Eradication of the smallpox virus through extensive global vaccination efforts has resulted in one of the most important breakthroughs in medical history, saving countless lives from the severe morbidity and mortality that is associated with this disease. Although smallpox is now extinct in nature, laboratory stocks of this virus still remain and the subject of smallpox vaccination has gained renewed attention due to the potential risk that smallpox may be used as a biological weapon by terrorists or rogue states. Despite having the longest history of any modern vaccine, there is still much to be learned about smallpox vaccination and the correlates of protection remain to be formally defined. This Commentary will discuss the strengths and weaknesses of traditional smallpox vaccination in comparison with immunization using modified vaccinia virus Ankura (MVA), a non-replicating virus with a strong safety record but weakened immunogenicity. Smallpox (Variola major) is a virus that no longer exists in the wild, but during its reign it caused 20–30% mortality in previously unvaccinated individuals and often left survivors with deeply pitted scars for life . The las"I have purposely selected several cases in which the disease [i.e. cowpox] had appeared at a very distant period previous to the experiments made with variolous matter, to show that the change produced in the constitution is not affected by time." [The first line of defense against smallpox is vaccination. Smallpox vaccination is highly effective at protecting against lethal infection and even if only partial protective immunity is attained, this often still results in survival and decreased viral spread to others  with an inoculum containing vaccinia virus. After the infection has reached a point in which a great deal of exudate was observed, the purulent lymph material was scraped from the infected cow, clarified, and lyophilized in the presence of antibiotics. The inoculum is later reconstituted with diluent containing 0.25% phenol to further decrease bacterial contamination . Unlike most other vaccines that are administered by subcutaneous or intramuscular injection, vaccinia virus replicates poorly under these conditions and optimal vaccination occurs by scarification of the virus inoculum onto the skin surface . Viral r6 doses), generalized vaccinia (242 cases/106 doses), eczema vaccinatum (39 cases/106 doses), vaccinia necrosum (1.5 cases/106 doses), encephalitis (12 cases/106 doses), or death (~1 death/106 doses) [6 doses) whereas the mortality rate for older children and adolescents aged 1–4 or 5–19 is approximately 0.5 deaths/106 doses  . The morsee also  and Dryvsee also .6 smallpox vaccinations [Myopericarditis is a recently identified adverse event that occurs at a rate of ~124 cases/10inations ,15. In 2inations . A retroinations . FollowiTo overcome some of the problems associated with the current calf lymph smallpox vaccine (Dryvax), an improved tissue culture-derived vaccine has been recently developed . Unlike Both Dryvax and ACAM1000 represent replication-competent smallpox vaccines and since these vaccines are based on the use of live viruses, there is always an inherent risk of severe adverse events or death  in vaccinees that have unknown or undisclosed immunodeficiencies at the time of vaccination. To overcome the hazards of replicating viruses, a highly attenuated strain of vaccinia, designated Modified Vaccinia virus Ankara (MVA) was developed by growing the virus for >500 passages on chicken embryo fibroblasts (CEF) and following the loss of about 15% of its parental genome, it no longer was capable of replicating in most mammalian cells, including human cells . Other sEdward Jenner developed the first test for vaccine efficacy when he immunized subjects with cowpox and then later challenged them with smallpox by inoculation. If a subject showed no secondary smallpox lesions indicative of systemic spread, then the individual was believed to have protective immunity. He noted that cowpox-vaccinated individuals who developed a pox-like lesion at the vaccination site were fully protected against smallpox challenge. To this day, most studies still use the identification of a "take" (vesicular or pustular lesion) as evidence of successful vaccination. However, there are rare cases of revaccinated subjects who present with vesicle formation after smallpox vaccination, but show no detectably boosted cellular or humoral immunity  and M.K.ex vivo including vaccinia-specific IFNγ ELISPOT assays [+ T cells can be quantitated regardless of their cytokine profiles, but the main disadvantages with this approach include the lack of identified CD4+ T cell/MHC Class II epitopes, the requirement for knowledge of the MHC haplotype of the subject, and the inability to measure the total antiviral T cell response, which may be directed against any number of immunodominant and subdominant peptide epitopes.Quantitative immunology is beginning to gain acceptance as a measurement of vaccine efficacy, although the Jennerian vesicle at the site of smallpox vaccination still remains the primary endpoint of successful vaccination in most studies ,23-25. OT assays ,23,26-28T assays -33, or pT assays . Each ofet al. [Direct quantitation of the antiviral immune response induced by smallpox vaccination has been critical for several recent advances in orthopoxvirus immunobiology – especially since smallpox has been eradicated and other human orthopoxvirus outbreaks are too small and sporadic  model of monkeypox infection. Using ELISA assays and neutralizing assays to measure vaccinia-specific antibody responses, and ICCS to measure antiviral T cell responses, the authors showed that antiviral immunity appeared similar between these three groups. Likewise, each of these groups were protected against lethal monkeypox challenge, although primates that only received MVA plus an MVA booster showed partial protection with 6/6 animals presenting with 1–36 monkeypox lesions (compared to >500 monkeypox lesions in the unvaccinated controls). This indicates that two MVA vaccinations are required to induce partial immunity whereas MVA followed by Dryvax immunization or a single Dryvax immunization each provides full immunity. The protective efficacy of a single dose of MVA is unknown, but based on the results of the Earl et al. study [Another study by Earl et al. , used qul. study  wherein l. study . This isl. study  includinl. study ,32,40. Al. study ,32 and tl. study . In thisl. study . Oddly, It is difficult to speculate the efficacy of MVA vaccination of humans as a method of protection against smallpox. It is possible that booster doses of MVA would provide strong enough immunity to at least protect against lethal smallpox, similar to its ability to protect primates from lethal monkeypox . On the per se. Further analysis indicated that approximately 10% of previously vaccinated household contacts of smallpox patients were actually infected with smallpox as demonstrated by isolation of infectious virus from pharyngeal mucosa, but only 4/34 (12%) of these subjects developed the clinical symptoms of smallpox [There is more than one definition of protective immunity against smallpox. There is protection against infection, protection against disease, and protection against death. Of these, one might argue that protection against lethal infection is the ultimate definition of protective immunity. However, protection against infection and protection against disease not only reduce the morbidity of an outbreak, but these high levels of protective immunity are also associated with reduced virus spread to others  have indicated that, as long as there is strong humoral immunity against vaccinia, T cell memory is dispensable for protective immunity [A major issue in the smallpox field is that there is no consensus on what is exactly required for protective immunity against this disease. In the age of quantitative immunology, we are beginning to find clues that might help answer this age-old question. In a study of >300 subjects, the levels of vaccinia-specific serum antibody and antiviral T cell responses were determined from 30 days to up to 75 years after smallpox vaccination . Antivirterature ,26,40. Ml memory . Based oimmunity , and it immunity -46. This10 ≥ 0.7 neutralizing titer) was also established in non-human primates by simply vaccinating groups of animals with different doses of the yellow fever vaccine, quantitating vaccine-induced antibody levels, and then challenging them with a lethal dose of yellow fever virus [Based on the overlapping roles for T cell and B cell memory on protective immunity, is there any chance that a consensus can be reached in regard to defining an immunological correlate of immunity? One way to address this issue is to examine historical studies in which an immunological correlate was identified. In this regard, there are two independent studies in which investigators showed that subjects with vaccinia-specific neutralizing antibody titers of >1:20  or ≥ 1:3er virus . This seTraditional smallpox vaccination has lead to the global eradication of smallpox but continues to be used today in an effort to thwart the potential use of smallpox as a biological weapon. Since this vaccine employs the use of a live virus, there is an inherent risk of adverse events, although these are generally quite rare. New generation smallpox vaccine candidates include MVA and other non-replicating poxviruses and although they demonstrate a high degree of safety, their immunogenicity appears to be substantially lower than traditional smallpox vaccination with live vaccinia virus. The role of MVA and traditional smallpox vaccination (or a combination thereof) in future vaccination campaigns has yet to be determined. However, developing a consensus on the definition of what is required for protective immunity and defining an immunological correlate of immunity would aid in the evaluation of current and future vaccine approaches.MVA modified vaccinia virus AnkuraGMP good manufacturing practicesHIV human immunodeficiency virusCEF chicken embryo fibroblastsHPV human papilloma virusFDA Federal Drug AdministrationJEV Japanese Encephalitis VirusIMV Intracellular mature virusEEV Extracellular enveloped virusELISA Enzyme-linked immunosorbent assayIFNγ Interferon-gammaELISPOT Enzyme-linked immunosorbent Spot assayICCS Intracellular cytokine stainingMHC Major histocompatibility complexVIG Vaccinia immune globulinWHO World Health OrganizationNIBSC National Institute of Biological Standards and ControlEU Elisa UnitsIU International UnitsOHSU and Dr. Slifka have a financial interest in Najít Technologies, Inc., a company that may have a commercial interest in the results of this research and technology. This potential conflict was disclosed to the OHSU Conflict of Interest in Research Committee and an approved management plan was implemented.


