Phylogeny of Alisma (Alismataceae) revisited: implications for polyploid evolution and species delimitation.

Alisma L. is a genus of aquatic and wetland plants belonging to family Alismataceae. At present, it is thought to contain ten species. Variation in ploidy level is known in the genus, with diploids, tetraploids and hexaploids recorded. Previous molecular phylogenetic studies of Alisma have generated a robust backbone that reveals important aspects of the evolutionary history of this cosmopolitan genus, yet questions remain unresolved about the formation of the polyploid taxa and the taxonomy of one particularly challenging, widely distributed species complex. Here we directly sequenced, or cloned and sequenced, nuclear DNA (nrITS and phyA) and chloroplast DNA (matK, ndhF, psbA-trnH and rbcL) of multiple samples of six putative species and two varieties, and conducted molecular phylogenetic analyses. Alisma canaliculatum and its two varieties known in East Asia and A. rariflorum endemic to Japan possess closely related but heterogeneous genomes, strongly indicating that the two species were generated from two diploid progenitors, and are possibly siblings of one another. This evolutionary event may have occurred in Japan. Alisma canaliculatum var. canaliculatum is segregated into two types, each of which are geographically slightly differentiated in Japan. We reconstructed a single phylogeny based on the multi-locus data using Homologizer and then applied species delimitation analysis (STACEY). This allowed us to discern A. orientale as apparently endemic to the Southeast Asian Massif and distinct from the widespread A. plantago-aquatica. The former species was most likely formed through parapatric speciation at the southern edge of the distribution of the latter.

The complete chloroplast genomes of two species in threatened monocot genus Caldesia in China.

Caldesia is a genus in the family Alismataceae mainly found in the tropical and temperate regions of the Northern hemisphere. In China, two species, Caldesia parnassifolia, and Caldesia grandis are recorded as critically endangered in sporadic regions. Available protection of the genetic resource of these threatened species has been impeded due to limited genomic information. Here, we sequence the whole chloroplast (cp) genome of the two Caldesia species using high throughput sequencing technology. The whole cp genomes of C. parnassifolia and C. grandis were 167,647 bp and 168,500 bp, respectively with a typical quadripartite structure. There were 115 unique genes with 81 protein-coding genes, 31 tRNA genes, and four rRNA genes. Both species showed a GC content of 37.1%. A duplication of two tRNA genes and a ~ 6 kb inversion region in the LSC was noted in both species. Mononucleotide simple sequence repeats (SSRs) A/T were most abundant for both Caldesia species. High nucleotide variability was recorded in ycf1 gene and trnK-UUU/rps16 intergenic spacer region. All RNA editing conversions were C-U in 23 and 24 protein-coding genes for C. parnassifolia and C. grandis, respectively. Phylogenetic analysis placed both Caldesia species as sister to Sagittaria lichuanensis. This study will be useful for further evolutionary, systematic researches and conservation of the genus Caldesia.

Generic phylogeny and historical biogeography of Alismataceae, inferred from multiple DNA sequences.

Alismataceae is an aquatic or semi-aquatic herb family with a subcosmopolitan distribution. The family is one of the oldest lineages within monocots and plays an important role in the systematics, biogeography and evolutionary processes of flowering plants. However, the generic relationships of the family are still a subject of debate, and its historical biogeography is less studied. In the present study, we carried out a comprehensive phylogenetic analysis based on multiple DNA sequences (nuclear: ITS; chloroplast: psbA, rbcL, matK, rpoB, rpoC1, trnK 5' intron and trnK 3' intron; mitochondria: cob and atp1). The result supports merging Limnocharitaceae into Alismataceae as one family. Two well-supported clades were obtained based on the combined ITS, psbA, rbcL and matK dataset. Clade B consists of Luronium, Damasonium, Baldellia and Alisma; and clade A consists of the remaining genera of Alismataceae as well as Limnocharitaceae. Biogeographic analysis and bayesian molecular dating suggested that Alismataceae originated in West Palearctic or Afrotropical area during the Late Cretaceous, and subsequently split into two clades. Clade A and clade B diversified in Afrotropical area and West Palearctic area, respectively. The intercontinental distribution of this family mainly resulted from dispersals involving migration across land bridges and long-distance dispersal.

Fine-scale spatial genetic structure of an endangered marsh herb, Caldesia grandis (Alismataceae).

