Fungi as Architects of the Rimstone Dams in 
Huanglong, NSD, Sichuan, China
Authors: Jie Xie, Gary Strobel, Wei-Feng Xu, Jie 
Chen, Hui-Shuang Ren, De-Jun An, and Brad Geary
The final publication is available at Springer via http://dx.doi.org/10.1007/s00248-016-0841-6.
Xie, Jie, Gary A. Strobel, Wei-Fang Xu, Jie Chen, Hui-Shuang Ren, De-Jun An, and Brad Geary. 
"Fungi as Architects of the Rimstone Dams in Huanglong, NSD, Sichuan, China." Microbial 
Ecology 73, no. 1 (January 2017): 29-38. DOI: 10.1007/s00248-016-0841-6.
Made available through Montana State University’s ScholarWorks 
scholarworks.montana.edu 
Fungi as Architects of the Rimstone Dams in Huanglong, NSD,
Sichuan, China
Jie Xie1 & Gary Strobel2 & Wei-Fang Xu1 & Jie Chen1 & Hui-Shuang Ren1 & De-Jun An3 &
Brad Geary4
Abstract The Huanglong park area of the Sichuan Province
of China is a unique scenic area of the world. It is known for
its thousands of aquamarine-colored pools that are formed
behind naturally formed rimstone dams of travertine (calcite)
along a cold water stream. The travertine, based on its crys-
talline structural analysis, is of biological origin. This makes
sense since the temperature of the waters of Huanglong varies
from 5 to 7 °C and thus geochemical crystallization does not
occur as it does in other locations around the world possessing
thermal pools whose structures are primarily formed through
cooling processes. Fungi and bacteria were discovered asso-
ciated with both leaves associated with the calcite dams as
well as in the older parts of well-established dams. Several
species of Phytium, a phycomycete and an endophyte,
accounted for over 45 % of all of the fungi successfully iso-
lated from the well-established dam samples and at least 85 %
in the floating leaf samples. Saprolegnia spp. (Phycomycetes)
along with Phoma spp. (Ascomycetes) were noted along with
Mortierella sp. as other dam-associated fungi. The fungal hy-
phae observed on dead leaf material as well as in the calcite
dams directly served as nucleation points for the formation of
crystalline CaCO3. Eventually, these crystals grow large
enough to fuse to make calcite plates which form the main
structural feature of all of the travertine dams in this area.
Interestingly, each of the individual crystals associated with
the dams has an associated hole in its core where a fungal
hypha used to reside as observed by scanning electronmicros-
copy. While diatoms were present in the analysis, they too
seem to contribute to the structure of the dams but in a minor
way. The only bacteria isolated from the older dam of this
aquatic environment were Pseudomonas spp. and their role
in dam formation is uncertain. Huanglong is a unique and
beautiful place, and the water features present in this area
can definitely be attributed to those fungal architects that en-
courage calcite crystal formation.
Keywords Aquatic fungi . Travertine . Calcium carbonate .
Crystals . Pools . China
Introduction
Only a few areas in the world are known to possess naturally
formed travertine (calcite) rimstone dams holding back beau-
tiful blue pools of cold water (5–7 °C). This is in sharp con-
trast to several hot water travertine water formations in such
places as Yellowstone National Park, USA; Badab-e Surt,
Iran; and Pamukkale, Turkey. The cold water travertine areas
are in Band-e Amir, Afghanistan, and certainly the most im-
pressive is the Huanglong Natural Scenic District of Sichuan,
China. The latter area is so impressive that it was declared a
World Heritage Site by United Nations Educational, Scientific
and Cultural Organization (UNESCO) in 1992. It is famous
for the colorful travertine landscape, diverse biological eco-
systems, and an extensive system of beautifully colored pools
created by travertine dams varying is height from several
* Gary Strobel
uplgs@montana.edu
1 State Key Laboratory of Silkworm Genome Biology, College of
Biotechnology, Southwest University, Chongqing 400715, People’s
Republic of China
2 Department of Plant Sciences, Montana State University,
Bozeman, MT 59717, USA
3 Huanglong National Scenic Resort Administrative Bureau, Songpan
Sichuan 623300, People’s Republic of China
4 Department of Plant and Wildlife Sciences, Brigham Young
University, Provo, UT 84602, USA
centimeters up to 5 m. The site of travertine deposition in
Huanglong Valley is located in Songpan County of
Northwestern Sichuan Province (32° 45 N, 103° 50 E). It is
also home to many endangered animal species including the
giant panda and the Sichuan golden snub-nosed monkey [1].
The water in the discharge area of Huanglong is extremely
high in calcium carbonate concentrations and this is apparent-
ly due to the dissolution of the deep basement limestone rock
through which the water in this area originates and flows [2].
This is a critical observation since this calcium carbonate
serves as the source material for the construction of the calcite
(travertine) rimstone dams of Huanglong. However, this con-
dition alone is not enough to account for the dramatic appear-
ance of nearly perpendicular walls made of impenetrable
layers of calcite making up the structure of each dam at
Huanglong. Overall, it appears that a combination of geo-
chemical, mineralogical, and microbiological factors contrib-
ute to the formation of calcium carbonate crystals in this nat-
ural cold aquatic environment (5–7 °C). It is well understood,
however, that that there are significant differences between
biogenic calcite and geologically formed calcite. Thus, if bio-
genic factors have taken part in the processes of travertine
formation this can ascertained by examining the crystal struc-
ture of the calcite. It turns out that the calcite in Huanglong
dams is a typical anisotropic lattice distortion, which means
that biogenic factors have contributed to its formation [3].
