Fish hosts, glochidia features and life cycle of the endemic freshwater pearl mussel Margaritifera dahurica from the Amur Basin

Margaritiferidae is a small freshwater bivalve family with 16 species. In spite of a small number of taxa and long-term history of research, several gaps in our knowledge on the freshwater pearl mussels still exist. Here we present the discovery of host fishes for Margaritifera dahurica, i.e. Lower Amur grayling, sharp-snouted lenok, and blunt-snouted lenok. The host fishes were studied in rivers of the Ussuri Basin. The identification of glochidia and fish hosts was confirmed by DNA analysis. The life cycle of M. dahurica and its glochidia are described for the first time. The SEM study of glochidia revealed that the rounded, unhooked Margaritifera dahurica larvae are similar to those of the other Margaritiferidae. Margaritifera dahurica is a tachytictic breeder, the larvae of which attach to fish gills during the Late August – September and finish the metamorphosis in June. Ancestral host reconstruction and a review of the salmonid - pearl mussel coevolution suggest that the ancestral host of the Margaritiferidae was a non-salmonid fish, while that of the genus Margaritifera most likely was an early salmonid species or their stem lineage. The overfishing of lenoks and graylings appears to be the most significant threat for this rare mussel species.

The study of parasites and hosts which have been connected to each other for tens of million years contributes significantly to our understanding of the evolutionary process [1][2][3][4] . A remarkable example of such associated species is presented in freshwater environments, in which freshwater mussels of the order Unionida (Bivalves) and fishes are closely connected to each other. Unionida, or naiades, use fishes as hosts for their larva (glochidium) during the parasitic stage. Our knowledge on the mussel-fish interactions is far from being complete, because host fishes of many mussel species are still unknown 5 . Such a knowledge gap leads to underestimating the role of fish in mussel conservation 6 and hampers the development of effective mussel conservation strategies.
The freshwater pearl mussels (Margaritiferidae) is a small freshwater bivalve family with only 16 species 7 but for less than half of these species the fish hosts are known. For instance, data on the hosts of Margaritifera dahurica (Middendorff, 1850), Pseudunio homsensis (Lea, 1865), P. marocanus (Pallary, 1918), and all five representatives of the genus Gibbosula Simpson, 1900 is absent or insufficient [7][8][9][10][11] . The identification of hosts is highly important for conservation of freshwater pearl mussels which are keystone species and ecosystem engineers in freshwaters. As other naiads, freshwater pearl mussels directly impact benthic processes as they burrow through sediments and ensure deposition of nutrients as filter feeders 12,13 .
One of the causes of freshwater pearl mussel population decline is the decreasing of abundance or local extinction of host fishes [14][15][16][17][18][19] . M. dahurica is listed in "The IUCN Red List of Threatened Species" under the "Data Deficient" category 20 . It means that reliable data on the population trends and abundance of this species is yet to be obtained. Our limited knowledge on the ecology and biology of M. dahurica enables to estimate possible  Table 3). Green filling indicates co-occurrence of both the mussel and the host, and yellow filling indicates freshwater basins, in which only Brachymystax populations were recorded. Red asterisks indicate sites from which fish host samples were collected: (1) Muraveika River (tributary of the Ussuri River), and (2) Komissarovka River (tributary of the Khanka Lake). The map has been created using ESRI ArcGIS 10 software (www.esri.com/arcgis). The base of the map has been compiled from free open sources such as Natural Earth Free Vector and Raster Map Data (http://www. naturalearthdata.com), Global Self-consistent Hierarchical High-resolution Geography, GSHHG (http://www. soest.hawaii.edu/pwessel/gshhg), HydroSHEDS (Lehner, B., Verdin, K., Jarvis, A. M. dahurica is the only species within the genus, the fish hosts of which are still unknown. Its nearest neighbor, M. margaritifera, use Salmonidae as a host for glochidia, like most of Margaritifera species 7 . It is logical to suppose that the larvae of M. dahurica also use salmonids as hosts. The lack of those data is the last white spot in our knowledge on fish hosts of freshwater pearl mussels parasitizing on salmonids. In the absence of these data general patterns of coevolution of freshwater pearl mussels and salmonids are debatable 24 and cannot be resolved. The aim of the present study is to describe the life cycle of M. dahurica and to improve our understanding of coevolution of the freshwater pearl mussels and their hosts. For this purpose, we: (1) identify the fish hosts of M. dahurica; (2) describe the morphology of the glochidium stage; (3) estimate reproductive timing of M. dahurica; and (4) reconstruct ancestral fish hosts for the freshwater pearl mussels on the basis of statistical approaches and multi-locus phylogeny.

