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Crocodile relationships are often discussed and debated to to where some groups need to be classified. Another topic is the conservation status of some of these mysterious beasts. These are both topics that are touched on in this study which was done to determine the relationship between the genus Crocodylus and critically endangered Gavialis gangeticus which was until recently considered part of the same family as Crocodylus. Next I looked with in the Crocodylus genus to see from where did these species originate as this will help to conserve the individual species and this will help under stand which species are likely able to hybridize as this will have grieve consequences. The sequence alignment and data analyses was all done in MEGA 5 version 5 which use Genbank as its data source. Genbank being a open source it was hard finding the correct sequences. But our result that I obtained from these test revieled that Gavialis gangeticus needs to be in its own family and that there is a few Crocodylus species that do hybridize especially in captivity. So we need a much better effort to conserve our fauna of crocodiles as many are on the IUCN red list of threatened species.
Key words: Crocodylus, G. gangeticus, Phylogenetic relationships of crocodiles, Neighbour-Joining, Minimum Evolution, Maximum Parsimony, UPGMA
Using molecular systematics have become popular to characterize species and their related families. It is also a helpful tool in helping conservation in many cases. By using molecular systematics one can easily and with very high certainty classify individuals in to the correct family and even sub specie level. So by means of these systems we are going to see the relationship with in the genus Crocodylus and the relationship between it and Gavialidae. By under standing the relationships one can do conservation work on each individual knowing its relationship to its relatives which in most cases share common habitats. Thus we had a look at "The IUCN red list of threatened species", to see which of the Crocodylus genus are under pressure. Here follows the Scientific name (Common name) Status:; Crocodylus acutus (American crocodile) Status: Vulnerable A1acÂ ver 2.3, Crocodylus intermedius (Orinoco crocodile) Status: Critically Endangered A1c, C2aÂ ver 2.3, Crocodylus johnsoni (Johnstone river crocodile) Status: Lower Risk/least concernÂ ver 2.3 , Crocodylus mindorensis (Philippine crocodile) Status: Critically Endangered A1c, C2aÂ ver 2.3 , Crocodylus moreletii (Morelet's crocodile) Status: Lower Risk/conservation dependentÂ ver 2.3, Crocodylus niloticus (Nile crocodile) Status: Lower Risk/least concernÂ ver 2.3 , Crocodylus novaeguineae (New Guinea crocodile) Status: Lower Risk/least concernÂ ver 2.3, Crocodylus palustris (Marsh crocodile) Status: Vulnerable A1a, C2aÂ ver 2.3, Crocodylus porosus (Australian saltwater crocodile) Status: Lower Risk/least concernÂ ver 2.3, Crocodylus rhombifer (Cuban crocodile) Status: Critically Endangered A2cdeÂ ver 3.1 , Crocodylus siamensis (Siamese crocodile) Status: Critically Endangered A1acÂ ver 2.3 (IUCN 2012). The out group status are also available and is as follows; Osteolaemus tetraspis (dwarf crocodile) Status: Vulnerable A2cdÂ ver 2.3, Gavialis gangeticus (Gharial) Status: Critically Endangered A2bc; C1Â ver 3.1Â and decreasing in the wild (IUCN 2012). I had a look at the families and genus level of these individuals an specifically chose the two outgroups as one with in the family (Osteolaemus tetraspis) and on out side the family (Gavialis gangeticus) of the genus Crocodylus, Crocodylinae (Janke 2008, Li et al. 2007)
Crocodile have a very interesting phylogeny as there is still a very hot debate on going to which families which species belong to (Janke 2008, Li et al. 2007). I looked at the relationship with in the Crocodylus genus as a very important conservation group as seven out of 11 species are either: Lower Risk/Conservation dependent, Vulnerable or Critically Endangered. This is also taking in to consideration where these individuals find themselves where most are loosing their natural habitat at a very rapid rate (Mazzotti et al. 2009). Most of these animals where and are hunted or breed for their skin as a leather product (Blake & Loveridge 1975). In some areas as the Everglades crocodiles are the flagship species of conservation and the success thereof is measured as how good these crocodiles to and if they are reproducing naturally (Mazzotti et al. 2009). Other area where the conservation of crocodiles is important is India, Indonesia, Cuba and Malaysia. In these areas the crocodiles are under high pressure as all of them are endangered (IUCN 2012). So how can this help conservation even more? Well in crimes where illegal trading takes place we are able to see if it is an endangered specie or not that is being trade with. In the case where the specie is at danger or vulnerable the data can be used as evidence to show to which species the persons are trading in. most countries can then implement the right legislation and begin with conservation plans. Some of these countries include Zimbabwe where there was a huge success in re-populating the Nile crocodile and the on going efforts in India (de Vos 1984, McGregor 2005)
So I then looked ate the relationship of the Gavialis gangeticus in relation to Crocodylus as it is been speculated that they are not near relatives and need to be separate families (Janke 2008, Li et al. 2007). I supported this assumption made in past literature as some work has shown that this is true (Janke 2008, Li et al. 2007). My work would help to show that this is true as I expect it to be the lowest and first divergence on any of the trees drawn. This would help establish how big the impact is on the existing populations and breeding programs can be worked out to insure a very healthy and stable population with a big variety of alleles.
