a metagenetic approach for revealing community structure of marine planktonic copepods j. hirai,* m....
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A metagenetic approach for revealing community structure of marine planktonic copepods
J. HIRAI,* M. KURIYAMA,* T. ICHIKAWA,* K. HIDAKA* and A. TSUDA†*National Research Institute of Fisheries Science, Fisheries Research Agency, 2-12-4 Fukuura, Kanazawa, Yokohama, Kanagawa236-8648, Japan, †Atmosphere and Ocean Research Institution, The University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa, Chiba277-8564, Japan
Molecular Ecology Resources (2015) 15, 68–80
Keywords: 28S rDNA: D2, 454 genome sequencer, biodiversity, Copepoda, metagenetics
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Introduction
Marine ecosystem Planktonic copepods: Most abundant, wide distributionArthropoda 388-522 Mya, 11500 morphological species (new, cryptic species)
Important: Marine food webs and biogeochemical cycles Indicator: sensitive to environmental changes (natural, anthropogenic stressors)
Þ Study their community structureÞ Understanding and monitoring changes in marine ecosystems
Horizontal distribution: locally studied, not global scalesDue to time-consuming morphological classification, requiring expertsCryptic species, difficulty to identify the immature stages etc.
Solution: DNA-based genetic analysis1) Sanger sequencing-> time-consuming & cost intensive for individual sorting and analysis 2) Metagenetic method-> useful for surveying species richness in metazoans-> MOUTs: molecular operational taxonomic units based on sequence similarity-> independent of morphological classification-> effective tool for rapidly and comprehensively revealing the community structure
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Introduction
DNA barcoding
To facilitate species identification based on similarity to known sequences in DBCommon molecular marker: COI, highly variable 5’ end of COI gene
Copepods: only a few studies used COI as a molecular markerCOI shows high evolutionary rate, difficulty in designing primer in a broad group
rRNA : common genetic marker for metagenetic analysis,having both variable and conserved region, Large subunit rDNA (LSU): more variable, used for species identificationD2 region of LSU (about 350bp): hypervariable region btw conserved regions-> suitable for designing universal primer, LSU region: available sequences in DB: Larger than COIUsed for metagenetic analysis of Haptophyta
In this study,Develop a metagenetic method for revealing community structure of copepods using 454 pyrosequencing Þ Rapid & comprehensive analysis of copepod community structure will be possiblePropose: new, efficient technique for assessing copepod community structure(diversity and distribution of planktonic copepods)
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Materials and methods4 plankton community samples(1 artificial (Art.), 3 field-collected (FC))1. Similarity threshold for MOTU clustering in Art.2. Apply new method to 3 FC samples(showing high species diversity, significant hydrographic variation observed)
* Compare: metagenetic vs. morphological analysis* Evaluation the accuracy of the new method
Artificial community samples33 species(subtropical regions off): Table1.3 orders, 17 families, 27 genera-> sanger/pyrosequencing
Sanger sequencing33 species: first antennaPrimer: LSUCop-D1F (50-GCGGAGGAAAAGAAAACAAC-30)Cop-D3R (50-CGATTAGTCTTTCGCCCCT-30), 1000bp
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Field-collected samples
O-line transect along 138°Subtropical: (31°00.00N, 137°59.90E Kuroshio: (32°54.70N, 138°00.60E)Slope: (33°52.00N, 137°44.00E).
* Vertical sampling- Depth: 0 to 200m / daytime- VMPS- Vertical multiple plankton sampler0.25m2 mouth-opening area100um mesh
* Sample preparation- 99% ethanol, 4°c maintain- After 24hours, replace to new EtOH- aliquots: morphological classifi-cation and metagenetic analysis
* Temperature-Salinity- CTD profiler
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Fig. S1. Vertical profiles of water temperature and salinity at 138°E (0–250 m depth).
