Geology Reference
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(2008). The genera Phormidium, Microcoleus as
well as Leptolyngbya as presently circumscribed
are polyphyletic in the 16S rDNA phylogenies
(Fig. 7); they are in urgent need of taxonomic revi-
sion. Oscillatoriales (sensu Anagnostidis &
Kom
´
rek 1988) that are associated with tufa for-
mation in freshwaters are often described as Phormi-
dium incrustatum, P. calcareum and P. foveolarum
(e.g. Freytet & Verrecchia 1998; Arp et al. 2001b;
Pentecost 2003). No cultures and no sequences are
yet available as references for P. incrustatum and
P. calcareum to clarify their distinction. However,
the P. incrustatum/calcareum morphotype has
been found very frequently in both karstwater
streams by microscopy (e.g. Fig. 5). It is possibly
represented by the 'Unidentified B' lineage in
the environmental rDNA clone libraries (Fig. 7)
because here the latter lineage was most frequently
recovered. Leptolyngbya foveolarum, although fre-
quently observed by microscopy in samples from
both hard water streams, may not have been recov-
ered in our molecular analyses yet. None of the ana-
lysed cyanobacterial clones appeared to be closely
related to the only available reference strain of
L. foveolarum (strain Kom
´
rek 1964/112; Fig. 7).
Our molecular analyses also failed to recover
cyanobacteria that represent the Aphanothece and
Hyella morphotypes (Fig. 5), i.e. no sequences
from members of Pleurocapsales/Chroococcales or
Chroococcidiopsis were retrieved at the WB5 site.
The Chamaesiphon morphotype, easy to detect by
its characteristic polar growth with formation of exo-
cytes, has been detected in resin-embedded biofilm
thin sections from the Deinschwanger Bach and
Erasbacher rivulet, where it formed a characteristic
biofilm constituent on spar-cemented moss surfaces.
The molecular analyses revealed Chamaesiphon in
the biofilm at the WB5 site, and it was also observed
in raw cultures developed from tufa biofilm material.
However, this morphotype could not be detected
in resin-embedded biofilm sections from WB5.
This might indicate that Chamaesiphon may not
always exhibit its typical growth form in the biofilms
and further underlines the importance of a molecular
investigation of cyanobacterial diversity of the tufa
biofilms.
Diatoms. The high abundance and microscopic
diversity of diatoms in biofilms associated with
tufa formation has already been recognized more
than hundred years ago by Reichelt (1899) and has
been reported continuously since then (von Pia
1934; Gr¨ninger 1965; Rott 1994; Reichardt 1995;
Freytet & Verrecchia 1998). A large variety of
diatoms has been reported from European tufa
biofilms, i.e. 29 genera with 169 species have been
distinguished by microscopic features of their frus-
tules (Rott 1994; Reichardt 1995; Freytet & Verrec-
chia 1998). Several diatom genera (e.g. Amphora
Ehrenberg ex K¨tzing, Gomphonema Ehrenberg,
Nitzschia Bory) are conspicuous in karstwater
stream biofilms as they exhibit calcite precipitation
around their stalks (e.g. Gomphonema olivaceum
var. calcareum (Cleve) Cleve-Euler; Winsborough
& Golubi
´
1987).
In an ongoing study the diatom diversity has
been assessed by a combination of both a culture
and a culture-independent approach. At the site
WB5 thirteen different lineages of raphid pennate
diatoms were detected (Fig. 8); they may represent
eight different genera and most likely 13 species
(Table 3). Biofilms at only two other tufa sampling
sites of both karstwater creeks have been found to
harbour a larger diatom diversity of their biofilms,
the range of diatom lineages per site has been
from 1 to 16 lineages. Taken all sites of both karst-
water streams together, a total of 17 different
lineages which occurred at two and more sites
have been recovered. They belong to a large mono-
phyletic clade representing the raphid pennate
diatoms in the 18S rDNA phylogenies, but also
three species of araphid pennate diatoms are found
(Brinkmann et al. 2007a, b). At phenetic sequence
similarities of 99% and higher, diatoms were ident-
ified at the species level (Table 3). Each diatom
lineage was represented one to five times in the 20
sequenced clones which indicated a low coverage,
i.e. that the detected diversity was still below the sat-
uration level. Four diatom species were also recov-
ered by cultures (Table 3); examples are shown in
Figure 9. Most of the WB5 diatoms were also recov-
ered from other sites of both karstwater streams;
only three species were found exclusively at the
Fig. 7. Maximum likelihood phylogenetic analysis of cyanobacteria from tufa biofilms and stromatolite laminae of
the Westerh
¨
fer Bach site WB5 (shown in bold) using 78 complete 16S rDNA sequences (1401 bp long; 667 variable/
466 informative sites). For cyanobacteria recovered from the tufa core, their location at a certain layer and the
percentage of clones from the 711 analysed clones of the core sample (BF, biofilm, CL 1-6, core layers 1 - 6; see
Fig. 10) is given. The tentative allocation of four morphotypes to certain cyanobacterial lineages is indicated. The
Tychonema/P. autumnale/Microcoleus clade has been drawn to a larger scale to show the genetic distances more
clearly. A circle indicates the various independent lineages to which members of the genera Phormidium, Leptolyngbya
and Microcoleus have been assigned. The sequences of Escherichia coli (AE000129), Bacillus subtilis (AJ276351) and
Chlorobium tepidum (M58468) were used to root the phylogeny, but have been pruned away from the graphic. For
details of cloning, sequencing and phylogenetic tree analysis see Appendix.
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