Comparative analysis of floral transition and floral organ formation in two contrasting species: Disocactus speciosus and D. eichlamii (Cactaceae).

KEY MESSAGE: Contrasting morphologies in Disocactus are the result of differential development of the vegetative and floral tissue where intercalary growth is involved, resulting in a complex structure, the floral axis. Species from the Cactaceae bear adaptations related with their growth in environments under hydric stress. These adaptations have translated into the reduction and modification of various structures such as leaves, stems, lateral branches, roots and the structuring of flowers in a so-called flower-shoot. While cacti flowers and fruits have a consistent structure with showy hermaphrodite or unisexual flowers that produce a fruit called cactidium, the developmental dynamics of vegetative and reproductive tissues comprising the reproductive unit have only been inferred through the analysis of pre-anthetic buds. Here we present a comparative analysis of two developmental series covering the early stages of flower formation and organ differentiation in Disocactus speciosus and Disocactus eichlamii, which have contrasting floral morphologies. We observe that within the areole, a shoot apical meristem commences to grow upward, producing lateral leaves with a spiral arrangement, rapidly transitioning to a floral meristem. The floral meristem produces tepal primordia and a staminal ring meristem from which numerous or few stamens develop in a centrifugal manner in D. speciosus and D. eichlamii, respectively. Also, the inferior ovary derives from the floral meristem flattening and an upward growth of the surrounding tissue of the underlying stem, producing the pericarpel. This structure is novel to cacti and lacks a clear anatomical delimitation with the carpel wall. Here, we present a first study that documents the early processes taking place during initial meristem determination related to pericarpel development and early floral organ formation in cacti until the establishment of mature floral organs.

Phylogenetic discordance and integrative species delimitation in the Mammillaria haageana species complex (Cactaceae).

Species complexes consist of very close phylogenetic relatives, where morphological similarities make it difficult to distinguish between them using traditional taxonomic methods. Here, we focused on the long-standing challenge of species delimitation in the Mammillaria haageana complex, a group that presents great morphological diversity that makes its taxonomy a puzzle. Our work integrates genomic, morphological, and ecological data to establish the taxonomic limits in the M. haageana complex, and we also studied the evolutionary relationships with the remainder of the M. ser. Supertextae species. Our genetic analyses, as well as morphological and ecological evidence, led us to propose that the M. haageana complex is made up of six distinct entities (M. acultzingensis, M. conspicua, M. haageana, M. lanigera, M. meissneri, and M. san-angelensis), mainly as a result of ecological speciation. A recent taxonomic proposal considered these taxa as a single species; therefore, we propose their recognition at the species level. Our results also show a high level of incomplete lineage sorting rather than reticulation, which is especially likely in recently diverged species such as those comprising M. ser. Supertextae. The species hypotheses proposed here may be useful in future extinction risk assessments and conservation strategies.

Phylogenomics and Biogeography of the Mammilloid Clade Revealed an Intricate Evolutionary History Arose in the Mexican Plateau.

Mexico harbors ~45% of world's cacti species richness. Their biogeography and phylogenomics were integrated to elucidate the evolutionary history of the genera Coryphantha, Escobaria, Mammillaria, Mammilloydia, Neolloydia, Ortegocactus, and Pelecyphora (Mammilloid Clade). We analyzed 52 orthologous loci from 142 complete genomes of chloroplast (103 taxa) to generate a cladogram and a chronogram; in the latter, the ancestral distribution was reconstructed with the Dispersal-Extinction-Cladogenesis model. The ancestor of these genera arose ~7 Mya on the Mexican Plateau, from which nine evolutionary lineages evolved. This region was the site of 52% of all the biogeographical processes. The lineages 2, 3 and 6 were responsible for the colonization of the arid southern territories. In the last 4 Mya, the Baja California Peninsula has been a region of prolific evolution, particularly for lineages 8 and 9. Dispersal was the most frequent process and vicariance had relevance in the isolation of cacti distributed in the south of Mexico. The 70 taxa sampled as Mammillaria were distributed in six distinct lineages; one of these presumably corresponded to this genus, which likely had its center of origin in the southern part of the Mexican Plateau. We recommend detailed studies to further determine the taxonomic circumscription of the seven genera.

