Climbing since the early Miocene: The fossil record of Paullinieae (Sapindaceae).

Paullinieae are a diverse group of tropical and subtropical climbing plants that belong to the soapberry family (Sapindaceae). The six genera in this tribe make up approximately one-quarter of the species in the family, but a sparse fossil record limits our understanding of their diversification. Here, we provide the first description of anatomically preserved fossils of Paullinieae and we re-evaluate other macrofossils that have been attributed to the tribe. We identified permineralized fossil roots in collections from the lower Miocene Cucaracha Formation where it was exposed along the Culebra Cut of the Panama Canal. We prepared the fossils using the cellulose acetate peel technique and compared the anatomy with that of extant Paullinieae. The fossil roots preserve a combination of characters found only in Paullinieae, including peripheral secondary vascular strands, vessel dimorphism, alternate intervessel pitting with coalescent apertures, heterocellular rays, and axial parenchyma strands of 2-4 cells, often with prismatic crystals. We also searched the paleontological literature for other occurrences of the tribe. We re-evaluated leaf fossils from western North America that have been assigned to extant genera in the tribe by comparing their morphology to herbarium specimens and cleared leaves. The fossil leaves that were assigned to Cardiospermum and Serjania from the Paleogene of western North America are likely Sapindaceae; however, they lack diagnostic characters necessary for inclusion in Paullinieae and should be excluded from those genera. Therefore, the fossils described here as Ampelorhiza heteroxylon gen. et sp. nov. are the oldest macrofossil evidence of Paullinieae. They provide direct evidence of the development of a vascular cambial variant associated with the climbing habit in Sapindaceae and provide strong evidence of the diversification of crown-group Paullinieae in the tropics by 18.5-19 million years ago.

Phylogenomics recovers monophyly and early Tertiary diversification of Dipteronia (Sapindaceae).

Dipteronia (Sapindaceae) is an ancient relict woody genus, and contains just two extant species endemic to Southwestern and Central China. As sharing numerous morphological characters, Dipteronia and Acer have long been considered as sister groups forming the traditional family Aceraceae. However, molecular phylogenetics has generally not resolved the phylogenetic placement of Dipteronia, especially not in its expected position as sister to Acer. In this study, we present large-scale, phylogenomic data sets, incorporating complete chloroplast (cp) genome sequences and transcriptome data from 13 Sapindaceae species, to resolve the phylogenetic relationship between Dipteronia and Acer. Moreover, the impact of long-branch attraction (LBA) artefacts and robustness of inferred topologies are assessed by long-branch excluding and coalescent-based methods. Corroborating classical morphology-based classifications, both cp genome and nuclear datasets (2466 co-orthologous genes and 273 co-SCNGs) recover Dipteronia and Acer as mutually monophyletic groups. In addition, our fossil-calibrated molecular phylogenies date the crown of the two extant Dipteronia species to the Paleocene/Eocene boundary, implying that these morphologically highly similar taxa are amongst the oldest 'living fossils' of the East Asian Flora.

The complete chloroplast genome sequence of Dodonaea viscosa: comparative and phylogenetic analyses.

The plant chloroplast (cp) genome is a highly conserved structure which is beneficial for evolution and systematic research. Currently, numerous complete cp genome sequences have been reported due to high throughput sequencing technology. However, there is no complete chloroplast genome of genus Dodonaea that has been reported before. To better understand the molecular basis of Dodonaea viscosa chloroplast, we used Illumina sequencing technology to sequence its complete genome. The whole length of the cp genome is 159,375 base pairs (bp), with a pair of inverted repeats (IRs) of 27,099 bp separated by a large single copy (LSC) 87,204 bp, and small single copy (SSC) 17,972 bp. The annotation analysis revealed a total of 115 unique genes of which 81 were protein coding, 30 tRNA, and four ribosomal RNA genes. Comparative genome analysis with other closely related Sapindaceae members showed conserved gene order in the inverted and single copy regions. Phylogenetic analysis clustered D. viscosa with other species of Sapindaceae with strong bootstrap support. Finally, a total of 249 SSRs were detected. Moreover, a comparison of the synonymous (Ks) and nonsynonymous (Ka) substitution rates in D. viscosa showed very low values. The availability of cp genome reported here provides a valuable genetic resource for comprehensive further studies in genetic variation, taxonomy and phylogenetic evolution of Sapindaceae family. In addition, SSR markers detected will be used in further phylogeographic and population structure studies of the species in this genus.

