Return of the prodigal son: morphology and molecular phylogenetic relationships of a new Antarctic fish leech (Hirudinea: Piscicolidae) imply a bipolar biogeographic pattern

The bipolar distribution of fish leeches (Piscicolidae) has been considered and discussed by leech biologists for a long time. All cases of putative bipolar ranges of related taxa that occur in cold and temperate waters of both hemispheres and are absent in the tropics have been morphology-based hypotheses. Here, we present, for the first time, an instance of bipolar distribution substantiated by morphological and molecular data. The latter include the mitochondrial genes 12S rRNA, COI, ND1 and tRNA Leu, and the nuclear 28S rRNA. A new genus and species of Antarctic piscicolids, Austroplatybdellina prodiga, is described. The new leech was part of a Boreal-Arctic monophyletic group that is informally called ‘classic platybdellins’. That clade is the core of the non-monophyletic subfamily Platybdellinae. Austroplatybdellina prodiga gen. nov. sp. nov. was further classified as a member of a monophyletic group along with two Boreal genera, Crangonobdella and Beringobdella, which share a number of systematically important morphological features with its newly described relative. It is hypothesized that the Boreal ancestor of the new leech crossed warm tropical waters and colonized the Antarctic. The colonization was relatively recent as the low genetic distance between A. prodiga and its Boreal sister species suggests. This migration can be viewed as a return of a Boreal descendant of the Antarctic ancestor of Piscicolidae to the area of origin of the entire family, which follows from the basal position of the Antarctic Megaliobdella szidati in the family phylogenetic tree. This evolutionary scenario is reflected in the species epithet of the new leech. https://zoobank.org/urn:lsid:zoobank.org:pub:DA548F19-922A-4B5D-B7F1-4C3AF57B9824 https://zoobank.org/urn:lsid:zoobank.org:act:A35C365A-A475-4F3E-B28B-C799F618215D https://zoobank.org/urn:lsid:zoobank.org:act:BCE6411F-E38A-4A99-A726-DE5C33D9D658

Although the traditional division of the family into three subfamilies Platybdellinae, Pontobdellinae, and Piscicolinae (Epshtein et al., 1994) has been challenged by recent molecular analyses that have revealed non-monophyly of each of these groups, the family Piscicolidae is a well-supported monophyletic group (Apakupakul et al., 1999;Utevsky et al., 2007;Williams & Burreson, 2006).Despite the fact that the morphology-based subfamilial taxonomy has been abandoned, the family has a deep phylogenetic structure that is paralleled by multiple morphological and geographic features.One of the clearly definable clades is the group of 'classic platybdellins' that share a number of common morphological characteristics.The clade contains the core of what was once considered Platybdellinae, including the type genus of the subfamily, Platybdella.The group is characterized by a distinct Boreal and Arctic distribution.The species of 'classic platybdellins' have been recorded from the Arctic as well as the high latitudes of the Atlantic and Pacific (Utevsky et al., 2007).The Boreal seas may be considered their centre of diversity and origin.
Historical ties between the Arctic and Boreal seas on one hand and the Antarctic and Subantarctic waters on the other have been an intriguing issue of biogeography for a long time (Briggs, 2003;Ludt, 2021;Moreau et al., 2019;Rogers, 2007).Several putative cases of the bipolar distribution of piscicolids have been hypothesized based on morphological similarities and biogeographic speculation (Sawyer, 1986;Utevsky et al., 2021).Here, we present the first evidence of a bipolar distribution of closely related fish leech genera based on both morphological and molecular data.The undescribed fish leeches were collected in the Ross Sea, Cosmonauts Sea, and Riiser-Larsen Sea.The specimens were examined using both morphological and molecular phylogenetic methodologies to determine their taxonomic position and reveal their evolutionary relationships.Our analyses revealed an outlet of 'classic platybdellins' in the Antarctic seas.This finding represents the first instance of the penetration of Boreal-Arctic piscicolids into the Antarctic.
Another four specimens (3 lots) were collected near Enderby Land and three more leeches (1 lot) were found near Princess Martha Coast during the Belorussia Antarctic expedition (O.The specimens were stored in 70% ethanol at À20 C for morphological and molecular characterization.
One specimen of the new species from lot 147 was embedded in paraplast; transverse sections were cut at 5 mm and stained with Mallory's triple stain to examine the reproductive, digestive, and coelomic systems.

