Genetic Variability in Balkan Paleoendemic Resurrection Plants Ramonda serbica and R. nathaliae Across Their Range and in the Zone of Sympatry.

The genus Ramonda includes three Paleoendemic and Tertiary relict species that survived in refugial habitats of the Balkan Peninsula (R. nathaliae and R. serbica) and the Iberian Peninsula (R. myconi). They are all "resurrection plants," a rare phenomenon among flowering plants in Europe. Ramonda myconi and R. nathaliae are diploids (2n = 2x = 48), while R. serbica is a hexaploid (2n = 6x = 144). The two Balkan species occur in sympatry in only two localities in eastern Serbia, where tetraploid potential hybrids (2n = 4x = 96) were found. This observation raised questions about the existence of gene flow between the two species and, more generally, about the evolutionary processes shaping their genetic diversity. To address this question, genetic markers (AFLP) and an estimate of genome size variation were used in a much larger sample and at a larger geographic scale than previously. The combination of AFLP markers and genome size results suggested ongoing processes of interspecific and interploidy hybridization in the two sites of sympatry. The data also showed that interspecific gene flow was strictly confined to sympatry. Elsewhere, both Ramonda species were characterized by low genetic diversity within populations and high population differentiation. This is consistent with the fact that the two species are highly fragmented into small and isolated populations, likely a consequence of their postglacial history. Within sympatry, enormous variability in cytotypes was observed, exceeding most reported cases of mixed ploidy in complex plant species (from 2x to >8x). The AFLP profiles of non-canonical ploidy levels indicated a diversity of origin pathways and that backcrosses probably occur between tetraploid interspecific hybrids and parental species. The question arises whether this diversity of cytotypes corresponds to a transient situation. If not, the question arises as to the genetic and ecological mechanisms that allow this diversity to be maintained over time.

Functional repertoire convergence of distantly related eukaryotic plankton lineages abundant in the sunlit ocean.

Marine planktonic eukaryotes play critical roles in global biogeochemical cycles and climate. However, their poor representation in culture collections limits our understanding of the evolutionary history and genomic underpinnings of planktonic ecosystems. Here, we used 280 billion Tara Oceans metagenomic reads from polar, temperate, and tropical sunlit oceans to reconstruct and manually curate more than 700 abundant and widespread eukaryotic environmental genomes ranging from 10 Mbp to 1.3 Gbp. This genomic resource covers a wide range of poorly characterized eukaryotic lineages that complement long-standing contributions from culture collections while better representing plankton in the upper layer of the oceans. We performed the first, to our knowledge, comprehensive genome-wide functional classification of abundant unicellular eukaryotic plankton, revealing four major groups connecting distantly related lineages. Neither trophic modes of plankton nor its vertical evolutionary history could completely explain the functional repertoire convergence of major eukaryotic lineages that coexisted within oceanic currents for millions of years.

Phylogenomics resolves deep subfamilial relationships in Malvaceae s.l.

Malvaceae s.l., the most diverse family within Malvales, includes well-known species of great economic importance like cotton, cacao, and durian. Despite numerous phylogenetic analyses employing multiple markers, relationships between several of its nine subfamilies, particularly within the largest lineage /Malvadendrina, remain unclear. In this study, we attempted to resolve the relationships within the major clades of Malvaceae s.l. using plastid genomes of 48 accessions representing all subfamilies. Maximum likelihood and Bayesian analyses recovered a fully resolved and well-supported topology confirming the split of the family into /Byttneriina (/Grewioideae +/Byttnerioideae) and /Malvadendrina. Within /Malvadendrina, /Helicteroideae occupied the earliest branching position, followed by /Sterculioideae, /Brownlowioideae, /Tiliodeae, and /Dombeyoideae formed a clade sister to /Malvatheca (/Malvoideae +/Bombacoideae), a grouping morphologically supported by the lack of androgynophore. Results from dating analyses suggest that all subfamilies originated during hot or warm phases in the Late Cretaceous to Paleocene. This study presents a well-supported phylogenetic framework for Malvaceae s.l. that will aid downstream revisions and evolutionary studies of this economically important plant family.

Chromosome-level reference genome of the soursop (Annona muricata): A new resource for Magnoliid research and tropical pomology.