The endangered marsh herb, Caldesia grandis, is native to China. We investigated the spatial structure of the genetic variation of three populations of C. grandis using RAPD markers and spatial autocorrelation analysis, based on the method of equal distance interval. A total of 157 individuals were sampled from four patches collected from the region of Hunan and Yunnan Provinces, China. Among the polymorphic bands generated by seven selective primers, polymorphic bands with frequencies ranging from 20 to 80% were used to calculate Moran's I spatial autocorrelation coefficient for each patch. We found significant spatial structure of genetic variation in the three patches in Bei Hai (BH) (patches BH-1 and BH-2) and Guai Hu (GH) (patch GH-1) populations of C. grandis (with significant positive autocorrelation within the short distance class). In contrast, the genetic variation in the Lang Pan Hu (LPH) population (patch LPH-1) was found to be randomly distributed. The different spatial distribution patterns may be attributed to environment differences. These results have implications for the conservation and management of this species, especially for sampling strategies for ex situ conservation.

Phylogenetics and phylogeography of the monocot genus Baldellia (Alismataceae): Mediterranean refugia, suture zones and implications for conservation.

Aquatic plants, and especially the emblematic genus Baldellia (Alismataceae), are among the most threatened organisms, due to unprecedented human-driven habitat destructions. Therefore protection plans are crucially needed and call for thoroughly documenting the genetic diversity and clarifying the taxonomy of this endangered genus. Our sampling included 282 individuals from 42 natural populations and covered the whole geographical range of the genus, across Europe and the Mediterranean. We combined sequencing of nuclear internal transcribed spacer (ITS) and chloroplastic trnL-ndhF regions with amplified fragment length polymorphism (AFLP) genotyping to investigate the Alismataceae phylogeny, and produce a phylogeography of Baldellia. Our phylogeny strongly supported the monophyly of Baldellia and placed it as the sister clade to Luronium and Alisma, therefore excluding, as previously supposed, a close genetic relatedness to the predominantly neotropical genus Echinodorus. The phylogeography of Baldellia outlined patterns consistent with a hypothesis considering glacial refugia located in the Iberian Peninsula and the Italy/Balkan region from which two distinct genetic lineages re-colonized Europe. These two lineages corresponded respectively to Baldellia ranunculoides (Italy/Balkan derived populations) and Baldellia repens (populations recovered from the Iberian Peninsula refuge), therefore supporting differences outlined between the two taxa in previous ecological and morphological studies. These results allowed clarifying taxonomic uncertainties by confirming the genetic distinctness of B. repens according to B. ranunculoides. A third lineage, Baldellia alpestris, originated and remained endemic to the mountainous regions of the Iberian Peninsula. Unexpectedly, B. repens populations collected in northern Africa, appeared to be genetically distinct from their European counterparts, this calls for further investigation to fully address their genetic and conservation status. Finally, we detected a large hybridization zone in northwestern Europe between B. repens and B. ranunculoides. These results were discussed in light of conservation approaches for Baldellia populations.

Genotoxicity and mutagenicity of Echinodorus macrophyllus (chapéu-de-couro) extracts

Echinodorus macrophyllus, commonly known as chapéu-de-couro, is a medicinal plant used in folk medicine to treat inflammation and rheumatic diseases. In this work, we used short-term bacterial assays based on the induction of SOS functions to examine the genotoxicity and mutagenicity of an aqueous extract of E. macrophyllus leaves. Whole extract and an ethyl acetate fraction showed similar genotoxicity and caused an ~70-fold increase in lysogenic induction. The extract also gave a positive result in the SOS chromotest with an increase of 12-fold in beta-Galactosidase enzymatic units. There was a strong trend towards base substitutions and frameshifts at purine sites in the mutations induced by the extract in Escherichia coli (CC103 and CC104 strains) and Salmonella typhimurium test strains (22-fold increase in histidine revertants in TA98 strain). Since reactive oxygen species may be implicated in aging process and in degenerative diseases, we used antioxidant compounds as catalase, thiourea and dipyridyl in the lysogenic induction test. All this compounds were able to reduce the induction factor observed in the treatment with chapéu-de-couro, thus suggesting that the genotoxicity and mutagenicity were attributable to the production of reactive oxygen species that targeted DNA purines.