It took nearly 200 years for biologists to redefine the plant/animal dichotomy set up by Linnaeus in 1758. Among the defining traits used in the new five kingdom model of the 20th century was the presence of a nucleus. Possession of a nucleus is one of the chief characteristics that earns an organism, even a single-celled organism, the name of eukaryote. Those not similarly blessed are prokaryotes. Biologists today classify life into three domains (of which microbes lay claim to two), yet the evolution of many fundamental features of eukaryotic biology remains a mystery.A pivotal moment in the evolution of early eukaryotes was the emergence of elaborate, interconnected membrane-bound compartments that make up the Golgi apparatus, endoplasmic reticulum, and nuclear envelope. The nuclear envelope, with its inner and outer membrane, forms a barrier between the cytoplasm and nucleus. Embedded in this envelope are nuclear pore complexes (NPCs), massive (over 400 subunits), cylindrically shaped protein assemblies that connect the outer and inner nuclear membranes via sharply curved sections of pore membranes. The NPC's central ring-like structure is sandwiched between a cytoplasmic ring, with fibrils extending into the cytoplasm, and a nuclear ring, with a “basket” extending into the nucleoplasm. NPCs police traffic flow between the nucleus and cytoplasm, routinely allowing entry to small molecules while providing only selective passage to macromolecules.How eukaryotes evolved complex membrane-mediated trafficking systems from their stripped down prokaryotic contemporaries is a fundamental question in biology. Michael Rout's team investigates one aspect of eukaryotic evolution—the origin and evolution of NPC proteins (nups)—by examining the structure of nups. In a new study, Rout and colleagues report the structure of a core building block of the NPC in yeast, and propose how the complex could have evolved from organisms with no such system.The researchers first tackled the structures of the seven protein components of a core NPC subcomplex, called the yNup84 subcomplex in yeast (and the vNup107-160 subcomplex in vertebrates). Rout and colleagues used algorithms that predict secondary structures to generate three-dimensional models of the component nups. Each nup, they found, consists mostly of either repeating alpha helixes , zigzagging beta sheets (in a beta-propeller fold), or a distinctive arrangement of an amino-terminal beta-propeller followed by a long stretch of alpha-solenoid. Next, the authors compared the structural conformations of the homologous nups found in humans and plants, and showed that the overall architecture of the subcomplex has been conserved throughout eukaryotic evolution.A search for evidence of the distinctive propeller/solenoid arrangement in other organisms shed light on the function and origin of the yNup87/vNup107-160 subcomplex. Neither bacterial nor archaebacterial proteins contain such an arrangement; it appears to exist only in eukaryotes. Moreover, proteins containing this arrangement function only as components of the coated vesicle complexes that operate in intracellular vesicular transport systems or as part of the NPC. That these complexes are linked by common architecture, the authors argue, suggests an “intimate connection between vesicle coating complexes and the yNup87/vNup107-160 subcomplex.” It's likely that both complexes function in curving membranes: when components of this subcomplex are disrupted in yeast, NPCs form abnormal clusters that impair nuclear membrane interactions.How did this shared molecular architecture evolve? Rout and colleagues propose that both nups and vesicle coating complexes developed from a common early eukaryotic ancestor—a primitive coating component with a simplified version of the repetitive folds described here. This molecular carpenter specialized in carving and remodeling membranes, and was repurposed to support the many specialized functions that facilitate molecular transport through the elaborately connected, highly specialized internal membrane systems of the modern eukaryote.


The exploitation of nature's machinery at length scales below the dimensions of a cell is an exciting challenge for biologists, chemists and physicists, while advances in our understanding of these biological motifs are now providing an opportunity to develop real single molecule devices for technological applications. Single molecule studies are already well advanced and biological molecular motors are being used to guide the design of nano-scale machines. However, controlling the specific functions of these devices in biological systems under changing conditions is difficult. In this review we describe the principles underlying the development of a molecular motor with numerous potential applications in nanotechnology and the use of specific synthetic polymers as prototypic molecular switches for control of the motor function. The molecular motor is a derivative of a TypeI Restriction-Modification (R-M) enzyme and the synthetic polymer is drawn from the class of materials that exhibit a temperature-dependent phase transition.The potential exploitation of single molecules as functional devices has been heralded as the dawn of new era in biotechnology and medicine. It is not surprising, therefore, that the efforts of numerous multidisciplinary teams ,2. have  Type I R-M enzymes are multifunctional, multisubunit enzymes that provide bacteria with protection against infection by DNA-based bacteriophage  They acc2M2S [et al. also showed that the EcoR124I R-M holoenzyme exists in equilibrium with a sub-assembly complex of stoichiometry R1M2S [2S) [Type I R-M enzymes fall into families based on complementation grouping, protein sequence similarities, gene order and related biochemical characteristics -8. Withi2M2S ,11,  ,14. HsdRM2S [2S) -18. HoweM2S [2S) .et al. [cis fashion indicating that the motor component of the HsdR subunit is able to 'grasp' adjacent DNA and pull this DNA through the enzyme-DNA-bound complex. According to the Studier model [The motor activity of Type I R-M enzymes is the mechanism through which random DNA cleavage is accomplished. Szczelkun et al.  showed ter model  cleavageer model ,22.DNA translocation has been assayed in bulk solution using protein-directed displacement of a DNA triplex and the kinetics of one-dimensional motion determined. The data shows processive DNA translocation followed by collision with the triplex and oligonucleotide displacement. A linear relationship between lag duration and inter-site distance gives a translocation velocity of 400 ± 32 bp/s at 20°C. Furthermore, this can only be explained by bi-directional translocation. An endonuclease with only one of the two HsdR subunits responsible for motion could still catalyse translocation. The reaction is less processive, but can 'reset' in either direction whenever the DNA is released  and HsdR(prrI), which provides a stable R1-complex, also gives a useful molecular motor that will not cleave the DNA that it translocates.As previously mentioned, the final step of the subunit assembly pathway of the Type I Restriction-Modification enzyme EcoR124I produces a weak endonuclease complex of stoichiometry R EcoprrI -25 Type et al. [via the HsdS subunit and that for some enzymes this may involve DNA [As can be seen from the above, DNA cleavage by Type I restriction enzymes occurs by means of a very unusual, and highly energy-dependent, mechanism. Therefore, these enzymes are believed to be involved not only as a defence mechanism for the bacterial cell, but also in some types of specialised recombination system controlling the flow of genes between bacterial strains ,27. A peet al.  detectedolve DNA .One of the major obstacles for the practical application of single molecule devices is the absence of control methods in biological media, where substrates or energy sources (such as ATP) are ubiquitous. Synthetic polymers offer a robust and highly flexible means by which devices based on single biological molecules can be controlled. They can also be used to link individual biomacromolecules to surfaces, package them or to control their specific functions, thus expanding the applicability of the natural molecules outside conventional biological environments.Moreover, a number of synthetic polymers have been recently developed that can potentially perform nanoscale operations in a manner identical to natural and engineered biopolymers. A key property of these materials is 'smart' behaviour, especially the ability to undergo conformational or phase changes in response to variations in temperature and/or pH. Synthetic polymers with these properties are being developed for applications ranging from microfluidic device formation,  through In the context of bio-nanotechnology we focus here on the uses of one particular subclass of smart materials, i.e. substituted polyacrylamides, but it should be noted that there are many more examples of synthetic polymers and engineered/modified biopolymers that exhibit responsive behaviour and new types and applications of smart materials are constantly being reported.Poly(N-isopropylacrylamide) (PNIPAm) is the prototypical smart polymer and is both readily available and of well-understood properties . PNIPAm Covalent attachment of single or multiple responsive polymer chains to biopolymers offers the possibility of exerting control over their biological activity as, in theory at least, the properties of the resultant polymer-biopolymer conjugate should be a simple additive function of those of the individual components. This principle is now being widely exploited in pharmaceutical development, as covalent attachment of, for example, PEG chains to therapeutic proteins has been shown to stabilize the proteins without losing their biological function -48. Polyvia site-directed mutagenesis was accessible for reaction with maleimide end-functionalised PNIPAm [Pioneering work in this area has been carried out by Hoffman, Stayton and co-workers, who engineered a mutant of cytochrome b5 such that a single cysteine introduced d PNIPAm . Since td PNIPAm ,51,50,51d PNIPAm .We are currently developing responsive polymers as a switch to control the EcoR124I motor function and are investigating this polymer-motor conjugate as part of an active drug delivery system. We aim for the practical demonstration of a nano-scale DNA packaging/separation and delivery system uniting the optimal features of both natural and synthetic molecules. In essence, we assemble a supramolecular device containing the molecular motor capable of binding and directionally translocating DNA through an impermeable barrier. To control the process of translocation in biological systems, where a constant supply of ATP is present, we have added to the motor subunit of EcoR124I the thermoresponsive poly(N-isopropylacrylamide) (PNIPAm), which, through its coil-globule transition, acts as a temperature-dependent switch controlling motor activity.via coupling of a maleimide-tipped linker on the synthetic polymer terminus to a cysteine residue. This residue has been selected, as it is both accessible and located close to the active centre on the R subunit of the motor. The protein-polymer conjugates are stable to extensive purification and, when combined with M2S complex, the activity of this conjugate motor system is similar to the native counterpart, but can be switched on or off as a result of thermally induced polymer phase transitions [PNIPAm copolymers with reactive end-groups are being attached to a preformed R subunit of the motor nsitions ,54.Thus the conjugation of the responsive polymer to the molecular motor generates a nano-scale, switchable device Fig , which cThe conjugation of the motor with synthetic polymers brings additional advantages. One such benefit arises from the ability to functionalise the polymer side chains or terminus in a way that allows attachment of the entire complex to surfaces for sensing and device applications.Therefore, although our