DNA extraction, amplification, and sequencing
Small pieces of muscle tissue were cut from the body of one specimen from lot 144 and from two specimens collected off the Mario Zucchelli Station.Genomic DNA was isolated using a GENE ELUTE Mammalian Genomic DNA Miniprep kit (Sigma-Aldrich, Steinheim, Germany).Mitochondrial cytochrome c oxidase subunit I gene (COI) and 12S rRNA gene fragments were amplified as described in Trontelj and Utevsky (2005).The amplifications of the mitochondrial tRNA Leu and NAD1 genes (nicotinamide adenine dinucleotide dehydrogenase subunit 1) followed Light and Siddall (1999).The nuclear 28S rRNA gene portion was amplified as described in Zaksek et al. (2007).The purified products were sequenced in both directions with amplification primers under BIGDYE terminator cycling conditions, purified by ethanol precipitation, and run on an Applied Biosystems 3730 xl sequencer by Macrogen.All fragments of genes were amplified and sequenced for one specimen from lot 144 and more COI sequences were generated for two specimens from the Mario Zucchelli Station.Sequence chromatograms were edited and assembled using ChromasPro 1.32 (Technelysium Pty., Queensland, Australia).The newly generated sequences of the samples marked as U7 (Enderby Land), TB3 and TB4 (both Terranova Bay) were submitted to the GenBank database.Their accession numbers are OQ160273, OQ160274, and OQ160275 for COI gene sequences of U7, TB3, and TB4, respectively, and OQ161991 (tRNA Leu and ND1), OQ161601 (12S), and OQ161573 (28S) for the specimen U7.
The online version of IQTREE v1.5.5 (Nguyen et al., 2015) was used to identify the optimal partitioning scheme and substitution models (Kalyaanamoorthy et al., 2017).Best-fit models were determined according to the Bayesian information criterion (BIC): for the first, TVM þ F þ I þ G for the second, and TN þ F þ G for the third position of the ND1 gene, TPM3 þ F þ G for tRNA Leu, and K2P þ G for 28S sequences.The phylogeny was inferred using maximum likelihood (ML) in the same software with branch support estimated using 1000 replicates of both the SH-like approximate likelihood-ratio test (SH-aLRT; Guindon et al., 2010) and the ultrafast bootstrapping algorithm (Minh et al., 2013).The phylogenetic tree was rooted at the outgroup with basal polytomy using PAUP Ã version 4.0a (Swofford, 2003).
The second analysis was done based on the dataset of all available GenBank COI sequences of 'classic platybdellins' and phylogenetically important piscicolid taxa plus the outgroup of non-piscicolid leeches; altogether 95 sequences (see Supplementary Materials).Problematic GenBank sequences lacking homology and/ or displaying frameshift gaps in the alignment were not included in the data set.The COI sequences were aligned using the online version of MAFFT v7 with the L-INS-i strategy selected by the 'Auto' option and checked for stop codons as already mentioned above.The lengths of the aligned COI sequences were 680 bp.The phylogenetic tree was built based on the maximum likelihood optimality criterion using the same methodology as for the multilocus dataset.Best-fit models were for the second, and TPM2u þ F þ G for the third codon position.After the phylogenetic analysis was conducted, the clade consisting of taxa that belong to 'classic platybdellins' (see Utevsky et al., 2007) was cut from the resulting tree (see Supplementary Material).Thus, the final dataset consisted of 39 samples.Some of their COI sequences were published in the studies as follows: Utevsky and Trontelj (2004), Williams and Burreson (2006), and Utevsky et al. (2007).The unpublished GenBank sequences were generated by Yanagimoto, T., Chow, S., Ichimura, M. (2016), and deWaard, J. R. (2017).
Within and between-group mean uncorrected distances and their standard errors were calculated using MEGA 11 (Tamura et al., 2021).

Ancestral state reconstruction related to geographic distribution
The geographic distribution of each piscicolid species was determined from previous studies (Utevsky et al., 2007).The possible ancestral states were reconstructed using Bayesian Binary MCMC (BBM) analysis implemented in RASP 4.3 (Yu et al., 2010(Yu et al., , 2015)).BBM analysis was run on the maximum likelihood tree based on the multilocus dataset for 50,000 generations using 10 chains and sampled every 100 generations.A fixed (JC) model was used to conduct the analysis.Only the piscicolid clade (ingroup) was chosen for the analysis.