However, there is little information on how any life form
may have contributed to travertine dam formation of
Huanglong [4]. Recently, the influence of diatoms on traver-
tine dam formation at the Huanglong Natural Scenic District
has been studied but no conclusive information on the direct
involvement of diatoms in the direct formation of the calcite
structure of the dams has been provided [3]. However, it is
possible that other microorganisms might be in the formation
of Huanglong travertine dams. These include aquatic mi-
crobes such as filamentous fungi and bacteria that have not
been explored or even considered as playing a role in dam
formation. Yet it is known that microbes, especially filamen-
tous fungi and some bacteria, can serve as nucleation sites for
calcite crystal formation in vitro [5–9].
Several interesting observations were made in
September of 2015, by the first two authors of this report.
As leaves were falling from the deciduous trees and float-
ing on the surface of the cold flowing waters in the
Huanglong area, they could each be observed supporting
mats of stringy filamentous fungi. These leaves then were
literally stopped in their movement as they hit each trav-
ertine dam wall and became immobile. The idea that these
leaves and their respective microflora were contributing to
dam formation was then hypothesized. Samples of leaves,
dam structures, and other features of the area were taken
and analyzed by a myriad of methods. Overall, it became
obvious that the travertine rimstone dams of Huanglong
were formed, in part, by the fungal hyphae associated
with the dead leaves floating on the cold calcite saturated
waters of this area. The numerous hyphae each serve as a
nucleation point sources for calcite crystal formation
which ultimately results in the rapid growth of the crystals
and the eventual fusing of structures to form larger calcite
plates. Thus, this report shows which fungi, bacteria, and
other microbial forms were recorded from the waters and
features of Huanglong and indicates how these organisms
contribute to one of the most beautiful and unique places
on this earth.
Materials and Methods
Sampling
Two different types of travertine sample were collected from
Zhengyancai pool of Huanglong Natural Scenic District (32°
44′ 26″N, 103° 49′ 51″ E). A soft sample (HL1) was collected
from the edge of the rimstone pool. It was yellow to brown
and appeared as a little Chinese dragon in the pool. The hard
sample (HL2) was collected by scraping the crusty surface of a
well-established calcite dam. Each sample was about 10 g. Yet
another sample consisted of leaves supporting massive fungal
growth caught on the dam face in the same area were collected
(HL3). The samples were immediately transported back to the
Southwest University in a sterile plastic sampling bottle and
stored at 4 °C until processed and examined.
Morphological Observations, Scanning Electron
Microscopy, and Edax
In order to acquire detailed information about the morpholog-
ical features of the Huanglong travertine, light microscopic
observations were performed on the soft sample (HL1) and
hard sample (HL2). The samples were transferred and mixed
in the sterile water on the slide and then examined with an
OLYMPUS FV1200 laser scanning microscope, and the sam-
ples were photographed. Samples were also prepared for scan-
ning electron microscopy (SEM). The SEM samples were
slowly dehydrated in ethanol and then critical point dried,
coated with gold palladium, and examined with an FEI
Helios Nanolab 600 dual beam instrument. The SEMwas also
used to do elemental analysis of the individual crystals asso-
ciated with fungal hyphae using an Edax energy dispersive X-
ray analyzer on the SEM [10].
Microbial Isolation and Storage
In this study, efforts were made to investigate the micro-
bial biodiversity associated with the dams forming the
aquamarine-colored pools of the Huanglong Valley using
a culture-dependent method rather than a strictly exclu-
sive metagenomics approach. The following procedures
were used to acquire the microbes associated with the soft
sample (HL1), hard sample (HL2), and leaf (HL3) which
were collected from the travertine pools. Firstly, the sam-
ple was cut into small fragments in sterilized petri dish
with surgical blades and then aseptically transferred to the
plate surface of oatmeal agar (OA), potato dextrose agar
(PDA), water agar (WA), and nutrient agar (NA). After
several days of incubation at 22 °C, hyphal tips of the
developing fungi were aseptically removed and placed
on the plate of PDA. For the bacteria, a small amount of
sample was obtained from the edge of the lawn and then
streaked onto the PDA medium. Thus, pure cultures were
obtained. The isolated fungi were preserved by growing a
pure fungal culture on sterile barley seeds and storing the
seeds at −70 °C. Fresh bacterial liquid cultures were
maintained in 15 % glycerol. All individual cultures were
appropriately labeled and stored at the author’s lab at the
State Key Laboratory of Silkworm Genome Biology,
College of Biotechnology, Southwest University,
Chongqing 400715, People’s Republic of China.
