Low-copy Genes Research Articles

Carica papaya L. sex chromosome review and physical mapping of the serk 2, svp-like and mdar 4 sequences

Physical mapping evidences the chromosome organization and structure. Despite the data about plant cytogenomics, physical mapping has been conducted from single-copy and/or low-copy genes for few species. Carica papaya cytogenomics has been accomplished from BAC-FISH and repeatome sequences. We aimed to map the serk 2, svp-like and mdar 4 sequences in C. papaya. The sequences were amplified and the amplicons sequenced, showing similarity in relation to serk 2, svp-like and mdar 4 genes. Carica papaya diploidy was confirmed and the mitotic chromosomes characterized. The chromosome 1 exhibited the secondary constriction pericentromeric to the centromere of the long arm. So, we concluded that it is the sex chromosomes. serk 2 was mapped in the long arm interstitial portion of the sex chromosomes, and the interphase nuclei showed two fluorescence signals. Considering these results and the sequencing data from the C. papaya sex chromosomes, svp-like and mdar 4 genes were mapped in the interstitial region of the sex chromosome long arm. Both sequences showed only one fluorescence signal in the interphase nuclei. The procedure adopted here can be reproduced for other single-copy and/or low-copy genes, allowing the construction of cytogenetic maps. In addition, we revisited the cytogenomics data about C. papaya sex chromosomes, presenting a revised point of view about the structure and evolution to these chromosomes.

Phylogenetic insight into ABCE gene subfamily in plants.

ATP-BINDING CASSETTE SUBFAMILY E MEMBER (ABCE) proteins are one of the most conserved proteins across eukaryotes and archaea. Yeast and most animals possess a single ABCE gene encoding the critical translational factor ABCE1. In several plant species, including Arabidopsis thaliana and Oryza sativa, two or more ABCE gene copies have been identified, however information related to plant ABCE gene family is still missing. In this study we retrieved ABCE gene sequences of 76 plant species from public genome databases and comprehensively analyzed them with the reference to A. thaliana ABCE2 gene (AtABCE2). Using bioinformatic approach we assessed the conservation and phylogeny of plant ABCEs. In addition, we performed haplotype analysis of AtABCE2 and its paralogue AtABCE1 using genomic sequences of 1,135 A. thaliana ecotypes. Plant ABCE proteins showed overall high sequence conservation, sharing at least 78% of amino acid sequence identity with AtABCE2. We found that over half of the selected species have two to eight ABCE genes, suggesting that in plants ABCE genes can be classified as a low-copy gene family, rather than a single-copy gene family. The phylogenetic trees of ABCE protein sequences and the corresponding coding sequences demonstrated that Brassicaceae and Poaceae families have independently undergone lineage-specific split of the ancestral ABCE gene. Other plant species have gained ABCE gene copies through more recent duplication events. We also noticed that ploidy level but not ancient whole genome duplications experienced by a species impacts ABCE gene family size. Deeper analysis of AtABCE2 and AtABCE1 from 1,135 A. thaliana ecotypes revealed four and 35 non-synonymous SNPs, respectively. The lower natural variation in AtABCE2 compared to AtABCE1 is in consistence with its crucial role for plant viability. Overall, while the sequence of the ABCE protein family is highly conserved in the plant kingdom, many plants have evolved to have more than one copy of this essential translational factor.

Ancient hybridization in Curcuma (Zingiberaceae)-Accelerator or brake in lineage diversifications?

Hybridization is a widespread phenomenon in the evolution of plants and exploring its role is crucial to understanding diversification processes of many taxonomic groups. Recently, more attention is focused on the role of ancient hybridization that has repeatedly been shown as triggers of evolutionary radiation, although in some cases, it can prevent further diversification. The causes, frequency, and consequences of ancient hybridization remain to be explored. Here, we present an account of several events of ancient hybridization in turmeric, the economically important plant genus Curcuma (Zingiberaceae), which harbors about 130 known species. We analyzed 1094 targeted low-copy genes and plastomes obtained by next-generation sequencing of 37 species of Curcuma, representing the known genetic diversity and spanning the geographical distribution of the genus. Using phylogenetic network analysis, we show that the entire genus Curcuma as well as its most speciose lineage arose via introgression from the genus Pyrgophyllum and one of the extinct lineages, respectively. We also document a single event of ancient hybridization, with C. vamana as a product, that represents an evolutionary dead end. We further discuss distinct circumstances of those hybridization events that deal mainly with (in)congruence in chromosome counts of the parental lineages.