The flowering plant family Annonaceae includes important commercially grown tropical crops, but development of promising species is hindered by a lack of genomic resources to build breeding programs. Annonaceae are part of the magnoliids, an ancient lineage of angiosperms for which evolutionary relationships with other major clades remain unclear. To provide resources to breeders and evolutionary researchers, we report a chromosome-level genome assembly of the soursop (Annona muricata). We assembled the genome using 444.32 Gb of DNA sequences (676× sequencing depth) from PacBio and Illumina short-reads, in combination with 10× Genomics and Bionano data (v1). A total of 949 scaffolds were assembled to a final size of 656.77 Mb, with a scaffold N50 of 3.43 Mb (v1), and then further improved to seven pseudo-chromosomes using Hi-C sequencing data (v2; scaffold N50: 93.2 Mb, total size in chromosomes: 639.6 Mb). Heterozygosity was very low (0.06%), while repeat sequences accounted for 54.87% of the genome, and 23,375 protein-coding genes with an average of 4.79 exons per gene were annotated using de novo, RNA-seq and homology-based approaches. Reconstruction of the historical population size showed a slow continuous contraction, probably related to Cenozoic climate changes. The soursop is the first genome assembled in Annonaceae, supporting further studies of floral evolution in magnoliids, providing an essential resource for delineating relationships of ancient angiosperm lineages. Both genome-assisted improvement and conservation efforts will be strengthened by the availability of the soursop genome. As a community resource, this assembly will further strengthen the role of Annonaceae as model species for research on the ecology, evolution and domestication potential of tropical species in pomology and agroforestry.

Genome-wide supermatrix analyses of maples (Acer, Sapindaceae) reveal recurring inter-continental migration, mass extinction, and rapid lineage divergence.

Acer (Sapindaceae) is an exceptional study system for understanding the evolutionary history, divergence, and assembly of broad-leaved deciduous forests at higher latitudes. Maples stand out due to their high diversity, disjunct distribution pattern across the northern continents, and rich fossil record dating back to the Paleocene. Using a genome-wide supermatrix combining plastomes and nuclear sequences (~585 kb) for 110 Acer taxa, we built a robust time-calibrated hypothesis investigating the evolution of maples, inferring ancestral ranges, reconstructing diversification rates over time, and exploring the impact of mass-extinction on lineage accumulation. Contrary to fossil evidence, our results indicate Acer first originated in the (north)eastern Palearctic region, which acted as a source for recurring outward migration. Warm conditions favored rapid Eocene-onward divergence, but ranges and diversity declined extensively as a result of the Plio-Pleistocene glacial cycles. These signals in genome-wide sequence data corroborate paleobotanical evidence for other major woody north-temperate groups, highlighting the significant (disparate) impact of climatic changes on the evolution, composition, and distribution of the vegetation in the northern hemisphere.

Plastome comparative genomics in maples resolves the infrageneric backbone relationships.

Maples (Acer) are among the most diverse and ecologically important tree genera of the north-temperate forests. They include species highly valued as ornamentals and as a source of timber and sugar products. Previous phylogenetic studies employing plastid markers have not provided sufficient resolution, particularly at deeper nodes, leaving the backbone of the maple plastid tree essentially unresolved. We provide the plastid genome sequences of 16 species of maples spanning the sectional diversity of the genus and explore the utility of these sequences as a source of information for genetic and phylogenetic studies in this group. We analyzed the distribution of different types of repeated sequences and the pattern of codon usage, and identified variable regions across the plastome. Maximum likelihood and Bayesian analyses using two partitioning strategies were performed with these and previously published sequences. The plastomes ranged in size from 155,212 to 157,023 bp and had structure and gene content except for Acer palmatum (sect. Palmata), which had longer inverted repeats and an additional copy of the rps19 gene. Two genes, rps2 and rpl22, were found to be truncated at different positions and might be non-functional in several species. Most dispersed repeats, SSRs, and overall variation were detected in the non-coding sequences of the LSC and SSC regions. Fifteen loci, most of which have not been used before in the genus, were identified as the most variable and potentially useful as molecular markers for barcoding and genetic studies. Both ML and Bayesian analyses produced similar results irrespective of the partitioning strategy used. The plastome-based tree largely supported the topology inferred in previous studies using cp markers while providing resolution to the backbone relationships but was highly incongruous with a recently published nuclear tree presenting an opportunity for further research to investigate the causes of discordance, and particularly the role of hybridization in the diversification of the genus. Plastome sequences are valuable tools to resolve deep-level relationships within Acer. The variable loci and SSRs identified in this study will facilitate the development of markers for ecological and evolutionary studies in the genus. This study underscores the potential of plastid genome sequences to improve our understanding of the evolution of maples.