Microbial Biodiversity Analysis
The microbial samples were individually analyzed by am-
plified the ITS sequences of fungal DNA and 16S rDNA
sequences of bacterial DNA. Genomic DNAwas extracted
from the colony grown on PDA medium after about
1 week. DNA templates were prepared with the
Prepman Ultra Sample Preparation Reagent (Applied
Biosystems, USA), according to the manufacturer’s
guidelines. The phylogenetic analysis of fungi was carried
out by acquisition of ITS-5.8S ribosomal gene sequence
using the polymerase chain reaction (PCR) with universal
ITS primers ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′)/
ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) or ITS5 (5′-
GGAAGTAAAAGTCGTAACAAGG-3′)/ITS4 (5′-TCCT
CCGCTTATTGATATGC-3′) [11, 12]. The PCR condi-
tions were set as follows: initial denaturing at 94 °C for
Fig. 1 The cold water pools of
Huanglong in Sichuan Province
of China. a The initiation of pool
formation by the accumulation of
leaves and other organic debris
deposited in an arching manner in
the stream bed. b A nearly
perpendicular calcite wall formed
by calcite deposition to form a
dam. cAmore mature calcite dam
that is over a meter in height. dAn
intricate system of calcite dams in
the midst of the stream bed. e A
giant wall calcite dam (est. 3 m)
over which a water falls has
developed. f A biological mat
located at the top of a calcite dam
containing filamentous fungal
filaments, bacteria, and algal
forms (mainly diatoms). Leaf
material supports microbial
growth
5 min, followed by 30 cycles of denaturing at 94 °C for
30 s, annealing at 55 °C for 30 s, extension at 72 °C for
45 s, and a final extension at 72 °C for 10 min. The
phylogenetic analyses of bacteria were carried out by ac-
quisition of 16S rDNA ribosomal gene sequence using
PCR with the universal pr imers 27F (5 ′-AGAG
TTTGATCCTGGCTCAG-3 ′) and 1500R (5 ′-GGTT
ACCTTGTTACGACTT-3′) [13]. The PCR conditions
were as follows: initial denaturing at 95 °C for 4 min,
followed by 30 cycles of denaturing at 94 °C for 30 s,
annealing at 50 °C for 45 s, extension at 72 °C for 60 s,
and a final extension at 72 °C for 8 min. PCR products
were purified and sequenced by Sangong Biotech
(Shanghai) Co., Ltd. The amplified sequences were car-
ried on the phylogenetic analyses on NCBI (http://blast.
ncbi.nlm.nih.gov/Blast.cgi). According to the blast results,
the microbial population distribution was assayed at the
genus level with designations to the genus level of
microbes having at least a 95 % or greater match to the
GenBank database. Furthermore, all sequences were
submitted to the GenBank. Accession numbers of the
strains were from KX378872 to KX378962.
Results
The Huanglong Area
The Huanglong Gou—Valley of the Yellow Dragon—is an
outstanding karst landscape within the southern part of the
Min Shan range which runs from the eastern Qinghai-
Tibetan plateau down to the Sichuan basin in SW China.
The Huanglong section of the reserve covers the catchment
of 22 tributaries of the upper Fujiang River which has its
source in the Snow Mountain Ridge. The Huanglong Valley
is one of these tributaries. The slopes above the valleys are
Fig. 2 Fungi serving as to
nucleate calcite crystal formation
from samples taken in
Huanglong, China. a Hard calcite
wall surface sample (sample HL-
2) was taken for observation and
some hyphae were associated
with individual crystals. b Surface
samples taken from the soft
calcite wall surface (sample HL-
1), showing many fungal hyphae
each with a corresponding calcite
crystal. c The wall is composed of
a myriad of fused calcite crystals
each in the range of 10–100 μm.
c, d Careful examination of each
crystal reveals the presence of a
small hole 3–4 μm in diameter
which is the exact diameter of a
fungal hyphae (arrow). e A
broken fungal hypha is seen
emerging from a hole in a calcite
crystal (arrow)
forested and steep. Above the tree line, the site is surrounded
bymountains; their strata tilted into jagged peaks, with several
glaciers. The pyramidal summit of Xuebaoding (Snow
Treasure Peak) is permanently snow-covered and carries the
easternmost glacier in China.
The 3.6-km-long Huanglong Valley consists of clusters
of over 3300 brightly colored travertine pools varying in
color depending upon the temperature of the water in each
pool to a certain extent (Fig. 1). For instance, some of the
warmer more stagnant pools support green algal growth
and are varying from yellowish to green while the major-
ity of the pools are cold and are aquamarine varying to
deep blue in color (see cover of this issue of ME) (Fig. 1).
In the area are terraces, travertine shoals, water rapids,
pools, and waterfalls. There are many caves in the valley.
Each of these features is basically related to a geological
source of calcite which originates in the tilted rock strata
of the area. It is comprised of largely carbonate Paleozoic
deposits over 4000 m thick and Mesozoic deposits of at
least 1000 m thick with a variety of sedimentary rocks in
a cataclastic sedimentation formation [14]. The water
flowing over and through this area contains saturating
amounts of CaCO3 [14].