Results
Discovery of fish hosts. Ten specimens of Lower Amur grayling Thymallus tugarinae Knizhin, Antonov, Safronov & Weiss, 2007 and one specimen of blunt-snouted lenok Brachymystax tumensis Mori, 1930 were caught in May 2017 in the Muraveika River, a tributary of the Ussuri River, Russian Far East (Fig. 1). Another two fish samples were collected in a small tributary of the Komissarovka River (tributary of the Khanka Lake) (Figs 1,  2). The first sample was caught in March 2017 and it contained eighteen sharp-snouted lenoks Brachymystax lenok, seven blunt-snouted lenoks, and one Lower Amur grayling. The second sample was caught in May 2017 and it contained eight blunt-snouted and three sharp-snouted lenoks. Two Lower Amur graylings were caught in another small unnamed tributary of the Komissarovka River. Both tributaries are close to each other.
Gills of all sampled fishes were examined for glochidia. The glochidia were identified using light microscopy on one of the ten Lower Amur graylings from the Muraveyka River, and on one of the three Lower Amur graylings from a tributary of the Komissarovka River. Both of these identifications were checked and confirmed by the DNA analyses (Table 1). Every lenok, that was caught in March 2017, was infested by glochidia according to the light microscopy investigation but was not checked by the DNA analysis. Among eleven lenoks caught in May 2017 in a tributary of the Komissarovka River, glochidia were identified, using light microscopy, on six fishes, i.e. on four blunt-snouted lenoks and two sharp-snouted lenoks. All these identifications were checked by the DNA analysis and the presence of glochidia was confirmed for five fishes. Glochidia on the sixth fish were recorded by microscopy, but the DNA identification was failed, likely due to a low number of the larvae. Encysted glochidia were attached to gill filaments and clearly visible on a fresh material (Fig. 3A). The COI and cyt-b partial sequences of infested fishes were examined to confirm species identification. Molecular identification of glochidia. DNA barcoding of glochidia observed on the fish gills reveals that Lower Amur grayling, sharp-snouted lenok and blunt-snouted lenok are host species for M. dahurica (Table 1). Among 11 lenoks caught in May in the Komissarovka river, infestation of 5 lenoks was confirmed with certainty thanks to the high concentration of glochidia DNA in the samples. Other fishes had a weak signal which did not allow us to say unambiguously whether it is the mussel DNA or not. Among 12 sampled Lower Amur graylings only three fishes were infested according to the DNA barcoding analysis (Table 1) Table 2). The glochidia of the latter are discharged into a river in mid-to late summer and developed in the host gills for a period up to 10 months 34 . Mature M. dahurica collected in the mid-July had empty marsupia. Aborted glochidia were collected at the end of August from the individuals that were disturbed during measurements, which means that specimens were ready to release glochidia in the nearest future. Encysted glochidia were observed in both fish samples from March and May. Some of glochidia registered in March were of similar size as free-living larvae after releasing from mussels, and others were up to four times larger. Glochidia observed at the end of May have had less variable length from 89.4 µm to 189.1 µm (min-max values). We found significant size differences between the samples of glochidia collected in September  Table 3). The two lenok species have been recognized as two different species recently, and we therefore cannot compare the distribution of M. dahurica and every of the two lenok species separately. The lenoks were observed in all the regions in which M. dahurica was recorded (Fig. 1, Supplementary Table 3). These fishes inhabit the Amur River drainage within the Russian territory 36,37 , China 38 and Mongolia 39 . The lenoks were also recorded in rivers of the southern part of the Okhotsk Sea coast and the Russian part of the Japan Sea coast 40,41 , e.g. in the Razdolnaya River 42 .