Targeted Nutritional Supplementation for Telogen Effluvium: Multicenter Study on Efficacy of a Hydrolyzed Collagen, Vitamin-, and Mineral-Based Induction and Maintenance Treatment.

Background: The condition of the hair is closely related to the nutritional state. Normal supply, uptake, and transport of nutrients are of fundamental importance in tissues with a high biosynthetic activity such as the hair follicle. Objective: The objective of the study was to evaluate the efficacy of a nutritional-based induction and maintenance treatment for telogen effluvium formulated with a combination of hydrolyzed collagen, amino acids, vitamins, and minerals. Patients and Methods: The clinical studies were conducted with each nutritional treatment individually, and both in sequential combination. Anagen/telogen ratio, hair density, and tolerability of treatment were assessed at baseline, 4 weeks of induction therapy, and another 12 weeks of maintenance treatment. Trichogram results showed a significant improvement of the anagen/telogen ratio between baseline and final visit at 16 weeks, with an increase of hair in anagen and a reduction of hair in telogen. Furthermore, a significant increase was observed in hair density. The effect size of the combination treatment was higher than that of each of the two products used separately as monotherapy. Conclusions: The study results provide a proof of concept for targeted nutritional supplementation for the treatment of telogen effluvium, with a special emphasis on the role of collagen, besides specific amino acids, vitamins, and minerals.

Phylogenetic and morphological analyses of Pilosocereus leucocephalus group s.s. (Cactaceae) reveal new taxonomical implications.

Pilosocereus is one of the Cactaceae family's most relevant genera in terms of the number of species and its wide geographical range in the Americas. Within Pilosocereus, five informal taxonomic groups have been recognized, one of which is P. leucocephalus group s.s., whose phylogenetic relationships remain unresolved. Therefore, our objectives are to recognize the circumscriptions of the species in P. leucocephalus group s.s. and to corroborate the monophyly and phylogenetic relationships of this group through a set of morphological and molecular characters. This study is based on representative sampling along the broad distribution of this group in Mexico and Central America using multivariate and phylogenetic analyses. The morphological characters identified to contribute to species recognition and group formation are branch diameter, areole length, the areole length-width ratio, the distance between areoles, the length of the longest radial spine, and branch and spines colors. The chloroplast markers rpl16, trnL-trnF, and petL-psbE and the nuclear marker AT1G18270 support the monophyly of the P. leucocephalus group s.s., and two probable synapomorphies are suggested, including one transversion in rpl16 and another in petL-psbE. Together, our results demonstrate that sampled species of P. leucocephalus group s.s. encompass six species distributed in Mexico and Central America: P. alensis and P. purpusii in the western region, P. chrysacanthus and P. collinsii in the central region, and P. gaumeri and P. leucocephalus in the eastern region. A taxonomic key to recognized species is provided.

Phylogenetic relationships in Coryphantha and implications on Pelecyphora and Escobaria (Cacteae, Cactoideae, Cactaceae).

The genus Coryphantha includes plants with globose to cylindrical stems bearing furrowed tubercles, flowers arising at the apex, and seeds with flattened testa cells. Coryphantha is the second richest genus in the tribe Cacteae. Nevertheless, the genus lacks a phylogenetic framework. The limits of Coryphantha with its sister genus Escobaria and the infrageneric classification of Coryphantha have not been evaluated in a phylogenetic study. In this study we analyzed five chloroplast regions (matK, rbcL, psbA-trnH, rpl16, and trnL-F) using Bayesian phylogenetic analysis. We included 44 species of Coryphantha and 43 additional species of the tribe Cacteae. Our results support the monophyly of Coryphantha by excluding C.macromeris. Escobaria + Pelecyphora + C.macromeris are corroborated as the sister group of Coryphantha. Within Coryphantha our phylogenetic analyses recovered two main clades containing seven subclades, and we propose to recognize those as two subgenera and seven sections, respectively. Also, a new delimitation of Pelecyphora including C.macromeris and all species previously included in Escobaria is proposed. To accommodate this new delimitation 25 new combinations are proposed. The seven subclades recovered within Coryphantha are morphologically and geographically congruent, and partially agree with the traditional classification of this genus.