Central to a study of the microbiology of the
Huanglong Valley and its travertine pools is an under-
standing of the botany of the area. Huanglong lies close
to the intersection of four floristic regions: Eastern Asia,
Himalaya, and the subtropical and tropical zones of the
Palaearctic. It is situated at the transition between the
eastern damp forest and the zone of mountainous
coniferous woods with meadow grassland and shrubs of
Qinghai-Tibetan Plateau. More than 1500 higher plants
are recorded for the site. About 65.8 % of the site is forest
covered, with much of the remainder being above the tree
line [15, 16]. Thus, the Huanglong area hosts a number of
species of Acer, mixed with Alnus nepalensis, Juglans
sigillata, and Betula alnoides. There are three major
spruce species, two fir species as well as a larch spe-
cies—Larix potaninii. Most interestingly, the area is home
to the most diverse species of Rhododendron in the world,
i.e., 16 species which is critical to this study since the
leaves of this plant possess a thick impervious cuticle,
readily float on water and serve as a food reservoir for
fungi and bacteria. Besides these species, the area also is
home to a myriad of orchids, several threatened species of
spruce, larch, magnolia, and cedar. Oddly, the area also
supports two bamboo species (Fargesia denudata and
F. scabrida) that are important food plants for indigenous
wild populations of the giant panda [15].
Travertine Pools and Their Formation
The saga begins with the fall season and the shedding
of leaves from deciduous trees of the types mentioned
above. As the wind blows, the leaves fall from the near-
by forests of Huanglong to the cold stream below. The
leaves float on the water surface and become lodged on
obstacles in their path in the form of branches, other
leaves, or quiet water that lead to a their deposit—usu-
ally in an arching manner as a function of water flow
Fig. 3 X-ray data set collected by the SEM on an Oxford Instrument
energy dispersive X-ray microanalysis instrument on sample HL-1
(biological mat-like material). The beam was focused on a calcite
crystal that had formed on a fungal hypha. The data set shows the
major elemental peaks with net integrations (counts) of carbon at 131.5,
oxygen at 129.2, and calcium at 382.3. The keV is on the Y-axis. Other
elements present are indicated on the graph. In another experiment done
on HL-2 (hard surface of a calcite dam sample), the beam also focused on
a calcite crystal and yielded virtually the same result
Table 1 Polygenetic analysis of Huanglong microbial isolates based on rDNA partial sequences
Taxon Loci used Strain number Accession number Source Isolation area
Botryotinia sp. ITS HL 1-1 KX378872 Soft travertine sample of Huanglong pool Sichuan, China
Botryotinia sp. ITS HL 1-2 KX378873 Soft travertine sample of Huanglong pool Sichuan, China
Botrytis sp. ITS HL 1-3 KX378874 Soft travertine sample of Huanglong pool Sichuan, China
Botrytis sp. ITS HL 1-4 KX378875 Soft travertine sample of Huanglong pool Sichuan, China
Mucor sp. ITS HL 1-5 KX378876 Soft travertine sample of Huanglong pool Sichuan, China
Phoma sp. ITS HL 1-6 KX378877 Soft travertine sample of Huanglong pool Sichuan, China
Pythium sp. ITS HL 1-7 KX378878 Soft travertine sample of Huanglong pool Sichuan, China
Pythium sp. ITS HL 1-8 KX378879 Soft travertine sample of Huanglong pool Sichuan, China
Pythium sp. ITS HL 1-9 KX378880 Soft travertine sample of Huanglong pool Sichuan, China
Saprolegnia sp. ITS HL 1-10 KX378881 Soft travertine sample of Huanglong pool Sichuan, China
Saprolegnia sp. ITS HL 1-11 KX378882 Soft travertine sample of Huanglong pool Sichuan, China
Saprolegnia sp. ITS HL 1-12 KX378883 Soft travertine sample of Huanglong pool Sichuan, China
Saprolegnia sp. ITS HL 1-13 KX378884 Soft travertine sample of Huanglong pool Sichuan, China
Saprolegnia sp. ITS HL 1-14 KX378885 Soft travertine sample of Huanglong pool Sichuan, China
Saprolegnia sp. ITS HL 1-15 KX378886 Soft travertine sample of Huanglong pool Sichuan, China
Saprolegniaceae sp. ITS HL 1-16 KX378887 Soft travertine sample of Huanglong pool Sichuan, China
Saprolegniaceae sp. ITS HL 1-17 KX378888 Soft travertine sample of Huanglong pool Sichuan, China
Mortierellales sp. ITS HL 1-18 KX378889 Soft travertine sample of Huanglong pool Sichuan, China
Arthrinium sp. ITS HL 2-1 KX378907 Hard travertine sample of Huanglong pool Sichuan, China
Bjerkandera sp. ITS HL 2-2 KX378908 Hard travertine sample of Huanglong pool Sichuan, China
Cladosporium sp. ITS HL 2-3 KX378909 Hard travertine sample of Huanglong pool Sichuan, China
Phoma sp. ITS HL 2-4 KX378910 Hard travertine sample of Huanglong pool Sichuan, China
Pythium sp. ITS HL 2-5 KX378911 Hard travertine sample of Huanglong pool Sichuan, China
