The lost world of Cuatro Ciénegas Basin, a relictual bacterial niche in a desert oasis

Barriers to microbial migrations can lead adaptive radiations and increased endemism. We propose that extreme unbalanced nutrient stoichiometry of essential nutrients can be a barrier to microbial immigration over geological timescales. At the oasis in the Cuatro Ciénegas Basin in Mexico, nutrient stoichiometric proportions are skewed given the low phosphorus availability in the ecosystem. We show that this endangered oasis can be a model for a lost world. The ancient niche of extreme unbalanced nutrient stoichiometry favoured survival of ancestral microorganisms. This extreme nutrient imbalance persisted due to environmental stability and low extinction rates, generating a diverse and unique bacterial community. Several endemic clades of Bacillus invaded the Cuatro Cienegas region in two geological times, the late Precambrian and the Jurassic. Other lineages of Bacillus, Clostridium and Bacteroidetes migrated into the basin in isolated events. Cuatro Ciénegas Basin conservation is vital to the understanding of early evolutionary and ecological processes.


Abstract 133 words
Barriers to immigration can lead to localized adaptive radiations and increased endemism. We propose that extreme oligotrophy can be a strong barrier to immigration over geological timescales, and facilitate the evolution of diverse and coevolved microbial communities. We show here that the endangered oasis of Cuatro Ciénegas Basin can be a model for a lost world, where the ancient niche of extreme oligotrophy favoured survival of ancestral microorganisms that persisted due to environmental stability and low extinction rates, generating a diverse and unique bacteria diversity.
Diversification/extinction rates in Bacillus showed several CCB endemic clades that diverged from the rest of Bacillus spp. in different times of the Paleozoic and Mesozoic, in contrast to more recent Bacillus, Clostridium and Bacteroidetes lineages. CCB conservation is vital to the understanding of early evolutionary and ecological processes.