Phylotranscriptomics supports numerous polyploidization events and phylogenetic relationships in Nicotiana.

Nicotiana L. (Solanaceae) is of great scientific and economic importance, and polyploidization has been pivotal in shaping this genus. Despite many previous studies on the Nicotiana phylogenetic relationship and hybridization, evidence from whole genome data is still lacking. In this study, we obtained 995 low-copy genes and plastid transcript fragments from the transcriptome datasets of 26 Nicotiana species, including all sections. We reconstructed the phylogenetic relationship and phylogenetic network of diploid species. The incongruence among gene trees showed that the formation of N. sylvestris involved incomplete lineage sorting. The nuclear-plastid discordance and nuclear introgression absence indicated that organelle capture from section Trigonophyllae was involved in forming section Petunioides. Furthermore, we analyzed the evolutionary origin of polyploid species and dated the time of hybridization events based on the analysis of PhyloNet, sequence similarity search, and phylogeny of subgenome approaches. Our results highly evidenced the hybrid origins of five polyploid sections, including sections Nicotiana, Repandae, Rusticae, Polydicliae, and Suaveolentes. Notably, we provide novel insights into the hybridization event of section Polydicliae and Suaveolentes. The section Polydicliae formed from a single hybridization event between maternal progenitor N. attenuata and paternal progenitor N. undulata; the N. sylvestris (paternal progenitor) and the N. glauca (maternal progenitor) were involved in the formation of section Suaveolentes. This study represents the first exploration of Nicotiana polyploidization events and phylogenetic relationships using the high-throughput RNA-seq approach. It will provide guidance for further studies in molecular systematics, population genetics, and ecological adaption studies in Nicotiana and other related species.

Malaria mitochondrial diagnosis: challenges and pitfalls

BackgroundHigh-copy genomic sequences could be used as PCR targets for the detection of Plasmodium infections, providing increased sensitivity over single- or low-copy genes. Mitochondrial genomes of malaria parasites are present in multiple copies in a single mitochondrion, and each parasite has many mitochondria. Here, we describe the development of seven species-specific qPCR assays for the diagnosis of Plasmodium vivax and Plasmodium falciparum, targeting coding and non-coding mitochondrial genomic regions.MethodsThe optimization of the qPCR protocols involved a gradient of annealing temperatures and concentrations of primers and probes, as well as the inclusion of PCR additives/enhancers [e.g., dimethyl sulfoxide (DMSO), glycerol, bovine serum albumin (BSA)] to improve the specificity of qPCR amplification.ResultsNon-specific amplification of other Plasmodium species and of human targets was observed in different levels for all assays. Regardless of the late Cq values for most non-specific amplifications, the application of a cutoff value did not completely exclude false-positive amplification, compromising the specificity and also the sensitivity of the assays.ConclusionsTherefore, although mitochondrial targets have higher sensitivity, they frequently lose specificity due to their high levels of sequence conservation. A screening to evaluate the cross-reaction between Plasmodium species and the non-specific amplification of human malaria-free samples must be performed for Plasmodium mitochondrial assays.

Notes to the Taxonomic Affiliation of the Bulbophyllym Sect. Physometra (Orchidaceae, Epidendroideae) Based on Molecular Phylogenetic Analyses.

To solve the taxonomic affiliation of Bulbophyllum physometrum, the only known species of the Bulbophyllym sect. Physometra (Orchidaceae, Epidendroideae), we conducted phylogenetic analyses based on nuclear markers, i.e., ITS and the low-copy gene Xdh, and the plastid region matK. We used Asian Bulbophyllum taxa, with a special focus on species from the sections Lemniscata and Blepharistes, i.e., the only Asian sections of this genus with bifoliate pseudobulbs, as in B. physometrum. Unexpectedly, the results of molecular phylogenetic analyses showed that B. physometrum is most probably more related to the representatives of the sections Hirtula and Sestochilos than Blepharistes or Lemniscata.

