Phylogeography of Eriotheca species complex: insights into the origin and range expansion of apomictic and polyploid trees in Neotropical Savannas.

Polyploidy and whole genome duplication are major evolutionary drivers in plants. Climate variations during the Pleistocene have influenced distribution and range expansion worldwide. Similar trends have been reported for Cerrado plants, but no attempt has been made to link phylogeography with ploidy and breeding changes. Thus, we aimed to (i) assess ploidy and genome size of Eriotheca estevesiae Carv.-Sobr., and compare it with E. pubescens (Mart.) Schott & Endl. (Both included into the Eriotheca Stellate Trichome Species Complex - ESTSC). (ii) Subsequently, we investigated their phylogeography to see whether genetic structure and range expansion trends were similar to those previously described for the Cerrado biome. Finally (iii), we discuss whether ESTSC phylogeographic patterns could be associated with geographic parthenogenesis processes. Common cytogenetic techniques and flow cytometry were used to confirm chromosome number and genome size of E. estevesiae. We used three cpDNA regions to analyse 14 ESTSC Cerrado populations, for which we also obtained ploidy level and breeding information. We investigated haplotype diversity, population structure and tested neutrality, aiming to reconstruct phylogeographic scenarios. We found three ploidy levels and eight cpDNA haplotypes in ESTSC, one shared by most populations. Haplotype and ploidy distribution corroborated that E. pubescens, the widely distributed polyploid and apomictic species, may have originated from northern diploid and probably sexual E. estevesiae. Matrilinear cpDNA links support the idea that apomixis and polyploidy in ESTSC may have allowed range expansion during the Pleistocene, in a process analogous to the geographic parthenogenesis described elsewhere.

Financial impact of healthcare-associated infections on intensive care units estimated for fifty Brazilian university hospitals affiliated to the unified health system.

BACKGROUND: Studies show that healthcare-associated infections (HAIs) represent a crucial issue in healthcare and can lead to substantial economic impacts in intensive care units (ICUs). AIM: To estimate direct costs associated with the most significant HAIs in 50 teaching hospitals in Brazil, affiliated to the unified health system (Sistema Único de Saúde: SUS). METHODS: A Monte Carlo simulation model was designed to estimate the direct costs of HAIs; first, epidemiologic and economic parameters were established for each HAI based on a cohort of 949 critical patients (800 without HAI and 149 with); second, simulation based on three Brazilian prevalence scenarios of HAIs in ICU patients (29.1%, 51.2%, and 61.6%) was used; and third, the annual direct costs of HAIs in 50 university hospitals were simulated. FINDINGS: Patients with HAIs had 16 additional days in the ICU, along with an extra direct cost of US$13.892, compared to those without HAIs. In one hypothetical scenario without HAI, the direct annual cost of hospital care for 26,649 inpatients in adult ICUs of 50 hospitals was US$112,924,421. There was an increase of approximately US$56 million in a scenario with 29.1%, and an increase of US$147 million in a scenario with 61.6%. The impact on the direct cost became significant starting at a 10% prevalence of HAIs, where US$2,824,817 is added for each 1% increase in prevalence. CONCLUSION: This analysis provides robust and updated estimates showing that HAI places a significant financial burden on the Brazilian healthcare system and contributes to a longer stay for inpatients.

Stomatal size, ploidy and polyembryony in Eriotheca Stellate Trichome Species Complex (Bombacoideae - Malvaceae) in the Cerrados of Brazil.

Geographic parthenogenesis, range expansion of apomictic plants after climate changes, has been described for Northern Hemisphere gametophytic apomicts. But similar trends have been observed for sporophytic apomicts of Cerrado, the savannas in Brazil. Eriotheca pubescens is a common Cerrado tree, an agamic complex of either hexaploid/polyembryonic apomicts or tetraploid/monoembryonic sexual individuals. Some populations have been described as a new species, Eriotheca estevesiae, all included in the Eriotheca Stellate Trichome Species Complex (ESTSC). Since breeding systems and ploidy are clearly associated with polyembryony and stomatal size, we used these ancillary features to map the reproductive and ploidy level traits of E. pubescens and E. estevesiae. Leaves and seeds were collected from individuals of 19 populations. Seeds were evaluated for the presence of polyembryony and leaves for stomatal measurements. Eight populations were monoembryonic while another eight were polyembryonic and for other three, the embryonic pattern was not readily verified. E. pubescens polyembryonic and hexaploid populations formed a homogeneous group, but monoembryonic plants were more variable. E. estevesiae populations were monoembryonic with smaller stomata. In contrast, some E. pubescens monoembryonic populations further south presented larger stomata. Despite these outliers, possibly mixed populations, stomatal size and embryonic pattern differed from northern to southern populations. Embryonic pattern and stomatal size indicated that northernmost populations of Eriotheca STSC (E. estevesiae) are diploid and sexual. Southernmost populations, mostly polyembryonic and with large stomata, are hexaploid and apomictic. This is in agreement with geographic parthenogenesis and range expansion of apomictic lineages to southern habitats available after the last glacial maximum.

