Biogeographic origins of southern African Silene (Caryophyllaceae).

Silene (Caryophyllaceae) is distributed predominantly in the northern Hemisphere, where it is most diverse around the Mediterranean Basin. The genus is also well represented in North Africa, extending into tropical, sub-Saharan and southern Africa. Eight native species are recognized in southern Africa, taxonomically placed in two sections: Elisanthe and Silene s.l. Although the taxonomy of the southern African taxa has recently been revised, their phylogenetic relationships and biogeographic history remain unclear. This study aims to infer the phylogenetic position and geographic origins of the southern African taxa. We generated DNA sequences of nuclear and plastid loci from several individuals belonging to all eight species of Silene recognized from southern Africa, and combined our DNA sequences with existing data representing species from major clades (i.e. sections) based on the recently revised Silene infrageneric taxonomy. We used a Bayesian coalescent species tree continuous diffusion approach to co-estimate the species tree and the ancestral areas of representative members of the genus. Our results show that the perennial southern African members of section Elisanthe form a strongly-supported clade with the Eurasian annual S. noctiflora and the Central Asian perennial S. turkestanica. The rest of the perennial species form a strongly-supported clade together with the annual S. aethiopica, which is nested in a larger Mediterranean clade comprising mostly annual species classified in section Silene s.l. Estimates of ancestral areas indicate a late Pleistocene dispersal to southern Africa from central and East Africa for the sub-Saharan members of section Silene s.l. The Elisanthe clade is inferred to have colonized southern Africa through long-distance dispersal from Eurasia during the late Pleistocene. Our findings support the hypothesis of a relatively recent colonization into southern Africa resulting from two independent dispersal events during the Pleistocene.

Evolutionary units delimitation and continental multilocus phylogeny of the hyperdiverse catfish genus Hypostomus.

With 149 currently recognized species, Hypostomus is one of the most species-rich catfish genera in the world, widely distributed over most of the Neotropical region. To clarify the evolutionary history of this genus, we reconstructed a comprehensive phylogeny of Hypostomus based on four nuclear and two mitochondrial markers. A total of 206 specimens collected from the main Neotropical rivers were included in the present study. Combining morphology and a Bayesian multispecies coalescent (MSC) approach, we recovered 85 previously recognized species plus 23 putative new species, organized into 118 'clusters'. We presented the Cluster Credibility (CC) index that provides numerical support for every hypothesis of cluster delimitation, facilitating delimitation decisions. We then examined the correspondence between the morphologically identified species and their inter-specific COI barcode pairwise divergence. The mean COI barcode divergence between morphological sisters species was 1.3 ± 1.2%, and only in 11% of the comparisons the divergence was ≥2%. This indicates that the COI barcode threshold of 2% classically used to delimit fish species would seriously underestimate the number of species in Hypostomus, advocating for a taxon-specific COI-based inter-specific divergence threshold to be used only when approximations of species richness are needed. The phylogeny of the 108 Hypostomus species, together with 35 additional outgroup species, confirms the monophyly of the genus. Four well-supported main lineages were retrieved, hereinafter called super-groups: Hypostomus cochliodon, H. hemiurus, H. auroguttatus, and H. plecostomus super-groups. We present a compilation of diagnostic characters for each super-group. Our phylogeny lays the foundation for future studies on biogeography and on macroevolution to better understand the successful radiation of this Neotropical fish genus.

Metapopulation Vicariance, Age of Island Taxa and Dispersal: A Case Study Using the Pacific Plant Genus Planchonella (Sapotaceae).

Oceanic islands originate from volcanism or tectonic activity without connections to continental landmasses, are colonized by organisms, and eventually vanish due to erosion and subsidence. Colonization of oceanic islands occurs through long-distance dispersals (LDDs) or metapopulation vicariance, the latter resulting in lineages being older than the islands they inhabit. If metapopulation vicariance is valid, island ages cannot be reliably used to provide maximum age constraints for molecular dating. We explore the relationships between the ages of members of a widespread plant genus (Planchonella, Sapotaceae) and their host islands across the Pacific to test various assumptions of dispersal and metapopulation vicariance. We sampled three nuclear DNA markers from 156 accessions representing some 100 Sapotaceae taxa, and analyzed these in BEAST with a relaxed clock to estimate divergence times and with a phylogeographic diffusion model to estimate range expansions over time. The phylogeny was calibrated with a secondary point (the root) and fossils from New Zealand. The dated phylogeny reveals that the ages of Planchonella species are, in most cases, consistent with the ages of the islands they inhabit. Planchonella is inferred to have originated in the Sahul Shelf region, to which it back-dispersed multiple times. Fiji has been an important source for range expansion in the Pacific for the past 23 myr. Our analyses reject metapopulation vicariance in all cases tested, including between oceanic islands, evolution of an endemic Fiji-Vanuatu flora, and westward rollback vicariance between Vanuatu and the Loyalty Islands. Repeated dispersal is the only mechanism able to explain the empirical data. The longest (8900 km) identified dispersal is between Palau in the Pacific and the Seychelles in the Indian Ocean, estimated at 2.2 Ma (0.4-4.8 Ma). The first split in a Hawaiian lineage (P. sandwicensis) matches the age of Necker Island (11.0 Ma), when its ancestor diverged into two species that are distinguished by purple and yellow fruits. Subsequent establishment across the Hawaiian archipelago supports, in part, progression rule colonization. In summary, we found no explanatory power in metapopulation vicariance and conclude that Planchonella has expanded its range across the Pacific by LDD. We contend that this will be seen in many other groups when analyzed in detail.

