit. In the present work, genome-wide and expression analyses of the YABBY gene family were performed in six species of the Fragaria genus: Fragaria × ananassa, F. daltoniana, F. nilgerrensis, F. pentaphylla, F. viridis, and F. vesca. The chromosomal location, synteny pattern, gene structure, and phylogenetic analyses were carried out. By combining RNA-seq data and RT-qPCR analysis we explored specific expression of YABBYs in F. × ananassa and F. vesca. We also analysed the promoter regions of FaYABBYs and performed MeJA application to F. × ananassa fruit to observe effects on gene expression. We identified and characterized 25 YABBY genes in F. × ananassa and six in each of the other five species, which belong to FIL/YAB3 (YABBY1), YAB2 (YABBY2), YAB5 (YABBY5), CRC, and INO clades previously described. Division of the YABBY1 clade into YABBY1.1 and YABBY1.2 subclades is reported. We observed differential expression according to tissue, where some FaYABBYs are expressed mainly in leaves and flowers and to a minor extent during fruit development of F. × ananassa. Specifically, the FaINO genes contain jasmonate-responsive cis-acting elements in their promoters which may be functional since FaINOs are upregulated in F. × ananassa fruit under MeJA treatment. This study suggests that YABBY TFs play an important role in the development- and environment-associated responses of the Fragaria genus.

A de novo genome assembly of Solanum bulbocastanum Dun., a Mexican diploid species reproductively isolated from the A-genome species, including cultivated potatoes.

Potato and its wild relatives are distributed mainly in the Mexican highlands and central Andes of South America. The South American A-genome species, including cultivated potatoes, are reproductively isolated from Mexican diploid species. Whole-genome sequencing has disclosed genome structure and similarity, mostly in cultivated potatoes and their closely related species. In this study, we generated a chromosome-scale assembly of the genome of a Mexican diploid species, Solanum bulbocastanum Dun., using PacBio long-read sequencing, optical mapping, and Hi-C scaffolding technologies. The final sequence assembly consisted of 737.9 Mb, among which 647.0 Mb were anchored to the 12 chromosomes. Compared with chromosome-scale assemblies of S. lycopersicum (tomato), S. etuberosum (non-tuber-bearing species with E-genome), S. verrucosum, S. chacoense, S. multidissectum, and S. phureja (all four are A-genome species), the S. bulbocastnum genome was the shortest. It contained fewer transposable elements (56.2%) than A-genome species. A cluster analysis was performed based on pairwise ratios of syntenic regions among the seven chromosome-scale assemblies, showing that the A-genome species were first clustered as a distinct group. Then, this group was clustered with S. bulbocastanum. Sequence similarity in 1,624 single-copy orthologous gene groups among 36 Solanum species and clones separated S. bulbocastanum as a specific group, including other Mexican diploid species, from the A-genome species. Therefore, the S. bulbocastanum genome differs in genome structure and gene sequences from the A-genome species. These findings provide important insights into understanding and utilizing the genetic diversity of S. bulbocastanum and the other Mexican diploid species in potato breeding.

Assessing small RNA profiles in potato diploid hybrid and its resynthesized allopolyploid reveals conserved abundance with distinct genomic distribution.