Evolution of flower allometry and pigmentation in Mammillaria haageana (Cactaceae).

BACKGROUND: A puzzle in evolution is the understanding of how the environment might drive subtle phenotypic variation, and whether this variation is adaptive. Under the neutral evolutionary theory, subtle phenotypes are almost neutral with little adaptive value. To test this idea, we studied the infraspecific variation in flower shape and color in Mammillaria haageana, a species with a wide geographical distribution and phenotypic variation, which populations are often recognized as infraspecific taxa. RESULTS: We collected samples from wild populations, kept them in the greenhouse for at least one reproductive year, and collected newly formed flowers. Our first objective was to characterize tepal natural variation in M. haageana through geometric morphometric and multivariate pigmentation analyses. We used landmark-based morphometrics to quantify the trends of shape variation and tepal color-patterns in 20 M. haageana accessions, belonging to five subspecies, plus 8 M. albilanata accessions for comparison as the sister species. We obtained eight geometric morphometric traits for tepal shape and color-patterns. We found broad variation in these traits between accessions belonging to the same subspecies, without taxonomic congruence with those infraspecific units. Also the phenetic cluster analysis showed different grouping patterns among accessions. When we correlated these phenotypes to the environment, we also found that solar radiation might explain the variation in tepal shape and color, suggesting that subtle variation in flower phenotypes might be adaptive. Finally we present anatomical sections in M. haageana subsp. san-angelensis to propose some of the underlying tepal structural features that may give rise to tepal variation. CONCLUSIONS: Our geometric morphometric approach of flower shape and color allowed us to identify the main trends of variation in each accession and putative subspecies, but also allowed us to correlate these variation to the environment, and propose anatomical mechanisms underlying this diversity of flower phenotypes.

Growth Patterns in Seedling Roots of the Pincushion Cactus Mammillaria Reveal Trends of Intra- and Inter-Specific Variation.

Genetic mechanisms controlling root development are well-understood in plant model species, and emerging frontier research is currently dissecting how some of these mechanisms control root development in cacti. Here we show the patterns of root architecture development in a gradient of divergent lineages, from populations to species in Mammillaria. First, we show the patterns of variation in natural variants of the species Mammillaria haageana. Then we compare this variation to closely related species within the Series Supertexta in Mammillaria (diverging for the last 2.1 million years) in which M. haageana is inserted. Finally, we compared these patterns of variation to what is found in a set of Mammillaria species belonging to different Series (diverging for the last 8 million years). When plants were grown in controlled environments, we found that the variation in root architecture observed at the intra-specific level, partially recapitulates the variation observed at the inter-specific level. These phenotypic outcomes at different evolutionary time-scales can be interpreted as macroevolution being the cumulative outcome of microevolutionary phenotypic divergence, such as the one observed in Mammillaria accessions and species.

Blurring the Boundaries between a Branch and a Flower: Potential Developmental Venues in CACTACEAE.

Flowers are defined as short shoots that carry reproductive organs. In Cactaceae, this term acquires another meaning, since the flower is interpreted as a branch with a perianth at the tip, with all reproductive organs embedded within the branch, thus giving way to a structure that has been called a "flower shoot". These organs have long attracted the attention of botanists and cactologists; however, the understanding of the morphogenetic processes during the development of these structures is far from clear. In this review, we present and discuss some classic flower concepts used to define floral structures in Cactaceae in the context of current advances in flower developmental genetics and evolution. Finally, we propose several hypotheses to explain the origin of these floral shoot structures in cacti, and we suggest future research approaches and methods that could be used to fill the gaps in our knowledge regarding the ontogenetic origin of the "flower" in the cactus family.

Evaluating the monophyly of Mammillaria series Supertextae (Cactaceae).