Ancestral host reconstruction. Our Bayesian Markov chain Monte Carlo (MCMC) modeling suggests that the ancestral host of the Margaritiferidae was a non-salmonid fish (probability = 98.9%) (Fig. 5). In contrast, the ancestral host of the genus Margaritifera most likely was an early salmonid species or their stem lineage (probability = 97.8%). The hypotheses of salmonid hosts of the most recent common ancestor (MRCA) of M. margaritifera + M. dahurica subclade and of the "Pacific" subclade were also fully supported by this www.nature.com/scientificreports www.nature.com/scientificreports/ model (probability = 100%) (Fig. 5). The interactions of M. hembeli and M. marrianae with non-salmonid hosts appear to be a secondary adaptation derived from a common ancestor of both species in North America (probability = 98.4%).  46 . Regarding size, the glochidia of M. dahurica fit well within the size range of larvae of other freshwater pearl mussels, with exception of P. auricularius having the largest larvae among the Margaritiferidae (Supplementary Table 1).

Discussion
Our findings reveal that M. dahurica is a tachytictic breeder with a long-term parasitic stage which passes throughout the winter (Fig. 4). According to Klishko 33 , M. dahurica starts to release glochidia when water temperature drops below 8-10 °C, which occurs at the end of September in Eastern Siberia. It is well known that reproductive timing in freshwater mussels shifts depending on water temperature 25,27,28 . In northern regions the glochidia release starts earlier, as we observed for M. dahurica in the Tyrma River, north of Khabarovsky Kray ( Table 2). The fact that the life cycle timing of M. dahurica is very similar to that of M. margaritifera is in agreement with the phylogenetic proximity of two species 47 . Representatives of the genus Thymallus have not yet been  Table 2  www.nature.com/scientificreports www.nature.com/scientificreports/ considered as potential hosts for the larvae of freshwater pearl mussels. Our discovery of M. dahurica glochidia parasitizing on Thymallus tugarinae expands the current knowledge on the host range in the Margaritiferidae.
Our study describes only general patterns of the life cycle of M. dahurica. However, the details are still incomplete and are in need of future studies. The data such as the age of maturity, dependence between temperature and timing of juveniles drop off from the host, certain dates of life cycle stages for different regions, and testing other putative host fishes, e.g. Siberian Taimen Hucho taimen (Pallas, 1773), are of great importance for the effective conservation planning for M. dahurica.

Co-occurrence of Margaritifera dahurica and the Brachymystax species.
Our results reveal that the host-parasite interaction between M. dahurica and lenoks corresponds well to the published data on the distribution and habitat preferences of these fish taxa (Supplementary Table 3). Sandy-gravel and gravel-pebble grounds at riffles and runs, typical habitats of the freshwater pearl mussels 35 , are also inhabited by lenoks 48 . However, lenoks share a wider distribution than M. dahurica. Besides the regions inhabited by the freshwater pearl mussel, these fishes are distributed in Siberia, Korean Peninsula 49,50 , and in several Eastern Chinese rivers 51 . The sharp-snouted lenok and the blunt-snouted lenok differentiated well by molecular data and morphology, i.e. shape of the head, number of gill rakers and osteological characters. Both species coexist in sympatry through the range including the Amur Basin with the Komissarovka and Myraveika rivers. Taxonomic position and phylogeny of these two lenok forms are actively discussed 41,52-57 , however we consider blunt-and sharp-snouted forms as two separate species based on significant genetic differences even in sympatric populations. Lenok from rivers of the Qinling Mountains, belonging to the Yangtze and Yellow River basins in China, was initially described as the subspecies Brachymystax lenok tsinlingensis Li, 1966, but was recently elevated to the species level based on molecular data 58 . This taxon also inhabits south of the Korean Peninsula 50 .
Coevolution of salmonid fishes and freshwater pearl mussels. Fossil records indicate that representatives of the family Margaritiferidae were abundant in Mesozoic water bodies of Southern Laurasia 7,47 and in the north of modern Africa 59,60 . Meanwhile, it is considered that salmonids originated not earlier than in the Cenozoic era 61,62 , and Mesozoic freshwater pearl mussels, most likely, used other hosts, like the recent Margaritiferidae from southern regions (Table 3 and Fig. 5).