Phylogenomic analyses of Camellia support reticulate evolution among major clades

Camellia (Theaceae) is a morphologically highly diverse genus of flowering plants and includes many famous species with high economic value, and the phylogeny of this genus is not fully resolved. We used 95 transcriptomes from 87 Camellia species and identified 1481 low-copy genes to conduct a detailed analysis of the phylogeny of this genus according to various data-screening criteria. The results show that, very different from the two existing classification systems of Camellia, 87 species are grouped into 8 main clades and two independent species, and that all 8 clades except Clade 8 were strongly supported by almost all the coalescent or concatenated trees using different gene subsets. However, the relationships among these clades were weakly supported and different from analyses using different gene subsets; furthermore, they do not agree with the phylogeny from chloroplast genomes of Camellia. Additional analyses support reticulate evolution (probably resulting from introgression or hybridization) among some major Camellia lineages, providing explanation for extensive gene tree conflicts. Furthermore, we inferred that together with the formation of East Asian subtropical evergreen broad-leaved forests, Camellia underwent a radiative divergence of major clades at 23 ∼ 19 Ma in the late Miocene then had a subsequent species burst at 10 ∼ 5 Ma. Principal component and cluster analyses provides new insights into morphological changes underlying the evolution of Camellia and a reference to further clarify subgenus and sections of this genus. The comprehensive study here including a nuclear phylogeny and other analyses reveal the rapid evolutionary history of Camellia.

Spatiotemporal process of long-distance seed dispersal in a pantropically distributed sea hibiscus group.

Long-distance dispersal (LDD) of seeds plays an essential role in the migration of plants to a new habitat and maintaining gene flow among geographically isolated populations. Pantropical plants with sea-drifted seeds, which have one of the largest distributions in all flowering plants, have achieved their global distribution by LDD. However, the spatiotemporal processes to achieve the wide distribution and the role of LDD in it have not yet been elucidated. In this study, we conducted phylogenomic analyses on the plastome, genome-wide nuclear SNP, and low-copy gene data of Hibiscus tiliaceus and its relatives. The dated phylogeny suggested that global expansion started approximately 4 million years ago (Ma), and species diversification occurred 1Ma. Plastome phylogeny confirmed the nonmonophyly of the haplotypes in the two widely distributed coastal species, H. tiliaceus and H. pernambucensis. In contrast, genome-wide nuclear SNP phylogenies demonstrated clear genetic segregation among species and/or geographical regions. Ancestral polymorphisms in chloroplast genomes shared among widely distributed species have remained below the range of rapid expansion and speciation of marginal populations. This study demonstrated that the LDD of sea-drifted seeds contributed to the rapid expansion and pantropical distribution of sea hibiscus in the last few million years, and adaptation to local environment or isolation by regional effect after LDD promoted speciation, suppressing gene flow.

A nuclear target sequence capture probe set for phylogeny reconstruction of the charismatic plant family Bignoniaceae.

The plant family Bignoniaceae is a conspicuous and charismatic element of the tropical flora. The family has a complex taxonomic history, with substantial changes in the classification of the group during the past two centuries. Recent re-classifications at the tribal and generic levels have been largely possible by the availability of molecular phylogenies reconstructed using Sanger sequencing data. However, our complete understanding of the systematics, evolution, and biogeography of the family remains incomplete, especially due to the low resolution and support of different portions of the Bignoniaceae phylogeny. To overcome these limitations and increase the amount of molecular data available for phylogeny reconstruction within this plant family, we developed a bait kit targeting 762 nuclear genes, including 329 genes selected specifically for the Bignoniaceae; 348 genes obtained from the Angiosperms353 with baits designed specifically for the family; and, 85 low-copy genes of known function. On average, 77.4% of the reads mapped to the targets, and 755 genes were obtained per species. After removing genes with putative paralogs, 677 loci were used for phylogenetic analyses. On-target genes were compared and combined in the Exon-Only dataset, and on-target + off-target regions were combined in the Supercontig dataset. We tested the performance of the bait kit at different taxonomic levels, from family to species-level, using 38 specimens of 36 different species of Bignoniaceae, representing: 1) six (out of eight) tribal level-clades (e.g., Bignonieae, Oroxyleae, Tabebuia Alliance, Paleotropical Clade, Tecomeae, and Jacarandeae), only Tourrettieae and Catalpeae were not sampled; 2) all 20 genera of Bignonieae; 3) seven (out of nine) species of Dolichandra (e.g., D. chodatii, D. cynanchoides, D. dentata, D. hispida, D. quadrivalvis, D. uncata, and D. uniguis-cati), only D. steyermarkii and D. unguiculata were not sampled; and 4) three individuals of Dolichandra unguis-cati. Our data reconstructed a well-supported phylogeny of the Bignoniaceae at different taxonomic scales, opening new perspectives for a comprehensive phylogenetic framework for the family as a whole.

