Species divergence and phylogeography of Corylus heterophylla Fisch complex (Betulaceae): Inferred from molecular, climatic and morphological data.

Historical geo-climatic changes have shaped the geographical distributions and genetic diversity of numerous plant taxa in East Asia, which promote species divergence and ultimately speciation. Here, we integrated multiple approaches, including molecular phylogeography, ecological niche modeling, and morphological traits to examine the nucleotide diversity and interspecific divergence within Corylus heterophylla complex (C. heterophylla, C. kweichowensis, and C. yunnanensis). These three sibling taxa harbored similar high levels of nucleotide diversity at the species level. The molecular data (SCNG and cpDNA) unanimously supported the division of C. heterophylla complex into two major clades, with C. yunnanensis diverged earlier from the complex, whereas C. heterophylla and C. kweichowensis could hardly be separated. The split between the two clades (c. 12.89 Ma) coincided with the formation of Sichuan Basin in the middle Miocene, while the divergence among and within the five subclades (YUN1-YUN3, HK1-HK2) occurred from the late Miocene to the Pleistocene. C. heterophylla of northern China experienced glacial contraction and interglacial expansion during the Quaternary, whereas C. kweichowensis and C. yunnanensis of southern China presented population expansion even during the last glacial maximum. Despite of high levels of genetic admixture between C. heterophylla and C. kweichowensis, significant ecological and morphological discrepancy as well as incomplete geographic isolation indicated that adaptive evolution triggered by divergent selection may have played important roles in incipient ecological speciation.

Draft genome of Puya raimondii (Bromeliaceae), the Queen of the Andes.

Puya raimondii, the Queen of the Andes, is an endangered high Andean species in the Bromeliaceae family. Here, we report its first genome to promote its conservation and evolutionary study. Comparative genomics showed P. raimondii diverged from Ananas comosus about 14.8 million years ago, and the long terminal repeats were likely to contribute to the genus diversification in last 3.5 million years. The gene families related to plant reproductive development and stress responses significantly expanded in the genome. At the same time, gene families involved in disease defense, photosynthesis and carbohydrate metabolism significantly contracted, which may be an evolutionary strategy to adapt to the harsh conditions in high Andes. The demographic history analysis revealed the P. raimondii population size sharply declined in the Pleistocene and then increased in the Holocene. We also designed and tested 46 pairs of universal primers for amplifying orthologous single-copy nuclear genes in Puya species.

Phylogenomic conflict analyses in the apple genus Malus s.l. reveal widespread hybridization and allopolyploidy driving diversification, with insights into the complex biogeographic history in the Northern Hemisphere.

Phylogenomic evidence from an increasing number of studies has demonstrated that different data sets and analytical approaches often reconstruct strongly supported but conflicting relationships. In this study, 785 single-copy nuclear genes and 75 complete plastomes were used to infer the phylogenetic relationships and estimate the historical biogeography of the apple genus Malus sensu lato, an economically important lineage disjunctly distributed in the Northern Hemisphere and involved in known and suspected hybridization and allopolyploidy events. The nuclear phylogeny recovered the monophyly of Malus s.l. (including Docynia); however, the genus was supported to be biphyletic in the plastid phylogeny. An ancient chloroplast capture event in the Eocene in western North America best explains the cytonuclear discordance. Our conflict analysis demonstrated that ILS, hybridization, and allopolyploidy could explain the widespread nuclear gene tree discordance. One deep hybridization event (Malus doumeri) and one recent event (Malus coronaria) were detected in Malus s.l. Furthermore, our historical biogeographic analysis integrating living and fossil data supported a widespread East Asian-western North American origin of Malus s.l. in the Eocene, followed by several extinction and dispersal events in the Northern Hemisphere. We also propose a general workflow for assessing phylogenomic discordance and biogeographic analysis using deep genome skimming data sets.

Evolutionary and domestication history of Cucurbita (pumpkin and squash) species inferred from 44 nuclear loci.

Phylogenetics can facilitate the study of plant domestication by resolving sister relationships between crops and their wild relatives, thereby identifying the ancestors of cultivated plants. Previous phylogenetic studies of the six Cucurbita crop lineages (pumpkins and squashes) and their wild relatives suggest histories of deep coalescence that complicate uncovering the genetic origins of the six crop taxa. We investigated the evolution of wild and domesticated Cucurbita using the most comprehensive and robust molecular-based phylogeny for Cucurbita to date based on 44 loci derived from introns of single-copy nuclear genes. We discovered novel relationships among Cucurbita species and recovered the first Cucurbita tree with well-supported resolution within species. Cucurbita comprises a clade of mesophytic annual species that includes all six crop taxa and a grade of xerophytic perennial species that represent the ancestral xerophytic habit of the genus. Based on phylogenetic resolution within-species we hypothesize that the magnitude of domestication bottlenecks varies among Cucurbita crop lineages. Our phylogeny clarifies how wild Cucurbita species are related to the domesticated taxa. We find close relationships between two wild species and crop lineages not previously identified. Expanded geographic sampling of key wild species is needed for improved understanding of the evolution of domesticated Cucurbita.