hybrid polymer-protein conjugate was originally aimed at gene targeting  this system may also be used in building automated nano-chip sensors, therapeutic and diagnostic devices, where DNA itself would be a target, or where DNA might be used as a 'conveyor-belt' for attached molecules. The strength of the molecular motor has proven sufficient to disrupt most protein-DNA interactions and thus numerous processes and applications where highly localised force is required can also be envisaged.in vitro and especially in vivo opens up a whole host of possibilities in medicine, including drug or DNA delivery (e.g. gene therapy), but also where protection of a therapeutic is required under one biological regime and release in another (e.g. prodrugs conjugated to DNA which can be released by nuclease-mediated degradation at the site of action). In addition, this system may allow the generation of switchable nanodevices and actuators, controllable by changes in the synthetic copolymer structure as well as ATP-mediated DNA motion and may pave the way for biofeedback-responsive nanosystems. It can be used for nano-scale isolation of various biochemical processes in separate compartments connected via a tightly controlled shuttle device.The use of synthetic polymers offers a number of possibilities, which otherwise could not be exploited or would be difficult to take advantage of, if purely biological systems were used. Moreover, the combination of the properties of molecular motors with "smart" polymers has hitherto been unexplored and represents a novel concept in nanotechnology, which could ultimately lead to a wholly new class of molecular devices. Nanoscale control of molecular transport In essence, this concept bridges the disciplines of chemistry and biology by using a biological motor to control chemistry and a synthetic polymer to regulate biological processes.KF conceived the idea of using the modified R-M enzyme as a molecular motor and carried out, with co-workers, the molecular studies of the motor components, SSP carried out the polymer synthesis, polymer-motor conjugations and functional studies, CA designed and participated in the synthesis of smart polymers and DCG conceived of the study. All authors participated in study design and coordination as well as the reading and approval of the final manuscript


The hit criterion is a key component of heuristic local alignment algorithms. It specifies a class of patterns assumed to witness a potential similarity, and this choice is decisive for the selectivity and sensitivity of the whole method.group criterion combining the advantages of the single-seed and double-seed approaches used in existing algorithms. Second, we introduce transition-constrained seeds that extend spaced seeds by the possibility of distinguishing transition and transversion mismatches. We provide analytical data as well as experimental results, obtained with the YASS software, supporting both improvements.In this paper, we propose two ways to improve the hit criterion. First, we define the .Proposed algorithmic ideas allow to obtain a significant gain in sensitivity of similarity search without increase in execution time. The method has been implemented in YASS software available at  The central problem is therefore to improve the trade-off between those opposite requirements.Sequence alignment is a fundamental problem in Bioinformatics. Despite of a big amount of efforts spent by researchers on designing efficient alignment methods, improving the alignment efficiency remains of primary importance. This is due to the continuously increasing amount of nucleotide sequence data, such as EST and newly sequenced genomic sequences, that need to be compared in order to detect similar regions occurring in them. Those comparisons are done routinely, and therefore need to be done very fast, preferably instantaneously on commonly used computers. On the other hand, they need to be precise, i.e. should report all, or at least a vast majority of interesting similarities that could be relevant in the underlying biological study. The latter requirement for the alignment method, called the hit) a potential similarity. Those patterns are formed by seeds which are small strings  that appear in both sequences. FASTA  = S2[j..j + k - 1] for some i ≤ m and j ≤ n, then these two k-words form a seed denoted <i, j>. Two functions on seeds are considered: For a seed <i, j>, the seed diagonal d is m + j - i. It can be seen as the distance between the k-words S1[i..i + k - 1] and S2[j..j + k - 1] if S2 is concatenated to S1, For two seeds <i1, j1> and <i2, j2>, where i1 <i2 and j1 <j2, the inter-seed distance D is the maximum between |i2 - i1| and |j2 - j1|. The problem considered in this Section is to derive conditions under which two seeds are likely to be a part of the same alignment, and therefore should be grouped together. More precisely, we want to be able to compute parameters ρ and δ such that two seeds <i1, j1> and <i2, j2> have a probability (1 - ε) to belong to the same similarity iffWe first introduce some notations used in this section. Let D ≤ ρ,     (1)d - d| ≤ δ.     (2)|k-words occur at close diagonals.The first inter-seed condition insures that the seeds are close enough to each other. The second seed diagonal condition requires that in both seeds, the two ρ and δ.We now describe statistical models used to compute parameters p for a match and (1 - p) for a mismatch. To estimate the inter-seed shift Dk, we have to estimate the distance between the starts of two successive runs of at least k matches in the Bernoulli sequence. It obeys the geometric distribution of order k called the Waiting time distribution [Consider two homologous DNA sequences that stem from a duplication of a common ancestor sequence, followed by independent individual substitution events. Under this assumption, the two sequences have an equal length and their alignment is a sequence of matched and mismatched pairs of nucleotides. We model this alignment by a Bernoulli sequence with the probability ribution ,26:ρ such that the probability  is (1 - ε) for some small ε.Using this formula, we compute k inside a Bernoulli sequence of length x. In a Bernoulli sequence of length x, the probability of the event Ip,x,r of having exactly r non-overlapping runs of matches of length at least k is given by the following recursive formula:Note that the Waiting time distribution allows us to estimate another useful parameter: the number of runs of matches of length at least r non-overlapping seeds of length at least k inside a repeat of size x. The recurrence starts with r = 0, in which case  and is computed through the Waiting time distribution.This gives the probability of having exactly  allows us to infer a lower bound on the number of non-overlapping seeds expected to be found inside a similarity region. In particular, we will use this bound as a first estimate of the group criterion introduced later.The distribution Indels (nucleotide insertions/deletions) are responsible for a diagonal shift of seeds viewed on a dotplot matrix. In other words, they introduce a possible difference between d and d. To estimate a typical shift size, we use a method similar to the one proposed in [posed in  for the q at each of l nucleotides separating two consecutive seeds. Under this assumption, estimating the diagonal shift produced by indels is done through a discrete one-dimensional random walk model, where the probability of moving left or right is equal to q, and the probability of staying in place is 1 - 2q. Our goal is to bound, with a given probability, the deviation from the starting point.Assume that an indel of an individual nucleotide occurs with an equal probability i after l steps is given by the following sum:The probability of ending the random walk at position  and consider the power Pl(x) = al.xl +…+ al-.xl-. Then the coefficient ai computes precisely the above formula, and therefore gives the probability of ending the random walk at position i after l steps. We then have to sum up coefficients ai for i = 0,1, -1, 2, -2,..., l, -l until we reach a given threshold probability (1 - ε). The obtained value l is then taken as the parameter δ used to bound the maximal diagonal shift between two seeds.A direct computation of multi-monomial coefficients quickly leads to a memory overflow, and to circumvent this, we use a technique based on generating functions. Consider the function


Acacia tree species. This study investigated germination of Acacia karroo, A. luederitzii and Dichrostachys cinerea, thought to be increasing in density, as well as the historically successful encroaching woody species, A. nilotica, in savanna grassland, Hluhluwe-iMfolozi Park, South Africa. A. karroo is thought to be replacing A. nilotica as the dominant microphyllous species in the park. We tested the hypothesis that observed increases in certain woody plants in a savanna were related to seed germination and seedling establishment. Germination is compared among species for burnt and unburnt seeds on burnt and unburnt plots at three different locations for both hot and cool fires.While fire has been used in some instances to control the increase of woody plants, it has also been reported that fire may cause an increase in certain fire-tolerant Acacia karroo showed higher germination  levels and better establishment . Seeds of the shrub Dichrostachys cinerea did not germinate in the field after fire and it is thought that some other germination cue is needed. On average, burning of A. karroo, A. nilotica and A. luederitzii seeds did not affect germination. There was a significant difference in the germination of burnt seeds on burnt sites (4.5%) and burnt seeds on unburnt plots (2.5%). Similarly, unburnt seeds on unburnt sites germinated better (4.9%) than unburnt seeds on burnt sites (2.8%).A. karroo and that fires may not be hot enough or may occur at the wrong time of year to control A. karroo establishment in HiP. Although germination and establishment of A. karroo was higher than for A. nilotica a competitive advantage after fire could not be shown.We conclude that a combination of factors may be responsible for the success of  Acacia karroo Hayne [A. nilotica (L.) Willd. Ex Del. subsp. kraussiana (Benth.) Brenan, [Dichrostachys cinerea (L.) Wight & Arn. and A. luederitzii Engl. var. retinens (Sim) Ross & Brenan are also thought to contribute to this phenomenon.The increasing density in the woody component of savannas has been widely reported -5 with soo Hayne ,7 and A. Brenan, ,9. in soIn hard seeded legumes dormancy is broken by rupturing part of the seed coat. The rupturing of the seed coat may be induced by heat from fire  enablingWhile some studies report that a decrease in grass cover favours the establishment of woody seedlings due to reduced competition ,21, otheAcacia species are shade intolerant resulting in decreased seedling establishment in shady areas [Acacia species have been found to be tolerant of low light conditions and may even experience increased seedling survival [Some dy areas ,23,24. Osurvival .Acacia densities [The frequency of fires may affect the direction of change in woody plant density . While iensities , it is aensities . This coA. nilotica, A. karroo, A. luederitzii and Dichrostachys cinerea in Hluhluwe-iMfolozi Park (HiP), where an increase in woody plant density over the past 40 years has been reported [A. karroo is apparently replacing A. nilotica as the dominant microphyllous element [This study investigated the direct (heat) and indirect  effects of fire on seed germination and seedling establishment of reported -28. It h element ,28.This study reports on the effects of burning, fire intensity and burning of sites on germination; burning, fire intensity, burning of sites and grass length (shade) on seedling establishment and specific species responses to treatments (treatment species interactions).D. cinerea germinated in the field and it was therefore excluded from the model for the field experiment. Testing for