Complex patterns of reticulate evolution in opportunistic weeds (Potentilla L., Rosaceae), as revealed by low-copy nuclear markers.

BACKGROUND: Most cinquefoils (Potentilla L., Rosaceae) are polyploids, ranging from tetraploid (4x) to dodecaploid (12x), diploids being a rare exception. Previous studies based on ribosomal and chloroplast data indicated that Norwegian cinquefoil (P. norvegica L.) has genetic material from two separate clades within Potentilla; the Argentea and the Ivesioid clades - and thus a possible history of hybridization and polyploidization (allopolyploidy). In order to trace the putative allopolyploid origin of the species, sequence data from low-copy, biparentally inherited, nuclear markers were used. Specimens covering the circumpolar distribution of P. norvegica and its two subspecies were included, along with the morphologically similar P. intermedia. Potentilla species of low ploidy level known to belong to other relevant clades were also included. RESULTS: Gene trees based on three low-copy nuclear markers, obtained by Bayesian Inference and Maximum Likelihood analyses, showed slightly different topologies. This is likely due to genomic reorganizations following genome duplication, but the gene trees were not in conflict with a species tree of presumably diploid taxa obtained by Multispecies Coalescent analysis. The results show that both P. norvegica and P. intermedia are allopolyploids with a shared evolutionary history involving at least four parental lineages, three from the Argentea clade and one from the Ivesioid clade. CONCLUSIONS: This is the first time that reticulate evolution has been proven in the genus Potentilla, and shows the importance of continuing working with low-copy markers in order to properly resolve its evolutionary history. Several hybridization events between the Argentea and Ivesioid clades may have given rise to the species of Wolf's grex Rivales. To better estimate when and where these hybridizations occurred, other Argentea, Ivesioid and Rivales species should be included in future studies.

Macronutrient balancing in free-ranging populations of moose.

At northern latitudes, large spatial and temporal variation in the nutritional composition of available foods poses challenges to wild herbivores trying to satisfy their nutrient requirements. Studies conducted in mostly captive settings have shown that animals from a variety of taxonomic groups deal with this challenge by adjusting the amounts and proportions of available food combinations to achieve a target nutrient balance. In this study, we used proportions-based nutritional geometry to analyze the nutritional composition of rumen samples collected in winter from 481 moose (Alces alces) in southern Sweden and examine whether free-ranging moose show comparable patterns of nutrient balancing. Our main hypothesis was that wild moose actively regulate their rumen nutrient composition to offset ecologically imposed variation in the nutritional composition of available foods. To test this, we assessed the macronutritional composition (protein, carbohydrates, and lipids) of rumen contents and commonly eaten foods, including supplementary feed, across populations with contrasting winter diets, spanning an area of approximately 10,000 km2. Our results suggest that moose balanced the macronutrient composition of their rumen, with the rumen contents having consistently similar proportional relationship between protein and nonstructural carbohydrates, despite differences in available (and eaten) foods. Furthermore, we found that rumen macronutrient balance was tightly related to ingested levels of dietary fiber (cellulose and hemicellulose), such that the greater the fiber content, the less protein was present in the rumen compared with nonstructural carbohydrates. Our results also suggest that moose benefit from access to a greater variety of trees, shrubs, herbs, and grasses, which provides them with a larger nutritional space to maneuver within. Our findings provide novel theoretical insights into a model species for ungulate nutritional ecology, while also generating data of direct relevance to wildlife and forest management, such as silvicultural or supplementary feeding practices.

Detecting destabilizing species in the phylogenetic backbone of Potentilla (Rosaceae) using low-copy nuclear markers.

The genus Potentilla (Rosaceae) has been subjected to several phylogenetic studies, but resolving its evolutionary history has proven challenging. Previous analyses recovered six, informally named, groups: the Argentea, Ivesioid, Fragarioides, Reptans, Alba and Anserina clades, but the relationships among some of these clades differ between data sets. The Reptans clade, which includes the type species of Potentilla, has been noticed to shift position between plastid and nuclear ribosomal data sets. We studied this incongruence by analysing four low-copy nuclear markers, in addition to chloroplast and nuclear ribosomal data, with a set of Bayesian phylogenetic and Multispecies Coalescent (MSC) analyses. A selective taxon removal strategy demonstrated that the included representatives from the Fragarioides clade, P. dickinsii and P. fragarioides, were the main sources of the instability seen in the trees. The Fragarioides species showed different relationships in each gene tree, and were only supported as a monophyletic group in a single marker when the Reptans clade was excluded from the analysis. The incongruences could not be explained by allopolyploidy, but rather by homoploid hybridization, incomplete lineage sorting or taxon sampling effects. When P. dickinsii and P. fragarioides were removed from the data set, a fully resolved, supported backbone phylogeny of Potentilla was obtained in the MSC analysis. Additionally, indications of autopolyploid origins of the Reptans and Ivesioid clades were discovered in the low-copy gene trees.

