Museomics help resolving the phylogeny of snowfinches (Aves, Passeridae, Montifringilla and allies).

Historical specimens from museum collections provide a valuable source of material also from remote areas or regions of conflict that are not easily accessible to scientists today. With this study, we are providing a taxon-complete phylogeny of snowfinches using historical DNA from whole skins of an endemic species from Afghanistan, the Afghan snowfinch, Pyrgilauda theresae. To resolve the strong conflict between previous phylogenetic hypotheses, we generated novel mitogenome sequences for selected taxa and genome-wide SNP data using ddRAD sequencing for all extant snowfinch species endemic to the Qinghai-Tibet Plateau (QTP) and for an extended intraspecific sampling of the sole Central and Western Palearctic snowfinch species (Montifringilla nivalis). Our phylogenetic reconstructions unanimously refuted the previously suggested paraphyly of genus Pyrgilauda. Misplacement of one species-level taxon (Onychostruthus tazcanowskii) in previous snowfinch phylogenies was undoubtedly inferred from chimeric mitogenomes that included heterospecific sequence information. Furthermore, comparison of novel and previously generated sequence data showed that the presumed sister-group relationship between M. nivalis and the QTP endemic M. henrici was suggested based on flawed taxonomy. Our phylogenetic reconstructions based on genome-wide SNP data and on mitogenomes were largely congruent and supported reciprocal monophyly of genera Montifringilla and Pyrgilauda with monotypic Onychostruthus being sister to the latter. The Afghan endemic P. theresae likely originated from a rather ancient Pliocene out-of-Tibet dispersal probably from a common ancestor with P. ruficollis. Our extended trans-Palearctic sampling for the white-winged snowfinch, M. nivalis, confirmed strong lineage divergence between an Asian and a European clade dated to 1.5 - 2.7 million years ago (mya). Genome-wide SNP data suggested subtle divergence among European samples from the Alps and from the Cantabrian mountains.

Diversity and biogeography of South American mud turtles elucidated by multilocus DNA sequencing (Testudines: Kinosternidae).

Kinosternon is the most speciose genus of extant turtles, with 22 currently recognized species, distributed across large parts of the Americas. Most species have small distributions, but K. leucostomum and K. scorpioides range from Mexico to South America. Previous studies have found discordance between mitochondrial and nuclear phylogenies in some kinosternid groups, with the current taxonomy following the nuclear-based results. Herein, based on extended molecular, geographic, and taxonomic sampling, we explore the phylogeographic structure and taxonomic limits for K. leucostomum and the K. scorpioides group and present a fossil-calibrated nuclear time tree for Kinosternon. Our results reveal contrasting differentiation patterns for the K. scorpioides group and K. leucostomum, despite overlapping distributions. Kinosternon leucostomum shows only shallow geographic divergence, whereas the K. scorpioides group is polyphyletic with up to 10 distinct taxa, some of them undescribed. We support the elevation of K. s. albogulare and K. s. cruentatum to species level. Given the deep divergence within the genus Kinosternon, we propose the recognition of three subgenera, Kinosternon, Cryptochelys and Thyrosternum, and the abandonment of the group-based classification, at least for the K. leucostomum and K. scorpioides groups. Our results show an initial split in Kinosternon that gave rise to two main radiations, one Nearctic and one mainly Neotropical. Most speciation events in Kinosternon occurred during the Quaternary and we hypothesize that they were mediated by both climatic and geological events. Additionally, our data imply that at least three South American colonizations occurred, two in the K. leucostomum group, and one in the K. scorpioides group. Additionally, we hypothesize that discordance between mitochondrial and nuclear phylogenetic signal is due to mitochondrial capture from an extinct kinosternine lineage.

Evolution of Transcriptomes in Early-Generation Hybrids of the Apomictic Ranunculus auricomus Complex (Ranunculaceae).

Hybridisation in plants may cause a shift from sexual to asexual seed formation (apomixis). Indeed, natural apomictic plants are usually hybrids, but it is still unclear how hybridisation could trigger the shift to apomixis. The genome evolution of older apomictic lineages is influenced by diverse processes such as polyploidy, mutation accumulation, and allelic sequence divergence. To disentangle the effects of hybridisation from these other factors, we analysed the transcriptomes of flowering buds from artificially produced, diploid F2 hybrids of the Ranunculus auricomus complex. The hybrids exhibited unreduced embryo sac formation (apospory) as one important component of apomixis, whereas their parental species were sexual. We revealed 2915 annotated single-copy genes that were mostly under purifying selection according to dN/dS ratios. However, pairwise comparisons revealed, after rigorous filtering, 79 genes under diversifying selection between hybrids and parents, whereby gene annotation assigned ten of them to reproductive processes. Four genes belong to the meiosis-sporogenesis phase (ASY1, APC1, MSP1, and XRI1) and represent, according to literature records, candidate genes for apospory. We conclude that hybridisation could combine novel (or existing) mutations in key developmental genes in certain hybrid lineages, and establish (together with altered gene expression profiles, as observed in other studies) a heritable regulatory mechanism for aposporous development.