Evolutionary cascades induced by large frugivores.

Large, fruit-eating vertebrates have been lost from many of the world's ecosystems. The ecological consequences of this defaunation can be severe, but the evolutionary consequences are nearly unknown because it remains unclear whether frugivores exert strong selection on fruit traits. I assessed the macroevolution of fruit traits in response to variation in the diversity and size of seed-dispersing vertebrates. Across the Indo-Malay Archipelago, many of the same plant lineages have been exposed to very different assemblages of seed-dispersing vertebrates. Phylogenetic analysis of >400 plant species in 41 genera and five families revealed that average fruit size tracks the taxonomic and functional diversity of frugivorous birds and mammals. Fruit size was 40.2-46.5% smaller in the Moluccas and Sulawesi (respectively), with relatively depauperate assemblages of mostly small-bodied animals, than in the Sunda Region (Borneo, Sumatra, and Peninsular Malaysia), with a highly diverse suite of large and small animals. Fruit color, however, was unrelated to vertebrate diversity or to the representation of birds versus mammals in the frugivore assemblage. Overhunting of large animals, nearly ubiquitous in tropical forests, could strongly alter selection pressures on plants, resulting in widespread, although trait-specific, morphologic changes.

A First Phylogeny of the Genus Dimocarpus and Suggestions for Revision of Some Taxa Based on Molecular and Morphological Evidence.

Dimocarpus longan, commonly known as the longan, belongs to the family Sapindaceae, and is one of the most economically important fruits commonly cultivated in several regions in Asia. There are various cultivars of longan throughout the Thai-Malay peninsula region, but until now no phylogenetic analysis has been undertaken to determine the genetic relatedness of these cultivars. To address this issue, 6 loci, namely ITS2, matK, rbcL, trnH-psbA, trnL-I and trnL-trnF were amplified and sequenced from 40 individuals consisting of 26 longan cultivars 2 types of lychee and 8 herbarium samples. The sequencing results were used to construct a phylogenetic tree using the neighbor-joining (NJ), maximum likelihood (ML) and Bayesian inference (BI) criteria. The tree showed cryptic groups of D. longan from the Thailand-Malaysia region (Dimocarpus longan spp.). This is the first report of the genetic relationship of Dimocarpus based on multi-locus molecular markers and morphological characteristics. Multiple sequence alignments, phylogenetic trees and species delimitation support that Dimocarpus longan spp. longan var. obtusus and Dimocarpus longan spp. malesianus var. malesianus should be placed into a higher order and are two additional species in the genus Dimocarpus. Therefore these two species require nomenclatural changes as Dimocarpus malesianus and Dimocarpus obtusus, respectively.

Formulation and evaluation of topical herbal gel for the treatment of arthritis in animal model

ABSTRACT The objective of the study is to formulate and evaluate a topical herbal gel containing Cardiospermum halicacabum and Vitex negundo leaf extracts for their anti-arthritic activity in rats. Twelve herbal gel formulations were prepared using 1.5% of gelling agents carbopol 934 (F1-F6) and carbopol 940 (F6-F12) and they were evaluated for physical appearance, net content, viscosity, extrudability, pH, spreadability, in vitro diffusion profile and primary skin irritation tests. The stability study for the topical herbal gel formulation was done as per ICH guidelines and anti-arthritic activity was evaluated by Freund's Complete Adjuvant (FCA) induced arthritis method. Assessment of body weight, paw volume, hematological and biochemical parameters, histopathological examination and In vitro determination of serum biomarkers were also carried out. Formulated gels were homogenous, stable and complied with the guidelines. Among the formulations, F4 showed better release (98.4 %) characteristics than other formulations. No erythema or edema was observed in the skin irritation test confirming the gel was non-toxic and safe. Topical application of the herbal gel F4 containing carbopol 934 displayed significant (p < 0.001) anti-arthritic activity compared to diseased rats. Reduction in paw volume, no agglutination in C - reactive protein and rheumatic factor, reduction in TNF level, regaining of normal hematological, and biochemical parameters, reduction in spleen and thymus weight and histopathological examination supported the anti-arthritic activity of the gel formulation.