Allopolyploidy in Fragariinae (Rosaceae): comparing four DNA sequence regions, with comments on classification.

Potential events of allopolyploidy may be indicated by incongruences between separate phylogenies based on plastid and nuclear gene sequences. We sequenced two plastid regions and two nuclear ribosomal regions for 34 ingroup taxa in Fragariinae (Rosaceae), and six outgroup taxa. We found five well supported incongruences that might indicate allopolyploidy events. The incongruences involved Aphanes arvensis, Potentilla miyabei, Potentilla cuneata, Fragaria vesca/moschata, and the Drymocallis clade. We evaluated the strength of conflict and conclude that allopolyploidy may be hypothesised in the four first cases. Phylogenies were estimated using Bayesian inference and analyses were evaluated using convergence diagnostics. Taxonomic implications are discussed for genera such as Alchemilla, Sibbaldianthe, Chamaerhodos, Drymocallis and Fragaria, and for the monospecific Sibbaldiopsis and Potaninia that are nested inside other genera. Two orphan Potentilla species, P. miyabei and P. cuneata are placed in Fragariinae. However, due to unresolved topological incongruences they are not reclassified in any genus.

Microsatellite markers for the biogeographically enigmatic sandmyrtle (Kalmia buxifolia, Phyllodoceae: Ericaceae).

PREMISE: Microsatellite markers were developed for sandmyrtle, Kalmia buxifolia (Ericaceae), to facilitate phylogeographic studies in this taxon and possibly many of its close relatives. METHODS AND RESULTS: Forty-eight primer pairs designed from paired-end Illumina MiSeq data were screened for robust amplification. Sixteen pairs were amplified again, but with fluorescently labeled primers to facilitate genotyping. Resulting chromatograms were evaluated for variability using three populations from Tennessee, North Carolina, and New Jersey, USA. Eleven primer pairs were reliable and polymorphic (mean 3.92 alleles), one was reliable but monomorphic, and four were not reliable. The markers exhibited lower heterozygosity (mean 0.246) than expected (mean 0.464). Cross-amplification in the remaining nine Kalmia species exhibited a phylogenetic pattern, suggesting broad applicability of the markers across the genus. CONCLUSIONS: These microsatellite markers will be useful in population genetics and species boundaries studies of K. buxifolia, K. procumbens, and likely all other Kalmia species.

Biodiversity and seasonal variation of the cyanobacterial assemblage in a rice paddy field in Fujian, China.

Cyanobacteria are one of the main components of the microbiota in rice paddy fields and significantly contribute to its fertilization. The diversity and changes of the cyanobacterial assemblage were investigated during a rice growth season and after harvest in a paddy field located in Fujian Province, China. The cyanobacterial populations were analyzed by a semi-nested PCR, followed by denaturing gradient gel electrophoresis analysis. Twenty-four phylotypes were identified from the denaturing gradient gel electrophoresis profiles. The number of cyanobacterial phylotypes showed a seasonal variation and reached a peak in September, both in the upper (0-5 cm) and the deeper (10-15 cm) soil fractions. Some cyanobacterial sequences were only present during the rice growth season, while others were only found after harvest.

Early Tertiary out-of-India dispersal of Crypteroniaceae: evidence from phylogeny and molecular dating.

Phylogenetic analyses and molecular dating estimates based on chloroplast DNA sequences were used to establish the relationships of the southern and Southeast Asian Crypteroniaceae and elucidate their biogeographic history. Maximum parsimony and likelihood analyses of rbcL sequences suggested that Crypteroniaceae should be restricted to Crypteronia, Axinandra, and Dactylocladus and that Crypteroniaceae, so defined, are sister to a clade formed by three small African taxa (Oliniaceae, Penaeaceae, and Rhynchocalycaceae) and the monotypic Central and South American Alzateaceae. Three molecular dating approaches (maximum-likelihood under a molecular clock, Langley-Fitch, and penalized-likelihood) were used to infer the age of Crypteroniaceae using both paleobotanic and geologic calibrations. Comparisons among these three methods revealed significant lineage effects in rbcL sequences. Clock-independent dating estimates suggested that divergence of Crypteroniaceae from its African and South American relatives coincided with the breakup of Gondwana, and that India likely served as a "raft" transporting Crypteroniaceae to Asia, with later expansion to Southeast Asia. To our knowledge, Crypteroniaceae are the first plant group for which the out-of-India hypothesis is well corroborated by molecular-based estimates of divergence times.

Molecular data and ploidal levels indicate several putative allopolyploidization events in the genus Potentilla (Rosaceae).

