OBSERVED DIFFERENCES IN THE MICROBIAL DIVERSITY BETWEEN SAMPLES COLLECTED BEFORE AND AFTER THE CAMP
A total of 1561 OTUs was identified from the total of the samples taken before camping against 1920 OTUs identified after (Table 2). Both archaea and bacteria were determined in this study area. Both before and after camping, archaea were observed with a relative abundance lower than that of bacteria. Before camping,the archaea in B, E, F, and D areas, were 0.35%, 3.77%, 0.28%, and 0.02% respectively. However, the points A and C registered an abundance rate less than 0.01% of archaea. Aftercamping, a decrease in the abundance of archaea was observed in areas B and C while it was increased in areas D and F.
Table 2
Alpha diversity of each sampling area.
sampling area
|
sampling period
|
chao1
|
observed_otus
|
shannon
|
simpson
|
A
|
NAR1
|
143,84
|
143
|
4,333666832
|
0,896421666
|
A
|
NAR2
|
141,0731707
|
141
|
3,212326362
|
0,772977034
|
B
|
NAR3
|
181,7142857
|
181
|
5,552910745
|
0,945450354
|
B
|
NAR4
|
342,185567
|
338
|
5,57312221
|
0,929612327
|
E
|
NAR5
|
253,4705882
|
248
|
5,290159065
|
0,914834489
|
E
|
NAR6
|
539,1818182
|
534
|
6,588622341
|
0,963566081
|
F
|
NAR9
|
438
|
433
|
6,620141902
|
0,9687542
|
F
|
NAR10
|
260,9710145
|
259
|
5,857635488
|
0,953874902
|
C
|
NAR11
|
99,32258065
|
99
|
2,721648905
|
0,700177309
|
C
|
NAR12
|
69,75
|
68
|
3,347061491
|
0,812636098
|
D
|
NAR13
|
461,0930233
|
457
|
6,3075618
|
0,95749345
|
D
|
NAR14
|
585,9482759
|
580
|
6,773397309
|
0,966900149
|
NAR1, NAR3, NAR5, NAR9, NAR11, and NAR13 represent data of the pre-camping period; NAR2, NAR4, NAR6, NAR10, NAR12, and NAR14 represent data of the post-camping period. |
Besides the archaea, bacteria identified in the samples taken before camping composed of 25 phyla dominated by Proteobacteria, Actinobacteria, Bacteroidetes, Firmicutes, Planctomycetes, Gemmatimonadetes, and Acidobacteria. It must be noted here that the indigenous microbial community of this part of Morca Cave under study was represented by the results observed on samples taken before the camping. The community composition of each specific sampling area is detailed and community differences between the two periods were evaluated on the class and genus levels.
The Chao1 of the Alpha diversity which indicates the species richness was noted to be sightly high before the camping period (143.8 against 141). Shannon index, which calculates the species diversity by considering both the richness and the equitability of the species, showed a high diversity of species before the camping period (4.3 against 3.2). It is the same for the Simpson index which shown that this site had a high microbial diversity before camping (0.89 against 0.77) (Table
2). The taxonomic analysis of this site revealed that there was no archaea in the two sampling periods. Even if mostbacteria in this area were unidentified at the genus level, significant differences were noted between the two periods.
Gamma-proteobacteria, Alpha-proteobacteria and Bacilli 46.29%, 29.18% and 10.59% respectively were the most dominant classes observed before the camp. Important differences were observed for Alpha-proteobacteria and Bacilli classes. While the Alpha-proteobacteria decresed by 21.7%, the Bacilli class significantly (p = 0,003) increased parallel to its phylum (Firmicutes) by 33.83% (Fig. 2). At genus level, Acinetobacter (23.25%), Brevundimonas (19.41%), Massilia (9.84%), Aerococcus (7.65%), Pseudomonas (5.34%) and wb1-P19 (4.48%) were the most abundant ones before the camping activities. Acinetobacteria increased by 7.37% while Brevundimonas and Pseudomonas decreased after the camping period by 17.97% and 5.34% respectivetly. In addition, Exiguobacterium belonging the class Bacilli, which was almost absent before the camping shown a significantly increase rate (33.3%).
