Transcriptional Activity of Aerobic and Anaerobic Ammonia-Oxidizing community in the Intertidal Sponge Cinachyrella Australiensis, Ambient Seawater, and Sediment

Microbial ammonia oxidation plays a central role in nitrogen cycling. Hitherto, four types of autotrophic ammonia-oxidizing microorganisms are identied, including aerobic ammonia-oxidizing archaea (AOA), aerobic partial-nitrication ammonia-oxidizing bacteria (parAOB), aerobic complete-nitrication AOB (comAOB), and anaerobic AOB (AnAOB). However, revelation and comparison of the active ammonia-oxidizing community in the marine sponges and their ambient environments is scarce. Here, transcribed ammonia oxidation phylomarker gene amoA of AOA, parAOB, and comAOB and hzsB of AnAOB were amplied to investigate the active ammonia-oxidizing populations in a representative marine sponge Cinachyrella australiensis, ambient seawater, and sediment niches. Ammonia-oxidizing population in C. australiensis consists of AOA, parAOB, and AnAOB, signicantly different from that in seawaters comprising of AOA and in sediments containing AOA, parAOB, comAOB, and AnAOB. The quantitative assay demonstrates that AOA amoA transcripts are exclusively detectable or higher in abundance than parAOB amoA, comAOB amoA, or AnAOB hzsB transcripts by orders of magnitude in C. australiensis, seawater, and sediment niches. This transcript-based analysis claries the remarkable niche differentiation of putatively active ammonia-oxidizing microbiota in C. australiensis and the ambient environments. Such a work further contributes to the understanding of in situ active ecological functions of sponge microsymbionts in nitrogen cycling.


Study Site and Sampling
The sponge individuals were collected at the low tide sites S1 (109°29′33″ E 18°15′37″ N), S2 (109°29′11″ E 18°15′37″ N), and S3 (109°26′18″ E 18°15′37″ N) from the intertidal zone of Hainan Island in the South China Sea at 8:00 -10:00 am in July 6 th , 2019. The seawater temperature was 27.2 -27.4°C. The sponge is solitary individual of globular size in appearance, typically up to 4 -6 cm diameter. In each site, triplicate samples of sponge tissue slice, ambient seawater (two liters each), and ambient sediment (0 -2 cm, ~ 5 g) were separately collected ( Fig. S1 A and B). Collections and pretreatments of sponge, seawater, and sediment samples referred to the reported strategy [19,20,43] before being transferred into the RNAprotect Bacteria Reagent (Qiagen, Hilden, Germany). Identi cation of the sponge species based on gross morphology and PCR con rmation of the mitochondrial coxl (cytochrome c oxidase subunit I) gene using the primer pair CinaF2/dgHCO2198 [44]. The ampli ed coxl clone gene showed 99% nucleotide sequence identity to the reported C. australiensis voucher LB_815 mitochondrial cox1 gene (JX177880) and was submitted to GenBank with the accession number MT913441. The time between sample acquisition and xation was no longer than 20 min to minimize RNA degradation [45]. All the RNA protector-xed samples were stored at -80°C before total RNA and DNA extraction within two weeks.
RNA, DNA Extraction and cDNA Synthesis RNA protector-xed C. australiensis, seawater, and sediment samples were ground in liquid nitrogen with a sterilized mortar and pestle. Both RNA and DNA were extracted from ground powders using the PrepRNA/DNA Mini Kit (Qiagen, Hilden, Germany) following the manufacturer's instruction. RNA and DNA were separately extracted from each sample of C. australiensis, seawater, and sediment. RNase-free DNase I (Fermentas, Hanover, USA) was used to digest the residual genomic DNA at 37°C for 60 min. RNA quality and integrity were checked by gel electrophoresis and by examining the A260/A280 ratio (ranging from 1.97 to 2.02) using a NanoDrop spectrophotometer (NanoDrop Technologies, Wilmington, USA). The nal RNA concentration and purity were quanti ed using the Qubit system (Invitrogen, Darmstadt, Germany). First-strand cDNA synthesis was performed using the SuperScript FirstStrand Synthesis System (Invitrogen, Carlsbad, USA). Each reaction volume was10 μl containing 100 ng RNA, 0.5 μl random hexamer primer (50 ng μl -1 ), 5 μl cDNA Synthesis Mix, and proper RNase-free water. This reaction system was incubated at 25°C for 10 min and then 50°C for 50 min and terminated at 85°C for 5 min. All cDNA aliquots were stored at -80°C before PCR ampli cation.