Several naturally occurring hybrids in Potentilla (Rosaceae) have been reported, but no molecular evidence has so far been available to test these hypotheses of hybridization. We have compared a nuclear and a chloroplast gene tree to identify topological incongruences that may indicate hybridization events in the genus. Furthermore, the monophyly and phylogenetic position of the proposed segregated genera Argentina, Ivesia and Horkelia have been tested. The systematic signal from the two morphological characters, style- and anther shape, has also been investigated by ancestral state reconstruction, to elucidate how well these characters concur with the results of the molecular phylogenies. Six major clades, Anserina, Alba, Fragarioides, Reptans, ivesioid and Argentea, have been identified within genus Potentilla. Horkelia, Ivesia and Horkeliella (the ivesioid clade), form a monophyletic group nested within Potentilla. Furthermore, the origin of the proposed segregated genus Argentina (the Anserina clade) is uncertain but not in conflict with a new generic status of the group. We also found style morphology to be an informative character that reflects the phylogenetic relationships within Potentilla. Five well-supported incongruences were found between the nuclear and the chloroplast phylogenies, and three of these involved polyploid taxa. However, further investigations, using low copy molecular markers, are required to infer the phylogeny of these species and to test the hypothesis of hybrid origin.

Molecular systematics and morphological character evolution of the Condamineeae (Rubiaceae).

PREMISE OF THE STUDY: The Condamineeae have in previous molecular studies been shown to be part of an early-divergent clade within the subfamily Ixoroideae, together with the tribes Calycophylleae, and Hippotideae, and genera of the former Cinchoneae and Rondeletieae. Generic relationships within this clade have, however, remained largely unresolved. • METHODS: In this study, the systematics of the Condamineeae was further examined by phylogenetic reconstruction of six cpDNA regions and one nrDNA region using parsimony and Bayesian Markov chain Monte Carlo inference. Morphological character evolution within the tribe was assessed by ancestral state reconstruction using likelihood optimization of characters onto Bayesian trees. • KEY RESULTS: Calycophylleae appears polyphyletic. "Hippotideae" is monophyletic but nested within the Condamineeae. The phylogenetic hypotheses presented support a resurrection of the genera Holtonia, Schizocalyx, and Semaphyllanthe. Furthermore, Bathysa is found to be polyphyletic, Tresanthera is found nested within Rustia, and the taxonomically disputed genus Dialypetalanthus is here shown to be sister to a Bothriospora-Wittmackanthus clade. Morphological ancestral state reconstructions indicate that protogyny have evolved at least two times within the tribe and that indehiscent fruits, loculicidal fruit dehiscence, and intrapetiolar stipules have evolved independently several times. The occurrence of calycophylls (leaf-like calyx lobes), poricidal anthers, and winged seeds also appear homoplastic within the tribe. • CONCLUSIONS: A diagnosis and delimitation of the tribe Condamineeae is presented, with taxonomic proposals to synonymize Tresanthera and to transfer several species of Bathysa as well as Phitopis to a resurrected Schizocalyx.

Assessing calibration uncertainty in molecular dating: the assignment of fossils to alternative calibration points.

Although recent methodological advances have allowed the incorporation of rate variation in molecular dating analyses, the calibration procedure, performed mainly through fossils, remains resistant to improvements. One source of uncertainty pertains to the assignment of fossils to specific nodes in a phylogeny, especially when alternative possibilities exist that can be equally justified on morphological grounds. Here we expand on a recently developed fossil cross-validation method to evaluate whether alternative nodal assignments of multiple fossils produce calibration sets that differ in their internal consistency. We use an enlarged Crypteroniaceae-centered phylogeny of Myrtales, six fossils, and 72 combinations of calibration points, termed calibration sets, to identify (i) the fossil assignments that produce the most internally consistent calibration sets and (ii) the mean ages, derived from these calibration sets, for the split of the Southeast Asian Crypteroniaceae from their West Gondwanan sister clade (node X). We found that a correlation exists between s values, devised to measure the consistency among the calibration points of a calibration set (Near and Sanderson, 2004), and nodal distances among calibration points. By ranking all sets according to the percent deviation of s from the regression line with nodal distance, we identified the sets with the highest level of corrected calibration-set consistency. These sets generated lower standard deviations associated with the ages of node X than sets characterized by lower corrected consistency. The three calibration sets with the highest corrected consistencies produced mean age estimates for node X of 79.70, 79.14, and 78.15 My. These timeframes are most compatible with the hypothesis that the Crypteroniaceae stem lineage dispersed from Africa to the Deccan plate as it drifted northward during the Late Cretaceous.

Phylogeny of symbiotic cyanobacteria within the genus Nostoc based on 16S rDNA sequence analyses.

