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 97.599% 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