KEY MESSAGE: The shock produced by the allopolyploidization process on a potato interspecific diploid hybrid displays a non-random remobilization of the small RNAs profile on a variety of genomic features. Allopolyploidy, a complex process involving interspecific hybridization and whole genome duplication, significantly impacts plant evolution, leading to the emergence of novel phenotypes. Polyploids often present phenotypic nuances that enhance adaptability, enabling them to compete better and occasionally to colonize new habitats. Whole-genome duplication represents a genomic "shock" that can trigger genetic and epigenetic changes that yield novel expression patterns. In this work, we investigate the polyploidization effect on a diploid interspecific hybrid obtained through the cross between the cultivated potato Solanum tuberosum and the wild potato Solanum kurtzianum, by assessing the small RNAs (sRNAs) profile of the parental diploid hybrid and its derived allopolyploid. Small RNAs are key components of the epigenetic mechanisms involved in silencing by RNA-directed DNA Methylation (RdDM). A sRNA sequencing (sRNA-Seq) analysis was performed to individually profile the 21 to 22 nucleotide (21 to 22-nt) and 24-nt sRNA size classes due to their unique mechanism of biogenesis and mode of function. The composition and distribution of different genomic features and differentially accumulated (DA) sRNAs were evaluated throughout the potato genome. We selected a subset of genes associated with DA sRNAs for messenger RNA (mRNA) expression analysis to assess potential impacts on the transcriptome. Interestingly, we noted that 24-nt DA sRNAs that exclusively mapped to exons were correlated with differentially expressed mRNAs between genotypes, while this behavior was not observed when 24-nt DA sRNAs were mapped to intronic regions. These findings collectively emphasize the nonstochastic nature of sRNA remobilization in response to the genomic shock induced by allopolyploidization.

Comparative analysis of molecular and morphological diversity in two diploid Paspalum species (Poaceae) with contrasting mating systems.

KEY MESSAGE: Interspecific comparison of two Paspalum species has demonstrated that mating systems (selfing and outcrossing) contribute to variation (genetically and morphologically) within species through similar but mutually exclusive processes. Mating systems play a key role in the genetic dynamics of populations. Studies show that populations of selfing plants have less genetic diversity than outcrossing plants. Yet, many such studies have ignored morphological diversity. Here, we compared the morphological and molecular diversity patterns in populations of two phylogenetically-related sexual diploids that differ in their mating system: self-sterile Paspalum indecorum and self-fertile P. pumilum. We assessed the morphological variation using 16 morpho-phenological characters and the molecular diversity using three combinations of AFLPs. We compared the morphological and molecular diversity within and among populations in each mating system. Contrary to expectations, selfers showed higher morphological variation within populations, mainly in vegetative and phenological traits, compared to outcrossers. The high morphological variation within populations of selfers led to a low differentiation among populations. At molecular level, selfing populations showed lower levels of genotypic and genetic diversity than outcrossing populations. As expected, selfers showed higher population structure than outcrossers (PhiST = 0.301 and PhiST = 0.108, respectively). Increased homozygous combinations for the same trait/locus enhance morphological variation and reduce molecular variation within populations in selfing P. pumilum. Thus, selfing outcomes are opposite when comparing morphological and molecular variation in P. pumilum. Meanwhile, pollen flow in obligate outcrossing populations of P. indecorum increases within-population molecular variation, but tends to homogenize phenotypes within-population. Pollen flow in obligate outcrossers tends to merge geographically closer populations; but isolation by distance can lead to a weak differentiation among distant populations of P. indecorum.

Heterozygosity analysis of spontaneous 2n female gametes and centromere mapping of the diploid Hevea brasiliensis based on full-sib triploid populations.

KEY MESSAGE: Unreduced megagametophytes via second-division restitution were confirmed through heterozygosity analysis, and four candidate physical centromeres of rubber were located for the first time. The evaluation of maternal heterozygosity restitution (MHR) is vital in identifying the mechanism of 2n gametogenesis and assessing the utilization value of 2n gametes. In this study, three full-sib triploid populations were employed to evaluate the MHR of 2n female gametes of rubber tree clone GT1 and to confirm their genetic derivation. The 2n female gametes of GT1 were derived from second-division restitution (SDR) and transmitted more than half of the parental heterozygosity. In addition, low recombination frequency markers were developed, and four candidate physical centromeres of rubber tree were located for the first time. The confirmation that 2n female gametes of rubber tree clone GT1 are derived from SDR provides insights into the molecular mechanisms of 2n gametogenesis. In addition, the identified centromere location will aid in the development of centromeric markers for the rapid identification of the 2n gametogenesis mechanism.

