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Naturalist Alfred Russel Wallace went on an expedition to Amazonas state in Brazil in 1848–52.Credit: Mondadori Portfolio via Getty

Best known for formulating the theory of evolution by natural selection, independently of Charles Darwin, Alfred Russel Wallace is an appealing if enigmatic figure. The appeal stems in part from his underdog status: poor and self-educated, Wallace had none of Darwin’s social and financial advantages. The enigma comes from his keen embrace of a range of eccentric non-scientific causes, including spiritualism, phrenology and anti-vaccination (for smallpox).Scientists do not like their scientific heroes to bear the taint of irrational thinking. Wallace’s enthusiasms have therefore contributed to him becoming marginalized in the history of evolutionary thought. Most people know about Darwin and the HMS Beagle. But what about Wallace and the Helen?The Helen story is worth revisiting because it shows Wallace at his resolute best. Despite numerous disastrous career setbacks — of which the Helen episode was the most severe — he persevered and eventually succeeded as a scientist.More than 150 years after Wallace’s experience on the Helen, doing science continues to be hard and can be disappointing. Wallace’s misadventure provides both perspective and an object lesson in how to navigate setbacks. His response to problems showcases his most inspiring traits: his commitment to science, his almost superhuman resilience and his refusal to mire himself in self-pity.Tropical explorationsIn his first job as a land surveyor, Wallace developed an interest in the plants he encountered as he tramped across the countryside. Then, in 1844 at the age of 21, he met Henry Walter Bates, who would later discover ‘Batesian mimicry’ (whereby members of a palatable prey species gain protection by mimicking an unpalatable one).Bates, two years Wallace’s junior, had a fixation with beetles, and he catalysed Wallace’s transformation from hobbyist naturalist to serious collector. Wallace’s new-found focus on beetles transcended mere entomological stamp-collecting; he developed an interest in some of the great scientific questions of the time. He was particularly inspired by the anonymously published Vestiges of the Natural History of Creation (1844) by Robert Chambers, which put forward a vision of a transmutational process, with life progressing from simple to complex.Without money or connections, Wallace and Bates aspired to careers in science at a time when the field was the preserve of the moneyed elite. They would have to fund their scientific explorations by collecting and selling specimens. After a hasty choice of destination — tropical South America — and a crash course in collecting methods, Wallace, aged 25, and Bates, aged 23, arrived in Belém, Brazil, in May 1848 (see ‘Doggedly determined’).
Doggedly determined

Alfred Russel Wallace tends to be unjustly relegated to a footnote in the Charles Darwin story. He was, in fact, a pioneering biologist who refused to let disadvantage or disaster prevent him from pursuing his scientific dreams.
January 1823: Alfred Russel Wallace is born in Usk in Wales.
May 1848: Wallace and Henry Walter Bates arrive in Belém, Brazil.
July 1852: Wallace boards the Helen, which catches fire three weeks later while at sea.
October 1852: Wallace reaches Deal, England, aboard the Jordeson.
March 1854: Wallace leaves Southampton for southeast Asia.
September 1855: Wallace’s first evolutionary paper describing his ‘Sarawak Law’ is published.
May 1856: Citing the Sarawak Law paper, geologist Charles Lyell alerts Darwin to the possibility that Wallace is developing ideas similar to Darwin’s.
February 1858: Wallace sends his paper on natural selection to Darwin from Ternate in the Maluku islands (Moluccas), Indonesia.
July 1858: The joint Darwin–Wallace paper is presented at the Linnean Society in London.
November 1859: Darwin’s On the Origin of Species is published.
March 1862: Wallace returns from southeast Asia.
November 1913: Wallace dies in Broadstone, England.

The two split up early on, with Wallace concentrating on the Amazon River’s northern tributary, the Rio Negro, and Bates on the southern fork, the Solimões.Collecting was challenging. The Amazon’s ubiquitous ants often deprived science of hard-won specimens. Crucial collecting materials also disappeared: Wallace once recovered from a bout of fever to discover that local people had drunk the cachaça (a Brazilian rum) he’d been using to pickle specimens. Transport was a constant headache, with travel upstream past rapids requiring unwieldy portages of canoes and cargo. And thanks to his collecting, the cargo became ever more voluminous and unwieldy.Wallace and Bates sporadically sent back shipments of material to their agent in London, Samuel Stevens, who publicized their adventures in scientific journals and sold their specimens, taking a 20% commission.
