A Direct-Digital 40 µA 100 kb/s Intracardiac Communication Receiver with 250 µs Startup Time for Low Duty-Cycle Leadless Pacemaker Synchronization.

The first commercial dual-chamber leadless pacemaker (LLPM) was introduced recently. The system combines two separate implants situated in the right atrium and the right ventricle of the heart. Implant synchronization is accomplished with conductive intracardiac communication (CIC) using the myocardium and blood as transmission channel. Successful implant synchronization of this dual-chamber LLPM has been demonstrated. However, the continuously active synchronization transceivers, consuming about 800 nA, cause a 25-45% reduction in the projected device longevity. This work proposes an alternative strategy for power-optimized LLPM synchronization, which is based on synchronous duty-cycling of the transceivers and direct-digital CIC (DD-CIC). In line with this strategy, a novel low-power DD-CIC receiver for short-packet communication based on Manchester-encoded data and with fast startup time is presented. The circuit was fabricated in 180 nm CMOS technology and analyzed with respect to sensitivity, current consumption and startup time under highly duty-cycled operation. The receiver achieves a sensitivity of 81.6±7.4 µV at a data rate of 100 kb/s, with an active current consumption of 39.1±0.6 µA and a startup time below 250 µs. Operating the receiver as specified by the proposed LLPM synchronization strategy reduces the current consumption to a measured average value of 73 nA. In conclusion, this work suggests synchronous duty-cycling for CIC-based implant synchronization as a promising concept to severely reduce the current consumption of contemporary dual-chamber LLPMs. Consequently, device longevity may be increased significantly, potentially reducing the frequency of costly and complication-prone re-interventions.

Functional characterization of Francisella tularensis subspecies holarctica genotypes during tick cell and macrophage infections using a proteogenomic approach.

Tularemia is a vector-borne disease caused by the Gram-negative bacterium Francisella tularensis. Known hosts and vectors in Europe are hare and ticks. F. tularensis is transmitted from ticks and animals, but also from the hydrotelluric environment and the consumption of contaminated water or food. A changing climate expands the range in which ticks can live and consequently might contribute to increasing case numbers of tularemia. Two subspecies of F. tularensis are human pathogenic. Francisella tularensis tularensis (Ftt) is endemic in North America, while Francisella tularensis holarctica (Fth) is the only subspecies causing tularemia in Europe. Ft is classified as a category A bioterrorism agent due to its low infectious dose, multiple modes of transmission, high infectivity and potential for airborne transmission and has become a global public health concern. In line with the European survey and previous phylogenetic studies, Switzerland shows the co-distribution of B.6 and B.12 strains with different geographical distribution and prevalence within the country. To establish itself in different host environments of ticks and mammals, F. tularensis presumably undergoes substantial changes on the transcriptomics and proteomic level. Here we investigate the transcriptomic and proteomic differences of five strains of Fth upon infection of rabbit macrophages and tick cells.

Phylogeography of Francisella tularensis subspecies holarctica and epidemiology of tularemia in Switzerland.

Tularemia, an endemic disease that mainly affects wild animals and humans, is caused by Francisella tularensis subsp. holarctica (Fth) in Switzerland. The Swiss Fth population consist of multiple different subclades which are distributed throughout the country. The aim of this study is to characterize the genetic diversity of Fth in Switzerland and to describe the phylogeographic relationship of isolates by single nucleotide polymorphism (SNP) analysis. This analysis is combined with human surveillance data from reported cases over the last 10 years and in vitro and in silico antibiotic resistance tests to provide insight into the epidemiology of tularemia in Switzerland. We sequenced the whole genomes of 52 Fth strains of human or tick origin collected in Switzerland between 2009 and 2022 and analyzed together with all publicly available sequencing data of Swiss and European Fth. Next, we performed a preliminary classification with the established canonical single nucleotide polymorphism nomenclature. Furthermore, we tested 20 isolates from all main Swiss clades for antimicrobial susceptibility against a panel of antimicrobial agents. All 52 sequenced isolates from Switzerland belong to major clade B.6, specifically subclades B.45 and B.46, previously described in Western Europe. We were able to accurately reconstruct the population structure according to the global phylogenetic framework. No resistance to clinically recommended antibiotics could be identified in vitro or in silico in the western B.6 strains.