Main
A "lost world" is both a poetic metaphor and a scientific pursuit; in both cases, it pertains the conservation or recreation of the deep past in a particular place. Scientists have looked for analogues of such world in environments possessing living microbial mats and stromatolites, since these structures were dominant for billions of years in the Proterozoic 1 . Nevertheless, in most cases, these communities represent more a physical metaphor of the past than an actually lost world, since they harbour mostly derived microbial lineages 2,3 . The exception to these observations seems to be the abundant and morphological diverse stromatolites and microbial mats from the endangered oasis of Cuatro Ciénegas Basin (CCB) in Northern Mexico. In this extremely diverse wetland 4 , the recycling of the deep aquifer by magmatic heat replicates many conditions of ancient oceans 5 including its extreme oligotrophy with phosphorous as low as 0.5 mM 6 . Spring water is also low in oxygen and rich in sulphur minerals 5,7 . These conditions represent niche variables that can explain the survival of marine as well as hydrothermal vent associated sulphur microbes 8 . Viral metagenomics confirm these observations, as these studies have shown substantial divergence from known sites and a similitude with marine habitats 9 . All these data have been hinting at a lost world scenario, since CCB, now it at the centre of the North American continent, was at its shore 35 Ma ago 8 .
If extremely low phosphorous levels were the initial conditions where these microbes evolved, bacteria have been, since their beginning, under strong selective pressure to adapt to these environmental conditions, generating parallel new ways to obtain the element that is rare. Nutrient conditions create an extreme stoichiometric bias in the N:P ratio (as high as 122:1 in our study site, Churince) at the community level 10 , but also an extreme imbalance at the bacterial cellular level (N:P 965:1 in a CCB Bacillus cereus group 11 ). Extreme oligotrophy, as well as rich sulphur conditions are a specific niche characteristic of the Precambrian ocean, that ended abruptly in the Phanerozoic Eon 542 Ma 12 . Moreover, using conservative time frames based on geological events, molecular clock studies have suggested that some strains of cultivable cyanobacteria 13 as well as of Bacillus 14 from CCB diverged many millions of years ago from their close relatives.
Hence, we propose that CCB is a microbial lost world, not just as a poetic metaphor, but as a true geographical site; a nutrient-deprived multidimensional niche isolated from the human environment by its peculiar conditions, as calcium carbonate rocks work as a buffer between the human activities and the turquoise blue ponds 5 .
What would make a lost world more than a metaphor? If we compare different models of diversification ( Fig. 1), we can observe that for cosmopolitan bacteria, such as Escherichia coli or Bacillus subtilis, the phrase "everything is everywhere and the environmental selects" could apply, since these microbes have an enormous population size and considerable migration rates (1a). A second model is isolation by distance, a pattern similar to the one observed for most macro-organisms. This isolation has been observed for the thermophile Crenarchaeota Sulfolobus islandicus 15 (Fig. 1b). The third model is that of isolation as in an island-like migration (Fig. 1c), and this pattern also occurs in microbes, as is the case for those in the lakes in the Pyrenees, explained by the island-like nature of each lake 16 . Here we suggest that there is a 4 th model, the lost world model (Fig. 1d), where a particular site is maintained as a "bubble-like" niche, a place where a community survived in relictual conditions. In this case, we would observe phylogenies where a few isolated microbes would exhibit long phylogenetic branches, and even complete lineages would appear separated and diversified from others in parallel bouts.
In order to distinguish between these four models in our system of study (CCB), we will first describe the microbial diversity at a small scale (ca. 1 km 2 ) and then zoomin even further into particular cultivated genera within the Gram positive. We studied aerobic (Bacillus) and anaerobic (Clostridiales and Bacteroidetes) lineages, to have a sample large enough to evaluate how much the CCB lineages diverged from those in the rest of the world.
Using NextGen 16S rRNA gene tags, we surveyed the microbial biodiversity in the Churince, a closed hydrological system 11 (and the most endangered site within CCB). We observed a vast diversity ( Figure 2) on a small scale (less than 1 km 2 ), in particular when we compared such diversity with other 343 16S rRNA studies and microbiomes. Those studies comprised several contrasting environments: human, plantassociated, soil, sediments, biofilm, marine biofilms and extreme environments like Yellowstone hot springs, Guerrero Negro salt flats, and Antarctica soils, all available in public databases (Table S1). Based on standard DNA similitude metrics, an operational taxonomic unit (OTU) is defined in general as 97% identity at the 16S rRNA gene.
The Churince's total Bacteria and Archaea richness are represented by a total of 5,167 OTUs assigned to samples from the water column, aquatic sediments and soil ( Fig. 2a). The assigned OTUs represented 60 different known phyla, three of which were Archaea. To compare with the other environments, we used the individual OTUs and then computed two alpha diversity indexes: Shannon, and Simpson (Fig. 2b).
The most diverse environments according to Shannon's diversity index are the aquatic sediments of different sites in the world; accordingly, the Churince's sediment had the highest Shannon value of our dataset. This was also supported by the Simpson's index, showing that water column < soil < sediment (Fig. 2b). Using both Shannon's and Simpson's diversity indexes, Churince showed a very high microbial diversity, even when compared with other microbial diversity hotspots, such as Pearl river in China, or Guerrero Negro in Mexico (Table S1).
To test for the lost world clade diversification, we zoomed-in into the diversity of CCB cultivated Bacillus, Clostridiales and Bacteroidetes. From our collection of approximately 2,500 cultivated Bacillus spp. from CCB, 265 unique sequences were obtained, selected at 97% identity, a very conservative estimate for Bacillus. In a global tree (Fig. 3), we observed that endemic CCB strains formed many exclusive (only feoun in CCB) lineages when we added them to the 1,019 OTUs reported for Bacillus spp.
from around the world (Fig. 3a), increasing by nearly 25% the number of known Bacillus species.
Within the Bacillus spp. from CCB, we can distinguish two diverse endemic lineages: one from sediment and another one closely related to marine Bacillus spp. None of the other lineages of Bacillus exclusive to CCB have been reported in any other site but they seem to constitute later events of arrival to the "island-like" unique niche and to have diversified locally. Bacteroidetes and Clostridiales strains were fewer and did not form monophyletic groups endemic to CCB ( Figure S1), even though the Bacteroidetes had a bout of diversification at the end of the Paleozoic ( Figure S2), suggesting also punctuated migrations and local radiations.
Environmental conditions are known barriers to dispersal and the extreme P limiting conditions at CCB could certainly constrain immigration from P demanding populations. This is the case of Patagonia's isolated and oligotrophic lakes that present unique but low diversity 17 . CCB is not only unique, but it also very diverse, despite its extreme oligotrophy. Our data showed that the small and endangered Churince system in CCB, contains 57 out of the 86 known Bacteria phyla, which is 66.3% of the world recorded bacterial diversity at phyla level (data from 342 analysed microbiomes, Table  2S). This diversity is only comparable to Pearl River in China 18 , where 48 microbial phyla are found. In contrast to CCB, Pearl River is a highly productive environment that receives inputs from multiple sources in the 2,400 km extension of China's third largest river, while Churince is a small hydrological system fed by a spring that extends 1 km at most.
The local scale makes the bacterial diversity of CCB even more interesting. It is possible that the reason for such diversity is not only due to local adaptation to the environment but to a "Red Queen" evolutionary process, where interactions among species shape diversity, causing an increase in localized adaptation and coevolution 19 .

