mussel ( m. trossulus ) specimens 5 to 6 cm long were collected from the intertidal zone at low tide ( noon ) on april 18 , 2015 at copper beach ( n 49 2241 w 123 they were transported to the laboratory in aerated seawater from copper beach . in the laboratory , 0.5 one drop of hemolymph was placed on a poly - l - lysine - coated slide and let sit for 1015 min to allow the cells to spread and attach to the slide . thereafter , the slide was viewed under a zeiss axiostar light microscope at 40 magnification . normal ( non - neoplastic ) specimens were those with greater than 90% cells with normal appearance , i.e. agranular or granular hemocytes with spread pseudopodia . fully leukemic ( diseased ) specimens were those with prolific amounts ( > 90% ) of round , non - adherent cells . hemolymph was added to 1.5 ml eppendorf tubes and spun at 900 g for 3 min . after the supernatant was withdrawn , the cells were re - suspended in absolute ethanol before shipping . excised tissues ( mantle , foot , and gills ) were preserved in absolute ethanol before shipping . of 28 individuals collected from west vancouver , two had high levels of neoplastic disease . a second set of m. trossulus samples were collected from esquimalt , vancouver island , b.c . , hemocyte and solid tissue samples were fixed in ethanol before dna extraction . of 250 individuals collected from esquimalt , nine were scored as potentially moderately or highly diseased , with seven samples available for analysis . cockles ( c. edule ) were collected from an intertidal bed named o sarrido ( 42 30 n , 8 49 w ) and golden carpet shell clams ( p. aureus ) and pullet shell clams ( v. corrugata ) were collected from a subtidal bed named o bohdo ( 42  32 n , 8 51 w ) ; both shellfish beds are located in the ria of arousa ( galicia , nw spain ) . once in the laboratory of cima , each cockle and clam was notched through the shell margin close to the posterior adductor muscle and hemolymph ( as much as possible ) was collected from the posterior adductor muscle using a 2-ml syringe with a 21 gauge needle . a small quantity ( ca . 100 l ) of hemolymph was used to produce a cell monolayer onto a slide by cytocentrifugation ( 92  g , 5 min , 4c ) , which was fixed , stained with hemacolor ( merck ) kit and examined with light microscopy for diagnosis of disseminated neoplasia ; the remaining hemolymph was preserved in absolute ethanol for molecular analysis . after hemolymph collection , molluscs were shucked and a small piece of mantle ( ca . 5  5 mm square ) was removed and preserved in absolute ethanol for molecular analysis ; additionally , an approximately 5 mm thick section of meat , containing gills , visceral mass , foot and mantle lobes , was fixed in davidson s solution and embedded in paraffin . neoplastic cells ( both a and b types ) had a unique morphology , and were clearly distinguished from normal cells , with much larger overall size , and much larger nucleus . the longest diameters of the cell and the nucleus of at least 10 neoplastic cells for each individual were measured by direct examination of histological sections with light microscopy using a reticle . two - tailed t - tests were used for comparisons of morphometric data from different individuals and types . cockles ( c. edule ) and golden carpet shell clams ( p. aureus ) were ranked according to a scale of disease severity based on hemolymph diagnosis : non - affected ( n0 , or n ) ; low severity ( n1 , or l ) , when individuals showed proportions of neoplastic cells lower than 15% in the hemolymph cell monolayers ; moderate severity ( n2 , or m ) , when the proportion ranged from 15% to 75% ; and high severity ( n3 , or h ) , when the proportion was higher than 75% . 74 golden carpet shell clams ( p. aureus ) were collected with 12 diagnosed with light , 10 with moderate , and 9 with heavy neoplasia . about 150 cockles ( c. edule ) were collected and a subset was analyzed for both disease and morphological type of neoplastic cells . of the 30 in this subset , two had type a neoplastic cells ( one light and the other moderate ) and one had low levels of type b neoplastic cells . we have also collected and analyzed hundreds of pullet clams ( v. corrugata ) from multiple beds throughout galicia from 1988 to the present time , including about 100 from the same bed in which the samples of p. aureus were collected for this study ( o bohdo ) . to this date , we have not observed any v. corrugata samples with neoplastic disease . one v. corrugata individual from a different location has been identified by a different researcher as harboring disseminated neoplasia , with a different morphology than that observed in p. aureus neoplasia ( personal communication , susana