Polyploidy: its consequences and enabling role in plant diversification and evolution.

Most, if not all, green plant (Virdiplantae) species including angiosperms and ferns are polyploids themselves or have ancient polyploid or whole genome duplication signatures in their genomes. Polyploids are not only restricted to our major crop species such as wheat, maize, potato and the brassicas, but also occur frequently in wild species and natural habitats. Polyploidy has thus been viewed as a major driver in evolution, and its influence on genome and chromosome evolution has been at the centre of many investigations. Mechanistic models of the newly structured genomes are being developed that incorporate aspects of sequence evolution or turnover (low-copy genes and regulatory sequences, as well as repetitive DNAs), modification of gene functions, the re-establishment of control of genes with multiple copies, and often meiotic chromosome pairing, recombination and restoration of fertility. World-wide interest in how green plants have evolved under different conditions - whether in small, isolated populations, or globally - suggests that gaining further insight into the contribution of polyploidy to plant speciation and adaptation to environmental changes is greatly needed. Forward-looking research and modelling, based on cytogenetics, expression studies, and genomics or genome sequencing analyses, discussed in this Special Issue of the Annals of Botany, consider how new polyploids behave and the pathways available for genome evolution. They address fundamental questions about the advantages and disadvantages of polyploidy, the consequences for evolution and speciation, and applied questions regarding the spread of polyploids in the environment and challenges in breeding and exploitation of wild relatives through introgression or resynthesis of polyploids. Chromosome number, genome size, repetitive DNA sequences, genes and regulatory sequences and their expression evolve following polyploidy - generating diversity and possible novel traits and enabling species diversification. There is the potential for ever more polyploids in natural, managed and disturbed environments under changing climates and new stresses.

A Multigene Phylogeny of Native American Hawkweeds (Hieracium Subgen. Chionoracium, Cichorieae, Asteraceae): Origin, Speciation Patterns, and Migration Routes.

Native American hawkweeds are mainly mountainous species that are distributed all over the New World. They are severely understudied with respect to their origin, colonization of the vast distribution area, and species relationships. Here, we attempt to reconstruct the evolutionary history of the group by applying seven molecular markers (plastid, nuclear ribosomal and low-copy genes). Phylogenetic analyses revealed that Chionoracium is a subgenus of the mainly Eurasian genus Hieracium, which originated from eastern European hawkweeds about 1.58–2.24 million years ago. Plastid DNA suggested a single origin of all Chionoracium species. They colonized the New World via Beringia and formed several distinct lineages in North America. Via one Central American lineage, the group colonized South America and radiated into more than a hundred species within about 0.8 million years, long after the closure of the Isthmus of Panama and the most recent uplift of the Andes. Despite some incongruences shown by different markers, most of them revealed the same crown groups of closely related taxa, which were, however, largely in conflict with traditional sectional classifications. We provide a basic framework for further elucidation of speciation patterns. A thorough taxonomic revision of Hieracium subgen. Chionoracium is recommended.

The relationship between transposable elements and ecological niches in the Greater Cape Floristic Region: A study on the genus Pteronia (Asteraceae).

Non-coding repetitive DNA (repeatome) is an active part of the nuclear genome, involved in its structure, evolution and function. It is dominated by transposable elements (TEs) and satellite DNA and is prone to the most rapid changes over time. The TEs activity presumably causes the global genome reorganization and may play an adaptive or regulatory role in response to environmental challenges. This assumption is applied here for the first time to plants from the Cape Floristic hotspot to determine whether changes in repetitive DNA are related to responses to a harsh, but extremely species-rich environment. The genus Pteronia (Asteraceae) serves as a suitable model group because it shows considerable variation in genome size at the diploid level and has high and nearly equal levels of endemism in the two main Cape biomes, Fynbos and Succulent Karoo. First, we constructed a phylogeny based on multiple low-copy genes that served as a phylogenetic framework for detecting quantitative and qualitative changes in the repeatome. Second, we performed a comparative analysis of the environments of two groups of Pteronia differing in their TEs bursts. Our results suggest that the environmental transition from the Succulent Karoo to the Fynbos is accompanied by TEs burst, which is likely also driving phylogenetic divergence. We thus hypothesize that analysis of rapidly evolving repeatome could serve as an important proxy for determining the molecular basis of lineage divergence in rapidly radiating groups.