Phylotranscriptomics Shed Light on Intrageneric Relationships and Historical Biogeography of Ceratozamia (Cycadales).

Ceratozamia Brongn. is one of the species-rich genera of Cycadales comprising 38 species that are mainly distributed in Mexico, with a few species reported from neighboring regions. Phylogenetic relationships within the genus need detailed investigation based on extensive datasets and reliable systematic approaches. Therefore, we used 30 of the known 38 species to reconstruct the phylogeny based on transcriptome data of 3954 single-copy nuclear genes (SCGs) via coalescent and concatenated approaches and three comparative datasets (nt/nt12/aa). Based on all these methods, Ceratozamia is divided into six phylogenetic subclades within three major clades. There were a few discrepancies regarding phylogenetic position of some species within these subclades. Using these phylogenetic trees, biogeographic history and morphological diversity of the genus are explored. Ceratozamia originated from ancestors in southern Mexico since the mid-Miocene. There is a distinct distribution pattern of species through the Trans-Mexican Volcanic Belt (TMVB), that act as a barrier for the species dispersal at TMVB and its southern and northern part. Limited dispersal events occurred during the late Miocene, and maximum diversification happened during the Pliocene epoch. Our study provides a new insight into phylogenetic relationships, the origin and dispersal routes, and morphological diversity of the genus Ceratozamia. We also explain how past climatic changes affected the diversification of this Mesoamerica-native genus.

On the Species Delimitation of the Maddenia Group of Prunus (Rosaceae): Evidence From Plastome and Nuclear Sequences and Morphology.

The recognition, identification, and differentiation of closely related plant species present significant and notorious challenges to taxonomists. The Maddenia group of Prunus, which comprises four to seven species, is an example of a group in which species delimitation and phylogenetic reconstruction have been difficult, due to the lack of clear morphological distinctions, limited sampling, and low informativeness of molecular evidence. Thus, the precise number of species in the group and the relationships among them remain unclear. Here, we used genome skimming to generate the DNA sequence data for 22 samples, including 17 Maddenia individuals and five outgroups in Amygdaloideae of Rosaceae, from which we assembled the plastome and 446 single-copy nuclear (SCN) genes for each sample. The phylogenetic relationships of the Maddenia group were then reconstructed using both concatenated and coalescent-based methods. We also identified eight highly variable regions and detected simple sequence repeats (SSRs) and repeat sequences in the Maddenia species plastomes. The phylogenetic analysis based on the complete plastomes strongly supported three main subclades in the Maddenia group of Prunus, while five subclades were recognized based on the nuclear tree. The phylogenetic network analysis detected six hybridization events. Integrating the nuclear and morphological evidence, we proposed to recognize five species within the Maddenia group, i.e., Prunus fujianensis, P. himalayana, P. gongshanensis, P. hypoleuca, and P. hypoxantha. Within this group, the first three species are well-supported, while the gene flow occurring throughout the Maddenia group seems to be especially frequent between P. hypoleuca and P. hypoxantha, eroding the barrier between them. The phylogenetic trees based on eight concatenated hypervariable regions had a similar topology with the complete plastomes, showing their potential as molecular markers and effective barcodes for further phylogeographic studies on Maddenia.

Comparison of taxon-specific versus general locus sets for targeted sequence capture in plant phylogenomics.

PREMISE OF THE STUDY: Targeted sequence capture can be used to efficiently gather sequence data for large numbers of loci, such as single-copy nuclear loci. Most published studies in plants have used taxon-specific locus sets developed individually for a clade using multiple genomic and transcriptomic resources. General locus sets can also be developed from loci that have been identified as single-copy and have orthologs in large clades of plants. METHODS: We identify and compare a taxon-specific locus set and three general locus sets (conserved ortholog set [COSII], shared single-copy nuclear [APVO SSC] genes, and pentatricopeptide repeat [PPR] genes) for targeted sequence capture in Buddleja (Scrophulariaceae) and outgroups. We evaluate their performance in terms of assembly success, sequence variability, and resolution and support of inferred phylogenetic trees. RESULTS: The taxon-specific locus set had the most target loci. Assembly success was high for all locus sets in Buddleja samples. For outgroups, general locus sets had greater assembly success. Taxon-specific and PPR loci had the highest average variability. The taxon-specific data set produced the best-supported tree, but all data sets showed improved resolution over previous non-sequence capture data sets. DISCUSSION: General locus sets can be a useful source of sequence capture targets, especially if multiple genomic resources are not available for a taxon.

Primers for Castilleja and their utility across Orobanchaceae: II. Single-copy nuclear loci.

