First freshwater mussel-associated piscicolid leech from East Asia

Parasites and symbionts of freshwater mussels are poorly understood, although a diverse assemblage of mussel-associated leeches (Glossiphoniidae) was recently described. Here, we report on the discovery of a fish leech (Piscicolidae) in the mantle cavity of the freshwater mussel Cristaria plicata (Unionidae) in the Russian Far East. It is the first member of this leech family being associated with freshwater molluscs. This leech does not match any known genus and species both morphologically and genetically, and is described here as Alexandrobdella makhrovi gen. & sp. nov. It uses mussels as shelter (and probably as a secondary host), while the Amur catfish Silurus asotus (Siluridae) seems to be the primary host. These novel findings indicate that mussel-associated leech assemblage contains at least one piscicolid species. Our fossil-calibrated phylogeny suggests that the crown group of Piscicolidae was originated in the Early Cretaceous. This primarily marine family shares at least five independent colonization events into freshwater environments.

. Type locality of Alexandrobdella makhrovi gen. & sp. nov. (A) Geographic position of the type locality (red star). The map was created using ESRI ArcGIS 10 software (https ://www.esri.com/arcgi s); the topographic base of the map was created with free open sources such as Natural Earth Free Vector and Raster Map Data (https ://www.natur alear thdat a.com), Global Self-consistent Hierarchical High-resolution Geography, GSHHG v2.3.7 (https ://www.soest .hawai i.edu/wesse l/gshhg ), and HydroSHEDS (https ://www.hydro sheds .org). (B) Coastal area of Lake Khanka at the type locality. Photo: Ilya V. Vikhrev. Table 1. List of COI and 18S rRNA gene sequences of the Hirudinea used in this study. N/A not available. *Data on environmental preferences was obtained from the IRMNG database (https ://www.irmng .org 33 and published sources 19,34 . www.nature.com/scientificreports/ Therefore, freshwater fish can be considered the primary hosts of Alexandrobdella, while the nature of its association with freshwater mussels (i.e., shelter only or a secondary host as well) remains largely unclear.
The deep subfamily-level nodes of our two-locus Bayesian phylogeny (COI + 18S rRNA) were rather weakly supported, while shallower nodes corresponding to generic and intra-generic clades shared high and moderate support values (BPP ≥ 0.95 and ≥ 0.75, respectively). Phylogenetically, Alexandrobdella makhrovi gen. & sp. nov. was recovered within a weakly supported clade that contains Limnotrachelobdella spp. and an unidentified www.nature.com/scientificreports/ marine leech species (Fig. 2). This group appears to be a part of the subfamily Piscicolinae, which was also weakly supported in our research.
Our ancestral trait modeling suggested that there were at least five independent colonization events of piscicolids to fresh and brackish water: (i) Piscicola + Baicalobdella + Cystobranchus respirans + Caspiobdella  Etymology. This genus is named for Dr. Alexander Makhrov, a prominent Russian ichthyologist and evolutionary biologist, and "bdella", the Greek word for leech.
Diagnosis. Body flattened dorso-ventrally, clearly divided into short trachelosome and elongated urosome (Fig. 3). Both trachelosome and urosome taper towards corresponding suckers. Suckers well developed. Pulsatile vesicles present, rather large, knob-like. One pair of large eyes on oral sucker. Segmental and caudal ocelli absent. Urosomal segments 7 (14) annulate. Five pairs of testisacs. Large, rectangular muscular organ (seminal receptacle) associated with bursa and ovisacs. Conductive tissue present. Conductive tissue cords conduct oviducts with the seminal receptacle (Fig. 4). Ovisacs massive, asymmetric, lobed. There are no freshwater genera of the Piscicolidae having such combination of morphological characters, although Limnotrachelobdella Epshtein, 1968 and Taimenobdella Epshtein, 1987 appear to be more closely related to the new genus externally. However, Alexandrobdella gen. nov. differs from Limnotrachelobdella Epshtein, 1968 by having smaller, knob-like pulsatile vesicles, a less distinct separation of the body into trachelosome and urosome, and a smaller posterior sucker. It can be distinguished from Taimenobdella Epshtein, 1987 by having one pair of eyes and larger pulsatile vesicles, and by the lack of segmental and caudal ocelli. Based on the reproductive system morphology (Fig. 4), the new genus can be distinguished from all the other Piscicolidae genera by having an exceptionally large seminal receptacle and massive, asymmetrical, lobed ovisacs. The Antarctic genus Trulliobdella Brinkmann, 1948 is partly similar to the new genus in that it shares oviducts connecting with the seminal receptacle by conductive tissue cords 18 . However, Alexandrobdella gen. nov. has a much larger, rectangular seminal receptacle and different morphology of ovisacs compared with those in Trulliobdella species.
Phylogenetic placement. It is clear that Alexandrobdella gen. nov. represents a highly divergent lineage, which is distant phylogenetically from other freshwater and marine piscicolid genera, the DNA sequences of which were available ( Fig. 2 and Table 1). This genus appears to be more closely related to Limnotrachelobdella and an unidentified marine leech Gen.2 indet. but these relationships were poorly supported in our phylogeny (BPP = 0.35-0.45).
Morphological diagnosis. External morphology: Small leech, body flattened dorso-ventrally, clearly divided into trachelosome and urosome. Body length with suckers up to 14.5 mm, width up to 3.4 mm (at the widest part of the urosome) ( Table 2). Skin smooth, without papillae, 11 pairs of knob-like pulsatile vesicles laterally (especially noticeable during pregnancy). Integuments weakly pigmented, light yellow in fixed specimens (translucent in living leeches) with a black and dark brown pigmentation. On dorsal side accumulations of pigment form dark brown transverse stripes, which together with unpigmented areas form a characteristic 'mosaic' pattern. Ventral side light yellow, almost unpigmented. Anterior sucker small, distinctly separated, approximately as broad as the width of trachelosome, eccentrically attached. One pair of eyes located on a brown stripe near the border with trachelosome. Posterior sucker medium sized (diameter approximately 1.5-2 times larger than that of the anterior sucker), eccentrically attached, eye-like spots absent, but yellow-orange spots present. Complete somite contains seven double rings. Anus separated from the posterior sucker by two annuli. Digestive system: Proboscis medium long, muscular. Oesophagus surrounded by fine salivary glands. The number of chambers of the crop in the sample was not determined. Posterior crop caeca fused incompletely, persist five fenestrae. Intestine with four chambers bearing lateral processes. Rectal dilatation located posteriorly to the posterior crop caeca. Reproductive system: Gonopores are separated by two annuli. Testisacs 5 pairs, relatively large, oval. Seminal reservoirs short. Ejaculatory ducts long, muscular, forming loops. Terminal parts of the ejaculatory ducts voluminous, spherical. Copulatory bursa small. Accessory glands not found. Ovisacs long, massive, consisting of some lobes, asymmetrical, located posteriorly of the voluminous seminal receptacle. Oviducts connected by conductive tissue cords with the seminal receptacle, which forms internal copulatory area. Externally, the copulatory area could appear as swelling of the ventral surface of clitellum.
Life style. At first glance, this new leech appears to be a mussel-associated species that uses the mantle cavity of a freshwater mussel as shelter. However, its relationship with freshwater mussels (i.e., shelter only or a hostparasite association) requires further research. The proportion of Cristaria plicata mussels infested by at least one leech in our sample from the type locality was 9.7%, and the intensity of leech infestation there was 0.10 ± 0.05 leeches per mussel (mean ± s.e.m.; N = 31 mussels and 3 leeches).
Distribution. This species is known only from its type locality in the southwestern part of Lake Khanka, Russian Far East (Fig. 1).

