This is the first molecular survey of the cpmA cistron diverseness in populations of cyanobacterium Chroococcidiopsis from utmost home grounds. The major findings of this survey are low nucleotide diverseness, low familial distinction among populations, high cistron diverseness and strong choice at the cpmA venue of Chroococcidiopsis sp.
3.7.1. Low degrees of nucleotide diverseness & A ; low population distinction globally ; perchance contributed by a high migration rate
Low degrees of nucleotide diverseness in the globally distributed Chroococcidiopsis sp.
Despite the rough environment and planetary distribution of the sampling locations, both the intra-population ( Table 3.5 ) and inter-population diverseness ( Table 3.6 ) of Chroococcidiopsis sp. were rather low. The nucleotide diverseness ( Iˆ ) averaged merely 0.0034 for the species as a whole. The theta ( Q ) value, another index of familial diverseness within a population, ranged between 0.004 and 0.007 and was besides instead low.
The mean Iˆ value ( 0.021 ) of three housekeeping cistrons ( atpD, glnII and recA ) of, a Gram negative dirt bacteria Bradyrhizobium was about six times higher than that of cistron cpmA of Chroococcidiopsis sp. ( mean Iˆ = 0.0034 ) ( Vinuesa et al. , 2005 ) . These cistrons of Bradyrhizobium are indispensable for nucleotide binding and contact action, nitrogen emphasis response and DNA fix. Likewise, the mean figure of nucleotide differences for the cpmA cistron of Chroococcidiopsis sp. ( K = 1.74 ) were ten to fifteen-fold lower than that for the above cistrons of Bradyrhizobium ( mean K = 10.94 ) ( Vinuesa et al. , 2005 ) . A comparing of the intra-population nucleotide diverseness in cistron cpmA sequences with that of the seven housekeeping cistrons of a cyanobacterium, Microcystis aeruginosa ( Tanabe et al. , 2007 ) , outputs similar consequences. The mean nucleotide diverseness reported for the cistrons of M. aeruginosa was 0.023, runing from 0.013 ( cistron recA ) to 0.043 ( cistron pgi ) ; this is more than ten-fold higher than for the cpmA cistron ( Table 3.5 ) .
Housekeeping cistrons are cistrons which are indispensable for any being ‘s activity. They are thought to be among the most conserved ( Jordan et al. , 2002 ) . The above information indicate that at least some circadian cistrons are probably more conserved than the housework cistrons.
Low inter-population distinction globally
The really low inter-population distinction of the globally distributed Chroococcidiopsis sp. ( mean GST and FST for the species were -0.0007 and 0.0079, severally ) observed in our survey is rather surprising. For illustration, the above mentioned Bradyrhizobium, which was sampled at much smaller geographical graduated table ( across Canarian Islands ) , had an mean GST = 0.061 ( Vinuesa et al. , 2005 ) . Furthermore, globally sampled strains of a cyanobacterium, Mastigocladus laminosus, showed rather high distinction at several nitrogen metamorphosis venue ( FST = 0.60 ) ( Miller et al. , 2007 ) .
The low GST and FST values for the cpmA cistron of Chroococcidiopsis sp. bespeak that the bulk of the fluctuation resides within populations. The important difference among the FST values between Chroococcidiopsis and M. laminosus is likely due to the differences in spacial isolation of the populations. Populations of M. laminosus were suggested to hold low cistron flow due to the dispersion barriers, whereas Chroococcidiopsis sp. seems to hold a high inter-population cistron flow ( average Nm = 31.25 ) .
Similar low interpopulation distinction despite the immense distribution country may be found in other species. For illustration, eukaryote Pinus sylvestris ( Scots pine ) is a chief wood species distributed across Eurasia, from Spain to Kamchatka Peninsula in Russia. However, it showed really low nucleotide diverseness in several functional cistrons, which was explained by low mutant rate and high cistron flow ( Dvornyk et al. , 2002a ) .
