Gut microbiota in homologous Chinese soft-shell turtle (Pelodiscus sinensis) under different habitats

Chinese soft-shell turtle (Pelodiscus sinensis) is an important commercial species for its high nutritive and medicinal value, but it has been a vulnerable species due to habitat loss. In this study, homologous juvenile turtles were allocated to lake, pond and paddy eld to investigate the habitat effects on turtles. The growth, morphology and gut microbial communities were monitored during the 4 months cultural period. It showed higher growth rate of turtles in paddy eld and pond. The appearance, visceral coecients, gut morphology and microbial communities in turtles were distinct among different habitats, which was mainly attributed to food abundance and living space. The diversity of gut microbial community was obviously higher in turtles from paddy eld than pond and lake. Signicant differences on dominant phyla, genera and species were found among initial samples and subsequent samples from different habitats. Firmicutes was the most abundant phylum in turtle gut sampled from greenhouse at initial and Proteobacteria was the most abundant phylum after cultivated in different habitats. The functional predictions indicated that both habitat type and sampling time had signicant effects on metabolic pathways, especially amino acid and carbohydrate metabolism.

provide capacious space, shelters and natural food for cultured animals. Cultured animals are able to prey on pest, meanwhile, activities of cultured animals could loosen the soil and provide organic fertilizer for paddies thus signi cantly decrease the utilization of chemical fertilizers and pesticides [24,25]. Therefore, the coculture mode has been considered as an economical and ecological culture mode in rice-growing regions.
Host genetics, diets and ambient environmental conditions could affect the composition of the complex gut microbiota [26,27]. It is di cult to fully disclose the diversity and dynamics of gut microbiota and identify keystone species for speci c functions [28]. In the present study, the homologous juvenile turtles in similar genotype and early life conditions were allocated to different habitats to investigate the difference in growth and morphology and analyze the diversity and variation of gut microbial communities within cultural periods. Efforts were also made to nd some functional microbes or representative communities as bio-markers to evaluate the physiological status of turtles in different habitats.

Turtle growth and morphology under different habitats
The mortality was negligible both in paddy elds and ponds during the experiment period. However, only a small number of turtles were caught from lake at 60d and no marked turtles were recaptured at 120d, resulting in incomplete statistics on mortality and growth for turtles in lake. We selected a small sample size for turtle resource protection purpose and the di cult in sample collection from nature lake, and the differences on growth, physiology and gut microbiota were distinct among different groups. The body weight of turtles in paddy elds and ponds were obviously higher than those in lake (p<0.05) and the divergence occurred in the early days. The growth rates of turtles were 0.76%/d, 0.68%/d and 0.40%/d for habitats of paddy elds, ponds and lake in rst 60d. It was 0.72%/d and 0.62%/d for turtles in paddy elds and ponds during the whole 120d. The hepato-smatic index and clumpy fat index were highest in ponds, secondly in paddy elds and lowest in lake (p<0.05). The gut-smatic index on weight (DSI W ) for turtles from pond was signi cantly higher than lake and paddy eld (p<0.05). Inversely, the gut-smatic index on length (DSI L ) was higher for turtles from paddy eld and lake compared to pond. Measured values are presented as mean ± standard deviation, the different superscript letters in same row indicated signi cant difference(p<0.05)( Table 1).
There was no obvious trauma for most of turtles from lake except occasional leeches parasitic on calipash. Meanwhile, more bruises or scars were observed for the turtles from ponds than paddy elds. The appearance such as color of carapace and plastron were different among turtles in different habitats. The carapace of turtles cultured in ponds presented bottle green, but the individuals from paddy elds presented bottle green with slight golden yellow, which were similar with turtles from lake. There was no signi cant difference on main somatotype index (p>0.05), but the calipash lateral width was relative higher for turtles from ponds than paddy elds (Table 1).