Escaping Darwin’s shadow: how Alfred Russel Wallace inspires Indigenous researchers
Wallace’s journeys on the Rio Negro and its tributaries took him into areas that had not yet been visited by Europeans. He saw (and collected) an extraordinary array of species, many of them new to science. He had a chance to observe and collect artefacts from several Indigenous groups with little or no previous contact with Europeans. As he travelled, Wallace capitalized on his surveying skills to map the terrain. But the remoteness took its toll. He made an “inward vow never to travel again in such wild, unpeopled districts without some civilised companion or attendant”1.Wallace was frequently ill, on one occasion nearly lethally so. His younger brother came out to join him as an assistant in 1849 but died of yellow fever two years later in Belém, on his way back to England. Wallace learnt that his brother was sick but had to wait many anxious months before news of his death made it upriver.In 1852, after four years of exploring and collecting, it was time for Wallace himself to head home. He envisaged a triumphant return. He would complement his collections of preserved organisms with a menagerie of living ones. Mr Wallace’s biological wonders would surely be the toast of scientific London.On 12 July in Belém, Wallace boarded the Helen, a freighter ship bound for London. The trip across the continent to Belém had not gone smoothly. The authorities in Manaus, Brazil, had had to be persuaded to release some of his earlier shipments meant for London, which they had impounded, making the final haul aboard the Helen even larger. But now all that remained was the long voyage back across the Atlantic. Wallace, who shared Captain Turner’s cabin, was the only passenger.Disaster strikesThree weeks into the voyage, Captain Turner interrupted Wallace’s morning routine to tell him that the ship was on fire.Friction caused by the rocking of the ship had ignited poorly stowed cargo. Attempts to intervene were counterproductive — removing the hold covers merely oxygenated the fire — and soon the ship became what Wallace later called “a most magnificent conflagration”1.Captain Turner gave the order to abandon ship, and the scramble to prepare two small wooden boats began. Having been stored on deck in the tropical sunshine, both boats leaked badly. The cook had to find corks to plug their hulls.Before he left the ship, Wallace “went down into the cabin, now suffocatingly hot and full of smoke, to see what was worth saving”1. He retrieved his “watch and a small tin box containing some shirts and a couple of old note-books, with some drawings of plants and animals, and scrambled up with them on deck”1. He tried to lower himself on a rope into one of the small boats, but fever-weakened, he ended up sliding down the rope, stripping the skin off his hands.

Some of Alfred Russel Wallace’s sketches were salvaged from the fire aboard the Helen on his return journey from South America in 1852.Credit: The Natural History Museum/Alamy

With fine weather, the best hope of rescue lay in other ships seeing the fire. The two boats duly circled the burning wreck for the next 24 hours, meaning that Wallace got to witness every moment of the tragedy. The animals he had brought with him on the long river journey across the continent, now free from their cages, sought refuge on the one part of the ship still untouched by the flames, the bowsprit. Wallace watched as the monkeys, parrots and more — his pets as well as his best hope of impressing London’s scientific elite — were incinerated.The hoped-for rescue did not immediately materialize, and Captain Turner turned the two open boats towards Bermuda, 1,100 kilometres away to the northwest.As the days ticked by, the situation became increasingly desperate. Water ran low and the tropical sun left Wallace’s “hands and face very much blistered”1. Wallace nevertheless remained upbeat, later recalling that during one night, he “saw several meteors, and in fact could not be in a better position for observing them, than lying on [his] back in a small boat in the middle of the Atlantic”1.Finally, ten days into the ordeal, salvation appeared on the horizon in the form of the Jordeson, a creaking and already overladen cargo ship bound for London.With the immediate crisis past, the magnitude of what had happened started to sink in. In a letter2 written aboard the Jordeson to botanist Richard Spruce (see go.nature.com/3prhbdk), Wallace tallied his catastrophic losses — “almost all the reward of my four years of privation & danger was lost” — and concluded with characteristic understatement, “I have some need of philosophic resignation to bear my fate with patience and equanimity.”