Pythium sp. ITS HL 2-6 KX378912 Hard travertine sample of Huanglong pool Sichuan, China
Pythium sp. ITS HL 2-7 KX378913 Hard travertine sample of Huanglong pool Sichuan, China
Pythium sp. ITS HL 2-8 KX378914 Hard travertine sample of Huanglong pool Sichuan, China
Pythium sp. ITS HL 2-9 KX378915 Hard travertine sample of Huanglong pool Sichuan, China
Saprolegnia sp. ITS HL 2-10 KX378916 Hard travertine sample of Huanglong pool Sichuan, China
Psiloglonium sp. ITS HL 2-11 KX378917 Hard travertine sample of Huanglong pool Sichuan, China
Pythium sp. ITS HL 3-1 KX378939 Leaf of Huanglong travertine pool Sichuan, China
Pythium sp. IT HL 3-2 KX378940 Leaf of Huanglong travertine pool Sichuan, China
Pythium sp. ITS HL 3-3 KX378941 Leaf of Huanglong travertine pool Sichuan, China
Pythium sp. ITS HL 3-4 KX378942 Leaf of Huanglong travertine pool Sichuan, China
Pythium sp. ITS HL 3-5 KX378943 Leaf of Huanglong travertine pool Sichuan, China
Phytopythium sp. ITS HL 3-6 KX378944 Leaf of Huanglong travertine pool Sichuan, China
Pythium sp. ITS HL 3-7 KX378945 Leaf of Huanglong travertine pool Sichuan, China
Aeromonas sp. 16S rDNA HL 1-19 KX378890 Soft travertine sample of Huanglong pool Sichuan, China
Bacillus sp. 16S rDNA HL 1-20 KX378891 Soft travertine sample of Huanglong pool Sichuan, China
Erwinia sp. 16S rDNA HL 1-21 KX378892 Soft travertine sample of Huanglong pool Sichuan, China
Hafnia sp. 16S rDNA HL 1-22 KX378893 Soft travertine sample of Huanglong pool Sichuan, China
Pseudomonas sp. 16S rDNA HL 1-23 KX378894 Soft travertine sample of Huanglong pool Sichuan, China
Pseudomonas sp. 16S rDNA HL 1-24 KX378895 Soft travertine sample of Huanglong pool Sichuan, China
Pseudomonas sp. 16S rDNA HL 1-25 KX378896 Soft travertine sample of Huanglong pool Sichuan, China
Pseudomonas sp. 16S rDNA HL 1-26 KX378897 Soft travertine sample of Huanglong pool Sichuan, China
Pseudomonas sp. 16S rDNA HL 1-27 KX378898 Soft travertine sample of Huanglong pool Sichuan, China
Pseudomonas sp. 16S rDNA HL 1-28 KX378899 Soft travertine sample of Huanglong pool Sichuan, China
Pseudomonas sp. 16S rDNA HL 1-29 KX378900 Soft travertine sample of Huanglong pool Sichuan, China
Pseudomonas sp. 16S rDNA HL 1-30 KX378901 Soft travertine sample of Huanglong pool Sichuan, China
Pseudomonas sp. 16S rDNA HL 1-31 KX378902 Soft travertine sample of Huanglong pool Sichuan, China
(Fig. 1a–c). The calcite dams eventually become larger
resulting in numerous deep pools or even water falls
(Fig. 1d, e). The dams in this area are formed mainly
by biological processes associated with the degradation
of the leaf material and the formation of fungal hyphae.
It is surmised that the leaves carrying endophytic fungi
or becoming infested with other aquatic fungi begin to
accumulate on the upper dam surface forming a
Table 1 (continued)
Taxon Loci used Strain number Accession number Source Isolation area
Pseudomonas sp. 16S rDNA HL 1-32 KX378903 Soft travertine sample of Huanglong pool Sichuan, China
Rahnella sp. 16S rDNA HL 1-33 KX378904 Soft travertine sample of Huanglong pool Sichuan, China
Rahnella sp. 16S rDNA HL 1-34 KX378905 Soft travertine sample of Huanglong pool Sichuan, China
Ewingella sp. 16S rDNA HL 1-35 KX378906 Soft travertine sample of Huanglong pool Sichuan, China
Pseudomonas sp. 16S rDNA HL 2-12 KX378918 Hard travertine sample of Huanglong pool Sichuan, China
Pseudomonas sp. 16S rDNA HL 2-13 KX378919 Hard travertine sample of Huanglong pool Sichuan, China
Pseudomonas sp. 16S rDNA HL 2-14 KX378920 Hard travertine sample of Huanglong pool Sichuan, China
Pseudomonas sp. 16S rDNA HL 2-15 KX378921 Hard travertine sample of Huanglong pool Sichuan, China
Pseudomonas sp. 16S rDNA HL 2-16 KX378922 Hard travertine sample of Huanglong pool Sichuan, China
Pseudomonas sp. 16S rDNA HL 2-17 KX378923 Hard travertine sample of Huanglong pool Sichuan, China
Pseudomonas sp. 16S rDNA HL 2-18 KX378924 Hard travertine sample of Huanglong pool Sichuan, China
Pseudomonas sp. 16S rDNA HL 2-19 KX378925 Hard travertine sample of Huanglong pool Sichuan, China
Pseudomonas sp. 16S rDNA HL 2-20 KX378926 Hard travertine sample of Huanglong pool Sichuan, China
Pseudomonas sp. 16S rDNA HL 2-21 KX378927 Hard travertine sample of Huanglong pool Sichuan, China
Pseudomonas sp. 16S rDNA HL 2-22 KX378928 Hard travertine sample of Huanglong pool Sichuan, China
Pseudomonas sp. 16S rDNA HL 2-23 KX378929 Hard travertine sample of Huanglong pool Sichuan, China
Pseudomonas sp. 16S rDNA HL 2-24 KX378930 Hard travertine sample of Huanglong pool Sichuan, China
Pseudomonas sp. 16S rDNA HL 2-25 KX378931 Hard travertine sample of Huanglong pool Sichuan, China
Pseudomonas sp. 16S rDNA HL 2-26 KX378932 Hard travertine sample of Huanglong pool Sichuan, China
