margaritiferidae is a family of medium-sized freshwater mussels , aquatic bivalve molluscs in the order unionoida .

they are known as freshwater pearl mussels , because the interior of the shell of these species has a thick layer of nacre or mother of pearl , and the mussels are thus capable of producing pearls .

margaritiferidae

margaritiferidae <EOT> ( mollusca : pelecypoda : margaritiferidae ) . zool j linn soc 69 : 257–270

( conrad , 1838 ) ( unionacea : margaritiferidae ) . nautilus 102 : 159–163

conrad ( bivalvia : margaritiferidae ) . conserv genet . 2004 ; 5 : 271–278 .

( bivalvia , margaritiferidae ) [ in russian ] . zoologicheskii zhurnal . 1993 ; 72 : 29–36 .

( bivalvia : margaritiferidae ) from japan [ in japanese ] . venus . 2007 ; 65 : 355–363 .

( bivalvia : margaritiferidae ) in japan [ in japanese ] . venus . 2009 ; 67 : 189–197 .

the origin and phylogeny of margaritiferidae ( bivalvia : unionoida ) . a synthesis of molecular and fossil data .

( bivalvia : margaritiferidae ) in the shinano river system , japan . venus . 2005 ; 64 : 63–70 .

( spengler , 1782 ) ( mollusca : margaritiferidae ) based on museum specimens . journal of conchology 37 : 49–59 .

( bivalvia : margaritiferidae ) of japan , with description of a new species . venus . 2005 ; 64 : 135–140 .

hass 1910 im oberen und mittleren donausystem ( bivalvia : unionidae , margaritiferidae ) . nachrichtenbl ersten vorarlberger malakol ges 1 : 20–40

( bivalvia : margaritiferidae ) , with reference to the existence of two distinct species . venus . 2005 ; 64 : 55–62 .

( spengler , 1793 ) ( bivalvia : margaritiferidae ) découvertes dans le sud - ouest de la france . malaco 6 : 294–297 .

kat pw ( 1983 ) conchiolin layers among the unionidae and margaritiferidae ( bivalvia ) : microsculptural characteristics and taxonomic implications . malacologia 24 : 298–311

smith dg ( 1986 ) the stomach anatomy of some eastern north american margaritiferidae ( unionoida : unionacea ) . am malacol bull 4 : 13–19

( bivalvia : margaritiferidae ) in the chubu - nougu river , nagano prefecture [ in japanese ] . venus . 2008 ; 67 : 61–71 .

the origin and phylogeny of margaritiferidae ( bivalvia : unionoida ) . a synthesis of molecular and fossil data . | laboratory of kevin j . roe

bogatov vv , prozorova la , starobogatov yi . the family margaritiferidae ( mollusca : bivalvia ) in russia . ruthenica . 2003 ; 13 : 41–52 .

all the phylogenies inferred in this study support the reciprocal monophyly of both ( unionidae + margaritiferidae ) f - and m - type lineages ( fig .

smith dg ( 1976b ) the distribution of the margaritiferidae : a review and a new synthesis . am malacol union inc bull 1976 : 42 ( abstr )

klishko ok . sakhalin - kurile species of pearl mussels ( bivalvia : margaritiferidae ) from transbaikalye . the korean journal of malacology . 2009 ; 25 : 237–242 .

smith dg . observations on the morphology and anatomy of margaritinopsis dahurica ( middendorff , 1850 ) ( unionoida : margaritiferidae ) . j conchol . 2001 ; 37 : 119–125 .

starobogatov y ( 1995 ) the pearly freshwater mussels ( mollusca , unionoida , margaritiferidae of russia . in : valovirta i , harding pt , kirne d ( eds ) proc 9

bogatov , v . v . , prozorova , l . a . , and starobogatov , y . i . , . the family margaritiferidae ( mollusca : bivalvia ) in russia ,

, a new species of unionacea ( bivalvia : margaritiferidae ) from the mobile - alabama - coosa and escambia river systems , alabama . occasional papers on mollusks . 1983 ; 4 : 299–304 .

, a new species of unionacea ( bivalvia : margaritiferidae ) from the mobile - alabama - coosa and escambia river systems , alabama . occas pap mollusks mus comp zool harv univ 4 : 299–304

machordom a , araujo r , erpenbeck d , ramos m - a . phylogeography and conservation genetics of endangered european margaritiferidae ( bivalvia : unionoidea ) . biol j linn soc . 2003 ; 78 : 235–252 .

smith dg . systematics and distribution of the recent margaritiferidae . in : bauer g , wachtler k editors . ecology and evolution of the freshwater mussels unionoida . heidelberg : springer verlag ; 2001 . pp . 33–49 .

sp . n . ( bivalvia , margaritiferidae ) a new species of pearl mussels from transbaikalye , with remarks on the natural history of far eastern najades [ in russian ] . vestnik zoologii . 2008 ; 42 : 291–302 .

smith dg ( 1983 ) on the so - called mantle muscle scars on shells of the margaritiferidae ( mollusca , pelecypoda ) , with observations on mantle - shell attachment in the unionoida and trigonioida . zool scr 12 : 67–71

smith dg . on the so - called mantle muscle scars on shells of the margaritiferidae ( mollusca , pelecypoda ) , with observations on mantle - shell attachment in the unionoida and trigonioida . zool scr . 1983 ; 12 : 67–71 .