Evolution of wood anatomical characters in Nepenthes and close relatives of Caryophyllales.

Background and Aims: Nepenthes attracts wide attention with its spectacularly shaped carnivorous pitchers, cultural value and horticultural curiosity. Despite the plant's iconic fascination, surprisingly little anatomical detail is known about the genus beyond its modified leaf tip traps. Here, the wood anatomical diversity of Nepenthes is explored. This diversity is further assessed with a phylogenetic framework to investigate whether the wood characters within the genus are relevant from an evolutionary or ecological perspective, or rather depend on differences in developmental stages, growth habits, substrates or precipitation. Methods: Observations were performed using light microscopy and scanning electron microscopy. Ancestral states of selected wood and pith characters were reconstructed using an existing molecular phylogeny for Nepenthes and a broader Caryophyllales framework. Pairwise comparisons were assessed for possible relationships between wood anatomy and developmental stages, growth habits, substrates and ecology. Key Results: Wood anatomy of Nepenthes is diffuse porous, with mainly solitary vessels showing simple, bordered perforation plates and alternate intervessel pits, fibres with distinctly bordered pits (occasionally septate), apotracheal axial parenchyma and co-occurring uni- and multiseriate rays often including silica bodies. Precipitation and growth habit (stem length) are linked with vessel density and multiseriate ray height, while soil type correlates with vessel diameter, vessel element length and maximum ray width. For Caryophyllales as a whole, silica grains, successive cambia and bordered perforation plates are the result of convergent evolution. Peculiar helical sculpturing patterns within various cell types occur uniquely within the insectivorous clade of non-core Caryophyllales. Conclusions: The wood anatomical variation in Nepenthes displays variation for some characters dependent on soil type, precipitation and stem length, but is largely conservative. The helical-banded fibre-sclereids that mainly occur idioblastically in pith and cortex are synapomorphic for Nepenthes , while other typical Nepenthes characters evolved convergently in different Caryophyllales lineages.

Species tree phylogeny and biogeography of the Neotropical genus Pradosia (Sapotaceae, Chrysophylloideae).

Recent phylogenetic studies in Sapotaceae have demonstrated that many genera need to be redefined to better correspond to natural groups. The Neotropical genus Pradosia is believed to be monophyletic and includes 26 recognized species. Here we reconstruct the generic phylogeny by a species-tree approach using (∗)BEAST, 21 recognized species (36 accessions), sequence data from three nuclear markers (ITS, ETS, and RPB2), a relaxed lognormal clock model, and a fossil calibration. We explore the evolution of five selected morphological characters, reconstruct the evolution of habitat (white-sand vs. clayish soils) preference, as well as space and time by using a recently developed continuous diffusion model in biogeography. We find Pradosia to be monophyletic in its current circumscription and to have originated in the Amazon basin at ∼47.5Ma. Selected morphological characters are useful to readily distinguish three clades. Preferences to white-sand and/or clay are somewhat important for the majority of species, but speciation has not been powered by habitat shifts. Pradosia brevipes is a relative young species (∼1.3Ma) that has evolved a unique geoxylic life strategy within Pradosia and is restricted to savannahs. Molecular dating and phylogenetic pattern indicate that Pradosia reached the Brazilian Atlantic coast at least three times: at 34.4Ma (P. longipedicellata), at 11.7Ma (P. kuhlmannii), and at 3.9Ma (weakly supported node within the red-flowered clade).

Phylogenetic properties of 50 nuclear loci in Medicago (Leguminosae) generated using multiplexed sequence capture and next-generation sequencing.