differences among treatments was based on the maximum number of seedlings for each species at each location over the 31-week period  indicating that the model was a good fit. Location and species were the only main effects significantly affecting germination  indicating that the model fitted the data well. Location and species were the only main effects significantly affecting establishment in the field Table . Acacia  species .Interaction terms, location × burn status, location × grass length, location × site burn status, burn status × site burn status and grass length × site burn status had a significant effect on establishment Table  Figure .Acacia karroo was 16.2 times more likely to establish than A. nilotica. Similarly A. luederitzii was 1.4 times more likely to establish than A. nilotica while A. karroo had 11.2 times more chance of establishing than A. luederitzii. Species differences in establishment for the various treatments are given in Table The odds of establishment were 8046.2 times less at Le Dube than at Nombali and 5850.5 times less at Le Dube than at Seme. 1.4 times more seedlings were likely to establish at Nombali than at Seme.D. cinerea in the field suggests that some disturbance other than fire is needed to cause a release from dormancy and commence germination.The lack of germination of A. karroo seeds germinated, which was higher than the other two species. Story [A. karroo, with 6.6% of seeds germinating under natural conditions in the field. He also found that A. karroo germination was erratic, with germinations still being recorded after 423 days. This was similar to what was found in this study, with the number of A. karroo seedlings still increasing until the end of the experiment. Acacia nilotica also showed dormancy with sporadic germination events over the 31-week period. Acacia luederitzii did not show dormancy with most germinations taking place in the first 3 weeks of the experiment. Acacia nilotica has a thick seed coat, which could account for it's poor level of germination. One would predict increased germination of burnt seeds due to a breaking of dormancy [Acacia species are temperature specific, suggesting a temperature threshold for germination [et al. [A. nilotica seeds to be highly vulnerable to fire with a 80% mortality of seeds on the soil surface. The current study, however, found no difference in germination between burnt and unburnt seed or seeds burnt at different temperatures. This finding is inconsistent with the recent study by Kanz [Germination of all species in the field was low. As the seeds relied on cotyledons for food, soil moisture may have been a limiting factor. As rainfall was not recorded, this should be kept in mind when interpreting the results. Five point one percent of s. Story  found sidormancy , but thimination ,20. This [et al.  found A. by Kanz  had simiAcacia drepanolobium in Kenya.Location had a significant effect on germination with Le Dube having very low germination overall and Seme having the most germinations. Germination at Nombali and Seme were similar. Site-specific effects may be attributed to various factors such as microclimate or soil type. Sites may also have different water infiltration rates and runoff, which may result in differences in germination levels. Okello and Young , howeverThe current study did not find a difference in the number of seedlings in burnt and unburnt patches. While neither burning of seeds nor burning of sites had any effect on germination, the interaction factor proved significant with unburnt seeds showing increased germination in unburnt sites as did burnt seeds in burnt sites. Kanz  also fouCeratotherium simum) grazing lawn with very short grass, which may lead to seeds losing moisture through more direct sunlight. This suggests a similar pattern to the seed burn × site burn interaction. The tall grass site at Seme had higher germination than any of the other tall or short grass sites. This may be due to possible site-specific effects mentioned earlier.Whilst more seeds germinated in short grass at both Le Dube and Nombali, those at the short-grass site (Seme) had higher levels of germination in tall grass sites. The short grass site at Seme is a white rhinoceros  may be detrimental to the germination of seeds  while colability . Tall grNo species factor interactions were observed suggesting that though species had different germination levels, they did not respond differently to the treatments.The same factors and interactions found to be significant influences on germination were found to influence establishment. This was expected as increased germination for these treatments would result in better establishment. The interaction patterns for most of the treatments, however, were different to those of the germination model. Owing to the low levels of germination, interspecific and intraspecific competition was thought to play a minor role in seedling establishment.A. karroo seedlings. This species is known to be dependent on moisture availability for survival [Le Dube again had the least seedlings at 31 weeks while Nombali had the best establishment. Seme, which had the highest level of germination, had establishment levels somewhere between that of the other two sites. It is again suggested that this may be due to soil or rainfall factors. Forty-five out of forty-eight seedlings established at Nombali and thirty-three out of thirty-five at Seme were survival  and thesA. karroo seedlings had established as opposed to six of A. nilotica and three of A. luederitzii. The high germination, but poor survival of A. luederitzii suggests that the absence of this species in the Hluhluwe section of HiP is not due to seed limitation or germinability, but possibly due to environmental factors decreasing its ability to establish. The differences in seedling survival between species are consistent with those reported by Kanz [A. karroo than A. nilotica.At week 31, 87  by Kanz  who founThe location × grass length interaction revealed the same patterns as for germination with regards to Nombali and Seme with Seme showing better establishment in tall grass and Nombali showing better establishment in short grass. There was no difference between establishment on tall and short grass at Le Dube. The short grass site at Nombali had the highest number of seedlings surviving at week 31.The grass length × site burn interaction displayed the same patterns as for the germination model, but this was not the case for the seed burn status × site burn status interaction. While unburnt seeds still did well on unburnt sites, burnt and unburnt seeds showed decreased establishment on burnt sites suggesting that, as a result of increased irradiance, burnt (open) sites may not hold sufficient moisture for seedlings to survive.Acacia species have been reported elsewhere [The interaction effects found to be significant for establishment only, both suggest the importance of fire temperature. Location × seed burn status and location × site burn status could both relate to the different grass lengths, and thus specific fire temperatures, at the three sites. Temperature sensitivity in lsewhere ,14,17,20A. karroo during the first year. Similarly, there was no difference in establishment of A. karroo in burnt or unburnt and tall or short grass sites. Smith & Goodman [A. nilotica seedlings, however, almost exclusively occurred away from canopy cover, suggesting an inability to establish in shaded environments. Acacia tortilis also showed a greater proportion of established seedlings in open than shaded areas [A. nilotica in tall and short grass, but its establishment was so low that no real prediction can be made.Chirara, Frost & Gwarazimba  found th Goodman  reporteded areas . We did A. karroo is strongly moisture dependent [A. karroo is more likely to invade moist rather than semi-arid grassland. This suggests that Hluhluwe Game Reserve, being an area with moist grassland, would be more prone to invasion by A. karroo. It has also been reported that A. karroo has the ability to withstand fire [A. karroo in the field and may be the reason for A. karroo's success over A. nilotica as the most important encroaching Acacia species in HiP at present. The literature does, however, suggest that high intensity fires may result in seed mortality [A. karroo seedlings survive fires from as little as 12 months of age [A. karroo. It has been reported that spring burns are the most effective )A. karro2 fenced protected area comprising the former Hluhluwe and iMfolozi Game Reserves, and the corridor of land that links the areas. The park has a moderate coastal climate, ranges in altitude from 60 – 750 m above sea level [The study was done in HiP, KwaZulu-Natal, South Africa . HiP is a 960 kmea level  and has ea level . Periodiea level . The ranea level .Most of Hluhluwe Game Reserve is found on rocks of the Ecca and Beaufort series with some basalt in the east . King 3 identifiThe main soils types associated with the Ecca and Beaufort series are Swartland and Sterkspruit, while areas of Shortlands, Milkwood and Bonheim series are found in association with the dolerite regions . They alThe vegetation in the park has been described as bushveld – savannah comprising five broad vegetation types . The thiAcacia luederitzii occurs in large numbers in certain areas of the iMfolozi part of the reserve but is mostly absent from the Hluhluwe and Corridor sections. Acacia nilotica, A. karroo and D. cinerea are found throughout the park. As opposed to the scattered trees found in iMfolozi, A. nilotica covers extensive areas of Hluhluwe and the Corridor and is usually found below the 300 m contour [A. karroo – D. cinerea induced thicket throughout the area, but particularly in the Corridor and Hluhluwe Reserves. Acacia luederitzii seeds used in this study were therefore collected in iMfolozi Game Reserve while those of the other species were collected in Hluhluwe.The field experiment took place in the Hluhluwe and Corridor sections of the HiP.  contour . Whatele contour  describeA. nilotica, A. karroo, A. leuderitzii and D. cinerea was tested in a field experiment. Seeds of all species were collected between May and August 2000. Parasitized seeds were extracted. Prior to planned management burns, six groups of seeds were placed in tall grass  and six in short grass (shorter than 0.10 m) at three locations . Tall grass produces hotter fires than short grass due to increased fuel load, which increases available heat energy [Dichrostachys cinerea seeds were only put out at Seme and Nombali. Each group contained 22 A. nilotica, 25 A. karroo, 10 A. leuderitzii and 10 D. cinerea seeds. Seeds were placed on the soil surface a day before each of the burns . This is considered the natural situation for the seeds with soil stored seed banks being virtually non-existent [The effect of fire, fire intensity and burning of sites on the germination of seeds of t energy . Sites wexistent . Seme anWe thus applied 96 possible seed treatment combinations for investigating factors affecting germination in the field (4 species × 2 burn treatments × 3 locations × 2 location burn treatments × 2 fire intensities).A. nilotica, A. karroo, A. leuderitzii and D. cinerea, data as on week 31 of the field experiment described above were used. Seedlings were considered to be established when they were rooted in the ground and the cotyledons replaced with leaves. Establishment was based on the total number of seeds.To test the effect of fire, fire intensity, burning of sites and grass length (shade) on seedling establishment of ®" [The "STATISTICA®"  Generali®" . In bothwhere = the log of variable 1 and 2 at different levels of the factors as given belowλ' = the overall mean effect of the categories = the effect of the jth species  = the effect of the kth location  = the effect of the lth seed burn status  = the effect of the mth grass length  = the effect of the nth site burn status  = the interaction effect between the jth species and the kth location = the interaction effect between