Metformin's Role in Hyperlactatemia and Lactic Acidosis in ICU Patients: A Systematic Review.

INTRODUCTION: Metformin-treated patients may experience severe hyperlactatemia or lactic acidosis (LA). LA often requires intensive-care-unit (ICU) treatment, and mortality rates are high. Here, we investigate the impact of renal dysfunction and renal replacement therapy (RRT) on the outcomes of critically ill patients with metformin-associated LA (MALA). Furthermore, we assessed associations between mortality and metformin dose, metformin plasma/serum concentrations, lactate level, and arterial pH. Finally, we investigated whether the recommended classification in MALA, metformin-unrelated LA, metformin-induced LA, and LA in metformin therapy appears useful in this regard. METHODS: We performed a retrospective analysis based on a systematic PubMed search for publications on hyperlactatemia/LA in metformin-treated ICU patients from January 1995 to February 2020. Case-level data including demographics and clinical conditions were extracted, and logistic regression analyses were performed. RESULTS: A total of 92 ICU patients were reported. Two of these patients had no comorbidities interfering with lactate metabolism. In the overall group, arterial pH, lactate levels, and metformin plasma/serum concentrations were similar in survivors versus non-survivors. Ingested daily metformin doses and plasma/serum creatinine levels were significantly higher in survivors versus non-survivors (p = 0.007 vs. p = 0.024, respectively). Higher plasma/serum creatinine levels, higher lactate levels, and lower arterial pH were all associated with patients receiving RRT (all p < 0.05). Overall mortality was 22% (20 out of 92 patients) and did not differ between the RRT and non-RRT groups. CONCLUSION: Mortality is high in ICU patients with metformin-associated hyperlactatemia/LA. Unexpectedly, higher ingested metformin dose and plasma/serum creatinine were associated with a better outcome. Survival was similar in patients with or without need for RRT.

Genome assembly, structural variants, and genetic differentiation between lake whitefish young species pairs (Coregonus sp.) with long and short reads.

Nascent pairs of ecologically differentiated species offer an opportunity to get a better glimpse at the genetic architecture of speciation. Of particular interest is our recent ability to consider a wider range of genomic variants, not only single-nucleotide polymorphisms (SNPs), thanks to long-read sequencing technology. We can now identify structural variants (SVs) such as insertions, deletions and other rearrangements, allowing further insights into the genetic architecture of speciation and how different types of variants are involved in species differentiation. Here, we investigated genomic patterns of differentiation between sympatric species pairs (Dwarf and Normal) belonging to the lake whitefish (Coregonus clupeaformis) species complex. We assembled the first reference genomes for both C. clupeaformis sp. Normal and C. clupeaformis sp. Dwarf, annotated the transposable elements and analysed the genomes in the light of related coregonid species. Next, we used a combination of long- and short-read sequencing to characterize SVs and genotype them at the population scale using genome-graph approaches, showing that SVs cover five times more of the genome than SNPs. We then integrated both SNPs and SVs to investigate the genetic architecture of species differentiation in two different lakes and highlighted an excess of shared outliers of differentiation. In particular, a large fraction of SVs differentiating the two species correspond to insertions or deletions of transposable elements (TEs), suggesting that TE accumulation may represent a key component of genetic divergence between the Dwarf and Normal species. Together, our results suggest that SVs may play an important role in speciation and that, by combining second- and third-generation sequencing, we now have the ability to integrate SVs into speciation genomics.

The emergence of supergenes from inversions in Atlantic salmon.

Supergenes link allelic combinations into non-recombining units known to play an essential role in maintaining adaptive genetic variation. However, because supergenes can be maintained over millions of years by balancing selection and typically exhibit strong recombination suppression, both the underlying functional variants and how the supergenes are formed are largely unknown. Particularly, questions remain over the importance of inversion breakpoint sequences and whether supergenes capture pre-existing adaptive variation or accumulate this following recombination suppression. To investigate the process of supergene formation, we identified inversion polymorphisms in Atlantic salmon by assembling eleven genomes with nanopore long-read sequencing technology. A genome assembly from the sister species, brown trout, was used to determine the standard state of the inversions. We found evidence for adaptive variation through genotype-environment associations, but not for the accumulation of deleterious mutations. One young 3 Mb inversion segregating in North American populations has captured adaptive variation that is still segregating within the standard arrangement of the inversion, while some adaptive variation has accumulated after the inversion. This inversion and two others had breakpoints disrupting genes. Three multigene inversions with matched repeat structures at the breakpoints did not show any supergene signatures, suggesting that shared breakpoint repeats may obstruct supergene formation. This article is part of the theme issue 'Genomic architecture of supergenes: causes and evolutionary consequences'.