The most ancient fossil representatives of the genus †Eosalmo (earliest known salmonids), that were found in British Columbia 62 and Kamchatka 61 , document that ancient salmonids existed in the northern part of the Pacific Ocean drainage in the Eocene. This record corresponds well with the time of appearance of freshwater pearl mussels in the region. These mussel taxa appeared at the northern coast of the Pacific Ocean for a first time near the Paleocene -Eocene boundary 7 . †Margaritifera herrei (Hannibal, 1912) was spread in waterbodies located on the territory of the modern California 63 , while †M. perdahurica (Yokoyama, 1932), †M. otatumei (Suzuki, 1942) and †M. owadaensis Noda, 1970 inhabited territory of the modern Hokkaido Island 47 . Ziuganov et al. 24 hypothesized that spreading of freshwater pearl mussels across the northern part of the Pacific drainages was likely related to the adaptation to parasitizing on salmonids.
During the mid-Eocene epoch (~42 Ma), freshwater pearl mussels appeared on the territory of the modern Yakutia, and then spread further to the west and south 7 . Moving southward, at the mid-Oligocene (~28 Ma), freshwater pearl mussels colonized the area of the modern Aral Sea and southeast shore of Lake Baikal. These freshwater pearl mussels were considered to be a single species, †M. martinsoni Modell, 1964 47 . Remarkably, www.nature.com/scientificreports www.nature.com/scientificreports/ Brachymystax representatives appeared in paleontological records in the Upper Oligocene, e.g. †B. bikinensis Sytchevskaya,1986 was discovered in the Ussuri River drainage 61 . Today, lenoks still inhabit the rivers of Yakutia 53 and the Baikal Lake Area 39 . Therefore, it is likely that lenoks were hosts of glochidia of M. martinsoni. The fact that endemic trout (Salmo trutta oxianus Kessler, 1874) inhabits the Aral Sea drainage 64 , can be an indirect evidence that ancestors of the genus Salmo might be possible hosts for the larvae of M. martinsoni in this area. The data on the distribution of M. martinsoni and its putative host fish supports the hypothesis that this fossil taxon could be considered the MRCA of M. dahurica and M. margaritifera 47 .
As it has been shown recently, M. dahurica forms a subclade together with M. margaritifera, which is distinct from a subclade containing the other freshwater pearl mussel species from the Pacific Ocean drainage 7,47,59 .The origin of M. margaritifera cannot be tracked by available paleontological data. However, Chepalyga 65 suggested that the fossil species †M. arca Chepalyga, 1965, described from the Upper Pliocene of the Dniester and Danube river valleys, can be an ancient stem lineage of M. margaritifera.
The primary hosts of M. margaritifera are Atlantic salmon Salmo salar Linnaeus 1758, brown trout Salmo trutta Linnaeus 1758, and Arctic charr Salvelinus alpinus (Linnaeus 1758) ( Table 3). Like †M. arca, representatives of the genus Salmo also appeared in paleontological records in the Late Pliocene -Pleistocene. For example, †Salmo derzhavini Vladimirov, 1946 inhabited the territory of the modern Armenia 66 . Likely, ancestors of M. margaritifera migrated to Europe together with ancestors of Salmo or Hucho, distributed in Europe, Siberia, and the Far East of Russia. However, Hucho appeared in Europe much earlier than †M. arca, i.e. in the late Miocene 67 . Moreover, the Danube salmon Hucho hucho (Linnaeus, 1758) was recorded as a least suitable host of M. margaritifera glochidia 68 . At the same time, Siberian Taimen H. taimen can be considered a potential host for the larvae of M. dahurica as far as taimens are close to lenoks by morphological and molecular data 41 . Balakirev et al. 69 even recorded a genetic introgression between these two genera. However, this hypothesis needs to be confirmed by means of an experimental approach.