Experiments of competition between strains of Bacillus from different sites within
Churince showed marked antagonisms against different strains from even sites few meters away, but tolerance and even cooperation between strains from exactly the same site 20 ; for instance, strains of endemic lineages of Bacillus from Churince require cross feeding and cooperation to obtain even basic amino-acids 21 . Hence, one potential explanation for the long-term survival of lost world Bacilli species in CCB would be their coevolution with the community members that exchanged nutrients with them in their particularly low nutrient niche following a "Black Queen" model 22  Our data showed that the total known phylogenetic diversity of Bacillus spp.
increased by ¼ the known species when we included the strains unique to CCB.
Moreover, a large part of this unique diversity belongs to early divergent clades. Even more so previously, some CCB Bacillus strains 14  Bacteroidetes and Clostridium in CCB seem to follow, like some of CCB Bacillus spp., an island model with more recent dispersal events, as the short branches in the phylogenies support a more cosmopolitan origin ( Figure S2). Similar patterns were found in a study of Bacillus spp. from diverse environments in India where cosmopolitan Bacillus spp. had short branches to sister species 24 . All our results suggest that extreme oligotrophy, along with community cohesion function like a "semipermeable" barrier to migration, where effective migration is possible, but rare.
Fossil evidence shows that stromatolites were still abundant between the Permian and Triassic boundary, in the site where the Tethys sea opened in the South-Western shores of Laurentia 22 where CCB was located at the onset of the Mesozoic 8 .
However, during the massive extinction event that marked the end of the Permian, stromatolites became rare, except on the western shores of the Tethys sea 22 . Microbial mats and stromatolites can be still found in other sites of the planet, but at CCB, aside from giving testimony to the past, microbial lineages have been safeguarded, bringing evidence for a lost world.
Even though CCB microbial communities have survived for an extended period of time, their particular niche conditions are being destroyed in the Anthropocene. This impact is even more poignant because CCB wetland has shrank 90% in the last 50 years, and its deep aquifer has been devastated by the use of fossil water in local agricultural practices. This deep niche change has already destroyed many of the microbial complex communities in Churince. However, we believe that this change can be reverted if the channels that drain the wetland are closed and the wetland recovers its water cycle. Conservation of the unique niche in CCB and similar sites is paramount for our understanding of the deep past as well as to predict and protect the future of our planet.
Espinosa-Asuar for making Figure 1 and Chris Dupont of JCVI for the phylogenetic analysis in Figure 2.

Microbial diversity context
We sampled ten sites during May 2011 in the Churince system of CCB in a 300 m long lagoon plus two more sites in the spring-head, ca. 1 km away. In each sample site we took 50 g of sediment and a gallon of water as well as a sample of both for biogeochemical variables, nutrients and minerals: C, N, P, Ca, Mg, 10 . We also sampled four types of vegetation from an established gradient and obtained composite soil samples. We extracted DNA from each sample using the same methodology 25 .
A total of 950,000 reads were sequenced; we required a minimum of 50,000 reads per site, with a minimum 500 bp length after Quality Control check. Not all samples produced the same amount of sequences, probably due to the natural low yield of DNA extraction in CCB water and sediments. Nevertheless, even at 97%, diversity is high, encompassing all the know phyla of Bacteria but a very low diversity and abundance of Archaea and mostly none of the cosmopolitan human related microbial taxa.
The 16S rRNA gene analysis was done as previously reported 26,27 . Briefly sequencing quality was processed and filtered using FASTQ and Fastx-toolkit, we filtered out any sequence with Phred < 30, length < 500 bp. Operational taxonomic units (OTUs) were clustered using cd-hit-est 28 with a 97% identity threshold cut-off. The OTUs were parsed into QIIME pipeline and the taxonomic assignments were done against Greengenes DB (v 13.8 29 ). Chimeras were removed after taxonomic assignments and detected by ChimeraSlayer 30 . Data management, diversity statistic, and plots were done using R phyloseq package 31 and ggplot2 and RColorBreweer R libraries. Pplacer was used to place the diversity into a reference tree (Figure 2)  Finally, we compared the distribution of rates along the tree of all lineages in order to assess the relative diversification rate differences in all lineages.