Ampli cation and Sequencing
The AOA amoA, AAOB amoA, CoAOB amoA, and AnAOB hzsB gene fragments were ampli ed, respectively, using the cDNA and DNA templates with the primers listed in Table 1. PCR ampli cations were performed in a total volume of 40 μl containing 2 μl cDNA or 2 ng DNA, 0.1 μM of each primer, and 20 μl TaqMasterMix (CoWin Biotech, Beijing, China) on a Thermocycler (Eppendorf, Hamburg, Germany) according to the following procedures: 95°C for 5 min; followed by 30 cycles at 95°C for 40 s, annealing (temperature referring to Table 1) for 30 s and 72°C for 30 s, and nally 72°C for 10 min. For negative control, a similar procedure was carried out using puri ed RNA to ensure that there was no genomic DNA contamination. PCR products originated from the triplicate samples of C. australiensis, seawater, or sediment from each sampling site were pooled to reduce potential ampli cation bias and maximize the transcript richness referring to the previous strategies [19]. The presence and sizes of these ampli cation products were estimated by gel electrophoresis (1.5% agarose gel). Since this study focused on the transcriptional activity of the ammonia-oxidizing community, the performance of DNA-based PCR ampli cation was to only test the presence of the targeted genes in the investigated biotopes, whose PCR products were not sequenced [19]. cDNA-based PCR products were gel-puri ed with MinElute Gel Extraction [49]. Phylotype representative sequences were taxonomically classi ed using BLASTn against NCBI Nucleotide database. Rarefaction curves and Good's coverage estimators were determined using the Mothur package [49] to estimate whether the sequencing depth is enough to cover most of the transcribed genes in each clone library.
One representative sequence from each phylotype and its closest sequence retrieved from the NCBI Nucleotide database were aligned using ClustalW implemented in the MEGA [46]. Maximum-likelihood (ML) tree was constructed by using the MEGA with the Kimura-2 parameter model according to a published guideline [50]. Bootstrap analysis was used to estimate the reliability of phylogenetic reconstructions (1000 replicates).

RT-qPCR Assays
RT-qPCR assays were performed using an ABI 7500 Fast Real-time qPCR platform (Applied Biosystems, Foster, USA), following the reported strategy on sponges [19]. Gene expression was tested using technical triplicates for each sample of C. australiensis, seawater, and sediment. PCR was performed in a total volume of 25 μl containing 12.5 μl of SYBR Premix Ex Taq™ II (Takara, Dalian, China), 1 μl of cDNA template (tenfold serial dilution), and 0.1 μM of each primer (Table 1). PCR thermocycling steps were set as follows: 95°C for 5 min and 40 cycles at 95°C for 45 s, annealing (temperature setting showed in Table   1) for 45 s, and 72°C for 45 s. For quanti cation, standard curves (log-linear R 2 > 0.99, E = 92% -110%) were generated using puri ed and quanti ed plasmids containing AOA amoA (sequence of the clone AOA-spg-1, GenBank ID MT925791), parAOB amoA (sequence of the clone parAOB-spg-1, GenBank ID MT925730), comAOB amoA (sequence of the clone comAOB-sed-1, GenBank ID MT925742), or AnAOB hzsB (sequence of the clone AnAOB-spg-1, GenBank ID MT925769) fragment in a dilution series that spanned from 10 1 to 10 7 gene copies per reaction. All standard dilutions were prepared in 10 ng μl −1 aqueous tRNA solution (Sigma-Aldrich, Steinheim, Germany). Plasmid DNA was extracted using the PurePlasmid 96 Kit (CoWin Biotech), and the plasmid concentration was measured using the Qubit system (Invitrogen). Since the sequences of the vector and PCR insert are known, copy numbers of transcribed amoA or hzsB genes were directly calculated according to the reported formula: copy numbers μl −1 = (A × 6.022 × 10 23 ) × (660 × B) −1 , where A is the plasmid concentration (g μl −1 ), B is the recombinant plasmid length (bp) containing the amoA or hzsB fragment, 6.022 × 10 23 is the Avogadro's number, and 660 is the average molecular weight of 1 bp [51]. For negative control, a similar procedure was performed using puri ed RNA to ensure that there was no genomic DNA contamination. After the qPCR assay, the speci city of ampli cation was veri ed by the generation of melting curves (in steps of 0.5°C for 5 s, with temperatures ranging from 60 to 95°C) and the qPCR product size and speci city were checked by 2% agarose gel electrophoresis.

