Reciprocal natural hybridization between Lycoris aurea and Lycoris radiata (Amaryllidaceae) identified by morphological, karyotypic and chloroplast genomic data.

BACKGROUND: Hybridization is considered as an important model of speciation, but the evolutionary process of natural hybridization is still poorly characterized in Lycoris. To reveal the phylogenetic relationship of two new putative natural hybrids in Lycoris, morphological, karyotypic and chloroplast genomic data of four Lycoris species were analyzed in this study. RESULTS: Two putative natural hybrids (2n = 18 = 4 m + 5t + 6st + 3 T) possessed obvious heterozygosity features of L. radiata (2n = 22 = 10t + 12st) and L. aurea (2n = 14 = 8 m + 6 T) in morphology (e.g. leaf shape and flower color), karyotype (e.g. chromosome numbers, CPD/DAPI bands, 45S rDNA-FISH signals etc.) and chloroplast genomes. Among four Lycoris species, the composition and structure features of chloroplast genomes between L. radiata and the putative natural hybrid 1 (L. hunanensis), while L. aurea and the hybrid 2, were completely the same or highly similar, respectively. However, the features of the cp genomes between L. radiata and the hybrid 2, while L. aurea and the hybrid 1, including IR-LSC/SSC boundaries, SSRs, SNPs, and SNVs etc., were significantly different, respectively. Combining the karyotypes and cp genomes analysis, we affirmed that the natural hybrid 1 originated from the natural hybridization of L. radiata (♀) × L. aurea (♂), while the natural hybrid 2 from the hybridization of L. radiata (♂) × L. aurea (♀). CONCLUSION: The strong evidences for natural hybridization between L. radiata (2n = 22) and L. aurea (2n = 14) were found based on morphological, karyotypic and chloroplast genomic data. Their reciprocal hybridization gave rise to two new taxa (2n = 18) of Lycoris. This study revealed the origin of two new species of Lycoris and strongly supported the role of natural hybridization that facilitated lineage diversification in this genus.

Coordination polymer nanoprobe integrated carbon dot and phenol red for turn-on fluorescence detection of urease activity.

The potential of coordination polymers (CPs) as a host of integrating multiple guest species to construct a fluorescence resonance energy transfer (FRET) nanoprobe was demonstrated. The ZnCPs built from zinc(II) and adenine was employed as a model of CPs to integrate carbon dot (CD) and phenol red (PR) for producing the FRET nanoprobe (CD/PR@ZnCPs). Benefiting from the confinement effect of ZnCPs, the integrated CD and PR can be brought in close proximity to favor the occurrence of FRET process from CD to PR, which leads to the quenching of CD fluorescence. However, the FRET process was disrupted upon the red-shift of PR absorption from 428 to 562 nm in alkaline medium, and consequently switches on the fluorescence of CD/PR@ZnCPs. Based on this finding, by utilizing urease to hydrolyze urea and mediate medium pH, a turn-on fluorescent method was established for the detection of urease activity. This fluorescent method has a linear response that covers 5 to 150 U/L urease with a detection limit of 0.74 U/L and exhibits an excellent selectivity over other enzymes. The successful determination of urease in saliva samples demonstrates the applicability of the fluorescent nanoprobe in complex biological matrix.

Development of microsatellite markers in Parakmeria nitida (Magnoliaceae).

PREMISE OF THE STUDY: We developed microsatellite makers for Parakmeria nitida to investigate its population structure and conservation genetics. METHODS AND RESULTS: A total of 25 microsatellite primer pairs were developed using the Fast Isolation by AFLP of Sequences COntaining repeats (FIASCO) protocol, and polymorphism was assessed in three natural populations of P. nitida. Among these markers, 11 were monomorphic and 14 showed polymorphism. CONCLUSIONS: These markers are potentially useful for future population genetic analyses of P. nitida and will serve as an important tool for conservation efforts.

Isolation and characterization of microsatellite markers from Clematoclethra scandens (Actinidiaceae).

PREMISE OF THE STUDY: Microsatellite makers were developed for Clematoclethra scandens to investigate its population genetics and speciation. METHODS AND RESULTS: A total of 36 microsatellite markers were isolated using the Fast Isolation by AFLP of Sequences COntaining repeats (FIASCO) method. Their polymorphisms were assessed in two natural populations. The results showed that 30 markers displayed prominent polymorphisms and six markers were monomorphic. CONCLUSIONS: These microsatellite loci will facilitate further studies on population genetics and speciation of C. scandens.

High nrDNA ITS polymorphism in the ancient extant seed plant Cycas: incomplete concerted evolution and the origin of pseudogenes.

Molecular studies of six species from the ancient extant seed plant Cycas, covering a wide range of its morphological diversity and all major areas of distribution, revealed a high level of intra-individual polymorphism of the internal transcribed spacer (ITS1, 5.8S, and ITS2) region, indicative of incomplete nrDNA concerted evolution. Through a range of comparisons of sequence characteristics to functional cDNA ITS copies, including sequence length and substitution variation, GC content, secondary structure stability, the presence of a conserved motif in the 5.8S gene, and evolutionary rates, the PCR amplified divergent genomic DNA ITS paralogs were identified as either putative pseudogenes, recombinants or functional paralogs. This incomplete ITS concerted evolution may be linked to the high number of nucleolar organizer regions in the Cycas genome, and the incomplete lineage sorting due to recent species divergence in the genus. Based on the distribution of a 14 bp deletion, an early evolutionary origin of the pseudogenes is indicated, possibly predating the diversification of Cycas. Due to their early origin combined with the unconstraint evolution of the ITS region in pseudogenes, they accumulate high levels of homoplastic mutations. This leads to random relationships among the pseudogenes due to long-branch attractions, whereas the phylogenetic relationships inferred from the functional ITS paralogs grouped the sequences in species specific clades (except for C. circinalis and C. rumphii). The findings of our extensive study will have a wide significance, for the evolution of these molecular sequences, and their utilization as a major marker for reconstructing phylogenies.