darriba , at intecmar ) . dna was extracted from ethanol - fixed hemocytes using dneasy blood and tissue kit ( qiagen ) . dna extraction of tissues used the same kit , but included an additional step in order to reduce the amount of pcr - inhibiting polysaccharides . after tissue lysis , 63 l of buffer p3 was added to lysate and allowed to precipitate for 5 min . lysate was spun 10 minutes at full speed at 4c , and the resulting supernatant was mixed with ethanol and added to the column , continuing with the standard protocol . pfuultra ii fusion hs dna polymerase ( agilent ) was used to amplify 10 ng of genomic dna for 35 cycles . extension was at 72c for 15 sec for mtcoi for all bivalves . for p. aureus , its was amplified ( using primers modified from ) with extension for 30 sec . despite multiple copies of its in genomic dna , a single sequence was observed for each normal p. aureus and v. corrugata , and a single pair of host / neoplasm sequences was observed in each diseased individual . pcr for ef1 followed the same program , with extension for 20 sec in mussels ( m. trossulus ) and clams ( p. aureus and v. corrugata ) and 1 min 30 sec in cockles ( c. edule ) . for amplification of steamer - like elements , a degenerate primer pair was designed to match conserved regions in reverse transcriptase and integrase of the ltr - retrotransposon , steamer , and 10 ng of dna was amplified for 35 cycles , annealing at 55c for 20 sec and extending for 1 min at 72c . when multiple alleles could not be resolved by direct sequencing , pcr products were cloned using the zero blunt topo kit ( invitrogen ) , and at least 6 colonies were sequenced . primer - binding regions were excluded from analysis and are not included in the sizes listed as sequenced . maximum likelihood phylogenetic trees were generated using phyml 3.0 , using the hky85 substitution model , with 100 bootstrap replicates , treating gaps in the alignment as missing data . trees were visualized using figtree v1.4 , with addition of markers at the branch termini . each phylogenetic tree based on alignment of sequence at a single locus ( figure 1c , 2e , 3b d ) includes a scale bar which shows genetic distance ( 01 ) based on the frequency of nucleotide divergence . microsatellites were amplified using primers for 12 loci , reported previously to be polymorphic in cockle ( c. edule ) populations . of the 12 primer pairs , 9 pairs amplified products in all cockle dna samples , with 12 alleles observed in normal samples and 14 neoplastic alleles observed . these were used in all further analyses , with fluorescent modifications on the 5 end of the forward primers as listed ( extended data table 2 ) . kod polymerase ( millipore ) was used to prevent ambiguity of untemplated addition of a residues by taq polymerase . allele sizes were identified to single - base precision by fragment analysis using florescent primers ( 6-fam , pet , ned , and vic ) using a 3730xl genetic analyzer with the liz-500 size standard , ( applied biosystems , operated by genewiz ) . peaks were called using peak scanner 2.0 ( applied biosystems ) , rounding sizes to whole bases , with some manual adjustment to keep alleles of the same size together . we used the r package poppr to generate distance matrices and phylogenetic trees using the bruvo s method with the infinite alleles model . the value of c ( repeat size ) was set to 0.00001 in order to calculate distances using the band sharing model , in which alleles are either identical sizes ( distance=0 ) or non - identical ( distance=1 ) . each allele was coded as a dominant marker , with a single variable for each allele observed at each position ( 104 total markers from 9 loci ) . for each allele ( a single size at a single locus ) , a sample is observed to be either present , absent , or the information is missing . for example , a sample with sizes 195 and 199 at locus a would be marked as  present  for marker a-195  and  a-199 ,  but  absent  for all other sizes at that locus ( like  a-203  and  a-207  ) . this analysis method ignores the uncertainty in the copy number of each locus in aneuploid cancer cells and allows for ambiguity at particular alleles . in cases where the hemocyte and tissue genotypes could be clearly differentiated , all alleles were analyzed ( h1 and h3 ) . in some cases ( m1 , m2 , m3 , and h2 ) , cancer - specific alleles could be detected in the hemocyte samples , but represented less than 50% of the total hemocyte dna , and the host tissue alleles obscured one or two positions ( depending on whether the normal genotype was homozygous or heterozygous at that locus ) . in these cases , since the tissue allele obscured a potential neoplastic allele , that size was coded as  