The high migration rate is likely one of the most of import factors for the low familial diverseness and distinction among the populations found in this survey. Other likely causes include high preservation of the cpmA cistron ( subdivision 3.7.3 ) and an spread outing population of Chroococcidiopsis sp. ( subdivision 3.7.4 ) .
3.7.2. Large figure of rare haplotypes in malice of the low nucleotide diverseness
Despite the low nucleotide diverseness, the haplotype diverseness in Chroococcidiopsis sp. was high ( H = 0.774 ) . Similar high cistron diverseness ( H = 0.951 ) was reported in a cyanobacterium Microcystis aeruginosa genotyped at seven housekeeping venue ( Tanabe et al. , 2007 ) . These blue-green algaes, Chroococcidiopsis sp. and M. aeruginosa have similar high cistron diverseness. However, the mean nucleotide diverseness ( Iˆ = 0.0034 ) for the cpmA cistron of Chroococcidiopsis sp. was about septuple lower than that reported for the multiple cistrons of M. aeruginosa ( mean Iˆ = 0.023 ) ( Tanabe et al. , 2007 ) .
The high cistron diverseness in M. aeruginosa ( Tanabe et al. , 2007 ) was instead unexpected, because it was thought that clonal beings such as bacteriums should see selective expanses at most loci ensuing in low cistron diverseness ( Atwood et al. , 1951 ) . In order to explicate such high cistron diverseness, Tanabe et Al. suggested the being of several ecologically distant populations of M. aeruginosa ( Tanabe et al. , 2007 ) . Higher familial diverseness is maintained if more ecologically distant populations exist within a individual species, as each distant population falls into its ain sequence-based bunch ( Cohan, 2002 ) . This possibility was besides supported by several distinguishable bunchs observed in the phyletic tree of M. aeruginosa ( Tanabe et al. , 2007 ) . However, this scenario is improbable for the cpmA cistron in Chroococcidiopsis sp, because distinction among the populations of Chroococcidiopsis sp. is really low and no resolved clades are observed in the phyletic tree ( Figure 3.4 ) .
The high figure of rare haplotypes may be an index of a quickly spread outing population, and is non needfully an index of high familial diverseness ( Slatkin & A ; Hudson, 1991 ) , as farther discussed in subdivision 3.7.4.
3.7.3. High preservation of the cpmA cistron
The old micro-site survey on the cpmA cistron ( Yau & A ; Dvornyk, unpublished informations ) of cyanobacterium Nostoc linckia from the “ Evolution Canyon ” revealed no polymorphism in the cyanophyte populations from both the stressful and the temperate inclines. On the other manus, genome-wide surveies of N. linckia from the same location showed significantly higher familial diverseness in the populations from the nerve-racking incline as compared to those from the temperate incline ( Krugman et al. , 2001 ; Satish et al. , 2001 ) . The high preservation of the cpmA cistron was besides supported by the low nucleotide diverseness observed in the cistron on a planetary graduated table.
Higher preservation for a cistron normally occurs if that cistron is indispensable harmonizing to the “ knockout-rate ” anticipation ( Jordan et al. , 2002 ) . This is because there is stronger sublimating choice moving on indispensable cistrons than that for less indispensable cistrons, which are more functionally excess. Stronger sublimating choice leads to take down rates of permutation, and, finally, to a higher grade of preservation of a cistron ; the degree of preservation is by and large related to the functional importance of that peculiar cistron ( Jordan et al. , 2002 ) .
The high preservation for circadian cistrons is supported by a survey of the kai cistrons of the N. linckia that revealed grounds for sublimating choice ( Dvornyk et al. , 2002b ) . Purifying choice was detected on the cpmA venue from our findings by dN/dS ratio, Tajima ‘s D, and Fu and Li ‘s D* and F* values. This besides supports the premise of high preservation of circadian cistrons ( mentioned in item in subdivision 3.7.5 ) .