Evolution’s red-hot radical
The Jordeson finally limped into Deal, England, on 1 October 1852. Wallace had been at sea for 80 days. His outward voyage with Bates had taken only 29 days.Wallace added a PS to his letter to Spruce. First there was immediate exhilaration about the return — “Such a dinner! Oh! beef steaks & damson tart”. But then came thoughts about the future: “Fifty times since I left Pará [Belém] have I vowed if I once reached England never to trust myself more on the ocean.” Even then, he noted that “good resolutions soon fade”.Stevens had thoughtfully taken out insurance. So Wallace had £200 (US$980 at the time) — a fraction of his collections’ actual value — to cover his costs for a year in London while he tried to salvage what he could from the disaster and make future plans.He rushed out two books, one a travelogue, the other a more technical account of the palm trees of the Amazon. Neither did well — 250 copies remained unsold a decade later from the travel book’s print run of 750. But he was getting his name out there. Stevens, too, had a done a good job of publicizing Wallace’s discoveries while Wallace had been away.Perhaps most crucially, the positive response of the UK Royal Geographical Society to his mapping work of the Rio Negro yielded a free steamship ticket to Singapore.In March 1854, less than 18 months since the Jordeson’s bedraggled arrival at Deal, Wallace departed from Southampton in England for what he would call the “central and controlling incident”2 of his life.Eight more years of perilous travel awaited. So, too, did the discoveries of what came to be known as Wallace’s Line (a boundary between the Asian and Australasian biogeographic regions) and of the theory of evolution by natural selection3,4.The scientific acclaim that greeted Wallace’s return from southeast Asia in 1862 was a just reward both for his contributions and for that phenomenal doggedness — his determination, despite everything, to be a scientist. More

Material characterization resultsTo investigate the structural composition of NMC-2, XRD analysis plots were performed. Figure 1a shows the XRD pattern of the NMC-2 composite before adsorption. The XRD pattern shows the corresponding strong and narrow peaks, from which it can be seen that the peaks of broad diffraction NMC-2 can correspond to the standard cards of Fe, C, Fe7C3, Fe2C, and FeC, indicating that the synthesized adsorbent is an iron-carbon composite. It can be indicated that mesoporous nitrogen-doped composites were formed during the carbonization process. During the experiments, it was found that the materials are magnetic, probably because of the presence of Fe, FeC, Fe7C3, Fe2C. Due to the magnetic properties of this type of material, rapid separation and recovery can be obtained under the conditions of an applied magnetic field, which allows easy separation of the adsorbent and metal ions from the wastewater15.Figure 1XRD and nitrogen adsorption and desorption tests on materials: (a) XRD pattern of NMC-2 adsorbent before adsorption, (b) pore size distribution of NMC-2, (c) nitrogen adsorption–desorption curve of NMC-2 adsorbent.Full size imageFrom the adsorption–desorption curves of adsorbent N2 in Fig. 1b, it can be seen that the NMC-2 isotherm belongs to the class IV curve, and the appearance of H3-type hysteresis loops is observed at the medium pressure end, and H3 is commonly found in aggregates with laminar structure, producing slit mesoporous or macroporous materials, which indicates that N2 condenses and accumulates in the pore channels, and these phenomena prove that NMC-2 is a porous material16. Figure 1c shows the pore size distribution of the adsorbent NMC-2 obtained according to the BJH calculation method, from which it can be seen that the pore size distribution is not uniform in the range, and most of them are concentrated below 20 nm, while according to Table 1, the specific surface area of the original sample of Fenton sludge and fly ash is 124.08 m2/g and 3.79 m2/g, respectively, and the specific surface area of NMC-2 is 228.65 m2/g. The Fenton The pore volume of the original samples of Fenton sludge and fly ash were 0.18 cm3/g and 0.006 cm3/g respectively, while the pore volume of NMC-2 was 0.24 cm3/g. The pore diameters of the original sample of Fenton sludge and fly ash were 5.72 nm and 6.70 nm respectively, while the pore diameter of NMC-2 was 4.22 nm. The above data indicated that the synthetic materials have increased the specific surface area and pore volume compared with the original samples, indicating that the doping of nitrogen can increase the specific surface area of the material. Since the pore size of mesoporous materials is 2–50 nm, NMC-2 is a porous material with main mesopores. Thanks to the large specific surface area provided by the mesopores, the material has a large number of active sites, and in addition, the mesopores can store more Cr(VI)16, which contributes to efficient removal.Table 1 Total pore-specific surface area, pore volume, and pore size of BJH adsorption and accumulation of Fenton sludge, fly ash and NMC-2.Full size tableThe morphological analysis of the material surface using SEM can see the surface structure and the pore structure of NMC-2. And Fig. 2a–d shows the swept electron microscope image of NMC-2. Figure 2a shows that the