Museomics for reconstructing historical floristic exchanges: Divergence of stone oaks across Wallacea.

Natural history collections and tropical tree diversity are both treasure troves of biological and evolutionary information, but their accessibility for scientific study is impeded by a number of properties. DNA in historical specimens is generally highly fragmented, complicating the recovery of high-grade genetic material. Furthermore, our understanding of hyperdiverse, wide-spread tree assemblages is obstructed by extensive species ranges, fragmented knowledge of tropical tree diversity and phenology, and a widespread lack of species-level diagnostic characters, prohibiting the collecting of readily identifiable specimens which can be used to build, revise or strengthen taxonomic frameworks. This, in turn, delays the application of downstream conservation action. A sizable component of botanical collections are sterile-thus eluding identification and are slowing down progress in systematic treatments of tropical biodiversity. With rapid advances in genomics and bioinformatic approaches to biodiversity research, museomics is emerging as a new field breathing life into natural collections that have been built up over centuries. Using MIGseq (multiplexed ISSR genotyping by sequencing), we generated 10,000s of short loci, for both freshly collected materials and museum specimens (aged >100 years) of Lithocarpus-a widespread tropical tree genus endemic to the Asian tropics. Loci recovery from historical and recently collected samples was not affected by sample age and preservation history of the study material, underscoring the reliability and flexibility of the MIGseq approach. Phylogenomic inference and biogeographic reconstruction across insular Asia, highlights repeated migration and diversification patterns between continental regions and islands. Results indicate that co-occurring insular species at the extremity of the distribution range are not monophyletic, raising the possibility of multiple independent dispersals along the outer edge of Wallacea. This suggests that dispersal of large seeded tree genera throughout Malesia and across Wallacea may have been less affected by large geographic distances and the presence of marine barriers than generally assumed. We demonstrate the utility of MIGseq in museomic studies using non-model taxa, presenting the first range-wide genomic assessment of Lithocarpus and tropical Fagaceae as a proof-of-concept. Our study shows the potential for developing innovative genomic approaches to improve the capture of novel evolutionary signals using valuable natural history collections of hyperdiverse taxa.

ECuADOR-Easy Curation of Angiosperm Duplicated Organellar Regions, a tool for cleaning and curating plastomes assembled from next generation sequencing pipelines.

BACKGROUND: With the rapid increase in availability of genomic resources offered by Next-Generation Sequencing (NGS) and the availability of free online genomic databases, efficient and standardized metadata curation approaches have become increasingly critical for the post-processing stages of biological data. Especially in organelle-based studies using circular chloroplast genome datasets, the assembly of the main structural regions in random order and orientation represents a major limitation in our ability to easily generate "ready-to-align" datasets for phylogenetic reconstruction, at both small and large taxonomic scales. In addition, current practices discard the most variable regions of the genomes to facilitate the alignment of the remaining coding regions. Nevertheless, no software is currently available to perform curation to such a degree, through simple detection, organization and positioning of the main plastome regions, making it a time-consuming and error-prone process. Here we introduce a fast and user friendly software ECuADOR, a Perl script specifically designed to automate the detection and reorganization of newly assembled plastomes obtained from any source available (NGS, sanger sequencing or assembler output). METHODS: ECuADOR uses a sliding-window approach to detect long repeated sequences in draft sequences, which then identifies the inverted repeat regions (IRs), even in case of artifactual breaks or sequencing errors and automates the rearrangement of the sequence to the widely used LSC-Irb-SSC-IRa order. This facilitates rapid post-editing steps such as creation of genome alignments, detection of variable regions, SNP detection and phylogenomic analyses. RESULTS: ECuADOR was successfully tested on plant families throughout the angiosperm phylogeny by curating 161 chloroplast datasets. ECuADOR first identified and reordered the central regions (LSC-Irb-SSC-IRa) for each dataset and then produced a new annotation for the chloroplast sequences. The process took less than 20 min with a maximum memory requirement of 150 MB and an accuracy of over 99%. CONCLUSIONS: ECuADOR is the sole de novo one-step recognition and re-ordination tool that provides facilitation in the post-processing analysis of the extra nuclear genomes from NGS data. The program is available at https://github.com/BiodivGenomic/ECuADOR/.