Discussion
First association of a piscicolid leech with freshwater mussels. Earlier research revealed that global mussel-associated leech assemblage includes at least 12 species belonging to the family Glossiphoniidae 2 . Our novel discovery of a piscicolid leech in the mantle cavity of a freshwater mussel from East Asia expands our knowledge of mussel-leech associations. The DNA analyses of the crop content of an Alexandrobdella makhrovi gen. & sp. nov. paratype indicated that the adult leech feeds on fish blood, i.e., uses the Amur catfish (and probably other freshwater fish species as well) as the primary host. However, nothing is known on the interactions between this leech and its host mussel. The mussel-associated glossiphoniids (Hemiclepsis and Batracobdelloides) use freshwater mussels as shelters and secondary hosts but adult leeches need to take one or several fish-blood meals to complete their life cycle 2 .
Freshwater piscicolid leeches are known to feed on fish blood almost exclusively 9,19 , while one species (Gonimosobdella virginica comb. nov.) was found to be a possible obligate egg feeder in nests of four fish taxa 20 . Conversely, the marine Piscicolidae share a much broader host range, with numerous species being associated with vertebrate and invertebrate taxa such as fishes, turtles, crustaceans, pycnogonids, and octopuses 7,21-24 . A brief review of the body of literature, outlined above, revealed that piscicolid leeches could use a variety of invertebrate animals as hosts, at least in marine environments. Hence, it is unclear whether Alexandrobdella feeds on its mussel host or uses it as available shelter only. Possible host-parasite relationships of the fish leech with freshwater mussels deserve further research efforts.