Numerous studies are dedicated to investigation of molecular phylogeny of salmonids, in particular evolutionary interactions between the genera Brachymystax, Hucho, and Salmo 70 . Osinov and Lebedev 71 , using a large dataset of protein-coding genes, revealed that Brachymystax, Hucho and Salmo belong to the same clade. An analysis of amino acid partial sequences of the COI returned the same result 70 . A genetic proximity of M. margaritifera and M. dahurica may be evidence of close evolutionary relations of their host-fishes, and correspond well to the findings of Osinov and Lebedev 71 and Artamonova et al. 70 .

Implications for conservation of Margaritifera dahurica and salmonids in the genus Brachymystax.
Most populations of M. dahurica inhabit clean mountain rivers in remote areas where sources of industrial, agricultural and recreational pollution are still lacking. Shell and pearl harvesting from M. dahurica's was praticed in the IX-V centuries BC 72,73 , but was prohibited in Russia in 1964 74 . Nowadays only illegal harvesting occurs occasionally in small amounts (personal observations). River damming bears a potential risk for several populations, but this threat requires additional research. Parasitizing of bitterling (Rhodeus sericeus (Pallas, 1776), Cyprinidae) embryos in gills of M. dahurica was mentioned among biological threats for this mussel species 23 . www.nature.com/scientificreports www.nature.com/scientificreports/ Now that, hosts of glochidia of M. dahurica have been discovered, it has become became clear that one of the main threats for this species is population decline of the Brachymystax species. Brachymystax lenok is listed as an endangered species in the "Red List of China's Vertebrates" 75 , and as a vulnerable species in the "Mongolian Red List of Fishes" 76 and "Korean Red List of Threatened Species" 77 . A few populations are listed in the "Red Data Book of the Russian Federation" 78 under the "endangered group of populations" category. All these red lists unite blunt-and sharp-snouted lenoks under the name Brachymystax lenok. Declines in the abundance of lenoks due to overfishing, water pollution and environmental changes 79 , in particular, destruction of their natural habitats caused by channel improvement 77 , was recorded in China. The main threat for lenoks in rivers of Russia is illegal fishing, which is especially intensive in small rivers of the Japan Sea basin in the Primorye Region 48 .

Mussel species Fish hosts
Finally, artificial breeding of lenoks is well developed at the present time [80][81][82] . We suggest that the artificial breeding of M. dahurica at fish hatcheries together with lenoks can be a possible approach for the future conservation of this freshwater pearl mussel species.

Methods
Data sampling. Glochidia were collected prior to the start of metamorphosis, right after release from mussels that were disturbed during measurements in the Tyrma River (Amur Basin) near the Tyrma settlement  7641°N, 131.4217°E). Rivers are located in the Primorye Region, Russian Far East. Investigated fishes were caught by a local fisherman using fishing net. Fishes that were caught in March were kept frozen until gills examination in the laboratory. Gills of every fish from all samples were examined by light microscopy. After that, a first gill arc was fixed in 5% formaldehyde to allow counting of the number of gill rakers. The rest of the fish gills, as well as snips of fish muscle soft tissues, were fixed in 96% ethanol for further DNA analyses. Fixed material was deposited in the collection of the Russian Museum of Biodiversity Hotspots (RMBH), Federal Center for Integrated Arctic Research, Russian Academy of Sciences, Arkhangelsk, Russia.