Trochodendron aralioides, the first chromosome-level draft genome in Trochodendrales and a valuable resource for basal eudicot research.

BACKGROUND: The wheel tree (Trochodendron aralioides) is one of only 2 species in the basal eudicot order Trochodendrales. Together with Tetracentron sinense, the family is unique in having secondary xylem without vessel elements, long considered to be a primitive character also found in Amborella and Winteraceae. Recent studies however have shown that Trochodendraceae belong to basal eudicots and demonstrate that this represents an evolutionary reversal for the group. Trochodendron aralioides is widespread in cultivation and popular for use in gardens and parks. FINDINGS: We assembled the T. aralioides genome using a total of 679.56 Gb of clean reads that were generated using both Pacific Biosciences and Illumina short-reads in combination with 10XGenomics and Hi-C data. Nineteen scaffolds corresponding to 19 chromosomes were assembled to a final size of 1.614 Gb with a scaffold N50 of 73.37 Mb in addition to 1,534 contigs. Repeat sequences accounted for 64.226% of the genome, and 35,328 protein-coding genes with an average of 5.09 exons per gene were annotated using de novo, RNA-sequencing, and homology-based approaches. According to a phylogenetic analysis of protein-coding genes, T. aralioides diverged in a basal position relative to core eudicots, ∼121.8-125.8 million years ago. CONCLUSIONS: Trochodendron aralioides is the first chromosome-scale genome assembled in the order Trochodendrales. It represents the largest genome assembled to date in the basal eudicot grade, as well as the closest order relative to the core-eudicots, as the position of Buxales remains unresolved. This genome will support further studies of wood morphology and floral evolution, and will be an essential resource for understanding rapid changes that took place at the base of the Eudicot tree. Finally, it can further genome-assisted improvement for cultivation and conservation efforts of the wheel tree.

A phylogenetic re-analysis of groupers with applications for ciguatera fish poisoning.

BACKGROUND: Ciguatera fish poisoning (CFP) is a significant public health problem due to dinoflagellates. It is responsible for one of the highest reported incidence of seafood-borne illness and Groupers are commonly reported as a source of CFP due to their position in the food chain. With the role of recent climate change on harmful algal blooms, CFP cases might become more frequent and more geographically widespread. Since there is no appropriate treatment for CFP, the most efficient solution is to regulate fish consumption. Such a strategy can only work if the fish sold are correctly identified, and it has been repeatedly shown that misidentifications and species substitutions occur in fish markets. METHODS: We provide here both a DNA-barcoding reference for groupers, and a new phylogenetic reconstruction based on five genes and a comprehensive taxonomical sampling. We analyse the correlation between geographic range of species and their susceptibility to ciguatera accumulation, and the co-occurrence of ciguatoxins in closely related species, using both character mapping and statistical methods. RESULTS: Misidentifications were encountered in public databases, precluding accurate species identifications. Epinephelinae now includes only twelve genera (vs. 15 previously). Comparisons with the ciguatera incidences show that in some genera most species are ciguateric, but statistical tests display only a moderate correlation with the phylogeny. Atlantic species were rarely contaminated, with ciguatera occurrences being restricted to the South Pacific. CONCLUSIONS: The recent changes in classification based on the reanalyses of the relationships within Epinephelidae have an impact on the interpretation of the ciguatera distribution in the genera. In this context and to improve the monitoring of fish trade and safety, we need to obtain extensive data on contamination at the species level. Accurate species identifications through DNA barcoding are thus an essential tool in controlling CFP since meal remnants in CFP cases can be easily identified with molecular tools.

The phylogeography of Eurasian Fraxinus species reveals ancient transcontinental reticulation.