surface of the material is not smooth, and there are more lint-like fiber structures. The fibers in Fig. 2b are loosely and irregularly arranged, which may be due to the irregular morphology caused by the small particles of the NMC-2 sample. As shown in Fig. 2c and Fig. 2a there are more pores generated on the surface of NMC-2, which may be due to the addition of K2CO3 to urea and, Fenton sludge solution to generate CO217.Figure 2SEM, TEM and EDS testing of materials: (a–d) SEM image of NMC-2 adsorbent, (e) TEM image of NMC-2; (g–i) TEM-EDS spectrum of NMC-2, (j) TEM-EDS spectra of NMC-2 obtained from.Full size imageThese pores can provide many active sites, which is consistent with the results derived in Fig. 1, where NMC-2 is a mesoporous-dominated porous material, and also demonstrates that the addition of urea can provide a nitrogen source for the material, providing abundant active sites. Figure 2j depicts the TEM of NMC-2. the TEM images show that the synthesized NMC-2 has a folded structure with a surface covered by a carbon film, and the HRTEM (Fig. 2e) also confirms this result with a lattice spacing of 0.13, 0.15, 0.20, 0.23, 0.24, and 0.25 nm, corresponding to the (4 5 2) and (1 0 2) of C, the (2 0 1) of FeC) surface, the (2 1 0) surface of Fe7C3, the (5 3 1) surface of Fe2C, and the (2 0 1) surface of FeC, which also confirms the synthesis of the above substances. The corresponding EDS spectra of the dark field diagram NMC-2 were obtained from Fig. 2j, and the EDS spectra proved the presence of various elements: carbon (C) (Fig. 2f) from fly ash, iron (Fe) (Fig. 2g) from Fenton sludge, nitrogen (N) (Fig. 2h) from urea, and the presence of (O) (Fig. 2i), further confirming the successful preparation of NMC-2.The type of functional groups and chemical bonding on the surface of the material can be analyzed by IR spectrogram analysis. Figure 3b shows the FTIR image of NMC-2 adsorbent 3440 cm−1 wide and strong absorption peak is due to the stretching vibration of –OH, there is a large amount of –OH present on the surface of the material; the peak appearing at 1640 cm−1 is –COOH. Characterization reveals that the –OH absorption peak is wider18,19. In addition, the absorptions at 1390 cm−1 and 1000 cm−1 were attributed to the bending of –OH vibrations of alcohols and phenol and the stretching vibration of C–O20. The above results indicate that the surface of NMC-2 contains a large number of oxygen-containing functional groups, and these functional groups can provide many active sites for the removal of Cr(VI). It was also found that the weak peaks corresponding to 573 cm−1 and 550 cm−1 were attributed to Fe–O groups21. The stretching of Fe–O may be due to the oxidation of loaded Fe0 and Fe2+ to Fe3+22. Figure 3a shows the Fenton sludge and fly ash FTIR images. It can be seen from the figure that the surfaces of Fenton sludge and fly ash contain a large number of oxygen-containing functional groups, the surface functional groups of the two raw materials are more abundant, and the functional groups of NMC-2 around 3441 cm−1, 1632 cm−1, and 1400 cm−1 are not significantly different from those of the raw materials, and the C–H stretching vibration peaks of NMC-2 around 873 cm−1 and 698 cm−1 is not obvious, which may be because the material the C–H bond on the surface of the raw material was oxidized to C–O in the process of synthesis.Figure 3FTIR testing of materials: (a) FTIR image of Fenton sludge, fly ash, (b) Ftir image of NMC-2 adsorbent.Full size imageCr(VI) adsorption experimentSelection of adsorbentTo select the best adsorbent, Cr(VI) adsorption tests were performed on four adsorbents. Figure 4a shows the effect of Fenton sludge and the urea addition on the adsorption efficiency. The Cr(VI) removal rates of the four adsorbents were ranked from low to high: MC-1  More

A map of the Amazon River and its tributaries, as published in Alfred Russel Wallace’s 1853 book.Credit: Mary Evans/Natural History Museum

Dzoodzo Baniwa, a member of an Indigenous community in Brazil’s Amazonas state, has been collecting data on the region’s biodiversity for around 15 years. He lives in a remote village called Canadá on the Ayari River, a tributary of the Içana, which in turn feeds the Rio Negro, one of the main branches of the Amazon. The nearest city, São Gabriel da Cachoeira, is a three-day trip by motor boat.Dzoodzo (who goes by his Indigenous name but is also known as Juvêncio Cardoso) takes inspiration for his work from many cross-cultural sources. A perhaps unexpected one is a 170-year-old book by the British naturalist Alfred Russel Wallace, who visited the Amazon and Negro rivers on his expeditions in 1848–52. A Narrative of Travels on the Amazon and Rio Negro gives detailed accounts of the wildlife and people Wallace encountered near Dzoodzo’s home, including the Guianan cock-of-the-rock (Rupicola rupicola), a bright orange bird that Wallace describes as “magnificent … sitting amidst the gloom, shining out like a mass of brilliant flame”1.Dzoodzo’s passion for local biodiversity is reflected in his work at Baniwa Eeno Hiepole School, an internationally praised education centre for Indigenous people. He dreams of one day turning it into a research institute and university that might increase scientific understanding of the region’s species, including R. rupicola.