Supernumerary B Chromosomes of Tetragonisca fiebrigi Share Repeat Content with Standard Chromosome Set of both T. fiebrigi and Tetragonisca angustula (Apidae: Meliponini).

The stingless bees Tetragonisca angustula and Tetragonisca fiebrigi are widely distributed in Brazil, and both are commonly known as "jataí." Our goal was to investigate the possible origin of the B chromosomes in T. fiebrigi, a cytotaxonomic trait that differentiates T. fiebrigi from T. angustula. We analyzed diploid chromosome number (2n), B chromosome incidence, patterns of constitutive heterochromatin, and in situ localization of different repetitive DNA probes in T. angustula and T. fiebrigi. Both species displayed 2n = 34, with similar karyotype structures. One to three B chromosomes were observed in T. fiebrigi only. Constitutive heterochromatin was distributed on one arm of all chromosomes in both species, and T. fiebrigi B chromosomes were mainly heterochromatic with one euchromatic extremity. The (GA)15 and (CAA)10 microsatellite probes marked the euchromatic arms of all chromosomes in both species without marking the B chromosomes. The 18S ribosomal DNA (rDNA) probe marked 10 chromosomes in T. angustula and 6 A chromosomes in T. fiebrigi with an additional marking on 1B in individuals with 3B. The Tan-Bsp68I repetitive DNA probe marked the heterochromatic portion of all T. fiebrigi A and B chromosomes. This probe also marked the heterochromatic portion of all T. angustula chromosomes; therefore, both alternative hypotheses to the B chromosome origin are possible: (i) from the A chromosome complement of T. fiebrigi (intraspecific origin); or (ii) a by-product of genome reshuffling following the hybridization between T. fiebrigi and T. angustula (interspecific origin).

Clonal diploid and autopolyploid breeding strategies to harness heterosis: insights from stochastic simulation.

KEY MESSAGE: Reciprocal recurrent selection sometimes increases genetic gain per unit cost in clonal diploids with heterosis due to dominance, but it typically does not benefit autopolyploids. Breeding can change the dominance as well as additive genetic value of populations, thus utilizing heterosis. A common hybrid breeding strategy is reciprocal recurrent selection (RRS), in which parents of hybrids are typically recycled within pools based on general combining ability. However, the relative performances of RRS and other breeding strategies have not been thoroughly compared. RRS can have relatively increased costs and longer cycle lengths, but these are sometimes outweighed by its ability to harness heterosis due to dominance. Here, we used stochastic simulation to compare genetic gain per unit cost of RRS, terminal crossing, recurrent selection on breeding value, and recurrent selection on cross performance considering different amounts of population heterosis due to dominance, relative cycle lengths, time horizons, estimation methods, selection intensities, and ploidy levels. In diploids with phenotypic selection at high intensity, whether RRS was the optimal breeding strategy depended on the initial population heterosis. However, in diploids with rapid-cycling genomic selection at high intensity, RRS was the optimal breeding strategy after 50 years over almost all amounts of initial population heterosis under the study assumptions. Diploid RRS required more population heterosis to outperform other strategies as its relative cycle length increased and as selection intensity and time horizon decreased. The optimal strategy depended on selection intensity, a proxy for inbreeding rate. Use of diploid fully inbred parents vs. outbred parents with RRS typically did not affect genetic gain. In autopolyploids, RRS typically did not outperform one-pool strategies regardless of the initial population heterosis.

Cytotaxonomy and Molecular Analyses of Mycteria americana (Ciconiidae: Ciconiiformes): Insights on Stork Phylogeny.