Next-generation sequencing technology has increased the capacity to generate molecular data for plant biological research, including phylogenetics, and can potentially contribute to resolving complex phylogenetic problems. The evolutionary history of Medicago L. (Leguminosae: Trifoliae) remains unresolved due to incongruence between published phylogenies. Identification of the processes causing this genealogical incongruence is essential for the inference of a correct species phylogeny of the genus and requires that more molecular data, preferably from low-copy nuclear genes, are obtained across different species. Here we report the development of 50 novel LCN markers in Medicago and assess the phylogenetic properties of each marker. We used the genomic resources available for Medicago truncatula Gaertn., hybridisation-based gene enrichment (sequence capture) techniques and Next-Generation Sequencing to generate sequences. This alternative proves to be a cost-effective approach to amplicon sequencing in phylogenetic studies at the genus or tribe level and allows for an increase in number and size of targeted loci. Substitution rate estimates for each of the 50 loci are provided, and an overview of the variation in substitution rates among a large number of low-copy nuclear genes in plants is presented for the first time. Aligned sequences of major species lineages of Medicago and its sister genus are made available and can be used in further probe development for sequence-capture of the same markers.

Estimating the Phanerozoic history of the Ascomycota lineages: combining fossil and molecular data.

The phylum Ascomycota is by far the largest group in the fungal kingdom. Ecologically important mutualistic associations such as mycorrhizae and lichens have evolved in this group, which are regarded as key innovations that supported the evolution of land plants. Only a few attempts have been made to date the origin of Ascomycota lineages by using molecular clock methods, which is primarily due to the lack of satisfactory fossil calibration data. For this reason we have evaluated all of the oldest available ascomycete fossils from amber (Albian to Miocene) and chert (Devonian and Maastrichtian). The fossils represent five major ascomycete classes (Coniocybomycetes, Dothideomycetes, Eurotiomycetes, Laboulbeniomycetes, and Lecanoromycetes). We have assembled a multi-gene data set (18SrDNA, 28SrDNA, RPB1 and RPB2) from a total of 145 taxa representing most groups of the Ascomycota and utilized fossil calibration points solely from within the ascomycetes to estimate divergence times of Ascomycota lineages with a Bayesian approach. Our results suggest an initial diversification of the Pezizomycotina in the Ordovician, followed by repeated splits of lineages throughout the Phanerozoic, and indicate that this continuous diversification was unaffected by mass extinctions. We suggest that the ecological diversity within each lineage ensured that at least some taxa of each group were able to survive global crises and rapidly recovered.

On the biogeography of Centipeda: a species-tree diffusion approach.

Reconstructing the biogeographic history of groups present in continuous arid landscapes is challenging due to the difficulties in defining discrete areas for analyses, and even more so when species largely overlap both in terms of geography and habitat preference. In this study, we use a novel approach to estimate ancestral areas for the small plant genus Centipeda. We apply continuous diffusion of geography by a relaxed random walk where each species is sampled from its extant distribution on an empirical distribution of time-calibrated species-trees. Using a distribution of previously published substitution rates of the internal transcribed spacer (ITS) for Asteraceae, we show how the evolution of Centipeda correlates with the temporal increase of aridity in the arid zone since the Pliocene. Geographic estimates of ancestral species show a consistent pattern of speciation of early lineages in the Lake Eyre region, with a division in more northerly and southerly groups since ∼840 ka. Summarizing the geographic slices of species-trees at the time of the latest speciation event (∼20 ka), indicates no presence of the genus in Australia west of the combined desert belt of the Nullabor Plain, the Great Victoria Desert, the Gibson Desert, and the Great Sandy Desert, or beyond the main continental shelf of Australia. The result indicates all western occurrences of the genus to be a result of recent dispersal rather than ancient vicariance. This study contributes to our understanding of the spatiotemporal processes shaping the flora of the arid zone, and offers a significant improvement in inference of ancestral areas for any organismal group distributed where it remains difficult to describe geography in terms of discrete areas.

Species tree phylogeny and character evolution in the genus Centipeda (Asteraceae): evidence from DNA sequences from coding and non-coding loci from the plastid and nuclear genomes.

A species tree phylogeny of the Australian/New Zealand genus Centipeda (Asteraceae) is estimated based on nucleotide sequence data. We analysed sequences of nuclear ribosomal DNA (ETS, ITS) and three plasmid loci (ndhF, psbA-trnH, and trnL-F) using the multi-species coalescent module in BEAST. A total of 129 individuals from all 10 recognised species of Centipeda were sampled throughout the species distribution ranges, including two subspecies. We conclude that the inferred species tree topology largely conform previous assumptions on species relationships. Centipeda racemosa (Snuffweed) is the sister to remaining species, which is also the only consistently perennial representative in the genus. Centipeda pleiocephala (Tall Sneezeweed) and C. nidiformis (Cotton Sneezeweed) constitute a species pair, as does C. borealis and C. minima (Spreading Sneezeweed), all sharing the symplesiomorphic characters of spherical capitulum and convex receptacle with C. racemosa. Another species group comprising C. thespidioides (Desert Sneezeweed), C. cunninghamii (Old man weed, or Common sneeze-weed), C. crateriformis is well-supported but then include the morphologically aberrant C. aotearoana, all sharing the character of having capitula that mature more slowly relative the subtending shoot. Centipeda elatinoides takes on a weakly supported intermediate position between the two mentioned groups, and is difficult to relate to any of the former groups based on morphological characters.