Pseudomonas sp. 16S rDNA HL 2-27 KX378933 Hard travertine sample of Huanglong pool Sichuan, China
Pseudomonas sp. 16S rDNA HL 2-28 KX378934 Hard travertine sample of Huanglong pool Sichuan, China
Pseudomonas sp. 16S rDNA HL 2-29 KX378935 Hard travertine sample of Huanglong pool Sichuan, China
Pseudomonas sp. 16S rDNA HL 2-30 KX378936 Hard travertine sample of Huanglong pool Sichuan, China
Pseudomonas sp. 16S rDNA HL 2-31 KX378937 Hard travertine sample of Huanglong pool Sichuan, China
Pseudomonas sp. 16S rDNA HL 2-32 KX378938 Hard travertine sample of Huanglong pool Sichuan, China
Pseudomonas sp. 16S rDNA HL 3-8 KX378946 Leaf sample of Huanglong travertine pool Sichuan, China
Hafnia sp. 16S rDNA HL 3-9 KX378947 Leaf sample of Huanglong travertine pool Sichuan, China
Pseudomonas sp. 16S rDNA HL 3-10 KX378948 Leaf sample of Huanglong travertine pool Sichuan, China
Pseudomonas sp. 16S rDNA HL 3-11 KX378949 Leaf sample of Huanglong travertine pool Sichuan, China
Pseudomonas sp. 16S rDNA HL 3-12 KX378950 Leaf sample of Huanglong travertine pool Sichuan, China
Hafnia sp. 16S rDNA HL 3-13 KX378951 Leaf sample of Huanglong travertine pool Sichuan, China
Pseudomonas sp. 16S rDNA HL 3-14 KX378952 Leaf sample of Huanglong travertine pool Sichuan, China
Hafnia sp. 16S rDNA HL 3-15 KX378953 Leaf sample of Huanglong travertine pool Sichuan, China
Pseudomonas sp. 16S rDNA HL 3-16 KX378954 Leaf sample of Huanglong travertine pool Sichuan, China
Pseudomonas sp. 16S rDNA HL 3-17 KX378955 Leaf sample of Huanglong travertine pool Sichuan, China
Pseudomonas sp. 16S rDNA HL 3-18 KX378956 Leaf sample of Huanglong travertine pool Sichuan, China
Pseudomonas sp. 16S rDNA HL 3-19 KX378957 Leaf sample of Huanglong travertine pool Sichuan, China
Ewingella sp. 16S rDNA HL 3-20 KX378958 Leaf sample of Huanglong travertine pool Sichuan, China
Pseudomonas sp. 16S rDNA HL 3-21 KX378959 Leaf sample of Huanglong travertine pool Sichuan, China
Pseudomonas sp. 16S rDNA HL 3-22 KX378960 Leaf sample of Huanglong travertine pool Sichuan, China
Pseudomonas sp. 16S rDNA HL 3-23 KX378961 Leaf sample of Huanglong travertine pool Sichuan, China
Ewingella sp. 16S rDNA HL 3-24 KX378962 Leaf sample of Huanglong travertine pool Sichuan, China
Placement at the genus level is based on at least 95 % identity of the listed genera to that in GenBank
biomatrix or biomat with these leaves being the base-
ment material (Fig. 1f). Eventually, the fungal hyphae
serve as sites for the nucleation of calcite crystals
(Fig. 2a, b). The calcite crystals merge into larger plates
and form much more substantial calcite structural walls
that act as dams for the calcite pools (Fig. 2c–e).
Morphological Features and Analysis of the Fungal
Associated Crystals
Using Edax energy dispersive X-ray analyzer on the SEM, it
was possible to do elemental analysis of the crystals forming
on fungal hyphae taken directly from the travertine dam sites,
i.e., samples HL1 and HL2. The data showed the major pres-
ence of calcium, oxygen, and carbon with trace amounts of
silicon, aluminum, magnesium, and sulfur (Fig. 3). The data
are consistent with the crystals being composed mainly of
calcite or calcium carbonate. The X-ray dispersive analyses
of both samples were nearly identical (Fig. 3).
The crystals associated with samples HL1 and HL2 were
examined by SEM they have the typical appearance of calcite
crystals, and in this case, they are anisotropic when viewed
with plane polarized light and this suggests that their origin is
biological. Also, of great interest is the appearance of one or
more discrete holes (and traces thereof) in virtually every cal-
cite crystal that was examined by SEM (Fig. 2c, d). The di-
ameter of the holes varied from 3 to 4 μmwhich approximates
the diameter of the hyphal strands commonly seen associated
with the holes in light and SEM observations (Fig. 2a–d). In
the SEM, it is worthy to note that some compression of the
a
b
58.82%
100.00%
70.59%
0%
20%
40%
60%
80%
100%
HL1 HL2 HL3
Ewingella
Rahnella
Pseudomonas
Hafnia
Erwinia
Bacillus
Aeromonas
16.67%
45.45%
85.71%
33.33%
9.09%
0.00%
20.00%
40.00%
60.00%
80.00%
100.00%
HL1 HL2 HL3
Phytopythium
Psiloglonium
Cladosporium
Bjerkandera
Arthrinium
Mortierellales
Saprolegniaceae
Saprolegnia
Pythium
Phoma
Mucor
Botrytis
Botryotinia
Fig. 4 Analysis of the microbial
communities of Huanglong
travertine samples at the genus
level. HL1 is the soft sample, HL2
is the hard sample, and HL3 is the
leaf. a Fungal analysis. b
Bacterial analysis
fungal hyphae did occur as a result of the high vacuum in the
chamber but holes with associated hyphae have been noted as
exemplified by a fungal hypha, as observed by SEM, to be
associated with a hole in a calcite crystal (Fig. 2e).
