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Reviewer no.1
We agree that the paper is descriptive, but not that it is overly so. We described what happened in those mats
when they were transplanted. Contrary to the reviewer´s opinion, if we shorten the description of the taxa
present, then we are cutting cross-referencing possibilities with other ecological reports (past and future) that
make use of the botanical species descriptions only. We can attest from our past experience that having had
solid descriptions of taxa in “process” papers, would have allowed for a much easier task of finding general
principles of function. In its absence, one is confounded in the uncertainty regarding the role of diversity (or
identity) in functional phenomena. There is nothing wrong with a good description.
On the other hand, the reviewer misses a discussion of the mechanism (s) that could have caused such changes.
We share such interest, but leaving the possible mechanisms for change out of the discussion was our original
intent. In reality our data cannot be used to discuss mechanisms at all, and any lengthy discussion about it
would be either trivial (i.e., a generic discussion of environmental differences at large) or highly speculative (if
we tried to pick our favorite environmental parameter). In this sense the data presented cannot be stretched to
prove or disprove something. We agree, however, that we could improve the paper with the inclusion of the
plausible reasons for community change in each case, including those proposed by the reviewer, and we have
done so now on page XX.
Reviewer no. 2.
General
The reviewer´s major concern is the lack of detail regarding the transplant environments, particularly regarding
possibility of contamination from the transplant environment. In fact, no particular procedures were carried out
to prevent or otherwise control the “contamination” from outside the inoculum. The incubations were all open
systems. This does however not diminish in the least the conclusions of the paper. Species that successfully
competed with the original population , did so because they were better suited to the new environment offered,
regardless if they originated from low level populations in the mat or were “contaminants”. But note that the mat
sampled in Solar Lake, being also an open system, has remained dominated by M. chthonoplastes for decades,
and perhaps for thousands of years. We agree with the reviewer that the possibility of novel populations
originating from other parts of Solar Lake is speculative. We have consequently toned down that paragraph. It is
however important to still note that populations morphologically resembling some of the “newcomers” have
been described from Solar Lake, suggesting that such possibility is certainly one to be considered. We now
leave the judgement to the reader.
Obviously, we caused some confusion by not explicitly stating what we meant with increases in diversity. In
fact both diversity (in Shannon´s sense) and Richness increased in some mats. This is now clarified in the text.
However, there is no apparent paradox in an increase in diversity, since this can be achieved by an increase in
the relative abundance of rare species (or DGGE bands) or, in open systems, by immigration of novel species.
BUT ALSO THE DECLINE OR DISAPPEARANCE OF A DOMIMANT species results in increased
Shannon´s diversity, with unchanged or decreased species richness.
Use of terms:
-biomass: has been substituted by biovolume which is what was measured. We had used biomass, since the
measurents were only relative (% of total counted) , and we had assumed a universal ratio of volume to mass.
But of course, there is no need for such assumption as the points are equally well made with the use of
biovolume.
The calculation of the biomass on page 14 was done as described exactly in the reference mentioned there
(Nübel et al.,1999), so there is no need to redescribe it again.
-cosmopolitan: (applied to M.chthonoplastes). We did not assert that this cyanobacterium is a cosmopolitan
SPECIES. Perhaps it is not a species (this depends on a definition). But the fact that virtually indistinguishable
populations of this cyanobacterium exist in various part of the world is a well-established fact that does not stem
from the results presented here, but has been published elsewhere. The study cited here (and in the text) did not
only include 16S RNA sequences, but also a battery of other tests including chemotaxony, biochemistry and
ultrastructure.
The variation in sequences obtained here is NOT large (i.e: 98% or higher similarity )
(checked and found correct 97.5-99% similarity) compared to, for example, Spirulina, Halothece, or
Trichodesmium, or other well studied ones, and much , much smaller than the differences found by Dave Ward
in thermophyllic Synechoccosus lividus populations. Undoubtedly there will be genetic variance within M
chthonoplastes, but the question is one of degree. We note that all new sequences presented here are
environmental, (not strains as surmised by the reviewer) originating from at least two consecutive rounds of
PCR (one from bulk DNA and a subsequent one from the DGGE band reamplifications). It cannot be
discounted that the variability encountered in M chthonoplastes stems partially from sequencing and
amplification errors. Studies conducted with cultured strains show identical or virtually identical sequences.
- polyphasic: we agree that “ polyphasic taxonomy” refers to the use of extensive batteries of test. We ( and
others) have been using the term “polyphasic approach” for the study of natural communities, inspired on the
former term. By necessity the number of “fingerprinting” test is bound to be smaller than in taxonomy, but the
principle remains the same. In fact even our “triphasic” approach is more extensive that usual.
Specific comments:
-We still like our title.
-insert suggestion: OK
-chemolithotrophs important?. Chemolithotrophs were most certainly present, but we disagree that they are net
primary producers. They may fix carbon, but this is only at the expense of the redox gradients created by the
cyanobacteria and the various decomposers. In a way they recycle the carbon already fixed and mineralized.
