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Discordance in Variation of the ITS Region and the Mitochondrial COI Gene in the Subterranean Amphipod Crangonyx islandicus

Discordance in Variation of the ITS Region and the Mitochondrial COI Gene in the Subterranean Amphipod Crangonyx islandicus
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  Discordance in Variation of the ITS Region and the MitochondrialCOI Gene in the Subterranean Amphipod  Crangonyx islandicus Etienne Kornobis  • Snæbjo ¨rn Pa´lsson Received: 4 April 2011/Accepted: 19 July 2011/Published online: 4 August 2011   Springer Science+Business Media, LLC 2011 Abstract  The amphipod  Crangonyx islandicus  is arecently discovered species endemic to Iceland. Popula-tions of   C. islandicus  are highly structured geographicallyand genetically. The COI and 16S mitochondrial genesconfine six monophyletic groups which have diverged forup to 5 million years within Iceland, and may present twocryptic species. To investigate the potential cryptic speciesstatus we analyse here the internal transcribed spacers(ITS1 and ITS2) and compare its variation with the patternsobtained with the mtDNA. The ITS regions present muchless divergence among the geographic regions in compar-ison with the mtDNA, distances based on ITS1 are corre-lated with the COI distances as well as with geographicdistances, but most of the variation is observed withinindividuals. The variation in the ITS region appears to havebeen shaped both by homogenization effect of concertedevolution and divergent evolution. A duplication of 269base pairs is found in the ITS1 of all individuals from thesouthern populations, its divergence from its paralogappears to predate the split of the different groups withinIceland but some evidence point to rapid diversificationafter the split. This duplication does not affect the sec-ondary structures found in the 3 0 and 5 0 ends of thesequence, suggested to have a role in the excision of theITS1. Compensatory base changes within the ITS2sequences which have been suggested to be a speciesindicator were not detected. Keywords  Crustacea    Ribosomal DNA    Internaltranscribed spacer    COI    Duplication    Populationstructure    Concerted evolution Introduction Crangonyx islandicus  is an endemic amphipod speciesdwelling in groundwater underneath lava fields in Iceland.Analysis of variation in the mitochondrial COI and16S genes has revealed cryptic species diversity within C. islandicus  (Kornobis et al. 2010). The populations arehighly structured geographically and confine at least sixmonophyletic groups which have diverged within Icelandfor up to 5 million years. The geographical patterns of thegenetic variation indicate that  C. islandicus  survivedrepeated glaciation periods in Iceland in sub-glacial refugiaat the tectonic plate boundary, probably facilitated bygeothermal activity. As convergent evolution in morpho-logical traits is common among subterranean species,molecular markers have been widely used for detection of cryptic diversity and species delimitation (e.g., Lefe´bureet al. 2006; Fisˇer et al. 2008). An application of the speciesscreening thresholds on  C. islandicus , developed by Hebertet al. (2004) and Witt et al. (2006), based on the COI variation gave evidence for two provisional species, onewith a wide distribution in Iceland, and a second one foundin northeastern Iceland (Kornobis et al. 2010).The mtDNA COI region has been the main marker usedin the Barcoding Life initiative (see for review: Savolainenet al. 2005). The use of a sole mtDNA marker may lead toan over- or under-estimation of species diversity (e.g., Roeand Sperling 2007), for example the COI gene displayshigh levels of variation even among conspecifics in Electronic supplementary material  The online version of thisarticle (doi:10.1007/s00239-011-9455-2) contains supplementarymaterial, which is available to authorized users.E. Kornobis ( & )    S. Pa´lssonDepartment of Life- and Environmental Sciences, Universityof Iceland, Askja, Sturlugata 7, 101 Reykjavik, Icelande-mail: etk1@hi.is  1 3 J Mol Evol (2011) 73:34–44DOI 10.1007/s00239-011-9455-2  copepod