Costs of healthcare-associated infections to the Brazilian public Unified Health System in a tertiary-care teaching hospital: a matched case-control study.

BACKGROUND: Little is known about the economic burden of healthcare-associated infections (HAIs) in Brazil. AIM: To analyse the costs of hospitalization by reimbursement from the Brazilian government, via the Brazilian Unified Health System (SUS) affiliation, and direct costs in the adult Intensive Care Unit (ICU). METHODS: The matched-pairs case-control study (83 patients with HAIs and 83 without HAIs) was performed at a referral tertiary-care teaching hospital in Brazil in January 2018. In order to calculate the HAI costs from the perspective of the payer, the total cost for each hospitalization was obtained through the Hospital's Billing Sector. Direct costs were calculated annually for 949 critical patients during 2018. FINDINGS: The reimbursement cost per hospitalization of patients with HAIs was 75% (US$2721) higher than patients without HAIs (US$1553). When a patient has an HAI, in addition to a longer length of stay (15 days), there was an extra increase (US$996) in the reimbursement cost per hospitalization. An HAI in the ICU was associated with a total direct cost eight times higher compared with patients who did not develop infections in this unit, US$11,776 × US$1329, respectively. The direct cost of hospitalization in the ICU without HAI was 56.5% less than the reimbursement (US$1329 × US$3052, respectively), whereas for the patient with an HAI, the direct cost was 111.5% above the reimbursement (US$11,776 × US$5569, respectively). CONCLUSION: HAIs contribute to a longer stay and an eight-fold increase in direct costs. It is necessary to reinforce programmes that prevent HAIs in Brazilian hospitals.

Mixed pollen load and late-acting self-incompatibility flexibility in Adenocalymma peregrinum (Miers) L.G. Lohmann (Bignonieae: Bignoniaceae).

Mixed cross and self-pollen load on the stigma (mixed pollination) of species with late-acting self-incompatibility system (LSI) can lead to self-fertilized seed production. This "cryptic self-fertility" may allow selfed seedling development in species otherwise largely self-sterile. Our aims were to check if mixed pollinations would lead to fruit set in LSI Adenocalymma peregrinum, and test for evidence of early-acting inbreeding depression in putative selfed seeds from mixed pollinations. Experimental pollinations were carried out in a natural population. Fruit and seed set from self-, cross and mixed pollinations were analysed. Further germination tests were carried out for the seeds obtained from treatments. Our results confirm self-incompatibility, and fruit set from cross-pollinations was three-fold that from mixed pollinations. This low fruit set in mixed pollinations is most likely due to a greater number of self- than cross-fertilized ovules, which promotes LSI action and pistil abortion. Likewise, higher percentage of empty seeds in surviving fruits from mixed pollinations compared with cross-pollinations is probably due to ovule discounting caused by self-fertilization. Moreover, germinability of seeds with developed embryos was lower in fruits from mixed than from cross-pollinations, and the non-viable seeds from mixed pollinations showed one-third of the mass of those from cross-pollinations. The great number of empty seeds, lower germinability, lower mass of non-viable seeds, and higher variation in seed mass distribution in mixed pollinations, strongly suggests early-acing inbreeding depression in putative selfed seeds. In this sense, LSI and inbreeding depression acting together probably constrain self-fertilized seedling establishment in A. peregrinum.

Pollen and stomata morphometrics and polyploidy in Eriotheca (Malvaceae-Bombacoideae).