Testing the monophyly of Aesculus L. and Billia Peyr., woody genera of tribe Hippocastaneae of the Sapindaceae.

Hippocastaneae is a well-supported clade in Sapindaceae that comprises 15+ species; 12+ in Aesculus, two in Billia, and one in Handeliodendron Rehder. The monophyly of Aesculus and Billia were widely assumed, but a recent molecular phylogenetic study of Sapindanceae used seven species of Aesculus and one each of Billia and Handeliodendron and showed that Billia and Handeliodendron were nested within Aesculus. Here, we tested the hypothesis that Aesculus and Billia are mutually monophyletic using phylogenetic analyses of seven molecular markers and 31 accessions of Hippocastaneae representing 14 species. We performed phylogenetic analyses using a dataset of concatenated genes as well as with coalescent method for constructing a species tree from individual gene trees. The analysis of seven concatenated markers and the species tree strongly supported the mutual monophyly of Aesculus and Billia. We also recovered support for the traditional arrangement of genera within Hippocastaneae: Aesculus and Billia comprising a clade that is sister to Handeliodendron. However, the relationships among the genera remain incompletely resolved.

Large scale patterns of genetic variation and differentiation in sugar maple from tropical Central America to temperate North America.

BACKGROUND: Geological events in the latter Cenozoic have influenced the distribution, abundance and genetic structure of tree populations in temperate and tropical North America. The biogeographical history of temperate vegetation that spans large ranges of latitude is complex, involving multiple latitudinal shifts that might have occurred via different migration routes. We determined the regional structuring of genetic variation of sugar maple (Acer saccharum subsp. saccharum) and its only subspecies in tropical America (Acer saccharum subsp. skutchii) using nuclear and chloroplast data. The studied populations span a geographic range from Maine, USA (46°N), to El Progreso, Guatemala (15°N). We examined genetic subdivisions, explored the locations of ancestral haplotypes, analyzed genetic data to explore the presence of a single or multiple glacial refugia, and tested whether genetic lineages are temporally consistent with a Pleistocene or older divergence. RESULTS: Nuclear and chloroplast data indicated that populations in midwestern USA and western Mexico were highly differentiated from populations in the rest of the sites. The time of the most recent common ancestor of the western Mexico haplotype lineage was dated to the Pliocene (5.9 Ma, 95% HPD: 4.3-7.3 Ma). Splits during the Pleistocene separated the rest of the phylogroups. The most frequent and widespread haplotype occurred in half of the sites (Guatemala, eastern Mexico, southeastern USA, and Ohio). Our data also suggested that multiple Pleistocene refugia (tropics-southeastern USA, midwestern, and northeastern USA), but not western Mexico (Jalisco), contributed to post-glacial northward expansion of ranges. Current southern Mexican and Guatemalan populations have reduced population sizes, genetic bottlenecks and tend toward homozygosity, as indicated using nuclear and chloroplast markers. CONCLUSIONS: The divergence of western Mexican populations from the rest of the sugar maples likely resulted from orographic and volcanic barriers to gene flow. Past connectivity among populations in the southeastern USA and eastern Mexico and Guatemala possible occurred through gene flow during the Pleistocene. The time to the most common ancestor values revealed that populations from the Midwest and Northeast USA represented different haplotype lineages, indicating major divergence of haplotypes lineages before the Last Glacial Maximum and suggesting the existence of multiple glacial refugia.

Fatty-Acid Composition of Seeds and Chemotaxonomic Evaluation of Sixteen Sapindaceae Species.

Circumscriptions for the Sapindaceae family and its infrafamilial relationships have been widely discussed. Certain groups are highly morphologically similar; thus, it is difficult to identify certain taxa. DNA Analyses have also indicated complex phylogenetic relationships, and it is difficult to relate such analyses to morphological data. Given the above concerns, this study aimed to investigate the fatty-acid profiles of the seed oils of 16 Sapindaceae species belonging to five tribes and to evaluate their potential chemotaxonomic significance. In total, eleven fatty acids were identified, and eicosenoic acid predominated in nine species. Multivariate analyses (principal component and cluster analyses) of the fatty-acid profiles of the seed oils allowed to separate them in two major clusters. The first cluster, characterized by oils with high eicosenoic acid levels, included all species belonging to the Paullinieae tribe (Cardiospermum, Paullinia, and Serjania species). In the second main cluster, the chemical similarity of the oils was lower, and the species belonged to different tribes. Nevertheless, the tree investigated Allophylus species (Thouinieae tribe) constituted a separate subcluster. Thus, the results showed that the fatty-acid composition of the seed oils of Sapindaceae species provide chemotaxonomic support for the separation of the Paullinieae tribe from the other tribes studied.