Chao1 index values for the two periods were 181.7 and 342.1 before and after camping respectively. These values shown an augmentation of species biomass after camping in this sampling area. The species diversity evaluated using Shannon and Simpson indices did not show important variation between these two periods: the Shannon index and the Simpson index before and after camping were 5.55 and 5.57, were 0.94 and 0.92 respectively (Table
2). In this area, both the Thermoplasmata (0.25%) and Nitrososphaeria (0.1%) classes of the archaea represented 0.35% of the microbial biomass. On the other hand, after the camp this microbial group was absent (Fig.
3).
In addition, the bacterial group before camping was dominated by Gamma-proteobacteria with a relative abundance rate of 47.01%, followed by Actinobacteria (16.64%), and Alpha-proteobacteria (13.12%). However, after the camping period, it was observed that while Gamma-proteobacteria had increased (11.98%), Actinobacteria shown a decrease of (11.25%) (Fig.
3). Genera
Pseudomonas (17.04%) and
Acinetobacter (14.18%),
Wb1-P19 (6.03%),
Variovorax (2.06%),
Sphingorhabdus (8.70%),
Nocardioides (2.36%),
Gaiella (1.61%),
Paeniglutamicibacter (1.31%) and
Pir4_lineage (1.01%) were the most abundant species before camping. At the end of camping,
Pseudomonas,
Acinetobacter, and
Sphingorhabdus decreased by 15.3%, 5.08%, and 6.59% while
wb1-P19 and
Limnobacter increased at the end of the camping period 11.59% and 15.68% respectively.
According to Chao1, Shannon and Simpson indices microbial biomass and diversity increased in this area after the camping (Table 2). This area shown the highest archaea biomass (3.7%) among all the sampling sites (Fig. 4). The following classes were identified Thermoplasmata (2.96%), Woesearchaeia (0.06%) and Nitrososphaeria (0.75%) classes with Candidatus Nitrososphaera being the sole genus. The highest rate of archaea remained uncultured archaeon. After the camping period, the abundance of each class decreased (Fig. 4).
The classes Actinobacteria (39.03%) and Gamma-proteobacteria (25.09%) dominated this site before the camping. Other classes like NC10 (7.47%), Alpha-proteobacteria (3.02%) and Planctomycetacia (1.71%) were also identified. However, after the camping it observed that Actinobacteria as well as Gamma-proteobacteria classes decreased by 34.03% and 10.34 respectively. On the other hand, a significant increment in Bacilli and Clostridia classes 44.21% and 8.49% respectively were noted after the camping (Fig. 4). It is also important to mention that there was a decrease in Nitrospira strains (0.28%) in this area. Genera Nocardioides, Variovorax, Alkanindiges, Acinetobacter, and Massilia of unidentified genus belonging the family Micrococcaceae of phylum Actinobacteria were identified before the camping with the respective rates of 19.58%, 8.22%, 2.93%, 2.49% and 2.09%. 19.08%. At this level, Nocardioides and Variovorax decreased by 19.58% and 8.22% respectively after the camping period. Furthermore, Exiguobacterium, Brevibacillus, and Bacillus also increased by 14.76%, 12.46%, and 4.72% respectively in the post-camping period with the respective increase rates.
The Table
2 show a lowering of species richness, through the Chao1 index values, in this area. Behind that, the Simpson and Shannon indices shown a drop of the microbial diversity after the camp with 0.96 and 6.58 before the camping period against 0.95 and 5.85 at the camping end, respectively. These values shown that this sampling area (before the camp) housed the most microbial diversity among all the studied points (Table
2). Archaea strains identified in this site before the camp period were composed of Thermoplasmata (0.03%), Woesearchaeia (0.03%) and Nitrososphaeria (0.21%) classes. Contrary to the diversity, the biomass of archaea (0.76%) observed before the camping was increased at the end of the camping. Thermoplasmata strains increased by 0.64% while strains of Woesearchaeia and Nitrososphaeria decreased after the camp with the respective decrease rates (0.03%) and (0.12%) (Fig.
