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Cyanobacteria from Brazilian building walls are distant relatives of aquatic genera.

Peter M. Gaylarde1, Cesar A.Crispim2, Brett A. Neilan3, Christine C. Gaylarde4*

1

2

Porto Alegre, Brazil

Curso de Pos-graduacao em Microbiologia Agricols e do Ambiente, Universidade Federal do Rio Grande do Sul (UFRGS), Brazil 3

4

University of New South Wales, Sydney, Australia

Dept. Biophysics, UFRGS, Porto Alegre, 91000-970, Brazil. Tel/Fax (+55) 51 336 6029 E-mail [email protected] *corresponding author

Running title: Cyanobacteria on Brazilian buildings

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Abstract

The 16S rDNA from 22 cyanobacteria isolated from biofilms on walls of modern and historic buildings in Brazil was partially sequenced (~350 bp) using specific primers. The cyanobacteria with the closest matching sequences were found using the BLAST tool. The sequences were combined with 52 other cyanobacterial sequences already deposited in public data banks and a dendrogram constructed, after deletion from each sequence of one of the variable 16S rDNA regions (V I). The newly sequenced organisms fitted well within their respective families, but their similarities to other members of the groups were generally low, less than 96%. Close matches were found only with one other terrestrial (hot dry desert) cyanobacterium, Microcoleus sociatus, and with Anabaena variabilis. Phylogenetic analysis suggested that the deletion of the hypervariable regions in the RNA structure is essential for meaningful evolutionary studies. The results support the standard phylogenetic tree based on morphology, but suggest that these terrestrial cyanobacteria are distant relatives of their equivalent aquatic genera and are, indeed, a distinct population.

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INTRODUCTION Phototrophic microorganisms growing on modern and historic buildings cause deterioration of the structure (Gaylarde and Gaylarde, 2004b). A large number of genera are present, and these grow in biofilms (microbial consortia) on stone and painted surfaces (Anagnostidis et al., 1983; Ortega-Calvo et al., 1995; Gaylarde and Gaylarde, 2000). In Latin America, the major biomass on these building surfaces is composed of cyanobacteria (Gaylarde and Gaylarde, 2004a). They lead to discoloration (aesthetic deterioration), as well as degradation of the surface (Warscheid et al., 1991; OrtegaCalvo et al., 1995). Cyanobacteria can survive repeated drying and rehydration cycles (Whitton, 1992) and high UV levels (Garcia-Pichel et al., 1992; Matsunaga et al., 1993; Chazal and Smith, 1994) and for these reasons they are particularly important organisms in biofilms on exposed surfaces in Latin America. Cyanobacteria (previously known as blue-green algae) are prokaryotic photosynthetic microorganisms, which are found worldwide. They have long been recognised as important soil and water organisms, where their activities of nitrogen fixation and toxin production are of special interest. Only more recently has their role in the deterioration of built structures been studied. Traditional methods for the detection of cyanobacteria are based on their culture on specific growth media. These isolation techniques were developed in the area of aquatic microbiology and later extended to terrestrial habitats (soil). They involve enrichment in liquid media, followed by isolation by micromanipulation or culture on solid medium (Rippka et al., 1979). However, these methods can result in the detection of artificially low numbers and diversity, due, in part, to the presence of inhibitory and predatory organisms, such as fungi, bacteria, and protozoa (Gaylarde

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and Gaylarde 2000). Many cyanobacterial species from dry environments are lost in culture because of the activity of browsing and inhibitory organisms, and, of course, many microorganisms present in the environment are not detected by current culture techniques (Ward et al., 1990). Although 16S rRNA gene sequencing has been used to study the relationship between cyanobacteria (Wilmotte et al., 1993; Nelissen et al., 1996; Turner, 1997), there has been practically no work on the molecular biology of cyanobacteria on buildings. Tomaselli et al. (2000) used amplified ribosomal DNA restriction analysis (ARDRA) on axenic cyanobacterial isolates from Italian monuments and constructed a dendrogram that provided some idea of biofilm diversity. This approach did not, however, overcome the problem of selective culture of organisms from a mixed population. Crispim et al. (2003) and Gaylarde et al. (2004) described a method to identify cyanobacteria in mixed biofilms on external walls of historic buildings that does not rely on isolation and this is the approach used here to examine the phylogenic relationships between these biofilm and other cyanobacteria.