Alfred Russel Wallace’s first expedition ended in flames
Wallace, who was born 200 years ago, on 8 January 1823, is best known for spurring Charles Darwin into finally publishing On the Origin of Species, after Wallace sent Darwin his own independent discovery of evolution by natural selection in 1858. Most of Wallace’s subsequent work drew on observations from his 1854–62 expeditions in southeast Asia; his earlier work in Amazonia is much less well known.Yet there are lessons from Wallace’s time in Brazil that are especially relevant for conservationists and other scientists today — notably, what can come from paying attention to what local people say about their own territory.Barriers and boundariesWallace made two key contributions that still shape thinking about Amazonia, the world’s most biodiverse region, which covers parts of Bolivia, Brazil, Colombia, Ecuador, Peru, Venezuela, Guyana, Suriname and French Guiana.On 14 December 1852, Wallace read out his manuscript ‘On the monkeys of the Amazon’ at a meeting of the Zoological Society of London. In this study, which was later published2, Wallace relays observations that form the basis of the most debated hypothesis for how Amazonian organisms diversified: the riverine barrier hypothesis.His paper refers to the large Amazonian rivers as spatial boundaries to the ranges of several primate species. “I soon found that the Amazon, the Rio Negro and the Madeira formed the limits beyond which certain species never passed,” he writes. Since 1852, Wallace’s observations that large rivers could act as geographical barriers that shape the distribution of species have been corroborated, criticized and debated by many. The phenomenon he described clearly holds for some groups, such as monkeys and birds3,4, but not for other groups, such as plants and insects5.Subsequent researchers have explored whether the distribution patterns of species, such as those observed by Wallace, indicate that the evolution of the Amazonian drainage system has itself driven the diversification of species6. Work in the past few years by geologists and biologists show that this drainage system, which includes some of the largest rivers in the world, is dynamic7, and that its rearrangements lead to changes in the distribution ranges of species8. Current species ranges thus hold information about how the Amazonian landscape has changed over time.

The Guianan cock-of-the-rock (Rupicola rupicola), which Wallace likened to a “brilliant flame”.Credit: Hein Nouwens/Getty

The second crucial observation made by Wallace, also in his 1852 paper, was that the composition of species varies in different regions. He describes how “several Guiana species come up to the Rio Negro and Amazon, but do not pass them; Brazilian species on the contrary reach but do not pass the Amazon to the north. Several Ecuador species from the east of the Andes reach down into the tongue of land between the Rio Negro and Upper Amazon, but pass neither of those rivers, and others from Peru are bounded on the north by the Upper Amazon, and on the east by the Madeira.” From these observations, he concluded that “there are four districts, the Guiana, the Ecuador, the Peru and the Brazil districts, whose boundaries on one side are determined by the rivers I have mentioned.”