Untying Gordian knots: unraveling reticulate polyploid plant evolution by genomic data using the large Ranunculus auricomus species complex.

Speciation via hybridization and polyploidization is a major evolutionary force in plant evolution but is still poorly understood for neopolyploid groups. Challenges are attributed to high heterozygosity, low genetic divergence, and missing information on progenitors, ploidy, and reproduction. We study the large Eurasian Ranunculus auricomus species complex and use a comprehensive workflow integrating reduced-representation sequencing (RRS) genomic data to unravel reticulate evolution, genome diversity and composition of polyploids. We rely on 97 312 restriction site-associated DNA sequencing (RAD-Seq) loci, 576 targeted nuclear genes (48 phased), and 71 plastid regions derived from 78 polyploid apomictic taxa and four diploid and one tetraploid putative sexual progenitor species. We applied (phylo)genomic structure, network, and single nucleotide polymorphism (SNP)-origin analyses. Results consistently showed only 3-5 supported and geographically structured polyploid genetic groups, each containing extant sexual and one unknown progenitor species. Combined analyses demonstrated predominantly allopolyploid origins, each involving 2-3 different diploid sexual progenitor species. Young allotetraploids were characterized by subgenome dominance and nonhybrid SNPs, suggesting substantial post-origin but little lineage-specific evolution. The biodiversity of neopolyploid complexes can result from multiple hybrid origins involving different progenitors and substantial post-origin evolution (e.g. homoeologous exchanges, hybrid segregation, gene flow). Reduced-representation sequencing genomic data including multi-approach information is efficient to delimit shallow reticulate relationships.

How challenging RADseq data turned out to favor coalescent-based species tree inference. A case study in Aichryson (Crassulaceae).

Analysing multiple genomic regions while incorporating detection and qualification of discordance among regions has become standard for understanding phylogenetic relationships. In plants, which usually have comparatively large genomes, this is feasible by the combination of reduced-representation library (RRL) methods and high-throughput sequencing enabling the cost effective acquisition of genomic data for thousands of loci from hundreds of samples. One popular RRL method is RADseq. A major disadvantage of established RADseq approaches is the rather short fragment and sequencing range, leading to loci of little individual phylogenetic information. This issue hampers the application of coalescent-based species tree inference. The modified RADseq protocol presented here targets ca. 5,000 loci of 300-600nt length, sequenced with the latest short-read-sequencing (SRS) technology, has the potential to overcome this drawback. To illustrate the advantages of this approach we use the study group Aichryson Webb & Berthelott (Crassulaceae), a plant genus that diversified on the Canary Islands. The data analysis approach used here aims at a careful quality control of the long loci dataset. It involves an informed selection of thresholds for accurate clustering, a thorough exploration of locus properties, such as locus length, coverage and variability, to identify potential biased data and a comparative phylogenetic inference of filtered datasets, accompanied by an evaluation of resulting BS support, gene and site concordance factor values, to improve overall resolution of the resulting phylogenetic trees. The final dataset contains variable loci with an average length of 373nt and facilitates species tree estimation using a coalescent-based summary approach. Additional improvements brought by the approach are critically discussed.

Gene Expression Profiles Suggest a Better Cold Acclimation of Polyploids in the Alpine Species Ranunculus kuepferi (Ranunculaceae).

Alpine habitats are shaped by harsh abiotic conditions and cold climates. Temperature stress can affect phenotypic plasticity, reproduction, and epigenetic profiles, which may affect acclimation and adaptation. Distribution patterns suggest that polyploidy seems to be advantageous under cold conditions. Nevertheless, whether temperature stress can induce gene expression changes in different cytotypes, and how the response is initialized through gene set pathways and epigenetic control remain vague for non-model plants. The perennial alpine plant Ranunculus kuepferi was used to investigate the effect of cold stress on gene expression profiles. Diploid and autotetraploid individuals were exposed to cold and warm conditions in climate growth chambers and analyzed via transcriptome sequencing and qRT-PCR. Overall, cold stress changed gene expression profiles of both cytotypes and induced cold acclimation. Diploids changed more gene set pathways than tetraploids, and suppressed pathways involved in ion/cation homeostasis. Tetraploids mostly activated gene set pathways related to cell wall and plasma membrane. An epigenetic background for gene regulation in response to temperature conditions is indicated. Results suggest that perennial alpine plants can respond to temperature extremes via altered gene expression. Tetraploids are better acclimated to cold conditions, enabling them to colonize colder climatic areas in the Alps.