Species identification of fishes and glochidia by molecular analysis. Fish gills of Lower Amur gray-
ling (Thymallus tugarinae), blunt-snouted lenok (Brachymystax tumensis) and sharp-snouted lenok (Brachymystax lenok) infested by glochidia were taken for species identification of the mussel larvae (Table 1). Samples were stored in 96% ethanol (1:5) and DNA was extracted using the phenol/chloroform extraction procedure 83 . The COI partial sequences of glochidia were amplified by polymerase chain reaction (PCR) using the universal primer LCO1490 84 and our own designed primer MarMIDL-R (5′-GAAAAAATAGCCAAGTCTACAG-3′). The primer MarMIDL-R was used to obtain a short PCR product (423 bp) that allows examination of partly degraded DNA. Fish identification was performed by amplification of the COI partial sequences using the universal primers FishF2 and FishR2 85 , and cyt-b partial sequences using the universal primers Glu L and Thr H 86  Morphological studies of glochidia. The morphology of free-living glochidia (N = 144) and encysted larvae (N = 35) on fish gills were studied using light and scanning electron microscopy. Fresh gills with encysted glochidia were photographed with a Canon EOS 7D camera (Canon Inc., Japan) with a reversed Jupiter 37 A 70 mm lens (KOMZ, Russia) mounted on the top of a microscope (LOMO C1Y4.2, Russia). Preparation of the fixed glochidia for microscopy followed the general scheme described by Hoggarth 88 with our modifications. Glochidial mass stored in 96% ethanol was washed by deionized water and kept at 57 °C in a solution of 390 ml phosphate buffer (pH = 6.86) and 10 mk proteinase K (concentration = 4 mg/ml). The samples were inspected every 15 min and removed from the thermostat when the first glochidia with open valves were observed. Thereafter glochidia were washed with deionized water and stored in 96% ethanol prior to examination. Length, height and width measurements of free-living glochidia and encysted larvae on frozen and 5% formaldehyde fixed fish gills were performed under a light microscope (Carl Zeiss Axio Lab.A1, ZEISS, Jena, Germany) using ZEN software.
Prior to scanning electron microscopy SEM, suspended glochidia were immediately frozen at −80 °C and then freeze-dried. The images of the samples were obtained with a SEM Sigma VP ZEISS instrument (ZEISS, Jena, Germany) (10 kV, InLens detector) in the Core Facility Center "Arctic" of Northern Arctic Federal University, Arkhangelsk, Russia (unique identifier RFMEFI59417X0013). A platinum-palladium coating with a thickness up to 5 nm was applied to the surface by means of a Q150TES device (QUORUM) to enhance the image contrast.
Mapping of the distribution ranges. The georeferenced distribution data set of Bolotov et al. 35 was used for M. dahurica. The reliable records of two lenok species (Brachymystax tumensis and B. lenok) were collected from the body of available literature (Supplementary Table 3). The map has been created using ESRI ArcGIS 10 software (www.esri.com/arcgis). www.nature.com/scientificreports www.nature.com/scientificreports/ Ancestral host reconstruction analyses. We applied a Bayesian MCMC analysis implemented in RASP v. 3.2 89 . As an input tree data, we used the multi-locus fossil-calibrated phylogeny of Lopes-Lima et al. 7 , which was calculated on the basis of the most complete sequence data set (five markers: COI -654 bp, 16S rRNA -475 bp, 18S rRNA -1778 bp, 28S rRNA -307 bp, and H3 -327 bp) sampled to date. Fish hosts of the Margaritiferidae were coded as follows: (a) non-salmonid hosts, and (b) salmonid hosts. The primary data on fish hosts has been collected from Lopes-Lima et al. 7 . The hosts of several species, i.e. Gibbosula laosensis (Lea, 1863), G. crassa (Wood, 1815), Pseudunio homsensis (Lea, 1865), and P. marocanus (Pallary, 1918), remain unknown. However, they most likely associated with non-salmonid fishes (Table 3), and we therefore assigned "b" code for these taxa. The analysis was computed with 500,000 generations (sampling every 100th generation) and 10 MCMC chains (temp = 0.1). Null distribution and two-host combination (i.e. "ab") were not allowed. We applied a 10% burn-in to exclude the pre-convergence part of the simulation.
Literature searching. The literature review was performed by searching through the ISI Web of Knowledge and Scopus databases using the following keywords: Brachymystax lenok, Thymallus tugarinae, Amur river fish, Primorski krai fish. Since these databases do not take into account the body of literature in Russian, we also looked for Cyrillic sources in RISC (Russian Index for Scientific Citation, https://elibrary.ru). Old publications were searched in libraries collections of the Russian State Library, Zoological Institute of the Russian Academy of Sciences, Russian Federal Research Institute of Fisheries and Oceanography, and the Berg State Research Institute on Lake and River Fisheries.

Data Availability
The sequences generated under this study are available from the GenBank database. Accession numbers for each specimen are presented in Table 1. The voucher specimens and fish host samples are available in the Russian Museum of Biodiversity Hotspots (RMBH), Federal Center for Integrated Arctic Research, Russian Academy of Sciences, Arkhangelsk, Russia.