MATERIALS AND METHODS Sampling and microbiological analysis. Samples of biofilms were taken from the external surfaces of modern and historic buildings in Porto Alegre, Ouro Preto, Tiradentes and São Paulo, Brazil, using the adhesive tape method (Gaylarde and Gaylarde, 1998; Shirakawa et al., 2002). All surfaces showed visible discoloration, generally grey/black, green, or orange/red in appearance. For microbiological analysis, tape samples were placed directly on solid Modified Knop’s Medium (MKM; Gaylarde and Gaylarde, 2004) and incubated at 25oC in an illuminated BOD incubator. Some organisms were isolated in liquid MKM by repeated subculture after an initial growth period on solid medium.

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Identification. Microorganisms in rehydrated biofilms (4h after contact with MKM) and in culture were identified by standard morphological methods (Holt et al., 1994; Castenholz, 2001). Cyanobacteria were also identified by molecular techniques, as briefly described in the next sections (Gaylarde et al., 2004). DNA extraction. Genomic DNA was extracted from some cyanobacterial isolates using lysozyme/proteinase K/SDS for lysis, followed by a phenol/chloroform/isoamyl alcohol extraction (Gaylarde et al., 2004). Extracted DNA was amplified using the PCR parameters described below. DNA amplification For the “direct” PCR, microcolonies or filaments from the rehydrated, or briefly incubated, biofilms and well separated filaments isolated from liquid medium were placed in 10 µl of TE in an Eppendorf and freeze-thawed five times in the freezer compartment of a domestic refrigerator (-18oC) and at room temperature (23oC). After the final thawing, dNTPs, primers, MgCl 2, buffer and Taq DNA polymerase were added directly in the appropriate amounts (Gaylarde et al., 2004) to the Eppendorf to give a final volume of 25 µl. The PCR reaction was carried out in an Applied Biosystems GeneAmp System 2400 (PE Applied Biosystems, Fullerton, CA) with the following programme: 92oC for 2 min, 30 cycles of 92 oC for 20 s, 55oC for 30 s, 72oC for 50 s and a final extension at 72 oC for 2 min. The products were visualized by electrophoresis in 1% agarose gels using ethidium bromide staining. Primers. 16S rRNA primers designed by Neilan et al. (1997, 2002) were used. The forward primer, 27F1, is a universal bacterial primer with the sequence 5'-AGAGTTTGATCCTGGCTCAG-3', based on primer 27F (Giovannoni, 1991) and is identical to A2 (Iteman et al., 1995). The reverse primer, 408R, is specific for cyanobacteria and has the sequence 5'-TTACAAYCCAARRRRCCTTCCTCCC-3'.

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These primers produce amplified fragments of around 400 bp. Primers were used at a concentration of 10 pmol/25µl PCR reaction. DNA sequencing. Purified PCR products were sequenced with the reverse primer 408R using Big Dye terminator technology (PE Applied Biosystems) and the sequencing gels were run and analysed by the University of New South Wales Genomic Analysis Facility. In some cases, both primers were used separately for sequencing. The consensus sequences were submitted to the BLAST search tool (http://www.ncbi.nlm.nih.gov/BLAST) and nearest matches and those conforming to the morphological appearance of the cells were recorded. Dendrograms were constructed for the sequences, along with others of similar groups selected randomly from public databases, using the CLUSTAL-X program (Higgins et al., 1992) and bootstrap with 1000 resampling events. An unrooted tree was constructed using a heuristic approach (Swofford et al., 1996). Close inspection of the sequences revealed that the hypervariable region, VI, differed not only in sequence but also in length and interfered with the CLUSTAL alignment. This section was therefore removed, the dendrogram reconstructed and the BLAST analyses repeated using these modified sequences. RESULTS AND DISCUSSION The cyanobacteria from our samples, and their similarities with public database organisms, are shown in Table 1. The results are shown with and without the sequence before base 101 (E. coli position), which includes VI (see Figure 1). Analysis of the secondary structure indicated that the bases that did not match deposited sequences were concentrated in variable region I. Since the alignments performed by the CLUSTAL-X program failed to retain the complementarity of the sequences where differences in length of VI occurred, the subsequent dendrogram was