Crocodiles show a very great diversity and we should try and keep this as each specie plays a very important role in our natural ecosystem. From the large Saltwater crocs to the dwarf crocodiles and even their cousins the caiman and alligators.
Materials and Methods
Phylogenetic and molecular analyses of the evolution of the Crocodylus genus was done by using MEGA version 5 (Tamura et al. 2011) a powerful computing program. This program, MEGA 5, uses Genbank as its genetic database for searching and finding appropriate genetic sequences. These sequences are then added to MEGA 5 and then aligned with in the program itself. MEGA 5 can be used to work out some statistical values and to construct phylogenetic trees with the aligned data with was collected from Genbank. The trees that were drawn in this program were Neighbour-joining (NJ), Minimum Evolution (ME), Maximum Parsimony (MP) and UPGMA (UP).
Ingroup and outgroup selection
I chose the following groups as my ingroups: Crocodylus acutus (American crocodile), Crocodylus intermedius (Orinoco crocodile), Crocodylus johnsoni (Johnstone river crocodile), Crocodylus mindorensis (Philippine crocodile), Crocodylus moreletii (Morelet's crocodile), Crocodylus niloticus (Nile crocodile), Crocodylus novaeguineae (New Guinea crocodile), Crocodylus palustris (Marsh crocodile), Crocodylus porosus (Australian saltwater crocodile), Crocodylus rhombifer (Cuban crocodile), Crocodylus siamensis (Siamese crocodile)
There was only two outgroups chosen:
Osteolaemus tetraspis (dwarf crocodile), Gavialis gangeticus (Gharial)
These two outgroups were chosen as they are part of different genus each and would give a very good presentation of the Crocodylus genus and their relation ship with in the genus itself. They were also chosen as at the next taxonomic level Crocodylinae is the sub-family level to which O. tetraspis belong and G. gangeticus belong to the a family Gavialidae which is a family outside Crocodylinae, and the others that was tested for where all in the ingroup to which the genus is Crocodylus.
Sequence for alignment
The full mitochondrion genome was chosen to get a much better picture of the relation ship between the genus and family levels as the all contain around 16100 base pairs which could be compared to one another. This would give me much more variable sites to work with and more parsimony informative sites. These sequences were al taken from natural or captive animals thus no DNA degradation would have taken place.
Constructing an aligned data set for analyses
In MEGA 5 when assembling the data it opens Genbank from within MEGA 5. To get the search going one need to insert Crocodylus AND full mt-genome in the search tab at the top of the screen. I then searched for a full mt-genome sequence from Crocodylus niloticus (Nile crocodile) and then added it to the data set in MEGA 5. I used the blast option within MEGA 5 to blast against the C. niloticus sequence. Then sequences close to but not exactly the same from different species were selected and added to the data set. The two outgroups I searched manually for by entering their scientific names AND full mt-genome in the search tab. I only picked one sequence from every entry.
The sequence statistics was obtained by generating variable sites (V) and Parsimony-informative sites (Pi) which then was recorded for the set of data that we obtained. Some nucleotide compositions were also generated these were the average values for the T, C, A, and G composition in the data set. 'R' statistic, transition/transversion ratio, could be generated using the nucleotide pair frequency test. p-distance was use as the model to generate and obtain the overall mean distance of the nucleotides evolution.
The model selection could only been done after all the appropriate statistical data was obtained some that the correct model of sequence evolution could be generated and selected. This model would be used in the phonetic analyses and was done by generating the hierarchical likelihood model selection test and selecting via the AICc (Akaike Information Criterion, corrected). The most apt model was the TN93+G+I for the data set at hand.
With all the obtained data in this data set we were able to construct a Neighbour-joining (NJ), Minimum Evolution (ME), Maximum Parsimony (MP) and UPGMA (Unweighted-Pair Group Method with Arrhythmic means) (UP) tree. All the SBL values were obtained for the appropriate trees. The MP tree was constructed and the RI, CI and CRI values was obtained from MEGA 5 for the different homoplasy indexes. The molecular clock was tested by means of Tajima's test and the log likelihood score was obtained this was done to test the heterogeneity.