smith dg , wall wp ( 1984 ) the margaritiferidae reinstated : a reply to davis and fulller ( 1981 ) , genetic relationships among recent unionacea ( bivalvia ) of north america . occas pap mollusks mus comp zool harv univ 4 : 321–330

klishko ok . some data on reproductive biology of the freshwater mussels ( margaritiferidae , unionidae ) and their relationships with bitterlings ( cyprinidae ) in transbaikalye [ in russian ] . the bulletin of the russian far east malacological society . 2012 ; 15 / 16 : 31–55 .

smith d . g . ( 2001 ) systematics and distribution of the recent margaritiferidae . in : bauer g . , wächtler k . ( eds ) ecology and evolution of the freshwater mussels unionoida . ecological studies ( analysis and synthesis ) , vol 145 . springer , berlin , heidelberg

citation : bolotov in , bespalaya yv , vikhrev iv , aksenova ov , aspholm pe , gofarov my , et al . ( 2015 ) taxonomy and distribution of freshwater pearl mussels ( unionoida : margaritiferidae ) of the russian far east . plos one 10 ( 5 ) : e0122408 . urltoken

huff sw , campbell d , gustafson dl , lydeard c , altaba cr , giribet g . investigations into the phylogenetic relationships of freshwater pearl mussels ( bivalvia : margaritiferidae ) based on molecular data : implications for their taxonomy and biogeography . j mollus stud . 2004 ; 70 : 379–388 .

diagrams of the five distinct gene orders detected in unionida . in the female f - type lineage , three gene orders are depicted : unionidae f - type 1 ( uf1 ) , unionidae f - type 2 ( uf2 ) and margaritiferidae f - type 1 ( mf1 ) . in the male m - type lineage , two gene arrangements are shown : unionidae m - type 1 ( um1 ) and margaritiferidae m - type 1 ( mm1 ) . continuous lines indicate different locations of genes between mitogenomes . grey box highlights gene rearrangement region between uf1 and uf2 . yellow boxes indicate main differences in gene arrangement between female and male mitogenomes , trna ( h ) location and rearrangement of atp8 - trna ( d ) region .

stephanie w . huff , david campbell , daniel l . gustafson , charles lydeard , cristian r . altaba , gonzalo giribet ; investigations into the phylogenetic relationships of freshwater pearl mussels ( bivalvia : margaritiferidae ) based on molecular data : implications for their taxonomy and biogeography , journal of molluscan studies , volume 70 , issue 4 , 1 november 2004 , pages 379–388 , urltoken

the family margaritiferidae includes 13 extant species , which are mainly distributed in temperate latitudes of the northern hemisphere [ 1 ] , [ 2 ] , [ 3 ] . smith [ 4 ] provided a detailed diagnosis of the family . recent species are known from north america , europe , northern africa , the middle east , and throughout much of southern and eastern asia [ 2 ] , [ 4 ] . the most ancient margaritiferidae fossils are known from the upper triassic and lower jurassic fluvio - lacustrine deposits in the sichuan , southeastern china [ 5 ] , [ 6 ] . the recent margaritiferids retain the simple , unfused mantle margins from the ancestral palaeoheterodont and several other ‘plesiomorphic’ features ; therefore , these species have been regarded as the basal unionoid family [ 3 ] , [ 7 ] .

the north american , european and northern african margaritiferidae are relatively well studied [ 8 ] , [ 4 ] , [ 9 ] , [ 10 ] , [ 11 ] in contrast to the asian representatives of the family . most of the references for far eastern freshwater pearl mussel populations are from japan [ 12 ] , [ 13 ] , [ 14 ] , [ 15 ] , [ 16 ] . recently , a description of a new japanese margaritiferidae species m . togakushiensis kondo & kobayashi , 2005 was published . this species was separated from m . laevis based on the results of long - term studies , specifically including differences in the host fish preference and genetic and morphological patterns [ 17 ] , [ 18 ] , [ 19 ] , [ 20 ] , [ 21 ] .

manuel lopes - lima , miguel m . fonseca , david c . aldridge , arthur e . bogan , han ming gan , mohamed ghamizi , ronaldo sousa , amílcar teixeira , simone varandas , david zanatta , alexandra zieritz , elsa froufe ; the first margaritiferidae male ( m - type ) mitogenome : mitochondrial gene order as a potential character for determining higher - order phylogeny within unionida ( bivalvia ) , journal of molluscan studies , volume 83 , issue 2 , 1 may 2017 , pages 249–252 , urltoken

shows the topology of the bi - nuc tree ; all other phylogenetic trees figures supplied on request ) . additionally , the monophyly of unionidae ( both f - and m - type ) , margaritiferidae ( f - type ) and all represented unionidae subfamilies are well supported in all inferred mtdna trees , with the exception of the unioninae , for which monophyly was only well supported in the bi - nuc tree . the remaining phylogenetic trees ( bi - aa , ml - nuc and ml - aa ) showed conflicting results regarding the position of the clade comprising

according to kondo & kobayashi [ 18 ] , the adductor muscle scar shape is one of the key features for m . togakushiensis identification . however , according to our data , this feature has a high variability and cannot be used for species identification among studied taxa . in the review of recent margaritiferidae by smith [ 4 ] , this feature was also not mentioned as a diagnostic . the adductor muscle scar shape was not used in the morphological descriptions of other unionoid species [ 10 ] , [ 86 ] , [ 95 ] , [ 96 ] , [ 97 ] .