3.7.4 Low familial diverseness in Chroococcidiopsis sp. explained by population enlargement and/or familial constrictions
Chroococcidiopsis sp. had low nucleotide diverseness and by and large low degree of distinction among the populations at the cpmA cistron. This can besides be a consequence of one or more recent familial constrictions followed by enlargement of the population.
Expanding population with one or more recent familial constrictions
At present, Chroococcidiopsis sp. has a big population size and is dominant in most utmost environments ( Friedmann, 1980 ) . Chroococcidiopsis sp. is one of the evolutionarily oldest blue-green algae, based on the morphological resemblance to certain Proterozoic microfossils ( Friedmann & A ; Ocampo-Friedmann, 1994 ) . Low nucleotide diverseness, low distinction, and multiple low frequence haplotypes are consistent with a possible population enlargement ( Bodkin et al. , 1999 ; Slatkin & A ; Hudson, 1991 ) .
We observed the negative values of Tajima ‘s D, and Fu and Li ‘s D* and F* test statistic in all populations. The parametric quantities of above two neutrality trials, yielded the statistically important ( P a‰¤ 0.05 ) norm for the species: D = -2.6443, D* = 4.7781 and F* = -4.7860 ) . This represents an surplus of low frequence polymorphism.
Tajima ‘s D, and Fu and Li ‘s D* and F* are sensitive trials for the frequence distribution of segregating sites. In a constant-size impersonal equilibrium population, Tajima ‘s D is expected to be about zero ( Tajima, 1989 ) . In footings of population size alterations, negative values of Tajima ‘s D are observed if the population has experienced one or more familial constrictions and/or is increasing in size ( Tajima, 1989 ) . A quickly increasing population would ensue in many haplotypes happening at low frequences, but with low overall base diverseness ( Slatkin & A ; Hudson, 1991 ) . This may be the instance observed for the globally distributed Chroococcidiopsis sp. populations ( Section 3.7.2 ) .
Expansion of a population normally occurs after one or more familial constrictions causes a diminution in familial diverseness in the population ( Robert, 1987 ) . A population constriction occurs, for illustration, when a little figure of persons ‘ groups attempt to happen a new population ( besides known as the ‘founder consequence ‘ ) . This may ensue in a important loss of familial fluctuation ( Leberg, 1992 ) . However, our findings indicate low familial fluctuation for all populations that are geographically rather distant ; therefore the likeliness of one or more recent familial constrictions in all locations at the same time is low.
Chroococcidiopsis sp. is more frequently found in utmost environments than in the temperate 1s ( Friedmann & A ; Ocampo-Friedmann, 1994 ) . It was proposed that this consequences from the inability of Chroococcidiopsis sp. to vie with more discriminating, specialized, or aggressive species abundant in temperate environments ( Friedmann & A ; Ocampo-Friedmann, 1994 ) . It is possible that, low fluctuation in Chroococcidiopsis sp. could be more suitable for utmost environments. Due to this possibility, the species may non be able to vie with beings of higher fluctuation in moderate environments.
The long evolutionary history of Chroococcidiopsis sp. and its copiousness in most utmost environments do non back up the possibility of recent constrictions. No such instances were reported for other blue-green algae as good ( Mes et al. , 2006 ) . Therefore, more surveies of multiple cistrons are required from Chroococcidiopsis sp. in order to analyze the population size alterations.
3.7.5. Choice as a factor of development at the cpmA venue of Chroococcidiopsis sp.
Purifying choice is the most common type of choice observed. For illustration, merely sublimating choice was detected for housekeeping cistrons of two strains of Bradyrhizobium sp. ( dN/dS ratio averaging 0.108 ) . Even at a planetary graduated table, housekeeping cistrons of another bacteria Bacillus Cereus, merely exhibited sublimating choice ( dN/dS = 0.031 ) ( Priest et al. , 2004 ) . However, in some instances, positive choice was besides detected. For illustration, Mes et Al. ( 2006 ) found that the petB and kaiC cistrons see this type of choice at some sites. Our findings indicate strong selective force per unit area upon the cpmA venue. Interestingly, both positive and purgatorial choices were detected in the different populations of Chroococcidiopsis sp.