Paleo-drainage basin connectivity predicts evolutionary relationships across three Southeast Asian biodiversity hotspots.

Understanding factors driving diversity across biodiversity hotspots is critical for formulating conservation priorities in the face of ongoing and escalating environmental deterioration. While biodiversity hotspots encompass a small fraction of Earth's land surface, more than half the world's plants and two-thirds of terrestrial vertebrate species are endemic to these hotspots. Tropical Southeast (SE) Asia displays extraordinary species richness, encompassing four biodiversity hotspots, though disentangling multiple potential drivers of species richness is confounded by the region's dynamic geological and climatic history. Here, we use multilocus molecular genetic data from dense multispecies sampling of freshwater fishes across three biodiversity hotspots, to test the effect of Quaternary climate change and resulting drainage rearrangements on aquatic faunal diversification. While Cenozoic geological processes have clearly shaped evolutionary history in SE Asian halfbeak fishes, we show that paleo-drainage re-arrangements resulting from Quaternary climate change played a significant role in the spatiotemporal evolution of lowland aquatic taxa, and provide priorities for conservation efforts.

Use of allele-specific sequencing primers is an efficient alternative to PCR subcloning of low-copy nuclear genes.

Direct Sanger sequencing of polymerase chain reaction (PCR)-amplified nuclear genes leads to polymorphic sequences when allelic variation is present. To overcome this problem, most researchers subclone the PCR products to separate alleles. An alternative is to directly sequence the separate alleles using allele-specific primers. We tested two methods to enhance the specificity of allele-specific primers for use in direct sequencing: using short primers and amplification refractory mutation system (ARMS) technique. By shortening the allele-specific primer to 15-13 nucleotides, the single mismatch in the ultimate base of the primer is enough to hinder the amplification of the nontarget allele in direct sequencing and recover only the targeted allele at high accuracy. The deliberate addition of a second mismatch, as implemented in the ARMS technique, was less successful and seems better suited for allele-specific amplification in regular PCR rather than in direct sequencing.

An open source chimera checker for the fungal ITS region.

The internal transcribed spacer (ITS) region of the nuclear ribosomal repeat unit holds a central position in the pursuit of the taxonomic affiliation of fungi recovered through environmental sampling. Newly generated fungal ITS sequences are typically compared against the International Nucleotide Sequence Databases for a species or genus name using the sequence similarity software suite blast. Such searches are not without complications however, and one of them is the presence of chimeric entries among the query or reference sequences. Chimeras are artificial sequences, generated unintentionally during the polymerase chain reaction step, that feature sequence data from two (or possibly more) distinct species. Available software solutions for chimera control do not readily target the fungal ITS region, but the present study introduces a blast-based open source software package (available at http://www.emerencia.org/chimerachecker.html) to examine newly generated fungal ITS sequences for the presence of potentially chimeric elements in batch mode. We used the software package on a random set of 12 300 environmental fungal ITS sequences in the public sequence databases and found 1.5% of the entries to be chimeric at the ordinal level after manual verification of the results. The proportion of chimeras in the sequence databases can be hypothesized to increase as emerging sequencing technologies drawing from pooled DNA samples are becoming important tools in molecular ecology research.

New insights into the phylogenetic relationships, character evolution, and phytogeographic patterns of Calceolaria (Calceolariaceae).

Biogeographical patterns and diversification processes in Andean and Patagonian flora are not yet well understood. Calceolaria is a highly diversified genus of these areas, representing one of the most specialized plant-pollinator systems because flowers produce nonvolatile oils, a very unusual floral reward. Phylogenetic analyses with molecular (ITS and matK) and morphological characters from 103 Calceolaria species were conducted to examine relationships, to understand biogeographic patterns, and to detect evolutionary patterns of floral and ecological characters. Total evidence analysis retrieved three major clades, which strongly correspond to the three previously recognized subgenera, although only subgenus Rosula was retrieved as a monophyletic group. A single historical event explains the expansion from the southern to central Andes, while different parallel evolutionary lines show a northward expansion from the central to northern Andes across the Huancabamba Deflection, an important geographical barrier in northern Peru. Polyploidy, acquisition of elaiophores, and a nototribic pollination mechanism are key aspects of the evolutionary history of Calceolaria. Pollination interactions were more frequently established with Centris than with Chalepogenus oil-collecting bee species. The repeated loss of the oil gland and shifts to pollen as the only reward suggest an evolutionary tendency from highly to moderately specialized pollination systems.