Procellariformes is the clade of birds which include millstones, petrels and their relations Stearns and Hoekstra, 2005. Albatrosss belong to the household Diomedeidae, these birds are durable and are a magnetic constituent of the pelagic avifauna ( Chambers et al. , 2009 ) . Albatrosss are big birds and hence breed less often ( Stearns and Hoekstra, 2005 ) , their per se slow rate of reproduction consequences in a monogamous brace raising merely 1 biddy every twelvemonth or every 2 old ages ( Chambers et al. , 2009 ) . Albatrosss are confronted by many menaces such as pollution of the marine environment and the activities of international fishing fleets ( Chambers et al. , 2009 ) . These jobs are farther compounded by the loss or break of engendering braces ( Chambers et al. , 2009 ) and engendering sites ( Stearns and Hoekstra, 2005 ) .
Genomic research has performed a important function in understanding inquiries such as version, speciation and population genetic sciences ( Backstrom et al. , 2008 ) , as a consequence of this it is thought that a “ stable evidence-based taxonomy is a critical demand for the effectual hereafter preservation of the millstones ” ( Chambers et al. , 2009 ) . As stated by Zink and Barrow-Clough ( 2008 ) , by “ understanding the evolutionary history of populations there has been a dramatically enhanced by the acquisition of molecular informations, uncovering the distribution of familial fluctuation within and among populations ” .
This fluctuation between populations is the consequence of speciation. Speciation may happen through four chief evolutionary procedures as stated by Mila et Al. ( 2010 ) , viz. “ micro-allopatric isolation of hereditary populations into at least three disjunct demographic units, which has resulted in divergency in impersonal genomic markers ; morphological distinction along altitudinal gradients through local version ; the visual aspect of distinguishable morphs which now exist ( or co-exist ) in different distributions ; and the care of these forms in topographic point over clip, through a combination of restricted dispersion and pre and/or post-zygotic mechanisms of generative isolation ” . It may even happen that all four evolutionary procedure occur. Once 2 taxa for good cease to interchange cistrons ( Chambers et al. , 2009 ) , familial, morphological and behavioural divergency will happen and finally ensuing in generative isolation. From this one can detect the forms of divergency and constructions in populations ( Mila et al. , 2010 )
Mitochondrial DNA cytochrome B ( cyt B ) is a cistron mark by and large used for inter-specific degrees of survey, intending comparing of species in the same genus or the same household ( Castresana, 2002 ) . It is moreover among the most extensively sequenced cistrons to day of the month across the craniates ( Johns and Avise, 1998 ) . Cytochrome B has a fast evolutionary rate of alteration ( Nunn et al. , 1996 ) and is a big protein-coding cistron, nevertheless, with this in head it has no indicant of interpolations or omissions ( Nunn and Stanley, 1998 ) within the cistron as this would ensue in loss of map of the cistron.
It was antecedently thought that household Diomedeidae was composed of 14 taxa ( Nunn et al. 1996 ) , nevertheless, recent phyletic analysis has shown that Diomedeidae consists of 24 taxon ( Chambers et al. , 2009 ) . Phylogenetic relationships can be found through assorted distinct methods, the most common being that of maximal parsimoniousness ( Nunn et al. , 1996 ) ( this method attempts to happen a phyletic tree which requires the fewest alterations, disregarding subdivision length information ( Omland et al. , 2008 ) ) , maximal likeliness methods with bootstrapping ( Nunn et al. , 1996 ) ( branch lengths are used, therefore a higher chance of alterations in the subdivisions represent longer spans of clip ( Omland et al. , 2008 ) ) . And in conclusion, the Bayesian phyletic analysis ( likewise to maximum likeliness, this theoretical account integrate more constituents of the existent uncertainness inherent in character function methods ( Omland et al. , 2008 ) ) .
Deducing molecular informations, one can analyze the planetary geographic scope size ( Webb and Gaston, 2000 ) . It has been found that avian scope sizes are non inactive but instead alteration over evolutionary clip ( Webb and Gaston, 2000 ) . It has farther been found that range-size transmutations is non random in birds with the exclusion of taxa such as island endemics and some threatened species ( Webb and Gaston, 2000 ) . “ In general, scope sizes appear to spread out comparatively quickly post speciation ; later, and possibly more bit by bit, they so decline as species age ” ( Webb and Gaston, 2000 ) .