missing  for the hemocyte genotype . the total distance between any two samples is calculated as the average of the pairwise differences at all allele sizes observed ( 104 pairwise comparisons if no data is missing ) . in pairwise comparisons for each allele , two samples either have a distance of 0 ( meaning that the particular allele size is either present in both samples or absent in both samples ) or a distance of 1 ( meaning that the particular allele size is present in one sample but absent in the other ) . if data are missing for one or both then that specific comparison is dropped with no contribution to the total distance between the two samples . the genetic distances calculated are based on the alleles observed at the 9 loci , so the absolute value of the scale itself is therefore dependent on the number of observed alleles that are included in the analysis . source data for the generation of the phylogenetic tree lists all observed alleles coded as present ( 1 ) , absent ( 2 ) , or missing ( 0 ) for each sample . as an alternate analysis method , the data were analyzed using bruvo s method as polyploid data with 9 loci , using only the individuals which could be confidently identified . in this alternate analysis , the topology of the resulting tree was not different  all nodes with bootstrap values above 50 were maintained . in this analysis , the genetic distance between the representatives of lineage 1 ( h3 ) and lineage 2 ( h1 ) was 0.676 , and the distances to the closest normal sample were 0.595 ( h3 to n5 ) and 0.648 ( h1 to n1 and n2 ) . pecies - specific qpcr of the ef1 locus in p. aureus and v. corrugata was done using faststart universal sybr green master mix ( roche ) . primers were designed to amplify the same region , with the 3 end of both forward and reverse primers on sites which differ between the two species ( extended data table 2 ) . standard curves for each primer set were generated using two control plasmids ( one containing the p. aureus ef1 fragment and p. aureus sle fragment and the other containing the fragments from v. corrugata ) . fragments were cloned with the zero blunt topo kit ( invitrogen ) , and plasmids were linearized with noti before qpcr . standard curve samples ( 10 through 10 copies per reaction ) and experimental samples ( 2.5 ng per reaction ) were done in triplicate . no amplification was detected with the p. aureus primers on the v. corrugata plasmid or with the v. corrugata primers on p. aureus plasmid ( using up to 10 copies per reaction ) . the fraction of v. corrugata in each sample was calculated as the copy number using v. corrugata - specific primers divided by copy number of p. aureus - plus v. corrugata - specific amplification . quantification of steamer - like elements in the three species was performed by the same method , using primers in the rt - in region of the retrotransposon ( based on sequence obtained through amplification using degenerate primers ) and control primers amplifying a region in ef1. all primers used to amplify sles and ef1 from genomic dna for cloning into standards and primers used for qpcr are listed in extended data table 2 . for each species , mussel ( m. trossulus ) specimens 5 to 6 cm long were collected from the intertidal zone at low tide ( noon ) on april 18 , 2015 at copper beach ( n 49 2241 w 123 they were transported to the laboratory in aerated seawater from copper beach . in the laboratory , 0.5 one drop of hemolymph was placed on a poly - l - lysine - coated slide and let sit for 1015 min to allow the cells to spread and attach to the slide . thereafter , the slide was viewed under a zeiss axiostar light microscope at 40 magnification . normal ( non - neoplastic ) specimens were those with greater than 90% cells with normal appearance , i.e. agranular or granular hemocytes with spread pseudopodia . fully leukemic ( diseased ) specimens were those with prolific amounts ( > 90% ) of round , non - adherent cells . hemolymph was added to 1.5 ml eppendorf tubes and spun at 900 g for 3 min . after the supernatant was withdrawn , the cells were re - suspended in absolute ethanol before shipping . excised tissues ( mantle , foot , and gills ) were preserved in absolute ethanol before shipping . of 28 individuals collected from west vancouver , two had high levels of neoplastic disease . a second set of m. trossulus samples were collected from esquimalt , vancouver island , b.c . , hemocyte and solid tissue samples were fixed in ethanol before dna extraction . of 250 individuals collected from esquimalt , nine were scored as potentially moderately or highly diseased , with seven samples available for analysis . cockles ( c. edule ) were collected from an intertidal bed named o sarrido ( 42 30 n , 8 49 w ) and golden carpet shell clams ( p. aureus ) and pullet shell clams ( v. corrugata ) were collected from a subtidal bed named o bohdo ( 42  32 n , 8 51 w ) ; both shellfish beds are located in the ria of arousa ( galicia , nw spain ) . once in the laboratory of cima , each cockle and clam was notched through the shell margin close to the posterior adductor muscle and hemolymph ( as much as possible ) was collected from the posterior adductor muscle using a 2-ml syringe with a 21 gauge needle . a small quantity ( ca . 