the mth grass length and the nth site burn status.The logit model may be written as a generalized linear model as follows:, , , , , , , , ,  and  are parameters to be estimated from the data and B, C, D, E and F refer to the explanatory variables species, location, burn status, grass length and site burnt status respectively. The estimated parameters for the GLZ were used to obtain the estimated parameters for the logit model. The estimated parameters of the odds were calculated for each factor or combination of factors (including the intercept) as the exponent of the estimated parameters of the logit model. The estimated odds of germination under any condition were then calculated as the product of the estimated parameter of the odds of the intercept (estimated geometric mean odds) and the factor or combination of factors in question. The odds of germination for significant treatment combinations were compared.where The predicted number of seeds germinating and seedlings establishing as calculated with the model based on presence/absence data, were seen as being appropriate for interpretation as summaries of the data. Thus, differences in the predicted mean number of seeds germinating and seedlings establishing  were illustrated graphically for each significant treatment combination.MW designed the experiment, participated in fieldwork, performed the statistical analysis and drafted the document. MJS participated in fieldwork, the coordination of the study and drafting of the document. JJM supervised the work and assisted in the drafting of the document. All authors read and approved the final manuscript.Acacia seeds in HiP. Gives parameters of the logit model and estimated odds of germination for the various levels of the factors used.The parameters of the logit model and odds, estimated odds of germination and the ratio of germination to non-germination for the factors included in the model for germination of certain Click here for fileAcacia species in HiP. Gives parameters of the logit model and estimated odds of establishment for the various levels of the factors used.The parameters of the logit model and odds, estimated odds of establishment and the ratio of establishment to non-establishment for the factors included in the model for establishment of certain Click here for fileOdds ratios for all significant interactions of the establishment model. Compares the odds of establishment between different levels of the factors used.Click here for file


Our purpose was to determine whether AFI<5 cm after preterm premature rupture of the membranes (PPROM) is associated with an increased risk of perinatal morbidity.We performed a prospective cohort study of 95 singleton pregnancies complicated by preterm premature rupture of the membranes (PPROM) with delivery between 26 and 34 weeks gestation.2 and Fisher exact tests. Continuous data were evaluated for normal distribution and tested for significance with the student t test.Patients were categorized in two groups on the basis of amniotic fluid index<5, (AFI<5 cm)(n = 26) or AFI ≥ 5 cm (n = 69). Categorical data were tested for significance with the χAll 2-sided p values < 0.05 were considered significant.Both groups were similar with respect to selected demographics, gestational age at rupture of the membranes, gestational age at the delivery, birth weight. Both groups were similar with respect to selected variable, latency until delivery, early onset neonatal sepsis, RDS and neonatal death. Patients with AFI<5 cm demonstrated greater frequency of C/S delivery for non reassuring fetal tests (23%vs 2.8%) (p = 0.001). Our study demonstrated that patients in group I had a significant increase in the frequency of clinical chorioamnionitis (P < 0/001). Post partum infections were not seen in 2 groups.An AFI<5 cm after PPROM between 26 and 34 weeks gestation is associated with an increased risk of maternal infections and frequency of C/S. Preterm premature rupture of the membranes (PPROM) is one of the most common complications of the pregnancy.Preterm PROM is an important cause of perinatal morbidity and mortality, particularly because it is associated with brief latency from membrane rupture to deliver, perinatal infection, and umbilical cord compression due to oligohydramnios. PPROM is multifactor in nature. In any given patient, one or more path physiologic processes may be evident. Choriodecidual infection or inflammation appears to play an important role in etiology of preterm PROM, especially at early gestational ages.It has also been proposed that amniotic fluid posse's certain bacteriostatic properties that protect against potential infections processes and that a decrease in amniotic fluid volume may impair the gravid women ability to combat such infections, this latter hypothesis was tested by Vintziuleos, et al .These investigations demonstrated that patients with olighhydramnios (AFI<5), were at greater risk of having chorioamnionitis and subsequent sepsis in the neonate ,8. Our pWe performed a prospective cohort study of infants delivered between 26 weeks – 34 weeks gestation after preterm premature rupture of the membranes.This study was performed at Vali-e-Asr hospital at the medical university of Tehran between October 2000 and February 2002.All patients were at between 26 and 34 of weeks of pregnancy as best estimated by LMP, and confirmed by ultrasonography. In all patients rupture of the membrane was diagnosed by sterile speculum examination using pooled fluid, fern test and Nitrazine paper test.Patients with clinical chorioamnionitis, nonreassuring fetal status, obstetrical indication for immediate delivery, major congenital anomalies and advanced labor , and a growth-restricted fetus at initial admission were excluded. 95 singleton pregnancies have observed until 34 weeks of gestation. At admission, all patients had an ultrasonographic examination, which included confirmation of the estimated gestational age, and cumulative 4 guardant AFI measurements, as previously described by Phelan et al. [All patients received antibiotic prophylaxis at admission consisting of Ampicillin plus Erythromycin for 7 days following. In addition all patients received a single course of Betamethasone, consisting of two 12 mg Betamethasone injections during the first 24 hours after admission. Fetal surveillance incorporated daily non-stress testing. For fetuses with non-reassuring, non stress test results, biophysical profile assessments were performed. Indications for delivery included: labor, the diagnosis of clinical chorioamnionitis or non-reassuring fetal test results. Eligible patients were subsequently categorized into 2 groups on the basis of the admission AFI measurement. Patients in group 1 were those with an AFI<5 cm, whereas those in group 2 had AFI ≥ 5 cm. The 2 groups were compared for demographic characteristics, the estimated gestation age at both rupture of the membranes and delivery, latency until delivery, mode of delivery, birth weight, the development of clinical chorioamnionitis, postpartum endometritis, early onset neonatal sepsis and respiratory distress syndrome.Neonatal sepsis was diagnosed by positive blood, urine, or cerebrospinal fluid cultures. Possible neonatal sepsis was diagnosed when two or more of the following criteria were present: white blood cell count less than 5000/mm3, polymorphonuclear counts less than 1800/mm3, ratio of bands to total neutrophil counts greater than 0.2. Early onset neonatal sepsis was defined as sepsis in a neonate with positive culture results or possible sepsis within the first 48 hours of life and prior to the antibiotic administration. The clinical diagnosis of chorioamnionitis was made in presence of two or more of the following criteria: maternal fever greater than 38 C, maternal tachycardia (120 beats per minute or more), leukocytosis , fetal tachycardia (greater than 160 beats per minute), uterine tenderness, and foul-smelling amniotic fluid.2 and Fisher exact tests. Continuous data were evaluated for normal distribution and tested for significance with the student t test.Categorical data were tested for significance with the χAll 2-sided p values <.05 were considered significant.A total of 95 patients with preterm premature rupture of membranes were included in the study;26 were included in group I (AFI<5 cm) and 69 in group II (AFI ≥ 5 cm). The 2 groups were similar with respect to maternal age, Parity, and gestational age at admission . Post partum infections were not seen in 2 groups.Early onset neonatal sepsis, respiratory distress syndrome (RDS), neonatal deaths were not significantly different between the 2 groups. Table Complications of preterm premature rupture of membranes count for approximately 25% to 33% of all preterm deliveries.Approximately, 75% of women will be delivered within 1 weeks of presentation ,11.Expectant management with antenatal antibiotics and corticosteroid administration are recommended the standard of care in the setting of PPROM at gestational ages of ≤ 34 ,4,9,10. Amniocentesis and amniotic fluid volume have been advocated as a useful adjunct for identifying these patients Several sOther investigators have demonstrated an association between the development of chorioamnionitis and a shorter latency in patients with PPROM -8. Post Our study demonstrated that the patients with oligohydramnios were more likely to undergo cesarean delivery because of non-reassuring fetal heart rate patterns and is consistent with the findings of these other studies ,6,8.This study didn't show an increased frequency of early onset sepsis in the group I (AFI<5 cm), because all newborn infants in the study were treated possible sepsis with clinical symptom and laboratory evidence. 7 of the included neonates had positive blood cultures or spinal fluid cultures; as a result, there was not sensitive mechanism for appropriately determining the diagnosis of early sepsis. The negative cultures in the neonates with possible sepsis may be related to inadequate culturing techniques or the inherent difficulty encountered by most laboratories in isolating anaerobic bacteria.Perhaps, Diagnosis of early onset neonatal sepsis and close observation for early signs of sepsis and more aggressive evaluation and early treatment for neonatal sepsis have decreased early onset sepsis in 2 groups. Preterm premature rupture of the membranes is associated with a significant decrease in the frequency of neonatal respiratory distress syndrome. In the Sims EJ study (2002), The frequency of respiratory distress syndrome in the neonate complicated with PPROM was (17%).11 This study evaluate the effect of AFI on the frequency of respiratory distress syndrome among two group that are complicated with PPROM. The frequency of respiratory distress syndrome in the neonate was not significantly lower in the group (II) than in the group I. (11/8% vs26/1%) (P < .01). The identification of oligohydramnios, defined as an AFI<5 cm, patients with preterm PPROM appear to indicate a significant risk of chorioamnionitis and early onset neonatal sepsis. These finding can aid in the counseling of patients with PPROM and may have several clinical application.Management of PPROM requires an accurate diagnosis as well as evaluation of costs and the risks and the benefits of continued pregnancy or expeditious delivery. It is important that the patient be well informed regarding the potential for subsequent maternal, fetal, and neonatal complications regardless of the management approach.The pre-publication history for this paper can be accessed here:


Joining a model for the molecular evolution of a protein family to the paleontological and geological records (geobiology), and then to the chemical structures of substrates, products, and protein folds, is emerging as a broad strategy for generating hypotheses concerning function in a post-genomic world. This strategy expands systems biology to a planetary context, necessary for a notion of fitness to underlie (as it must) any discussion of function within a biomolecular system.Sus scrofa that encode cytochrome P450 aromatases–enzymes that convert androgens into estrogens. The evolutionary history of the vertebrate aromatase gene family was reconstructed. Transition redundant exchange silent substitution metrics were used to interpolate dates for the divergence of family members, the paleontological record was consulted to identify changes in physiology that correlated in time with the change in molecular behavior, and new aromatase sequences from peccary were obtained. Metrics that detect changing function in proteins were then applied, including KA/KS values and those that exploit structural biology. These identified specific amino acid replacements that were associated with changing substrate and product specificity during the time of presumed adaptive change. The combined analysis suggests that aromatase paralogs arose in pigs as a result of selection for Suoidea with larger litters than their ancestors, and permitted the Suoidea to survive the global climatic trauma that began in the Eocene.Here, we report an example of such an expansion, where tools from planetary biology were used to analyze three genes from the pig This combination of bioinformatics analysis, molecular evolution, paleontology, cladistics, global climatology, structural biology, and organic chemistry serves as a paradigm in planetary biology. As the geological, paleontological, and genomic records improve, this approach should become widely useful to make systems biology statements about high-level function for biomolecular systems. The emergence of complete genomes for many organisms, including humans, has created the need for hypotheses concerning the "function" of specific genes that encode specific proteins. While "function" is interpreted by different workers in different ways , DarwiniSystems hypotheses can be supported by information from many areas. Geology, paleontology, and genomics, for example, provide three records that capture the natural history of past life on Earth. At the same time, structural biology, genetics, and organic chemistry describe the structures, behaviors and reactivities of proteins that allow them to support present life. It has been appreciated that a combination of these six types of analysis provides insights into functional behavior of proteins that cannot be provided by any of these alone . Over thThe aromatase family of proteins offers an interesting system to illustrate the power of this combination as a way to create hypotheses regarding protein function within a system . These hAromatases are cytochrome P450-dependent enzymes that use dioxygen to catalyze a multistep transformation of an androgenic steroid (such as testosterone) to an estrogenic steroid (such as estradiol) Figure . The proDifferent numbers of aromatase genes are found in different vertebrates. Two aromatase genes are known in teleost fish ,7. Only Sus scrofa). Three different mRNA molecules had been reported in different tissues from pig [A still different phenomenology is observed in the pig  and ox (Bos taurus) were constructed for transitions at the silent sites of two-fold redundant codon systems where the encoded amino acid was conserved between the species [f2 = 0.87, corresponding to a TREx distance of kt = 0.332. As the fossil record constrains the date of divergence of these two lineages to be 60 ± 5 Ma [Sus scrofa and Bos taurus project an equilibrium value for f2 = 0.54 [-9 transitions/silent site/year during the time interval that separates these lineages.To calibrate the silent TREx clock, inter-taxa histograms relating pig , birds versus mammals (f2 = 0.612), primates versus ungulates (f2 = 0.823), and horses versus artiodactyls (f2 = 0.828). Assuming a time-invariant single lineage first order rate constant of 3.6 × 10-9 changes/site/year and an equilibrium f2 of 0.54, the corresponding dates of divergence are calculated to be 435, 258, 67, and 65 Ma respectively, with the oldest dates being the least precise. The last three of these dates of divergence are similar to those suggested by the paleontological record [f2 values. A tree for the artiodactyl lineage was constructed from the corresponding TREx distances . Briefly, the rate constants for transitions and transversions are more different than the two rate constants for purine-purine and pyrimidine-pyrimidine transitions. Further, nucleotide frequencies can be used to calibrate the end equilibrium points for two-fold redundant codon systems directly, and this permits an "approach to equilibrium" formalism, well known in chemical kinetics, to be applied [The TREx clock is not widely used. It may, however, provide more accurate dates in regions where synonymous transitions have not equilibrated than conventional clocks that combine data from synonymous transitions and synonymous transversions, or from non-synonymous changes. A comparison of different clocks will be provided in detail elsewhere , corresponding to a range in the time of divergence of 26–38 Ma. The TREx distances from the divergence of all of the porcine aromatases and the modern forms ranges from 0.082–0.116, corresponding to dates of divergence in the range of 27–39 Ma. This suggests that the three aromatase paralogs diverged in the late Eocene to mid Oligocene.Sus scrofa. Both peccary and babirusa seminal plasma  was probed by PCR (Polymerase Chain Reaction) amplification using exon 4-specific primers [To further correlate the duplication of the genes with the fossil record, genomic DNA was analyzed from relatives of  primers . Bands h primers ,34.Diacodexis, from the early Eocene ca. 55 Ma) [These data are consistent with an evolutionary model that holds that the ancestor of pig and oxen (approximated in the fossil record by . 55 Ma)  containeSus). Note that there has long been the tacit assumption that large litters in suoids represent the primitive artiodactyl condition. Large litters are primitive for mammals in general, and because suoids are plesiomorphic in some anatomical conditions relative to other artiodactyls , they have been assumed to be plesiomorphic in other respects.This does, however, suggest that the emergence of the aromatase paralogs was approximately contemporaneous with the emergence of a litter in the Suoidea larger than that found in the ancestral artiodactyl condition. While ruminant and camelid artiodactyls have only one-two young per litter, suoids in general have at least two young per litter (as seen in peccaries) and most suines (true pigs) routinely have three-four young  shows that they also only had one-two young [Other data suggest that small litters are in fact the primitive artiodactyl condition. Tragulids (mouse deer or chevrotains) are surviving small, primitive ruminants that are not too dissimilar from wo young ,37. A clA/KS value), strongly suggests functional change when the value is significantly greater than unity [A/KS values were reconstructed for individual branches of the evolutionary tree derived from the Darwin bioinformatics workbench (see Methods) using a distance matrix and ancestral states constructed by the method of Messier and Stewart [A/KS value of 0.35. A higher KA/KS value of 0.85 is found in the episodes of evolution near when the pig aromatases diverged. While a KA/KS value of 0.85 does not require the conclusion that positive selection occurred during the emergence of these aromatase paralogs, an inference based on the magnitude of KA/KS in one branch, relative to the KA/KS value for typical branches [The approximate correlation in time of the aromatase divergence in Suoidea with the enlargement of litters in Suoidea suggests, as a hypothesis, that the two are functionally related. To expand on this hypothesis, we sought genomic signatures of functional change within the aromatase paralogs. The number of non-synonymous changes in the gene divided by the number of the synonymous changes, normalized for the number of non-synonymous and synonymous sites  was again found in the divergence of the swine isoforms on the branch leading to the ancestor of the placental and embryonic enzymes following their divergence from the pig ovarian enzyme. The distribution of substitutions along this branch is consistent with altered functional constraints for the placental and embryonic enzymes compared with their extinct and extant counterparts  , a homolA/KS values  involving pig paralogs was not random over the protein surface. Rather, the sites are clustered near the substrate binding pocket, and in a region of the surface believed to contact the co-reductant protein, as identified by mutagenesis experiments in the homolog [In contrast, the distribution of sites accepting amino acid replacements during the episode of rapid sequence evolution of branch 1 . The events of interest (such as the extinction of dinosaurs) are frequently distant in time, or require a passing of time (as for speciation), making them difficult to reproduce in a laboratory. The scale of the concepts involved  also does not lend these concepts to laboratory models and laboratory-controlled tests. Further, a reductionist approach, even when available, will not necessarily generate data that are relevant to the big issue that concerns the natural historian. The emphasis on data and proof has ameliorated the worst excesses of storytelling in natural history, with enormous positive impact.In part to make this connection, natural historians have sought to change the research paradigm in their field to favor quantitative data directed towards the "proof" of hypotheses over "story telling". Proving hypotheses is difficult in natural history  for a protein that had been selected for functional reasons. Today, the typical researcher knows the structure of many proteins, and wishes to select one for expression and study based on a hypothesis about its potential function.Here, the fact that any definition of function, which must make reference to fitness, requires some systems, ecological, or planetary context, makes the natural historian a natural source of hypotheses. Their full reductionist armamentarium is available in the laboratory to test and explore any hypothesis that the natural historian might provide. The biomedical researchers may like some guidance from the natural historian to narrow the broad selection, or to shorten the random walk, if only slightly.For this purpose, the forswearing by natural historians of storytelling has come at a most inopportune time. To the modern natural historian, creating hypothesis can easily be regarded as "storytelling". They are reluctant to do so, and may criticize as atavistic colleagues who do.This has created a vacuum in the scientific community. Very few laboratories exist that can draw upon an expertise in natural history to generate stories that create hypotheses for the researcher working in experimental biochemistry and molecular biology.This article is designed in part to illustrate how this vacuum might be filled. Here, we do not just tell a story based on natural history, or even a story based on natural history supplemented with physiology and molecular sequence data. Rather, we show how the addition of other data, including data from X-ray crystallography, can make a story sufficiently rich that it can be viewed as being internally consistent with a wide range of independent data drawn from independent sources. This creates a hypothesis that is more than a story, even if it is less than proven.Sus. Steroid production by the porcine embryo is tightly controlled by the transient expression of aromatase and 17-hydroxylase (P450C17) between days 10 and 13 [With aromatase, the congruence of our different analyses makes a compelling suggestion that the three aromatase paralogs in pigs arose by two duplication events in the late Eocene or early Oligocene. The emergence of the aromatase paralogs corresponded approximately in time to the emergence of larger litter size in suines. This implies that the two duplication events are functionally related to the larger litter sizes. This inference