Statistical Analysis
Data acquisition of the qPCR assay was performed using the 7500 System SDS Software Version 1.2 (Applied Biosystems). One-way analysis of variance (ANOVA) was performed to evaluate the abundance variations of the amoA or hzsB transcripts between C. australiensis, seawater, and sediment niches using the commands in SPSS 19.0. The thetaYC matrix distances based on the amoA or hzsB transcript sequences were calculated via Mothur commands and were visualized by the principal co-ordinate analysis (PCoA) in Canoco 5.0. Comparison between the amoA or hzsB-harboring communities from C. australiensis, seawater, and sediment niches were analyzed using the analysis of similarity statistics (ANOSIM) on thetaYC indices through Mothur. Statistical differences were determined at the level of α = 0.05.

Rarefaction Curves and Good's Coverage Values
PCR and sequencing result showed that AOA amoA was detected from C. australiensis, seawater, and sediment niches; parAOB amoA and AnAOB hzsB were revealed from C. australiensis and sediment niches, whereas comAOB amoA was only uncovered from sediment niches (Fig. S2). Rarefaction curve analysis showed that most of the amoA and hzsB transcript curves reach an asymptote based on 3% or 5% nucleotide sequence dissimilarity (Fig. S3), consistent with the Good's Coverage values ranging from 76.9% − 100% (Table S1). Therefore, enough amoA and hzsB transcript clones were sequenced to represent their diversity in corresponding clone libraries.

Community Compositions Of Active Aop
Most of the AOA amoA transcripts were taxonomically classi ed into the Nitrosopumilus, Nitrosotenuis, Nitrososphaera, Nitrosopelagicus, and Cenarchaeum taxa, and the remaining ones (7.3% of total) cannot gather into a de nite taxon (Unclassi ed). As shown in Fig. 1A, AOA population in C. australiensis was composed of Nitrosopumilus, Nitrosopelagicus, and Cenarchaeum. Such a composition differed from that in sediment niches (consisting of Nitrosopumilus, Cenarchaeum, and Unclassi ed) and in seawater niches (consisting of Nitrosopumilus, Nitrosotenuis, Nitrosopelagicus, Cenarchaeum, and Unclassi ed). Similarly, parAOB or AnAOB compositions were different between C. australiensis and sediment niches. Thus, the parAOB population consisted of Nitrosospira in C. australiensis and Nitrosospira and Nitrosomonas in sediment niches (Fig. 1B); while the AnAOB population comprised Kuenenia and Scalindua in C. australiensis and Brocadia, Scalindua, and Jettenia in sediment niches (Fig. 1D). Moreover, Nitrospira Clade A and Clade B composed the comAOB population in sediment niches with Clade A taking a dominant proportion (95.6% of total) (Fig. 1C).

Community Dissimilarity Of Aop Communities Between Different Niches
PCoA based on the thetaYC matrix distance showed that AOA populations within C. australiensis, seawater, and sediment niches were clearly separated by axes PC1 (explaining 71.2% of the variation) and PC2 (explaining 18.3% of the variation) ( Fig. 2A). Similarly, the parAOB or AnAOB populations from C. australiensis and sediment niches were clearly separated by axes PC1 (explaining 52.6% or 68.5% of the variation) and PC2 (explaining 16.8% or 20.4% of the variation) ( Fig. 2B and C). ANOSIM results based on thetaYC indices showed that the AOA, parAOB, or AnAOB populations within C. australiensis, seawater, and sediment niches was signi cantly different from each other (r = 0.812-0.639, p = 0.002-0.028 < 0.05).