the dimensions of the shell are the least reliable morphological traits in unionoida , upon which to base a description [ 4 ] , [ 32 ] due to high variability and the dependence on environmental gradients [ 4 ] , [ 33 ] , [ 34 ] , [ 100 ] , [ 101 ] . however , sometimes , the shell dimensions and shape are useful for distinguishing several margaritiferidae species . for example , m . middendorffi has a small shell length ( < 100 mm ) ( fig 10 ) , and m . dahurica has the largest shell length ( up to 196 . 5 mm ) among the far eastern pearl mussels ( fig 5 ) .

the analysis of the mitochondrial coi gene showed that c . monodonta forms a separate clade , which is basal within margaritiferidae . m . dahurica , m . margaritifera , m . middendorffi , m . laevis and m . falcata cluster together in a well - supported clade ( bpp 1 . 00 ) ( fig 2b ) . among these , our bayesian inference strongly supports the sister relationships within two pairs of species , namely m . dahurica and m . margaritifera as the first pair ( bpp 1 . 00 ) , and m . middendorffi and m . laevis as the second pair ( bpp 0 . 97 ) . m . falcata shows a closer affinity to m . middendorffi and m . laevis ( bpp 0 . 93 ) . lastly , the coi phylogeny confirms that m . auricularia and m . marocana are sister taxa ( bpp 0 . 98 ) .

the length of the two newly sequenced mitogenomes of m . marocana , 16 , 001 bp for the female haplotype and 17 , 562 bp for the male haplotype , is within the typical range for each sex - specific haplotype within unionida . the sequenced haplotypes include the 13 pcgs typically found in metazoan mitochondrial genomes , the sex - specific orf described for all unionida mitogenomes with dui system ( breton et al . , 2009 , 2011a ) and 22 transfer rna ( trna ) and two ribosomal rna ( rrna ) genes . the m - type genome is the largest sequenced to date within the unionida . m - type genomes are generally larger than f - type genomes due to the larger size of the pcg cox2 and m - orf in m - type genomes compared with cox2 and f - orf in f - type genomes ( breton et al . , 2009 ) . four intergenic regions were identified in the m . marocana m - type genome between the following gene pairs : nad3–trna ( a ) 106 bp , trna ( h ) –trna ( q ) 411 bp , nd4l–trna ( d ) 255 bp and trna ( d ) –atp8 498 bp . these regions were analysed to search for the m - orf . the results of the blast search ( altschul et al . , 1997 ) retrieved a significant hit with another margaritiferidae m - orf ( margaritifera monodonta , e - value = 4e–34 ) and a fickett test score of 1 . 201 ( fickett , 1982 ; a score > 0 . 95 means the sequence is probably coding ) , suggesting that the m - orf is located in the region between the genes nd4l and trna ( d ) .

genus margaritinopsis accepted as margaritanopsis f . haas , 1910 accepted as margaritifera schumacher , 1815 ( incorrect subsequent spelling )

henderson j . ( 1929 ) . non - marine mollusca of oregon and washington . the university of colorado studies . 17 ( 2 ) : 47 - 190 . page ( s ) : 53 [ details ]

invertebase . ( 2015 ) . authority files of u . s . and canadian land and freshwater mollusks developed for the invertebase project ( invertebase . org ) . [ details ]

( of margaritanidae ortmann , 1910 ) graf , d . ; cummings , k . ( 2007 ) . review of the systematics and global diversity of freshwater mussel species ( bivalvia : unionoida ) . journal of molluscan studies . 73 ( 4 ) : 291 - 314 . [ details ]

( of margaritanidae ortmann , 1910 ) invertebase . ( 2015 ) . authority files of u . s . and canadian land and freshwater mollusks developed for the invertebase project ( invertebase . org ) . [ details ]

( of cumberlandiidae heard & guckert , 1970 ) graf , d . ; cummings , k . ( 2007 ) . review of the systematics and global diversity of freshwater mussel species ( bivalvia : unionoida ) . journal of molluscan studies . 73 ( 4 ) : 291 - 314 . [ details ]

( of cumberlandiidae heard & guckert , 1970 ) invertebase . ( 2015 ) . authority files of u . s . and canadian land and freshwater mollusks developed for the invertebase project ( invertebase . org ) . [ details ]

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copyright : © 2015 bolotov et al . this is an open access article distributed under the terms of the creative commons attribution license , which permits unrestricted use , distribution , and reproduction in any medium , provided the original author and source are credited

data availability : data are available from : rmbh - russian museum of the biodiversity hotspots of institute of environmental problems of the north of the ural branch of russian academy of science , arkhangelsk , 163000 , russia . inrec - institute of natural resources , ecology and cryology of the siberian branch of russian academy of sciences , chita , 672000 , russia . zisp - zoological institute of the russian academy of sciences , st . petersburg , 199034 , russia . smf - forschungsinstitut und natur - museum senckenberg , senckenberg - anlage 25 , 6000 frankfurt - am - main 1 , germany .

funding : this study was supported by the federal agency for scientific organisations ( no . 0410 - 2014 - 0028 ) , the ural branch of russian academy of sciences ( nos . 15 - 12 - 5 - 3 and 15 - 2 - 5 - 7 ) , grants from the president of russia ( no . md - 6465 . 2014 . 5 and мк - 4735 . 2015 . 4 ) and the russian foundation for basic research ( nos . 14 - 04 - 98801 and 15 - 04 - 05638 ) and the ministry of science and education of russia ( no . p362 ) . the funders had no role in study design , data collection and analysis , decision to publish , or preparation of the manuscript .

