Advertisement
Browse Subject Areas
?

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

Taxonomy and Distribution of Freshwater Pearl Mussels (Unionoida: Margaritiferidae) of the Russian Far East

  • Ivan N. Bolotov,

    Affiliation Institute of Ecological Problems of the North of the Ural Branch of the Russian Academy of Sciences, Arkhangelsk, Russia

  • Yulia V. Bespalaya,

    Affiliation Institute of Ecological Problems of the North of the Ural Branch of the Russian Academy of Sciences, Arkhangelsk, Russia

  • Ilya V. Vikhrev ,

    vikhrevilja@gmail.com

    Affiliation Institute of Ecological Problems of the North of the Ural Branch of the Russian Academy of Sciences, Arkhangelsk, Russia

  • Olga V. Aksenova,

    Affiliation Institute of Ecological Problems of the North of the Ural Branch of the Russian Academy of Sciences, Arkhangelsk, Russia

  • Paul E. Aspholm,

    Affiliation Norwegian Institute for Agricultural and Environmental Research (Bioforsk), Svanhovd, Svanvik, Norway

  • Mikhail Y. Gofarov,

    Affiliation Institute of Ecological Problems of the North of the Ural Branch of the Russian Academy of Sciences, Arkhangelsk, Russia

  • Olga K. Klishko,

    Affiliation Institute of Natural Resources, Ecology and Cryology of the Siberian Branch of the Russian Academy of Sciences, Chita, Russia

  • Yulia S. Kolosova,

    Affiliation Institute of Ecological Problems of the North of the Ural Branch of the Russian Academy of Sciences, Arkhangelsk, Russia

  • Alexander V. Kondakov,

    Affiliation Institute of Ecological Problems of the North of the Ural Branch of the Russian Academy of Sciences, Arkhangelsk, Russia

  • Artyom A. Lyubas,

    Affiliation Institute of Ecological Problems of the North of the Ural Branch of the Russian Academy of Sciences, Arkhangelsk, Russia

  • Inga S. Paltser,

    Affiliation Institute of Ecological Problems of the North of the Ural Branch of the Russian Academy of Sciences, Arkhangelsk, Russia

  • Ekaterina S. Konopleva,

    Affiliation Institute of Ecological Problems of the North of the Ural Branch of the Russian Academy of Sciences, Arkhangelsk, Russia

  • Sakboworn Tumpeesuwan,

    Affiliation Department of Biology, Faculty of Science, Maha Sarakham University, Maha Sarakham, Thailand

  • Nikita I. Bolotov,

    Affiliation Institute of Ecological Problems of the North of the Ural Branch of the Russian Academy of Sciences, Arkhangelsk, Russia

  • Irina S. Voroshilova

    Affiliation I.D. Papanin Institute of the Biology of Inland Waters of the Russian Academy of Sciences, Yaroslavl oblast, Nekouzsky district, Borok, Russia

Taxonomy and Distribution of Freshwater Pearl Mussels (Unionoida: Margaritiferidae) of the Russian Far East

  • Ivan N. Bolotov, 
  • Yulia V. Bespalaya, 
  • Ilya V. Vikhrev, 
  • Olga V. Aksenova, 
  • Paul E. Aspholm, 
  • Mikhail Y. Gofarov, 
  • Olga K. Klishko, 
  • Yulia S. Kolosova, 
  • Alexander V. Kondakov, 
  • Artyom A. Lyubas
PLOS
x

Abstract

The freshwater pearl mussel family Margaritiferidae includes 13 extant species, which are all listed by IUCN as endangered or vulnerable taxa. In this study, an extensive spatial sampling of Margaritifera spp. across the Russian Far East (Amur Basin, Kamchatka Peninsula, Kurile Archipelago and Sakhalin Island) was conducted for a revision of their taxonomy and distribution ranges. Based on their DNA sequences, shell and soft tissue morphology, three valid species were identified: Margaritifera dahurica (Middendorff, 1850), M. laevis (Haas, 1910) and M. middendorffi (Rosén, 1926). M. dahurica ranges across the Amur basin and some of the nearest river systems. M. laevis is distributed in Japan, Sakhalin Island and the Kurile Archipelago. M. middendorffi was previously considered an endemic species of the Kamchatka. However, it is widespread in the rivers of Kamchatka, Sakhalin Island, the Kurile Islands (across the Bussol Strait, which is the most significant biogeographical boundary within the archipelago), and, likely, in Japan. The Japanese species M. togakushiensis Kondo & Kobayashi, 2005 seems to be conspecific with M. middendorffi because of similar morphological patterns, small shell size (<100 mm long) and overlapped ranges, but it is in need of a separate revision. Phylogenetic analysis reveals that two NW Pacific margaritiferid species, M. laevis and M. middendorffi, formed a monophyletic 18S rDNA clade together with the North American species M. marrianae and M. falcata. The patterns that were found in these Margaritifera spp. are similar to those of freshwater fishes, indicating multiple colonizations of Eastern Asia by different mitochondrial lineages, including an ancient Beringian exchange between freshwater faunas across the Pacific.

Introduction

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].

Reliable data on the Far Eastern Russian Margaritiferidae are relatively poor. Several old studies contain most of the available information [22], [23], [24], [25]. Unfortunately, later taxonomic revisions of the Russian Margaritiferidae are based on the so-called comparatory method that uses a single character to differentiate species, the frontal curvature of the shell [26], [27], [28], [29], [30]. This method does not account for the simple axiom about bivalve shell flexibility that shell shape has intraspecific variation according to environmental gradients [31], [32], [33], [34]. Therefore, any works that are based on the comparatory method usually cannot be used as reliable references for mussel taxonomy, although they provide a wide variety of material for species synonymy. For example, five comparatory Margaritiferidae “taxa” from the Amur Basin, were described [26], [28], [29]. Some authors published interesting data about the reproductive biology of M. dahurica and its relationships with bitterlings in Transbaikalia [35], [36]. Important studies of the Russian Margaritiferidae [8] clarified their taxonomy and provided some information on population size and biology, but ignored the comparatory method. Akiyama et al. [37] presented data with respect to two recorded Margaritifera species on Sakhalin Island.

Smith [4] summarized the available data for all of the Margaritiferidae species, including the Asian representatives, and revised the family based on morphological and anatomical data. In addition, Smith conducted an analysis of each species’ distribution using relatively detailed range maps. An analysis of the molecular phylogeny of Margaritiferidae shows that an ancient history of this Laurasian family is very difficult to interpret [10], [38]. Referenced authors have associated the problems with a pattern of extinction and contraction of an ancient Margaritiferidae fauna with the peripheral isolation of a formerly widespread taxon, fish host dispersal or even host switching. In cited papers, the sequences of only two Asian species (M. dahurica and M. laevis) were used. The Margaritiferidae taxonomy that was proposed by Smith [4], was not confirmed by recent molecular data [10], [38], [39].