-Halothece is not italicized because it is not a generic name but just the name of a phylogenetic cluster
-Vacuole, as suggested, would be incorrect. Vacuole refers to a membrane-bound eukaryotic organelle The
correct name is gas vesicle, meaning a proteinaceous prokaryotic structure containing gas.
- more data on environmental conditions: as requested we include a longer description of the experimental
ponds, temperature ranges and “low light”
The low light intensity of the (M) mat was measured and found to be ( ranging between 10-20µE). The
measurment was done in the place were the mat was kept.
Reviewer 3
The major concern of this reviewer is the limitation of the analyses to the “photosynthetically active layer” as
defined by active oxygenic photosynthesis. He/She contends that cyanobacteria also may be present in deeper
layers of the mat, and argues that this part of the population is being missed in our analyses.
We principally agree with this contention. However, most cyanobacteria that are found in deeper
layers, where no visible light penetrates, can only be doing sulfide-dependent anoxygenic photosynthesis or just
having a minimal metabolism. They represent the buried layers of previous phases of growth, and they are not
likely to be very responsive to changes. We could have included deeper layers in the analyses, but then the
following rather serious problem would have been introduced: the importance of the “active” population
becomes diluted in a background of signatures from dormant, old populations. Because the sensitivity of (any)
community fingerprinting methods is limited, this may lead to missing secondary populations from the active
layer, and to a much reduced ability to detect community changes, which was the purpose of the study. Not
delimiting the study to the photosynthetically active layer would thus have resulted in more detriment than
benefit. This point is now made in the experimental section.
This notwithstanding, the point is well taken that such deeper populations may have acted here (and
may act in Nature) as reservoirs of biodiversity. It is in fact possible that some of the population changes
detected may have had its origins in buried cyanobacteria. This point is now made in the discussion
Yes the Phormidium-like population had to come from somewhere (see response to Revierwer 2). We
(purposefully) stated that we did not observe it in our samples of Solar Lake , not that it was not there, since we
only counted subsamples. All is meant is that, compared to the other morphotypes, it must have been either
rare or absent. Maybe it would have been interesting, as the reviewer suggests, to proceed with template
dilutions, or to keep counting samples to see if we could fish the Phormidium or its sequence out of Solar Lake,
but this would not have changed our conclusions in the least: first it was rare and then it was common. We fail
to see how dilutions of template could have aided in identifying our mistery cyanobacterium in the FB, since ,
even without it, we could fish out the sequence. The main problem is that we have a sequence without an
attached morphotype to it.
1. One observation related to the phormidium was that, though it was microscopically very very
dominnt in the FB mat (we could observe it every where spreading), but if you look at the DGGE band intensity
you notice that it was not very strong which means that this cynobacterium does not break easily during DNA
extraction and therefore the DNA content was realtively not much. This means if the phormidium like was there
in SL but very rare then it would have been very difficult to get a DGGE signal related to that organism.
2. (in Fig1 We called the culture TFEP-A which i renamed in the second paper to TFEP-4. Do you
think it is better to change it so that when we refer to it then it is clear which one we are taking about???)
3. The additional band over SL2 was not a real band. It could be a smear of DNA, which was
concentrated there and appeared like a real band. Cutting that band and re-amplifying it gave always all other
bands in that lane without it. Trials were made to obtain i from other PCR amplificates but it was not at all there
(see for example the other two lanes in the SL DGGE picture). This suggests that this “band-like” was simply an
artifact in that particular PCR reaction.
4. Description of the mat conditions:
Description of the experimental pond in Eilat (as described in the extension proposal)
The experimental pond contains 48m2 (this is also the size of the pond) of cyanobacterial mat inoculated from a
total 10m2 of source mats that were collected from the Solar Lake in the years between 1995-1998. The pond is
fed by oligotrophic seawater from the nearby Gulf of aqaba, which is allowed to evaporate until it has a salinity
of 90±5 ‰. The ponds (will be) kept at this salinity either by manual control or autamatically. The proper
development of the mat in this system requires a persistently low nutrient load in the overlying brine less than
5µm NO3-, not detectable NH4+, and less than 0.1µm PO43-. The nutrient na d chlorophyll a levels (will be)
monitored on a bi-weekly basis.
The parameters were measured everyday during our visit to the ponds in April 99. The temperature range was
between 22 in the morning to 30 in the mid-day. The salinity range was 85-100‰.
So these parameters can be true for the (FB) mat but not to the (EP) mat which was collected in Nov. 1998 and
sent immediately to the MPI in brine filled boxes along with (SL) mat sample.(I dont have exact information
about the parameters in the experimental pond at the time of sampling ).
The same is also true for the (SL) mat which was collected in Nov. 1998 and sent immediately. By refering to
Andreas Wieland thesis
The temperature was 28C and the salinity was 105ppt (for a sample collected in Nov 96).
...................................33C..................................114ppt(for a sample collected in June 97).
....................................28C ...............................86ppt (......................................in APril 97)
....................................30C ....................................83...........................................in NOv 97