Approximately 70% of the angiosperm species are polyploid, an important phenomenon in the evolution of those plants. But ploidy estimates have often been hindered because of the small size and large number of chromosomes in many tropical groups. Since polyploidy affects cell size, morphometric analyses of pollen grains and stomata have been used to infer ploidy level. Polyploidy is present in many species of the Cerrado, the Neotropical savanna region in Central Brazil, and has been linked to apomixis in some taxa. Eriotheca gracilipes and Eriotheca pubescens are common tree species in this region, and present cytotypes that form reproductive mosaics. Hexaploid individuals (2n = 6x = 276) are polyembryonic and apomictic, while tetraploid and diploid individuals (2n = 2x = 92, 2n = 4x = 184) are sexual and monoembryonic. We tested whether morphometric analysis can be used to estimate ploidy levels in E. gracilipes and E. pubescens individuals. Pollen material from diploid and hexaploid individuals of E. gracilipes, and tetraploid and hexaploid individuals of E. pubescens, were fixed in 50% FAA, and expanded leaves were dried in silica gel. Pollen grains and stomata of at least five individuals from each population were measured. The results demonstrate that all measures were significantly different among cytotypes. Individuals with higher levels of ploidy (hexaploid) all presented measurements that were higher than those with lower levels (diploid and tetraploid). There was no overlap between ploidy levels in each species at 95% confidence interval. Thus, the size of the pollen grains and stomata are effective parameters for analysis of ploidy levels in E. gracilipes and E. pubescens.

Polyembryony in Melastomataceae from Brazilian Cerrado: multiple embryos in a small world.

Polyembryony has been commonly associated with apomixis in the angiosperms and seems to be more common than expected, even in biomes where sexual reproduction processes are predominant. Recent studies in Cerrado, the Neotropical savannas of Central Brazil, showed high frequencies of apomixis and polyembryony and indicated these processes as reproductive and evolutionary alternatives for plants in these areas. In this sense, we investigated the occurrence of polyembryony and its relationships with ecological (season and type of dispersal, ploidy, species distribution and breeding system) and taxonomic (tribe) factors in the Melastomataceae, a mostly tropical family already known for its high frequency of apomixis and very common in Cerrado. We collected seeds from 69 populations of 53 species, which were sown in germination chambers. After seed germination, the presence and number of seedlings per seed were evaluated as a method to estimate polyembryony. We encountered 18 species (33.96%) with polyembryony (more than one seedling, or gemellar seedlings, originated per seed) concentrated in species of the tribe Miconieae (64%) and Microlicieae (16.67%), but absent in Melastomeae. Monoembryony was present only in sexual species, while all apomictic species were polyembryonic. In Miconia, the polyembryony was correlated with polyploidy, and monoembryony with diploid species. Polyembryony was more common among species with wide distribution in the Cerrado region, which indicates that the presence of gemellar seedlings is important for establishment and survival of the group in the Cerrado biome.

Polyembryony and apomixis in Eriotheca pubescens (Malvaceae - Bombacoideae).

Apomixis and adventitious polyembryony have been reported for several species of Bombacoideae, including Eriotheca pubescens, a tree species of the Neotropical savanna (Cerrado) areas in Brazil. However, the origin of polyembryonic seeds and their importance for the reproduction of the species remained to be shown. Here, we analyzed the early embryology of this species to establish the apomictic origin of extranumerary embryos. We also observed the geographic distribution of polyembryony in E. pubescens, and tested if apomixis was related to the source of pollen (self or cross) and population density. Moreover, we tested if polyembryonic apomictic embryos would develop normally into seedlings. In the observed seed primordia, after a relatively long quiescent period, the zygote developed into a sexual embryo concurrently with adventitious apomictic embryos which developed from nucellus cells. Adventitious embryos develop faster than sexual ones and are morphologically similar, so that 44 days after anthesis it was virtually impossible to distinguish and trace the fate of the sexual embryo. Polyembryony is widely distributed in populations some 400 km distant, and only one strictly monoembryonic individual was observed during the study. The number of embryos per seed varied between fruits and individuals but was significantly higher in seeds from cross-pollinations than from selfs, although fruit and seed set after crosses were much lower than after selfs. Embryo development into seedlings depended on their weight at germination, but polyembryonic seeds germinated and produced up to seven seedlings per seed in greenhouse conditions. Adventitious embryony and apomictic seedlings would explain the mostly clonal populations suggested by molecular studies.