thus , the available information on the asian freshwater pearl mussels is limited . their taxonomy is not completely clear and reliable data on the species’ ranges are sparse . however , freshwater pearl mussels are extremely demanding regarding habitat quality and can exist only in a narrow range of environmental conditions [ 11 ] . the majority of these species are listed by iucn as endangered or vulnerable taxa [ 8 ] , [ 9 ] , [ 10 ] , [ 11 ] , [ 40 ] . freshwater mussels have experienced one of the highest rates of extinction of any group of organisms in the past 100 years [ 41 ] . therefore , reliable information on the taxonomy and distribution ranges is still needed for the conservation of the margaritifera species .

we conducted field studies within several regions of eastern russia in the period 2004–2012 ( see fig 1 for the location of the study areas ) . sampling on the russian federation territory was permitted within the framework of the special grants of the russian foundation for basic research ( rfbr , no . 12 - 04 - 00594 ) , the ministry of science and education of russia ( no . № p362 ) , and the scientific program of the ural branch of russian academy of sciences ( no . 12 - p - 5 - 1014 ) . sampling on the protected sites of the kunashir island was permitted by the directorate of the state nature protected area “kurilsky” ( scientific agreement no . 5 / 11 of 17 . 08 / 2010 between the institute of ecological problems of the north of the ural branch of russian academy of sciences and the kurilsky nature reserve ) .

1—kamchatka peninsula ( 2012 , i . bolotov , y . bespalaya , i . vikhrev , m . gofarov ) , 2—central sakhalin ( 2012 , same team ) , 3—southern sakhalin ( 2011–2012 , same team & y . kolosova , o . aksenova ) , 4—kunashir island ( 2011 , same team & y . kolosova , o . aksenova ) , 5—primorye ( 2012 , same team ) , 6—transbaikalia ( 2004–2011 , o . k . klishko ) . data on the studied river sites are presented in s1 – s4 tables .

for molecular analyses , the tissue samples were collected from 52 live bivalves in 15 localities ( see s1 table ) . samples were immediately preserved in 96 % ethanol . dead shells were collected from each field site for morphological investigation ; additional shells from some museum collections were studied ( see s2 – s4 tables ) . a total of 554 shells from 44 localities were studied ( 426 from our collection and 128 museum specimens ) .

the total length of the live specimens and dead shells were measured to the nearest 0 . 1 mm with dial calipers [ 8 ] . the comparative morphologies of the shells were analyzed using shape and structure of the pseudo - cardinal and lateral teeth in the valves , shell shape , umbo position and the patterns of distribution of mantle attachment scars on the inner shell surface . we calculated the plane projection squares of the inner surface of the right valve for the estimation of the density of mantle attachment scars . ten specimens of each species were taken for analysis . the squares were calculated using shape v . 1 . 3 software [ 42 ] . the scars were counted by the gnu image manipulation program ( gimp ) v . 2 . 8 . a normality test of the obtained density values and a one - way anova welsh’s test ( s5 table ) were calculated using statistica v . 10 software .

the soft tissue morphologies were analyzed and compared based on the shape and structure of the inhalant siphon , gills and labial palps . shell and soft tissue photos were taken using a dslr camera ( canon eos 7d , japan ) with a 24–70 mm lens ( sigma af 24–70 mm f / 2 . 8 if ex dg aspherical hsm , japan ) , and photos of the shell structure details and inhalant siphons were taken using a stereomicroscope ( leica m2c , germany ) and a dslr camera with a 100 mm macro lens ( canon macro lens ef 100 mm 1 : 2 , 8 l is usm , japan ) . descriptions of the shell morphologies were based on an analysis of the collected specimens and museum samples using the original species descriptions [ 18 ] , [ 38 ] , [ 43 ] , [ 44 ] , [ 45 ] , [ 46 ] and a few other works [ 23 ] , [ 24 ] , [ 47 ] , [ 48 ] .

we studied the type specimens for the following taxa : unio ( alasmodonta ) dahuricus middendorff , 1850 ; margaritana middendorffi rosén , 1926 ( including specimens of unio ( alasmodonta ) complanatus middendorff , 1851 ) ; and m . sachalinensis zhadin , 1938 ( zisp ) . the type series of several comparatory “taxa” were also investigated , including dahurinaia sujfunensis moskvicheva , 1973 ; d . kurilensis zatravkin & starobogatov , 1984 ; d . tiunovae bogatov & zatravkin , 1988 ; d . komarovi bogatov et al . , 2003 ; d . ussuriensis bogatov et al . , 2003 ; d . prozorovae bogatov et al . , 2003 ; d . transbaicalica klishko , 2008 ; kurilinaia kamchatica bogatov et al . , 2003 ; and k . zatravkini bogatov et al . , 2003 ( zisp ) . in general , the zisp systematic catalog has a very complicated numeration because v . v . bogatov has transferred many specimens from different samples , including true type series , to his newly described comparatory “taxa” .

the mapping was based on the data of our field studies ( see s2 – s4 tables ) . we determined the precise coordinates of investigated river sites using a geographical positioning system ( gps ) . data on other localities were obtained from museum collections and reliable published studies ( see s2 – s4 tables ) . in the cases where a particular map scale did not allow separated precision pointing of closely situated localities , we marked those localities one by one with points and joined them under the one number in the respective table . the arrangement of the localities was digitized and mapped using esri arcgis 10 . the presumed error of determination of the locality coordinates is around ±1–2 km , because published records and collection labels are usually ascribed to an approximate location . the layers of digital maps were added from standard esri data & maps 10 dataset .