The intent of this survey is to find the correlativity between the geographical distribution of the household Diomedeidae, its phyletic tree and the ‘IUCN ruddy list of threatened species ‘ categorization. Furthermore, utilizing this correlativity to assistance in the preservation of the species by placing which species is lower familial diverseness ( Abbott and Double, 2003 ) due to a recent variegation.
Materials and method
Ingroup, outgroup and mark cistron choice
The plans used in this survey was MEGA version 5 ( Tamura et al. 2011 ) and Genbank ( www.ncbi.nlm.nih.gov ) , a familial database which searches for familial sequences antecedently published.
The survey species was the members of the Family Diomedeidae, this included sub-families Diomedea, Phoebastria, Phoebetria and Thalassarche ( Table 1 ) . The outgroup selected was Grus Antigone. It was decided that the degree of this survey would be up to that of Family degree, at this degree of survey the relationships between the assorted members of household Diomedeidae can easy be visualized. For this survey, it was decided that the cytochrome B cistron would be suited for our probe, this was due to it being a good mitochondrial DNA ( mtDNA ) mark for inter-specific degrees of survey. Cytochrome B is a coding cistron and is of a fixed length.
Constructing a information set
MEGA version 5 ( Tamura et al. , 2011 ) was unfastened and a new alliance was created, Genbank database was so used for seeking for informations on the survey species of involvement and placing the corresponding mark cistron cytochrome B, the selected entry ideally should be greater than 300 bases in length. Once the mention sequence was found and inserted into MEGA 5 ( Tamura et al. , 2011 ) , a nucleotide-nucleotide BLAST hunt was performed, with Blastn option selected as ‘other ‘ and non human genome sequences. Through this procedure, closely-related, homologous sequence Genbank entries was obtained and was used in roll uping the clique dataset. Once all the cliques were compiled and sequence alliance was performed and the sequence on the terminals for which there was non homologous informations was trimmed off.
Sequence statistics, theoretical account choice and phenetic analyses
Sequence statistic, exemplary choice and phonic analysis were performed following. The aligned sequence of the household Diomedeidae and outgroup and Grus Antigone was analyzed and statistics such as variable sites ( V ) , parsimony enlightening sites ( Pi ) , nucleotide composing ( i.e. average A, T, C and G values ) and the R-statistic, which is the transition/transversion ratio was recorded. Next the overall average distance ( p-distance ) was performed in order to find the highest and lowest sequence divergency values between any two taxa, in other words, to find the nucleotide development.
On the footing of the collected informations, a sequence development theoretical account for the phonic analysis was chosen, this was determined by executing a hierarchical likeliness theoretical account of choice trial. The evolution of a ‘neighbor fall ining ‘ tree was constructed with analysis penchant of pairwise omission and bootstrapping between 1000 and 100A 000. The phyletic building was repeated for a ‘Minimum development ‘ tree with analysis penchant of pairwise omission and bootstrapping between 1000 and 100A 000.
Parsimony analysis, likeliness analysis, rate of heterogeneousness and molecular clock
Parsimony ( Cladistic ) Analysis, Likelihood analyses, proving for rate heterogeneousness ( Tajima ‘s 3 taxon trial and likeliness molecular clock trial ) and enforcing a molecular clock was performed. The evolution of a ‘Maximum parsimoniousness ‘ tree was constructed with analysis penchant of pairwise omission and bootstrapping between 100 and 1000. The consistence index ( CI ) , keeping index ( RI ) and rescaled consistence index ( RCI ) and ‘tree length ‘ values for the different homoplasy indexes were recorded. The evolution of a ‘Maximum likeliness ‘ tree was constructed with analysis penchant of pairwise omission and bootstrapping between 100 and 1000. The log likeliness ( log L ) tonss and concluding figure of trees obtained with this mark were recorded. Deducing a ‘neighbor fall ining ‘ tree with analysis penchants of pairwise omissions and nucleotide theoretical account set to ‘p-distance ‘ a Tajima ‘s comparative rate trial was preformed for enforcing a molecular clock. The ‘evolutionary rate ‘ value was so set to 0.02 ( 2 % sequence divergency per million old ages ) , this converted Tajima ‘s comparative rate trial to a UPGMA ( Unweighted-Pair Group Method with Arrhythmic agencies ) tree.