Divergence time analysis
The COI gene tree did not uncover the expected position of Notostomum laeve Levinsen, 1882 as the sister species of Notostomum cyclostomum (Johansson, 1898) within the clade of 'classic platybdellins' (see below).This suggests an unconcerted evolution of COI in N. laeve, which precludes the use of that fragment in the timetree analysis.Therefore, the multilocus dataset with no COI fragment of N. laeve was employed for molecular dating.The substitution models of the reduced dataset did not differ from those already found for the original matrix.The ML tree built based on the modified matrix had the same topology as the original ML tree had.
A timetree was inferred by applying the RelTime method (Tamura et al., 2012(Tamura et al., , 2018) ) as implemented in MEGA 11 to the ML phylogenetic tree whose branch lengths were calculated using the ML method and the General Time Reversible substitution model (Nei & Kumar, 2000).The timetree was computed using the exponential model of probability distribution (offset 2.90 and k 1.000) and 1 calibration constraint attributed the first closure of the Bering strait (3 Ma) (Loeza-Quintana &ÁAdamowicz, 2018), which led to the divergence of the Arctic N. laeve and the North Pacific N. cyclostomum (see Sawyer, 1986).The Tao et al. (2020) method was used to set minimum and maximum time boundaries on nodes for which calibration densities were provided.Confidence intervals were computed using the Tao et al. (2020) method.A discrete Gamma distribution was used to model evolutionary rate differences among sites (þG).The rate variation model allowed for some sites to be evolutionarily invariable (þI).

Diagnosis
Small leeches.Trachelosome subcylindrical, urosome flattened.Anterior sucker small, its diameter smaller than maximum width of trachelosome.Posterior sucker medium-sized, three times or more wider than anterior sucker, disc-shaped.Eyes on anterior sucker and eyelike spots on posterior sucker lacking.No appendages on body surface.Oesophageal diverticula present, posterior crop caeca fused.Testisacs five pairs, common atrium vestigial, accessory glands lacking, bursa lacking, two separate male pores.Female reproductive system with copulatory area and conductive tissue, vagina long, female gonopore large, ovisacs loop-like.Main communication of coelomic system lacking, no marginal lacunae and pulsatile vesicles, lateral lacunae and voluminous lacunae around testisacs present.ETYMOLOGY: The species epithet prodiga is an allusion to the Parable of the Prodigal Son implying that he returns home, to the Antarctic, after long travelling in the Arctic and Boreal seas.
DIAGNOSIS: Body with transverse pigment stripes.Posterior sucker wider than maximum body width.Crop wide; intestine two-chambered, bearing two pairs of caeca; rectum long.Atrial cornua with well-developed glandular tissue inside, expanding posterior to paired gonopores.
Female reproductive system with conductive tissue.Main communication of coelomic system not confluent in ganglion region.Accessory communication encompassing testisacs and reaching both dorsal and ventral lacunae.
Anterior sucker small, separated from trachelosome by constriction; its diameter (0.70-1.54 mm) less than largest width of trachelosome and urosome (ethanol fixation and preservation resulted in its strong compression).Mouthpore central.No additional formations (tubercles and papillae) on anterior sucker.No eyes in most of preserved specimens.(One specimen from the Ross Sea has unclear spots that may be two pairs of eyes located on the sucker and one pair on the trachelosome, Fig. 4d.) Posterior sucker medium-sized, clearly separated from urosome, flat, elliptical, directed ventrally.Width of posterior sucker (2.89-4.39mm) about three times width of anterior sucker; its length 2.80-6.05mm in state of contraction.(In living individuals, the posterior sucker may be larger in size and wider than the maximum body width.)BODY COLOURATION: The pattern of colouration results from the different arrangements of the three types of chromatophores: brown, black, and white.Their composition makes the colour of the leeches quite characteristic.Even preserved specimens from Terranova Bay (Ross Sea) maintain their colouration for a longer period of time (Fig. 4).The dorsal side is much darker than the ventral side due to brown and black chromatophores densely arranged there.
There are five transverse dark brown-black spots in the form of stripes and four white spots in the form of white transverse stripes on the trachelosome.The white stripe in the clitellum area is the longest.Densely arranged chromatophores cause the dark colour, more often brown than black.Most often, brown chromatophores do not have protrusions and only their bodies remain.Black chromatophores, which are present in definitely fewer numbers, have relatively long protrusions.