Mammillaria (Cactaceae) taxonomy has been historically problematic due to the morphological variability and sympatry of the species. This has led to several proposals for infrageneric classification, including subgeneric, section and series categories. Mammillaria ser. Supertextae is one of 15 series and is made up of a variable set of species that are mainly distributed in southern Mexico and Central America. However, the phylogenetic relationships within M. ser. Supertextae and its relationship to other Mammillaria taxa are far from fully understood. Here we attempt to elucidate these relationships using complete terminal sampling and newly obtained chloroplast marker sequences and comparing them to Mammillaria species sequences from GenBank. Our phylogenetic analyses showed that M. ser. Supertextae comprises a well-supported monophyletic group that diverged approximately 2.1 Mya and has M. ser. Polyacanthae as its sister group; however, relationships within M. ser. Supertextae remain unresolved. The topology obtained within M. ser. Supertextae must also be interpreted under the distribution shared by these taxa, but it is difficult to differentiate ancestral polymorphisms from possible introgression, given the short time elapsed and the markers used. Our results show that the infrageneric units of M. haageana and M. albilanata can be considered independent evolutionary units. We also suggest that the relationship between M. haageana and M. albilanata is convoluted because their distribution overlaps (mainly towards southern Mexico), with genetic differences that possibly indicate they represent more than two taxonomic entities. One possible explanation is that there could still be gene flow between these taxa, and we might be witnessing an ongoing speciation process.

The importance of environmental conditions in maintaining lineage identity in Epithelantha (Cactaceae).

The use of environmental variables to explain the evolution of lineages has gained relevance in recent studies. Additionally, it has allowed the recognition of species by adding more characters to morphological and molecular information. This study focuses on identifying environmental and landscape variables that have acted as barriers that could have influenced the evolution of Epithelantha species and its close genera.Our results show that soil pH, isothermality, temperature seasonality, and annual precipitation have a significant phylogenetic signal for Epithelantha. Soil type and landforms are also relevant as ecological barriers that maintain the identity of Epithelantha species.The variables associated with the soil (pH) have influenced the evolution of Epithelantha and probably in other genera of Cactaceae. Additionally, Epithelantha is frequent in the piedmont and haplic kastanozems. Bioclimatic variables reinforce the recognition of E. micromeris, and E. cryptica as independent species. Therefore, ecology can be considered as a factor to explain the high level of endemism in Cactaceae.

Identification of High Molecular Variation Loci in Complete Chloroplast Genomes of Mammillaria (Cactaceae, Caryophyllales).

In plants, partial DNA sequences of chloroplasts have been widely used in evolutionary studies. However, the Cactaceae family (1500-1800 species) lacks molecular markers that allow a phylogenetic resolution between species and genera. In order to identify sequences with high variation levels, we compared previously reported complete chloroplast genomes of seven species of Mammillaria. We identified repeated sequences (RSs) and two types of DNA variation: short sequence repeats (SSRs) and divergent homologous loci. The species with the highest number of RSs was M. solisioides (256), whereas M. pectinifera contained the highest amount of SSRs (84). In contrast, M. zephyranthoides contained the lowest number (35) of both RSs and SSRs. In addition, five of the SSRs were found in the seven species, but only three of them showed variation. A total of 180 homologous loci were identified among the seven species. Out of these, 20 loci showed a molecular variation of 5% to 31%, and 12 had a length within the range of 150 to 1000 bp. We conclude that the high levels of variation at the reported loci represent valuable knowledge that may help to resolve phylogenetic relationships and that may potentially be convenient as molecular markers for population genetics and phylogeographic studies.

Unraveling Reticulate Evolution in Opuntia (Cactaceae) From Southern Mexico.

The process of hybridization occurs in approximately 40% of vascular plants, and this exchange of genetic material between non-conspecific individuals occurs unequally among plant lineages, being more frequent in certain groups such as Opuntia (Cactaceae). This genus is known for multiple taxonomic controversies due to widespread polyploidy and probable hybrid origin of several of its species. Southern Mexico species of this genus have been poorly studied despite their great diversity in regions such as the Tehuacán-Cuicatlán Valley which contains around 12% of recognized Mexico's native Opuntia species. In this work, we focus on testing the hybrid status of two putative hybrids from this region, Opuntia tehuacana and Opuntia pilifera, and estimate if hybridization occurs among sampled southern opuntias using two newly identified nuclear intron markers to construct phylogenetic networks with HyDe and Dsuite and perform invariant analysis under the coalescent model with HyDe and Dsuite. For the test of hybrid origin in O. tehuacana, our results could not recover hybridization as proposed in the literature, but we found introgression into O. tehuacana individuals involving O. decumbens and O. huajuapensis. Regarding O. pilifera, we identified O. decumbens as probable parental species, supported by our analysis, which sustains the previous hybridization hypothesis between Nopalea and Basilares clades. Finally, we suggest new hybridization and introgression cases among southern Mexican species involving O. tehuantepecana and O. depressa as parental species of O. velutina and O. decumbens.