The cytochrome B dataset was 1008 bases in length and comprised of 25 taxa. Of these 1008 bases, 279 sites were variable ( V ) and 195 sites were parsimony enlightening sites ( Pi ) . Directly from the dataset, it was through empirical observation determined that the mean base frequences were T=25.3, C=34.1, A=27.8, G=12.8 ( Table 2 ) . The R-statistic, which is the transition/transversion ratio was recorded to be 5.32 and the overall average distance or ‘d ‘ statistic was 0.078 ( 7.8 % ) .
The best fit theoretical account of sequence development selected under AIC ( Akaike Information Criterion = 7468.845 ) for the information set at manus was the GTR ( General Time Reversible ) + G ( Gama distribution theoretical accounts =0.21 and I=n/a ) in MEGA 5 ( Nei and Kumar, 2000 ; Tamura et al. , 2011 ) ( Table 4 ) . Under the GTR+G theoretical account of sequence development, the mean base frequences were estimated to be T=25.3, C=34.1, A=27.8, G=12.8. The R-statistic, which is the transition/transversion ratio was recorded to be 9.38 and the overall average distance or ‘d ‘ statistic was 0.086 ( 8.6 % ) .
The Minimum Evolution tree ( Figure 2 ) and the Neighbor-Joining tree ( Figure 1 ( Appendix ) ) are about identical, it was decided to include the Minimum Evolution tree in the consequence ( Figure 2 ) . The ground for this choice was due to the fact that in this method the amount of all the subdivision length estimations ( S ) is computed for all topologies and the topology with the smallest S value is selected as the best tree, it is considered optimum.
A bootstrap of 10 000 was performed in order to find the assurance interval on the trial of Minimum Evolution tree ( Figure 2 ) of Diomedeidae ( Felsenstein, 1985 ) . It must be stated that 10 000 bootstrap reproductions, MEGA 5 ( Tamura et al. , 2011 ) automatically changed the bootstrap of 100A 000 to a bootstrap of 10 000. A individual best tree with an SBL ( Sum of Branch Lengths ) of 0.45873726 was constructed for the Minimum Evolution tree ( Figure 2 ) . Therefore, it can be stated that Grus Antigone is the outgroup of the ingroup Family Diomedeidae by being on separate subdivisions.
The following tree created was the Maximum Parsimony tree ( Figure 3 ) . Like the Minimum Evolution tree, the Maximum Parsimony tree demonstrates that Grus Antigone is the outgroup of the ingroup Family Diomedeidae by being on separate subdivisions. The different homoplasy indexes recorded for the Maximum Parsimony tree were as follows: the consistence index ( CI ) is 0.663848, the keeping index ( RI ) is 0.851679, and the rescaled consistence index is 0.565385 for all sites ( with a RC value of 0.501764 for all sites and parsimony-informative sites ) .
The Maximum likeliness incorporates explicit theoretical accounts of sequence development and allows statistical trials of evolutionary hypotheses. This means that the likeliness of detecting a given sequence dataset for a specific permutation theoretical account is maximized for each topology and the topology that give the highest maximal likeliness and the highest Log likelihood value of -3697.77 ( Figure 4 ) is selected as the concluding tree.
The equality of evolutionary rate was determined between sequences A ( U48950 Phoebastria nigripes ) and B ( AF 076091 Thalassarche carteri ) , with sequence C ( FJ769854 Grus Antigone ) used as an outgroup in Tajima ‘s comparative rate trial ( Table 5 ) . The ‘clock trial ‘ utilizing Tajima ‘s comparative rate trial ‘ had a P value of 0.05371, the void hypothesis of equal rates between line of descents was non rejected.With a P value greater than 0.05, the molecular clock ( Table 7 ) was imposed and a UPGMA ( Unweighted-Pair Group Method with Arrhythmic agencies ) tree was created ( Figure 7 ) . The evolutionary rate was set 0.02 ( 2 % sequence divergency per million old ages ) , as specified for Cytochrome B ( Figure 7 ) .
Grus Antigone and Diomedeidae species last shared a common ascendant 2.5735 million old ages ago ( Figure 7 ) . The 2nd molecular clock trial utilizing the Likelihood trial for rate of heterogeneousness was rejected ; therefore the molecular clock could non be imposed.