Evolution’s red-hot radical
Even though Amazonia is presented as a single, large, green ellipse in most world maps, it is actually a heterogeneous place, with each region and habitat type holding a distinct set of species9,10. The four districts proposed by Wallace are bounded by the region’s largest rivers: the Amazon, Negro and Madeira. But further studies of species ranges since then have revealed more districts, now called areas of endemism, some of which are also bounded by these and other large Amazonian rivers, such as the Tapajós, Xingu and Tocantins9,11.This recognition of spatial heterogeneity in Amazonian species distributions — first accomplished by Wallace — is essential for today’s research, conservation and planning10. Each area of endemism includes species that occur only in that area. And different areas of endemism are affected differently by anthropogenic impacts, such as deforestation, fires and development10. More than half of Amazonia is now within federal or state reserves or Indigenous lands — territories that are recognized by current governments as belonging to Indigenous people. But nearly half of the region’s areas of endemism are located in the south of the region, close to the agricultural frontier, and the species they contain are severely threatened by habitat loss10 (see also www.raisg.org/en).Local knowledgeAlthough Wallace’s writings indicate that in many ways he admired most of the Indigenous people he met, especially those from the upper Rio Negro basin, he still viewed Indigenous people through the European colonial lens of his time. In A Narrative of Travels on the Amazon and Rio Negro1, Wallace describes the Indigenous communities he encountered as “in an equally low state of civilization” — albeit seemingly “capable of being formed, by education and good government, into a peaceable and civilized community”.Yet he did better than many of his contemporaries when it came to respecting local knowledge. In his 1852 paper, for example, Wallace notes that his fellow European naturalists often give vague information about the locality of their collected specimens, and fail to specify such localities in relation to river margins. By contrast, he writes, the “native hunters are perfectly acquainted” with the impact of rivers on the distribution of species, “and always cross over the river when they want to procure particular animals, which are found even on the river’s bank on one side, but never by any chance on the other.” Likewise, in his 1853 book1, Wallace frequently corroborates his findings with information he has obtained from Indigenous people — for example, about the habitat preferences of umbrellabirds (Cephalopterus ornatus) or of “cow-fish” (manatees; Trichechus inunguis).Considering the vastness and complexity of Amazonia, it is hard to see how Wallace could have gained the insights he did after working in the region for only four years, had he not paid close attention to local knowledge.
The other beetle-hunter
Amazonian Indigenous peoples have had to endure invasion of their lands, enslavement, violence from invaders and the imposition of other languages and cultures. Despite this, numerous Indigenous researchers wish to expand their knowledge about Amazonia by combining Indigenous and European world views. Meanwhile, a better understanding of how the Amazonian socio-ecological system is organized, and how it is being affected by climate change and local and regional impacts12, hinges on the ability of researchers worldwide to learn from and to be led by Indigenous scientists.The 98 Indigenous lands in the Rio Negro basin cover more than 33 million hectares (see go.nature.com/3wkkftu). If the hopes of Dzoodzo and others to build a research institute and university for the region are met, school students will no longer have to leave their homeland to pursue higher education. The community would have a way to document its own knowledge and that of its ancestors in a more systematic way. And the legitimization of Indigenous people’s research efforts in the legal and academic frameworks recognized by non-Indigenous scientists — such as through the awarding of degrees — would make it easier for Indigenous researchers to partner with other organizations, both nationally and internationally.Indigenous people in the Rio Negro basin today are no longer objects of observation — they have taken charge of their own research using tools from different cultures. Indeed, Dzoodzo is turning to Wallace’s writings, in part, to learn more about how his own ancestors lived.Perhaps the thread between Wallace and Dzoodzo, spanning so many years and such disparate cultures, could seed new kinds of partnership in which learning is reciprocal and for the benefit of all. More

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In a previous study18, we used bioinformatics tools to analyze the metagenome and the amplicon 16S sequences to gain an insight into microbial diversity in Moroccan lagoons, namely Marchica and Oualidia. 