Composition and diversity of turtle gut microbiota
The grouping details for samples from different habitats, cultured days and intestinal segment were listed in Table 2. The gut samples A total of 1 723 158 valid bacterial 16S rRNA gene reads were obtined and 4 901 OTUs were identi ed from all samples. The observed total OTUs varied in 64~822. The total number of OTUs was signi cantly less in initial groups IF and IL, and more in groups F1F and F1L from paddy elds at 60d. The number was 17~48 on OTUs more than 0.01% of total OTUs (Table S1). Signi cant differences were found in OTU composition among groups (Fig S1). Guts sampled at 120d had few unique OTUs, both in former and later part. The alpha diversity was calculated according to the composition and relative abundance of OTUs. Generally, the alpha diversity indices of microbes in later gut were higher than those in former gut. Besides, it was obviously lower in initial turtle guts from hothouse (p<0.05), whereas obviously higher in turtle gut sampled from paddy elds than ponds and lake (Fig.1). The species and number of OTUs varied signi cantly at 60d, different from that relatively harmonious at 120d. The microbial abundance was higher in samples from paddy elds than lake and ponds during the experiment. The microbial community presented relatively high similarity in guts sampled at same time. The PCA (Principal Component Analysis) showed high microbial community similarity in guts from the same individual or group, and signi cant discrepancy in samples from different habitats and sampling time (Fig.2). Generally, both sampling time and living habitats affected the variation of gut microbial communities.

Dominant microbes
The recognized microbes belonged to 27 phyla, 59 classes, 97 orders, 151 families, 219 genera from all the samples based on GreenGene. The phylum and genus level were emphasized in analysis. Bacteroidetes, Firmicutes, Fusobacteria and Proteobacteria were the most dominant phyla, accounting for more than 95% of the total bacteria in all samples. Firmicutes was the most abundant phylum in turtle gut sampled from hothouse at initial, while Proteobacteria was the most abundant phylum after cultivated in different habitats then followed by Bacteroidetes. Firmicutes and Fusobacteria commonly existed at 60d but rarely present at 120d in all the three habitats (Fig.3a). Additionally, the unidenti ed bacteria were more in lake compared to paddy elds and ponds.
There was signi cant difference on dominant genera among initial samples and subsequent samples from different habitats. The dominant genera in initial samples were an unclassi ed genus belonging to Bacteroidales, Romboutsia, Cetobacterium, Weissella, Lactococcus, Lactobacillus, Clostridium, Edwardsiella, Plesiomonas, and Sarcina. As for samples from the mentioned three habitats, the dominant genera were Cetobacterium, Chryseobacterium,Clostridium, Epulopiscium, Flavobacterium, Helicobacter, Pseudomonas, Stenotrophomonas and another unclassi ed genus belonging to Xanthomonadaceae. The abundance of dominant genera varied with different habitats, sampling time and gut location. For turtles sampled from paddy elds, the most dominant genus in foregut sampling at 60d was Clostridium and in hindgut was Cetobacterium, while at 120d, the most dominant genus was Stenotrophomonas both in former and later gut. For turtles sampled from pond, the most dominant genus at 60d was Flavobacterium and Cetobacterium in former and later gut, while at 120d, the most dominant genus was also Stenotrophomonas. For turtles sampled from lake, the most dominant genus at 60d was Flavobacterium and Cetobacterium in former and later gut, respectively (Fig.3b).

Microbial community in different habitats and sampling time
The microbial community was relatively complex at 60d, especially in former gut. At 60d, the species of microbes were signi cantly more in eld, following by pond and lake. There were 140 common species (8.2%) in former gut from the three different habitats (Fig.4a), thereinto, Flavobacterium sp., Pseudomonas sp., Chryseobacterium sp. and two species belonging to Xanthomonadaceae were relatively abundant. Cetobacterium somerae was more abundant in paddy eld than pond and lake. For later gut, there were 205 common species (8.1%) in the three different habitats (Fig.4b). Among these, one specie belonging to Bacteroidaceae was abundant in all habitats. Cetobacterium somerae, Epulopiscium sp., Pseudomonas sp., Stenotrophomonas sp. and Flavobacterium sp. were more abundant in paddy eld and lake than in pond, while Clostridium sp. and Epulopiscium sp. were relatively abundant in pond. Meanwhile, Chryseobacterium sp., Parabacteroides sp., Sphingobacterium faecium, Clostridium perfringens, Pseudomonas sp., Bacteroides sp. and Pseudomonas sp. commonly existed in samples from lake and paddy eld but did not appear in pond. At 120d, the speci c microbes were more in pond (74%) than paddy eld (33.4%) for former gut and the common species accounted for 18.6%, the speci c microbes were more in paddy eld (44%) than pond (34.4%) for later gut and the common species accounted for 26.1% (Fig.S2).
The LEfSe analysis was also conducted to identify representative microbes among various groups. For initial groups, representative genera were Weissella, Cetobacterium, Chryseobacterium, Epulopiscium, Escherichia, Flavobacterium, Lactococcus, Leuconostoc, Plesiomonas, Romboutsia, Sarcina and Stenotrophomonas. For groups cultured in different habitats, it showed F1L contained more species differed from other groups including Cetobacterium, Lactobacillaceae, Bacteroides, Parabacteroides, Plesiomonas and several species belonging to phyla Firmicutes presented higher LDA score. For F1F, representative genera were Sutterella, Bacteroides and Clostridiales. For samples from lake, Xanthomonadaceae and Pseudomonadales were representative genera, especially at 60d. The representative microbes in ponds were numerous and belonging to various phyla, especially the phyla Proteobacteria, also there were some unassigned species were indicated in pond (Fig.S3).