Highest Temperature, Salinity
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Pyrosequencing
4 ethanol-preserved samples (one: artificial, ¼ aliquot field collected samples)* Remove all large noncopepod samples using 2mm mesh* Filtered onto 100um mesh
Primer: 400bp w D2 region, highly conserved region; successful amplification of LSU-D2 region in > 100 copepod species
LSU Cop-D2F (50-AGACCGATAGCAAACAAGTAC-30)LSU Cop-D2R (50-GTCCGTGTTTCAAGACGG-30)
Chimera: major cause of overestimation of diversitySolution:- Low number of cycles, - Long extension time, - Low concentrations of template DNA
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Quality filtering
To minimize overestimation of MOTUs
Criteria1. Contained no ambiguous Ns2. Comprised 300-420bp w/o primer sites3. Contained ≤ 5 homopolymers4. No MID adaptor mismatch5. No more than three mismatches per primer6. Average quality score > 277. contained primer sitesbased on LSU sequences of copepods in GenBank
Merge: Forward & Reverse sequences in mothurClassify: based on the Reference data set using the naïve Bayesian classifier > 70%Align: sequences classified as subclass Copepod, MaffFiltering: using single-linkage preclusteringChimeras: remove them using UCHIMEReference Data: copepod sequences w LSU-D2 region in GenBank + 100 LSU-D2 seqs.artificial & field-collected samplesTaxonomic information: Boxshall & Halsey(2004), SILVA
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MOTU analysis of the artificial community sample
Clustered at 95-99% similarity thresholdIn-del region: removed to minimize overestimation of MOTUs in the distant cal. Due to Homopolymer: most common error of 454 pyrosequencingOnly MOTUs w ≥3 Sequence readsMOTUs numbers: calculated for each similarity threshold 95~99%33 reference sequence vs. MOTUs : NJ tree build using MEGA5
No. of sequence reads for MOTU/ DW of the identified reference species in Art.logDW =2.891(Log PL)-7.467
Pearson’s product-moment correlation coefficients(r) 1: positive 0: no correlation -1: negativeBtw proportion of DW and sequence reads using SPSS
Fig. S3. Relationship between per-centage of dry weight and sequence reads at 97% similarity in the artificial community analysis (r = 0.638, p < 0.01).
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MOTU analysis of field-collected samples
Quality-filtered sequence:Slope: 10611, Kuroshio: 6221, Subtropical: 12500 reads: low, many short fragments6221 reads was used for MOTU clustering for comparison of No. of 3 sites MOTUs ?
Clustered into MOTUs (97 similarity threshold)Classified into taxonomic order using a naïve Bayesian classifierCalanoid: classified at family level< 70% threshold : unclassified MOTU
Taxonomic composition of MOTUs(1)A nonbiomass-based approach: use only No. of the MOTUs(2)Biomass-based approach: MOTUs including the No. of sequence reads
Detection of biomass-dominant species: representative sequence of the top 6 MOTUs(those with the highest numbers of sequence reads) and blasted
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Morphological analysis of field-collected samples
Morphological classification: Quantitative sample aliquots for calanoid copepods: depends on the size
Biomass(DW) estimation: logDW =2.891(Log PL)-7.467
Total number of species, biomass of each speciesNumbers of species and total biomass per familyBiomass-dominant species=> Compared with the value obtained from metagenetic analysis
MOTUs from metagenetic analysis vs. LSU-D2 sequences of morphologically identified species
Pearson’s product-moment correlation coefficients(r) Btw sequence reads of MOTUs and biomass
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Results
Artificial community sample analysis33 reference sequences: D2 region, 405-408
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Asymptote: suggesting sufficient sampling coverage
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99%, 98%: overestimation 96%, 95%: underestimation97%: the closest match to the true MOTUs numbers
Nonselected MOTUs: probably present as gut contents of predatory copepods
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MOTU no.
Reads
Best hit Iden-tity
Acces-sion no.