16S rRNA gene classification revealed a high percentage of bacteria in both lagoons. On average, bacteria accounted for 90% of the total prokaryotes in Marchica and ~ 70% in Oualidia. The five phyla that were the most abundant in both lagoons, Marchica and Oualidia, respectively, were Proteobacteria (53.62%, 29.18%), Bacteroidetes (16.46%, 43.49%), Cyanobacteria (0.53%, 34.35%), Verrucomicrobia (1.75%, 15.82%), and Actinobacteria (7.42%, 13.98%). At the genus level, we found that the highest assigned hits were attributed to Synechococcus, which was highly abundant in Marchica (32%) compared to Oualidia (0.07%) in 2014. This amount dropped to 22% in Marchica and 0.04% in Oualidia in 2015. Hence, in this study we performed the analysis of the Synechococcus genus community using oligotyping to investigate their dynamics and understand their co-occurrence and covariation in space and time within fragile ecosystems such as lagoons.We may divide our results into two emerging Synechococcus communities: one dominated in 2014 and the other was less present in 2015, each composed of different cooccurring Synechococcus oligotypes. The abundant Synechococcus community in Marchica in 2014 consisted of clades I, 5.3, III, IV, and VII. These clades are typically found in either warmer or more oligotrophic environments19,20. This result is in accordance with Marchica’s environmental characteristics; it is an oligotrophic ecosystem with high primary production and warmer water in summer21. The community included clades CB5 and WPC1 in Marchica 2014 and 2015 when the number of Synechococcus reads was lower. Strains belonging to the CB5 clade lack phycourobilin (PUB), contain one motile strain22,23, are present in temperate coastal waters and are prevalent in polar/subpolar waters24,25,26. WPC1 strains are observed in open-ocean and near-shore waters1,24,27. Clades IV and I usually co-occur and are more prevalent in cold coastal waters19,28,29,30. Interestingly, Clade III was prominent in Marchica. This clade is known to be motile and restricted to warm, oligotrophic water19,20,30. Although at a smaller read number, clade III was also observed in Oualidia, where the temperature is cooler compared to Marchica. Furthermore, we found that clade III growth has been shown to be severely affected at low temperatures30. Moreover, representatives of both clades I and IV were present in Oualidia in both the summers of 2014 and 2015. Some Synechococcus strains, which are known to prefer cooler water temperatures and salinities, were in higher relative abundance in the waters of Marchica. This result agrees with a previous study showing that Synechococcus isolates of clades I and IV exhibited temperature preferences31. Their growth rates were marginally lower at low temperatures in strains from clades I and IV, which were dominant in temperate regions.Nitrate levels are typically low or undetectable in these lagoons, which allows the persistence of clades that would not typically thrive in coastal waters at other times of the year. In 2014, the nitrate concentration was higher than the average of 10 mg/l, which could be due to increased agricultural activities and wastewater treatment plant effluent21. The decreasing nitrate concentration in Marchica in 2015 could be explained by the newly installed inlet in 2010, which was designed to improve water exchange with the open sea and reduce the amount of suspended matter21. Temperature and salinity have a large effect on nitrate in marine ecosystems32; the highest nitrate degradation rates were observed at 35 °C and at increasing salinity rates. Therefore, we expected to see correlations between salinity, temperature and nitrate concentrations. Interestingly, clades CB5 in Marchica and IV in Oualidia increased in relative abundance in summer 2015 compared to 2014, when the nitrate concentration decreased. Moreover, the Synechococcus microbial community diversity and density are variables depending on the variations in the physical and chemical parameters. These parameters are strongly influenced by the marine waters passing through the artificial inlets, which have an impact on the internal hydrodynamics of both lagoons and hence the distribution and co-occurrence of Synechococcus strains. In addition, anthropogenic activities also have a great influence on Synechococcales population growth and interactions with their viruses33,34.This study revealed some differences between Marchica and Oualidia in identified Synechococcus clades. The Marchica lagoon showed more heterogeneity (clades I, II, III, IV, VII, VIII, 5.3, WPC1, CB5, and IX) than the Oualidia lagoon, where fewer clades were identified (I, III, IV, and VII). There was a clear variation in the pattern of correlation between oligotypes of the same or different clades for both the 2014 and 2015 samplings. Furthermore, we observed complex patterns of co-occurrence among oligotypes; in 2014 (clades I, III, IV, 5.3, VII), and in 2015, we found clades CB5 and WPC1. In Oualidia, values decreased in comparison to Marchica in both 2014 and 2015 summer samplings, following a pattern of co-occurrence, especially for both clades I and IV in both sampling years. Many studies have shown that the relative proportions of cooccurring Synechococcus populations to each other at the clade and subclade levels vary in space and time based on environmental factors such as seasonal temperature fluctuations, nutrient availability and upwelling, circulation patterns, and abundance of other phytoplankton8.We presume that the greater variability in oligotype co-occurrence behavior observed in Marchica Lagoon, especially in the summer of 2014, could be due to the higher abundance and diversity of Synechococcus oligotypes, physico-chemical parameter fluctuations or rehabilitation of the lagoon.Less abundant oligotypes could also be considered potential bioindicators of Synechococcus genetic diversity. Their seasonal occurrence might contribute to changing ecological and biogeochemical characteristics of the marine environment35. The Synechococcus relative abundance count revealed that the Marchica Synechococcus community included the least abundant oligotypes in 2015. For instance, O7 and O8 were detected in 2014 and were absent in 2015 (Table 1). It is unclear which factors served to constrain the relative abundances of these least present oligotypes, but temperature and salinity could have an impact on their distribution in Marchica (Fig. 4) and the opposite for Oualidia, which are cooler-temperature adapted ones. We noticed that the relative abundance of cooccurring Synechococcus was not constant. For instance, oligotype 4 belonging to Clade IV