Figure 2: Minimal Evolution tree. Through the usage of the Minimum Evolution tree evolutionary history of the can be inferred. The Minimal Evolution tree was obtained utilizing 1008 bases matching to the cytochrome B cistron part. The Minimal Evolution algorithm with a the Tamura-Nei theoretical account of sequence development was used to deduce the evolution ( the Tamura-Nei theoretical account being the indistinguishable theoretical account to that of the GTR+G theoretical account ) , with the nodal supports being assessed following 10A 000 parametric bootstrap reproductions. The ME tree was searched utilizing the Close-Neighbor-Interchange ( CNI ) algorithm at a hunt degree of 0. The Neighbor-joining algorithm was used to bring forth the initial tree. The analysis involved 25 nucleotide sequences with all equivocal sequences being removed for each sequence brace. The optimal tree with the Sum of Branch Lengths ( SBL ) is 0.45873726. The tree was rooted with the outgroup Grus Antigone and the ingroup Family Diomedeidae.
Figure 3: Maximal parsimony analysis of taxa. Through the usage of the Maximum Parsimony tree evolutionary history of the can be inferred. The consensus Maximum Parsimony tree was obtained from the six most penurious trees utilizing homologous dataset of 1008 bases of the cytochrome B cistron of the chondriosome. Branchs matching to dividers reproduced in less than 50 % trees are collapsed. The consistence index ( CI ) is 0.663848, the keeping index ( RI ) is 0.851679 and the composite index ( RC ) is 0.565385 ( with a RC value of 0.501764 for all sites and parsimony-informative sites ) . The Maximum Parsimony tree was obtained utilizing the Close-Neighbor-Interchange algorithm with hunt degree 1 in which the initial trees were obtained with the random add-on of sequences ( 10 replicates ) . The analysis involved 25 nucleotide sequences with all equivocal sequences being removed for each sequence brace.
Figure 4: Molecular Phylogenetic analysis by Maximum Likelihood method
Through the usage of the Maximum Likelihood tree evolutionary history of the can be inferred based on the Dataset specific theoretical account. The consensus Maximum Likelihood tree was obtained utilizing 1008 bases matching to the cytochrome B cistron part. The tree with the highest log likeliness ( -3734.5355 ) is shown supra. A distinct Gamma distribution was used to pattern evolutionary rate differences among sites ( 5 classs ( +G, parametric quantity = 0.5341 ) ) .
Figure 7: Unweighted-Pair Group Method with Arrhythmic agencies ( UPGMA ) tree with native distribution of the members of Family Diomedeidae
The molecular clock trial was performed by comparing the Maximum Likelihood value for the given topology with and without the molecular clock restraints. Differences in evolutionary rates among sites were modeled utilizing a distinct Gamma ( G ) distribution. The void hypothesis of equal evolutionary rate throughout the tree was non rejected at a 5 % significance degree ( P & lt ; 0.05 ) . The ‘clock trial ‘ was performed utilizing Tajima ‘s comparative rate trial ‘ had a P value of 0.05371. The evolutionary rate was set 0.02 ( 2 % sequence divergency per million old ages ) , as specified for Cytochrome B.
The information set of 24 species of Diomedeidae and the outgroup Grus Antigone contains 279 variable sites. Therefore, 27.7 % of the full cytochrome B cistron ( Nunn et al, 1996 ) is variable, bespeaking that familial diverseness is low in this group. The truth of tree buildings decreases with a relative addition in variable sites ( Grievink et al. 2010 ) . Furthermore, as familial fluctuation addition, in other words an addition in variable sites, there is an addition in the rate of evolutionary alteration ( Sterns and Hoekstra, 2005 ) . It is understood that as the familial diverseness of a species increases, so does the stableness of the species ( Bininda-Emonds et al. , 1999 ) . 195 of the 279 variable sites were parsimony-informative, this means that 195 of the variable sites have undergone mutational alterations. A tree constructed with 195 penurious enlightening sites will be the most penurious tree with the least figure of alterations.
The uncorrected ( p-distance ) pairwise comparing is 7.8 % , bespeaking that the highest and lowest sequence divergency value between any two taxa is low. This indicates that members in the household Diomedeidae are closely related ( Kholodova, 2009 ) . The best fit theoretical account of sequence development selected under AIC ( Akaike Information Criterion = 7468.845 ) for the dataset was the GTR ( General Time Reversible ) + G ( Gamma distribution theoretical accounts ) in MEGA 5 ( Tamura et al. , 2011 ) . The GTR+G theoretical account of sequence development was selected because of the unequal base-pair frequences, the mean base frequences were estimated to be: A= 27.8, T=25.3, C=34.1 and G= 12.8. This indicates that there is a base prejudice towards AT. The estimated value of transition/transversion prejudice ( R ) is 5.38, bespeaking a prejudice in passage compared to transversion ( Tamura et al. , 2011 ) . The Gamma distribution value was 0.21 and evolutionarily invariable ( +I ) was non applicable.