is consistent with the physiological impact of estrogen synthesis by these paralogs in 0 and 13 ,21,52. I0 and 13 . AdequatThis is consistent with a structural biological analysis that correlates specific amino acid replacements with specific changes in the substrate and product specificity of the protein . InteresA/KS ratio of 0.93 would not, by itself, compel any particular interpretation. Its implications are greater given the relatively low KA/KS ratios of other branches of the tree. But the addition of crystallographic information, itself not compelling, makes a combination that is more compelling.It should be noted that the hypothesis is supported by the combination of data that individually would not have strength past storytelling. Thus, the KA/KS ratio  compel the inference of an episode of adaptive change. Intriguingly, these changes are also clustered in the same regions of the structure as those changing along branch 1 leading to the stem fetal/placental enzyme, near the substrate and co-reductant binding sites. This implies that the substrate/product specificity of the ancestral aromatase protein was not like that of either the human or the pig placental forms, but rather reflects features that arose convergently in these two species [Further, this hypothesis generation itself generates discoveries that might lead to their own hypotheses. An analysis of the evolutionary branches separating pigs and humans suggests an additional episode of adaptive change. The branch leading to the ancestor of human aromatase (branch 3) has a remarkably high K species .Notably, four of the sites  that undergo replacement during the emergence of pig placental aromatase from the last common ancestor are the same as four that arose in the emergence of the human aromatase from its last common ancestor. Of these, the amino acid replacements are identical at two sites . The probability associated with randomly observing this pattern is extremely low (0.000021) . An addiIt is important to point out that even an analysis this broad is likely to cover only a small part of a complicated reproductive endocrinology that must be associated with larger litter sizes. For example, the exact nature of the products produced by individual aromatases remains controversial, and may be different in laboratory studies depending on the conditions where they are studied ,60-62. Tet al. [Further, an elegant recent study by Corbin et al.  identifiet al. [In fact, some data suggest that uterine exposure to androgens severely decreases litter size and embryonic survival during the time of maternal recognition of pregnancy . This iset al.  that theDiacodexis, see Figure It goes without saying that still more factors might be associated with an increase in litter size from one-two  young, a reproductive strategy apparently not available to larger, cursorial (running-adapted) ungulates, which give birth to precocial  young that are fully locomotory at birth [Nevertheless, the first aromatase duplication, shared by pigs and peccaries, appears to have happened in the late Eocene (recognizing the error associated with these dates), around 35 Ma Figure . This waat birth .The second aromatase duplication, with the ensuing capacity to produce multiple young, probably occurred within the family Suidae, some time during the Oligocene. The molecular data suggest dates of divergence between porcine fetal and placental aromatases as between 27–38 Ma, and the earliest known suid is of early Oligocene age , around b places this data at ca. 18 Ma (data not shown). Clearly, further study is warranted.This type of speculation opens questions. For example, the babirusa (an Indonesian pig) is reported to have average litters of one-two individuals ,69. WhilThe aromatase family offers an example where a combination of phylogenetic analysis, molecular evolutionary analysis, and chemical analysis set within the context of the paleontological and geological records, and supported by contemporary bioinformatics and molecular modeling tools, permits a higher order level of hypothesis generation concerning the function of proteins. Rather than simply an Enzyme Commission number (E.C. 1.14.14.1 for aromatase), a description of catalytic activity (the enzyme oxidizes testosterone), or a description of the regulatory pattern (the protein expressed between day 10 and 13), this type of analysis can generate a truly functional hypothesis: that the embryonic enzyme oxidizes testosterone as a way of managing the larger litter sizes that emerged in the Suoidea during a time of dramatic planetary cooling (ca. 35 Ma).Such hypotheses set a higher bar, and a more useful standard, for the field of systems biology. Evolutionary theory holds that the only mechanism for obtaining functional behavior in a biological system is natural selection. Selection, based on a frequently poorly defined concept of "fitness", is determined by a context that not only includes the cell and tissue, but also the organism, the ecosystem, and a changing planet . One caninter alia, from a study of Indo-European languages. Proto-Indo-European (PIE) language had words for "pig"  following their domestication. Evidence for ancient domestication of pigs comes, ah, etc. ), indicaah, etc. . With thcbrg@inf.ethz.ch.Calculations were done under the RedHat Linux 6.3 operating system on an Intel-Pentium III instrument using Blackdown's Java-SDK 1.1.8. PAML calculations were done on an IBM PC using the Unix operating system. Sequence analyses were aided by the DARWIN bioinformatics package . The DARInitially, pairwise alignments were constructed for the aromatase protein sequences available in the database. An evolutionary distance in PAM units was calculated for each pair by applying the PamEstimator-package from DARWIN using an empirical log-odds matrix. From this, a preliminary evolutionary tree was built for the mammalian sequences, with branch lengths along internal nodes calculated to minimize a least-squares distance. The sequences of the ancestral genes and proteins at branch points in the tree were then reconstructed. From there, mutations  at both the DNA level and protein level were assigned to individual branches in the tree using the method of Fitch .n) of two-fold redundant amino acids  that are conserved in the aligned pairs was determined. The number of those amino acids that are encoded by the same codon (c) was determined, and the fraction (f2 = c/n) of the codons that are the same were then tabulated /(1-Equil)), where Equil is the f2 value expected after a large number of nucleotide substitutions have occurred at the synonymous sites [The evolutionary history of the aromatase family was then analyzed using the transition redundant exchange (TREx) metric based on an analysis of two-fold redundant codon systems ,78. Thesable see ). The TRus sites .The DNA sequences for aromatase were phylogenetically analyzed using a maximum likelihood framework in PAUP 4.0* (beta 10) , with thf2 value characteristic of orthologs determined [For inter-taxon analyses, families in the MasterCatalog  were identified that contained at least one representative protein from both of the taxa of interest. For these families, all inter-taxa pairs of genes were extracted, together with the pairwise protein sequence alignment. A pairwise alignment of the DNA sequences was then generated to follow the protein sequence alignment. If a family contained more than one sequence of a species belonging to one of the taxa analyzed, then those sequences were checked to determine whether they were duplicate entries into the database. If this was the case, only one of the duplicate sequences was retained in the analysis. A histogram of inter-taxa pairs was constructed, and the termined . This waCodon biases were obtained from the CUTG (Codon Usage Tabulated from GenBank) made available by the Kazusa DNA Research Institute Foundation, Japan .A/KS values to individual branches. Tests of parallel evolution were conducted using Converge [Pairwise TREx distances were used to generate lengths for the branches connecting the swine paralogs using the minimum evolution criterion in PAUP. This preliminary analysis was followed by a maximum likelihood analysis for the complete dataset using the PAML program . This inConverge , implemeSecondary structural data based on homology modeling for aromatases were generated using the DARWIN bioinformatics package, and in agreement with previous studies ,84. RendEAG performed the evolutionary, statistical and structural analyses, and prepared the manuscript. LGG cloned genes as part of his Masters work, and called the evolutionary problem to the attention of SAB. TL provided computational infrastructure. RCMS and FAS initiated the work with suid reproductive endocrinology, and supervised LGG. DRS and DAL did the initial bioinformatics analysis. CMJ provided paleontological expertise, constructed the cladogram, and helped prepare the manuscript. SAB has developed planetary biological analysis as a paradigm for generating hypotheses about the biological function of proteins, and prepared the manuscript.Illustration of planetary biology. This figure illustrates the concepts of planetary biology as they relate to combining genomic, paleontological, chemical and ecological records to understand the history of the biosphere.Click here for fileAn analysis of silent nucleotide substitutions in vertebrate aromatases. The first five columns from the left indicate the index number of sequence 1 compared with sequence 2, the fraction of sites at conserved two-fold redundant coding systems that are identical (f2), the number of such sites that are conserved (c2), and the number of such sites overall (n2). The remaining columns report analogous data: for silent sites in codon systems where a change at the third nucleotide is silent only if the change is a pyrimidine-pyrimidine transition ; in silent sites where a change at the third nucleotide is silent only if the change is a purine-purine transition ; for the silent sites at three-fold redundant codon systems ; and for the silent sites at four-fold redundant codon systems .Click here for file


Most self-pollinating plants are annuals. According to the 'time-limitation' hypothesis, this association between selfing and the annual life cycle has evolved as a consequence of strong r-selection, involving severe time-limitation for completing the life cycle. Under this model, selection from frequent density-independent mortality in ephemeral habitats minimizes time to flower maturation, with selfing as a trade-off, and / or selection minimizes the time between flower maturation and ovule fertilization, in which case selfing has a direct fitness benefit. Predictions arising from this hypothesis were evaluated using phylogenetically-independent contrasts of several life history traits in predominantly selfing versus outcrossing annuals from a data base of 118 species distributed across 14 families. Data for life history traits specifically related to maturation and pollination times were obtained by monitoring the start and completion of different stages of reproductive development in a greenhouse study of selfing and outcrossing annuals from an unbiased sample of 25 species involving five pair-wise family comparisons and four pair-wise genus comparisons.Selfing annuals in general had significantly shorter plant heights, smaller flowers, shorter bud development times, shorter flower longevity and smaller seed sizes compared with their outcrossing annual relatives. Age at first flower did not differ significantly between selfing and outcrossing annuals.This is the first multi-species study to report these general life-history differences between selfers and outcrossers among annuals exclusively. The results are all explained more parsimoniously by selection associated with time-limitation than by selection associated with pollinator/mate limitation. The shorter bud development time reported here for selfing annuals is predicted explicitly by the time-limitation