showed higher values in summer 2014 (974 reads) in Marchica compared to summer 2015 (319 reads), and the opposite was observed in Oualidia, with a lower abundance compared to Marchica. Increased values of cooccurring clade I oligotypes (14, 26, and 6) were detected in the summer of 2014 in both lagoons.Figure 4Principle component analysis of Synechococcus oligotype relative abundance. The plot is generated using the relative abundance of each oligotype, T temperature, S Salinity, and NO3− Nitrate. Each point represents an oligotype. Colors represent the year of sampling; red for 2014 and blue for 2015. The shape of point indicates the sampling site; rounded points refer to Marchica lagoon, and triangles refer to Oualidia. Circles represent the normal distribution of oligotypes; the red circle refers to 2014, and the blue one refers to 2015.Full size imageIn comparing our results with a study from Little Sippewissett Marsh (LSM)8 that used oligotyping to investigate the distribution of the genus Synechococcus in space and time sequencing the V4-V6 hypervariable region of the 16S rRNA gene, we found 31 oligotypes, while they identified 12. In both studies, the proportion of Synechococcus oligotypes increased in summer and in coastal waters compared to estuaries. In addition, Clades I and IV were more abundant in saline conditions, such as Marchica Lagoon. However, these clades were found in greater relative abundances at cold temperatures, in contrast to our study, where they were identified in Marchica’s warm waters. Moreover, clade CB5 tended to be prominent at relatively warm temperatures (17–20 °C)6. In our work, it was not prevalent either in cooler or warmer water. Notably, the relative abundance of rare oligotypes was higher in warm hypersaline estuary waters8,18, while in our case study, they occurred in cooler moderately saline Oualidia waters.The dominance of a certain clade could have many different ecological ramifications, especially as the clades can be incredibly diverse in their growth, loss, nutrient utilization and other attributes. The dominant clade’s growth and loss patterns will set the stage for the population dynamics. For instance, if the dominant clade only blooms in a given environmental factor such as temperature, light, or salinity, it will then affect the timing of blooms, and follow-on the effects of subsequent grazing, lysis or even biogeochemical cycling. Even if the population is diverse, the dynamics as a whole will be a composite response of each individual clade’s ecophysiology, making it important to understand their composition and how it changes over space and time.While the rpoC1 gene is a higher resolution diversity marker36, 16S amplicon data can be used for exploring the entire bacterial assemblage including Synechococcus clade designations via oligotyping35. The latter has a great advantage in answering unexplained diversity contained in taxa using 16S rRNA gene sequences. Nevertheless, it has some limitations, as it acts optimally only when performed on taxa that are closely related. Regarding distantly related taxa, the high number of increased-entropy locations makes the supervision steps difficult. In addition, although oligotyping does not rely on clustering conditions or availability of existing reads within reference databases, it demands preliminary operational taxonomic unit clustering to find closely related species appropriate for the analysis. This method is under continuous improvement to better exploit the information within subtle variations in 16S rRNA gene sequences5.In conclusion, we explored the patterns of Synechococcus diversity in space and time using an oligotyping approach to examine these populations in lagoon waters of Mediterranean Marchica and Atlantic Oualidia, in Morocco. Patterns that have been observed at the clade and subclade levels, such as Synechococcus, relative abundance and the co-occurrence of groups from different clades, were shown to occur among oligotypes. The Marchica Lagoon showed a heterogeneous Synechococcus diversity compared to Oualidia in summer 2014. Thirty-one Synechococcus oligotypes were identified. Two distinct communities emerged in the 2014 and 2015 summer samplings, abundant and rare Synechococcus species, each comprising cooccurring Synechococcus oligotypes from different clades. Network analysis showed that six oligotypes were exclusive to Marchica Lagoon. The identified clades I, III, IV, VII, and 5.3 in Marchica were in accordance with its environmental characteristics. In addition, the relative abundance of some cooccurring Synechococcus strains was not constant over time and space (e.g., clades I and IV). Using gene oligotyping, we illustrated some of the challenges associated with the identification of novel Synechococcus strains or studied their co-occurrence in space and time. Oligotyping has been instrumental in discriminating closely related Synechococcus strains. However, this study leaves open questions about how samples differ by location and whether locations differ from year to year. Do cooccurring oligotypes interact with each other and to what extent do they correlate with physicochemical parameters? What triggers the coexistence of clades I and IV with clade III in warm water or 5.3 with VII, which do not know much about. Finally, how do relative abundances change over seasons. Hence, future work needs to consider additional stations and seasons to provide better statistical support for our findings and to better understand their correlation with physical and chemical environmental parameters. Other factors were not considered in this study, such as nutrient availability, chlorophyll, irradiance, viral lysis, and greater sequencing depth, which could also influence the observed seasonal dynamics. More

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