On the urosome, there are 13 transverse brown-black and 13 white stripes.However, both brown-black and white stripes tend to merge just in front of the posterior  sucker and then there are fewer (10 or 11) of these stripes.The dark brown and black stripes vary in length and take from two to seven annuli.The white stripes are arranged in the same way.There are neither brown nor black chromatophores on the suckers, which are white with no discernible photoreceptors in preserved specimens.
DIGESTIVE SYSTEM: Proboscis short, not longer than anterior ganglion mass.Oesophageal diverticula short, beginning at first third of oesophagus between anterior ganglion mass and first ganglion of ventral nerve cord, not reaching middle of proboscis.Oesophagus wide.Crop wide, with four well-developed wide chambers, first chamber reduced and its place taken by ovisacs.Posterior crop caeca wide, completely fused (no fenestrae between them), forming posterior chamber with two pouches directed posteriorly.Intestine with two chambers; the first with long lateral pouches; the second wide, extending for several segments, with a pair of pouches directed posteriorly.Rectum long (Fig. 5).
REPRODUCTIVE SYSTEM: Male reproductive system (Fig. 6) with five pairs of testisacs; first pair of maximum number (6) of testisacs lacking and replaced by ovisacs.Seminal reservoirs thin, long, twisted many times and reaching anterior portions of ovisacs.Ejaculatory ducts thin, abruptly turning into large ovoid atrial cornua.Atrial cornua filled with glandular tissue; lumen inside atrial cornua not discernible.Accessory glands lacking.Common atrium vestigial.Copulatory bursa lacking, two closely located male gonopores present.The male pores are separated by a crest-like outgrowth of the upper wall of the atrium.The crest extends into a cavity situated posterior to the male pores.This cavity with the crest inside is called the 'crested organ' (Fig. 7).
Female reproductive system (Fig. 6) with very long thick-walled vagina (Fig. 8) extended from above 'crested organ' anteriorly to female gonopore posteriorly.Copulatory area located around female gonopore.Anterior portions of ovisacs and distal extremity of vagina surrounded by conductive tissue.Conductive tissue well-developed, connected by a system of channels with vagina and ovisacs, and penetrating their anteriormost parts (Fig. 6, a1, a2).Ovisacs loop-like, expanding backwards, extended to 7th ganglion of ventral nerve cord.
COELOMIC SYSTEM: Dorsal, ventral, and lateral lacunae present (Fig. 9).Main communication not completely confluent, dorsal lacuna expanded in ganglion area and connected to lateral lacuna.Additional communication completely confluent, dorsal and ventral lacunae connected to lacuna of testisac; the latter connected to lateral lacuna by a narrow channel.
Austrobdella translucens resembles the new species in size and overall body shape.However, in contrast to the new species, in A. translucens, the urosome is clearly separated from the trachelosome by 'shoulders'.The anterior sucker is also small and clearly separated from the trachelosome and, unlike the new species, has a pair of eyes.The width of the posterior sucker is less than the maximum width of the urosome and much larger than the anterior sucker.The digestive systems also have differences.The proboscis of A. translucens has a greater length and starts from the 3rd ganglion of the ventral nerve cord.Oesophageal diverticula have a different location-between 4th and 5th ganglia of the ventral nerve cord.The crop is also different from the new speciesit has six caeca.The intestines of both species are similar-they have developed chambers; the rectum is long in both species.Unlike the new species, the posterior crop caeca are not completely fused and have developed lateral pouches.The structure of the male reproductive system of A. translucens has significant differencesa well-developed atrium, long bursa and accessory glands.Its male gonopore is not paired, and the 'crested organ' is lacking.There is no conductive tissue in the female reproductive system.There are long ovisacs that reach the posteriormost pair of testisacs; the long vagina is similar to that of the new  species.The copulatory area is lacking.Significant differences are observed in the structure of the coelomic system -A.translucens has marginal channels, but no lateral lacunae as in the new species.