Phylogeny of amoA and hzsB Transcript Phylotypes
All the AOA amoA transcript phylotypes were most similar to the uncultured environmental sequences from sponges, seawaters, and sediments (Table S3) and phylogenetically fell into six clusters (Fig. 3). Basically, ve phylotypes fell into the Nitrosopumilus cluster; these ve phylotypes related to Nitrosopumilus spp. and the uncultured sequences from aquarium bio lter, marine sediments, and seawaters. One phylotype gathered into the Nitrosotenuis cluster and was similar to Ca. Nitrosotenuis spp. and the uncultured sequences from the Black Sea. Another phylotype falling into the Nitrososphaera cluster was related to Ca. Nitrososphaera spp. and the uncultured sequence from marine sediment. Four phylotypes gathered into the Nitrosopelagicus cluster; these phylotypes were closely related to Ca. Nitrosopelagicus brevis, Crenarchaeote SCGC AAA288-J14, and the uncultured sequences from seawater and sponge. Another four phylotypes were ascribed into the Cenarchaeum cluster which were closely related to Cenarchaeum symbiosum and the uncultured sequences from seawater and sponge. Besides, the remaining four phylotypes gathered into the Unclassi ed cluster; these phylotypes were similar to the uncultured sequences from marine sediments and seawaters. Thus, a complex active AOA population was uncovered from C. australiensis, seawater, and sediment niches. Analysis of the Proportion of AOA amoA phylotypes in each niche showed that, The phylotypes accounting for the highest proportion in turn fell into the Cenarchaeum, Nitrosopumilus, and Nitrosopelagicus cluster in C. australiensis, sediment, and seawater niches, respectively.
The parAOB amoA transcript phylotypes which were most similar to the uncultured environmental sequences (Table S3) fell into the Nitrosospira and Nitrosomonas clusters (Fig. 4A). Eleven phylotypes fell into the Nitrosospira cluster and were mainly related to Nitrosospira spp. and the uncultured sequences from various sponges and marine sediments; while the remaining three sediment-derived phylotypes gathered into the Nitrosomonas cluster and were related to Nitrosomonas spp. and the uncultured sequences from marine sediments and nitrifying granules (Fig. 4A). The phylotype accounting for the highest proportion in turn gathered into the Nitrosospira and Nitrosomonas cluster in C. australiensis and sediment niches, respectively.
Specially, comAOB amoA transcripts were most similar to the uncultured environmental sequences from marine sediments and other niches (Table S3) and gathered into the Nitrospira Clade A and B clusters (Fig. 4B), according to their distinct divergence [4]. Clade A included 12 phylotypes which were closely related to the sequences from various types of sediments, wastewater treatment plant, bio lter sand, and the known comAOB species, i.e., Ca. Nitrospira nitri cans, Ca. Nitrospira nitrosa, Ca. Nitrospira inopinata, and Nitrospira moscoviensis. The Clade B containing two phylotypes were similar to the wetland derived sequences and the Ca. Nitrospira sp. comreactor17. Thus, a complex comAOB population with transcriptional activity inhabits the surface sediment adjacent to the sponge C. australiensis. The phylotype accounting for the highest proportion gathered into the Nitrospira Clade A cluster in sediment niches.
All the AnAOB hzsB transcript phylotypes were most similar to the uncultured environmental sequences from the South China Sea and other soil or sediment environments (Table S3) and fell into four clusters (Fig. 5). Brie y, the Brocadia cluster including seven phylotypes were related to the sequences from granular sludge and different types of wetlands. The Jettenia cluster included one phylotype related to the sequence from paddy soil. Two phylotype fell into the Kuenenia cluster and were similar to the sequence from South China Sea sediment, wastewater treatment plant, and Ca. Kuenenia stuttgartiensis; while the Scalindua cluster included 12 phylotypes which were closely related to Ca. Scalindua rubra and the South China Sea sediment-derived sequences. The phylotypes accounting for the highest proportion in turn gathered into the Scalindua and Brocadia cluster in C. australiensis and sediment niches, respectively. Taken together, the amoA and hzsB transcript phylotypes fell into different clusters and were clustered with the uncultured environmental sequences from marine sponges, seawaters, sediments, and other aquatic niches.