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determined by bootstrapping after deletion of VI and the preceding part of the sequence. The dendrogram is shown in Figure 2. The percentage match of sequences with those of other organisms in the public databases increased in all cases after this modification. Our results demonstrate that the removal of the V I region of the genome improved the similarity levels of the new sequences to those in public databases. Some workers (Stackebrandt et al., 1991; Heuer et al., 1999) have suggested that phylogenetic studies should be carried out on partial sequences of the 16S rRNA variable regions, since they represent areas where evolutionary change may take place in an otherwise exceptionally conserved genome. Others, however, recommend the removal of the variable regions after their identification with dedicated programs (Crosbie et al., 2003). Cyanobacteria are notable for the short length of their 16S rDNA genome compared to other eubacterial groups; substantial deletions between E. coli positions 68 to 101, 197 to 220 and 452 to 480 are consistently present. However, it is only in the case of region 68 to 101 (VI) that the length of the deletion is variable (Figures 3a-c). The length of VI does not vary systematically between aquatic and terrestrial cyanobacteria; however, several of the terrestrial organisms have very distinct sequences in this region, not found in related, but non-terrestrial, genera. We suggest that variable regions, if they have any utility, should be used only for phylogenic studies at the genus level, especially since alignment methods do not work for regions of variable length. The dendrogram produced from the modified sequences (Figure 2) shows that the 16S rRNA-based phylogeny is broadly in agreement with accepted phylogenies based on morphology and that the cyanobacteria from buildings group well with their aquatic

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neighbours at the family or genus level. The small number of organisms of subgroup I (Chroococcales) are scattered throughout the diagram, showing that morphological taxonomy is inadequate to treat this group realistically. Turner (1997) stated that the genus Synechococcus is polyphyletic, emphasising this point. Epilithic cyanobacteria (including those from painted stone surfaces) must be well adapted to survive frequent desiccation, high salt levels and extremes of temperature. The epilithic environment must be considered as a very distinct ecological niche. Terrestrial cyanobacteria from painted or unpainted stone surfaces in Brazil form a distinct population that differs from the better-studied aquatic members of this group. Major differences include the fact that cyanobacteria found on solid surfaces include no members of genera in which the production of gas vacuoles is considered a fundamental character and that in the tropics (or at altitude) the cells frequently have thick sheaths, which, like the cell cytoplasm, may be heavily pigmented. Figure 4(ad), in the web-based supplementary material, shows this feature, as well as the range of morphological variation of Scytonema spp. ccg16, ccg25 and ccg26, which show a very close relationship and cluster tightly in the dendrogram. These Scytonema spp. differ from other environmental organisms that cluster with Scytonema hofmannii in that dark pigments are present in the cell cytoplasm, false branches are more common and consistently geminate and on media rich in nitrogen heterocysts are frequent. A thick, pigmented sheath is normal in both groups in the environment and this usually becomes thin and colourless in culture. A similar grouping, with a tight cluster of terrestrial strains of Nostoc, is seen in the dendrogram; these sequences are rooted in the same branch as deposited sequences of Nostoc, Anabaena and Nodularia, but are clearly distant.

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One of our isolates most closely matched the M. sociatus 16S rDNA short sequence deposited by Garcia-Pichel et al. (2001); however it was identified morphologically as Plectonema (Table 1). Boyer et al. (2003) studied 31 isolates of Microcoleus from desert crusts and found the M. vaginatus clade was most similar to published sequences from Trichodesmium and Arthrospira. They concluded that the genus requires revision and our result emphasises this. The M. vaginatus morphotype isolated from terrestrial environments must be regarded as a very diverse group with a highly conserved 16S rDNA sequence. This analysis once again highlights the problems of attempting to use morphological features for phylogenic analysis, since organisms with very close 16S rDNA sequences may have very different appearances. Almost all of the cyanobacteria show changes in morphology in response to environmental conditions, and in the case of the heterocystous cyanobacteria and the baeocyte-forming groups, this morphological variation is always large. We recognise Plectonema as any filamentous, non-heterocystous cyanobacterium, growing within a sheath, which produces false branches at some stage of growth. Since the branching character includes a wide range of stable morphotypes, defined by the width and length of the cells, their shape and colour, this is almost certain to represent several genera, which will be phylogenically distant and, indeed, the existence of the genus Plectonema has been questioned (Castenholz, 2001). The dendrogram shows that these terrestrial cyanobacteria fit well to the taxonomic positions of related genera, but also shows that their genetic distance from other members of their morphologically identified genera is large. The table confirms this result. The physiological adaptations for survival in the dry environment of terrestrial surfaces would lead one to expect the emergence of novel taxa. The ribosomal