Epsteinia alba is twice as long, and the proportions of its body are significantly different from the proportions of the new species.The trachelosome is relatively shorter, and the urosome is clearly separated and forms 'shoulders'.Suckers are small, and the width of the posterior sucker is much smaller than the largest width of the urosome.The digestive system has well-developed oesophageal diverticula, the intestine with five broad chambers, and no posterior crop caeca.The male reproductive system is similar to the reproductive system of the new species-there are developed atrial cornua, a small atrium, and a short copulatory bursa.However, the 'crested organ' is missing.Male and female gonopores located in depressions and separated by three annuli.The copulatory area is located in the depression around the female gonopore.The female reproductive system differs significantly from that of the new species-the conductive tissue is lacking and the vagina is much shorter.The structure of the coelomic system is unknown.
Unlike the new species, T. glabra has external pulsatile vesicles on the central segments of the urosome.This is a fundamental difference.The vesicles are connected with paired lateral lacunae and main communication in the ganglion region of a urosomal segment.The main and accessory communications are fully developedthe dorsal and ventral lacunae are connected by ducts in the ganglion region and in the area of the testisacs.There are also differences in the structure of the digestive systemthe crop is tubular, and the posterior crop caeca are not completely fused, with fenestrae.The reproductive system is insufficiently studied.The shape of the body can change significantlyshrinks and stretches.
Members of the genus Trulliobdella, like the previous species, have lateral pulsatile vesicles on the central segments of the urosome.The vesicles are small in size and may go unnoticed, especially after fixation in alcohol.The lateral vesicles are connected to the lateral lacunae and main communication in the ganglion area.Unlike the new species, both communications (main and accessory) are not confluent.The digestive system differs significantly from that of the new species in having the developed chambers of the crop with lateral pouches, the intestine with five chambers with lateral pouches, incompletely fused posterior crop caeca with four fenestrae and five chambers with long lateral pouches.The reproductive system has some features similar to those of the new species.The atrial cornua are voluminous, the common atrium is much larger than that of the new species and the copulatory bursa is weakly developed.With the exception of T. bacilliformis (Brinkmann, 1948), there is a formation similar to the 'crested organ', which can turn outward.The male gonopore is not paired.The female reproductive system  has short strands of conductive tissue that extend to a wide folded vagina, which passes into a deep copulatory area that can turn outwards.Unlike the new species, the anterior sucker and adjacent trachelosome annuli have clusters of dotted eyes.Eye-like formations are present on the trachelosome, urosome, and posterior sucker.
A number of external and anatomical features found in A. prodiga are present in some members of the family Piscicolidae outside Antarctica.The medium-sized leech Beringobdella rectangulata (Levinsen, 1882) from the North Pacific also has a subcylindrical trachelosome and flattened urosome, a small anterior sucker and a much larger muscular posterior sucker (1.7 times larger than the anterior) whose width is almost equal to the width of the urosome (Utevsky, 2008).In contrast to the new species, B. rectangulata has two pairs of eyes on the anterior sucker and one on the first annuli of the trachelosome.The male reproductive system also contains well-developed atrial cornua, a small common atrium and a short copulatory bursa, but the male gonopore is not doubled.In many specimens, both male and female gonopores are located in a hollow (genital cloaca).Between the gonopores there is a tubercle, which functions as the copulatory zone.The female reproductive system contains long ovisacs and strands of conductive tissue and an elongated vagina.The coelomic system is much more reduced and consists of a ventral lacuna, which expands at the ganglion of the ventral nerve cord and gives processes back and forth.Beringobdella rectangulata parasitizes on the inner surface of the gill covers of the Far Eastern pollock Theragra chalcogramma (Pallas, 1814) and the Far Eastern cod Gadus macrocephalus Tilesius, 1810 (Epshtein, 1973;Nagasawa, 1988;Utevsky, 2008).
The Boreal-Arctic Crangonobdella fabricii (Malm, 1863) and the Boreal C. maculosa Utevsky, 2005 are similar to A. prodiga in many respects of their morphology, including their colouration, body shape, small common atrium and bursa.The long thick-walled vagina of C. maculosa is similar to that of the new species.In both species, the vagina appears to evolve due to the invagination of the external surface surrounding the female gonopore.It is not unlikely that the well-developed vagina may play the role of the receptacle of spermatophores produced by a partner individual (see Utevsky, 2005).However, C. fabricii and C. maculosa have a single male gonopore in contrast to the double one of the new species.The conductive tissue of C. fabricii is much more developed than that of A. prodiga.The coelomic systems of the leeches of the genus Crangonobdella are more reduced than that of A. prodiga.The presence of lateral tubercles on the urosome distinguishes both species of the genus Crangonobdella from the new leech (see Utevsky, 2005).