Positive choice in populations AC, AH and SH
The dN/dS ratio was above 1 in three populations, AC, AH and SH. The dN/dS trial requires a instead strong signal in order to observe choice and is rather conservative ( Plotkin et al. , 2004 ) . Therefore the several ratio is seldom above 1 meaning positive, diversifying choice. For illustration, a large-scale database hunt performed by Endo et Al. ( 1996 ) identified merely 17 cistrons that are under positive choice out of 3,595 functional cistrons.
Although rare, illustrations of positive choice have been reported. For case, 55 protein coding cistrons has been listed by Yang and Bielawski ( 2000 ) and two cistrons of blue-green algae has been reported elsewhere ( Mes et al. , 2006 ) detected by the dN/dS ratio. Positive choice consequences in choice of favourable mutants or selective expanses take downing familial diverseness ( Galtier et al. , 2000 ) . A comprehensive scrutiny of 12 protein coding cistrons of blue-green algae revealed positive choice at the kaiC cistron of Microcoleus chthonoplastes and the rbcX cistron of Anabaena and Aphanizomenon sp. ( Mes et al. , 2006 ) . The kaiBC operon, which encodes two nucleus circadian cistrons, kaiB and kaiC, was besides reported to be under positive choice in another cyanobacterium, Nostoc linckia ( Dvornyk et al. , 2002b ) .
Purifying choice in populations AE, CH and TH
The negative Tajima ‘s D, and Fu and Li ‘s F* and D* ( Table 3.7 ) suggest strong purifying choice moving on the cpmA cistron in the studied populations. For the populations, AE, CH and TH, every bit good as the norm of the species, the dN/dS ratio was below 1 connoting sublimating choice. Purifying choice is common in which hurtful mutants are eliminated to continue functional familial characteristics due to being conserved over clip. These functional familial characteristics can include protein coding cistrons or regulative sequences.
Selective neutrality trials affected by population size alterations, possible cause of contradiction between dN/dS and neutrality trial values
The sum and form of DNA fluctuation in a population may be affected by natural choice. However, other than natural choice, alterations in population size and construction are chief factors that affect DNA fluctuation ( Tajima, 1989 ) . dN/dS ratio above 1 for three of the populations implied positive choice. However, negative values of Tajima ‘s D, and Fu and Li ‘s D* and F* for all populations implied sublimating choice in our survey. Tajima ‘s, and Fu and Li ‘s trial indices, are powerful but can be affected by alterations in population size and hence can give inaccurate reading of choice ( Tajima, 1989 ; Fu and Li, 1996 ) .
In an spread outing population, there would be a inclination for Tajima ‘s D values to be negative and frailty versa ( Tajima, 1989 ) . For illustration, the dN/dS ratio above 1 of the AMA1 ( Apical Membrane Antigen 1 ) cistron in Plasmodium falciparum indicates positive choice ; nevertheless, the ascertained positive values of Tajima ‘s D, and Fu and Li ‘s D* and F* suggest equilibrating choice moving upon the cistron ( Polley and Conway, 2001 ) . Thus the two attacks of trials for choice provided contradictory consequences. This contradiction between neutrality trial values and dN/dS ratio was besides present in our findings. The difference in the consequences obtained from these two attacks was explained by a recent decrease in the P. falciparum population, as alterations in population size can do skewed neutrality trial values ( Polley and Conway, 2001 ) .