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genome of terrestrial cyanobacteria is distinct from those of related aquatic genera and this indicates prolonged isolation of the two groups.

Acknowledgements We wish to thank the Brazilian agency CNPq for funding for materials and a postgraduate grant to CAC. BAN thanks the Australian Research Council for financial support.

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NEILAN, B.A., BURNS, B.P., RELMAN D. and LOWE, D.R. (2002). Molecular identification of cyanobacteria associated with stromatolites from distinct geographical locations. Astrobiol. 2, 271-280. NELISSEN, B., DE BAERE, R., WILMOTTE, A. and DE WACHTER, R. (1996). Phylogenetic relationships of nonaxenic filamentous cyanobacterial strains based on 16S rRNA sequence analysis. J. Mol. Evol. 42, 194-200. ORTEGA-CALVO, J.J., ARINO, X., HERNANDEZ-MARINE, M. and SAIZ JIMENEZ, C. (1995). Factors affecting the weathering and colonization of monuments by phototrophic microorganisms. Sci. Total Environ. 167, 329-341. ORTEGA-MORALES, O., GUEZENNEC, J., HERNANDEZ-DUQUE, G., GAYLARDE, C.C. and GAYLARDE, P.M. (2000). Phototrophic biofilms on ancient Mayan buildings in Yucatan, Mexico. Current Microbiol. 40, 81-85. RIPPKA, R., DERUELLES, J., WATERBURY, J.B., HERDMAN, M. and STANIER, R.Y. (1979). Generic assignments, strain histories and properties of pure cultures of cyanobacteria. J. Gen. Microbiol. 111, 1-61. SHIRAKAWA, M.A., GAYLARDE, C.C., GAYLARDE, P.M., JOHN, V. and GAMBALE, W. (2002). Fungal colonization and succession on newly painted buildings and the effect of biocide. FEMS Microbiol. Ecol. 39, 165-173. STACKEBRANDT, E., WITT, D., KEMMERLING, C., KROPPENSTEDT, R and LIESACK, W. (1991). Designation of streptomycete 16S and 23S rRNA-based target regions for oligonucleotide probes. Appl. Environ. Microbiol., 57, 1468-1477 SUZUKI, M., RAPPE, M.S. and GIOVANNONI, S.J. (1998). Kinetic bias in estimates of coastal picoplankton community structure obtained by measurements of small-subunit rRNA gene PCR amplicon length heterogeneity. Appl. Environ. Microbiol. 64, 4522–4529.

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SWOFFORD, D.L., OLSEN, G.J., WADDELL, P.J. and HILLIS, D.N. (1996). Phylogenetic inference. In: Molecular Systematics, Hillis, D.M., Moritz, C. & Mable, B. (eds.), Sinauer Ass. Ltd., Sunderland, MA, Chap. 11, pp. 407-514. TOMASELLI, L., LAMENTI, G., BOSCO, M. and TIANO, P. (2000). Biodiversity of photosynthetic microorganisms dwelling on stone monuments. Internat. Biodet. Biodeg. 46, 251-258. TURNER, S. (1997). Molecular systematics of oxygenic photosynthetic bacteria. Plant Syst. Evol. Suppl. 11, 13-52. WARD, D.M., WELLER, R. and BATESON, M.M. (1990). 16S rRNA sequences reveal numerous uncultured microorganisms in a natural community. Nature 345, 6365. WARSCHEID, T., OELTING, M. and KRUMBEIN, W.E. (1991). Physico-chemical aspects of deterioration process on rocks with special regard to organic pollution. Internat. Biodet. 28, 37-48. WHITTON, B.A. (1992). Diversity, ecology and taxonomy of the cyanobacteria. In: Mann, N.H., Carr, N.G. (Eds.), Photosynthetic Prokaryotes, Plenum Press, New York, pp 1-51. WILMOTTE, A., VAN DER AUWERA, G. and DE WACHTER, R. (1993). Structure of the 16S rRNA of the thermophilic cyanobacterium Chlorogloeopsis HTF (Mastigoclaudus laminosus HTF') strain PCC 7518 and phylogenetic analysis. FEBS Lett. 317, 96-100.