The Boreal-Arctic leech Platybdella anarrhichae (Diesing, 1859) is similar to the new species in body shape.However, P. anarrhichae lacks any colouration pattern in contrast to the A. prodiga.Moreover, P. anarrhichae has a single male genital opening.It differs from A. prodiga in the lack of the copulatory area and conductive tissue.Crop chambers of P. anarrhichae are not developed and posterior crop caeca are not completely fused, with large fenestrae between them.Its coelomic system is reduced in contrast to the new leech described herein (see Utevsky, 2010).

Phylogeny
Phylogenetic analyses of both the multilocus (Fig. 10) and COI (Fig. 11) datasets placed A. prodiga within the well-supported monophyletic group of 'classic platybdellins'.This clade includes the following genera: Platybdella, Heptacyclus, Oceanobdella, Crangonobdella, Beringobdella, and Notostomum.However, the COI sequence of Notostomum laeve Levinsen, 1882 was clustered outside this monophyletic group.Austroplatybdellina prodiga was further assigned to the clade that corresponds in part to the tribe Crangonobdellini consisting of the genera Crangonobdella and Beringobdella plus some unpublished samples from Spitzbergen.Beringobdella rectangulata is sister to A. prodiga according to the analysis of COI sequences, and C. maculosa took that place in the multilocus tree with no sequences of Beringobdella.The new species is the only Antarctic member of those Boreal-Arctic clades.According to Epshtein et al. (1994), the Antarctic genera Trulliobdella, Cryobdellina, and Antarctobdella (the latter was synonymized with Trulliobdella by Utevsky et al., 2007) are classified as members of the tribe Crangonobdellini.Our analyses challenge this classification as Trulliobdella capitis Brinkmann, 1947 was clustered outside Crangonobdella, Beringonobdella, and Austroplatybdellina.
Both analyses found that Pisicicolidae is a well-supported monophyletic group, and the Antarctic Megaliobdella szidati is the earliest splitting species of the piscicolid family tree.
The COI sequences of all three A. prodiga individuals are identical over its known range.The uncorrected genetic distances between the genera of the tribe Crangonobdellini (Table 1), including the new leech, are close to the range of the within-group mean distances of the platybdellin genera Heptacyclus (0.076 ± 0.007) and Oceanobdella (0.080 ± 0.009).

Ancestral areas
The biogeographic ancestral states reconstructed via BBM analysis are shown in Fig. 12.The last common ancestor (LCA) of all analysed piscicolid species was estimated to be Antractic with a of 91.6%.The LCAs both of all 'classic platybdellins' and the clade of A. prodiga plus C. maculosa were Arctic and Boreal with probabilities of 99.1% and 97.4%, respectively.The reconstruction at the node of the LCA of A. prodiga and C. maculosa indicates a trans-equatorial dispersal event from the Arctic and Boreal areas to Antarctica with a probability of 97.4%.

Molecular dating of the divergence of Austroplatybdellina prodiga and its closest relative
The timetree analysis found that the divergence time of A. prodiga and its North Pacific relative C. maculosa is 1.76 Ma with 0.95% confidence intervals of 3.62-0.86Ma.This range largely falls within the Pleistocene, which is the period of a series of global coolings.During a cooling of tropical waters, the ancestor of A. prodiga crossed the equator and reached the Antarctic Seas.
The divergence time of the monophyletic group of 'classic platybdellins' is 6.30 Ma (9.79-4.06Ma).

Discussion
Both morphological and molecular characters suggest that the leech described herein should be assigned to a new genus and a new species of the family Piscicolidae.Austroplatybdellina prodiga is similar in many respects to leeches of the Boreal genera Crangonobdella and Beringobdella (Epshtein, 1973;Utevsky, 2005).The common morphological characters include a flattened body separated into the trachelosome and urosome, weakly developed or lacking bursa, a long vagina and the presence of conductive tissue and a copulatory area.The new leech differs from its relatives in a pair of closely located male genital openings, a character that is very unusual for leeches (see Lukin, 1976;Sawyer, 1986;Selensky, 1915), and in its loop-like ovisacs.In addition, C. fabricii and C. maculosa can be easily distinguished from A. prodiga by tubercles on the margins of the urosome.Beringobdella rectangulata differs from the new species in having a muscular posterior sucker and in a number of details of the anatomy of its reproductive system.Austroplatybdellina prodiga has a much more developed coelomic system in contrast to the very reduced coeloms of its relatives.Obviously, the Boreal Crangonobdella and Beringobdella on the one hand Our phylogenetic analyses substantiated in part the monophyly of the tribe Crangonobdellini (Figs 10 and 11), which was erected based on some characters of the reproductive system (Epshtein et al., 1994) to include Crangonobdella, Beringobdella, and a few Antarctic genera.