Cloning and molecular characterization of phospholipase D (PLD) delta gene from longan (Dimocarpus longan Lour.).

Longan (Dimocarpus longan Lour.) is a non-climacteric fruit with a short postharvest life. The regulation of phospholipase D (PLD) activity closely relates to postharvest browning and senescence of longan fruit. In this study, a novel cDNA clone of longan PLDδ (LgPLDδ) was obtained and registered in GenBank (accession No. JF791814). The deduced amino acid sequence possessed all of the three typical domains of plant PLDs, a C2 domain and two catalytic HxKxxxxD motifs. The tertiary structure of LgPLDδ was further predicted. The western blot result showed that the LgPLDδ protein was specifically recognized by PLDδ antibody. The Q-RT-PCR (real-time quantitative PCR) result showed that the level of LgPLDδ mRNA expression was higher in senescent tissues than in developing tissues, which was also high in postharvest fruit. The western-blotting result further certified the different expression of LgPLDδ. These results provided a scientific basis for further investigating the mechanism of postharvest longan fruit adapting to environmental stress.

Identification of a PTC-containing DlRan transcript and its differential expression during somatic embryogenesis in Dimocarpus longan.

RAN (Ras-related nuclear protein) plays crucial roles in multiple cellular processes in yeast, animals and plants. Here we present a DlRan gene and its alternative splicing transcripts containing premature terminator codons (PTCs), identified from embryogenic cultures in longan. Multiple alignment and splicing pattern analyses indicated that DlRan-1 transcript harboring PTC was the consequence of alternative splicing. The accumulation of DlRan PTC-containing transcripts increased significantly when the embryogenic calli were treated with the translation inhibitor, cycloheximide, indicating that DlRan-1 may be targeted by NMD. The analysis of expression profiles of DlRan transcripts revealed that differential expression levels of the alternative spliced DlRan transcripts occurred during the development of embryogenic callus, globular-shaped embryos, and cotyledon-shaped embryos, respectively, in the longan somatic embryogenesis, and were in consistent with the embryo development in corresponding wild-type transcripts. The present work offers evidence to speculate that the alternatively spliced PTC-containing transcripts can be functional and may shed light on expression regulation of DlRan during development of the longan somatic embryos.

Fruits of Koelreuteria (Sapindaceae) from the Cenozoic throughout the northern hemisphere: their ecological, evolutionary, and biogeographic implications.

PREMISE OF THE STUDY: Koelreuteria (Sapindaceae) has four extant deciduous tree species, disjunctly distributed in eastern Asia and the Fiji Islands. While K. paniculata is widely cultivated, the biogeographic origin and evolutionary history of Koelreuteria remain unclear. METHODS: Fruits, pollen, wood, and leaves of closely related extant taxa were examined in comparison with fossil remains to evaluate the fossil record and biogeographic history of Koelreuteria. KEY RESULTS: Overall, characters of capsular fruits are more diagnostic than other organs for this genus. We describe two new species of fruit remains from the Eocene, K. taoana sp. nov. (northeastern China and far eastern Russia) and K. dilcheri sp. nov. (western United States), and give emended descriptions of three species: K. allenii (Lesq.) W. N. Edwards (early-late Eocene of the United States), K. macroptera (Kováts) W. N. Edwards (late Oligocene-early Pliocene of Europe), and K. miointegrifoliola Hu et R. W. Chaney (Miocene of eastern Asia). CONCLUSIONS: Reliable fossil records of capsules and ring-porous wood indicate that Koelreuteria may have originated in North Pacific-Rim area of the northern hemisphere by the early Eocene, representing an early temperate lineage in Sapindaceae adapted for wind dispersal. The fossils herein place a minimum age (ca. 52 Ma) for the divergence of Koelreuteria from tropical genera that appear more basal in the molecular phylogeny of Sapindaceae. Regional extinctions after the Eocene in North America and the Pliocene in Europe, reduced the range of Koelreuteria to eastern Asia, where three species occur today. The present distribution of another species in the Southern Pacific may be explained by long-distance dispersal.