Functional predictions
The nearest sequenced taxon index (NSTI) were developed to quantify the availability of nearby genome representatives for groups (Table S2). Totally 41 predicted functional categories which represented 7 pathway maps in KEGG level 2 were indicated by PICRUSt.
Cultural periods had signi cant effect on metabolism especially the amino acid and carbohydrate metabolism, membrane transport as well as replication and repair. At 60d, the functional microbiota related to amino acid and carbohydrate metabolism was distinct in lake compared to pond and paddy eld (Fig.S4).

Discussion
Turtles had same general microbiota regardless of origin, body size and habitats, and also presented fast adaption after allocated to different habitats [29]. The differentiation on growth, behavior and physiology of the homologous turtles appeared under different living habitats in a short period. Environmental changes can substantially in uence the gut microbiome [30,31]. The differences might be attributed to living space [6], water quality, food composition and abundance [32,33], prey and predation conditions for different habitats [34,35]. Considering the similarity of natural conditions as geographical location, climate, rainfall and temperature for the mentioned three habitats, the food intake and relative living space might be the main factors determining the growth and physiology of turtles in this study [36,37]. Wild turtles are predominantly carnivorous and prey on small sh, mollusks, crustaceans, insects or their larvae, occasionally some plant seeds, but the food abundance was affected by water environment, competitor or enemy [38,39].
In present study, turtles in paddy elds and ponds were regularly fed with arti cial feeds, but no feeds were provided for turtles in lake during the experiment. Besides, gastropods and insect larvae commonly existed as supplementary food in lake and paddy eld, but rarely in ponds [40]. The stocking density in lake was undoubted lower than in paddy elds and ponds and the lake environment was relatively stable with capacious water and less disturbance. Otherwise, more competitors existed in the lake, predators and parasite, but negligible inter-speci c competition [41]. Paddy eld in this study was a complicated habitat of which environmental features were comprised of common eld and pond. The paddy eld provided spacious living space and rice plants served as shelter for turtles. The high growth rate of turtles might be attributed to relative low stocking density in paddy eld and enough food. He et al. (2017) demonstrated the taste of turtles cultured in paddy eld was better than turtles in ponds based on texture and chewiness of meat, which might also due to the broad space of paddy elds for turtle activities [18]. All of these indicated the extensive living space of paddy elds could promote the growth and quality with proper amount of food.
Food and feeding strategy obviously affected the morphology and function of digestive system [42], previous study demonstrated that the relative gut length was shorter in stress conditions such as food shortage [35]. Su cient feeds might enhance digestive function and promote the development of gut at earlier feeding stage, but continuous regular feeding with su cient food might decline the appetite and digestive activities, along with changes of gut morphology and structure [43,44]. The gut presented obvious adaptation to habitat, the relative length of gut was signi cant lower in ponds compared to paddy elds and lake. It might be related to the complicate food composition in lake and paddy eld which increased nutrient absorption and prolonged intestinal transit time [45,46]. Although the turtles in ponds were fed apparently satiation during the experiment, the xed and simple arti cial feeding might not be accorded with ingestion habit for turtles and the food species or types also in uenced the internal environment and gut microbial communities [47].
Gut microbiota was closely associated with host physiological metabolism, nutrients utilization, nutritional status, immunity, even the body health [48,49]. The microbes originally derived from parents, then living environment played important roles in forming procession of gut microbial community and micro-ecological system [21,50]. There were signi cant differences in gut microbial composition under different habitats [51,52]. In general, the microbial population was less diverse in diseased organism compared to healthy ones. The gut microbial species were more in paddy eld and pond than lake at 60d, while the species were fewer and no obvious differences were found among three habitats at 120d. It might due to obvious reduction of the feed intake at 120d. The composition and abundance of gut microbial communities varied under different habitats to adapt the heterogeneous habitats [53]. Food was deemed to a main factor which in uenced the gut morphology, homeostasis and microbiota, which provided nutrients for body, also act as fermentation substrate for gut microbes [54,55]. The microbial gut communities varied a lot when fed with diets in different compositions [56].
Ambient water conditions like temperature and diets change affected microbiome composition [57,58], suitable diet was conducive to improve the intestinal environment and increasing the abundance of probiotics [49]. The PICRUSt functional predictions revealed both the cultural periods (different seasons) and habitats had signi cant effects on metabolism, especially the amino acid and carbohydrate metabolism, which also indicated the key role of food intake on gut microbial community [59]. Meanwhile, gut microbiota would further in uence the metabolic activity of host [60].