A bootstrap analysis with 10A 000 reproductions was performed on the ‘Neighbor-joining ‘ tree and the ‘Minimum evolutionary ‘ tree. This method of trying with replacing sites indicated that there is a high estimation of assurance in both trees. Minimum evolutionary Heuristic Method used was the Close-Neighbor-Interchange ( CNI ) . The maximal parsimoniousness analysis produced six every bit most penurious trees, a consensus tree was obtained by ciphering a consensus with a ‘cut-off ‘ value of 50 % . In order to find if there is homoplasy, the consistence index needs to be calculated, if CI is non equal 1 there is homoplasy and as the CI value decrease the homoplasy additions. The consistence index [ CI ] [ excepting uninformative characters ] is 0.663848 and the keeping index [ RI ] is 0.851679 ( Nunn and Stanley ) . This indicates that there is homoplasy and the RI indicates that the maximal possible homoplasy. The rescaled consistence index ( RCI ) is 0.565385 for complete homoplasious characters in all the sites. Maximum parsimony Search Method used was the Close-Neighbor-Interchange ( CNI ) on Random Trees. Through the usage of the Maximum parsimoniousness it is besides seen that the tree is partially resolved with Diomedea exulans, Thalassarche steadi and Thalassarche cauta cauta sharing a soft polytomy.
The concluding ‘Maximum Likelihood ‘ tree or tree with the most likely evolutionary result selected was the tree with the highest maximal log-likelihood which was -3697.77. Tajima ‘s comparative rate trial was performed to find the equality of evolutionary rate between sequences A ( U48950 Phoebastria nigripes ) and B ( AF 076091 Thalassarche carteri ) , with sequence C ( FJ769854 Grus Antigone ) used as an outgroup in Tajima ‘s comparative rate trial. The I‡2 trial statistic was 3.72 ( P = 0.05371 ) . Due to the P-value being higher than 0.05 the void hypothesis of equal rates between line of descents is non rejected. It was found that 819 sequences were indistinguishable in all 3 sites, it was besides found that 7 sequences were divergent in all 3 sites. It was further found that Phoebastria nigripes 39 alone differences, Thalassarche carteri had 58 alone differences and in conclusion Grus Antigone has 85 alone differences.
As the first molecular clock trial was non rejected a UPGMA tree could be inferred. Through the usage of this tree it can be seen that Grus Antigone and the Family Diomedeidae shared common ascendant 2.5735 million old ages ago. In the UPGMA tree it is seen that the terminal nodes of Phoebastria albatrus, Phoebastria irrorata, and Phoebastria immutabilis and Phoebastria nigripes portion a soft polytomy, bespeaking that with respect to divergence clip the tree is partially resolved.
All species of household Diomedeidae scope from ‘near threatened ‘ to ‘critically endangered ‘ . By integrating both the distribution and the UPGMA tree one can loosely do the tax write-off that as the species ‘ native scope distribution decreases their ruddy list class and standard additions with concern. This can be seen in critically endangered species such as Diomedea dabbenena ( located merely in Argentina, Brazil, Namibia, Saint Helena, South Africa and Uruguay ) , Diomedea amsterdamensis ( Amsterdam Island ) and Phoebastria irrorata ( Chile, Colombia, Ecuador ( Galapagos ) and Peru ) . Thus species which are do n’t hold a wide distribution have a greater chance of traveling nonextant due to an increased likeliness of geographical isolation or a lessening in cistron flow in the population will roll up familial, behavioural or morphological difference. It is stated that species range sizes appear to spread out comparatively quickly post speciation, followed by a period of bit by bit range enlargement and so a diminution in scope as species age ( Webb and Gaston, 2000 ) . It can be noted from the UPGMA tree, that more frequently than non, species which diverged long ago are of an increased concern in the ‘IUCN ruddy list of threatened species ‘ . However, this is non a concrete correlativity due to the many anthropogenetic ( i.e. piscaries, habitat invasion etc. ) and planetary factors ( i.e. planetary heating, nest and egg predation etc. ) act uponing these species.
Therefore in decision, there is a correlativity between the geographical distribution and phyletic relationships of Family Diomedeidae, nevertheless, one can non easy find a correlativity between the ‘IUCN ruddy list of threatened species ‘ and geographical distribution and phyletic relationship.