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Legends for figures Figure 1 The structure of E. coli 16S rRNA from base 28 to base 408 is shown with variable regions VI and VII marked. The simplified structure is modified from Cannone et al., (2002), in such a way that the E. coli 16S rRNA will show variation with respect to cyanobacteria; the modification has been made with reference to all bacterial sequences. Suzuki et al. (1998) state that VI runs from 72 to 101, inclusive, whereas our observations show that it runs from 69 to 100 in all cyanobacteria. The pairing between bases at positions 69 and 99 is generally conserved in cyanobacteria. VII runs from 176 to 221 (Suzuki et al., 1998). Two helical regions, denominated Helix 1 (136 to 142 and 221 to 227, inclusive) and Helix 2 (154 to 156 and 165 to 167, inclusive), show marked variability, in which, nevertheless, complementarity is highly conserved. Within VII, bases 195 to 197 are conserved in all the bacteria from deposited sequences that we have examined; this region is frequently complimentary to 180-182 in cyanobacteria. Two regions, V IIa and VIIb, show features characteristic of cyanobacteria. VIIa runs from 183 to 193 and its variability is very large. VIIb (198 to 205 and 214 to 219) is deleted in cyanobacteria. This same deletion is shared with bacteria of the genus Clostridium and its length varies in other bacterial genera. Wide bars show canonical pairing (G:C and T:U), narrow bars show G:U pairs and dots mark other non-canonical pairs which contribute to the secondary structure. Lines mark the E. coli positions, which are numbered at every tenth base.

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Figure 2 Dendrogram constructed from sequences starting at E. coli position 101. The position of Leptolyngbya sp. ccg40 is marked to indicate that this short sequence clusters with its nearest relatives, but that the genetic distance is increased because of the short sequence used to give the match. This indicates that bootstrapping is robust for sequences of variable length, but shows that the degree of relatedness cannot be estimated when such variation is inherent. In the coloured diagram (supplementary web material), black = subgroup I, red = subgroup II, blue = subgroup III and green = heterocystous cyanobacteria. The colours group well together, even though the stems of the tree do not always give the same message.

Figure 3 This figure shows the variation in length of V I in cyanobacteria. The diagrams show that when helical regions are present they are well paired. 3a. Nostoc ccg19, showing a VI helix of typical cyanobacterial length. 3b. A Phormidium murrayii sequence, showing minimum sequence length 3c. The longest V I helix found, Nostochopsis lobatus.

Figure 4 (Coloured photos available in supplementary Web material) Photomicrographs showing Scytonema ccg25 (4a), Scytonema ccg16 (4b) and Scytonema ccg26 (4c and d), all grown on MKM. The bar markers are 10µm.

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Caption for Table 1 This summarises the morphological identity of the organisms sequenced and the nearest matches of the sequences to deposited organisms in the BLAST database. The nearest matches to the morphological identifications are also given. The similarities are for the full sequence and for the short sequence from E. coli position 101 onward, together with the percentage similarities with and without the sequence preceding base 101.