Most of previous studies focused on factors that affected gut microbial community, such as genotype, rearing conditions and diets [61][62][63]. However, the causality between microbial community and speci c diseases were ambiguous [64,65]. Healthy individuals often had intricate and stable gut microbial community and the pathogenic bacteria might disturb the homeostasis and microbial balance which may present as reduction of gut microbial species and richness. Adversely, in several recent studies, more bacteria and higher alpha diversity were observed in diseased intestines compared to healthy ones and the richness of bacteria couldn't utterly indicate the health status [66]. The representative microbes which could re ect the balance of microbial communities and contribute to intestinal health should be concerned, and it might also vary in different species or life stage.
For turtles in this study, the dominant phyla were Proteobacteria, Bacteroidetes, Firmicutes and Fusobacteria in different habitats, which were similar with other freshwater sh as crucian carp (Carassius auratus), grass carp (Ctenopharynodon idellus), bighead carp (Hypophthalmichthysnobilis) [67] and marine turtles such as green turtles (Chelonia mydas) [29]. Previous studies indicated there was clear difference in composition between aquaculture reared and wild aquatic animals: in the wild species, Proteobacteria was always the most abundant phylum; whereas Firmicutes were the most abundant phylum in the aquaculture reared species [68,69]. For turtles in this study, it was also found that Firmicutes was the most abundant phylum in turtle gut sampled from greenhouse at initial intensive aquaculture condition, whereas Proteobacteria was the most abundant phylum after cultivated in pond, lake and paddy eld, especially in gut sampled at 120d. It also indicated that gut microbiota of turtles had both intrinsic and distinct environmental characteristic. Aeromonas, Chryseobacterium and Citrobacter commonly existed in European pond turtles kept in breeding centers and there were obvious differences in bacteria composition and abundance for turtles in different ages [70]. The composition and abundance of gut bacteria also varied in different physical status, the virulence and prevalence of pathogens were always suppressed in healthy individuals [71]. Cetobacterium, Cyanobacterium and Clostridiaceae were more abundant in healthy sh, whereas Aeromonas, Vibrio and Shewanella OTUs were more abundant in diseased individuals [72]. Enterococcus spp. and Citrobacter spp. were the dominant bacteria in healthy turtles, while Citrobacter spp., Aeromonas spp. and Bacillus spp. dominated in the diseased ones [73]. Lactococcus garvieae, Citrobacter freundii and Edwardsiella tarda were commonly existed pathogenic bacteria in water environment [74]. In this study, Edwardsiella spp. occasionally existed in samples from ponds, but rarely found in paddy elds and lake. Aeromonas spp. and Citrobacter spp. were almost absent in all samples. Bacillus spp. were more abundant in paddy elds than lake and ponds at 60d. The Pseudomonas spp. widely existed and were rich in most of samples except later gut from ponds at 60d. In addition, the nonpathogenic bacterium as Enterococcus faecium, Enterococcus hirae, Haemophilus segnis, Ochrobactrum anthropi and Pseudomonas spp. could also induce fester of carapace and plastron when the cultural environment became worse. It revealed the relationship between gut microbial communities and body health were not static and the formation of gut microbial community was mutual adaptation with internal and external environment. Therefore, the relationship among microbial communities in gut, cultural water and soil should also be detected to reveal the adaptation for turtles in different habitats. It was necessary to optimize feeding regime and cultural conditions to improve the economic and environmental sustainability of aquaculture. Burgeoning modes as cultured in reconstructive outdoor ponds and paddy elds occurred to replace the hothouse cultivation, especially in later life stage before coming into the market. In this study, turtles cultured in paddy elds presented maximum growth rate. Meanwhile, it could keep the rice yield and increase the value of turtles with remarkable decrease of fertilizers and pesticides utilization. All of these indicated that the co-culture mode was economic and ecological. The co-culture mode could be optimized by reasonable soil, water and fertilizer, especially the nitrogen fertilizer and feeding regime of turtles on basis of digestibility that could minimize nutrient outputs and decline the environmental impacts in intensive culture [75,76]. The rice-turtle coculture was an economic and ecological integrated culture mode which might play important roles in paddy elds environment protection and food security, due to the sharply declined utilization of chemical fertilizer and pesticide compared to traditional planting modes. The mutual promotion for eld environment and turtle health were preliminarily detected in present study but the effectiveness and potentiality should be investigated more systematically in future work.