Discussion
An active AOP community composing of AOA, parAOB, and AnAOB was revealed in the sponge C. australiensis. Such a AOP community was signi cantly different from that in ambient seawater (consisting of AOA) and sediment niches (consisting of AOA, parAOB, comAOB, and AnAOB). Such a nding was consistent with previous studies that microorganism compositions were signi cantly varied between sponges, environmental seawaters, and sediments [43,52]. Therefore, marine sponges may have eco-physiological preferences for speci c microbes distinctive from ambient seawater and sediment niches [53].
AOA are ubiquitous and signi cant contributors to nitrogen cycling in marine environments [54]. Nitrosopumilus, Nitrosopelagicus, Nitrosotenuis, Nitrososphaera, and Cenarchaeum AOA were detected (Fig. 1). The active AOA population in C. australiensis mainly consisting of Nitrosopumilus, Nitrosopelagicus, and Cenarchaeum differed from that in sediment niches (consisting of Nitrosopumilus, Cenarchaeum) and in seawater niches (consisting of Nitrosopumilus, Nitrosotenuis, Nitrosopelagicus, Cenarchaeum) (Figs. 1A and 2A). Generally, there is an obvious niche differentiation of AOA population between C. australiensis, and their ambient seawater and sediment niches. Similar condition has been found in another case that the South China Sea sponges harbored an AOA population (Nitrosopelagicus and Cenarchaeum) differed from that in ambient seawaters (Nitrosopumilus) [19]. Among the detected AOA lineages, Nitrosopumilus was detected from C. australiensis, seawater, and sediment niches (Fig. 1A), in agreement with previous ndings that Nitrosopumilus is prevalent in ammonia oxidation in marine sponges (Fig. S4), seawaters, and sediments [19,55,56]. Nitrosopelagicus and Cenarchaeum were detected from C. australiensis and seawater niches (Fig. 1A), consonant with former reports that these two AOA lineages are commonly found in various sponges (Fig. S4) and seawaters [24,57]. Nitrosotenuis and Nitrososphaera were detected from seawater and sediment niches, respectively (Fig. 1A), consistent with previous revelations that Nitrosotenuis can be found widely distributed in seawaters [58] while Nitrososphaera represents an AOA genus frequently found in marine sediments [59].
Generally, there is an obvious niche differentiation of putative active AOA population among C. australiensis, ambient seawater, and sediment niches.
The parAOB also contribute to ammonia oxidation in marine environments [60]. The parAOB population in C. australiensis comprised of Nitrosospira distinctive from that in sediment niches consisting of Nitrosospira and Nitrosomonas (Figs. 1B and 2B). Conclusion of the reported sponge-associated parAOB amoA sequences showed that, Nitrosospira amoA sequences were reported in diverse sponges (Fig. S5), while Nitrosomonas amoA homologues were only uncovered from few sponges, e.g., Neamphius huxleyi and Placospongia sp. (Fig. S5), indicating that Nitrosospira rather than Nitrosomonas may the dominate the parAOB population in sponges. Many studies also demonstrated that Nitrosomonas and Nitrosospira parAOB have also been uncovered from marine sediments [61-63], in agreement with our nding (Fig. 1). Whereas the negative detection of parAOB in seawaters different from previous ndings that parAOB residence in seawaters [17,64,65]. Therefore, the presence of parAOB in marine environments may be varied in different geographic areas and a speci c niche, e.g., sponge, seawater, or sediment niche may harbor a speci c parAOB population.
A comAOB population composed of Nitrospira Clade A and B lineages (Fig. 3) was detected in sediments ( Fig. 1C and Fig. S2). Such a comAOB amoA population composition were also found in marine coastal waters and sediments [31][32][33]66, 67] and river waters and sediments [68]. Such a comAOB population differs from that in eutrophic lake sediments [69] and river tidal at sediments [31] where only Nitrospira clade A were detected. The comAOB amoA was undetectable in seawaters, in agreement with the ndings in former studies [4,70]. Besides, no comAOB amoA fragment was detected from C. australiensis, and no reports have con rmed the presence of comAOB in sponges so far. Therefore, whether the comAOB resident in sponges needs further investigation. These ndings indicate the niche differentiation of comAOB in different environments.
AnAOB greatly contributes to marine nitrogen removal from the oceans [71]. The AnAOB population in C. australiensis niches comprised of Kuenenia and Scalindua (Fig. 1) which was different from the Brocadia AnAOB population in the sponge Mycale laxissima [20]. Moreover, AnAOB population in sediment niches were divergent from that in C. australiensis niches (Fig. 2C) and consisted of Brocadia, Jettenia, Kuenenia, and Scalindua (Fig. 1D), consistent with that in China's coastal wetlands [72], but different from that in surface sediments of the Bohai Sea [73] and north marginal seas of China [74] which was composed of Scalindua, Jettenia, and Anammoxoglobus. Conclusively, the ecological niche segregation of AnAOB was prevalent in marine environments, such as in sponges, seawaters, and sediments.
Quantitative surveys of amoA or hzsB transcripts demonstrated that AOA amoA transcripts were either exclusively detectable or were higher in abundance than parAOB amoA, comAOB amoA and AnAOB hzsB transcripts by orders of magnitude in C. australiensis, seawater, and sediment niches (Fig. 6). Such a nding indicated the dominant transcriptional expression of AOA in these niches. Similar condition has been revealed from the cold-water sponges where higher amoA transcripts abundance of AOA than that of parAOB were detected [12]. Based on the nitri cation kinetics of pure cultures, ammonia a nity of ammonia-oxidizing species ranking from high to low is marine AOA, comAOB, terrestrial AOA, and parAOB [75]. A higher ammonia a nity of AOA would contribute to their survival in the oligotrophic ocean. Meanwhile, although comAOB showed a higher ammonia a nity than parAOB, both parAOB and comAOB were uncovered in sediment niches, while parAOB rather than comAOB were uncovered in C. australiensis. Therefore, besides ammonia contents, other environmental factors may also in uence the residence of comAOB in sponges, which need further investigation.
Generally, microbially mediated ammonia oxidation process by phylogenetically divergent AOP lineages in the sponge C. australiensis, ambient seawater, and sediment niches. Certainly, some aspects need further study. For example, the comAOB amoA sequence was not detected from C. australiensis. Therefore, more sponge species from distant geographic scales over different sampling timepoints can be investigated to verify the residence of comAOB in sponges. Additionally, the investigated C. australiensis, ambient seawater, and sediment niches were only collected at one timepoint rather than serial timepoints. Temporal change in gene expression is possible, such as the AOA and parAOB amoA transcripts in coastal sediments [76]. Therefore, more data from different sponges in time series will give us a better insight into the ammonia oxidation and other nitrogen metabolic processes.