species (Goetze 2003). As single gene trees andspecies trees have shown incongruencies (see Roe andSperling 2007), additional DNA barcodes have been pro-posed such as the ITS2 to identify species and have provedto be highly effective (Yao et al. 2010). The internaltranscribed spacer regions (ITS1 and ITS2) of the ribo-somal RNA have been commonly used as suitable markersfor molecular taxonomy (Tang et al. 2003) includingcrustacean species (Chu et al. 2001; Ota et al. 2010). Criteria based on the variation within the ITS2 region, suchas the occurrence of compensatory base changes (CBC) arehighly informative for species identification for plants andfungi (Mu¨ller et al. 2007). Variation within the ITS1 hasbeen suggested to be suitable for intraspecies analysis andhas been used to assess divergence between populationse.g., in the crustacean  Penaeus japonicus  (Chu et al. 2001).The ITS2 region has been shown to be less informative forintraspecies variation than ITS1 (Areekit et al. 2009), thusthe variation in both regions might be particularly infor-mative for genetic analyses of   C. islandicus  populations.Most rRNA genes in prokaryotes and eukaryotes aresubject to concerted evolution (Dover et al. 1982; Ganleyand Kobayashi 2007, see for review Nei and Rooney 2005), where the member genes are assumed to evolve as an unitin concert within a gene family. Homogenization of themembers of the gene family results from unequal cross-overs and gene conversion (see Nei and Rooney 2005) andaffects the intraspecific variability (e.g., Bower et al. 2008),opposite to the effect of divergent evolution, following adeath–birth process, where duplications or intraspecificparalogs will differ more from each other than interspecificorthologs (Ota and Nei 1994; Ambrose and Crease 2010). The different mechanisms of evolution for the mitochon-drial gene COI and the ITS regions are known to have ledto different patterns in intraspecific variations for variousspecies (e.g., Hansen et al. 2006; Navajas et al. 1998; Carlini et al. 2009). A difference in variation of nuclear andmitochondrial markers is expected due to the haploidy anduniparental inheritance of the latter, resulting in four timessmaller effective population size than for a diploid nuclearmarker. In addition, lack of recombination within themtDNA is expected to reduce the variation due to back-ground selection (Charlesworth et al. 1993) and hitch-hiking (Maynard-Smith and Haig 1974). This difference of mtDNA and nuclear markers may be further augmentedwhen considering a nuclear gene family, with multiplegene copies (Mano and Innan 2008).The aims of this study are twofold: first, to compare thegenetic structures observed in an earlier study for the COIand 16S mitochondrial genes among  C. islandicus  popu-lations to the patterns of variation for the nuclear ITS1 andITS2 genes, and second to evaluate the cryptic speciesdiversity within  C. islandicus. Materials and Methods Molecular Work The nuclear ITS region was amplified using primers ITS1F and 28S R (Table A1 in Supplementary Material) from18 individuals already extracted by Kornobis et al. (2010).Sampling locations of the specimens are presented inFig. 1. PCR amplifications were performed in 20  l l, con-taining 0.15 mM dNTPs, 0.1% tween, 0.5  l g/  l l of BSA,1 9  Taq buffer, 0.35  l M primers, 0.5 unit of Taq(New England Biolabs), and 10–100 ng of template.Amplifications were obtained with the PCR conditions:94  C 4 min, followed by 39 cycles of 94  C 30 s, anneal-ing temperature ranged from 50 to 60  C depending on thesamples, for 45 s, 72  C 1.5 min, with a final elongationstep at 72  C for 10 min. PCR products were cut out of thegel and purified using the Nucleospin Extract II Kit(Macherey–Nagel). They were then ligated to TOPOvector (TOPO TA Cloning Kit, Invitrogen) and cloned inchemically competent cells DH5 a TM -T1 R . Plasmids puri-fied using the Nucleospin Plasmid Kit (Macherey–Nagel)were sequenced in both directions and with internalprimers (Table A1 in Supplementary Material), one to fourclones per individual, using ABI BigDye Terminator v3.1(Applied Biosystems) and ran on ABI PRISM TM 3100Genetic Analyser. Raw sequences were checked and editedusing BioEdit 7.0.9.0. (Hall 1999). Sequences have beensubmitted to GenBank under accession number fromJN258055 to JN258095. 