100 l ) of hemolymph was used to produce a cell monolayer onto a slide by cytocentrifugation ( 92  g , 5 min , 4c ) , which was fixed , stained with hemacolor ( merck ) kit and examined with light microscopy for diagnosis of disseminated neoplasia ; the remaining hemolymph was preserved in absolute ethanol for molecular analysis . after hemolymph collection , molluscs were shucked and a small piece of mantle ( ca . 5  5 mm square ) was removed and preserved in absolute ethanol for molecular analysis ; additionally , an approximately 5 mm thick section of meat , containing gills , visceral mass , foot and mantle lobes , was fixed in davidson s solution and embedded in paraffin . neoplastic cells ( both a and b types ) had a unique morphology , and were clearly distinguished from normal cells , with much larger overall size , and much larger nucleus . the longest diameters of the cell and the nucleus of at least 10 neoplastic cells for each individual were measured by direct examination of histological sections with light microscopy using a reticle . two - tailed t - tests were used for comparisons of morphometric data from different individuals and types . cockles ( c. edule ) and golden carpet shell clams ( p. aureus ) were ranked according to a scale of disease severity based on hemolymph diagnosis : non - affected ( n0 , or n ) ; low severity ( n1 , or l ) , when individuals showed proportions of neoplastic cells lower than 15% in the hemolymph cell monolayers ; moderate severity ( n2 , or m ) , when the proportion ranged from 15% to 75% ; and high severity ( n3 , or h ) , when the proportion was higher than 75% . 74 golden carpet shell clams ( p. aureus ) were collected with 12 diagnosed with light , 10 with moderate , and 9 with heavy neoplasia . about 150 cockles ( c. edule ) were collected and a subset was analyzed for both disease and morphological type of neoplastic cells . of the 30 in this subset , two had type a neoplastic cells ( one light and the other moderate ) and one had low levels of type b neoplastic cells . we have also collected and analyzed hundreds of pullet clams ( v. corrugata ) from multiple beds throughout galicia from 1988 to the present time , including about 100 from the same bed in which the samples of p. aureus were collected for this study ( o bohdo ) . to this date , we have not observed any v. corrugata samples with neoplastic disease . one v. corrugata individual from a different location has been identified by a different researcher as harboring disseminated neoplasia , with a different morphology than that observed in p. aureus neoplasia ( personal communication , susana darriba , at intecmar ) . dna was extracted from ethanol - fixed hemocytes using dneasy blood and tissue kit ( qiagen ) . dna extraction of tissues used the same kit , but included an additional step in order to reduce the amount of pcr - inhibiting polysaccharides . after tissue lysis , 63 l of buffer p3 was added to lysate and allowed to precipitate for 5 min . lysate was spun 10 minutes at full speed at 4c , and the resulting supernatant was mixed with ethanol and added to the column , continuing with the standard protocol . pfuultra ii fusion hs dna polymerase ( agilent ) was used to amplify 10 ng of genomic dna for 35 cycles . extension was at 72c for 15 sec for mtcoi for all bivalves . for p. aureus , its was amplified ( using primers modified from ) with extension for 30 sec . despite multiple copies of its in genomic dna , a single sequence was observed for each normal p. aureus and v. corrugata , and a single pair of host / neoplasm sequences was observed in each diseased individual . pcr for ef1 followed the same program , with extension for 20 sec in mussels ( m. trossulus ) and clams ( p. aureus and v. corrugata ) and 1 min 30 sec in cockles ( c. edule ) . for amplification of steamer - like elements , a degenerate primer pair was designed to match conserved regions in reverse transcriptase and integrase of the ltr - retrotransposon , steamer , and 10 ng of dna was amplified for 35 cycles , annealing at 55c for 20 sec and extending for 1 min at 72c . when multiple alleles could not be resolved by direct sequencing , pcr products were