When the data set was aligned and trimmed I found that there 16311 nucleotides in length that was composed by 13 taxa of which 5734/16311 were variable sites (V) and 2973/16311 of variable sites that were parsimony informative (Pi). In the nucleotide base composition result I found that the average base pairs were T=24.8; C=28.8; A=31.5; G=14.9 with a transition/transversion ratio (R statistic) of 2.99 and the overall mean distance measured in 'd' statistic was 0.108 (10.8%). In the results of the hierarchical likelihood model selection test I used the AICc to choose the best apt model and this was accomplished by searching for the model with the lowest value. The TN93+G+I Model of sequence evolution (Tamura-Nei) was the chosen model as it had the lowest value. The Invariant parameter value was 0.374 and the Gamma parameter value was 0.569. Neighbour-Joining (NJ) (Fig. 1), Minimum Evolution (ME) (Fig. 2), Maximum Parsimony (MP) (Fig. 3) and a UPGMA (UP) (Fig. 4) tree was drawn and all showed similar results with a slight variation in the bootstrap values. These variations were very small as all the branches of the trees with a bootstrap value less than 50% were collapsed. In the NJ tree (Fig. 1) one can clearly see that the Crocodylus genus is split into two major linages which diverged in to seven sub linages. The two chosen out groups are still as expected on their own (Fig. 1). The Sum of Branch Lengths (SBL) was 0.84424731 for the NJ tree (Fig. 1). The ME tree (Fig. 2) was constructed on a SBL that was 0.84386914. This tree (Fig. 2) had a very similar shape and the only difference was some bootstrap values. In the MP tree (Fig. 3) there was a bit of uncertainty around the placement of C. moreletii but it still was placed in the same linage as the NJ (Fig. 1) an ME (Fig. 2) trees had placed it. The MP tree constructs many trees and chooses the one with the least amount of change in it. I got two trees that were very similar but I chose the best fit tree by looking which had the higher bootstrap values. This tree (Fig. 3) also split the Crocodylus in to seven separate linages which shows us the same as the NJ tree (Fig. 1) and ME tree (Fig. 2). It did however swap the out groups around. I obtained the different homoplasy indexes for the MP tree and these were as follow: consistency index (CI) = 0.653600; retention index (RI) = 0.471171; composite index = 0.307957(for all sites). For the UP tree I first ran a clock test using Tajima's relative rate test which was not rejected at a p-value of 0.32302 with 1 degree of freedom thus meaning I could use the UP tree (Fig. 4). I ran a second molecular clock using the Likelihood test for the rate of heterogeneity; this was rejected though thus a molecular clock could not be carried out. I was not able to exceed 10 000 bootstrap replications, as the program automatically changed it from 100 000 replications to 10Â 000 when entering 100 000.
Figure 1. Neighbour-Joining Tree. This is the optimal tree for the Neighbour-Joining tree and has a branch length sum of 0.33980092 (Saitou and Nei 1987). This tree is the result of 10 000 bootstrap replications and the percentage of trees where the associated taxa clustered together is shown by the values next to the branches (Felsenstein 1985). Bootstrap values less than 50% were collapsed. Tamura-Nei method was used to compute the evolutionary distances (Tamura 1992). A gamma distribution (gamma parameter value = 0.57) was used to model the rate of variation amongst sites. Thirteen nucleotide sequences were used in the analysis of the Neighbour-Joining tree (Tamura et al. 2011). The final dataset had a total of 16311 positions (Tamura et al. 2011).
Figure 2. Minimum Evolution tree. The Neighbour-Joining tree was used to generate the initial tree. This tree is the result of 10 000 bootstraps. Branches with less than 50% bootstrap values are collapsed. The percentage associated with the taxa are clustered together are shown next to the branches. Tamura-Nei method was used to compute the evolutionary distances (Tamura 1992). The rate variation among sites was modelled with a gamma distribution (shape parameter = 0.57). Thirteen nucleotide sequences were used in the analysis. There were a total of 16311 positions in the final dataset.
Figure 3. Maximum Parsimony Tree. There were two most parsimonious trees, of which the above tree is tree number one with the best bootstrap cut off values. This tree is the result of 10 00 bootstrap replications and the percentage of trees where the associated taxa clustered together is shown by the values next to the branches (Felsenstein 1985). Bootstrap values less than 50% were collapsed. Thirteen nucleotide sequences were used in the analysis of the Maximum Parsimony tree (Tamura et al. 2011). The final dataset had a total of 16 311 positions (Tamura et al. 2011).