5).
In addition, the bacterial group composed of 20 phyla with a highly diversified class level dominated by Gamma-proteobacteria (52.72%) at the pre-camping period. The Bacilli (9.61%), Alpha-proteobacteria (7.37%), NC10 (6.02%), Actinobacteria (2.66%), and Planctomycetacia (2.36%) have also identified at abundance rates >1%. Important variations were noted between the two sampling periods on Gamma-proteobacteria and Bacilli classes which shown respectively a decrease rate of 12.61% and an increase rate of 11.49% (Fig. 5). In this second point near the toilet area, the camping end was also marked by a decrease of members of Nitrospira strains (0.18%). In addition, genera Pseudomonas (9.99%), wb1-P19 (9.17%), Massilia (4.48%), Bacillus (4.45%), Candidatus Methylomirabilis (3.43%), Acinetobacter (2.95%) and Brevibacillus (2.76%) were identified before the installation of the camp. The significant change at this level was observed on the genus Acinetobacter which shown an increase rate of 13.5% after the camp.
Results of alpha diversity analyses shown that this site has the lowest microbial count and diversity (Table 2). The Chao1 index before the camp was 99.3 while after the camp was 69.75. Contrary to that, the diversity of this area increased at the end of the camp. The Shannon and Simpson indexes were respectively 2.72 and 0.70 before the camp period against 3.34 and 0.81 at the end of the camp (Table 2). Before as well as after the camp, archaea strains were absent (0.0%) in this site. The bacterial community was dominated by strains of Gamma-proteobacteria (69.51%) and Alpha-proteobacteria (37.92%). However, their abundances decreased by respectively 31.92% and 18.47% at the end of the camping. Strains of Bacilli class became dominants in this area by showing an increase rate of 49.01% (Fig. 6). Furthermore, our results revealed that Acinetobacter (56.37%), Brevundimonas (23.36%) and Massilia (7.71%) dominated the area before the camping period. At the end of the camping period, genera Acinetobacter and Brevundimonas were observed with respective decrease rates of 28.11% and 22.65% contrary to the genera Exiguobacterium and Sphingomonas which shown significant increase rates of respectively 36.54% and 5.24%.
Compared to all the other studied points, this site shown the highest species richness at the two sampling periods. The Chao1 index, of the Alpha diversity, was 461.09 before camping and 585.9% at the end of the camping period (Table
2). Both Shannon and Simpson indices shown an augmentation of the diversity after the camping period (Table
2). Before the camp, the abundance of archaeal community in this site was weak and almost composed by strains of Nitrososphaeria class (0.02%). The increase of diversity and biomass of this community after the camp period is marked by the increase of Nitrososphaeria strains (0.34%) and the appearance of Thermoplasmata (0.10%). On the other hand, the bacterial class level at the pre-camping period, is largely dominated by Gamma-proteobacteria (54.61%) followed by Alpha-proteobacteria (9.88%). However, at the post-camping period, the class Gamma-proteobacteria decreases by 37.58% while Bacilli and Actinobacteria classes increased significantly with (28.86%) and (8.3%) as respective rates (Fig.
7). The genera
wb1-P19 (14.26%),
Massilia (8.70%) and
Acinetobacter (5.76%) are the most dominant at the pre-camping period.
Pseudomonas, Sphingomonas, Brevundimonas, Bacillus, Terrimonas are also identified at the genus level with relative percentage rates between 5% and 1%. At the post-camping period,
Massilia and
Acinetobacter were nearly absent (0.0%) and (0.03%) while
Bacillus and
Micromonospora shown increase rates of 8.18% and 6.13% respectively.