Cryptic taxonomic diversity and high-latitude melanism in the glossiphoniid leech assemblage from the Eurasian Arctic
Suborder Glossiphoniiformes Tessler & de Carle, 2018Family Glossiphoniidae Vaillant, 1890Comments. Our two-locus phylogeny reveals the presence of two large clades, corresponding to the subfamilies Glossiphoniinae and Haementeriinae (Fig. 1). The subfamily Theromyzinae Sawyer, 1986, delineated by some authors2,10,22, was not supported as a distant phylogenetic clade and their representatives are clustered within the monophyletic Glossiphoniinae. The same pattern was recovered by earlier phylogenetic reconstructions3,30,33,34. These data indicate that Theromyzinae may represent a synonym of the latter subfamily. However, a subfamily-level revision of the Glossiphoniidae is beyond the framework of the present study.Subfamily Glossiphoniinae Vaillant, 1890Genus Alboglossiphonia Lukin, 1976Type species: Alboglossiphonia heteroclita (Linnaeus, 1761) (= Hirudo heteroclita Linnaeus, 1761; by original designation).Arctic occurrences. Our results reveal that members of this genus are not common inhabitants of the Arctic but two species, A. heteroclita (Linnaeus, 1761) and A. sibirica sp. nov., cross the Arctic Circle on the Yamal Peninsula through the Ob and Taz rivers (Table 1). Previously, it was shown that A. heteroclita occurs in the lower Ob Basin, northern edge of Western Siberia23.Comments. This genus contains inconspicuous minute leeches and is characterized by a nearly global distribution1. It definitely requires an integrative taxonomic revision. Available genetic evidence (Fig. 1 and Supplementary Fig. S1) reveals that the North American populations of what was traditionally assigned to A. heteroclita should be considered a separate species, A. pallida (Verrill, 1872) (type locality: West River near New Haven, Connecticut, USA)35,36. Other species, which occurs in Siberia and the Far East, was tentatively assigned to Alboglossiphonia cf. papillosa (Braun, 1805) based on a darker pigmentation of its dorsum37,38 but it represents a separate North Asian species, which is described here.
Alboglossiphonia sibirica Bolotov, Eliseeva, Klass & Kondakov sp. nov = Alboglossiphonia heteroclita Lukin (1957): 27339 (identification error). = Alboglossiphonia heteroclita papillosa Kaygorodova et al. (2014): 337; Kaygorodova (2015): 4140 (identification error). = Alboglossiphonia cf. papillosa Klass et al. (2018): 2638 (identification error).Figures 4a, 5a, 7a, Supplementary Figs. S2a, S3a, S4, Supplementary Table S2.LSID: https://zoobank.org/urn:lsid:zoobank.org:act:19B581C3-E912-487C-B9EC-8E50DDEFD380.Holotype. RMBH Hir_0542_2-H (non-sequenced), RUSSIA: Lake Torfyanka, 43.0761° N, 131.9620° E, Vladivostok, Primorye, August 12, 2021, Y. E. Chapurina leg.Paratypes (N = 13). RUSSIA: 1 specimen RMBH Hir_0542_2 (sequenced: COI sequence acc. No. ON873332), the type locality, the same date, and collector; 1 specimen RMBH Hir_0396 (non-sequenced), an oxbow lake of Taz River, near Tazovsky settlement, 67.5063° N, 78.6751° E, Yamal-Nenets Region, August 22, 2019, E. S. Babushkin leg.; 1 specimen RMBH Hir_0394 (DNA voucher; sequenced: COI sequence acc. No. ON548508), Vitim River, 57.2010° N, 116.4300° E, Lena River basin, Vitimsky Nature Reserve, Irkutsk Region, July 12, 2019, E. S. Babushkin leg.; 4 specimens RMBH Hir_0013 (3 sequenced with DNA vouchers and one placed on 36 permanent slides as a series of slices; COI sequence acc. No. MH286267, MH286268, and MH286269; 18S rRNA sequence acc. No. MH286273), between zooids of a bryozoan colony, small floodplain lake of the Lena River near Yakutsk, 62.3076° N, 129.8999° E, Yakutia Republic, August 20, 2017, I. N. Bolotov leg.; 1 specimen RMBH Hir_0417_2 (DNA voucher; sequenced: COI sequence acc. No. ON548511), Oron Lake, Gnilaya Kurya Bay, 57.1750° N, 116.4031° E, Lena River basin, Vitimsky Nature Reserve, Irkutsk Region, July 1, 2019, E. S. Babushkin leg.; 1 specimen