Conclusion
The juvenile Chinese soft-shelled turtles could adapt to different habitats including natural lake, arti cial ponds and paddy elds. The divergence on growth, appearance, physiological characteristics and gut microbial communities could be observed within a relative short term. The species of microbes were signi cantly diverse in paddy eld than in pond and lake. The diversity and abundance of gut microbes were also higher for turtles from paddy eld than from lake and pond. Signi cant divergence was found in summer, whereas relatively harmonious was detected in late autumn. The abundance of dominant phyla and genera were obviously different in various habitats in speci c sampling time. Sampling time and habitat had signi cant effects on turtle metabolism, especially the amino acid and carbohydrate metabolism. The rice-turtle co-culture was a potential ecological and economic farming mode which would play important roles in wild turtle protection, food security and paddy eld environment improvement.

Experimental habitats and turtles rearing
The turtles (Pelodiscus sinensis, Janpanese strain) were intensive breeding in a standardized farm Xijiang Aquaculture co. LTD, located in Anqing, China. The turtles were stocked at cement tanks in hothouse with relative stable conditions (temperature was 30.0±1.0°C and water depth was about 0.5 m) before being allocated to different experimental habitats. The turtles were fed apparently satiation once a day with commercial feed containing 46% crude protein (Haihuang, Hangzhou, China). Thereafter, thousands of juvenile turtles in similar size of approximately 340 g were purchased and randomly divided into three groups that allocated to different experimental culture habitats as follows. Natural Lake (L): Bohu Lake was located in Anqing, Anhui Province, China (E116°22′, N30°13′), which belonged to the Yangtze River basin. It covered 217 km 2 and the average water depth was about 3.5 m during July to October. It was abundant in sh, shell sh and other aquatic species. Two thousand marked turtles were released to the lake and no arti cial feeds were provided. The arti cial releasing would be conducive to the recovery of wild turtle population.
Arti cial Pond (P): The quadrate arti cial ponds equipped with feeding and basking facilities was located in a standard aquafarm  5m deep), which was about 10% of the total eld area. Two hundred turtles were allocated to each paddy eld. The turtles were fed with commercial feed twice a day same as ponds.The rearing experiment was conducted for 120 days from July to November. Air temperature was monitored at 11:00 AM every day during experiment, which varied in range of 22.5°C~35.8°C. Water temperature, pH and dissolved oxygen were monitored daily with a multi-parameter water quality analyzer (YSI ProPlus, Yellow Springs, Oh, USA). Besides, ammonium nitrogen, nitrite and nitrate nitrogen were measured weekly. Water were partially changed when it became worse for ponds and small ponds in paddy elds. The change interval was about 20 days in summer and 30 days in autumn.