Recovery and genetic characterization of black queen cell virus.

Black queen cell virus (BQCV) is a severe threat to the honeybee (Apis mellifera) worldwide. Although several BQCV strains have been reported in China, the molecular basis for BQCV pathogenicity has not been well understood. Thus, a reverse genetic system of BQCV is required for studying viral replication and its pathogenic mechanism. Here, the complete genome sequence of BQCV was obtained from honeybees using reverse transcription PCR (RT-PCR), namely a BQCV China-GS1 strain (KY741959). Then, a phylogenetic tree was built to analyse the genetic relationships among BQCV strains from different regions. Our results showed that the BQCV China-GS1 contained two ORFs, consistent with the known reference strains, except for the BQCV China-JL1 strain (KP119603). Furthermore, the infectious clone of BQCV was constructed based on BQCV China-GS1 using a low copy vector pACYC177 and gene recombination. Due to the lack of culture cells for bee viruses, we infected the healthy bees with infectious clone of BQCV, and the rescued BQCV resulted in the recovery of recombinant virus, which induced higher mortality than those of the control group. Immune response after inoculated with BQCV further confirmed that the infectious clone of BQCV caused the cellular and humoral immune response of honeybee (A. mellifera). In conclusion, the full nucleotide sequence of BQCV China-GS1 strain was determined, and the infectious clone of BQCV was constructed in this study. These data will improve the understanding of pathogenesis and the host immune responses to viral infection.

Molecular Evolution and Functional Divergence of Stress-Responsive Cu/Zn Superoxide Dismutases in Plants.

Superoxide dismutases (SODs), a family of antioxidant enzymes, are the first line of defense against oxidative damage and are ubiquitous in every cell of all plant types. The Cu/Zn SOD, one of three types of SODs present in plant species, is involved in many of the biological functions of plants in response to abiotic and biotic stresses. Here, we carried out a comprehensive analysis of the Cu/Zn SOD gene family in different plant species, ranging from lower plants to higher plants, and further investigated their organization, sequence features, and expression patterns in response to biotic and abiotic stresses. Our results show that plant Cu/Zn SODs can be divided into two subfamilies (group I and group II). Group II appeared to be conserved only as single- or low-copy genes in all lineages, whereas group I genes underwent at least two duplication events, resulting in multiple gene copies and forming three different subgroups (group Ia, group Ib, and group Ic). We also found that, among these genes, two important events—the loss of introns and the loss of and variation in signal peptides—occurred over the long course of their evolution, indicating that they were involved in shifts in subcellular localization from the chloroplast to cytosol or peroxisome and underwent functional divergence. In addition, expression patterns of Cu/Zn SOD genes from Arabidopsis thaliana and Solanum lycopersicum were tested in different tissues/organs and developmental stages and under different abiotic stresses. The results indicate that the Cu/Zn SOD gene family possesses potential functional divergence and may play vital roles in ROS scavenging in response to various stresses in plants. This study will help establish a foundation for further understanding these genes’ function during stress responses.

The full-length transcriptome sequencing of three sturgeons reveals the occurrence of whole genome duplication event.

Sturgeon is one of the ancient fish species with the fossil records over 200 million years ago. However, the sturgeon genomes are rather complicated, owing to several rounds of genome polyploidizations, which makes it difficult to assemble a high quality genome so that only a few studies analyze their whole genome evolutions (WGDs). This study applied the PacBio sequencing technology to sequence the full-length transcriptomes of blood samples of octoploid Huso dauricus, octoploid Acipenser schrenckii and tetraploid Acipenser stellatus. A total of non-redundant 33,407 transcript assemblies of the three sturgeons were finally generated and fully annotated with different gene function databases, as well as the predictions of simple sequence repeats, transcription factors and long non-coding RNAs. Data valuation exhibited that annotations of the three transcriptomes had the higher quality than those of the previously sequenced sturgeon genomes. A phylogeny of eight typical sturgeons was constructed using the low copy genes, which illustrated that Acipenser and Huso might share a single origin. The sturgeon WGDs were parallelly timed by calculating the divergence times of 2-member gene clusters and heterozygous-SNP-carried genes, which both suggested the occurrence of the WGDs later than 150 mya. The full-length transcriptome sequences were supposed to be the reliable reference data for the study of sturgeon.