1713221 8411192 591218 A’ABCDEF 24 22 20 18 16 14    6   3 .   5   6   4 .   5   6   5 .   5   6   6 .   5 Longitude (°W)    L  a   t   i   t  u   d  e   (   °   N   ) Fig. 1  Sampling locations of the groundwater amphipod  Crangonyxislandicus  in Iceland analysed in this study.  Numbers  and  capitalletters  refer to sites and different monophyletic groups defined by theCOI and 16S variation in Kornobis et al. (2010). The volcanic activezone is displayed in  gray . Glaciers are in  light gray . Longitudes andlatitudes are shown in degrees on the  x - and  y -axes, respectivelyJ Mol Evol (2011) 73:34–44 35  1 3  Alignment, Annotation, and Secondary StructureComplete sequences were trimmed into separate datasets18S, ITS1, 5.8S and ITS2. 18S, ITS1, and 5.8S wereannotated by using annotations available for the amphipodspecies  Diporeia hoyi , as well as by detecting highly con-served regions (18S and 5.8S) in an alignment encompass-ing other Crangonyctoidean species (in prep.). The ITS2region was defined using the MCMC analysis available atthe ITS2 Database III (Koetschan et al. 2010) choosing theeukaryote model, with maximum  E   values \ 0.1 (since thesuggested value of   E  \ 0.001 did not lead to successfulannotation for all the sequences), and a minimum size of 150 nt (as suggested in the ITS2 annotation software).The ITS1 and ITS2 sequences were aligned usingRNAsalsa 0.8.1 (Stocsits et al. 2009) to produce simulta-neously the alignment and secondary structure for eachsequence. The consensus structure constrain, necessary asinput for a RNAsalsa run, was obtained with RNAalifold(Bernhart et al. 2008). After checking nakedly, no ambig-uous parts in the alignments were found. The highly con-served 18S, 5.8S, and 28S regions were aligned by eye inBioEdit (Hall 1999). The program 4SALE (Seibel et al.2008) was used to plot the consensus secondary structures,and to identify potential cryptic species by searching forCBC in the ITS2 region. Mountain plots (see Hofacker2003) were drawn to compare the different structures.Genetic Diversity, Comparison with COI mtDNADiversity and Population StructureVariability indices (i.e., segregating sites, phylogeneticallyinformative sites, haplotype, and nucleotide diversity) weresummarized using DnaSP v5 (Librado and Rozas 2009),the APE (Paradis et al. 2004), and PEGAS (Paradis 2010) packages implemented in R (R Core Development Team2005). The relationships between ITS1 haplotypes werecharacterized by a reduced median network (Bandelt et al.1995) computed in Network v4.6 (available at http://www.fluxus-engineering.com/sharenet.htm) and redrawn in R.The optimal model of evolution for each dataset waschosen according to the maximum likelihood tree withrespect to substitutions, transition/transversion ratio, pro-portion of invariant sites, and the gamma distributionparameters selected with PhyML (Guindon and Gascuel2003) and implemented in the R-package APE. The AkaikeInformation Criterion (AIC) (Akaike 1974) was used todetect the model which best fitted the data. Two types of genetic distances were calculated, one based on the bestfitted model of nucleotide mutations and a second onebased on differences in number of tandem repeats and indelevents, each considered as a point mutation.The correlation of the ITS1 and ITS2 pairwise geneticdistances, and the genetic distances based on mtDNA COI(Kornobis et al. 2010) were tested with a Mantel test, usingthe ade4 package (Dray and Dufour 2007) in R. In order toavoid bias in the Mantel test due to different samplingeffort between locations, we tested also the correlation on asubset of distances, including solely two clones (whenavailable) per location.To characterize the structure among populations, a stan-dard AMOVA based on pairwise distances and haplotypefrequencies were computed using Arlequin 3.5 (Excoffierand Lischer 2010). The observed variation was partitioned,among mtDNA monophyletic groups, as defined by Korn-obis et al. (2010), among individuals