Mes et Al. ( 2006 ) obtained negative values of Tajima ‘s D in the kaiC and petB cistrons of a cyanobacterium Microcoleus chthonoplastes. These cistrons are indispensable for circadian ordinance and photosynthesis, severally. The negative D may bespeak either sublimating choice or spread outing population size, or both. However, the dN/dS ratio was higher for locus kaiC than petB ( Mes et al. , 2006 ) . Since, dN/dS ratio is instead selective and unswayed by population size alterations ; this suggests that these two cistrons from M. chthonoplastes are under different selective force per unit areas. This was confirmed by the important grounds found for positive choice at venue kaiC by McDonald-Kreitman trials, which was non found for the petB venue. This determination was deemed to be of import as it allowed the separation of gene-specific forces ( such as positive choice ) and genome-specific forces ( population size alterations and familial constrictions ) ( Mes et al. , 2006 ) .
We discovered similar differences in selective force per unit areas for different populations, identified by the neutrality trial values and dN/dS ratio. However, informations on other cistrons from Chroococcidiopsis sp. are non available to separate between gene-specific forces and genome-specific forces on the population. Therefore, in order to separate between selective force per unit areas and population size alterations in Chroococcidiopsis sp. , more cistrons need to be studied. The M. chthonoplastes informations set is an first-class illustration of a survey on multiple cistrons from a individual cyanobacterium which identified forces that affect genomic diverseness without the cognition on population size alterations.
Sing the power of the dN/dS ratio as it stays unaffected by population alterations and the restrictions of the neutrality trials used, it can be assumed that the cpmA venue in Chroococcidiopsis sp. is most likely under selective force per unit areas for all populations ; some with rare positive choice ( AC, AH and SH populations ) .
Significant neutrality trial values rejecting the impersonal theory theoretical account at equilibrium for populations TH, CH and the norm of the species, back uping high preservation of the cpmA cistron
Significant values of Tajima ‘s D, every bit good as Fu and Li ‘s F* and D* ( Table 3.7 ) imply that the populations TH and CH, every bit good as the overall species are non indiscriminately germinating for the cpmA venue. Significant negative Tajima ‘s D, and Fu and Li ‘s F* and D* normally suggest either equilibrating choice runing on the population or a demographically spread outing population ( Tajima, 1989 ) . However, the dN/dS ratio clearly indicates positive choice for the three populations, AC, AH and SH. It is possible that, even though positive choice is runing in some populations, it is non strong plenty to get the better of the effects of sublimating choice in the norm of the species. Since skewing of the Tajima ‘s D and Fu and Li ‘s F* and D* , and enlargement of population is so a possibility, the overestimate of the neutrality trial values can non be ruled out.
The strong purifying choice observed on the species can besides be an index of higher preservation of the cpmA cistron and circadian cistrons proposed in subdivision 3.7.3. Comparisons of several functional cistrons from other bacteriums with the cpmA cistron of Chroococcidiopsis sp. revealed low nucleotide diverseness and distinction. Although familial diverseness was higher for multiple cistrons in Microcystis aeruginosa, Tajima ‘s D was observed to be about close to nothing for all cistrons meaning weak selective forces moving upon those cistrons ( Tanabe et al. , 2007 ) . This could connote the cpmA cistron of Chroococcidiopsis sp. are more conserved, and hence have more selective force per unit area than the functional N metamorphosis cistrons in M. aeruginosa.
3.7.6 Contrast among microbic communities in a micro-site due to changing degrees of environmental emphasis
Cyanobacterias are singular for their ability to boom in diverse environments with fluctuating emphasis factors, such as temperature, H2O handiness and light strength ( Schopf, 1996 ) . A survey on the cyanophyte circadian cistrons under contrasting environments with long-run microclimatic emphasis revealed that lasting ecological emphasis may ensue in a higher mutant rate and higher familial polymorphism ( Dvornyk et al. , 2002b ) . From our findings it could non be deduced if emphasis caused higher polymorphism in the cpmA cistron of Chroococcidiopsis sp. , as samples that were used in our survey were merely from nerve-racking sites and non from temperate. However, fluctuation was observed in the polymorphism degree in the cpmA cistron sequences of six different populations from four sites. This indicates differences in the emphasis degrees that these six populations are exposed to, even for populations within a home ground.