Fungal Diversity of Huanglong Travertine Pool
and the Fungal Architects
There were at least 18 individual fungal isolates made from
sample HL1 and were distributed among eight different fungal
genera with a domination of Saprolegnia spp. (33.33 %) and
Pythium spp. (16.67 %) which are both phycomycetes (water
molds) (Table 1, Fig. 4a). In the case of HL2, there were 11
fungal isolates and they were distributed among seven genera
with Pythium spp. being dominant and accounting for 45.45 %
of the isolates. The remainder of the isolates was represented by
Saprolegnia spp. and a variety of others including Phoma sp.
and Mortierella sp. (Table 1, Fig. 4a). Furthermore, seven iso-
lates were obtained from sample HL 3 and theywere dominated
by Pythium spp. (85.71 %) (Table 1, Fig. 4a). The cultural
characteristics of the major fungi isolated from the travertine
samples HL1, HL2, and HL3 are distinct and characteristic for
these genera. Pythium spp. having hyaline hyphae and produc-
ing a rapid growth on PDA (covering a 90-mm petri dish within
9 days) resulting in a whitish-erumpent mycelium (Fig. 5a–c)
while Saprolegnia spp. resembles Pythium sp. but with a plain,
non-erumpent surface (Fig. 5d, e). Phoma sp. on the other hand
makes a brownish-black mycelium having a whitish halo and
commonly associated with the mycelium are pycnidia (fruiting
bodies) (Fig. 5f). Confirmation of the identity of each fungal
isolate was further made by comparing its ITS sequence data
with that in GenBank. In each case, at least a 95 % match was
found and all sequence information was deposited in GenBank.
Bacteria Associated with the Pool Samples
In sample HL1, there were seven genera of bacteria with
Pseudomonas being the dominant one (58.82 %) from 17
isolates (Table 1, Fig. 4b). However, in HL2, the only bacte-
rium isolated was the genus Pseudomonas (Table 1, Fig. 4b).
Thus, it appeared as if the maturation of the calcite crystals, as
in HL2, there was a marked reduction in the bacterial diversity
of the sample which is in contrast to a sample having rapid
crystal growth as per HL1. Furthermore, of the 17 bacteria
isolates that were isolated from the leaves associated with
the travertine pool, the genus Pseudomonas was still the one
that dominated (70.59 %) (Table 1, Fig. 4b).
Conclusions
While ITS sequence technology and cultural identity work
provided information on the various fungal and bacterial gen-
era associated with the calcite dams and floating leaves, these
data were not substantial enough to provide information on
species identity. However, it appears that the phycomycetous
fungi, i.e., Pythium and Saprolegnia were the dominant fungi
recovered from each sample type (Fig. 4). And it seems that
multiple species of these fungi are present but more work will
be needed to substantiate this observation. Fungi, of this type,
are commonly considered as water molds and have a prefer-
ence for substrates that are aquatically based [17]. Other fun-
gal species were recovered from the three sample types includ-
ing Phoma sp., Mucor sp., Botrytis sp., and others.
It also appears that other microbial forms are associated
with the rimstone calcite dams and these include diatoms (ob-
served in some SEMs) and bacteria. Bacteria were found in
each of the three samples with uneven distribution of genera.
Interestingly, the most frequently isolated bacterial genus was
Pseudomonas. It is not understood if the bacteria make a sig-
nificant impact upon the development of the structure of the
calcite dams. However, it is the case that each of the
Fig. 5 Individual fungal cultures grown for 9 days on PDA. They were
each isolated from Huanglong samples HL1, HL2, and HL3. They are
each represented by the strain acquisition number as per Table 1. a
Pythium spp. 1–7. b Pythium spp. 2–5. c Pythium spp. 3–7. d
Saprolegnia spp. 1–15. e Saprolegnia spp. 2–10. f Phoma spp. 1–6
pseudomonad has significant antifungal activity towards the
fungal species associatedwith the calcite dams as well as other
plant-associated fungi (Xie unpublished). This antifungal ac-
tivity may in some way help regulate fungal development so
as to have appropriate fungal growth as a function of available
nutrient supply.
The main thesis of the paper for the involvement of fungi as
architects of the dams at Huanglong seems to have substantial
support via the data provided in this report. That is, fungal
hyphae are seen on virtually every leaf floating in the valley
stream. Examination of the hyphae on these leaves reveals the
presence of crystals forming on these hyphae (Fig. 2).