RMBH Hir_0409_1 (sequenced: COI sequence acc. No. ON548509), a roadside ditch in Knevichi settlement, 43.3886° N, 132.1880° E, Primorye, September 10, 2020, O. V. Aksenova et al. leg.; 1 specimen RMBH Hir_0413 (sequenced: COI sequence acc. No. ON548510), a puddle near railway at Artem city, 43.3794° N, 132.2188° E, Primorye, September 10, 2020, O. V. Aksenova et al. leg.; 1 specimen RMBH Hir_0003_3 (DNA voucher; sequenced: COI sequence acc. No. MN393256), Tumnin River, 49.9451° N, 139.9181° E, Khabarovsk Region, July 14, 2014, I. N. Bolotov & I. V. Vikhrev leg.; 1 specimen RMBH Hir_0509_1 (sequenced: COI sequence acc. No. ON548516), a reservoir on the Bolshoy Alim River, near Tolstovka settlement, 50.1981° N, 127.9431° E, Amur Region, July 3, 2021, O. V. Aksenova et al. leg.; 1 specimen RMBH Hir_0510_1 (DNA voucher; sequenced: COI sequence acc. No. ON548517), an oxbow lake of Bureya River, near Novospassk, 49.6756° N, 129.7343° E, Amur Region, July 3, 2021, O. V. Aksenova et al. leg.Etymology. The name of this species reflects its broad distribution in Siberia.Differential diagnosis. Small leech, which could be distinguished from other congeners by a combination of the following characters: dorsum covered by numerous small, shallow, and indistinct papillae, light yellow, with multiple dark spots and short dashes arranged to 18–24 longitudinal rows; these spots and dashes merged into longitudinal lines in the anterior half of the body (the dark markings pattern often lost in ethanol-preserved animals). Externally, the new species is similar to A. heteroclita, A. hyalina (O. F. Müller, 1773), and A. striata (Apáthy, 1888). However, all these species do not have numerous dark spots and short dashes arranged to multiple longitudinal rows. Additionally, A. heteroclita differs from the new species by having a median row of segmentally arranged dark spots and a smooth dorsum without papillae. A. hyalina differs from A. sibirica sp. nov. by the general lack of dark pigmentation. A. striata differs from the new species by having a median row of segmentally arranged dark transverse stripes and a smooth dorsum without papillae.Molecular diagnosis. The new species represents a separate genetic lineage but is more closely related to A. heteroclita (mean pairwise COI p-distance = 5.1%; range 4.9–5.4%). The intraspecific pairwise COI p-distance ranges from 0.0 to 2.1% (mean ± s.e.m. = 1.31 ± 0.10%; N = 14 sequences and 91 pairwise distance values). The GenBank acc. numbers of reference DNA sequences (COI and 18S rRNA) are given in Supplementary Table S2 and Supplementary Datasets S1–S2.Description. Small leech (body length up to 11.9 mm). Measurements of the type series are given in Supplementary Table S2. Body broad, leaf-like, ovate. Dorsum with numerous small, shallow, and indistinct papillae. Posterior sucker small, circular (maximum diameter of 2.25 mm), ventrally directed. Proboscis pore in the center of anterior sucker. Coloration of living animals: body dirty yellow with multiple brown spots and dashes arranged to longitudinal rows; in the anterior half of the body, these spots and dashes merged into longitudinal lines. Coloration of ethanol-preserved animals: body light yellow; dorsum with multiple dark spots and short dashes arranged to 18–24 longitudinal rows; these spots and dashes merged into longitudinal lines in the anterior half of the body but the dark markings pattern often lost due to preservation. Three pairs of eyespots; the eyespots of the distal pair joined into a single spot; the eyespots of the next two pairs are spaced apart and fused together. Venter light yellow or whitish. Total number of annuli: 70. Somites I–IV joined to form a head region, somites V–XXIV triannulate, somites XXV–XXVII uniannulate. Gonopores joined and open in the furrow XIIa1/a2. Reproductive system: 6 pairs of large, bag-like testisacs inter-segmentally from XIII/XIV to XIX/XX; atrium small, spherical, the atrial cornua