To investigate the biogeographical history of ashes species of the Eurasian section Fraxinus and to test the hypothesis of ancient reticulations, we sequenced nuclear DNA (nETS and nITS, 1075 bp) for 533 samples and scored AFLP for 63 samples of Eurasian ashes within the section Fraxinus. The nITS phylogeny retrieved the classical view of the evolution of the section, whereas nETS phylogeny indicated an unexpected separation of F. angustifolia in two paraphyletic groups, respectively found in southeastern Europe and in the other parts of the Mediterranean basin. In the nETS phylogeny, the former group was closely related to F. excelsior, whereas the later was closely related to F. mandshurica, a species which is restricted nowadays to northeastern Asia. This topological incongruence between the two loci indicated the occurrence of an ancient reticulation between European and Asian ash species. Several other ancient reticulation events between the two European species and the other species of the section were supported by the posterior predictive checking method. Some of these reticulation events would have occurred during the Miocene, when climatic variations may have lead these species to expand their distribution range and come into contact. The recurrent reticulations observed among Eurasian ash species indicate that they should be considered as conspecific taxa, with subspecific status for some groups. Altogether, the results of the present study provide a rare documented evidence for the occurrence of multiple ancient reticulations within a group of temperate tree taxa with modern disjunct distributions in Eurasia. These ancient reticulation events indicate that the speciation process is slow in ashes, necessitating long periods of geographical isolation. The implications for speciation processes in temperate trees with similar life history and reproductive biology are discussed.

The 'Pseudorhabdosynochus cupatus group' (Monogenea: Diplectanidae) on Epinephelus fasciatus, E. howlandi, E. rivulatus and E. merra (Perciformes: Serranidae) off New Caledonia, with descriptions of Pseudorhabdosynochus cyathus n. sp. and P. calathus n. sp.

Pseudorhabdosynochus cupatus (Young, 1969) is characterised by small lamellosquamodiscs made up of central telescopic rings and peripheral rows of rodlets, and a sclerotised vagina in the shape of a thin-walled coiled tube with three accessory chambers. Two new species of Pseudorhabdosynochus are herein described from epinepheline fish off New Caledonia, South Pacific, and are considered part of the 'P. cupatus group'. P. cyathus n. sp. from Epinephelus howlandi has lamellosquamodiscs made up of four telescopic rings and four rows of separate rodlets; there are dorsal tegumental scales. This species was found only in young hosts; older hosts harbour P. venus Hinsinger & Justine, 2006. P. calathus n. sp. from Epinephelus rivulatus has lamellosquamodiscs made up of four telescopic rings and five rows of separate rodlets; the tegument is smooth. The three species, P. cupatus, P. calathus and P. cyathus, are strictly species specific; although morphologically very similar, they can be distinguished by features of the tegumental scales and measurements of the vagina and haptoral hard parts. P. melanesiensis (Laird, 1958) from E. merra is close to the 'P. cupatus group' according to the morphology of its sclerotised vagina but is distinguished by its squamodiscs which lack telescopic rings. Rare specimens found in E. merra are tentatively attributed to P. coioidesis Bu et al., 1999. A paratype of P. coioidesis is figured for comparison. E. merra, E. fasciatus, E. rivulatus and E. howlandi each have a Pseudorhabdosynochus species of the P. cupatus lineage: these are the dominant species in each host. In addition, E. merra, E. fasciatus and E. howlandi harbour a rare species of Pseudorhabdosynochus, respectively P. cf. coioidesis, P. caledonicus Justine, 2005 and P. venus; these rare species are morphologically unrelated to each other and to the 'P. cupatus group'.

Pseudorhabdosynochus venus n. sp. (Monogenea: Diplectanidae) from Epinephelus howlandi (Perciformes: Serranidae) off New Caledonia.

Pseudorhabdosynochus venus n. sp. is described from specimens collected from the gills of Epinephelus howlandi off Nouméa, New Caledonia, South Pacific. The male sclerotised quadriloculate organ of P. venus, 61-101 microm in internal length, has an anterior chamber with a thin anterior wall, a very short cone and a short posterior tube. The sclerotised vagina, 50-67 microm in total length, is composed of an anterior open trumpet, an S-shaped canal, a tear-shaped principal chamber and a spherical accessory chamber; all parts are heavily sclerotised. The two squamodiscs have 10-11 rows of separate rodlets and no central closed row of rodlets. P. venus is differentiated from all other species of Pseudorhabdosynochus by the spectacular morphology of its sclerotised vagina. It is the first diplectanid described from E. howlandi.