cloned using the zero blunt topo kit ( invitrogen ) , and at least 6 colonies were sequenced . primer - binding regions were excluded from analysis and are not included in the sizes listed as sequenced . maximum likelihood phylogenetic trees were generated using phyml 3.0 , using the hky85 substitution model , with 100 bootstrap replicates , treating gaps in the alignment as missing data . trees were visualized using figtree v1.4 , with addition of markers at the branch termini . each phylogenetic tree based on alignment of sequence at a single locus ( figure 1c , 2e , 3b d ) includes a scale bar which shows genetic distance ( 01 ) based on the frequency of nucleotide divergence . microsatellites were amplified using primers for 12 loci , reported previously to be polymorphic in cockle ( c. edule ) populations . of the 12 primer pairs , 9 pairs amplified products in all cockle dna samples , with 12 alleles observed in normal samples and 14 neoplastic alleles observed . these were used in all further analyses , with fluorescent modifications on the 5 end of the forward primers as listed ( extended data table 2 ) . kod polymerase ( millipore ) was used to prevent ambiguity of untemplated addition of a residues by taq polymerase . allele sizes were identified to single - base precision by fragment analysis using florescent primers ( 6-fam , pet , ned , and vic ) using a 3730xl genetic analyzer with the liz-500 size standard , ( applied biosystems , operated by genewiz ) . peaks were called using peak scanner 2.0 ( applied biosystems ) , rounding sizes to whole bases , with some manual adjustment to keep alleles of the same size together . we used the r package poppr to generate distance matrices and phylogenetic trees using the bruvo s method with the infinite alleles model . the value of c ( repeat size ) was set to 0.00001 in order to calculate distances using the band sharing model , in which alleles are either identical sizes ( distance=0 ) or non - identical ( distance=1 ) . each allele was coded as a dominant marker , with a single variable for each allele observed at each position ( 104 total markers from 9 loci ) . for each allele ( a single size at a single locus ) , a sample is observed to be either present , absent , or the information is missing . for example , a sample with sizes 195 and 199 at locus a would be marked as  present  for marker a-195  and  a-199 ,  but  absent  for all other sizes at that locus ( like  a-203  and  a-207  ) . this analysis method ignores the uncertainty in the copy number of each locus in aneuploid cancer cells and allows for ambiguity at particular alleles . in cases where the hemocyte and tissue genotypes could be clearly differentiated , all alleles were analyzed ( h1 and h3 ) . in some cases ( m1 , m2 , m3 , and h2 ) , cancer - specific alleles could be detected in the hemocyte samples , but represented less than 50% of the total hemocyte dna , and the host tissue alleles obscured one or two positions ( depending on whether the normal genotype was homozygous or heterozygous at that locus ) . in these cases , since the tissue allele obscured a potential neoplastic allele , that size was coded as  missing  for the hemocyte genotype . the total distance between any two samples is calculated as the average of the pairwise differences at all allele sizes observed ( 104 pairwise comparisons if no data is missing ) . in pairwise comparisons for each allele , two samples either have a distance of 0 ( meaning that the particular allele size is either present in both samples or absent in both samples ) or a distance of 1 ( meaning that the particular allele size is present in one sample but absent in the other ) . if data are missing for one or both then that specific comparison is dropped with no contribution to the total distance between the two samples . the genetic distances calculated are based on the alleles observed at the 9 loci , so the absolute value of the scale itself is therefore dependent on the number of observed alleles that are included in the analysis . source data for the generation of the phylogenetic tree lists all observed alleles coded as present ( 1 ) , absent ( 2 ) , or missing ( 0 ) for each sample . as an alternate analysis method , the data were analyzed using bruvo s method as polyploid data with 9 loci , using only the individuals which could be confidently identified . in this alternate analysis , the topology of the resulting tree was not different  all nodes with bootstrap values above 50 were maintained . in this analysis , the genetic distance between the representatives of lineage 1 ( h3 ) and lineage 2 ( h1 ) was 0.676 , and the distances to the closest normal sample were 0.595 ( h3 to n5 ) and 0.648 ( h1 to n1 and