Taxonomic issues.
A new genus and species, Alexandrobdella makhrovi gen. & sp. nov., are introduced here for a mussel-associated fish leech from East Asia. This leech clearly differs from other members of the family by having an enormous seminal receptacle, which posteriorly connects with large, asymmetrical, lobed ovisacs. In its turn, the proximal parts of oviducts connected with the seminal receptacle by conductive tissue cords.
Based on our two-locus phylogeny, two freshwater Cystobranchus and one marine Calliobdella species from North America are transferred here to Gonimosobdella (Table 1), as it was already suggested in a dissertation 19 .
The new combinations are proposed as follows: Gonimosobdella salmositica (Meyer, 1946) comb. nov., G. virginica (Hoffman, 1964) comb. nov., and G. vivida (Verrill, 1872)  www.nature.com/scientificreports/ Multiple colonization events of marine fish leeches into fresh water. Our ancestral trait modeling supports the hypothesis that the Piscicolidae is primarily a marine group of leeches 14,17 . The crown group of this clade was likely originated in the Early Cretaceous. There were at least five independent colonization events of the marine fish leeches to fresh and brackish water environments since the Late Cretaceous. Multiple independent colonization events of piscicolids to oceanic trenches were also recorded, with subsequent morphological and ecological adaptations to extreme deep-sea environments 8,25 .

Methods
Data sampling and molecular analyses. Leeches were collected from the mantle cavity of living freshwater mussels Cristaria plicata (Bivalvia: Unionidae) that were found near the shore of Lake Khanka after a strong storm. Their habitat was a shallow littoral area of the lake with silty-sand bottom (Fig. 1). The mussel specimens were opened with an extractor and their mantle cavity was visually examined for leeches and other inhabitants that were sampled by forceps 2 . During the 2-day period (24-25.v.2017), we examined 31 specimens of Cristaria plicata, and collected three individuals of Alexandrobdella makhrovi gen. & sp. nov. The leech infestation prevalence (%) and intensity of leech parasitism estimates were calculated using the equations described in our previous papers 1,2 .
We obtained sequences of the mitochondrial cytochrome c oxidase subunit I (COI) and the nuclear small subunit of ribosomal RNA (18S rRNA) gene fragments from one specimen of the novel species (paratype RMBH Hir_0084) using the laboratory protocols and primers followed those described in our earlier work 2 .
Divergence time estimates and statistical biogeography. We sampled a comprehensive two-locus (COI + 18S rRNA) molecular dataset with 43 Piscicolidae species (Table 1). Additionally, sequences of two Ozobranchidae species were collected as outgroup. Each partition was aligned separately using the MUSCLE algorithm of MEGA7 26 . The 18S rRNA alignment was processed with GBlocks v0.91b 27 using a set of available options for less stringent selection to eliminate hypervariable flanking and poorly aligned regions from the alignment (in summary, 74% of the initial alignment were excluded). The COI (665 bp) and 18S rRNA (1698 bp) alignments were joined to a combined alignment using FaBox v1.5 28 . Divergence time was estimated using BEAST v1.10.4 29 . The best-fit evolutionary model HKY + G + I was applied to each partition. To dating the phylogeny, we used an external mean molecular rate for the COI (6.25 × 10 −9 subst./site/year) and 18S rRNA (1.99 × 10 −10 subst./site/year) genes that were obtained based on a comprehensive fossil-calibrated phylogeny of the Hirudinea 2 . A lognormal relaxed clock algorithm and the Yule speciation process were applied as the tree priors. Two independent runs, each with 25,000,000 generations (sampling every 5000 cycles) were performed at the San Diego Supercomputer Center (SDSC, University of California, San Diego, USA) through the CIPRES Science Gateway 30 . The resulting log files were checked for convergence of the MCMC chains with Tracer v1.7 31 .
The ESS values for all parameters were recorded > 200. The sets of time-calibrated trees obtained from the two runs were joined through LogCombiner v1.10.4 29 with 10% burn-in. The maximum clade credibility tree was constructed based on 9000 binary time-calibrated trees using TreeAnnotator v1.10.4 29 .
Ancestral trait modeling was calculated using Bayesian Binary MCMC algorithm implemented in RASP v3.2 32 based on the set of 9000 time-calibrated binary trees and the user-specified maximum clade credibility tree obtained in the previous BEAST analyses (see above). We coded three possible types of environmental preferences of leech species as follows: (a) freshwater, (b) marine, and (ab) euryhaline. The analysis was run with the following settings: 500,000 generations, sampling every 100th generation, 10 MCMC chains with temperature = 0.1 and 10% burn-in. Null distribution was not allowed.
Nomenclatural acts. The electronic edition of this article conforms to the requirements of the amended International Code of Zoological Nomenclature (ICZN), 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 (https ://zooba nk.org), the online registration system for the ICZN. The LSID for this publication is as follows: https ://zooba nk.org/urn:lsid:zooba nk.org:pub:33993 F3F-FFD1-4688-8219-3F6D9 1D81A 05. The electronic edition of this paper was published in a journal with an ISSN, and has been archived and is available from PubMed Central.

Data availability
The type series of the new species is available in the RMBH-Russian Museum of Biodiversity Hotspots, Federal Center for Integrated Arctic Research of the Ural Branch of the Russian Academy of Sciences, Arkhangelsk, Russia. The sequences used in this study are presented in GenBank.