Only three genera, Crangonobdella, Beringobdella, and Austroplatybdellina, are a monophyletic group that shares diagnostic characteristics of the tribe.A number of unpublished GenBank sequences, which were clustered inside that clade, await a proper morphological examination and taxonomic decision.The genetic diversity within the clade is comparable to those within the platybdellin genera Heptacyclus and Oceanobdella.However, we do not change the current classification and retain traditional naming given the substantial biological differences between Crangonobdella, Beringobdella, and Austroplatybdellina and the important distinguishing features of their reproductive systems, which usually justifies separate generic assignments in Piscicolidae (Epshtein, 1984;Epshtein et al., 1994).Both species of Crangonobdella are characteristic in their symbiotic associations with marine arthropods whose coverings are used for cocoon deposition.However, C. fabricii and C. maculosa feed on fish blood and do not consume the body liquids of arthropods (Utevsky, 2005;Utevsky & Sorbe, 2012).Beringobdella rectangulata is a common parasite of gadid fish in the North Pacific.Its muscular posterior sucker embeds deeply into the inner surface of the host operculum (Epshtein, 1973;Nagasawa, 1988).More surveys are needed to study the biology of A. prodiga.Anyway, it is clear that the members of the clade demonstrate diverse life strategies and morphological adaptations that are reflected by the current classification.
Another piscicolid tribe, Platybdellini, consisting of Platybdella, Heptacyclus, and Oceanobdella, which differs from Crangonobdellini in the lack of both the copulatory area and the well-developed conductive tissue (Epshtein et al., 1994), was not found to be monophyletic according to our phylogenetic analyses of COI and multilocus datasets.It should be noted that COI appears to work well at the level of genus.Most of the genera of 'classic platybdellins' were corroborated by the phylogenetic analysis.It was found that the North Pacific P. anarrhichae (DQ414335) is not conspecific to its North Atlantic and Arctic relatives and should be assigned to another species and genus.
The 'classic platybdellins' are a monophyletic group of Arctic and Boreal piscicolids that include the genera Platybdella, Heptacyclus, Oceanobdella, Crangonobdella, Beringobdella, Austroplatybdellina, and Notostomum.They share a number of common morphological characters and geographic patterns of distribution.All of the members of this group have rather a simple coelomic system with no pulsatile vesicles.None of the species of the clade has accessory glands on the atrium (Epshtein et al., 1994).The reconstruction of their ancestral geographic area (BBM analysis, Fig. 12) implies that 'classic platybdellins' have evolved and diversified in the Boreal waters of the North Pacific and the North Atlantic and the Arctic, while they have been never recorded for tropical, Subantarctic and Antarctic waters (Epshtein et al., 1994;Utevsky et al., 2007;2007).This research found that a leech with the body plan of 'classic platybdellins' occurs in the Antarctic.Moreover, for the first time, molecular phylogenetic analysis corroborated that conclusion.We may hypothesize that the area of warm water in the tropics was interrupted during glaciations so that cryophilic organisms crossed the strip and found themselves in the cold waters of the southern hemisphere (Ludt, 2021).According to this scenario, an ancestor of A. prodiga migrated southward.The colonization was relatively recent, given the low genetic distance between A. prodiga and its Boreal sister species.This is also suggested by our molecular dating of the divergence of A. prodiga and C. maculosa based on the calibration of the divergence of two platybdellin sister species, the Arctic N. laeve and the North Pacific N. cyclostomum, which is explained by the closure of the Bering Strait (Sawyer, 1986) approximately 3 million years ago (Loeza-Quintana &ÁAdamowicz, 2018).The trans-equatorial dispersal of the Boreal ancestor of A. prodiga dates back to the Pleistocene when a series of glaciations occurred and tropical waters might be less hostile to cryophilic organisms.However, this hypothesis is challenged by another calibration based on fossilized hirudinid cocoons (Bolotov et al., 2020).The molecular rate for the COI (6.25 Â 10 À9 subst./site/year)implies an earlier divergence of Boreal and Antarctic platybdellins that pre-dates the most recent glacial cycles.If so, the transequatorial dispersal may be assigned to the late Miocene global cooling (see Martinot et al., 2022).Obviously, more fossils and further calibrations can resolve this contradiction.Perhaps, other scenarios, e.g. a potential deep-sea route (Moreau et al., 2019;Strugnell al., 2008), cannot explain the geographic distribution of Crangonobdella, Beringobdella, and Austroplatybdellina and may be applied to the geographic distribution of deep-sea leeches such as members of the genus Pterobdellina (see Utevsky et al., 2021).Interestingly, the opposite direction of migration was the case as well.The Boreal Pterobdellina jenseni Bennike & Bruun, 1939 appears to be a descendant of southern migrants that colonized northern cold waters (Utevsky et al., 2021).