Figure 4. UPGMA (Unweighted-Pair Group Method with Arrhythmic means) Tree. The Neighbour-Joining method was indirectly used to construct the evolutionary history of the UPGMA tree (Saitou and Nei 1987). This tree is the result of 10 000 bootstrap replications and the percentage of trees where the associated taxa clustered together is shown by the values next to the branches (Felsenstein 1985). Bootstrap values less than 50% were all collapsed. The tree is drawn to scale measured in MYA (Millions of Years Ago) with intervals of 0.05 MYA. Tamura-Nei method was used to compute the evolutionary distances (Tamura 1992). A gamma distribution (gamma parameter value = 0.57) was used to model the rate of variation amongst sites. Thirteen nucleotide sequences were used in the analysis of the UPGMA tree (Tamura et al. 2011). The final dataset had a total of 16 311 positions (Tamura et al. 2011).
Changes occur in the evolutionary process and that is derived by variable sites and the increase in these sites helps the evolutionary process even more (Grievink et al. 2010). The more variable sites in a proportionate manner the less accurate the construction of the tree become (Grievink et al. 2010). So it will benefit a specie to increase the genetic diversity within because this in turn leads to better survival due to a better fitness (Groom et al. 2006). The percentage variable sites were reasonably high at 35.2% thus showing a good genetic diversity threw the changes in the evolutionary process of Crocodylus. With a very high count of parsimony informative sites of 2973 would show how much change have occurred with in the genus and the outgroup (Stearns and Hoekstra 2005). Within the transition/transversion ratio the high number indicated that transition was occurring more frequent the transversion with in this data set. This high value shows a bias toward transition (Tamura et al. 2007). With the overall mean distance at only 10.8% the individuals are very close in the genus level (Kholodova 2009). In the NJ tree (Fig. 1) I could see that the length of the branches in the final tree was enough on the topology of the tree that was drawn (Saitu and Nei 1987). Crocodylus acutus, Crocodylus intermedius, Crocodylus johnsoni, Crocodylus mindorensis, Crocodylus moreletii, Crocodylus niloticus (, Crocodylus novaeguineae, Crocodylus palustris, Crocodylus porosus, Crocodylus rhombifer and Crocodylus siamensis where all very close in relation to each other according to the HJ tree (Saitu and Nei 1987). The bootstrap value level was set at 50% so to give a view to within certain relationships with in Crocodylus (Saitu and Nei 1987). There is a bit of homoplasy though as the CI value is 0.653600 which is less than one (Lipscomb 1998). This phenomenon is a result of the similarity of certain independent events (Stearns and Hoekstra 2005). We need to know that with the increase in homoplasy the chances are there that we can loose genetic variation and thus loose speciation ultimately (Groom et al. 2006, Stearns and Hoekstra 2005).
` Thus by looking at the data form all the trees (Fig. 1-4) we can clearly see the relationship to each of the Crocodylus genus individuals. They are also very close linages as to how they are geographically distributed which one could expect as the evolutionary line are close to each other. The statement of some earlier literature thought that crocodiles came from Africa seem to be wrong. As two other independent studies also revealed the relation ship between the African, New World and Indopacific species (Man et al. 2011, Meredith et al. 2011, Oaks 2011). I support these authors in saying that the crocodile specie was initially from Australia thus Indopacific (Man et al. 2011, Meredith et al. 2011, Oaks 2011). The best conclusion is that C. niloticus crossed to the New World and this is why it is closer related to the New World species C. moreletii, C. intermedius, C. acutus and C. rhombifer (Meredith et al. 2011). So for conservation it is important to note that hybridization can occur with in some of these species and that we need to take care other wise we might loose one or even both hybridizing species and form a new specie at the expense of two species (Meganathan et al. 2010). So the right conservation strategies are crucial for the survival of some of our endangered crocodiles. Thus it is my belief that by knowing their genetic relationship we can prevent this with proper breeding programs.
In this study we could also touch on another big talking point in the Crocodilian group as to where does G. gangeticus belong? Well I would support most recent studies which say they are a sister group of the genus Crocodylus but even more so they thus need to be in their own family (Janke 2008, Li et al. 2007). In all the trees (Fig. 1-4) we could see that, only in the MP tree (Fig. 3) it was different, that the G. gangeticus was under the O. tetraspis in all the trees which suggest that it is even further out of the family as O. tetraspis has already been classified as its own family (Janke 2008, Li et al. 2007). So we can now say with ease that G. gangeticus is in its own family with Tomistoma (Janke 2008).
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