New Acetophenones and Chromenes from the Leaves of Melicope barbigera A. Gray.

The dichloromethane extract from leaves of Melicope barbigera (Rutaceae), endemic to the Hawaiian island of Kaua'i, yielded four new and three previously known acetophenones and 2H-chromenes, all found for the first time in M. barbigera. The structures of the new compounds obtained from the dichloromethane extract after purification by chromatographic methods were unambiguously elucidated by spectroscopic analyses including 1D/2D NMR spectroscopy and HRESIMS. The absolute configuration was determined by modified Mosher's method. Compounds 2, 4 and the mixture of 6 and 7 exhibited moderate cytotoxic activities against the human ovarian cancer cell line A2780 with IC50 values of 30.0 and 75.7 µM for 2 and 4, respectively, in a nuclear shrinkage cytotoxicity assay.

Phylogenomics unravels Quaternary vicariance and allopatric speciation patterns in temperate-montane plant species: A case study on the Ranunculus auricomus species complex.

The time frame and geographical patterns of diversification processes in European temperate-montane herbs are still not well understood. We used the sexual species of the Ranunculus auricomus complex as a model system to understand how vicariance versus dispersal processes in the context of Pleistocene climatic fluctuations have triggered speciation in temperate-montane plant species. We used target enrichment sequence data from about 600 nuclear genes and coalescent-based species tree inference methods to resolve phylogenetic relationships among the sexual taxa of the complex. We estimated absolute divergence times and, using ancestral range reconstruction, we tested if speciation was enhanced by vicariance or by dispersal processes. Phylogenetic relationships among taxa were fully resolved with some incongruence in the position of the tetraploid R. marsicus. Speciation events took place in a very short time at the end of the Mid-Pleistocene Transition (830-580 thousand years ago [ka]). A second wave of intraspecific geographical differentiation occurred at the end of the Riss glaciation or during the Eemian interglacial between 200 and 100 ka. Ancestral range reconstruction suggests a widespread European ancestor of the R. auricomus complex. Vicariance has triggered allopatric speciation in temperate-montane plant species during the climatic deterioration that occurred during the last phase of the Mid-Pleistocene Transition. Vegetation restructuring from forest into tundra could have confined these forest species into isolated glacial macro- and microrefugia. During subsequent warming periods, range expansions of these species could have been hampered by apomictic derivatives and by other congeneric competitors in the same habitat.

Phylogeny of Hawaiian Melicope (Rutaceae): RAD-seq Resolves Species Relationships and Reveals Ancient Introgression.

Hawaiian Melicope are one of the major adaptive radiations of the Hawaiian Islands comprising 54 endemic species. The lineage is monophyletic with an estimated crown age predating the rise of the current high islands. Phylogenetic inference based on Sanger sequencing has not been sufficient to resolve species or deeper level relationships. Here, we apply restriction site-associated DNA sequencing (RAD-seq) to the lineage to infer phylogenetic relationships. We employ Quartet Sampling to assess information content and statistical support, and to quantify discordance as well as partitioned ABBA-BABA tests to uncover evidence of introgression. Our new results drastically improved resolution of relationships within Hawaiian Melicope. The lineage is divided into five fully supported main clades, two of which correspond to morphologically circumscribed infrageneric groups. We provide evidence for both ancestral and current hybridization events. We confirm the necessity for a taxonomic revision of the Melicope section Pelea, as well as a re-evaluation of several species complexes by combining genomic and morphological data.

Phylogeny and biogeography of the pantropical genus Zanthoxylum and its closest relatives in the proto-Rutaceae group (Rutaceae).

Zanthoxylum L. (prickly ash) is the only genus in the Citrus L. family (Rutaceae) with a pantropical distribution. We present the first detailed phylogenetic and biogeographic study of the genus and its close relatives in the proto-Rutaceae group. Our phylogenetic analyses based on two plastid and two nuclear markers show that the genus Toddalia Juss. is nested within Zanthoxylum, that earlier generic and intrageneric classifications need revision, and that the homochlamydeous flowers of the temperate species of Zanthoxylum are the result of a reduction from heterochlamydeous flowers. The biogeographic analyses reveal a Eurasian origin of Zanthoxylum in the Paleocene or Eocene with successive intercontinental or long-range migrations. Zanthoxylum likely crossed the North Atlantic Land Bridges to colonize the Americas in the Eocene, and migrated back to the Old World probably via the Bering Land Bridge in the Oligocene or Miocene. Zanthoxylum also colonized several Pacific Islands and the Hawaiian clade shows phylogenetic incongruence between the plastid and nuclear datasets, suggesting hybridization. The Hawaiian species are one of the rare examples of endemic Hawaiian lineages that are older than the current main islands.