within monophyleticgroup and within individuals. Due to the limited number of samples obtained for ITS1 and ITS2, the only mtDNAmonophyletic groups considered were A, A 0 , D, and E.Results were compared with a corresponding result from anAMOVA based on COI pairwise distances computed inArlequin. To avoid a bias due to larger samples of the COIthan for the ITS1 and ITS2, the AMOVA presents theaverage of an analysis of ten random samples of the COIsequences where the number of sequences is equal to thenumber of sequenced clones obtained for the ITS1. Results Sequence VariationIn total, 41 complete sequences were obtained for the ITS1region and 30 for the ITS2 and 5.8S. Forty partialsequences were obtained from the 3 0 end of the 18S gene.For the mitochondrial genes, a total of 128 sequences of theCOI (658 bp) and 103 of the 16S (414 bp) were previouslyobtained by Kornobis et al. (2010). Diversity indices aresummarized in Table 1 for each DNA region. The ITS1region is highly variable in length, ranging from 363 to672 bp. The length variation is mainly due to a 269 bpduplication (called ‘‘RII’’ in this article) solely present insouthern populations (Tables 1, 2; Fig. 2), and was iden- tified in a dot matrix view using http://blast.ncbi.nlm.nih.gov. Interestingly, the long version of 672 bp of the ITS1contains an open reading frame (ORF) of 107 amino acidslocated in the middle of the sequence (Fig. 2). Minorlength variation is also due to variation in number of TGAtandem repeats surrounding RII and CT repeats (Table 2;Fig. 2). The nucleotide diversity of ITS1 and ITS2 nuclearregions were much less variable than observed for the COIand 16S mitochondrial genes but showed more variationthan the 18S and 5.8S regions (Table 1). The number of haplotypes for the ITS1 and ITS2 is however large com-pared to the sample size and the number of haplotypes 36 J Mol Evol (2011) 73:34–44  1 3  observed for the COI and 16S sequences. The nucleotidediversity within monophyletic groups, defined by COI and16S genealogy (Kornobis et al. 2010), is higher for the ITS1and ITS2 than for the COI (Table 2). Nucleotide diversityamong clones within individuals ranges from 0 to 1.7%.Based on the AIC, the best fit model was the HKY(Hasegawa et al. 1985) for the ITS1 and the F84 (Felsen-stein and Churchill 1996) for the ITS2. The TN93 model(Tamura and Nei 1993), the most similar substitutionmodel to HKY and implemented in R, was used for pair-wise distance calculations for the ITS1.Secondary Structure of the ITS RegionsTwo main secondary structures are observed for the ITS1within  C. islandicus  (Fig. 3) due to the occurrence of the269 bp duplication, otherwise the structures are similar(Fig. 4). The slight differences in length of the helicesobserved in Fig. 4 can be either explained by single pointmutations or variation in the number of repeats in themicrosatellites TGA1 and TGA2. The occurrence of theinsertinthesouthernpopulationisnotaffectingthestructureof the common region RI (Figs. 3, 4). The ITS1 secondary structure presents three conserved helices for all individualsstudied (Fig. 3). Interestingly, two of these helices areoccurring at the 5 0 and 3 0 ends of the ITS1. One CBC wasobserved in the ITS1 sequence comparing one clone fromone individual from northern Iceland to the other sequences.The few variable sites observed in the ITS2 sequencesdo not lead to noticeable changes in the secondary struc-tures predicted by RNAsalsa. The secondary structure of the ITS2 is characterized by two long helices at the 5 0 and3 0 end of the sequence with terminal ramifications. NoCBC were observed among the ITS2 sequences.Geographic PatternsMost of the variation of the ITS1 and ITS2 is found withinindividuals (Table 3), or 59 and 75% for the ITS1 and 82and 93% for the ITS2, depending on how the variation isanalyzed. The difference is larger when the variationamong individuals within groups is also considered. This isclearly different from the results obtained from the mtDNACOI sequences where only 12.55% of the variation iswithin sample locations. However, the geographic struc-ture, observed with mtDNA genes, holds to a certaindegree when