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Figure 1

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Figure 2

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Figure 3 (a)

(b)

(c)

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Figure 4

(a)

(b)

(c)

(d)

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Table 1 Code

Town

Nearest match(es)

ccg08 ccg10 ccg13 ccg 16

Morphological identity Lyngbya Nostoc Nostoc Scytonema

POA OP OP POA

ccg 19

Nostoc

SP

ccg 23

Tolypothrix

POA

ccg 24

Tolypothrix

POA

ccg 25

Scytonema

POA

ccg 26

Scytonema

POA

ccg 28

Lyngbya

POA

ccg 29

Plectonema

POA

ccg 32 ccg 34 ccg 38

Plectonema Leptolyngbya Lyngbya

POA POA POA

ccg 40 ccg 44

Leptolyngbya Tolypothrix

OP POA

ccg 46

Subsection II

T

ccg 48

Chlorogloeopsis OP

ccg 49 ccg 50

Nostoc OP Chlorogloeopsis OP

ccg 51 ccg 52

Leptolyngbya Leptolyngbya

Phormidium murrayii Nostoc sp. PCC 9229 Nostoc sp. Mastigocladopsis repens MORA Nostoc sp. 8941 Scytonema sp. IAM M-262 Anabaena variabilis Nostoc sp. PCC 7120 Nostoc sp. PCC 7120 Tolypothrix sp. IAM M-259 Nostoc sp. PCC 7120 Tolypothrix sp. IAM M-259 Mastigocladopsis repens MORA Nostoc sp. 8941 Scytonema sp. IAM M-262 Mastigocladopsis repens MORA Nostoc sp. 8941 Scytonema sp. IAM M-262 Leptolyngbya sp. PCC 73110 L. foveolarum Microcoleus sociatus MPI 96MS.KID Phormidium sp. ETS-05 Plectonema boryanum Leptolyngbya sp. Phormidium murrayii Symphyonema sp. 1269-1 Leptolyngbya PCC7104 Cyanospira rippkae Tolypothrix sp. IAM M-259 Chroococcidiopsis sp. BB96.1 Chroococcidiopsis sp. BB79.2 Nostoc sp. 'Azolla cyanobiont' Chlorogloeopsis sp. PCC 6718 Nostoc sp. PCC 9229 Nostoc sp. 'Azolla cyanobiont' Chlorogloeopsis sp. PCC 6718 Leptolyngbya PCC7104 Leptolyngbya PCC7104

OP OP

Short sequence Full sequence Difference between Matches Mismatches Matches Mismatches mismatches 253 11 282 17 6 254 8 303 15 7 255 7 308 16 9 246 16 n.a. n.a. n.a. 244 18 296 23 5 240 22 292 27 5 251 2 305 3 1 247 6 299 11 5 259 13 310 19 6 250 22 301 28 6 249 13 300 19 6 240 22 291 28 6 243 13 n.a. n.a. n.a. 241 18 293 23 5 237 22 289 27 5 257 14 n.a. n.a. n.a. 255 16 307 21 5 251 20 303 25 5 208 22 258 29 7 207 23 257 30 7 270 13 n.a. n.a. n.a. 266 16 316 23 7 225 22 276 28 6 252 8 308 11 3 241 10 272 14 4 229 22 278 31 9 231 12 244 14 2 251 12 303 18 6 243 20 294 26 6 246 17 n.a. n.a. n.a. 245 19 297 24 5 268 12 319 18 6 259 21 310 27 6 251 3 297 12 9 268 13 319 19 6 259 22 310 28 6 268 7 321 9 2 263 11 315 14 3

Percentage match Short Full 95.8 94.3 96.9 95.3 97.3 95.1 93.9 n.a. 93.1 92.8 91.6 91.5 99.2 99.0 97.6 96.5 95.2 94.2 91.9 91.5 95.0 94.0 91.6 91.2 93.8 n.a. 93.1 92.7 91.5 91.5 94.8 n.a. 94.1 93.6 92.6 92.4 90.4 89.9 90.0 89.5 98.9 n.a. 94.3 93.2 91.1 90.8 96.9 96.6 96.0 95.1 91.2 90.0 95.1 94.6 95.4 94.4 92.4 91.9 93.5 n.a. 92.8 92.5 95.7 94.7 92.5 92.0 98.8 96.1 95.4 94.4 92.2 91.7 97.5 97.3 96.0 95.7

POA = Porto Alegre; OP = Ouro Preto; SP = Sao Paulo; T = Tiradentes; n.a. = not available (short sequence deposited in data bank)

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