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.

Based on the most spatially comprehensive sampling of the Margaritiferidae that has ever been conducted in the Far Eastern rivers, we here address three questions concerning the taxonomy and distribution of these species:

  1. How many species are living in the Far Eastern Russian rivers? What are their recent detailed ranges?
  2. What are the phylogenetic relationships of the Margaritiferidae from the Russian Far East?
  3. Is it possible to obtain a reliable identification of these species by using a morphological pattern?

Methods

Sampling

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).

thumbnail
Fig 1. Map of location of the field study areas.

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 S1S4 Tables.

https://doi.org/10.1371/journal.pone.0122408.g001

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 S2S4 Tables). A total of 554 shells from 44 localities were studied (426 from our collection and 128 museum specimens).

Morphological studies

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”.

Species range mapping

The mapping was based on the data of our field studies (see S2S4 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 S2S4 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.

PCR amplification and DNA sequencing

The present study includes new molecular data for 53 Margaritiferidae specimens (S1 Table). 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 [49] 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 M. dahurica, and C1-N-2329 was used for two other Margaritiferidae species [49], [50]. The newly designed primers 18S-IF (5´-TTCCTTAGATCGTACAATCCTAC-3´) and 18S-IR (5´-TCCTATTCCATTATTCCATGC-3´) 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 MgCl2), 0.8 units of Taq DNA polymerase (SibEnzyme Ltd., Novosibirsk, Russia), and H2O, 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, [51], [52]). In addition, 25 sequences were obtained from NCBI’s GenBank, including two sequences of Unio pictorum as outgroup (S6 Table).

Sequence alignment and phylogenetic analyses

The alignment of the COI and 18S sequences was performed using the ClustalW algorithm implemented in MEGA6 [53]. For the phylogenetic analyses, each sequence of the aligned datasets was trimmed, leaving a 654-bp COI and a 681-bp 18S fragment. The sequence datasets were collapsed into haplotypes using an online FASTA sequence toolbox (FaBox 1.41, [54]). The analyses were performed using 24 COI and 8 18S unique haplotypes, including an outgroup. The GTR+I model of sequence evolution was used for the COI gene sequences, and the K2+I model was used for the 18S gene sequences based on the corrected Akaike Information Criterion for small sample sizes (AICc) in MEGA6 [53]. Phylogenetic relationships were reconstructed for each gene separately, based on Bayesian inference as implemented in the software package MrBayes version 3.2.2 [55]. Four Markov chains, one cold and three heated (temperature = 0.1), were run simultaneously for 1,000,000 generations. The trees were sampled every 100th generation. After completing the Markov Chain Monte Carlo (MCMC) analysis, the first 2,500 trees (25%) were discarded as burn-in, and the majority-rule consensus tree was calculated from the remaining 7,500 trees. The convergence of the MCMC chains to a stationary distribution was checked visually based on the plotted posterior estimates using an MCMC trace analysis tool (Tracer version 1.6, [56]). The effective sample size (ESS) for each parameter that was sampled from the MCMC analysis was observed to be greater than 1000. The combined set of trees showed a smooth frequency plot. The resulting phylogenies were constructed using a tree figure drawing tool (FigTree software version 1.4.0 [57]).

Nomenclatural acts

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 “http://zoobank.org/”. 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.

Deposition of examined material

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.

Results

Phylogenetic analyses

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.

thumbnail
Fig 2. Bayesian phylogeny of Margaritifera spp. haplotypes.

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.

https://doi.org/10.1371/journal.pone.0122408.g002

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).

Shell and soft body morphology

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 general plan of the whole soft body of Margaritifera species is shown in Fig 3. The soft body morphology has more similarity than does the shell morphology among the studied taxa. The mantle color generally is colored cream to cafe au lait with black or brown edges (Fig 3). The gills are cream or light brown. The anterior margin inner gills are slightly longer and wider (higher) than the outer gills (Fig 3). The inner and outer gills are the same size in the middle of the posterior portion. The foot is massive, cream and dark brown distally (Fig 3). The labial palps are half-round, cream to cafe au lait, and convex dorsally. The palps have a smooth external surface and a finely canaliculated inner side. Three apertures are visible in the posterior portion: the supra-anal aperture, the exhalant and the inhalant siphons. This margin of the mantle has dark brown color with a small fold of the exhalant and a large fold of the inhalant siphon (Fig 4). The exhalant and the inhalant siphons are divided by the epithelial fold. The papillae of the inhalant siphon decrease in height in the direction of the ventral margin and are represented by monodactylous outgrowths. The morphological differences between the structures of the inhalant siphon papillae within Margaritifera spp. are presented below in the description of the each species.

thumbnail
Fig 3. General plan of soft tissue of three Far Eastern margaritiferid species.

A—Margaritifera dahurica (Middendorff, 1850). B—M. laevis (Haas, 1910). C—M. middendorffi (Rosén, 1926). Scale bar—1 cm.

https://doi.org/10.1371/journal.pone.0122408.g003

thumbnail
Fig 4. Inhalant siphon morphology of three Far Eastern margaritiferid species.

A—Margaritifera dahurica (Middendorff, 1850). B—M. middendorffi (Rosén, 1926). C—M. laevis (Haas, 1910). Scale bar—2 mm.

https://doi.org/10.1371/journal.pone.0122408.g004

Taxonomic account

Family MARGARITIFERIDAE

Margaritifera Schumacher, 1816

Margaritifera dahurica (Middendorff, 1850)—Amurean freshwater pearl mussel

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.

Margaritinopsis dahurica (Middendorff, 1850): [4]: 42.

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).

Figs 3A, 4A, 5, 6A, 7A, 8 & 9A.

thumbnail
Fig 5. Shells of Margaritifera dahurica (Middendorff, 1850).

A—Lectotype (ZISP: no. 7a). B—specimen from Komarovka River, Razdolnaya River basin, Primorye. Photos by I.V. Vikhrev. Scale bar—2 cm.

https://doi.org/10.1371/journal.pone.0122408.g005

thumbnail
Fig 6. Teeth morphology of three Far Eastern margaritiferid species.

A—Margaritifera dahurica (Middendorff, 1850). B—M. middendorffi (Rosén, 1926). C—M. laevis (Haas, 1910). Scale bar—1 cm.

https://doi.org/10.1371/journal.pone.0122408.g006

thumbnail
Fig 7. 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).

https://doi.org/10.1371/journal.pone.0122408.g007

thumbnail
Fig 8. Range map of Margaritifera dahurica (Middendorff, 1850).

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.

https://doi.org/10.1371/journal.pone.0122408.g008

thumbnail
Fig 9. Typical habitats of the Eastern Asian margaritiferid species.

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.

https://doi.org/10.1371/journal.pone.0122408.g009

Type locality.

“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).

Type.

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 35; 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].

Morphology.