Molecular cytogenetic characterization and phylogenetic analysis of four Miscanthus species (Poaceae).

Chromosomes of four Miscanthus (Andersson, 1855) species including M. sinensis (Andersson, 1855), M. floridulus (Schumann & Lauterb, 1901), M. sacchariflorus (Hackel, 1882) and M. lutarioriparius (Chen & Renvoize, 2005) were analyzed using sequentially combined PI and DAPI (CPD) staining and fluorescence in situ hybridization (FISH) with 45S rDNA probe. To elucidate the phylogenetic relationship among the four Miscanthus species, the homology of repetitive sequences among the four species was analyzed by comparative genomic in situ hybridization (cGISH). Subsequently four Miscanthus species were clustered based on the internal transcribed spacer (ITS) of 45S rDNA. Molecular cytogenetic karyotypes of the four Miscanthus species were established for the first time using chromosome measurements, fluorochrome bands and 45S rDNA FISH signals, which will provide a cytogenetic tool for the identification of these four species. All the four have the karyotype formula of Miscanthus species, which is 2n = 2x = 38 = 34m(2SAT) + 4sm, and one pair of 45S rDNA sites. The latter were shown as strong red bands by CPD staining. A non-rDNA CPD band emerged in M. floridulus and some blue DAPI bands appeared in M. sinensis and M. floridulus. The hybridization signals of M. floridulus genomic DNA to the chromosomes of M. sinensis and M. lutarioriparius genomic DNA to the chromosomes of M. sacchariflorus were stronger and more evenly distributed than other combinations. Molecular phylogenetic trees showed that M. sinensis and M. floridulus were closest relatives, and M. sacchariflorus and M. lutarioriparius were also closely related. These findings were consistent with the phylogenetic relationships inferred from the cGISH patterns.

Nuclear ribosomal ITS functional paralogs resolve the phylogenetic relationships of a late-Miocene radiation cycad Cycas (Cycadaceae).

Cycas is the most widespread and diverse genus among the ancient cycads, but the extant species could be the product of late Miocene rapid radiations. Taxonomic treatments to date for this genus are quite controversial, which makes it difficult to elucidate its evolutionary history. We cloned 161 genomic ITS sequences from 31 species representing all sections of Cycas. The divergent ITS paralogs were examined within each species and identified as putative pseudogenes, recombinants and functional paralogs. Functional paralogs were used to reconstruct phylogenetic relationships with pseudogene sequences as molecular outgroups, since an unambiguous ITS sequence alignment with their closest relatives, the Zamiaceae, is unachievable. A fully resolved and highly supported tree topology was obtained at the section level, with two major clades including six minor clades. The results fully supported the classification scheme proposed by Hill (2004) at the section level, with the minor clades representing his six sections. The two major clades could be recognised as two subgenera. The obtained pattern of phylogenetic relationships, combined with the different seed dispersal capabilities and paleogeography, allowed us to propose a late Miocene rapid radiation of Cycas that might have been promoted by vicariant events associated with the complex topography and orogeny of South China and adjacent regions. In contrast, transoceanic dispersals might have played an important role in the rapid diversification of sect. Cycas, whose members have evolved a spongy layer in their seeds aiding water dispersals.

Genetic differentiation and relationships of populations in the Cycas balansae complex (Cycadaceae) and its conservation implications.

BACKGROUND AND AIMS: The Cycas balansae complex is arguably a controversial group with regard to species delineation. Some taxonomists recognize a single polymorphic species while others distinguish five narrowly defined ones. The unresolved taxonomy has the potential to bring about significant problems for species conservation. Thus, an investigation to examine the genetic diversity and differentiation in the C. balansae complex was performed to determine the relationship of populations and to test whether the morphologically defined segregations represent genetically distinct units. METHODS: Inter-simple sequence repeat (ISSR) markers were employed to assess the genetic diversity in the C. balansae complex with a sample of 158 individuals from all extant populations in China. KEY RESULTS: ISSR markers revealed low genetic diversity in all populations studied (H(E) and H(O) averaged 0.0639 and 0.0798 at the population level, respectively). Phenetic analysis showed that the C. balansae complex grouped into five clusters closely corresponding to the narrowly defined C. balansae, C. parvula, C. shiwandashanica, C. tanqingii and C. simplicipinna. CONCLUSIONS: ISSR data suggest that the C. balansae complex has evolved into five genetically distinct units. These might be derived from a relatively widespread common ancestor through multiple vicariant events including geographical isolation resulting from the collision of the Indian plate with the Eurasian plate and from Pleistocene glaciations. In conservation, attention should be paid to each genetic unit.

ISSR variation in the endemic and endangered plant Cycas guizhouensis (Cycadaceae).

BACKGROUND AND AIMS: Cycas guizhouensis (Cycadaceae) is a rare and endangered species endemic to the southwest of China. An investigation was undertaken into the genetic variation of wild populations. METHODS: ISSR markers were used to determine the genetic variation within and between 12 extant populations of this species. KEY RESULTS: Low genetic diversity (at population level, P = 14.21 %, H(E) = 0.0597; at species level, P = 35.90 %, H(T) = 0.1082) and a high degree of differentiation among populations (G(ST) = 0.4321) were detected. CONCLUSIONS: This genetic structure is considered to be due to the combined effects of slow biochemical evolution, genetic drift, inbreeding and limited gene flow between populations. Based on these findings, strategies are proposed for the genetic conservation and management of the species.