A phylogenetic analysis of selected symbiotic Nostoc strain sequences and available database 16S rDNA sequences of both symbiotic and free-living cyanobacteria was carried out using maximum likelihood and Bayesian inference techniques. Most of the symbiotic strains fell into well separated clades. One clade consisted of a mixture of symbiotic and free-living isolates. This clade includes Nostoc sp. strain PCC 73102, the reference strain proposed for Nostoc punctiforme. A separate symbiotic clade with isolates exclusively from Gunnera species was also obtained, suggesting that not all symbiotic Nostoc species can be assigned to N. punctiforme. Moreover, isolates from Azolla filiculoides and one from Gunnera dentata were well nested within a clade comprising most of the Anabaena sequences. This result supports the affiliation of the Azolla isolates with the genus Anabaena and shows that strains within this genus can form symbioses with additional hosts. Furthermore, these symbiotic strains produced hormogonia, thereby verifying that hormogonia formation is not absent in Anabaena and cannot be used as a criterion to distinguish it from Nostoc.

Ancient allopolyploid speciation in Geinae (Rosaceae): evidence from nuclear granule-bound starch synthase (GBSSI) gene sequences.

A nuclear low-copy gene phylogeny provides strong evidence for the hybrid origin of seven polyploid species in Geinae (Rosaceae). In a gene tree, alleles at homologous loci in an allopolyploid species are expected to be sisters to orthologues in the ancestral taxa rather than to each other. Alleles at a duplicated locus in an autopolyploid, however, are expected to be more closely related to each other than they are to any orthologous copies in closely related species. We cloned and sequenced about 1.9 kilobases from the 5' end of the GBSSI-1 gene from two diploid, one tetraploid, and six hexaploid species. Each of the three loci in the hexaploid species forms a separate group, two of which are more closely related to copies in other species than they are to each other. This finding indicates that the hexaploid lineage evolved through two consecutive allopolyploidization events. Based on the GBSSI-1 gene tree, we hypothesized that there was an initial hybridization between a diploid species from the ancestral lineage of Coluria and Waldsteinia and an unknown diploid species to form the tetraploid Geum heterocarpum lineage. Backcrossing of G. heterocarpum with a representative of the unknown diploid lineage then resulted in a hexaploid lineage that has radiated considerably since its origin, comprising at least 40 extant species with various morphologies. A penalized likelihood analysis indicated that Geinae may be about 17 million years old, implying that the hypothesized allopolyploid speciation events are relatively ancient. Six of the 22 cloned Geinae GBSSI-1 copies in this study, which all are duplicate copies in polyploid taxa, may have become pseudogenes. We compared the GBSSI-1 phylogeny with one from chloroplast data and explored implications for the evolution of some fruit characters.

Molecular phylogeny of the extinct cave lion Panthera leo spelaea.

To reconstruct the phylogenetic position of the extinct cave lion (Panthera leo spelaea), we sequenced 1 kb of the mitochondrial cytochrome b gene from two Pleistocene cave lion DNA samples (47 and 32 ky B.P.). Phylogenetic analysis shows that the ancient sequences form a clade that is most closely related to the extant lions from Africa and Asia; at the same time, cave lions appear to be highly distinct from their living relatives. Our data show that these cave lion sequences represent lineages that were isolated from lions in Africa and Asia since their dispersal over Europe about 600 ky B.P., as they are not found among our sample of extant populations. The cave lion lineages presented here went extinct without mitochondrial descendants on other continents. The high sequence divergence in the cytochrome b gene between cave and modern lions is notable.

Phylogenetic position and biogeography of Hillebrandia sandwicensis (Begoniaceae): a rare Hawaiian relict.

The Begoniaceae consist of two genera, Begonia, with approximately 1400 species that are widely distributed in the tropics, and Hillebrandia, with one species that is endemic to the Hawaiian Islands and the only member of the family native to those islands. To help explain the history of Hillebrandia on the Hawaiian Archipelago, phylogenetic relationships of the Begoniaceae and the Cucurbitales were inferred using sequence data from 18S, rbcL, and ITS, and the minimal age of both Begonia and the Begoniaceae were indirectly estimated. The analyses strongly support the placement of Hillebrandia as the sister group to the rest of the Begoniaceae and indicate that the Hillebrandia lineage is at least 51-65 million years old, an age that predates the current Hawaiian Islands by about 20 million years. Evidence that Hillebrandia sandwicensis has survived on the Hawaiian Archipelago by island hopping from older, now denuded islands to younger, more mountainous islands is presented. Various scenarios for the origin of ancestor to Hillebrandia are considered. The geographic origin of source populations unfortunately remains obscure; however, we suggest a boreotropic or a Malesian-Pacific origin is most likely. Hillebrandia represents the first example in the well-studied Hawaiian flora of a relict genus.