a sample size comparable to the one obtainedfor the ITS1 and ITS2 was used for the COI (Table 3).When the indel variation (RII and microsatellites) wereconsidered as point mutations, 23% of the ITS1 variation isamong groups defined by the mtDNA genealogy (Fig. 5).This value is reduced to 8% when the indels were notconsidered. Including indels in the partition of the ITS2variation did not result in larger differentiation amonggroups (Table 3).The pairwise ITS1 genetic distances increase withgeographic distances (Mantel test,  P \ 0.001, see Fig. 6a)but not for the ITS2 ( P  =  0.84). This correlation for theITS1 stands even when the Mantel test was conductedsolely on two clones per location ( P \ 0.001). Although,the ITS1 pairwise genetic distances are much smaller thanthe COI distances they were correlated (Mantel test, P \ 0.05, see Fig. 6b), variation in ITS2 was not relatedto the COI genetic distances ( P  =  0.92). Considerablevariation is observed in the genetic distances of the ITS1for a given COI genetic distance (Fig. 6b) which is partlyexplained by the large variation witnessed within indi-viduals for the ITS1. COI nucleotide variation for thecomplete dataset is more than three times higher than theITS2 nucleotide variation and the proportion of informa-tive sites for the COI is also much higher (Table 1). Thisvariation in the COI led to the identification of cleargeographic groups, whereas in this study most of theclusters are inferred from the ITS1 variations and aremuch less geographically structured (Fig. 7). Although,most of the variation in ITS1 is found within individuals,the genetic variation among ITS1 sequences supportthe pattern of geographic structure observed with the Table 1  Comparison between nuclear rDNA regions and mitochondrial COI and 16S genes18S (41) ITS1 (41) 5.8S (30) ITS2 (30) COI (128) 16S (103)bp 144 363–672 309–311 613–618 658 414 S   4 44 10 53 89 24  IS   0 8 1 2 69 21 p  0.398% 0.746% 0.364% 0.646% 2.146% 1.033% k   5 26 4 24 35 17  H   18.80% 90.10% 14.30% 95.20% 93.30% 90.50%Numbers in parentheses present the total number of clones (nucDNA) or haplotypes (mtDNA) bp  length of the fragment in base pairs,  S   number of segregating sites,  IS   number of phylogenetically informative sites,  p  nucleotide diversity, k   number of haplotypes,  H   haplotype diversityJ Mol Evol (2011) 73:34–44 37  1 3  mitochondrial genes (Figs. 5, 7). The E and F mtDNA groups from the North are distinct from the A, A 0 , B, andC mtDNA groups from the western volcanic zone incentral and southwestern Iceland by a single mutation(Figs. 1, 5, and 7). The occurrence of the 269-bp indel (RII in the ITS1) and the length variation of the tandemrepeats TGA1 and TGA2 supports further the distinctionbetween the northern and southern regions (Table 2;Fig. 1). Except for one individual, the D group from theeastern volcanic zone in southern Iceland is also differentfrom the central and southwestern populations (A, A 0 , B,and C) by a single mutation (Fig. 1, 7). Most of the segregating sites in the other regions, 18S, 5.8S, andITS2, are singletons and their only effect on the network (Fig. 7) are to increase the length of the branches alreadydrawn. 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     5 ,     6     9 ,     1     0 ,     1     1 ,     1     2     7 ,     8     1     1     5     1     0     2     1     0 .     6     4     0 .     4     2     6     3     6     1     1     0 .     6     0     0 .     4     5     1     1     4 .     2     4     6 .     5     2     0 .     2     9     0 .     2     7     A              0      ?      5 ,     6     9 ,     1     0 ,     1     1     6 ,     7     1     0     3     1     0     1     8     0 .     5     7     0 .     4     0     1     0     3     1     0     2     5     0 .     8     6     0 .     4     9     1     0     5 .     2     4     6 .     6     9     0 .     3     4     0 .     3     1     B      ?      6     9 ,     1     0     7     1     1     1     N     A     N     A     N     A     1     1     1     N     A     N     A     N     A     1     1     N     A     N     A     N     A     C      ?      