The genetic uniformity of A. prodiga in individuals separated by thousands of kilometres suggests a recent and fast dispersal of this single mtDNA haplotype around the Antarctic continent.A shallow genetic structure across vast areas is not unexpected for ectoparasites that can migrate while attached to highly mobile hosts (Trontelj et al., 2012;Utevsky et al., 2021).Nonetheless, with only three sequenced individuals it is too early to speculate about possible demographic scenarios that might explain the complete lack of genetic diversity of the COI gene.
At a larger evolutionary scale, piscicolids originated in the Antarctic as our reconstruction of biogeographic ancestral states suggests (Fig. 12).Therefore, the southward migrations from the Boreal seas to the cold waters of the Antarctic may be viewed as a return to the area of origin of the family Piscicolidae, which is reflected in the species epithet of the new leech.
In conclusion, a new genus and a new species of fish leeches that belongs to a Boreal clade were revealed in the Antarctic.This finding is the first case of both the morphologically and molecularly supported bipolar distribution of closely related taxa of Piscicolidae.Pie charts on nodes show the probabilities of possible ancestral states with numbers representing the highest probabilities (%).Ã , ancestral state with a relative probability <5%.
Austroplatybdellina prodiga sp.nov.https://zoobank.org/urn:lsid:zoobank.org:act:BCE6411F-E38A-4A99-A726-DE5C33D9D658 HOLOTYPE: SMNH 006462 (lot 344) deposited to the State Museum of Natural History of the National Academy of Sciences of Ukraine, Lviv, Ukraine.PARATYPE: þffi-RX (¼ZB-KCh) 12 (lot 344) deposited to the Zoological Museum of the Department of Zoology of Ivan Franko National University of Lviv, Lviv, Ukraine.Other specimens are stored in the collection of the Department of Zoology and Animal Ecology of V. N. Karazin Kharkiv National University and Aleksander Bielecki's personal collection.TYPE LOCALITY: Princess Martha Coast (Riiser-Larsen Sea).

Figure 10 .
Figure 10.Phylogenetic relationships of Austroplatybdellina prodiga gen.nov.sp.nov.inferred using the maximum likelihood optimality criterion based on the concatenated dataset consisting of newly generated sequences of A. prodiga and COI, 12S, ND1, tRNA Leu, and 28S fragments obtained by Utevsky et al. (2007).Values of both the SH-like approximate likelihood-ratio test (SH-aLRT) and ultrafast bootstrapping are shown for clades.

Figure 11 .
Figure 11.The clade of 'classic platybdellins' cut from the COI tree of Piscicolidae and outgroup taxa.Values of both the SH-like approximate likelihood-ratio test (SH-aLRT) and ultrafast bootstrapping are shown for clades.

Figure 12 .
Figure 12.Ancestral distributions reconstructed using Bayesian Binary Markov chain Monte Carlo analysis.Nodes are numbered.Pie charts on nodes show the probabilities of possible ancestral states with numbers representing the highest probabilities (%).Ã , ancestral state with a relative probability <5%.
Austroplatybdellina prodiga sp.nov.ETYMOLOGY: The genus name consists of the Latin root Austro, meaning 'southern' and platybdellina referring to the monophyletic group of the 'classic platybdellins'.

Table 1 .
Uncorrected between-group mean distances and their standard errors for 'classical platybdellin' genera based on COI sequences.