Eventually, the crystals fuse into the matrix of the dam and
individual holes exist in each crystal indicating the location of
the individual fungal hypha. The fusion of all of the crystals
results in massive calcite dams at Huangong (Fig. 1). The fact
that the crystals are calcite is strongly supported by the SEM-
Edax study showing the presence of the elements making up
CaCO3 (Fig. 3). The mechanism of calcite crystal formation is
likely regulated by the presence of calcium-binding proteins
on the hyphal surfaces [18, 19]. While microbial induced
calcite-crystal formation has been noted in vitro, this appears
to be the first and most noteworthy example of microbial
induced calcite dam formation in nature.
Acknowledgments The authors acknowledge the financial support of
the Natural Science Foundation of Chongqing (cstc2015jcyjys80001) to
Jie Xie.
References
1. Wang J, Liu S, Ran J, Wang C, Shen L, Jiang P, Guo G (2004)
Effects of annual net primary productivity of forest ecosystem and
habitat complexity on species diversity of small mammals. Acta
Theriol Sin 24(4):298–303
2. Yoshimura K, Liu Z, Cao J, Yuan D, Inokura Y, Noto M (2004)
Deep source CO2 in natural waters and its role in extensive tufa
deposition in the Huanglong Ravines, Sichuan, China. Chem Geol
205:141–153
3. Sun S, Dong F, Ehrlich H, ZhaoX, LiuM, Dai Q, Li Q, AnD,Dong
H (2014) Metabolic influence of psychrophilic diatoms on traver-
tines at the Huanglong natural scenic district of China. Int J Environ
Res Public Health 11:13084–13096
4. Li QF, Dong FQ, Dai QW, Hou TT, An DJ, Tang S (2012) The
microbial factor of travertine deposition between Yellowstone
National Park (YNP), USA and Huanglong Scenic, Sichuan. Adv
Mater Res 518–523:136–139
5. Li Q, Csetenyi L, Gadd GM (2014) Biomineralization of metal
carbonates by Neurospora crassa. Environ Sci Technol 48:
14409–14416
6. Cao C, Jiang J, Sun H, Huang Y, Fand T, Lian B (2016) Carbonate
mineral formation under the influence of limestone-colonizing
actinobacteria: morphology and polymorphism. Front Microbiol
7:366. doi:10.3389/fmicb.2016.00366
7. Chahal N, Rajor A, Siddique R (2011) Calcium carbonate precipi-
tation by different bacterial strains. Afr J Biotechnol 10(42):8359–
8372
8. Kolo K, Keppens E, Préat A, Claeys P (2007) Experimental obser-
vations on fungal diagenesis of carbonate substrates. J Geophys Res
112:G01007. doi:10.1029/2006JG000203
9. Bindschedler S, Cailleau G, Verrecchia E (2016) Role of fungi
in the biomineralization of calcite. Minerals 6(2):41.
doi:10.3390/min6020041
10. John JB, Lonnie DR (1998) Electronmicroscopy. Jones andBartlett
Pub, Inc, Sudbury
11. White TJ, Bruns T, Lee S, Taylor J (1990) Amplification and direct
sequencing of fungal ribosomal RNA genes for phylogenetics. In:
Innis MA, Gelfand DH, Sninsky JJ, White TJ (eds) PCR protocols:
a guide to methods and applications. Academic Press, Inc, San
Diego, pp 315–322
12. Xie J, Strobel GA, Mends MT, Hilmer J, Nigg J, Geary B (2013)
Collophora aceris, a novel antimycotic producing endophyte asso-
ciated with Douglas Maple. Micorbial Ecol 66(4):784–795
13. Primieri S, Costa MD, Stroschein MRD, Stocco P, Santos JCP,
Antunes PM (2016) Variability in symbiotic effectiveness of N2
fixing bacteria in Mimosa scabrella. Appl Soil Ecol 102:19–25
14. Wang H, Yan H, Liu Z (2014) Contrasts in variations of the carbon
and oxygen isotopic composition of travertines formed in pools and
a ramp stream at Huanglong Ravine, China: implications for paleo-
climatic interpretations. Geochim Cosmochim Acta 125:34–48
15. McGinley M (2009) Huanglong national scenic area, China. The
Encyc loped ia of Ear th onl ine : h t tp : / /www.eoear th .
org/view/article/153576/
16. Huang B, Luo Y, Yu F, Tang S, Dong L, An D (2007) Interspecific
relationships of dominant species in orchid communities of forest
vegetation in Huanglong Valley, Sichuan, China. J Plant Ecol
(Chinese version) 31(5):865–872
17. Van Den Berg AH, Mclaggan D, Dieguez-Uribeondo J, VanWest P
(2013) The impact of the water moulds Saprolegnia diclina and
Saprolegnia parasitica on natural ecosystems and the aquaculture
industry. Fungal Biol Rev 27:33–42
18. Deborah SF, Joseph H (2005) Calcineurin-binding protein Cbp1
directs the specificity of calcineurin-dependent hyphal elongation
during mating. Eukaryot Cell 4(9):1526–1538
19. Stephenson KS, Gow NA, Davidson FA, Gadd GM (2013)
Regulation of vectorial supply of vesicles to the hyphal tip
determines thigmotropism in Neurospora crassa. Fungal Biol
118(3):287–294