The shell is equivalve and inequilateral, large (max length 196.5 mm, N = 221), elongate-oval and flat. The anterior margin is rounded, smoothly following the ventral margin, which is slightly concave in the middle nearer to the ventral edge (Fig 5). The posterior margin is wide, oval and rounded-triangular. The dorsal margin is slightly curved in passing from the posterior margin. The umbos are not projecting. The right and the left valves have only pseudo-cardinal teeth (Fig 6). Lateral teeth are absent or reduced to faintly visible rudimentary lamellae. The right valve has a pseudo-cardinal tooth, which is pyramidal, strong and canted with deep streaks and serrated edge. The anterior pseudo-cardinal tooth of the left valve is inconspicuous, often reduced and similar to a pointed tubercle; the posterior pseudo-cardinal tooth is triangular and slight with unpronounced streaks. The anterior adductor muscle scar is deep and clear; the posterior adductor muscle scar not deep and more weakly pronounced. Mantle attachment scars are deep, rounded and well pronounced and their sharpness varies from rounded to bean-like (Fig 7A). The nacre is white with oil spots. The aperture of the inhalant siphon (Fig 4A) of M. dahurica has large, tightly fitting papillae. The papillate outgrowths are small and tightly grouped (in the form of inflorescences) on the apex of the papillae. The papillae are branched from the edges of the mantle.

Distribution.

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).

Habitats.

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.

Host fishes.

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

Remarks.

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.

Material examined.

See S1 and S2 Tables.

Margaritifera middendorffi (Rosén, 1926)—Middendorff’s freshwater pearl mussel

Unio (Alasmodonta) complanatus Middendorff, 1851 (ident. err., non Dillwyn, 1817): [44]: 273–274, pl. 27, Figs 1–6.

Unio margaritiferus Simpson 1895, partim (ident. err., non Linnaeus, 1758): [59]: 328.

Margaritana middendorffi Rosén, 1926: [46]: 269–270, [23]: 112–114, Fig 36, [24]: 289, Fig 251.

Margaritana margaritifera middendorffi Rosén 1926: [61]: 12.

Dahurinaia middendorffi (Rosén, 1926): [66]: 16.

Dauhrinaia middendorffii Buyanovsky, 1993 (inadv. err.): [67]: 29.

Margaritinopsis middendorffi (Rosén, 1926): [4: 42].

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.

Figs 3C, 4B, 6B, 7B, 9B, 10 & 11.

thumbnail
Fig 10. Shells of Margaritifera togakushiensis (Kondo and Kobayashi, 2005) and Margaritifera middendorffi (Rosén, 1926).

A—Holotype of M. togakushiensis [18]: 137, figs 58. B—Lectotype of M. middendorffi (ZISP: no. 6). Photo by I. V. Vikhrev. Scale bar—2 cm.

https://doi.org/10.1371/journal.pone.0122408.g010

thumbnail
Fig 11. Range map of Margaritifera middendorffi (Rosén, 1926) and Margaritifera togakushiensis (Kondo and Kobayashi, 2005).

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.

https://doi.org/10.1371/journal.pone.0122408.g011

Type locality.

“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.

Type.

Middendorff [44]: 273–274, pl. 27, Figs 16 described this species as Unio (Alasmodonta) complanatus and pictured two specimens of the species. In his protologue, Rosén [46]: 270 fully noted the Middendorff description and indicated the shell size of one Middendorff specimen as 93 mm in length, 48 mm in height and 32 mm in width. Rosén did not see these specimens but used their images from the work of Middendorff. In addition, Rosén [46] was used for the description of two other specimens that were collected by Mr. Ruvinsky. The larger specimen had a shell length of 79 mm, a shell height of 40 mm and a shell width of 28 mm; the smaller specimen had shell dimensions of 56.25 mm, 33.75 mm and 20 mm, respectively (its left valve had a 3-mm-diameter pear-shaped pearl with a dirty light brown color). Rosén had only this material for the species description, as noted in the paper. Thus, these four specimens constitute a type series of M. middendorffi; all of these specimens are deposited in the ZISP. Bogatov et al. [26]: 45 designated the specimen as no. 6, which was described and pictured by Middendorff [44]: 273–274, pl. 27, Figs 36, as a lectotype. However, Bogatov et al. [26]: 45 erroneously considered 16 specimens as paralectotypes because they included many additional specimens from two type localities into the type series. At the same time, Bogatov et al. [26] replaced both of Rosén’s specimens (true paralectotypes) by a type series of their new comparatory “taxon” Kurilinaia kamchatica (one of these specimens was assigned as a holotype of this comparatory “species”). However, we found that only three of these specimens were really paralectotypes of M. middendorffi (by ZISP catalogue: “paralectotype of M. middendorffi no. 1”; “holotype of Kurilinaia kamchatica no. 1”; “paratype of K. kamchatica no. 2”).

Morphology.

The shell is small relative to other margariferid species (max length 93 mm, N = 193), oval, slightly convex and rhomboid in shape (Fig 10). The anterior margin is rounded, smoothly following the slightly curved ventral margin. The posterior margin is angular. The dorsal margin is slightly curved with a prominent umbo. Two pseudo-cardinal teeth and rudiments of two lateral teeth are in the left valve, while a pseudo-cardinal tooth and a rudimentary lateral tooth are in the right valve. The right valve has a thin triangular pseudo-cardinal tooth serrated at the apex. A deep triangular groove passing through a thickened fold is arranged behind the pseudo-cardinal tooth. The anterior pseudo-cardinal tooth in the left valve is short; the posterior pseudo-cardinal tooth is trapezoidal or rarely triangular with deep streaks and is serrated at the apex. The lateral teeth in both valves are presented as thin rudimentary lamellae. The anterior adductor muscle scar is deep; the posterior scar is pronounced more weakly. The mantle attachment scars are not well pronounced (Figs 10 and 7B). The nacre is pink in the anterior edge and bluish-silvery in the posterior edge with oil spots. The papillae of the inhalant siphon are large and differently digitated. The outgrowths on the apex of the papillae of M. middendorffi are slightly branched in comparison with those of M. laevis and M. dahurica (Fig 4B). The papillae are branched below the edges of the mantle.

Distribution.

Rivers of the Kamchatka Peninsula, Kurile Archipelago, Sakhalin Island (Fig 11 & S3 Table), and likely of Japan.

Habitats.

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).

Host fishes.

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].

Remarks.

Middendorff [44] erroneously identified this species as Unio complanatus.

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.

Material examined.

See S1 & S3 Tables.

Margaritifera laevis (Haas, 1910)—Japanese freshwater pearl mussel

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.

Ptychorhynchus laevis Haas, 1910: [45]: 498.

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.

Margaritinopsis laevis (Haas, 1910): [4]: 42.

Kurilinaia kurilensis (Zatravkin & Starobogatov): [26]: 42, [27]: 25.