) . genomic dna was extracted from the foot or mantle tissues using the diatom dna prep 200 reagents kit ( “laboratoriya isogen” llc , russia ) following the manufacturer’s protocol . the standard forward primer lco 1490 [

] was used to amplify the mitochondrial coi gene from every species . as reverse coi primers , hco 2198 in a modified version ( without the first 6 bases at the 5´ end ) was applied to

) were used to amplify the nuclear 18s rrna gene from every species . the pcr mix contained approximately 200 ng of total cellular dna , 10 pmol of each primer , 200 μmol of each dntp , 2 . 5 μl of pcr buffer ( with 10×2 mmol mgcl

) , 0 . 8 units of taq dna polymerase ( sibenzyme ltd . , novosibirsk , russia ) , and h

o , which was added up to a final volume of 25 μl . thermocycling included one cycle at 95°c ( 4 min ) , followed by 30–35 cycles of 95°c ( 50 sec ) , 52°c ( 50 sec ) , and 72°c ( 50 sec ) and a final extension at 72°c ( 5 min ) . forward and reverse sequencing was performed on an automatic sequencer ( abi prism 3730 , applied biosystems ) using the abi prism bigdye terminator v . 3 . 1 reagent kit . the resulting sequences were checked using a sequence alignment editor ( bioedit version 7 . 2 . 5 , [

the electronic edition of this article conforms to the requirements of the amended international code of zoological nomenclature , and hence the new names contained herein are available under that code from the electronic edition of this article . this published work and the nomenclatural acts it contains have been registered in zoobank , the online registration system for the iczn . the zoobank lsids ( life science identifiers ) can be resolved and the associated information viewed through any standard web browser by appending the lsid to the prefix “ urltoken ” . the lsid for this publication is : urn : lsid : zoobank . org : pub : 46c93315 - 18e0 - 45dd - b121 - 1f225d6e200f . the electronic edition of this work was published in a journal with an issn , and has been archived and is available from the following digital repositories : pubmed central and lockss .

rmbh—russian museum of the biodiversity hotspots of institute of ecological problems of the north of the ural branch of russian academy of science , arkhangelsk , 163000 , russia .

inrec—institute of natural resources , ecology and cryology of the siberian branch of russian academy of sciences , chita , 672000 , russia .

zisp—zoological institute of the russian academy of sciences , st . petersburg , 199034 , russia .

smf—forschungsinstitut und natur - museum senckenberg , senckenberg - anlage 25 , 6000 frankfurt - am - main 1 , germany .

the bayesian analysis of the nuclear 18s rrna gene revealed that the nw pacific species m . middendorffi and m . laevis belong to a well - supported monophyletic clade ( bpp 1 . 00 ) together with the north american species m . marrianae and m . falcata ( fig 2a ) . among these species , m . middendorffi is a sister to m . marrianae ( bpp 1 . 00 ) . m . dahurica , m . auricularia and c . monodonta cluster together in an unresolved clade ( bpp 0 . 79 ) , but there is a sister relationship between the two latter species ( bpp 0 . 99 ) . m . dahurica and m . auricularia both have an intra - specific variation of the nuclear 18s rrna gene with two close haplotypes in each species .

the scale bar indicates the branch length . asterisks : posterior probabilities ≥0 . 95 ; other significant support node values are mentioned in the figure . for detailed locality and specimen data for analyzed haplotypes , see the supplementary materials ( s1 and s2 tables ) . a—the 18s rdna gene dataset . b—the coi gene dataset .

the structure of hinge teeth and the shape and relative dimensions of the shell have specific characteristics that are described in detail below in the taxonomic account of each species . the density of mantle scars has no significant differences ( welch’s test : f = 2 . 45 , df = 16 , 38 , p = 0 . 12 ; ( s5 table ) ) whereas muscle scar has a high variability among studied taxa .

a— margaritifera dahurica ( middendorff , 1850 ) . b— m . laevis ( haas , 1910 ) . c— m . middendorffi ( rosén , 1926 ) . scale bar—1 cm .

a— margaritifera dahurica ( middendorff , 1850 ) . b— m . middendorffi ( rosén , 1926 ) . c— m . laevis ( haas , 1910 ) . scale bar—2 mm .

unio ( alasmodonta ) dahuricus middendorff , 1850 : [ 43 ] : 109 , [ 44 ] : 275–276 , pl . 26 , figs 3–5 .

unio ( margaritana ) dahuricus middendorff , 1850 : [ 58 ] : 699–700 .

unio margaritiferus simpson 1895 , partim ( ident . err . , non linnaeus , 1758 ) : [ 59 ] : 328 .

margaritana dahurica ( middendorff , 1850 ) : [ 23 ] : 109–112 , fig 35 , [ 24 ] : 289 , fig 250 .

margaritifera margaritifera dahurica ( middendorff , 1850 ) : [ 60 ] : 120 , [ 61 ] : 11 .

dahurinaia sujfunensis moskvicheva , 1973 ( comparatory “taxa” ) : [ 62 ] : 1468 , fig 3 , e - h .

dahurinaia sujfunica moskvicheva , 1973 ( inadv . err . in the species description ) . [ 62 ] : 1468 .