Measurement and sampling
Turtles were randomly collected at initial 0d and 60d, then totally collected as much as possible at 120d. The turtles collected was randomly numbered from different habitats, the investigator who selected individuals for analysis was unaware of the grouping details, and an other investigator (also unaware of grouping details) conducted the anaesthetic and anatomy procedure. Every three male individuals with no trauma, bruises or scars from each habitat and cultural periods were collected for sampling. The turtles were anesthetized after 48h fasting by intramuscular injection with Tiletamine and Zolazepam (1:1) at dosage of 30 mg/kg, the turtles were in deep anesthesia and unconscious within 15-20 min after injection from left foreleg. The somatotype index including body weight, carapace length, carapace width and calipash lateral width were measured. Then turtles were quickly decapitated in unconscious state and dissected by a sharp bone shears, the livers, clumpy fat, guts were carefully removed on ice and weighted under sterile condition. Gut length, i.e. the length from end of esophagus to end of rectum was also measured without external tension. The gastric area (expressed as former gut "F") and rectum (expressed as later gut "L") were separated, rapidly frozen in liquid nitrogen, and then stored at -80°C until DNA extraction for microbial analysis. The grouping details were listed in Table 2 for 3 minutes, followed by 25 cycles of 30 sec at 95°C, 30 sec at 55°C, 30 sec at 72°C with a post-ampli cation extension of 10 min at 72°C. The products were con rmed by agarose gel electrophoresis (Peiqing, Shanghai, China). AMPure XP beads (Beckman Coulter, Indianapolis,IN,USA) and fresh 80% EtOH were used to purify the 16S V4 and V5 amplicon away from free primers and primer dimer species for index PCR. Dual indices and Illumina sequencing adapters were attached by using the Nextera XT Index Kit (FC-131-1002, Illumina, San Diego,CA,USA). Perform PCR on a thermal cycler using the following program: 95°C for 3 minutes, followed by 8 cycles of 30 sec at 95°C, 30 seconds at 55°C, 30 sec at 72°C with a post-ampli cation extension of 5 min at 72°C. AMPure XP beads were used as cleaning up the nal library before quanti cation, normalization and pooling. The puri ed bacterial DNA samples were sent to Sangon Biotech co., Ltd (Shanghai, China) for Illumina Miseq Sequencing.

16S Metagenomics sequencing analysis
Methods were mainly referenced to those mentioned by Campos et al. (2018) and Abdelrhman et al. (2016) [29,77]. The obtained DNA reads were compiled in FastQC version 0.11.5 for further processing. QIIME version 1.9.1 was used for performing microbiome analysis from raw DNA sequencing data, including demultiplexing and quality ltering, OTU picking, taxonomic assignment, and phylogenetic reconstruction, diversity analyses and visualizations. The barcode and primer sequences were cut off after the samples are loaded, read pairs were merged using PANDAseq assembler version 2.10 for raw tags, the pairs sequences would be ltered if there was no overlap between them. And then the chimeras and host sequences were further ltered for clean tags. Singletons were removed before operational taxonomical units (OTUs) clustering (with an identity threshold of 97%). The valid data were clustered into OTU using UPARSE. The rarefaction curves for each sample were produced and diversity values were estimated. The distances among samples were calculated according the abundance and the samples were clustered on OTUs to evaluate the similarity. The cluster dendrogram and a phylogenetic tree were also built. Speci c differences in community composition were determined using PCA based on Bray-Curtis distance matrix. OTUs were taxonomically classi ed using USEARCH (a unique sequence analysis tool) version 5.2.236 against GreenGenes databases and compiled into each taxonomic level. Meanwhile, the composition, abundance and diversity analysis on OTUs were conducted for the species richness and evenness, mutual or proper traits on OTUs for various samples or groups. Test of the signi cance of difference on OTUs composition were conducted LEfSe analysis to nd the various species. The prediction of microbial community function was conducted by using PICRUSt to evaluate the abundance of function genes for samples.

Statistical analysis
All differences among biometric measurements were determined by analysis of variance using SPSS20.0. The measured data were subjected to one-way analysis of variance (ANOVA). Differences among treatments were tested by Tukey's multiple range test and results of p < 0.05 were deemed statistically signi cant. Duncan's multiple comparison was carried out to determine the difference among repeated groups. All statistics on gut microbiota were conducted by using R (version 3.2.2).

Consent for publication
Not applicable.
Availability of data and materials All data generated or analysed during this study are included in this published article [and its supplementary information les]. Raw sequence data on 16 s RNA gene had been submitted to the NCBI Sequence Read Archive (SRA) with the accession number PRJNA639398 (http://trace.ncbi.nlm.nih.gov/Traces/sra/).

Competing interests
The authors declare that they have no competing interests.