Kurilinaia laevis (Haas, 1910): [26]: 45.

Kurilinaia zatravkini Bogatov et al., 2003: partim (comparatory “taxa”): [26]: 49–50, Figs 4B & 4F.

Figs 3B, 4C, 6C, 7C, 9C, 12 & 13.

thumbnail
Fig 12. Shells of Margaritifera laevis (Haas, 1910).

A—Sennaya River, Kunashir Island. B—Tym’ River, Sakhalin Island. Photos by I. V. Vikhrev. Scale bar—2 cm.

https://doi.org/10.1371/journal.pone.0122408.g012

thumbnail
Fig 13. Range map of Margaritifera laevis (Haas, 1910).

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.

https://doi.org/10.1371/journal.pone.0122408.g013

Type locality.

“Sakhalin Island.”

Type.

Type specimen (holotype) of Haas [45] is deposited in the Senckenberg Museum, Frankfurt (no. SMF3626) [39].

Morphology.

The shell is large (max length 139.5 mm, N = 140) and elongate-elliptical and sometimes is compressed laterally in large specimens (Fig 12). The anterior margin is rounded; the dorsal margin is slightly curved, almost parallel to the ventral margin and smoothly follows to the posterior margin. The ventral margin is slightly concave or straight. The umbos are not projected. Two pseudo-cardinal teeth and the rudiments of two lateral teeth are in the left valve, while a pseudo-cardinal tooth and a rudimentary lateral tooth are in the right valve. The right valve has a pseudo-cardinal tooth that is strong, high, triangular and serrated. A triangular groove is arranged between the pseudo-cardinal teeth of the left valve. The left valve has an anterior pseudo-cardinal tooth that is triangular, smaller and shorter than the posterior one, which is crenulated from the side groove. The posterior pseudo-cardinal tooth of the left valve is high, pyramidal and serrated peripherally. The lateral teeth in both valves are presented as rudimentary narrow lamellae that are slightly serrated in the distal part. The anterior adductor muscle scar is deep; the posterior scar is unclear. The mantle attachment scars are well pronounced, numerous, sharply rounded with a characteristic groove and arranged diagonally to the shell (Figs 7C and 12). The nacre always has oil spots and is brilliant-violet, white or white in the periphery and pink in the middle of the valve. The inhalant siphon structure of M. laevis is generally similar to that of M. dahurica (Fig 4C). However, we noted the following distinctive features: the papillae of the inhalant siphon of M. laevis are high and branched significantly lower of the mantle edge.

Distribution.

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).

Habitats.

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).

Host fishes.

The masu salmon (Oncorhynchus masu (Brevoort, 1856)) [18].

Remarks.

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].

Material examined.

See S1 & S4 Tables.

Discussion

How many species are distributed in the rivers of the Russian Far East? What are their detailed recent ranges?

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 km2, 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.

M. middendorffi is also a widespread species that inhabits many rivers across the Northwestern Pacific, from Kunashir Island to the Kamchatka (see Fig 11). Previously, this taxon was considered a local endemic for Kamchatka [4], [23], [24], [25], [31]. It is interesting that M. middendorffi ranges across the Bussol Strait, lying between the Urup and Simushir Islands (Central Kurile), which is the most significant biogeographical boundary within the Kurile Archipelago [84]. M. middendorffi and M. togakushiensis seem to be conspecific because of similar morphological patterns, small shell size (<100 mm) and overlapped ranges. However, the complicated relationships between these poorly known Far Eastern Margaritifera taxa preclude final taxonomic solution, which must be carefully verified using sequences for specimens from the type locality of M. togakushiensis (type series or newly collected topotypes).

The number of M. middendorffi localities increases northward. For example, on Kunashir Island, only three populations were found, but this species successfully inhabits the cold subarctic rivers of the Kamchatka and Northern Kuriles. Using data from the literature, this species ranges only in the rivers of the Western Kamchatka, Okhotsk Sea Basin [4], [8], [25]. This phenomenon is explained by the fact that these rivers in the Pleistocene were tributaries of the Paleo-Penzhina River system [25]. Therefore, the report by Eyerdam [85] of an occurrence of Margaritifera shell in the downstream part of the river Kamchatka (Pacific drainage) was questioned [25]. However, we found a reliable sample of shells from the Paratunka River of Eastern Kamchatka, which flows into the Pacific Ocean (see S3 Table, loc. no. 8). These data indicate that the Margaritifera record from the Kamchatka River is most likely true and that this species has a wider range on the Kamchatka Peninsula, but a large part of the region is difficult to access and is still poorly investigated.

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].

What are the phylogenetic relationships of the Margaritiferidae from the Russian Far East?

Based on the nuclear 18S rRNA gene, M. laevis and M. middendorffi cluster together with two North American species, M. falcata and M. marrianae. These results support the hypothesis of an ancient Beringian exchange between the freshwater mussel faunas of Northeastern Asia and North America [86]. These data also correspond to results of molecular studies of cyprinid fishes of Eurasia and North America, where the Far Eastern phoxinins have dispersed from North America to the Far East across the Beringia land bridge during the Late Cretaceous or Early Paleocene [87]. It is interesting, that recent populations of M. marrianae are isolated in a few tributaries of the Alabama and the Conecuh (Escambia) rivers [4], [88]. It is known that North American Margaritiferidae are not limited strictly to drainages of the Great Basin and Pacific Coast but also occur in parts of the upper Missouri drainage, where they are represented by one species M. falcata [86].

According to the COI gene sequences, we found a similar “Beringian” clade, formed of M. laevis, M. middendorffi and M. falcata. Unfortunately, the position of M. marrianae based on the COI phylogeny remains uncertain, because there are only three very short COI sequences for this species in NCBI’s Genbank, which are unsuitable for phylogenetic assessment. However, the minimal COI distances were found between M. laevis and two other species, M. marrianae (4.7%) and M. middendorffi (5.5%), which is in agreement with results, obtained from 18S sequences. M. dahurica is the sister species of M. margaritifera, which is distributed across Western Europe and in the river basins of the Atlantic coast of North America [4], [8], [10], [38], [65]. Similar patterns have been found for several freshwater fishes that have closely related representatives from Amur Basin and European rivers, with a huge gap in the Siberian Plain [89], [90], [91], [92], [93]. Most Eastern Asian species have very low intraspecific COI divergence, particularly M. dahurica and M. middendorffi. For each of these species, only two COI haplotypes were observed. For twelve examined M. margaritifera populations, a lack of differentiation was observed in the COI and 16S genes, and these populations may be considered as a metapopulation [9]. A survey of five populations of North American species Margaritifera hembeli (Conrad, 1838) showed extensive allozyme monomorphism [94]. These authors mentioned that low genetic diversity appears to be characteristic of Margaritiferidae, as an ANOVA indicated that mussels of the family Margaritiferidae (only M. auricularia, M. margaritifera and M. hembeli were tested) have a significantly lower heterozygosity level than do mussels of the family Unionidae. These authors assumed that, although bottlenecks are known to cause low genetic variability, Margaritiferidae might exhibit metapopulation structure with extinction/re-colonization dynamics leading to low genetic variability. Meanwhile, both COI and microsatellite loci have substantially high levels of genetic variation between C. monodonta populations that are, most likely, associated with the Pleistocene history of the Mississippi Basin [65]. However, the genetic diversity within each of the populations was low, indicating high gene flow after isolation during the last glacial period.