MOTU 1 1415 Pareucalanus attenuatus 99%AB796416
MOTU 2 1028 Subeucalanus subtenuis 100%AB796417
MOTU 3 811 Calanus sinicus 100%AB796406
MOTU 4 591 Euchirella messinensis 99%AB796401
MOTU 5 523 Eucalanus californicus 100%AB796414
MOTU 6 262 Undeuchaeta major 100%AB796403
MOTU 7 187 Centropages sp. 99%AB796413
MOTU 8 93 Paraeuchaeta media 99%AB796418
MOTU 9 88 Euchirella curticauda 99%AB796400
MOTU 10
87 Neocalanus gracilis 99%AB796410
MOTU 11
61 Temora discaudata 99%AB796428
MOTU 13
53 Mesocalanus tenuicornis 99%AB796407
MOTU 14
52Pleuromamma abdomi-nalis
99%AB796423
MOTU 15
39 Pontellina plumata 99%AB796426
MOTU 16
31 Aetideus acutus 99%AB796399
MOTU 17
24 Gaetanus minor 99%AB796402
MOTU 18
18 Candacia curta 100%AB796412
MOTU 19
17 Lucicutia flavicornis 100%AB796419
MOTU 20
11 Corycaeus sp. 99%AB796430
MOTU 21
10 Oithona sp. 100%AB796429
MOTU 23
10 Mecynocera clausi 100%AB796420
MOTU 26
7 Haloptilus sp. 99%AB796405
MOTU 27
7 Paracalanus sp. 99%AB796425
MOTU 30
4 Scolecithrix danae 99%AB796427
MOTU 31
4 Cosmocalanus darwinii 99%AB796408
MOTU 33
3 Calocalanus sp. 99%AB796411
MOTU 34
3 Pareucalanus sp. 99%AB796415
MOTU 35
3 Metridia brevicauda 100%AB796421
Table S1. BLAST results of selected MO-TUs in the artificial community analysis.
Fig. S2. Unrooted NJ tree of the artificial community for comparison between reference sequences of 33 species and MOTUs at the 97% similarity threshold. Scale bar indicates p-distance. Reference sequences obtained by Sanger sequencing are indicated by red circles and rep-resentative sequences of MOTUs are indicated by blue squares. Values in parentheses represent numbers of sequence reads in each MOTU.
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Fig. S3. Relationship between percentage of dry weight and sequence reads at 97% similarity in the artificial community anal-ysis (r = 0.638, p < 0.01).
The number of sequence reads & DW of each speciesÞ Correlation (not strong)
High biomass(DW) tends to contain large num-bers of sequence reads
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Field-collected sample analysis110 copepod MOTUs, 97% similarity70 of these were classified into calanoid copepods73 calanoid copepod species: morphologically identified
MOTU number≒ Species richnessNo. of calanoid MOTUs > Obs. Morphological species
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59.4, 65.6, 63.3% calanoida
11.5-15.6%
3.1-3.3%
16.7-20.3%
59.4-65.6% dominant 3sites
49.9, 85.1, 62.9% calanoida49.3, 11.0, 35.2% cy-clopoida
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Fig. S4. Unrooted neighbor-joining tree of the field-collected samples for comparison between morphological species and MOTUs at 97% similarity. Scale bar indicates p-distance. Sequences of morphologically identified species are indicated by red circles and representative sequences of MOTUs are indicated by blue squares.