Although molecular information for the wood stork (Mycteria americana) has been well described, data concerning their karyotypical organization and phylogenetic relationships with other storks are still scarce. Thus, we aimed to analyze the chromosomal organization and diversification of M. americana, and provide evolutionary insights based on phylogenetic data of Ciconiidae. For this, we applied both classical and molecular cytogenetic techniques to define the pattern of distribution of heterochromatic blocks and their chromosomal homology with Gallus gallus (GGA). Maximum likelihood analyses and Bayesian inferences (680 bp COI and 1007 bp Cytb genes) were used to determine their phylogenetic relationship with other storks. The results confirmed 2n = 72, and the heterochromatin distribution pattern was restricted to centromeric regions of the chromosomes. FISH experiments identified fusion and fission events involving chromosomes homologous to GGA macrochromosome pairs, some of which were previously found in other species of Ciconiidae, possibly corresponding to synapomorphies for the group. Phylogenetic analyses resulted in a tree that recovered only Ciconinii as a monophyletic group, while Mycteriini and Leptoptlini tribes were configured as paraphyletic clades. In addition, the association between phylogenetic and cytogenetic data corroborates the hypothesis of a reduction in the diploid number throughout the evolution of Ciconiidae.

Sperberacris, a new Neotropical genus of Gomphocerinae (Orthoptera: Acridoidea: Acrididae) from State of Alagoas, Brazil with chromosome complement.

Sperberacris, a new Neotropical genus of Amblytropidiini. Sperberacris muriciensis n. sp. (type species) from the municipality of Murici, State of Alagoas, Brazil is described. The new genus differs from other tribe congenera by the epiproct suboval, furculae divided into four long sclerotized lobes, cerci curved inward, apex truncated and sclerotized at the tip and convex bridge of the epiphallus with two large black sclerotized spots. The new species presents a diploid number of 2n = 23, X0♂/24, XX♀, and the karyotype is made up of telo/acrocentric chromosomes, with one dot-like B chromosome occurring in some nuclei. Morphological descriptions are provided along with illustrations emphasizing the most significant diagnostic features of external morphology and male genitalia. Information on type specimens, material examined, and distribution provided.

Morphological and anatomical characterization of yellow diploid potato flower for effective breeding program.

The diploid yellow potato (Solanum tuberosum L. Phureja Group) is an important plant genetic resource. In this study, we report for the first time the characterization of anther development and pollen formation in the cultivar Criolla Colombia. The description of morphological and histological characters of buds and flowers at different developmental stages permitted to identify ten main stages, from the differentiation of the male cells of the sporangium, meiosis, microspores formation and maturation, to the release of mature pollen. In addition, the results provide a graphic guide of the development of the anther, through the sequential and orderly formation of the epidermis, the endothecium, the middle layer and the nutritive layer or tapetum. This microanatomical information will be useful for work focused on androgenesis and identification of gene regulation in floral biology and gamete formation. Therefore, this study determined that to efficiently obtain haploids, flower buds between 5 and 8.9 mm long (stage 6 to 8) should be used, in which tetrads and microspores are in the early uninucleate and binucleate stage.

Comparative repeatome analysis reveals new evidence on genome evolution in wild diploid Arachis (Fabaceae) species.

MAIN CONCLUSION: Opposing changes in the abundance of satellite DNA and long terminal repeat (LTR) retroelements are the main contributors to the variation in genome size and heterochromatin amount in Arachis diploids. The South American genus Arachis (Fabaceae) comprises 83 species organized in nine taxonomic sections. Among them, section Arachis is characterized by species with a wide genome and karyotype diversity. Such diversity is determined mainly by the amount and composition of repetitive DNA. Here we performed computational analysis on low coverage genome sequencing to infer the dynamics of changes in major repeat families that led to the differentiation of genomes in diploid species (x = 10) of genus Arachis, focusing on section Arachis. Estimated repeat content ranged from 62.50 to 71.68% of the genomes. Species with different genome composition tended to have different landscapes of repeated sequences. Athila family retrotransposons were the most abundant and variable lineage among Arachis repeatomes, with peaks of transpositional activity inferred at different times in the evolution of the species. Satellite DNAs (satDNAs) were less abundant, but differentially represented among species. High rates of evolution of an AT-rich superfamily of satDNAs led to the differential accumulation of heterochromatin in Arachis genomes. The relationship between genome size variation and the repetitive content is complex. However, largest genomes presented a higher accumulation of LTR elements and lower contents of satDNAs. In contrast, species with lowest genome sizes tended to accumulate satDNAs in detriment of LTR elements. Phylogenetic analysis based on repetitive DNA supported the genome arrangement of section Arachis. Altogether, our results provide the most comprehensive picture on the repeatome dynamics that led to the genome differentiation of Arachis species.