hypothesis for the fitness benefit of selfing . Support for the time-limitation hypothesis is also evident from published surveys: whereas selfers and outcrossers are about equally represented among annual species as a whole, selfers occur in much higher frequencies among the annual species found in two of the most severely time-limited habitats where flowering plants grow – deserts and cultivated habitats. Inter-limited ,4-8. , versus time-limitation . Accordingly, these two hypotheses for selfing involve very different assumptions and predictions.A recent third hypothesis, the 'time-limitation' hypothesis, predicts that both selfing and the annual life cycle are concurrent products of strong 'r-selection' associated with high density-independent mortality risk in ephemeral habitats with a severely limited period of time available to complete the life cycle . Both thdirect fitness benefit; abbreviating the time between anthesis and ovule fertilization may ensure that there is enough remaining time in the growing season (after ovule fertilization) to allow complete seed and fruit maturation [The time-limitation hypothesis has direct and indirect components. The indirect component predicts higher selfing rates in annuals as a trade-off of selection for earlier reproductive maturity in annuals ,13 Figu. More raturation  Fig 1b)direct fiHowever, the two components of time-limitation cannot be separated clearly, as they operate simultaneously; i.e., earlier onset of flowering, shorter flower development time, smaller flowers and selfing can all be interpreted to have direct fitness benefits because they may all contribute directly to accelerating the life cycle . Indeed,The time-limitation hypothesis remains untested. Some recent studies have explored the rapid growth and maturation time of annuals in terms of bud development rates and ontogeny -15. HoweThe objective of the present study was to compare, for annuals exclusively, life history traits associated with selfing versus outcrossing using several species from a wide range of plant families. Phylogenetically-independent contrasts (PIC) were used to control for confounding effects due to common ancestry among species . Using aData on the timing of life history stages  were also obtained from a greenhouse study of 25 annual species involving 5 families. The time-limitation hypothesis predicts that selfers should produce mature flowers more quickly and should have shorter flowering times.Based on phylogenetically-independent contrasts, selfing annuals had significantly shorter plant heights  or flower longevity (P = 0.328). Age at first flower was not significantly related to mating system for any of the family or genus comparisons .Bud development time Figure  and flowThere is a rich body of theory and empirical work on the evolution of selfing in flowering plants [e.g. ,4-7,9,10for annuals the time-limitation hypothesis provides a more parsimonious explanation for the differences in traits between selfers and outcrossers. We consider each of these traits in turn below.At the same time, these results are not inconsistent with the predictions of selection resulting from pollinator/mate-limitation associated with the traditional reproductive assurance hypothesis. Just as with many situations where two different mechanisms can potentially produce the same outcome/pattern, it is not easy here to clearly distinguish between the roles of "pollinator/mate-limitation" and "time-limitation". Nevertheless there are two important contributions from our study: First, in reporting significant life history differences between selfers and outcrossers, our multi-species study is unique in its comparison of monocarpic annual species exclusively. All previous multi-species studies of trait comparisons between selfers and outcrossers have involved variable mixes of monocarpic and longer-lived polycarpic species. Second, by comparing annuals exclusively, our results provide indirect support for the time-limitation hypothesis, not by rejecting the role of pollinator/mate-limitation, but rather by representing a system in which it is more plausible to argue for the role of time-limitation; i.e., compared with pollinator/mate-limitation, time-limitation as a selection factor favoring selfing is likely to have been much stronger, more persistent and more widespread. The strength of this argument lies in the fact that the annual life history is unequivocally a product of some type of time-limitation favoring an abbreviated life cycle, which is promoted by (among other things) selfing (as opposed to outcrossing) Fig. . It is mCrepis tectorum. Arroyo [Limnanthes floccosa flowered earlier than the outcrossing L. alba, as predicted by the time-limitation hypothesis. The results for flowering times in the present study may be confounded by the controlled greenhouse environment of constant day-length, temperature and moisture regime. In the field, flowering times may be triggered by environmental cues. L. floccosa, for example, uses soil moisture to trigger the early onset of flowering, thus escaping the detrimental effects of soil desiccation during seed development [Taller plants may attract more pollinators and, hence, experience greater outcrossing rates ,21. The . Arroyo , howeverelopment . Note alOne of the most well established trends of predominantly self-fertilizing species is their reduced flower sizes compared with outcrossing species ,17. The Under the time-limitation hypothesis, smaller flowers and selfing may be tradeoffs of selection for precocity Figure , or smalMimulus guttatus [Clarkia xantiana [C. tembloriensis [Arenaria uniflora develop over a longer period of time than observed in outcrossing populations. In the field, selfing populations of A. uniflora were also observed to flower at the same time or even later than outcrossing populations [A. uniflora may have arisen through reproductive assurance in response to competition for pollinators [vis-à-vis the fitness benefits of selfing.Selfers had significantly shorter bud development times in all but one of the independent family contrasts Figure  and all guttatus  and in Cxantiana . Howeveroriensis . Hill, Loriensis  reportedulations , suggestlinators . The evolinators ,3,14, eaTrifolium hirtum; Fabaceae), with a large proportion of flowers open for only one day, which is common amongst self-fertilizing species [The families and genera in which selfers had shorter bud development times also had significantly shorter flower longevities Figure . In fact species . The pre species . This fi species . If timemaximum flower longevity, since there were no pollinators in the greenhouse, nor was hand pollination conducted. Pollination has been shown to induce floral senescence in numerous species [It is important to note that our data measure  species . This efStrong r-selection associated with the annual life form presumably favors wide dispersal mechanisms (for colonizing new and distant sites) which may be conferred by small seed sizes . The repCrepis tectorum took an average of 16 days for fruit maturation, whereas outcrossing individuals of the same species required 43.3 days. Small seed size may also be simply a trade-off of selection for high fecundity, also favored by strong r-selection [An alternative explanation, however, is offered by an extension of the time-limitation hypothesis: strong r-selection favors an acceleration of all stages of the life cycle Figure , includielection .While most selfers are annuals, it is not the case that most annuals are selfers. An unbiased literature survey  suggestsClarkia xantiana [Limnanthes alba and its selfing relative L. floccosa [L. alba was reduced by one sixth, whereas the seed set of L. floccosa found in the same area was virtually unaffected by the identical drought [While rigorous tests of these predictions have yet to be explored, some preliminary support is available from published surveys. From a representative sample of Mediterranean annuals , we findxantiana  and betwfloccosa . Since t drought .The association between 'weediness' and self-fertilization has also been noted ,30. An eBotanists have long known that selfing is particularly associated with the annual life cycle in flowering plants . The preThe effect of time-limitation under strong r-selection is to minimize the duration of the life cycle, with selfing favored directly Figure  and/or ivis-à-vis Figure Designing empirical studies that clearly distinguish between mechanisms involving time-limitation versus pollinator/mate limitation remain a challenge but we anticipate that our results and our discussion of these issues may help to inspire further research along these lines. Future studies may be designed to test more directly the role of limited pollination time (The literature was surveyed to obtain breeding information (i.e. selfing versus outcrossing) for as many annuals species as possible. For each species, data on plant height, flower size, and seed size were obtained where possible from standard floras and other published literature. A complete database was assembled for 118 species from both Europe and North America, involving 14 families  was known and could also be obtained as germinable seeds. Seeds were obtained from a variety of sources; Herbiseed, Rancho Santa Ana Botanic Gardens, Chiltern Seeds, S&S Seeds, and the National Plant Germplasm System. Our search lead to an unbiased sample of 25 candidate species, allowing five pair-wise family comparisons and four pair-wise genus comparisons (Table ©). Pots were placed in a greenhouse and watered daily until the appearance of their first true leaves. Subsequently, they were watered uniformly every second day to ensure that the soil was kept moist. Some species were germinated in a petri-dish in a growth chamber , after which they were transplanted into pots and placed in the greenhouse. Each species was replicated five times, with one plant per pot. Pots were arranged randomly on benches at a density of 1 pot per 0.093 m2.Most species were germinated in 15 cm pots filled to 3 cm below the top with standard potting soil  were used to supplement the light exposure to 16 hours of light per day. The greenhouse was kept at an average temperature of 23.1°C during the day and dropped to 20.0°C at night. The plants were fertilized every 2 weeks with 200 ml per pot of a 2g/L concentration of 20–20–20 N-P-K fertilizer.For each plant, age at first flower, bud development time, and flower longevity were measured. Emergence of the first pair of true leaves, after the cotyledons, was considered day 1 of the plant's life. Age at first flower was measured in days from day 1 to when the first flower opened on each plant. Bud development time (n = 3 buds per plant) was calculated as the number of days from the first appearance of a new bud until the flower opened. The same three buds on each plant were then monitored every day after opening, and the number of days until the flower senesced (flower longevity) was recorded for each. A flower was considered to be senesced when the corolla wilted, fell apart, or became discolored, as designated by Primack . Any floFor bud development time and flower longevity, the three replicate measurements for each plant were averaged, and then these values for the five replicate plants were averaged to obtain a mean value for the species. The data were analyzed with a standard least squares one-way analysis of variance (ANOVA) model, with a post-hoc contrast between selfers and outcrossers. These analyses were done for each family and genus separately in order to control for phylogeny at these levels. In cases where the data were non-normal, a log-transformation was applied which corrected the distribution.RS collected the data, performed most of the analyses, participated in the design of the study, and wrote the first draft as a B.Sc.(Hons) thesis. LWA conceived of the study, participated in its design and coordination, and wrote the final draft for submission to BMC. Both authors read and approved the final manuscript.