dahurinaia tiunovae bogatov & zatravkin , 1988 ( comparatory “taxa” ) : [ 63 ] : 156–158 , fig 1 , a - b .

dahurinaia komarovi bogatov et al . , 2003 ( comparatory “taxa” ) : [ 26 ] : 45–47 , figs 3a & 3d .

dahurinaia ussuriensis bogatov et al . , 2003 ( comparatory “taxa” ) : [ 26 ] : 47 , figs 3b & 3g .

dahurinaia prozorovae bogatov et al . , 2003 ( comparatory “taxa” ) : [ 26 ] : 48 , figs 3c & 3i .

dahurinaia transbaicalica klishko , 2008 ( comparatory “taxa” ) : [ 28 ] : 292–296 , figs 1–4 , 5a .

dahurinaia ( kurilinaia ) laevis klishko , 2009 ( ident . err . , non haas , 1910 ) : [ 64 ] : 237–238 , figs 1–2 .

dahurinaia ( kurilinaia ) zatravkini klishko , 2009 ( ident . err . , non bogatov et al . , 2003 ) : [ 64 ] : 238–239 , fig 3 .

margaritifera dahurica inoue et al . , 2013 ( inadv . err . ) : [ 65 ] : fig 3 ( a ) .

a—lectotype ( zisp : no . 7a ) . b—specimen from komarovka river , razdolnaya river basin , primorye . photos by i . v . vikhrev . scale bar—2 cm .

a— margaritifera dahurica ( middendorff , 1850 ) . b— m . middendorffi ( rosén , 1926 ) . c— m . laevis ( haas , 1910 ) . scale bar—1 cm .

microphotographs of the mantle attachment scars on shells of four eastern asian margaritiferid species .

a— margaritifera dahurica ( middendorff , 1850 ) . b— m . middendorffi ( rosén , 1926 ) . c— m . laevis ( haas , 1910 ) .

green circles are representing recent viable populations ( observed since 2000 ) , white circles—old records ( until 2000 ) . question mark is indicated an uncertain record from the langry river [ 69 ] , [ 26 ] . grey areas are indicated an approximate modern species range ( it is shown only for the large river systems ) . species locality numbers on the map correspond to numbers in s2 table .

a— margaritifera dahurica ( middendorff , 1850 ) : the ilystaya river , primorsky kray . b— m . middendorffi ( rosén , 1926 ) : the nachilova river , kamchatka . c— m . middendorffi ( rosén , 1926 ) & m . laevis ( haas , 1910 ) : the golovnina river , kunashir island . photos by y . v . bespalaya , y . s . kolosova & i . v . vikhrev .

“transbaikalien” [ 43 ] : 109 ; “am zusammenflusse des argunj mit der schilka” [ 44 ] : 276 ( confluence of the rivers argun and shilka , the upper amur basin : see fig 8 & s2 table , locality no . 36 ) .

the middendorff’s type series includes two specimens that are deposited in the zisp collection . bogatov et al . [ 26 ] : 45 designate specimen no . 7a as a lectotype , because it was pictured by middendorff [ 44 ] : pl . 26 , figs 3 – 5 ; and assigned the second specimen no . 7b ( single valve ) as a paralectotype . the lectotype shell has 105 mm length , 32 mm height and 25 mm width . it is noteworthy , that bogatov et al . [ 26 ] were not using the protologue of middendorff [ 43 ] for their revision , and cited only middendorff’s later work [ 44 ] .

the amur basin ( within the territories of russia , mongolia and china ) , the razdolnaya basin ( upper tributaries ) , the peschernaya ( kulumbe ) river , the iska river and the arey lake ( fig 8 & s2 table ) .

different types of watercourses , such as small streams , small and medium sized rivers , as well as large rivers ( rivers ussuri , arkhara , shilka , etc . ) ( fig 9a ) . the sole lake population was found in arey lake , transbaikalia , siberia , russia [ 28 ] ; but divers recorded only a few dead shells here in the year 2013 ( o . k . klishko , pers . comm . ) . populations prefer sand - gravel and gravel - pebble grounds at the riffles and runs . sometimes , individual specimens were observed on the clay and silt - sand bottom of the pool river sites .

not known . actually , a list of seven salmonid species , which can serve as hosts of the m . dahurica [ 36 ] , has not been verified experimentally .

all of the referred comparatory “taxa” of the genus dahurinaia were synonymized with m . dahurica based on shell morphology [ 4 ] , [ 8 ] , [ 31 ] , [ 39 ] . our molecular data confirmed this decision . for example , bogatov [ 29 ] noted that the single known dahurinaia sujfunensis population inhabits razdolnaya basin , which is separate from the amur drainage . bogatov reports that “…between the amur basin and basins of rivers of the south of primorsky kray , no common species of large bivalves has been found yet , which is explained by the historic development of these basins” [ 29 ] : 674 . however , according to new molecular and morphological data , the specimens from the razdolnaya drainage are identical to typical m . dahurica specimens from different parts of the river amur basin .

unio ( alasmodonta ) complanatus middendorff , 1851 ( ident . err . , non dillwyn , 1817 ) : [ 44 ] : 273–274 , pl . 27 , figs 1–6 .

dauhrinaia middendorffii buyanovsky , 1993 ( inadv . err . ) : [ 67 ] : 29 .

kurilinaia kamchatica bogatov et al . , 2003 ( comparatory “taxa” ) : [ 26 ] : 48 , figs 4a & 4c .