Dysphagia Post-Extubation Affects Long-Term Mortality in Mixed Adult ICU Patients-Data From a Large Prospective Observational Study With Systematic Dysphagia Screening.

Data on long-term effects of post-extubation dysphagia is lacking. We investigate mid- and long-term clinical outcomes in a large sample of ICU patients with systematic dysphagia screening. DESIGN: Outcome analysis with a follow-up of 6 years or death (whichever occurred earlier) of ICU patients from a prospective observational trial (Dysphagia in Mechanically Ventilated ICU Patients study) with systematic dysphagia screening. SETTING: ICU of a tertiary care academic center. PATIENTS: Nine-hundred thirty-three mixed medical-surgical ICU patients (median age, 66 yr; interquartile range [IQR], 54-74, Acute Physiology and Chronic Health Evaluation II score 19 [IQR, 14-24], 71% male). INTERVENTIONS: ICU patients were followed up for a mean follow-up period of 1,731 ± 772 days (4.7 ± 2.1 yr). Primary outcome measures were 180-day and 360-day all-cause mortality in ICU patients with versus without dysphagia. MEASUREMENTS AND MAIN RESULTS: Two-hundred seventy-three patients died (29.3%) during the observational interval (n = 76 lost to follow-up). In dysphagia screening positive versus negative ICU patients, mortality at 180 days was 16% versus 5.8% (excess mortality 10.2%), whereas mortality at 360 days was 25% versus 9.1% (excess mortality 15.9%). Adjustment for confounders in a Cox model revealed a significant association of dysphagia with all-cause mortality in a time-dependent manner. The risk of death in ICU patients with versus without post-extubation dysphagia declined from about 2.5 times higher to about equal risk for both groups over the first year (i.e. 1.03 yr) post-ICU admission (at 360 d: hazard ratio [HR], 1.03; 95% CI, 0.42-3.70). The mean mortality HR for the first year post-ICU admission was HR 2.09 (95% CI, 1.34-3.24; p = 0.0009). CONCLUSIONS: Long-term follow-up of a large cohort of medical-surgical adult ICU patients systematically screened for dysphagia showed that dysphagia is associated with increased hazards for death for up to 1 year after ICU admission. Our data underline effects of post-extubation dysphagia on long-term clinical outcomes in affected critically ill patients.

Fluid Overload Phenotypes in Critical Illness-A Machine Learning Approach.

BACKGROUND: The detrimental impact of fluid overload (FO) on intensive care unit (ICU) morbidity and mortality is well known. However, research to identify subgroups of patients particularly prone to fluid overload is scarce. The aim of this cohort study was to derive "FO phenotypes" in the critically ill by using machine learning techniques. METHODS: Retrospective single center study including adult intensive care patients with a length of stay of ≥3 days and sufficient data to compute FO. Data was analyzed by multivariable logistic regression, fast and frugal trees (FFT), classification decision trees (DT), and a random forest (RF) model. RESULTS: Out of 1772 included patients, 387 (21.8%) met the FO definition. The random forest model had the highest area under the curve (AUC) (0.84, 95% CI 0.79-0.86), followed by multivariable logistic regression (0.81, 95% CI 0.77-0.86), FFT (0.75, 95% CI 0.69-0.79) and DT (0.73, 95% CI 0.68-0.78) to predict FO. The most important predictors identified in all models were lactate and bicarbonate at admission and postsurgical ICU admission. Sepsis/septic shock was identified as a risk factor in the MV and RF analysis. CONCLUSION: The FO phenotypes consist of patients admitted after surgery or with sepsis/septic shock with high lactate and low bicarbonate.

Insertion of an endogenous Jaagsiekte sheep retrovirus element into the BCO2 - gene abolishes its function and leads to yellow discoloration of adipose tissue in Norwegian Spælsau (Ovis aries).