Target enrichment improves phylogenetic resolution in the genus Zanthoxylum (Rutaceae) and indicates both incomplete lineage sorting and hybridization events.

Zanthoxylum is the only pantropical genus within Rutaceae, with a few species native to temperate eastern Asia and North America. Efforts using Sanger sequencing failed to resolve the backbone phylogeny of Zanthoxylum. In this study, we employed target-enrichment high-throughput sequencing to improve resolution. Gene trees were examined for concordance and sectional classifications of Zanthoxylum were evaluated. Off-target reads were investigated to identify putative single-copy markers for bait refinement, and low-copy markers for evidence of putative hybridization events. A custom bait set targeting 354 genes, with a median of 321 bp, was designed for Zanthoxylum and applied to 44 Zanthoxylum species and one Tetradium species as the outgroup. Illumina reads were processed via the HybPhyloMaker pipeline. Phylogenetic inferences were conducted using coalescent and maximum likelihood methods based on concatenated datasets. Concordance was assessed using quartet sampling. Additional phylogenetic analyses were performed on putative single and low-copy genes extracted from off-target reads. Four major clades are supported within Zanthoxylum: the African clade, the Z. asiaticum clade, the Asian-Pacific-Australian clade and the American-eastern Asian clade. While overall support has improved, regions of conflict are similar to those previously observed. Gene tree discordances indicate a hybridization event in the ancestor of the Hawaiian lineage, and incomplete lineage sorting in the American backbone. Off-target putative single-copy genes largely confirm on-target results, and putative low-copy genes provide additional evidence for hybridization in the Hawaiian lineage. Only two of the five sections of Zanthoxylum are resolved as monophyletic. Target enrichment is suitable for assessing phylogenetic relationships in Zanthoxylum. Our phylogenetic analyses reveal that current sectional classifications need revision. Quartet tree concordance indicates several instances of reticulate evolution. Off-target reads are proven useful to identify additional phylogenetically informative regions for bait refinement or gene tree based approaches.

Development and phylogenetic utilities of a new set of single-/low-copy nuclear genes in Senecioneae (Asteraceae), with new insights into the tribal position and the relationships within subtribe Tussilagininae

The tribe Senecioneae is one of the largest tribes in Asteraceae, with a nearly cosmopolitan distribution. Despite great efforts devoted to elucidate the evolution of Senecioneae, many questions still remain concerning the systematics of this group, from the tribal circumscription and position to species relationships in many genera. The hybridization-based target enrichment method of next-generation sequencing has been accepted as a promising approach to resolve phylogenetic problems. We herein develop a set of single-/low-copy genes for Senecioneae, and test their phylogenetic utilities. Our results demonstrate that these genes work highly efficiently for Senecioneae, with a high average gene recovery of 98.8% across the tribe and recovering robust phylogenetic hypotheses at different levels. In particular, the delimitation of the Senecioneae has been confirmed to include Abrotanella and exclude Doronicum, with the former sister to core Senecioneae and the latter shown to be more closely related to Calenduleae. Moreover, Doronicum and Calenduleae are inferred to be the closest relatives of Senecioneae, which is a new hypothesis well supported by statistical topology tests, morphological evidence, and the profile of pyrrolizidine alkaloids, a special kind of chemical characters generally used to define Senecioneae. Furthermore, this study suggests a complex reticulation history in the diversification of Senecioneae, accounting for the prevalence of polyploid groups in the tribe. With subtribe Tussilagininae s.str. as a case study showing a more evident pattern of gene duplication, we further explored reconstructing the phylogeny in the groups with high ploidy levels. Our results also demonstrate that tree topologies based on sorted paralogous copies are stable across different methods of phylogenetic inference, and more congruent with the morphological evidence and the results of previous phylogenetic studies.