kurilinaia zatravkini bogatov et al . , 2003 , partim ( comparatory “taxa” ) : [ 26 ] : 49–50 , figs 4b & 4f .

margaritifera laevis huff et al . , 2004 ( ident . err . , non haas , 1910 ) : [ 38 ] : 381 , genbank acc . no . ay579124 .

a—holotype of m . togakushiensis [ 18 ] : 137 , figs 5 – 8 . b—lectotype of m . middendorffi ( zisp : no . 6 ) . photo by i . v . vikhrev . scale bar—2 cm .

circles— m . middendorffi locations , squares— m . togakushiensis locations . green circles and squares are representing recent viable populations ( observed since 2000 ) , white circles—old records ( until 2001 ) , yellow squares—records without exact dates . grey areas indicate approximate modern species ranges ( showing only the large river systems ) . species locality numbers on the map correspond to numbers in s3 table .

“reka golyshka” [ the river golygina , kamchatka peninsula : see fig 11 & s3 table , locality no . 1 ] [ 46 ] : 269 ; “kamtschatka ; im see mjäkéshino des südendes ( lopatka ) dieser halbinsel” ( kamchatka ; the lake mekeshino on the southernmost extremity of the peninsula : see fig 11 & s3 table , locality no . 4 ) [ 44 ] : 274 .

rivers of the kamchatka peninsula , kurile archipelago , sakhalin island ( fig 11 & s3 table ) , and likely of japan .

small streams , small - and medium - sized rivers ( fig 9b ) . usually , populations were recorded on sand - gravel grounds of the runs but , in the extreme north of the range , the species inhabited only pool river sites . in some rivers of the sakhalin and kunashir islands , far east of russia , the species coexists with m . laevis ( see fig 9b & s3 table ) .

not known . kondo & kobayashi [ 18 ] stated salvelinus leucomaenis as a host fish for m . togakushiensis . as far as we suppose m . togakushiensis and m . middendorffi conspecific , they may have the same host fish . [ 18 ] .

then , simpson [ 59 ] : 328 noted that middendorf’s specimens are “…without lateral teeth , and appear to be a stunted form of unio margaritiferus ” . finally , rosén [ 46 ] described m . middendorffi as a separate species and not as an intra - specific form of m . margaritifera . however , haas [ 61 ] , [ 60 ] incorrectly cited rosén’s name as margaritana margaritifera middendorffi . kurilinaia kamchatica bogatov et al . , 2003 is a comparatory “species” that was recently synonymized with m . middendorffi [ 31 ] . kurilinaia zatravkini bogatov et al . , 2003 is also a comparatory “species” ; its holotype belongs to m . laevis , but among the paratypes , we found four specimens of m . middendorffi ( zisp : nos . 5 , 5a , 7 and 8 ) . records of margaritifera specimens on kunashir island [ 68 ] : 134 can pertain to m . middendorffi as well as to m . laevis . therefore , some published references [ 8 ] , [ 67 ] , [ 69 ] , [ 70 ] with records from the sakhalin and southern kurile islands cannot be used without revision of the specimen samples .

margaritana dahurica kobelt , 1879 ( ident . err . , non middendorff , 1850 ) : [ 71 ] : 427–428 , pl . 13 , figs 1–2 .

unio margaritifer schrenck , 1867 , partim ( ident . err . , non linnaeus , 1758 ) : [ 58 ] : 700–704 .

unio margaritiferus simpson , 1895 , partim ( ident . err . , non linnaeus , 1758 ) : [ 59 ] : 303 .

margaritana sachalinensis zhadin , 1938 : [ 23 ] : 114–115 , fig 37 , [ 24 ] : 289–291 , fig 252 .

margaritifera margaritifera laevis ( haas , 1910 ) : [ 60 ] : 120 , [ 61 ] : 12 .

dahurinaia kurilensis zatravkin & starobogatov , 1984 ( comparatory “taxa” ) : [ 72 ] : 1789–1790 , figs 11–14 , [ 66 ] : 21 .

dahurinaia shigini zatravkin & bogatov , 1987 : ( comparatory “taxa” ) : [ 66 ] : 23–24 , fig 4b .

margaritifera ( dahurinaia ) kunahiriensis habe , 1991 ( inadv . err . ) : [ 73 ] : 3 .

dauhrinaia kurilensis buyanovsky , 1993 ( inadv . err . ) : [ 67 ] : 29 .

margaritana sacchariensis bába , 2000 ( inadv . err . ) : [ 74 ] : 133 .

kurilinaia kurilensis ( zatravkin & starobogatov ) : [ 26 ] : 42 , [ 27 ] : 25 .

kurilinaia zatravkini bogatov et al . , 2003 : partim ( comparatory “taxa” ) : [ 26 ] : 49–50 , figs 4b & 4f .

a—sennaya river , kunashir island . b—tym’ river , sakhalin island . photos by i . v . vikhrev . scale bar—2 cm .

green circles are representing recent viable populations ( observed since 2000 ) , white circles—old records ( until 2001 ) , yellow circles—records without exact dates . grey areas indicate the approximate modern species range ( showing only the large river systems ) . species locality numbers on the map correspond to numbers in s4 table .

type specimen ( holotype ) of haas [ 45 ] is deposited in the senckenberg museum , frankfurt ( no . smf3626 ) [ 39 ] .