5     9     7     1     1     1     N     A     N     A     N     A     1     1     1     N     A     N     A     N     A     1     1     N     A     N     A     N     A     D      ?      5 ,     6     9 ,     1     0     7     9     4     9     2     2     0 .     7     9     0 .     4     8     3     2     3     6     0 .     6     5     0 .     6     0     9     3 .     0     9     4 .     0     7     0 .     2     3     0 .     2     5     E    -      4     0     7 ,     8     6     3     2     2     0 .     1     8     0 .     2     5     6     3     5     5     0 .     2     7     0 .     3     0     6     2 .     7     8     1 .     9     6     0 .     1     1     0 .     1     9     F    -      4     0     7     3     1     3     8     1 .     2     9     0 .     8     5     3     1     2     2     0 .     2     2     0 .     3     5     3     2 .     2     6     1 .     4     4     0 .     1     6     0 .     3     0     I   n     d   e     l   r   e   g     i   o   n   s   c   o   r   r   e   s   p   o   n     d    t   o   r   e   g     i   o   n   s     i   n     F     i   g .     2 .     R     I     I     i   s   c   o     d   e     d   a   s   p   r   e   s   e   n    t     (      ?      )   o   r   a     b   s   e   n    t     (    -      ) .     T     G     A     1 ,     T     G     A     2   a   n     d     C     T   s     h   o   w   s    t     h   e   n   u   m     b   e   r   s   o     f    t   a   n     d   e   m   r   e   p   e   a    t   s   o     f    t     h   e   c   o   r   r   e   s   p   o   n     d     i   n   g   m     i   c   r   o   s   a    t   e     l     l     i    t   e   s .     T     h   e     C     O     I   v   a     l   u   e   s   p   r   e   s   e   n    t    t     h   e   a   v   e   r   a   g   e   v   a     l   u   e   s   c   a     l   c   u     l   a    t   e     d     f   o   r     1     0     0   s   a   m   p     l   e   s   o     f    t     h   e   o   r     i   g     i   n   a     l     d   a    t   a   s   e    t   w     i    t     h   n   u   m     b   e   r   o     f     i   n     d     i   v     i     d   u   a     l   s   c   o   r   r   e   s   p   o   n     d     i   n   g    t   o    t     h   e   n   u   m     b   e   r   o     f   c     l   o   n   e   s   o     b    t   a     i   n   e     d     f   o   r    t     h   e     I     T     S     1      N       c    n   u   m     b   e   r   o     f   c     l   o   n   e   s   ;      N         i    n   u   m     b   e   r   o     f     i   n     d     i   v     i     d   u   a     l   s   ;      K    n   u   m     b   e   r   o     f     h   a   p     l   o    t   y   p   e   s   ;      S    n   u   m     b   e   r   o     f   s   e   g   r   e   g   a    t     i   n   g   s     i    t   e   s   ;      p    n   u   c     l   e   o    t     i     d   e     d     i   v   e   r   s     i    t   y ,      S     D    s    t   a   n     d   a   r     d     d   e   v     i   a    t     i   o   n   o     f    t     h   e   n   u   c     l   e   o    t     i     d   e     d     i   v   e   r   s     i    t   y R ΙΙ  R Ι ORF18S 28SITS1 5.8S ITS2 TGA1 TGA2 CT Fig. 2  The internal transcribed region of the nuclear rRNA complexin  C. islandicus.  Location of the ITS1 and ITS2 region is shown withrespect to the rRNA genes 18S, 5.8S, and 28S. Regions RI and RIIrepresent a duplicated region. RII is only present in southernpopulations. The ORF region corresponds to a 107-amino acid openreading frame. Microsatellites are displayed in  light gray  and aredesignated as TGA1, TGA2, and CT Fig. 3  The secondary structure of the ITS1 in  C. islandicus . Theconsensus secondary structure obtained with RNAsalsa is displayedfor the northern ( a ) and southern ( b ) populations. The location of thetwo microsatellites is indicated by TGA1 and TGA2. The structuresframed by  broken lines  (numbered 1–3) represent the only threehelices which are conserved among all individuals studied. The asterisk   and the  arrow  indicate, respectively, the 5 0 end of the ITS1and its orientation (5 0 –3 0 ). The duplicated region (RII) in  b  isdisplayed in  light gray 38 J Mol Evol (2011) 73:34–44  1 3
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