South-polar populations ; from same home ground yet with different selective forces on the cpmA venue
The three populations from the Antarctic Balham Valley used in our survey were AC, AE and AH. They differ by their microbic community construction ( Table 3.2 ) . Although their nucleotide diverseness ( P ) , cistron diverseness ( Hd ) , K and theta ( Q ) values were similar, the dN/dS ratio was rather different. The AE population had the lowest dN/dS ( 0.37 ) as compared to those of the AC and AH populations ( 1.06 and 1.64, severally ) .
Although, the three South-polar populations were from the same home ground with similar environmental conditions, UV radiation is assumed to be extremely different. This is because AE consisted of settlements populating pore infinites deep within stones, whereas AC and AH consisted of settlements populating the surface and underneath the stones ( Figure 3.1 ) . Sing the localisation of these three types of microbic communities, it could be assumed that the least solar radiation would be received by AE, followed by AH and AC.
Previous surveies on blue-green algaes under emphasis showed that UV radiation is one of the major emphasis factors in nerve-racking environments ( Dillon et al. , 2002 ) . Besides, recombination frequences and mutant rate tend to increase under nerve-racking conditions ( Hoffmann & A ; Hercus, 2000 ) . Therefore, if endolithic communities were exposed to lower emphasis degrees than the chasmolithic and hypolithic communities, they should hold lower polymorphism. K and theta ( Q ) values back up this premise, as these were found to be slightly lower for AE population compared to that of AC and AH.
From the dN/dS ratio, AE populations were detected to be under strong purifying choice, whereas AC and AH both were detected to be under strong positive choice. Our informations suggests that, there is selective remotion of hurtful allelomorphs in AE population by sublimating choice that could ensue in a lessening in fluctuation. On the other manus, AC and AH populations were detected to hold amino acerb alterations fixed at a higher rate than synonymous alterations by positive choice, leting adaptative molecular development. It is thought that evolutionary rates are higher in more nerve-racking environments ( Nevo, 1997 ) . Therefore it could be due to the exposure of higher UV emphasis, that AC and AH populations are under positive choice for cpmA venue. This allows these populations to accommodate to the rough environment by deriving fittingness of the protein, CpmA.
It was reported from a survey on microbic diverseness in Antarctic Balham Valley by Indicating et Al. ( 2009 ) , that even though the three microbic communities ( AC, AE and AH ) were present in the same home ground, they were extremely different from each other ( with an mean FST value of 0.088 ) , which supports our observation in differences in polymorphism degrees in the cpmA cistron for the three microbic communities. In add-on, our findings reveal different selective forces moving upon the three microbic communities in the Antarctic Balham Valley. We have non found informations in the available literature that report different selective forces for same venue. This is why our determination can hold to be of import in apprehension of the nexus between environmental emphasis and selective force per unit area.
Compared with other housekeeping cistrons of bacteriums, nucleotide diverseness and distinction was instead low in the cpmA cistron of Chroococcidiopsis sp. A high cistron flow and/or population enlargement with one or more familial constrictions, supported by big Nm value, are considered to be plausible grounds behind the low familial distinction among globally separated populations. Chroococcidiopsis sp. were detected to be under strong selective forces ( with both rare positive choice and sublimating choice ) for the same venue. A strong purifying choice found for the whole species, and low fluctuation in the cpmA cistron allowed us to suggest a high preservation of the cpmA cistron. In add-on, our findings reveal different selective forces moving upon three microbic communities in the Antarctic Balham Valley, on the same venue. More molecular surveies of multiple cistrons from Chroococcidiopsis sp. populations and/or from other cyanobacterium species globally, are required to corroborate the high migration rate and/or population size enlargement.