BACKGROUND: The accumulation of carotenoids in adipose tissue leading to yellow fat is, in sheep, a heritable recessive trait that can be attributed to a nonsense mutation in the beta-carotene oxygenase 2 (BCO2) gene. However, not all sheep breeds suffering from yellow fat have this nonsense mutation, meaning that other functional mechanisms must exist. We investigated one such breed, the Norwegian spælsau. RESULTS: In spælsau we detected an aberration in BCO2 mRNA. Nanopore sequencing of genomic DNA revealed the insertion of a 7.9 kb endogenous Jaagsiekte Sheep Retrovirus (enJSRV) sequence in the first intron of the BCO2 gene. Close examination of its cDNA revealed that the BCO2 genes first exon was spliced together with enJSRV-sequence immediately downstream of a potential -AG splice acceptor site at enJSRV position 415. The hybrid protein product consists of 29 amino acids coded by the BCO2 exon 1, one amino acid coded by the junction sequence, followed by 28 amino acids arbitrary coded for by the enJSRV-sequence, before a translation stop codon is reached. CONCLUSIONS: Considering that the functional BCO2 protein consists of 575 amino acids, it is unlikely that the 58 amino acid BCO2/enJSRV hybrid protein can display any enzymatic function. The existence of this novel BCO2 allele represents an alternative functional mechanism accounting for BCO2 inactivation and is a perfect example of the potential benefits for searching for structural variants using long-read sequencing data.

A genome-wide association study, supported by a new chromosome-level genome assembly, suggests sox2 as a main driver of the undifferentiatiated ZZ/ZW sex determination of turbot (Scophthalmus maximus).

BACKGROUND: Understanding sex determination (SD) across taxa is a major challenge for evolutionary biology. The new genomic tools are paving the way to identify genomic features underlying SD in fish, a group frequently showing limited sex chromosome differentiation and high SD evolutionary turnover. Turbot (Scophthalmus maximus) is a commercially important flatfish with an undifferentiated ZW/ZZ SD system and remarkable sexual dimorphism. Here we describe a new long-read turbot genome assembly used to disentangle the genetic architecture of turbot SD by combining genomics and classical genetics approaches. RESULTS: The new turbot genome assembly consists of 145 contigs (N50 = 22.9 Mb), 27 of them representing >95% of its estimated genome size. A genome wide association study (GWAS) identified a ~ 6.8 Mb region on chromosome 12 associated with sex in 69.4% of the 36 families analyzed. The highest associated markers flanked sox2, the only gene in the region showing differential expression between sexes before gonad differentiation. A single SNP showed consistent differences between Z and W chromosomes. The analysis of a broad sample of families suggested the presence of additional genetic and/or environmental factors on turbot SD. CONCLUSIONS: The new chromosome-level turbot genome assembly, one of the most contiguous fish assemblies to date, facilitated the identification of sox2 as a consistent candidate gene putatively driving SD in this species. This chromosome SD system barely showed any signs of differentiation, and other factors beyond the main QTL seem to control SD in a certain proportion of families.

A Nanopore Based Chromosome-Level Assembly Representing Atlantic Cod from the Celtic Sea.

Currently available genome assemblies for Atlantic cod (Gadus morhua) have been constructed from fish belonging to the Northeast Arctic Cod (NEAC) population; a migratory population feeding in the Barents Sea. These assemblies have been crucial for the development of genetic markers which have been used to study population differentiation and adaptive evolution in Atlantic cod, pinpointing four discrete islands of genomic divergence located on linkage groups 1, 2, 7 and 12. In this paper, we present a high-quality reference genome from a male Atlantic cod representing a southern population inhabiting the Celtic sea. The genome assembly (gadMor_Celtic) was produced from long-read nanopore data and has a combined contig length of 686 Mb with an N50 of 10 Mb. Integrating contigs with genetic linkage mapping information enabled us to construct 23 chromosome sequences which mapped with high confidence to the latest NEAC population assembly (gadMor3) and allowed us to characterize, to an extent not previously reported large chromosomal inversions on linkage groups 1, 2, 7 and 12. In most cases, inversion breakpoints could be located within single nanopore contigs. Our results suggest the presence of inversions in Celtic cod on linkage groups 6, 11 and 21, although these remain to be confirmed. Further, we identified a specific repetitive element that is relatively enriched at predicted centromeric regions. Our gadMor_Celtic assembly provides a resource representing a 'southern' cod population which is complementary to the existing 'northern' population based genome assemblies and represents the first step toward developing pan-genomic resources for Atlantic cod.