twisted anteriorly; paired ejaculatory ducts twisted, short; paired ovisacs narrow, very short. Digestive system: proboscis sheath massive, long, thick; salivary glands diffuse; crop with 6 pairs of crop caeca: 1st-5th uniform, bag-like, 6th pair (posterior caeca) with 3 blind processes; intestine with 4 pairs of rather short processes and an ovate extention after the last pair of processes.Distribution. North Asia: Western Siberia, Eastern Siberia, the Russian Far East, and Mongolia39.Habitats and ecology. This species is known to occur in a broad range of freshwater environments such as rivers, oxbow lakes, large to small natural lakes, reservoirs, road ditches, and even puddles (Supplementary Dataset S2). An unusual example of its association with a bryozoan species was described from Eastern Siberia38. Probably, the record of an Alboglossiphonia leech in the mantle cavity of an unidentified lymnaeid snail from the Altai Mountains, Russia41 could also be attributed to this species. The life cycle of the new species is unknown.Genus Glossiphonia Johnson, 1816Type species: Glossiphonia complanata (Linnaeus, 1758) (= Hirudo complanata Linnaeus, 1758; by subsequent designation).Arctic occurrences. Representatives of this genus are the most remarkable component of the Arctic Glossiphoniidae fauna. Altogether seven species were recorded north of the Arctic Circle, two of which are new to science and described here (Table 1).Comments. In general, sequenced representatives of the genus Glossiphonia could phylogenetically be delineated to three species groups (or subgenera): (1) the complanata-group (= subgenus Glossiphonia s. str.); (2) the verrucata-group (= subgenus Boreobdella Johansson, 1929; type species: Clepsine verrucata Müller, 1844); and (3) the concolor-group (= subgenus Paratorix Lukin & Epstein, 1960; type species: Torix baicalensis Stschegolew, 1922) (Table 1, Fig. 1 and Supplementary Fig. S1).
Glossiphonia arctica Bolotov, Eliseeva, Klass & Kondakov sp. novFigures 4B, 5b,c, 7c, Supplementary Figs. S2b, S3b, S5, Supplementary Table S2.LSID: https://zoobank.org/urn:lsid:zoobank.org:act:FADF0993-A946-413A-9680-25BA0F9BE90D.Holotype. RMBH Hir_0457_2_1-H (sequenced: COI sequence acc. No. ON810735; 18S rRNA sequence acc. No. ON819028), RUSSIA: a large lake near Sob’ railway station, 67.0480°N, 65.6316°E, Polar Urals, June 23, 2021, A. V. Kondakov et al. leg.Paratypes (N = 18). 18 specimens RMBH Hir_0457 (two specimens sequenced: COI sequence acc. No. ON810736 and ON810737; 18S rRNA sequence acc. No. ON819029; one specimen placed on 18 permanent slides as a series of slices), the type locality, the same date, and collectors.Etymology. The name of the new species indicates that its type locality is situated in the Arctic Region.Differential diagnosis. Medium-sized leech, which could be distinguished from other congeners by a combination of the following characters: dorsum with four rows of ovate, broad but very shallow and indistinct papillae on annulus a2 (outer paramedian and inner paramarginal series); each papilla bears ovate light yellow or white spot; dorsal black markings pattern absent. Externally, the new species is similar to G. mollissima. However, the latter species differs from G. arctica sp. nov. by having larger papillae and a well-developed black markings pattern dorsally.Molecular diagnosis. The new species represents a separate genetic lineage belonging to the verrucata-group (Fig. 1). The pairwise COI p-distance of the new species from other congeners varies from 7.0 to 12.4%. The intraspecific pairwise COI p-distance ranges from 0.0 to 0.2% (mean ± s.e.m. = 0.10 ± 0.05%; N = 3 sequences and 3 pairwise distance values). The GenBank acc. numbers of reference DNA sequences (COI and 18S rRNA) are given in Supplementary Table S2 and Supplementary Datasets S1–S2.Description. Medium-sized leech (body length up to 13.3 mm). Measurements of the type series are given in Supplementary Table S2. Body