Is it possible to reliably identify the Far Eastern species using morphological patterns?

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].

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).

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 structure and expression of pseudo-cardinal and lateral teeth have a low interspecific variability (Fig 6) and could be reliable characteristics for identification of Margaritiferidae species [4]. The specific characteristics of the structure of the hinge plate in the right valve of M. middendorffi, namely the arranged deep groove passing through a thickened fold, are typical only for this species and are present in adults, as well as juvenile individuals. Zhadin [23] indicated this key feature, but it was not mentioned within hinge descriptions in later studies of the species [86]. The reduced anterior pseudo-cardinal tooth of the left valve is the basic distinctive feature for M. dahurica that has been noted in numerous studies [4], [23], [24], [28], [43], [98]. The lateral teeth of M. laevis are present as thin rudimental lamellae in both of the valves [4], [18], [23], [24]. In all other species of the Far Eastern pearl mussels, lateral teeth are absent or are present as weakly pronounced rudimentary lamellae.

According to Smith [4], [99], the mantle attachment scars on the inner surface of the valves are formed by modified epithelial cells, and specific patterns of the distribution and density of the scars can be identified among taxa. Therefore, the presence of mantle-attachment scars in M. marocana was sufficient to separate M. marocana and M. auricularia [10]. However, our data do not confirm significance of this character for species identification within Margaritiferidae. Fig 7 shows that the mantle scar shape has a high intra-specific variability that overlaps with the differences between taxa. Based on visual assessments, it seems that M. middendorffi has weaker and less frequent scars than M. dahurica or M. laevis.

In addition to shell morphology, the soft body details were also analyzed. The structure of inhalant siphon papillae is often used for species identification [28], [34], [95], [96], [97]. The Far Eastern pearl mussels have differences in this organ. The length of papillae, their frequency, the characteristics of papillae outgrowth, and the position with respect to the mantle edge can be patterns for species identification. Our data confirm the previous description of the M. dahurica inhalant siphon [28], [34], whose papillae are large and have variable widths. The papillae in the siphon of M. dahurica are branched over the mantle edges, in contrast to the papillae of M. laevis and M. middendorffi. In addition, the papillae of M. laevis are more branched but in other respects, their structure is similar to that of the papillae of M. dahurica. Similarly to the mantle attachment scar pattern, the structure of the inhalant siphon of M. middendorffi is the most distinct; its papillae are less branched and thinner. The structure of the gills and labial palps have no specific features and, despite some differences, could not be used to identify Margaritifera spp.

Conclusions

The present study focuses on the freshwater pearl mussels of the genus Margaritifera. This study provides novel insight into the taxonomy of the Far Eastern Margaritiferidae species, their re-description based on morphological patterns, the molecular identification of each species and reliable data for the species ranges, including maps and locality information with precise coordinates. Our results provide an important framework for further research on the phylogeny, ecology, life history and biogeography of one of the most stenobiontic freshwater faunas. Future studies should focus on the unexplored areas of Eastern Asia, including the Russian Far East (the northern region of the Khabarovsky kray, the Magadan oblast, and the Koryakia and Chukotka districts), Korea and Eastern China. Currently, we have negligible information on the freshwater bivalves of these huge regions, which is one of the largest gaps in the current biogeography of the freshwater pearl mussels. Thus, further research should form a basis for understanding the evolutionary history and biogeographical patterns of these freshwater “living fossils” of ancient Laurasian origin.

Supporting Information

S1 Table. List of sequenced Margaritifera specimens including species, localities and voucher details as well as NCBI GenBank accession numbers.

https://doi.org/10.1371/journal.pone.0122408.s001

(DOC)

S2 Table. List of known localities of Margaritifera dahurica (Middendorff, 1850).

https://doi.org/10.1371/journal.pone.0122408.s002

(DOC)

S3 Table. List of known localities of Margaritifera middendorffi (Rosén, 1926) and Margaritifera togakushiensis (Kondo and Kobayashi, 2005).

https://doi.org/10.1371/journal.pone.0122408.s003

(DOC)

S4 Table. List of known localities of Margaritifera laevis (Haas, 1910).

https://doi.org/10.1371/journal.pone.0122408.s004

(DOC)

S5 Table. Variance and significance tests results for mantle attachment scars density within three margaritiferid species.

https://doi.org/10.1371/journal.pone.0122408.s005

(DOC)

S6 Table. Additional species sequences that were used in the analyses with NCBI Genbank accession numbers.

https://doi.org/10.1371/journal.pone.0122408.s006

(DOC)

Acknowledgments

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.

Author Contributions

Conceived and designed the experiments: INB YVB IVV. Performed the experiments: INB YVB IVV OVA MYG OKK YSK AVK ISP ESK ST NIB ISV. Analyzed the data: INB YVB IVV AVK. Contributed reagents/materials/analysis tools: YVB IVV OVA PEA MYG OKK YSK AVK AAL ISP ESK ST NIB ISV. Wrote the paper: INB YVB IVV AVK.

