This manuscript reports several genetic analyses of Ludwigia sp. having different ploidy levels with the aim to establish their relationships and to understand the origin of hexa and decaploids. The authors infer a genealogy of species based on their genomic composition revealed by GISH. Genome size measurement, morphological observation and experimental crosses are also used to confirm the characteristics and limits of the taxa. The work which has been done is adequate with the main objectives.  Comprehensive cytogenetic studies are rather rare although essential to decipher the origin and evolution of polyploids. Here they offer a different view of evolutionary relationships among taxa than the molecular phylogenetic studies. Such data and results will be very precious for future genomic studies. In my opinion there no major issues in the Ms, however, the structure of the text and the text itself could be improved to facilitate its understanding. 

Firstly, the authors should explain, or remind the reader, that with GISH methods, the inference is based on the blocking DNA rather than the probe.

Secondly, the role of morphological observations is unclear as the authors report many features but without any morphometric analysis (measurement or coding of discrete variables and multivariate analyses). Without these morphometric analyses, it is difficult to assess the effects and even the purpose of the morphological observations. Perhaps the authors could use it as a simple verification step to prove that the identification is correct, this would allow them to reduce the size of their text.

Thirdly, the inference by GISH of the origin of the two 4X is clear but it is less clear for the 6X and 10X, the authors need to give more explanation on the relevance of intensity variation (page 13).

Finally, we are left wanting more from the experiment cross, perhaps a figure summarizing which are the crosses that always led to a dead-end could be informative to make hypotheses on what may have happened. Currently, a simple, fast reading may lead to the inference that all the crosses failed, but that’s not true.

Minor concerns :

page 2, l. 44 : so what is diploid if all plants have experienced at least one polyploidy event ?

page 5 l. 117: the word phylogeny should be kept for inference based on phylogenetic methods (e.g. cladistic). Genomic relationships or genealogy is better here.

page 8 l. 191 : how many genomic DNA ?

page 9 l. 222 : does it mean that the experimental crosses were done after GISH and based on GISH inferences ?

page 10 l. 227 : did you used a hood on flower to prevent non-controlled pollination ?

Figure 1 : improve quality of the pictures, it is pixelized.

Table 1 : estimator of variance are needed (Sd or 95% CI)

GISH  results :  The explanation of the diploid GISH is confusing. I do not understand why and how Lpm and Lh could be “genetically close” but could “correspond to different genomes”. Perhaps I miss it but the overall strategy leading to table 2 should be explained. For example why not blocking the 4X La genome by the 2X Lpm genome ? 

Page 14, l. 329 : “no plants survived at 90 days after seedling” : is it due to their genomic composition or to another (external) factor ? do you compare the survival of seedlings among inter and intra specific crosses ?

P 15 l. 349 and after : it’s true that morphometrics done on plants grown in common garden are very precious however morphometrics require measurement or at least standardized observation of discrete variables and analyses (e.g discriminant analysis). For example, the differences between Lh and Lpm need to be confirmed by the data. 

Page 20 l. 475-478 : Although I agree on the fact that cytogenetics is crucial, it does not replace molecular phylogenetics or phylogenomics. Both are complementary and both are expensive if the salary costs are considered.

The paper is generally well designed, the structure and origin of the polyploid complex is approached using combination of multiple methodical approaches. There are, however, several issues in the manuscript that should be addressed while preparing its revised version.

(1) The authors should adopt a single taxonomic concept that should be referred to throughout the paper. The current version, where the same taxon is in one paragraph referred to as L. grandiflora subsp. grandiflora and in the other one as L. grandiflora (or in one as L. grandiflora subsp. hexapetala and in the other one as L. hexapetala) is very confusing.

(2) When counting species and subspecies one should not call all of them species (the term “taxa” instead of “species” would be more appropriate). E.g., “one hexaploid species (2n=6x=48) (Ludwigia grandiflora subsp. grandiflora); and one decaploid species (2n=10x=80) (Ludwigia grandiflora subsp. hexapetala)” [lines 85-87] – these are not two separate species, but two subspecies of the same species, i.e., two taxa. The same problem is here: ”It is not easy to distinguish between the hexaploid and decaploid species morphologically and both have previously been treated as a single species (Ludwigia uruguayensis (Cambess.) H. Hara; Zardini et al., 1991)” [lines 87-89] – in the concept adopted in the previous sentence, these are two subspecies of the same species.

“Taxon name” should be used instead of “Species name” should be used also in the Table 1.

(3) Line 117: “phylogenic origin” – what is the exact meaning of this term? Would just “origin” be sufficient?

(4) The description of methods of Chromosome counting, Genome size estimation by flow cytometry, and Genomic in situ hybridization is too long, it can be shortened, providing the reference to some other papers. On the other hand, the description of morphological measurements is not sufficient. The number and origin (locality) of measured plants for each taxon and morphological character should be provided (to document the representativeness of the measurements).

(5) line 142: “Morphological observations for each species were randomly made” – this is unclear (meaning of “randomly”??).

(6) Locality details (at least geographical coordinates of the locality of origin) and number of analyzed plants should be provided for each chromosome number count and genome size measurement for each taxon.

(7) Lines 225 (“produced flowers in continuous on a shoot”), 228 (“for each of other species”) – unclear meaning (language).

(8) Lines 404, 421: “hexaploid” instead of “hexaploidy”.