A de novo genome assembly of Solanum verrucosum Schlechtendal, a Mexican diploid species geographically isolated from other diploid A-genome species of potato relatives.

There are over 100 known species of cultivated potatoes and their wild relatives. Many of these species, including cultivated potatoes, share the A genome; these species are mainly distributed in South America and are reproductively isolated from Mexican diploid species. The only diploid A-genome species distributed in Mexico is Solanum verrucosum Schlechtendal, which is also a maternal progenitor of Mexican polyploid species. In this study, we constructed a high-quality de novo assembly of the S. verrucosum genome using PacBio long-read sequencing and Hi-C scaffolding technologies. A monohaploid clone (2n = x = 12) of S. verrucosum was used to reduce assembly difficulty due to the heterozygous nature of the species. The final sequence assembly consisted of 780.2 Mb of sequence, 684.0 Mb of which were anchored to the 12 chromosomes, with a scaffold N50 of 55.2 Mb. Putative centromeres were identified using publicly available data obtained via chromatin immunoprecipitation sequencing against a centromere-specific histone 3 protein. Transposable elements accounted for approximately 61.8% (482.1 Mb) of the genome, and 46,904 genes were functionally annotated. High gene synteny and similarity were revealed among the genomes of S. verrucosum, Solanum commersonii, Solanum chacoense, Solanum phureja, Solanum tuberosum, and Solanum lycopersicum. The reference-quality S. verrucosum genome will provide new insights into the evolution of Mexican polyploid species and contribute to potato breeding programs.

Rapid karyotypic evolution with high diploid number variation in a rare genus of bromeligenous frogs.

Bromeligenous Crossodactylodes is a leptodactylid genus closely related to Paratelmatobius and Scythrophrys. The diploid number in all karyotyped species of these two latter genera is 24, which diverges from the modal diploid number (2n = 22) in the family. Here, we analyzed three species of Crossodactylodes and found karyotypes with 2n = 30, 2n = 32, and 2n = 36, diploid numbers that have not been reported in any other diploid leptodactylid species to date. Reconstruction of the ancestral chromosome number indicated that the diploid number changed from 22 to 24 in the common ancestor of Crossodactylodes, Paratelmatobius, and Scythrophrys, and that progressive increases in diploid number have occurred in Crossodactylodes. The large number of telocentric/subtelocentric chromosomes in karyotypes with higher diploid numbers raises the possibility that centric fissions may have occurred during the evolution of Paratelmatobiinae. Three metacentric chromosomes, probably involved in fission events, were inferred to be present in the common ancestor of all species of Crossodactylodes, but in C. bokermanni. Chromosome mapping of the satellite DNA PcP190 suggests homology between one arm of metacentric chromosome 1 of Crossodactylodes sp. 3 and telocentric chromosome 2 of C. itambe, supporting one of the presumed centric fission events.

Higher frequency of legitimate pollinators and fruit set of autotetraploid trees of Libidibia ferrea (Leguminosae) compared to diploids in a mixed tropical urban population.