Publisher Correction: Sex-dependent dominance maintains migration supergene in rainbow trout.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Sex-dependent dominance maintains migration supergene in rainbow trout.

Males and females often differ in their fitness optima for shared traits that have a shared genetic basis, leading to sexual conflict. Morphologically differentiated sex chromosomes can resolve this conflict and protect sexually antagonistic variation, but they accumulate deleterious mutations. However, how sexual conflict is resolved in species that lack differentiated sex chromosomes is largely unknown. Here we present a chromosome-anchored genome assembly for rainbow trout (Oncorhynchus mykiss) and characterize a 55-Mb double-inversion supergene that mediates sex-specific migratory tendency through sex-dependent dominance reversal, an alternative mechanism for resolving sexual conflict. The double inversion contains key photosensory, circadian rhythm, adiposity and sex-related genes and displays a latitudinal frequency cline, indicating environmentally dependent selection. Our results show sex-dependent dominance reversal across a large autosomal supergene, a mechanism for sexual conflict resolution capable of protecting sexually antagonistic variation while avoiding the homozygous lethality and deleterious mutations associated with typical heteromorphic sex chromosomes.

Pseudogenization and Resurrection of a Speciation Gene.

A persistent question in evolutionary biology is how complex phenotypes evolve and whether phenotypic transitions are reversible. Multiple losses of floral pigmentation have been documented in the angiosperms, but color re-gain has not yet been described, supporting that re-gain is unlikely. Pollinator-mediated selection in Petunia has resulted in several color shifts comprised of both losses and gains of color. The R2R3-MYB transcription factor AN2 has been identified as a major locus responsible for shifts in pollinator preference. Whereas the loss of visible color has previously been attributed to repeated pseudogenization of AN2, here, we describe the mechanism of an independent re-gain of floral color via AN2 evolution. In P. secreta, purple color is restored through the improbable resurrection of AN2 gene function from a non-functional AN2-ancestor by a single reading-frame-restoring mutation. Thus, floral color evolution in Petunia is mechanistically dependent on AN2 functionality, highlighting its role as a hotspot in color transitions and a speciation gene for the genus.

Gain and Loss of Floral Scent Production through Changes in Structural Genes during Pollinator-Mediated Speciation.

The interactions of plants with their pollinators are thought to be a driving force in the evolution of angiosperms. Adaptation to a new pollinator involves coordinated changes in multiple floral traits controlled by multiple genes. Surprisingly, such complex genetic shifts have happened numerous times during evolution. Here we report on the genetic basis of the changes in one such trait, floral scent emission, in the genus Petunia (Solanaceae). The increase in the quantity and complexity of the volatiles during the shift from bee to hawkmoth pollination was due to de novo expression of the genes encoding benzoic acid/salicylic acid carboxyl methyltransferase (BSMT) and benzoyl-CoA:benzylalcohol/2-phenylethanol benzoyltransferase (BPBT) together with moderately increased transcript levels for most enzymes of the phenylpropanoid/benzenoid pathway. Loss of cinnamate-CoA ligase (CNL) function as well as a reduction in the expression of the MYB transcription factor ODO1 explain the loss of scent during the transition from moth to hummingbird pollination. The CNL gene in the hummingbird-adapted species is inactive due to a stop codon, but also appears to have undergone further degradation over time. Therefore, we propose that loss of scent happened relatively early in the transition toward hummingbird pollination, and probably preceded the loss of UV-absorbing flavonols. The discovery that CNL is also involved in the loss of scent during the transition from outcrossing to selfing in Capsella (Brassicaceae) (see the accompanying paper) raises interesting questions about the possible causes of deep evolutionary conservation of the targets of evolutionary change.

Insight into the evolution of the Solanaceae from the parental genomes of Petunia hybrida.