References

  1. 1. Graf DL, Cummings KS. Review of the systematics and global diversity of freshwater mussel species (Bivalvia: Unionoida). J Mollus Stud. 2007; 73: 291–314.
  2. 2. Bogan AE, Roe KJ. Freshwater bivalve (Unioniformes) diversity, systematics, and evolution: status and future directions. J N Am Benthol Soc. 2008; 27: 349–369.
  3. 3. Graf DL. Patterns of freshwater bivalve global diversity and the state of phylogenetic studies on the Unionoida, Sphaeriidae, and Cyrenidae. Am Malacol Bull. 2013; 31: 135–153.
  4. 4. 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.
  5. 5. Liu X-Z. On some newly discovered non-marine pelecypods from the Late Triassic Wuzhongshan Formation in Sichuan Basin [in Chinese]. Bulletin of the Chengdu Institute of Geology and Mineral Resources, Chinese Academy of Geological Sciences. 1981; 2: 121–136.
  6. 6. Fang Z-J, Chen J, Chen C, Sha J, Lan X, Wen S. Supraspecific taxa of the Bivalvia first named, described, and published in China (1927–2007). The University of Kansas Paleontological Contributions, New Series. 2009; 17: 1–157.
  7. 7. Graf DL, Cummings KS. Palaeoheterodont diversity (Mollusca: Trigonioida + Unionoida): what we know and what we wish we knew about freshwater mussel evolution. Zool J Linn Soc Lond. 2006; 148: 343–394.
  8. 8. Ziuganov V, Zotin A, Nezlin L, Tretiakov V. The freshwater pearl mussels and their relationships with salmonid fish. Moscow: VNIRO, Russian Federal Institute of Fisheries and Oceanography; 1994. 135 p.
  9. 9. 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.
  10. 10. Araujo R, Toledo C, Van Damme D, Ghamizi M, Machordom A. Margaritifera marocana (Pallary, 1918): a valid species inhabiting Moroccan rivers. J Mollus Stud. 2007; 75: 95–101.
  11. 11. Geist J. Strategies for the conservation of endangered freshwater pearl mussels (Margaritifera margaritifera L.): a synthesis of conservation genetics and ecology. Hydrobiologia. 2010; 644: 69–88.
  12. 12. Akiyama Y, Iwakuma T. Survival of glochidial larvae of the freshwater pearl mussel, Margaritifera laevis (Bivalvia: Unionoida), at different temperatures: a comparison between two populations with and without recruitment. Zool Sci. 2007; 24: 890–893. pmid:17960993
  13. 13. Akiyama YB, Iwakuma T. Growth parameters of endangered freshwater pearl mussel (Margaritifera laevis, Unionoida). Fund Appl Limnol. 2009; 175: 295–305.
  14. 14. Kobayashi O, Kondo T. Age determination of the freshwater pearl mussel Margaritifera laevis (Bivalvia: Margaritiferidae) in the Chubu-Nougu River, Nagano Prefecture [in Japanese]. Venus. 2008; 67: 61–71.
  15. 15. Kondo T. Monograph of Unionoida in Japan (Mollusca: Bivalvia) [in Japanese]. Special publication of the Malacological Society of Japan. 2008; 3: 1–69.
  16. 16. Uchiyama R, Kondo T. Margaritifera laevis, M. togakushiensis (Margaritiferidae). In: Animal Distribution Atlas of Japan. The Biodiversity Center of Japan, the Nature Conservation Bureau, the Ministry of the Environment, Japan; 2010. p. 43
  17. 17. Kurihara Y, Sakai H, Kitano S, Kobayashi O, Goto A. Genetic and morphological divergence in the freshwater pearl mussel, Margaritifera laevis (Bivalvia: Margaritiferidae), with reference to the existence of two distinct species. Venus. 2005; 64: 55–62.
  18. 18. Kondo T, Kobayashi O. Revision of the Genus Margaritifera (Bivalvia: Margaritiferidae) of Japan, with description of a New Species. Venus. 2005; 64: 135–140.
  19. 19. Kobayashi O, Kondo T. Difference in host preference between two populations of the freshwater pearl mussel Margaritifera laevis (Bivalvia: Margaritiferidae) in the Shinano river system, Japan. Venus. 2005; 64: 63–70.
  20. 20. Kobayashi O, Kondo T. Comparative morphology of glochidia and juveniles between two species of freshwater pearl mussel Margaritifera (Bivalvia: Margaritiferidae) from Japan [in Japanese]. Venus. 2007; 65: 355–363.
  21. 21. Kobayashi O, Kondo T. Reproductive ecology of the freshwater pearl mussel Margaritifera togakushiensis (Bivalvia: Margaritiferidae) in Japan [in Japanese]. Venus. 2009; 67: 189–197.
  22. 22. Buldovsky AT. About harvested freshwater mussels of the Soviet Far East [in Russian]. Proceedings of the Far Eastern Branch of the USSR Academy of Sciences. 1935; 12: 39–65.
  23. 23. Zhadin VI. Fam. Unionidae [in Russian]. Faune de l’URSS, New Series. 1938; 4: 1–170. pmid:25389754
  24. 24. Zhadin VI. Mollusks of fresh and brackish waters of the USSR [in Russian]. Identification guides on the USSR fauna, issued by Zoological Institute of the USSR Academy of Sciences. 1952; 46: 1–376.
  25. 25. Kurenkov II. On distribution of the Kamchatka freshwater pearl mussel [in Russian]. Questions of Kamchatka geography. 1966; 4: 110–112.
  26. 26. Bogatov VV, Prozorova LA, Starobogatov YI. The family Margaritiferidae (Mollusca: Bivalvia) in Russia. Ruthenica. 2003; 13: 41–52.
  27. 27. Starobogatov YI, Prozorova LA, Bogatov VV, Saenko EM. Mollusks [in Russian]. In: Freshwater invertebrates of Russia and adjacent territories: an identification guide. Vol. 6: Mollusks, Polychaetes, and Nemertines. St. Petersburg: Nauka Publ.; 2004. pp. 9–422.
  28. 28. Klishko OK. Dahurinaia transbaicalica 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.
  29. 29. Bogatov VV. Pearl Mussels (Bivalvia, Margaritiferidae, Dahurinaia) from the Amur River Basin. Biol Bull. 2012; 39: 672–675.
  30. 30. Bogatov VV. A lengthy discussion concerning the composition of the genus Margaritifera Schum., 1915 [sic] (Mollusca, Bivalvia). Biol Bull+. 2013; 40: 488–481.
  31. 31. Graf DL. Palearctic freshwater mussel (Mollusca: Bivalvia: Unionoida) diversity and the comparatory method as a species concept. P Acad Nat Sci Phila. 2007; 156: 71–88.
  32. 32. Preston SJ, Harrison A, Lundy M, Roberts D, Beddoe N, Rogowski D. Square pegs in round holes—the implications of shell shape variation on the translocation of adult Margaritifera margaritifera (L.). Aquat Conserv. 2010; 20: 568–573.
  33. 33. Bolotov IN, Makhrov AA, Bespalaya YuV, Vikhrev IV, Aksenova OV, Aspholm PE, et al. The results of comparatory method testing: frontal section curvature of shell valve could not be a systematic indicator for freshwater pearl mussels of Margaritifera genus. Biol Bull. 2013; 40: 221–231.