Karyotype evolution and phylogenetic analyses in the genus Cardiospermum L. (Paullinieae, Sapindaceae).

Cardiospermum L. belongs to the Paullinieae tribe (Sapindaceae) and comprises 16 species. Of these, 12 species are present in South America and all occur in Brazil. Cardiospermum shows the most variable chromosome number of the tribe. Phylogenetic relationships within the genus Cardiospermum, especially with other species of the tribe, are poorly understood. This research focuses on characterisation of the karyotypic features of Cardiospermum using conventional cytogenetic methods, CMA/DAPI chromosome banding and fluorescence in situ hybridisation (FISH). To elucidate the phylogeny of the genus, the nuclear markers ITS1 and ITS2 were sequenced and analysed using maximum parsimony and Bayesian inference. Cardiospermum shows important diversity in basic numbers, with x = 7, 9, 10, 11 and 12. All species studied have metacentric and submetacentric chromosomes, some species have subtelocentric chromosomes, while telocentric chromosomes are absent. The interphase nuclei differentiate the Cardiospermum species into two groups. The CMA(3) /DAPI chromosome banding revealed the presence of an AT-rich terminal region in C. corindum, C. grandiflorum and C. urvilleoides, whereas GC-rich regions were found in C. grandiflorum, C. halicacabum var. halicacabum, C. halicacabum var. microcarpum, C. heringeri and C. integerrimum. FISH revealed syntenic and non-syntenic distribution of the 18-5.8-26S and 5S rDNA. The syntenic distribution always occurred in the short arms of the same chromosome in all of the species. The phylogenetic relationships reveal, in part, the taxonomic arrangement of the genus Cardiospermum.

Comparative performance of supertree algorithms in large data sets using the soapberry family (Sapindaceae) as a case study.

For the last 2 decades, supertree reconstruction has been an active field of research and has seen the development of a large number of major algorithms. Because of the growing popularity of the supertree methods, it has become necessary to evaluate the performance of these algorithms to determine which are the best options (especially with regard to the supermatrix approach that is widely used). In this study, seven of the most commonly used supertree methods are investigated by using a large empirical data set (in terms of number of taxa and molecular markers) from the worldwide flowering plant family Sapindaceae. Supertree methods were evaluated using several criteria: similarity of the supertrees with the input trees, similarity between the supertrees and the total evidence tree, level of resolution of the supertree and computational time required by the algorithm. Additional analyses were also conducted on a reduced data set to test if the performance levels were affected by the heuristic searches rather than the algorithms themselves. Based on our results, two main groups of supertree methods were identified: on one hand, the matrix representation with parsimony (MRP), MinFlip, and MinCut methods performed well according to our criteria, whereas the average consensus, split fit, and most similar supertree methods showed a poorer performance or at least did not behave the same way as the total evidence tree. Results for the super distance matrix, that is, the most recent approach tested here, were promising with at least one derived method performing as well as MRP, MinFlip, and MinCut. The output of each method was only slightly improved when applied to the reduced data set, suggesting a correct behavior of the heuristic searches and a relatively low sensitivity of the algorithms to data set sizes and missing data. Results also showed that the MRP analyses could reach a high level of quality even when using a simple heuristic search strategy, with the exception of MRP with Purvis coding scheme and reversible parsimony. The future of supertrees lies in the implementation of a standardized heuristic search for all methods and the increase in computing power to handle large data sets. The latter would prove to be particularly useful for promising approaches such as the maximum quartet fit method that yet requires substantial computing power.

Plastid and nuclear DNA markers reveal intricate relationships at subfamilial and tribal levels in the soapberry family (Sapindaceae).