The chloroplast genome of the pincushion cactus Mammilllaria haageana subsp. san-a ngelensis, a Mexican endangered species.

The genus Mammillaria occupies diverse habitats and exhibits diverse growth patterns and a large range of morphologies. Most of the species of this genus are used as ornamental plants and are subject to mass habitat loss. Due to these factors, they are being submitted to selective pressure that might affect conservational efforts and management plans. We obtained the 133 gene chloroplast genome as part of the project of sequencing the complete genome of pincushion cactus, including 88 protein-coding genes, 8 rRNA genes, and 37 tRNA genes. The phylogenetic tree indicates the pincushion cactus is a sister species of M. supertexta and M. huitzilopochtli.

De Novo Assembly Discovered Novel Structures in Genome of Plastids and Revealed Divergent Inverted Repeats in Mammillaria (Cactaceae, Caryophyllales).

The complete sequence of chloroplast genome (cpDNA) has been documented for single large columnar species of Cactaceae, lacking inverted repeats (IRs). We sequenced cpDNA for seven species of the short-globose cacti of Mammillaria and de novo assembly revealed three novel structures in land plants. These structures have a large single copy (LSC) that is 2.5 to 10 times larger than the small single copy (SSC), and two IRs that contain strong differences in length and gene composition. Structure 1 is distinguished by short IRs of <1 kb composed by rpl23-trnI-CAU-ycf2; with a total length of 110,189 bp and 113 genes. In structure 2, each IR is approximately 7.2 kb and is composed of 11 genes and one Intergenic Spacer-(psbK-trnQ)-trnQ-UUG-rps16-trnK-UUU-matK-trnK-UUU-psbA-trnH-GUG-rpl2-rpl23-trnI-CAU-ycf2; with a total size of 116,175 bp and 120 genes. Structure 3 has divergent IRs of approximately 14.1 kb, where IRA is composed of 20 genes: psbA-trnH-GUG-rpl23-trnI-CAU-ycf2-ndhB-rps7-rps12-trnV-GAC-rrn16-ycf68-trnI-GAU-trnA-AGC-rrn23-rrn4.5-rrn5-trnR-ACG-trnN-GUU-ndhF-rpl32; and IRB is identical to the IRA, but lacks rpl23. This structure has 131 genes and, by pseudogenization, it is shown to have the shortest cpDNA, of just 107,343 bp. Our findings show that Mammillaria bears an unusual structural diversity of cpDNA, which supports the elucidation of the evolutionary processes involved in cacti lineages.

Cell-based product classification procedure: What can be done differently to improve decisions on borderline products?

In June 2015, European Medicines Agency/Committee for Advanced Therapies (CAT) released the new version of the reflection paper on classification of advanced therapy medicinal products (ATMPs) established to address questions of borderline cases in which classification of a product based on genes, cells or tissues is unclear. The paper shows CAT's understanding of substantial manipulation and essential function(s) criteria that define the legal scope of cell-based medicinal products. This article aims to define the authors' viewpoint on the reflection paper. ATMP classification has intrinsic weaknesses derived from the lack of clarity of the evolving concepts of substantial manipulation and essential function(s) as stated in the EU Regulation, leading to the risk of differing interpretations and misclassification. This might result in the broadening of ATMP scope at the expense of other products such as cell/tissue transplants and blood products, or even putting some present and future clinical practice at risk of being classified as ATMP. Because of the major organizational, economic and regulatory implications of product classification, we advocate for increased interaction between CAT and competent authorities (CAs) for medicines, blood and blood components and tissues and cells or for the creation of working groups including representatives of all parties as recently suggested by several CAs.

