I found this article useful for species characterization: http://www.ncbi.nlm.nih.gov/pubmed/19700448 IJSEM has some other articles on that topic, like this one: http://www.ncbi.nlm.nih.gov/pubmed/24505072

To be sure whether you have a new species or not and want to introduce the isolate as a new species, you need to perform a phenotypic characterization, including morphology, biochemical tests, fatty acid analysis, etc, especially considering the close similarity in 16S rRNA sequence. Have you checked the G-C content of your isolate and the reference strain?

I think you need a combination of phenotype & genotype characterisation to decide if its a new species or not.

good luck  

I found this article useful for species characterization: http://www.ncbi.nlm.nih.gov/pubmed/19700448 IJSEM has some other articles on that topic, like this one: http://www.ncbi.nlm.nih.gov/pubmed/24505072

To be sure whether you have a new species or not and want to introduce the isolate as a new species, you need to perform a phenotypic characterization, including morphology, biochemical tests, fatty acid analysis, etc, especially considering the close similarity in 16S rRNA sequence. Have you checked the G-C content of your isolate and the reference strain?

I think you have to follow the suggestions of Christian,you will need a combination of Phenotypic and Genetic characterisation of your isolate and compare your findings with the referenced strain. I think you should be fine afterwards. Best of luck.   

Phenotypic & geotypic characterization & its comparison with reference strain is essential. Check the G-C content as well.

The relationship between 16S rRNA similarity and DNA-DNA hybridization is not necessarily that of a linear correlation. 70% DNA-DNA hybridization is a recommended threshold for species delineation, so you likely have isolated a new species. Note, it can happen that 16S rRNA is >98% and DNA-DNA hybridization is

16S rRNA similarity is good at figuring out if a bacterial species is within the same genus, but for many bacterial groups can not be used to determine species-level identification. 

The new genomic/bioinformatic tools that are in use when comparing novel species include ANI, AAI, TETRA (tetranucleotide) and dDDH (digital DNA-DNA hybridization, which I think you referred to as GGDH?). Its sounds like you mostly determined these already, however, your confidence intervals are extremely wide. 87.5% +/- 4.5% ANI puts an upper limit of 92%, which is close to the species cutoff. 89.9% +/- 13.3% for AAI indicates an upper range of 103.1%, which is impossible. I would first recheck these numbers. I use JSpeciesWS for my ANI and TETRA analysis (the http://jspecies.ribohost.com/jspeciesws/#Home). It does not provide AAI analysis, but from my understanding, you only need AAI when your ANI results are 80% or below, which is not the case you provide above. For dDDH I use the online tool at GGDC http://ggdc.dsmz.de/.

In addition to ANI, TETRA and dDDH, you could include a phylogenetic tree, either based on whole genomes (since you have the sequence) or a MLSA approach that has been validated within genus you are studying. G&C analysis across the genome is easy and should also be included.

As far as whether or not you need to include fatty acid analysis and a long series of biochemical comparisons; I don't know if these are required if you have the full genome. Before the genomic area, those were necessary when naming a new species. But assuming that speciation is the result of genomic evolution, a comprehensive genomic comparison alongside basic phenotypic characterization should be adequate. 

I work with Pseudomonas bacteria, so here is a paper within my field that discusses the relationship between ANI, TETRA, dDDH, phylogenetics and species-cutoff.

http://journal.frontiersin.org/article/10.3389/fmicb.2015.00214/full

you could use the below species identification tool if you have genome sequence

http://vm-lux.embl.de/~mende/specI//

Thank you all for the answers. The arcicles and links you mentioned will be also helpfull.

Brittan Starr Scales - GGDH was just a typo, I meant GGDC available from DSMZ. 

I work with Pseudomonas mandelii, which belongs to wide P.fuorescens group. Problem in my case is that the genome of a type strain is not sequenced yet. There are only 3 genomes available within this subgroup, but they are not considered as type strains. Does it influence the reliability of the analysis?

G-C content of  "my" strain is slightly higher (0.4-0.6%) than in those 3.

I encounter Pseudomonas mandelii in my analysis all the time. I also work within the P. fluorescens species complex.

You are correct; there are no P. mandelii type strains fully sequenced at this time, but that does not have to limit your analysis. I would recommend looking at the type of analysis provided in the Gomila paper I provided above and in Garrido-Sanz paper just published February of this year, provided below. The second paper focuses on the phylogenomics of the P. fluorescens species complex and provides all the tools necessary in determining whether your strains fall within a previously determined species-group or are a new species entirely.

Repeat the analysis in these papers, including as much, if not all, of the P. fluorescens species complex genomes that are fully sequenced at this time. When you re-create the phylogenetic tree using ANI, TETRA, GGDC and MLSA, you will be able to see where your strains fall within the complex. If they form their own branch (one that is highly supported by confidence intervals) away from all previously determine species-groups, then they are a new species. But if they fall within a previously determined species clade- even if they have differing GC content- they are NOT a new species. 

Be aware that the P. fluorescens species complex contains a huge amount of genetic diversity and members of the same species can differ quite a bit by GC, or ANI, or MLSA but not ALL of them at the same time, which is why you have to use multiple metrics. The Mulet 2010 paper, the first to really put P. fluorescens into a species complex, discusses the huge genetic diversity among these strains. 

Gomila:  http://journal.frontiersin.org/article/10.3389/fmicb.2015.00214/full

Garrido-Sanz: http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0150183

Mulet: https://www.ncbi.nlm.nih.gov/pubmed/20192968

If you can't access these papers via the links, I have copies of them I can give you. Let me know if you have any more questions. 

For say that you have discovered new bacterial species, do you have to do is a Analysis polifasic, how Abdulhadi Albaser say... you need make polifasic analysis (fenotype and genotype) . Why a analysis polifasic? The razon lies in that although two (or more) complete genome look alike each other the expression can be different. If you have the genome complete of the "new bacteria", you can find codificants region that mark the difference at phenotipic level... search it. Good luck.

I agree with Brittan Starr Scales. Also notice that the genomes currently published as P. mandelii (strains 36MFCvi1.1 and JR-1) are in quite distant branches as shown on our latest work (Garrido-Sanz et al, 2016).  And the same happens to the strain PD30. This and the dDDH values via GGDC show that indeed these strains are different species. On the other hand, our multilocus analysis have shown that P. mandelii JR-1 is the closest one to the P. mandelii type strain.

Looking at the values of ANI and GGDC you got, and assuming the subject genome you’ve used is one of the published P. mandelii genomes, I’m certain your genome belongs to the P. mandelii subgroup as the values are above the group thresholds published in Garrido-Sanz et al, 2016. Unfortunately, none of the type strains of the five species included in this group is sequenced (P. lini, P. migulae, P. mandelii, P. arsenicoxydans and P. fredekirsbergensis). I would perform a MLSA with at least the genomes belonging to the P. mandelii group, including type strains. Housekeeping genes for these type strains are available via PseudoMLSA database as described in Mulet et al., 2010 and in Gomila et al., 2015. If your genome is not close to any of the type strains, meaning it doesn’t cluster together with a previous described species, and it has sufficient statistical support, your strain is a new species. I’m not sure how to proceed in case you want to describe a new species or the requirements for that. You can also use GGDC to see if there is any other unclassified genome that cluster together with your genome.

Thank you all once again, it was very helpful!

I agree with Prof. Gosh with what he said. Just hace the 16r RNA sequence, align it  using BLAST and then use the BLAST data to search the phylogenetic tree for your new bacterial species