The economically important soapberry family (Sapindaceae) comprises about 1900 species mainly found in the tropical regions of the world, with only a few genera being restricted to temperate areas. The infrafamilial classification of the Sapindaceae and its relationships to the closely related Aceraceae and Hippocastanaceae - which have now been included in an expanded definition of Sapindaceae (i.e., subfamily Hippocastanoideae) - have been debated for decades. Here we present a phylogenetic analysis of Sapindaceae based on eight DNA sequence regions from the plastid and nuclear genomes and including 85 of the 141 genera defined within the family. Our study comprises 997 new sequences of Sapindaceae from 152 specimens. Despite presenting 18.6% of missing data our complete data set produced a topology fully congruent with the one obtained from a subset without missing data, but including fewer markers. The use of additional information therefore led to a consistent result in the relative position of clades and allowed the definition of a new phylogenetic hypothesis. Our results confirm a high level of paraphyly and polyphyly at the subfamilial and tribal levels and even contest the monophyletic status of several genera. Our study confirms that the Chinese monotypic genus Xanthoceras is sister to the rest of the family, in which subfamily Hippocastanoideae is sister to a clade comprising subfamilies Dodonaeoideae and Sapindoideae. On the basis of the strong support demonstrated in Sapindoideae, Dodonaeoideae and Hippocastanoideae as well as in 14 subclades, we propose and discuss informal groupings as basis for a new classification of Sapindaceae.

Comparative study of the evolution of nuclear ribosomal spacers incorporating secondary structure analyzes within Dodonaeoideae, Hippocastanoideae and Xanthoceroideae (Sapindaceae).

Ribosomal DNA internal transcribed spacers (ITS) and partial external transcribed spacers (ETSf) are popularly used to infer evolutionary hypotheses. However, there is generally little consideration given to the secondary structures of these small RNA molecules and their potential effects on sequence alignment and phylogenetic analyzes. Intergeneric relationships amongst three of the four major lineages in the Sapindaceae, the Dodonaeoideae, Hippcastanoideae and Xanthoceroideae were assessed by firstly, generating secondary structure predictions for ITS and partial ETSf sequences, and then these predictions were used to assist alignment of the sequences. Secondly, the alignment was analyzed using RNA specific models of sequence evolution that account for the variation in nucleotide evolution in the independent loops and covariating stems regions of the ribosomal spacers. These models and phylogeny drawn from these analyzes were compared with that from analyzes using 'traditional' 4-state models and previous plastid analyzes. These analyzes identified that paired-site models developed to deal specifically with stem structures in RNA encoding sequences more appropriately account for the evolutionary history of the sequences than traditional 4-state substitution models.

Occurrence of physical dormancy in seeds of Australian Sapindaceae: a survey of 14 species in nine genera.

BACKGROUND AND AIMS: Sapindaceae is one of 16 angiosperm families whose seeds have physical dormancy (PY). However, the extent and nature of PY within this family is poorly known. The primary aims of this study were: (1) to evaluate seed characteristics and determine presence (or not) of PY within nine genera of Australian Sapindaceae; and (2) to compare the frequency of PY across the phylogenetic tree within Australian Sapindaceae. METHODS: Viability, imbibition and seed characteristics were assessed for 14 taxa from nine genera of Sapindaceae. For five species of Dodonaea, optimal conditions for germination and dormancy break were evaluated. An in situ burial experiment was performed on D. hackettiana seeds to identify the factor(s) responsible for overcoming PY. Classes of dormancy and of non-dormancy for 26 genera of Sapindaceae were mapped onto a phylogenetic tree for the family. KEY RESULTS: Mean seed viability across all taxa was 69.7 %. Embryos were fully developed and folded (seven genera) or bent (two genera); no endosperm was present. Seeds of all five Dodonaea spp. and of Distichostemon hispidulus had PY. Hot-water treatment released PY in these six species. Optimal germination temperature for seeds of the four Dodonaea spp. that germinated was 15-20 degrees C. Following 5 months burial in soil, 36.4 % of D. hackettiana seeds had lost PY and germinated by the beginning of the winter wet season (May). Laboratory and field data indicate that dormancy was broken by warm, moist temperatures (> or =50 degrees C) during summer. CONCLUSIONS: PY occurs infrequently in genera of Sapindaceae native to Australia. Seeds of Dodonaea and Distichostemon had PY, whereas those of the other seven genera did not. Seeds of these two genera and of Diplopeltis (a previous study) are the only three of the 20 native Australian genera of Sapindaceae for which germination has been studied that have PY; all three belong to subfamily Dodonaeoideae.