sakhalin island , south kurile islands ( kunashir , iturup and shikotan ) , honshu and hokkaido islands ( fig 13 ) . correspondence on the species occurrence in the upper amur basin , transbaikalia [ 64 ] is based on an erroneous identification ; however , all of these specimens belong to m . dahurica based on morphological and molecular data ( see s1 table ) .

small streams , small - and medium - sized rivers . usually , populations were recorded on sand - gravel grounds at the run and pool sites . in several rivers of sakhalin , kunashir , honshu and hokkaido islands , islands , the species coexists with m . middendorffi ( see fig 9c & s4 table ) .

the masu salmon ( oncorhynchus masu ( brevoort , 1856 ) ) [ 18 ] .

in the main , species synonymy was provided in previous reviews [ 4 ] , [ 18 ] , [ 31 ] , [ 39 ] . m . perdahurica ( yokoyama , 1932 ) , m . otatumei ( suzuki , 1942 ) and m . owadaensis ( noda , 1970 ) , three fossil cenozoic margaritiferid species from japan , were also assumed as m . laevis representatives [ 39 ] . however , the condition of specimens of these species is very poor [ 75 ] , [ 76 ] , [ 77 ] , [ 78 ] . it is impossible to reliably compare these fossils with shells of recent margaritiferids . moreover , most of the known fossil specimens , including m . perdahurica and m . otatumei , are ancient and belong to the eocene or oligocene by stratigraphic classification [ 78 ] , [ 79 ] . it is unlikely that these fossils belong to recent species , but thorough revision of all specimens and collections of fossil margaritiferids would be necessary , in order to clarify the relationships of these bivalves [ 80 ] .

three margaritifera species inhabit the rivers of the russian far east , as discerned from morphological and molecular data : m . dahurica ( middendorff , 1850 ) , m . middendorffi ( rosén , 1926 ) and m . laevis ( haas , 1910 ) . however , the rivers of northeastern russia , where large mainland regions remain unexplored , are still not completely studied ; therefore the same species that were previously noted by smith [ 4 ] or additional species , may inhabit the north of the khabarovsky kray , the magadan oblast , and the koryakia and chukotka districts . these areas are difficult to access and require future studies .

m . dahurica has the largest range among the eastern asian species in the genus ( see fig 8 ) . this species is found in almost all of the major tributaries of amur basin , an area of approximately two million km 2 , as well as in razdolnaya basin and in two separate small rivers . a similar distribution pattern was found in some freshwater fish species , among which approximately 16 were endemic to this area [ 81 ] . we found only a single old record of m . dahurica from sungari river , the largest chinese tributary of the amur , and a few occurrences from mongolia . however , this species is likely widely distributed in part of the amur river system within the territory of those countries . dashi - dorgi [ 82 ] reported m . dahurica as one of the most common mussel species in the rivers of eastern mongolia . this species is not found in the rivers of the kurile islands and the most of sakhalin island . bogatov [ 69 ] and bogatov et al . [ 26 ] reported a few specimens from the langry river , which is situated on the extreme northwest of the sakhalin island . it is possible that because this river empties into the amur estuary , this river might have belonged to the ancient system of the paleo - amur [ 81 ] . the range of some amurean fish species also includes rivers in northwestern sakhalin island [ 83 ] . nevertheless , the species identification is in need of revision .

from our data , the range of m . laevis is significantly narrower than the distribution of the previous species ( see fig 13 ) . this species has a more southerly distribution , and has not been found north of central sakhalin . however , the host fish oncorhynchus masu is much more widespread northward up to kamchatka peninsula . most of the known localities of m . laevis are situated on the honshu island , and their number is linearly reduced to the north . a few fish species have a distribution resembling this , including the sakhalin taimen ( hucho perryi ( brevoort , 1856 ) ) [ 83 ] .

in this study , we analyzed the applicability of specific shell features that were indicated in previous studies for reliable species identification . three pearl mussel species were identified using a complex of morphology patterns and molecular data . the development and expression of hinge teeth , the shape and the relative dimensions of the shell , the mantle attachment scars , and the muscle scar shape are the most used conchological key features [ 4 ] , [ 10 ] , [ 18 ] , [ 23 ] , [ 24 ] , [ 34 ] , [ 38 ] , [ 47 ] , [ 43 ] , [ 44 ] , [ 45 ] , [ 46 ] , [ 95 ] , [ 96 ] , [ 97 ] , [ 98 ] , [ 99 ] .

s1 table . list of sequenced margaritifera specimens including species , localities and voucher details as well as ncbi genbank accession numbers .

s2 table . list of known localities of margaritifera dahurica ( middendorff , 1850 ) .

s3 table . list of known localities of margaritifera middendorffi ( rosén , 1926 ) and margaritifera togakushiensis ( kondo and kobayashi , 2005 ) .

s4 table . list of known localities of margaritifera laevis ( haas , 1910 ) .

s5 table . variance and significance tests results for mantle attachment scars density within three margaritiferid species .

s6 table . additional species sequences that were used in the analyses with ncbi genbank accession numbers .

we are thankful to to dr . p . v . kijashko and l . l . jarochnovich ( zoological institute of russian academy of sciences , st . petersburg , russia ) for assistance in studies of margaritifera specimens in zisp collection . the authors express their gratitude to mr . e . p . dekin ( russia ) , mr . m . a . antipin ( state nature protected area “kurilsky” , russia ) . special thanks go to mr . o . n . bespaliy ( russia ) for his excellent fieldwork assistance .

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