  34. 34. Klishko OK. Pearl Mussels of the Genus Dahurinaia (Bivalvia, Margaritiferidae): Differently Sized Groups of Margaritifera dahurica Middendorff, 1850. Biol Bull. 2014; 41: 434–443.
  35. 35. 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.
  36. 36. Klishko OK, Bogan AE. The conservation status of the freshwater Pearl Mussel Margarititifera dahurica in Far Eastern Russia. Ellipsaria. 2013; 15: 31–33.
  37. 37. Akiyama B, Kimura R, Nomoto K, Usui T, Machida Y. New record of the freshwater pearl mussel Margaritifera togakushiensis from northern Sakhalin, the Russian Far East. Venus. 2013; 71: 191–198. pmid:23542829
  38. 38. 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.
  39. 39. Graf DL, Cummings KS. The freshwater mussels (Unionoida) of the World (and other less consequential bivalves), updated 8 August 2013. MUSSEL Project Web Site. Available: http://www.mussel-project.net. Accessed 4 October 2014.
  40. 40. Bogan AE. Freshwater bivalve extinctions (Mollusca: Unionoida): a search for causes. Am Zool. 1993; 33: 599–609.
  41. 41. Haag WR. Past and future patterns of freshwater mussel extinctions in North America during the Holocene. In: Turvey S., editor. Holocene Extinctions. New York: Oxford University Press; 2009. pp. 107–128.
  42. 42. Iwata H, Ukai Y SHAPE: A computer program package for quantitative evaluation of biological shapes based on elliptic Fourier descriptors. J Hered. 2002; 93: 384–385. pmid:12547931
  43. 43. Middendorff ATv. Beschreibung einiger neuer Mollusken-Arten, nebst einem Blicke auf den geographischen Charakter der Land- und Süsswasser-Mollusken Nord-Asiens. Bulletin de la Classe Physico-mathématique de l'Académie Impériale des Sciences de St.-Pétersbourg. 1850; 9: 108–112.
  44. 44. Middendorff ATv. Wirbellose Thiere: Annulaten. Echinodermen. Insecten. Krebse. Mollusken. Parasiten. Reise in den Äussersten Norden und Osten Sibiriens, Zoologie. 1851; 2: 163–508. pmid:24870494
  45. 45. Haas F. New Unionidae from East Asia. The Annals and Magazine of Natural History, 8th series. 1910; 6: 496–499.
  46. 46. Rosén OV. Terrestrial and freshwater mollusks, collected by Kamchatka Expedition of F. P. Riabushinsky in 1908–1909 [in Russian]. Annuaire du Musée Zoologique de l’Academie des Sciences de l’URSS. 1926; 27: 261–274.
  47. 47. Brandt RAM. The non-marine aquatic mollusca of Thailand. Archiv für Mollusckenkunde. 1974; 105: 1–423. pmid:24715412
  48. 48. Sayenko EM. New data on soft parts morphology of the anodontine bivalves from Russia [in Russian]. The Bulletin of the Russian Far East Malacological Society. 2007; 11: 100–106.
  49. 49. Folmer O, Black M, Hoeh W, Lutz R, Vrijenhoek R. DNA primers for amplification of mitochondrial cytochrome C oxidase subunit I from diverse metazoan invertebrates. Molecular Marine Biology and Biotechnology. 1994; 3: 294–299. pmid:7881515
  50. 50. Simon C, Frati F, Beckenbach A, Crespi B, Liu H, Flook P. Evolution, weighting, and phylogenetic utility of mitochondrial gene sequences and a compilation of conserved polymerase chain reaction primers. Ann Entomol Soc Am. 1994; 87: 651–701.
  51. 51. Hall TA. BioEdit: a user-friendly biological sequence alignment editor and analysis. Department of Microbiology, North Carolina State University. 1999.
  52. 52. Hall TA. BioEdit version 7.2.5. 2013. Available: http://www.mbio.ncsu.edu/bioedit/bioedit.html. Accessed 1 May 2014.
  53. 53. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Molecular Biology and Evolution. 2013; 30: 2725–2729. pmid:24132122
  54. 54. Villesen P. FaBox: an online toolbox for fasta sequences. Mol Ecol Notes. 2007; 7 (6): 965–968.
  55. 55. Ronquist F, Teslenko M, van der Mark P, Ayres DL, Darling A, Höhna S, et al. MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Syst Biol. 2012; 61: 539–542. pmid:22357727
  56. 56. Rambaut A, Suchard M, Drummond A. J. Tracer v1.6. 2013. Available: http://beast.bio.ed.ac.uk/software/tracer/. Accessed 1 June 2014.
  57. 57. Rambaut A. FigTree. Tree Figure Drawing Tool Version 1.4.0. 2012. Available: http://tree.bio.ed.ac.uk/software/figtree/. Accessed 1 June 2014.
  58. 58. Lv Schrenck. Süsswasser- und Land-Mollusken. Reisen und Forschungen im Amur-Lande. 1867; 2: 605–726.
  59. 59. Simpson CT. The classification and geographical distribution of the pearly freshwater mussels. Proceedings of the United States National Museum. 1895; 28: 295–343.
  60. 60. Haas F. A tentative classification of the Palearctic unionids. Zoological Series of Field Museum of Natural History. 1940; 24: 115–141.
  61. 61. Haas F. Superfamilia Unionacea. Das Tierreich. 1969; 88: 1–663.
  62. 62. Moskvicheva IM. Najades (Bivalvia, Unionoidea) of Amur River basin and Primorye territory [in Russian]. Zoologicheskii Zhurnal. 1973; 52: 1458–1471.
  63. 63. Bogatov VV, Zatravkin MN. New species of the order Unioniformes (Mollusca Bivalvia) from the south of Soviet Far East. Proceedings of the Zoological Institute of the USSR Academy of Sciences. 1988; 171: 155–168.
  64. 64. Klishko OK. Sakhalin-Kurile species of pearl mussels (Bivalvia: Margaritiferidae) from Transbaikalye. The Korean Journal of Malacology. 2009; 25: 237–242.
  65. 65. Inoue K, Monroe EM, Elderkin CL, Berg DJ. Phylogeographic and population genetic analyses reveal Pleistocene isolation followed by high gene flow in a wide ranging, but endangered, freshwater mussel. Heredity (online publication). 2013.
  66. 66. Zatravkin MN, Bogatov VV. Large bivalve molluscs in fresh and brackish waters of the Far East of the USSR: An identification guide [in Russian]. Vladivostok: Far Eastern Branch of the USSR Academy of Sciences; 1987. p. 152.
  67. 67. Buyanovsky AI. On the ecology of the Far-East pearl mussels Dauhrinaia [sic] middendorffii [sic] and Dauhrinaia [sic] kurilensis (Bivalvia, Margaritiferidae) [in Russian]. Zoologicheskii Zhurnal. 1993; 72: 29–36.
  68. 68. Miyadi D. Bottom fauna of the lakes in Kunashiri-sima of the South Kurile Islands. Internationale Revue der Gesamten Hydrobiologie und Hydrographie. 1938; 37: 125–163.