Conclusion
In this study, the community structure and abundance of active ammonia-oxidizing microbiota in the sponge C. australiensis, ambient seawaters, and sediments were investigated using transcript-based strategies. Active AOP community selectively consisting of AOA, parAOB, comAOB, or AnAOB was uncovered inhabiting C. australiensis, seawater, and sediment niches. The AOP community in C. australiensis was signi cantly different from that in ambient seawater and sediment niches. AOA amoA transcripts were exclusive or higher in abundance than parAOB amoA, comAOB amoA, or AnAOB hzsB transcripts by orders of magnitude in each niche type. This study indicates the obvious divergence of AOP population structures and signi cant variations of AOP transcriptional expressions among C. australiensis, ambient seawater, and sediment niches. This research would extend our understanding of the putative metabolic activity and ecological functions of the ammonia-oxidizing microbiota within marine sponges and the ambient environments.

Declarations
Author contribution Study conception and design: FG, SL, LZ, and HT; acquisition of data: FG, SL, LZ, and HT; bioinformatic analysis: FG and SL; analysis and interpretation of data: FG; drafting of manuscript: FG, SL, LZ, and HT; critical revision: FG, SL, LZ, and HT.
Con ict of interest The authors declare no competing interests.    Phylogenetic maximum-likelihood tree of AOA amoA transcript phylotypes retrieved from the sponge C.
australiensis, ambient seawater, and sediment niches. Scale bar represents 20% sequence divergence per homologous position. Bootstrap values more than 50% of 1000 replicates are shown. Phylotypes retrieved from C. australiensis, seawater and sediment niches were marked with purple, blue, red, and yellow, respectively. The outgroup symbolized by an arrow represented the amoA sequence of Nitrosomonas europaea ATCC 19178 (JN099309). AOA, ammonia-oxidizing archaea Phylogenetic maximum-likelihood tree of parAOB amoA (A) and comAOB amoA (B) transcript phylotypes retrieved from the sponge C. australiensis, ambient seawater, and sediment niches. Scale bar represents 10% sequence divergence per homologous position. Bootstrap values more than 50% of 1000 replicates are shown. Phylotypes retrieved from C. australiensis, and sediment niches were marked with blue and yellow, respectively. The outgroup symbolized by an arrow represented the amoA sequence of Ca.
australiensis, ambient seawater, and sediment niches. Scale bar represents 5% sequence divergence per homologous position. Bootstrap values more than 50% of 1000 replicates are shown. Phylotypes retrieved from C. australiensis, seawater and sediment niches were marked with blue and yellow, respectively. The outgroup symbolized by an arrow represented the hzsB sequence of Planctomycetes bacterium (A3D13_04020). AnAOB, anaerobic ammonia-oxidizing bacteria