n2 ) . pecies - specific qpcr of the ef1 locus in p. aureus and v. corrugata was done using faststart universal sybr green master mix ( roche ) . primers were designed to amplify the same region , with the 3 end of both forward and reverse primers on sites which differ between the two species ( extended data table 2 ) . standard curves for each primer set were generated using two control plasmids ( one containing the p. aureus ef1 fragment and p. aureus sle fragment and the other containing the fragments from v. corrugata ) . fragments were cloned with the zero blunt topo kit ( invitrogen ) , and plasmids were linearized with noti before qpcr . standard curve samples ( 10 through 10 copies per reaction ) and experimental samples ( 2.5 ng per reaction ) were done in triplicate . no amplification was detected with the p. aureus primers on the v. corrugata plasmid or with the v. corrugata primers on p. aureus plasmid ( using up to 10 copies per reaction ) . the fraction of v. corrugata in each sample was calculated as the copy number using v. corrugata - specific primers divided by copy number of p. aureus - plus v. corrugata - specific amplification . quantification of steamer - like elements in the three species was performed by the same method , using primers in the rt - in region of the retrotransposon ( based on sequence obtained through amplification using degenerate primers ) and control primers amplifying a region in ef1. all primers used to amplify sles and ef1 from genomic dna for cloning into standards and primers used for qpcr are listed in extended data table 2 . for each species , a partial region of the mtcoi gene was amplified from genomic dna of solid tissue and hemocytes from mussels ( m. trossulus ) and directly sequenced ( figure 1b ) . trace images show a region flanking a representative snp marked with an open triangle ( tissue ) or closed triangle ( hemocytes ) . a b , in normal mussels , tissue and hemocyte alleles match ( with g496 at all positions ) . c  e , in mussels with disseminated neoplasia , the tissue and hemocyte alleles are different . neoplastic hemocytes have a at position 496 and g in tissue , with some a observable in tissue , likely due to infiltration of neoplastic hemocytes . d , fragments from the sle reverse transcriptase region and ef1 genes were cloned from each species . haploid copy numbers of steamer - like elements ( sle ) were quantified by determining the ratio of sle / ef1a in genomic dna from hemocytes . single species - specifc sles were analyzed in ( a ) mussels ( m. trossulus ) and ( b ) cockles ( c. edule ) . ( c , d ) in golden carpet shell clams , one sle ( sle - pa ) was cloned from a normal p. aureus ( clam n2 ) and a different one ( sle - vc ) was cloned from neoplastic cells ( clam h2 ) . both sles could be found in both species , and qpcr analysis confirmed that sle - pa is more highly amplified in p. aureus and has fewer copies in v. corrugata and in the neoplastic cells derived from v. corrugata . morphometric analysis of type a and type b cockle ( c. edule ) neoplasia cell diameter is statistically different between types a and b ( p < 0.0001 ) . nuclear diameter is statistically different between types a and b ( p < 0.0001 ) ; for all pairwise comparisons between type a and type b individuals p < 0.05
most cancers arise from oncogenic changes in the genomes of somatic cells , and while the cells may migrate by metastasis , they remain within that single individual . natural transmission of cancer cells from one individual to another has been observed in two distinctive cases in mammals ( tasmanian devils1 and dogs2,3 ) , but these are generally considered to be rare exceptions in nature . the discovery of transmissible cancer in soft - shell clams ( mya arenaria)4 suggested that this phenomenon might be more widespread . 
 here we analyzed disseminated neoplasia in mussels ( mytilus trossulus ) , cockles ( cerastoderma edule ) , and golden carpet shell clams ( polititapes aureus ) and found that neoplasias in all three species are attributable to independent transmissible cancer lineages . in mussels and cockles , the cancer lineages are derived from their respective host species , but unexpectedly , cancer cells in p. aureus are all derived from venerupis corrugata , a different species living in the same geographic area . 
 no cases of disseminated neoplasia have thus far been found in v. corrugata from the same region . 
 these findings show that transmission of cancer cells in the marine environment is common in multiple species , that it has originated many times , and that while most transmissible cancers were found spreading within the species of origin , cross - species transmission of cancer cells can occur .