  69. 69. Bogatov VV. New data on Unioniformes of the Sakhalin Island. [in Russian]. The Bulletin of the Russian Far East Malacological Society. 2001; 5: 71–77.
  70. 70. Prozorova LA, Bogatov VV, Sayenko EM. New data on the freshwater mollusk fauna of Sakhalin Island [in Russian]. In: Flora and fauna of Sakhalin Island (Materials of International Sakhalin Project), vol. 1 (Storozhenko S. Yu., ed.) Vladivostok: The Far Eastern Branch of the Russian Academy of Sciences, Dalnauka Publ.; 2004. pp. 138–144.
  71. 71. Kobelt W. Fauna japonica extramarina. Abhandlungen der Senckenbergischen Naturforschenden Gesellschaft. 1879; 11: 285–496.
  72. 72. Zatravkin MN, Starobogatov YI. New species of the superfamily Unionoidea (Bivalvia, Unioniformes) from the Soviet Far East [in Russian]. Zoologicheskii Zhurnal. 1984; 63: 1785–1791.
  73. 73. Habe T. Catalogue of non-marine molluscs in Japan. Hitachiobi. 1991; 56: 3–7.
  74. 74. Bába K. An area-analytical zoogeographical classification of Palearctic Unionaceae species. Bolletino Malacologica. 2000; 36: 133–140.
  75. 75. Yokoyama M. Tertiary Mollusca from the coalfield of Uryu, Ishikari. Journal of the Faculty of Science, Imperial University of Tokyo, Section. 1932; 23: 221–224.
  76. 76. Yokoyama M. Tertiary fossils from various localities in Japan. Part III. Special Papers of the Palaeontological Society of Japan. 1959; 5: pl. 86.
  77. 77. Suzuki K. Notes on some Tertiary non-marine mollusca from North Nippon [in Japanese]. Journal of the Geological Society of Japan. 1944; 51: 100–109.
  78. 78. Noda H. Freshwater molluscs from the coal-bearing Owada formation, Southeast Rumoi, Hokkaido, Japan. Transactions and Proceedings of the Palaeontological Society of Japan. 1970; 77: 235–242.
  79. 79. Honda Y. Paleogene molluscan faunas from Hokkaido, northern Japan. In: Kotaka T, editor. Japanese Cenozoic Molluscs—Their Origin and Migration. Palaeontological Society of Japan (special papers, no. 29). 1986; pp. 3–16 pmid:21545707
  80. 80. Scholz H, Tietz O, Büchner J. Unionoid bivalves from the Miocene Berzdorf Basin (eastern Germany): taxonomic remarks and implications for palaeoecology and palaeoclimatology. Neues Jahrbuch für Geologie und Paläontologie. Abhandlungen. 2007; 244: 43–51.
  81. 81. Bogutskaya NG, Naseka AM, Shedko SV, Vasil'eva ED, Chereshnev IA. The fishes of the Amur River: updated check-list and zoogeography. Ichthyological Exploration of Freshwaters. 2008; 19: 301–366.
  82. 82. Dashi-Dorgi A. To the knowledge of the waterbodies and hydrofauna of the eastern and northern Mongolia (Amur and Selenga rivers basin on the territory of the Mongolia People Republic). Abstract of thesis of the dissertation (Biology). Irkutsk: Irkutsk State University; 1953. 20 p.
  83. 83. Reshetnikov YuS (ed.) The fishes in nature reserves of Russia. Vol. 1. Moscow: KMK Publishers; 2010. 627 p.
  84. 84. Pietsch TW, Bogatov VV, Amaoka K, Zhuravlev YuI, Barkalov VYu, Gage S, et al. Biodiversity and biogeography of the islands of the Kuril Archipelago. J Biogeogr. 2003; 30: 1297–1310.
  85. 85. Eyerdam WJ. A collection of freshwater shells from Kamchatka. The Nautilus. 1938; 52 (2): 56–59.
  86. 86. Taylor DW. Aspects of freshwater mollusc ecological biogeography. Palaeogeogr Palaeocl. 1988; 62: 511–576.
  87. 87. Imoto JM, Saitoh K, Sasaki T, Yonezawa T, Adachi J, Kartavtsev YP, et al. Phylogeny and biogeography of highly diverged freshwater fish species (Leuciscinae, Cyprinidae, Teleostei) inferred from mitochondrial genome analysis. Gene. 2013; 514: 112–124. pmid:23174367
  88. 88. Johnson RI. Margaritifera marrianae, 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.
  89. 89. Banarescu P. Zoogeography of Fresh Waters. Volume 2: Distribution and dispersal of freshwater animals in North America and Eurasia. Wiesbaden: AULA-Verlag GmbH; 1992. 511 p.
  90. 90. Grande T, Laten H, López JA. Phylogenetic relationships of extant Esocid species (Teleostei: Salmoniformes) based on morphological and molecular characters. Copeia. 2004; 4: 743–757.
  91. 91. Bohlen J, Slechtová V, Bogutskaya N, Freyhof J. Across Siberia and over Europe: Phylogenetic relationships of the freshwater fish genus Rhodeus in Europe and the phylogenetic position of R. sericeus from the River Amur. Mol Phylogenet Evol. 2006; 40: 856–865. pmid:16757185
  92. 92. Zaki SAH, Jordan WC, Reichard M, Przybylski M, Smith C. A morphological and genetic analysis of the European bitterling species complex. Biological Journal of the Linnean Society. 2008; 95: 337–347.
  93. 93. Zeng Q, Wang Z, Peng Z. Mitochondrial genome of Silurus asotus (Teleostei: Siluriformes). Mitochondrial DNA. 2011; 22: 162–164. pmid:22165828
  94. 94. Curole JP, Foltz DW, Brown KM. Extensive allozyme monomorphism in a threatened species of freshwater mussel, Margaritifera hembeli Conrad (Bivalvia: Margaritiferidae). Conserv Genet. 2004; 5: 271–278.
  95. 95. Araujo R, Gomez I, Machordom A. The identity and biology of Unio mancus Lamarck, 1819 (= U. elongatulus) (Bivalvia: Unionidae) in the Iberian Peninsula. J Mollus Stud. 2005; 71: 25–31.
  96. 96. Reis J, Araujo R. Redescription of Unio tumidiformis Castro, 1885 (Bivalvia, Unionidae), an endemism from the south-western Iberian Peninsula. J Nat Hist. 2009; 43: 31–32.
  97. 97. Khalloufi N, Toledo C, Machordom A, Boumaïza M, Araujo R. The Unionids of Tunisia: taxonomy and phylogenetic relationships, with redescription of Unio ravoisieri Deshayes, 1847 and U. durieui Deshayes, 1847. J Mollus Stud. 2011; 77: 103–115.
  98. 98. Smith DG. Observations on the morphology and anatomy of Margaritinopsis dahurica(Middendorff, 1850) (Unionoida: Margaritiferidae). J Conchol. 2001; 37: 119–125.
  99. 99. 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.
  100. 100. Melnychenko RK, Janovich LN, Korniushin AV. Changeability of the shells’ morfometrical characteristics, peculiarities of ecology and reproduction of the species complex U. crassus (Bivalvia, Unionidae) in the Fauna of Ukraine [In Russian]. Vestnik zoologii. 2004; 38: 19–35.
  101. 101. Zieritz A, Aldridge DC. Identification of ecophenotypic trends within three European freshwater mussel species (Bivalvia: Unionoida) using traditional and modern morphometric techniques. Biol J Linn Soc. 2009; 98: 814–825.