PREMISE OF THE STUDY: We developed primers targeting nuclear loci in Castilleja with the goal of reconstructing the evolutionary history of this challenging clade. These primers were tested across other major clades in Orobanchaceae to assess their broader utility. METHODS AND RESULTS: We assembled low-coverage genomes for three taxa in Castilleja and developed primer combinations for the single-copy conserved ortholog set (COSII) and the pentatricopeptide repeat (PPR) gene family. These primer combinations were designed to take advantage of the Fluidigm microfluidic PCR platform and are well suited for high-throughput sequencing applications. Eighty-seven primers were designed for Castilleja, and 27 were found to have broader utility in Orobanchaceae. CONCLUSIONS: These results demonstrate the utility of these primers, not only across Castilleja, but for other lineages within Orobanchaceae as well. This expanded molecular toolkit will be an asset to future phylogenetic studies in Castilleja and throughout Orobanchaceae.

Marker Development for Phylogenomics: The Case of Orobanchaceae, a Plant Family with Contrasting Nutritional Modes.

Phylogenomic approaches, employing next-generation sequencing (NGS) techniques, have revolutionized systematic and evolutionary biology. Target enrichment is an efficient and cost-effective method in phylogenomics and is becoming increasingly popular. Depending on availability and quality of reference data as well as on biological features of the study system, (semi-)automated identification of suitable markers will require specific bioinformatic pipelines. Here, we established a highly flexible bioinformatic pipeline, BaitsFinder, to identify putative orthologous single copy genes (SCGs) and to construct bait sequences in a single workflow. Additionally, this pipeline has been constructed to be able to cope with challenging data sets, such as the nutritionally heterogeneous plant family Orobanchaceae. To this end, we used transcriptome data of differing quality available for four Orobanchaceae species and, as reference, SCG data from monkeyflower (Erythranthe guttata, syn. Mimulus g.; 1,915 genes) and tomato (Solanum lycopersicum; 391 genes). Depending on whether gaps were permitted in initial blast searches of the four Orobanchaceae species against the reference, our pipeline identified 1,307 and 981 SCGs with average length of 994 bp and 775 bp, respectively. Automated bait sequence construction (using 2× tiling) resulted in 38,170 and 21,856 bait sequences, respectively. In comparison to the recently published MarkerMiner 1.0 pipeline BaitsFinder identified about 1.6 times as many SCGs (of at least 900 bp length). Skipping steps specific to analyses of Orobanchaceae, BaitsFinder was successfully used in a group of non-parasitic plants (three Asteraceae species and, as reference, SCG data from Arabidopsis thaliana based on previously compiled SCGs). Thus, BaitsFinder is expected to be broadly applicable in groups, where only transcriptomes or partial genome data of differing quality are available.

MarkerMiner 1.0: A new application for phylogenetic marker development using angiosperm transcriptomes.

PREMISE OF THE STUDY: Targeted sequencing using next-generation sequencing (NGS) platforms offers enormous potential for plant systematics by enabling economical acquisition of multilocus data sets that can resolve difficult phylogenetic problems. However, because discovery of single-copy nuclear (SCN) loci from NGS data requires both bioinformatics skills and access to high-performance computing resources, the application of NGS data has been limited. METHODS AND RESULTS: We developed MarkerMiner 1.0, a fully automated, open-access bioinformatic workflow and application for discovery of SCN loci in angiosperms. Our new tool identified as many as 1993 SCN loci from transcriptomic data sampled as part of four independent test cases representing marker development projects at different phylogenetic scales. CONCLUSIONS: MarkerMiner is an easy-to-use and effective tool for discovery of putative SCN loci. It can be run locally or via the Web, and its tabular and alignment outputs facilitate efficient downstream assessments of phylogenetic utility, locus selection, intron-exon boundary prediction, and primer or probe development.

Comparative transcriptome resources of eleven Primulina species, a group of 'stone plants' from a biodiversity hot spot.

The genus Primulina is an emerging model system in studying the drivers and mechanisms of species diversification, for its high species richness and endemism, together with high degree of habitat specialization. In this study, we sequenced transcriptomes for eleven Primulina species across the phylogeny of the genus using the Illumina HiSeq 2000 platform. A total of 336 million clean reads were processed into 355 573 unigenes with a mean length of 1336 bp and an N50 value of 2191 bp after pooling and reassembling twelve individual pre-assembled unigene sets. Of these unigenes, 249 973 (70%) were successfully annotated and 256 601 (72%) were identified as coding sequences (CDSs). We identified a total of 38 279 simple sequence repeats (SSRs) and 367 123 single nucleotide polymorphisms (SNPs). Marker validation assay revealed that 354 (27.3%) of the 1296 SSR and 795 (39.6%) of the 2008 SNP loci showed successful genotyping performance and exhibited expected polymorphism profiles. We screened 834 putative single-copy nuclear genes and proved their high effectiveness in phylogeny construction and estimation of ancestral population parameters. We identified a total of 85 candidate orthologs under positive selection for 46 of the 66 species pairs. This study provided an efficient application of RNA-seq in development of genomic resources for a group of 'stone plants' from south China Karst regions, a biodiversity hot spot of the World. The assembled unigenes with annotations and the massive gene-associated molecular markers would help guide further molecular systematic, population genetic and ecological genomics studies in Primulina and its relatives.