64 morphological species-> 47 MOTUs, 97% similarity9 morphological species-> not detected in MOTUs23 MOTUs-> not correspond to Morphological species
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Proportion of sequence reads-> proportion of DWpositive correlation
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Family level species richness Significant correlated with morphological analyses of calanoid copepods in all 3 sites(slope r: 0.691, Kuroshio r=0.878, subtropical r= 0.808)
Fig. 6 Comparison btw metagenetic & morphological analaysis of calanoid copepods
Family level % of sequence reads & DWSignificant correlation with FC (slope r: 0.843, Kuroshio r=0.802, subtropical r= 0.921)Large proportion – High biomass
underestimation
In Kuroshio currentHigh species richness
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The correlation btw No. of sequence reads & DWUsefulness of the number of reads as a proxy for biomass
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99% similarity threshold: high species-level resolution for detection of dominant species
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Discussion
MOTUs (LSU-D2)in metagenetic analysis: Reflect Species compositionProxy for species richness97% similarity: suitable for surveying the community structure of pelagic copepodsusing LSU regions
LSU: simple to design primer,Slow evolution rate: underestimation with insufficient taxonomic resolution97%: high species resolution97% similarity MOTUs clustering: avoid Artificial inflation of diversity; Haptophyta
99%: ideal for species identification, not proper for evaluating species richnessInflation in the No. of MOTUs => small numbers of sequence reads: =>no significant effect on dominant MOTUs
Art.: (Metridia venusta & Oncaea sp.): not detected in the MOTUs, NematodaWhy: Insufficient quantity of template DNA, PCR bias
Gut contents of carnivorous copepods
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Discussion
Discrepancy btw Biomass & No. of sequence reads: primer mismatches, length of amplicons & copy numbers of rRNA
Art. s: - primer mismatch-> PCR efficiency; to minimize mismatch 3’ region of each primer is important- sequence length (OK)- Sequence reads: suggested to be a proxy for biomass, not strong (bias):Correlation btw Biomass & No. of sequence reads in NGS: SSU region study
FC.s:High species richness in the warm, western-boundary Kuroshio Current Copepods diversity: strongly correlated with temperatureHigher diversity: warm oligotrophic oceansHighest species richness: affected by the Kuroshio Current (HT and S)Kuroshio Current: - transport plankton from lower latitudes- Increase species diversity in the western North Pacific
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Discussion
No. of MOTUs > morphological speciesMorphological identified by only adult copepods
Metagenetic analysis: immature stages, possible cryptic species, cut contents=> Higher estimates of species richnessMorphological species with large biomass: successful detection with MOTUs in FC.s.
MOTUs O, Morphological species (X): small sequence reads: rare speciesOther possibility: rare MOTUs = artefacts, pseudogenes, remnants of extracellular DNA in the water
Rarefaction curve: no fully stabilizeNo. of sequence reads ∝ larger numbers of MOUTs
MMGH sample
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Discussion
Proportion of sequence reads Vs. DW: correlated
Discrepancies btw sequence reads & DW: - methodological biases of metagenetic & morphological analysis
MOTU classification at family level (Fig.6)Species richness of taxa: difficult to identify morphologically(ex: Paracalanidae, & Scolecitrichidae): small size, subtle morphological dif-ferences
Hydrographic area: Kuroshio & Subtropical station: Paracalanidae (genus Calocalanus)
Underestimation: Acartiidae (primer mismatches, short sequence lengths, phylogeny)Clausocalanidae (small genetic distance Clausocalanidae & Calanidae)Þ 97% similarity threshold: not good!
This metagenetic anlaysis: optimized in wide range of copepod taxaSolutionDifferent methods for data analysis and different molecular marker should be selected (Acartiidae, Calanidae, Clausocalanidae)
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Discussion
Sequence reads ∝ biomass composition at the family level: (Fig.6,7)
Morphological analysis: time-consuming sorting, dissection
* Metagenetic analysis: all individuals, immature stages, And rapid detection of biomass-dominant taxa, Dominant taxa: valuable insight into the composition of the copepod com-munity: important to understanding copepod community structure and envi-ronmental conditions
C. sinicus, P. parvus : dominant at the Slope stationKnown to Key species and important prey for planktivorous fish in this region
Detect species richness and biomass of small copepods (Oithona, Para-calanus, Clausocalanus – underestimated)99% similarity: proper to detect dominant species
Rapid means of obtaining valuable information on copepod community structureMust be improved, LSU Reference DB accumulationCalanoid copepods (specific ecological characters): classification to the genus level-> easily adapted to field-collected samples on the global scale.