In mixed-ploidy populations, newly formed polyploids initially occur at low frequencies when compared to diploids. However, polyploidy may lead to morphological and phenological changes, which promote reproductive isolation and favor polyploid establishment and reproductive success. Additionally, previous studies have shown that polyploidy can confer some adaptive advantages to organisms in stressful environments. Here, we investigate variation in reproductive phenology, floral traits and reproductive success between diploid and autotetraploid trees of Libidibia ferrea (Mart. Ex Tul.) L.P. Queiroz (Leguminosae) in a mixed tropical urban population, a stressful environment. We assessed ploidy levels, flowering and fruiting phenology, flowering synchrony, floral and reproductive biology, pollination and fruit and seed set. We tested the hypothesis that autotetraploid individuals have a higher frequency of pollinators and higher fruit and seed set per inflorescence (as a proxy of reproductive success) than diploids in an urban green space. Libidibia ferrea is a good model to test our hypothesis because it is self-incompatible (i.e. relies on pollinators to set fruits). In the urban ecosystem studied, we found that diploids flowered for 6-7 months/year and autotetraploids for 3-5 months/year. Flowering synchrony was low between and within cytotypes and even though autotetraploids and diploids exhibited some overlap in flowering period, diploids flowered alone for 2-3 months. Autotetraploids had significantly more flowers per inflorescences, larger flowers and larger pollen grains (as expected for polyploids), but also a higher frequency of visits by legitimate pollinators including two exclusive ones, and higher fruit and seed set per inflorescence when compared to diploids, despite having a shorter flowering period. Our findings reveal some advantages for polyploids over their related diploids in a tropical urban green space. Also, our results highlight the need for more studies that seek to understand abiotic mechanisms affecting reproductive output of polyploids in urban ecosystems.

Robust and efficient software for reference-free genomic diversity analysis of genotyping-by-sequencing data on diploid and polyploid species.

Genotyping-by-sequencing (GBS) is a widely used and cost-effective technique for obtaining large numbers of genetic markers from populations by sequencing regions adjacent to restriction cut sites. Although a standard reference-based pipeline can be followed to analyse GBS reads, a reference genome is still not available for a large number of species. Hence, reference-free approaches are required to generate the genetic variability information that can be obtained from a GBS experiment. Unfortunately, available tools to perform de novo analysis of GBS reads face issues of usability, accuracy and performance. Furthermore, few available tools are suitable for analysing data sets from polyploid species. In this manuscript, we describe a novel algorithm to perform reference-free variant detection and genotyping from GBS reads. Nonexact searches on a dynamic hash table of consensus sequences allow for efficient read clustering and sorting. This algorithm was integrated in the Next Generation Sequencing Experience Platform (NGSEP) to integrate the state-of-the-art variant detector already implemented in this tool. We performed benchmark experiments with three different empirical data sets of plants and animals with different population structures and ploidies, and sequenced with different GBS protocols at different read depths. These experiments show that NGSEP has comparable and in some cases better accuracy and always better computational efficiency compared to existing solutions. We expect that this new development will be useful for many research groups conducting population genetic studies in a wide variety of species.

Chromosomal view of Lippia alba, a tropical polyploid complex under genome stabilization process.

Lippia alba is a phenotypically variable tropical shrub thought to comprise a young autopolyploid complex. Chromosome numbers in L. alba include 2n = 30, 38, 45, 60, and 90. High levels of chemical and phenotypic variation associated with economic and medicinal importance were reported. However, the genetic background including chromosome composition remains under-explored. Furthermore, the occurrence of at least four ploidal levels in L. alba and the lack of data for polyploid plants in tropical areas also merit further study of L. alba. Here we assessed the chromosome composition using two new satellite repeats (CL98 and CL66) applied as FISH probes to mitotic chromosomes, and we proposed to calculate the degree of homozygosis for CL66 satDNA (named as index h) and to associate it to meiotic instability. The CL98 mapping showed few variations in both number of signals and position. However, the levels of structural homozygosity for a satellite repeat CL66 were very variable. The numbers of CL66-bearing-chromosomes were under-represented in tetraploids relative to diploids implying that CL66 arrays have been lost in tetraploid lineages as a result of increased meiotic instability. High percentage of irregularities was observed in meiotic cells, especially in polyploids. L. alba complex comprised a mixture of homomorphic and heteromorphic chromosomes. Overall, the polyploid complex presents features typical of both young and older stable polyploids. It seems that L. alba genome is still in the process of stabilization.