High proportion of cactus species threatened with extinction.

A high proportion of plant species is predicted to be threatened with extinction in the near future. However, the threat status of only a small number has been evaluated compared with key animal groups, rendering the magnitude and nature of the risks plants face unclear. Here we report the results of a global species assessment for the largest plant taxon evaluated to date under the International Union for Conservation of Nature (IUCN) Red List Categories and Criteria, the iconic Cactaceae (cacti). We show that cacti are among the most threatened taxonomic groups assessed to date, with 31% of the 1,478 evaluated species threatened, demonstrating the high anthropogenic pressures on biodiversity in arid lands. The distribution of threatened species and the predominant threatening processes and drivers are different to those described for other taxa. The most significant threat processes comprise land conversion to agriculture and aquaculture, collection as biological resources, and residential and commercial development. The dominant drivers of extinction risk are the unscrupulous collection of live plants and seeds for horticultural trade and private ornamental collections, smallholder livestock ranching and smallholder annual agriculture. Our findings demonstrate that global species assessments are readily achievable for major groups of plants with relatively moderate resources, and highlight different conservation priorities and actions to those derived from species assessments of key animal groups.

How and why does the areole meristem move in Echinocereus (Cactaceae)?

BACKGROUND AND AIMS: In Cactaceae, the areole is the organ that forms the leaves, spines and buds. Apparently, the genus Echinocereus develops enclosed buds that break through the epidermis of the stem adjacent to the areole; this trait most likely represents a synapomorphy of Echinocereus. The development of the areole is investigated here in order to understand the anatomical modifications that lead to internal bud development and to supplement anatomical knowledge of plants that do not behave according to classical shoot theory. METHODS: The external morphology of the areole was documented and the anatomy was studied using tissue clearing, scanning electron microscopy and light microscopy for 50 species that represent the recognized clades and sections of the traditional classification of the genus, including Morangaya pensilis (Echinocereus pensilis). KEY RESULTS: In Echinocereus, the areole is sealed by the periderm, and the areole meristem is moved and enclosed by the differential growth of the epidermis and surrounding cortex. The enclosed areole meristem is differentiated in a vegetative or floral bud, which develops internally and breaks through the epidermis of the stem. In Morangaya pensilis, the areole is not sealed by the periderm and the areole meristem is not enclosed. CONCLUSIONS: The enclosed areole meristem and internal bud development are understood to be an adaptation to protect the meristem and the bud from low temperatures. The anatomical evidence supports the hypothesis that the enclosed bud represents one synapomorphy for Echinocereus and also supports the exclusion of Morangaya from Echinocereus.

Proteomic analysis of plasma from patients with systemic lupus erythematosus: increased presence of haptoglobin alpha2 polypeptide chains over the alpha1 isoforms.

In the present study plasma samples from 15 systemic lupus erythematosus (SLE) patients and 16 healthy controls of initially unknown haptoglobin (Hp) phenotype were separated by 2-DE, and tryptic digests of the excised Hpalpha polypeptide chain spots were analyzed by MALDI-TOF-MS. Selected tryptic peptides were sequenced by nano-(n)ESI-IT MS/MS. The six major Hp phenotypes were present, although with distinct frequencies in controls and SLE patients. Thus, there were an increased proportion of SLE patients with Hp 2-2, or Hp 2-1S phenotypes. The Hp phenotype distribution resulted in allele frequencies of 0 625 (Hp(2)), 0.281 (Hp(1S)), and 0.093 (Hp(1F)) in healthy controls, correlating fairly well with the allele frequencies of European populations. In contrast, the Hp allele frequencies of the SLE patients were 0.733 (Hp(2)), 0.233 (Hp(1S)), and 0.033 (Hp1(1F)), which clearly indicated an increased frequency of Hp(2), a similar proportion of Hp(1S) and a diminished proportion of Hp(1F) in SLE patients compared with that in healthy controls. Preferential Hpalpha2 expression in SLE patients may contribute to some of the clinical manifestations of the disease such as hypergammaglobulinemia, systemic vasculitis, and cardiovascular disorders.