New genetic markers for Sapotaceae phylogenomics: More than 600 nuclear genes applicable from family to population levels

Some tropical plant families, such as the Sapotaceae, have a complex taxonomy, which can be resolved using Next Generation Sequencing (NGS). For most groups however, methodological protocols are still missing. Here we identified 531 monocopy genes and 227 Short Tandem Repeats (STR) markers and tested them on Sapotaceae using target capture and NGS. The probes were designed using two genome skimming samples from Capurodendron delphinense and Bemangidia lowryi, both from the Tseboneae tribe, as well as the published Manilkara zapota transcriptome from the Sapotoideae tribe. We combined our probes with 261 additional ones previously published and designed for the entire angiosperm group. On a total of 792 low-copy genes, 638 showed no signs of paralogy and were used to build a phylogeny of the family with 231 individuals from all main lineages. A highly supported topology was obtained at high taxonomic ranks but also at the species level. This phylogeny revealed the existence of more than 20 putative new species. Single nucleotide polymorphisms (SNPs) extracted from the 638 genes were able to distinguish lineages within a species complex and to highlight geographical structuration. STR were recovered efficiently for the species used as reference (C. delphinense) but the recovery rate decreased dramatically with the phylogenetic distance to the focal species. Altogether, the new loci will help reaching a sound taxonomic understanding of the family Sapotaceae for which many circumscriptions and relationships are still debated, at the species, genus and tribe levels.

Genesis, Evolution, and Genetic Diversity of the Hexaploid, Narrow Endemic Centaurea tentudaica

Within the genus Centaurea L., polyploidy is very common, and it is believed that, as to all angiosperms, it was key in the history of its diversification and evolution. Centaurea tentudaica is a hexaploid from subsect. Chamaecyanus of unknown origin. In this study, we examined the possible autopolyploid or allopolyploid origin using allozymes and sequences of three molecular markers: nuclear-ribosomic region ETS, and low-copy genes AGT1 and PgiC. We also included three species geographically and morphologically close to C. tentudaica: C. amblensis, C. galianoi, and C. ornata. Neighbor-Net and Bayesian analyses show a close relationship between C. amblensis and C. tentudaica and no relationship to any of the other species, which suggest that C. tentudaica is an autopolyploid of C. amblensis. Allozyme banding pattern also supports the autopolyploidy hypothesis and shows high levels of genetic diversity in the polyploid, which could suggest multiple origins by recurrent crosses of tetraploid and diploid cytotypes of C. amblensis. Environmental niche modeling was used to analyze the distribution of the possible parental species during the present, Last Glacial Maximum (LGM), Last Interglacial Period (LIG), and Penultimate Glacial Maximum (PGM) environmental conditions. Supporting the molecular suggestions that C. tentudaica originated from C. amblensis, environmental niche modeling confirms that past distribution of C. amblensis overlapped with the distribution of C. tentudaica.

Subspecies divergence and pronounced phylogenetic incongruence in the East-Asia-endemic shrubMagnolia sieboldii

The biogeographic patterns of the East-Asia-endemic shrub Magnolia sieboldii, in which the range of the subsp. sieboldii is interposed with the disjunct distribution of subsp. japonica, implies a complex evolutionary history, involving rapid speciation and hybridization. Here, we aim to reveal the evolutionary and phylogeographic histories of the species with a particular focus on the time of subspecies divergence, the hypothesis of secondary hybridization and the Pleistocene survival of each subspecies, using a combination of genetic analyses and ecological niche modelling. Genetic variation, genetic structures and phylogenetic relationships were elucidated based on nuclear low-copy genes, chloroplast DNA, and nuclear simple sequence repeats (SSRs). A scenario selection analysis and divergence time estimation were performed using coalescent simulation in DIYABC and *BEAST. Ecological niche modelling and a test of niche differentiation were performed using Maxent and ENMTools. All marker types showed deep, but pronouncedly incongruent, west-east genetic divergences, with the subspecies being delineated only by the nuclear low-copy genes. Phylogenetic tree topologies suggested that ancient hybridization and introgression were likely to have occurred; however, this scenario did not receive significant support in the DIYABC analysis. The subspecies differentiated their niches, but both showed a dependence on high humidity and were predicted to have persisted during the last glacial cycle by maintaining a stable latitudinal distribution via migration to lower altitudes. We found a deep genetic divergence and a pronounced phylogenetic incongruence among the two subspecies of M. sieboldii, which may have been driven by major paleogeographic and paleoclimatic events that have occurred since the Neogene in East Asia, including global cooling, climate oscillations and the formation of land bridges. Both subspecies were, however, considered to persist in situ in stable climatic conditions during the late Pleistocene.