Genomic regions associated with physiological, biochemical and yield-related responses under water deficit in diploid potato at the tuber initiation stage revealed by GWAS.

Water deficit, which is increasing with climate change, is a serious threat to agricultural sustainability worldwide. Dissection of the genetic architecture of water deficit responses is highly desirable for developing water-deficit tolerant potato cultivars and enhancing the resilience of existing cultivars. This study examined genetic variation in response to water deficit in a panel of diploid potato and identified the QTL governing this trait via a genome-wide association study (GWAS). A panel of 104 diploid potato accessions were evaluated under both well-watered and water deficit treatments at tuber initiation stage. Drought stress index (DTI) was calculated to assess tolerance of the diploid potato genotypes to water deficit. The GWAS was conducted using a matrix of 47K single nucleotide polymorphisms (SNP), recently available for this population. We are reporting 38 QTL, seven for well-watered conditions, twenty-two for water deficit conditions and nine for DTI which explain between 12.6% and 44.1% of the phenotypic variance. A set of 6 QTL were found to be associated with more than one variable. Marker WDP-9.21 was found associated with tuber fresh weigh under WD and gene annotation analysis revealed co-localization with the Glucan/water dikinase (GWD) gene. Of the nine QTL detected from DTI on chromosomes 2,3,5,8,10 and 12, three candidate genes with a feasible role in water deficit response were identified. The findings of this study can be used in marker-assisted selection (MAS) for water- deficit tolerance breeding in potato.

Cytogenetic approaches provide evidence of a conserved diploid number and cytological differences between Proceratophrys species (Anura: Odontophrynidae).

Taxonomic and cytogenetic aspects of Proceratophrys have not been thoroughly clarified in the literature; thus, the objective of the present study was to provide unprecedent karyotype data regarding P. schirchi, P. laticeps and P. melanopogon. Additionally, the karyotype of P. boiei (2n = 22) and its ZZ / ZW sex chromosome system was analyzed for different populations of southeastern and southern Brazil. All Proceratophrys species have a diploid number of 2n = 22 chromosomes. In P. schirchi, a strong C-band was found in the long arm in one of the homologues of the pair 8 in female metaphasic cells, denoting that this chromosome pair could represent the heteromorphic sex chromosome in a ZZ / ZW sex system. Despite the conserved number of chromosomes, there are considerable chromosomal differences in P. melanopogon and P. boiei (southern Brazil), strongly differentiating them cytogenetically from other species of the genus. Moreover, with the confirmation of chromosomal heteromorphism related to sexual differentiation in P. boiei and the possible description of this system in P. schirchi, the Proceratophrys genus can be regarded as an interesting group for evolutionary studies and sex chromosome differentiation in anurans.

Species diversification in the lowlands of mid-latitude South America: Turnera sidoides subsp. carnea as a case study.

The lowlands of mid-latitude South America comprise complex temperate ecoregions characterized by a unique biodiversity. However, the processes responsible for shaping its species diversity are still largely unknown. Turnera sidoides subsp. carnea is a variable subspecies occurring in the lowlands of northeastern Argentina and Uruguay, extending to southern Paraguay and Brazil. It constitutes a good model to perform evolutionary studies. Here we used an integrative approach to understand the process of diversification within this subspecies and to increase the knowledge concerning patterns and processes responsible for shaping the species diversity in the temperate lowlands of South America. The results provided strong evidences that this subspecies is an autopolyploid complex per se, being in an active process of intrasubspecific diversification. Morphological and genetic data show that the diversity of T. sidoides subsp. carnea is in congruence with the great past and present abiotic and biotic variability of the mid-latitude South American lowlands. The evolutionary history of this subspecies is consistent with past fragmentation and allopatric differentiation at diploid level. Geographic isolation and local adaptation would have promoted strong morphological, ecological, and genetic differentiation, resulting in two morphotypes and different genetic groups indicative of incipient speciation.