Petunia hybrida is a popular bedding plant that has a long history as a genetic model system. We report the whole-genome sequencing and assembly of inbred derivatives of its two wild parents, P. axillaris N and P. inflata S6. The assemblies include 91.3% and 90.2% coverage of their diploid genomes (1.4 Gb; 2n = 14) containing 32,928 and 36,697 protein-coding genes, respectively. The genomes reveal that the Petunia lineage has experienced at least two rounds of hexaploidization: the older gamma event, which is shared with most Eudicots, and a more recent Solanaceae event that is shared with tomato and other solanaceous species. Transcription factors involved in the shift from bee to moth pollination reside in particularly dynamic regions of the genome, which may have been key to the remarkable diversity of floral colour patterns and pollination systems. The high-quality genome sequences will enhance the value of Petunia as a model system for research on unique biological phenomena such as small RNAs, symbiosis, self-incompatibility and circadian rhythms.

MYB-FL controls gain and loss of floral UV absorbance, a key trait affecting pollinator preference and reproductive isolation.

Adaptations to new pollinators involve multiple floral traits, each requiring coordinated changes in multiple genes. Despite this genetic complexity, shifts in pollination syndromes have happened frequently during angiosperm evolution. Here we study the genetic basis of floral UV absorbance, a key trait for attracting nocturnal pollinators. In Petunia, mutations in a single gene, MYB-FL, explain two transitions in UV absorbance. A gain of UV absorbance in the transition from bee to moth pollination was determined by a cis-regulatory mutation, whereas a frameshift mutation caused subsequent loss of UV absorbance during the transition from moth to hummingbird pollination. The functional differences in MYB-FL provide insight into the process of speciation and clarify phylogenetic relationships between nascent species.

Impaired glucose metabolism and type 2 diabetes in apparently healthy senior citizens.

STUDY PRINCIPLE: To estimate the prevalence of unknown impaired glucose metabolism, also referred to as prediabetes (PreD), and unknown type 2 diabetes mellitus (T2DM) among subjectively healthy Swiss senior citizens. The fasting plasma glucose (FPG) and glycated haemoglobin A(1c) (HbA(1c)) levels were used for screening. A total of 1 362 subjects were included (613 men and 749 women; age range 60-99 years). Subjects with known T2DM were excluded. METHODS: The FPG was processed immediately for analysis under standardised preanalytical conditions in a cross-sectional cohort study; plasma glucose levels were measured by means of the hexokinase procedure, and HbA(1c) was measured chromatographically and classified using the current American Diabetes Association (ADA) criteria. RESULTS: The crude prevalence of individuals unaware of having prediabetic FPG or HbA(1c) levels, was 64.5% (n = 878). Analogously, unknown T2DM was found in 8.4% (n = 114) On the basis of HbA(1c) criteria alone, significantly more subjects with unknown fasting glucose impairment and laboratory T2DM could be identified than with the FPG. The prevalence of PreD as well as of T2DM increased with age. The mean HOMA indices (homeostasis model assessment) for the different age groups, between 2.12 and 2.59, are consistent with clinically hidden disease and are in agreement with the largely orderly Body Mass Indices found in the normal range. CONCLUSIONS: Laboratory evidence of impaired glucose metabolism and, to a lesser extent, unknown T2DM, has a high prevalence among subjectively healthy older Swiss individuals. Laboratory identification of people with unknown out-of-range glucose values and overt diabetic hyperglycaemia might improve the prognosis by delaying the emergence of overt disease.

Tight genetic linkage of prezygotic barrier loci creates a multifunctional speciation island in Petunia.

Most flowering plants depend on animal vectors for pollination and seed dispersal. Differential pollinator preferences lead to premating isolation and thus reduced gene flow between interbreeding plant populations. Sets of floral traits, adapted to attract specific pollinator guilds, are called pollination syndromes. Shifts in pollination syndromes have occurred surprisingly frequently, considering that they must involve coordinated changes in multiple genes affecting multiple floral traits. Although the identification of individual genes specifying single pollination syndrome traits is in progress in many species, little is known about the genetic architecture of coadapted pollination syndrome traits and how they are embedded within the genome. Here we describe the tight genetic linkage of loci specifying five major pollination syndrome traits in the genus Petunia: visible color, UV absorption, floral scent production, pistil length, and stamen length. Comparison with other Solanaceae indicates that, in P. exserta and P. axillaris, loci specifying these floral traits have specifically become clustered into a multifunctional "speciation island". Such an arrangement promotes linkage disequilibrium and avoids the dissolution of pollination syndromes by recombination. We suggest that tight genetic linkage provides a mechanism for rapid switches between distinct pollination syndromes in response to changes in pollinator availabilities.