broad, leaf-like, ovate. Dorsum with four rows of ovate, broad but very shallow and indistinct papillae on annulus a2 (outer paramedian and inner paramarginal series). Posterior sucker small, circular (maximum diameter of 1.9 mm), ventrally directed. Proboscis pore in the center of anterior sucker. Coloration of living animals: body almost transparent, light brown, with multiple yellowish pigment cells. Coloration of ethanol-preserved animals: dorsum beige to light brown, with darker broad inner paramedian lines and light yellowish areas laterally and anteriorly; ovate light yellow or white spots at each papillae on annulus a2 arranged into four longitudinal rows (outer paramedian and inner paramarginal), sometimes with a few white spots between them. Three pairs of ovate eyespots arranged to two parallel rows; in some specimens eyes on each side are joined to a single large spot. Venter whitish to light brown, sometimes with irregular brownish shading. Total number of annuli: 70. Somites I–III uniannulate, IV biannulate, V–XXIV triannulate, XXV biannulate, XXVII uniannulate. The male and female genital pores are separated by two annuli and are located in the furrows XIa3/XIIa1 and XIIa2/a3, respectively. Reproductive system: 6 pairs of spherical testisacs inter-segmentally from XIII/XIV to XVIII/XIX; atrium spherical, the atrial cornua large, twisted anteriorly; paired ejaculatory ducts very long, extending to XVIII; paired ovisacs massive, long, with multiple lobes, arranged as loops, extending to XVIII (pregnant specimen with eggs). Digestive system: proboscis sheath massive, thick, elongated; esophagus narrow; salivary glands diffuse; crop with 7 pairs of crop caeca: 1st-6th uniform, bag-like, 7th pair (posterior caeca) with 4 blind processes and several smaller lobes; intestine enlarged, with 4 pairs of large, long, bag-like processes, expanding distally, each with several short lobes; a large circular extension after the last pair of processes.Distribution. Polar Urals (not known beyond the type locality).Habitats and ecology. The type series of this species was collected from a natural mountain lake with stony bottom. The leeches were recorded beneath flat stones (Fig. 3b); their feeding behavior and life cycle remain unknown.
Glossiphonia taymyrensis Bolotov, Eliseeva, Klass & Kondakov sp. novFigures 4E, 5d, 7b, Supplementary Figs. S2h, S3c, S6, Supplementary Table S2.LSID: https://zoobank.org/urn:lsid:zoobank.org:act:40269BF4-FE1C-4269-A7CC-41020789DC44.Holotype. RMBH Hir_0258_1-H (sequenced: COI sequence acc. No. ON810695), RUSSIA: small lake near Dudinka on Taymyr Peninsula, 69.4008°N, 86.3384°E, July 16, 2018, O. V. Aksenova et al. leg.Paratypes (N = 8). RUSSIA: 2 specimens RMBH Hir_0263_1 and RMBH Hir_0264_3 (sequenced: COI sequence acc. No. ON810701 and ON810705; 18S rRNA sequence acc. No. ON819017), the type locality, the same date, and collectors; 2 specimens RMBH Hir_0256_1 (one sequenced and one placed on 20 permanent slides as a series of slices; COI sequence acc. No. ON810693), small lake near Dudinka on Taymyr Peninsula, 69.3987° N, 86.3505° E, July 16, 2018, O. V. Aksenova et al. leg.; 1 specimen RMBH Hir_0261_2 (sequenced: COI sequence acc. No. ON810699; 18S rRNA sequence acc. No. ON819016), small lake near Dudinka on Taymyr Peninsula, 69.4014° N, 86.3250° E, July 16, 2018, O. V. Aksenova et al. leg.; 1 specimen RMBH Hir_0265_2 (sequenced: COI sequence acc. No. ON810706; 18S rRNA sequence acc. No. ON819021), Bolgokhtokh River near Dudinka, Taymyr Peninsula, 69.3780° N, 87.2215° E, July 21, 2018, O. V. Aksenova et al. leg.; 1 specimen RMBH Hir_0488 (sequenced: COI sequence acc. No. ON810755), a lake on Putorana Plateau, 68.7607° N, 91.9014° E, July, 2021, E. S. Chertoprud leg.; 1 specimen RMBH Hir_0449 (sequenced: COI sequence acc. No. ON810731), Pyzas River near Ust-Kabyrza settlement, 52.8277° N, 88.3973° E, Tashtagolsky District, Kemerovo Region, July 23, 2020, E. S. Babushkin & M. V. Vinarski leg.Etymology. The new species is named after the Taymyr Peninsula, where the majority of the type specimens were collected.Differential diagnosis. Small leech with broad, leaf-like, ovate body; three pairs of eyespots (distal pair joined; next two pairs separate); dorsal papillae absent; dorsal coloration with two inner paramedian rows of black spots, sometimes joining into unclear dashed lines; two annuli between the male (XIa3/XIIa1) and female (XIIa2/a3) genital pores. The new species largely resembles G. complanata but could be distinguished from it by having a smooth dorsum, without clear papillae. These taxa seem to have non-overlapping, allopatric ranges and, hence, could be separated on the basis of geographic criteria. However, the DNA approach seems to be the most appropriate way to distinguish these two species.Molecular diagnosis. The new species represents a separate genetic lineage belonging to the complanata-group (Fig. 1). The pairwise COI p-distance of the new species from other congeners varies from 6.0 to 12.2%. The intraspecific pairwise COI p-distance ranges from 0.0 to 1.1% (mean ± s.e.m. = 0.52 ± 0.07%; N = 8 sequences and 28 pairwise distance values). The GenBank acc. numbers of reference DNA sequences (COI and 18S rRNA) are given in Supplementary Table S2 and Supplementary Datasets S1–S2.Description. Small leech (body length up to 11.3 mm). Measurements of the type series are given in Supplementary Table S2. Body broad, leaf-like, ovate. Dorsum smooth, without clear papillae. Posterior sucker ovate (maximum diameter of 3.0 mm), ventrally directed. Proboscis pore in the center of anterior sucker. Coloration of living animals: not examined. Coloration of ethanol-preserved animals: (1) typical form having beige to light brown ground color without light spots but with darker brown coloration between inner paramedian lines; (2) melanic forms having dark brown ground color with four rows of large yellow spots (outer paramedian and marginal series) and yellow median stripe anteriorly (f. ‘maculosa’) or with strongly reduced yellow markings pattern. In all forms, there are two inner paramedian rows of black spots, sometimes joining into unclear dashed lines. Three pairs of ovate eyespots; the eyespots of the distal pair joined into a single spot; the eyespots of the next two pairs separate and are spaced apart. In the typical form, venter light yellow, with paired brown median and outer paramedian lines, which may be reduced to series of narrow brown longitudinal stripes. In melanic forms, ventral markings is more developed, with a series of brown longitudinal lines from median to inner paramarginal position and outer paramarginal brown spots. Posterior sucker with dense brown spots in melanic forms and with scarce brown spots in typical form. Total number of annuli: 68. Somites I–IV uniannulate, V–XXIV triannulate, XXV biannulate, XXVI–XXVII uniannulate. The male and female genital pores are separated by two annuli and are located in the furrows XIa3/XIIa1 and XIIa2/a3, respectively. Reproductive system: 6 pairs of spherical testisacs inter-segmentally from XII/XIII to XVIII/XIX; atrium ovate, the atrial cornua directed laterally; paired ejaculatory ducts twisted, short; paired ovisacs short, thick (undeveloped). Digestive system: salivary glands diffuse; proboscis sheath moderately thick; esophagus ovate; crop with 6 pairs of massive, bag-like, uniform crop caeca; intestine with 4 pairs of processes.Distribution. Western and Eastern Siberia.Habitats and ecology. The new species was recorded from natural lakes and rivers (Supplementary Dataset S2); its feeding behavior and life cycle are unknown.Genus Hyperboreomyzon Bolotov, Eliseeva, Klass & Kondakov gen. novLSID: https://zoobank.org/urn:lsid:zoobank.org:act:298FF41E-AF0D-4442-9F82-3022B8094A67.Type species: Hyperboreomyzon polaris gen. & sp. nov.Etymology. This name is compiled using two Greek words: ‘Hyperborea’ (meaning a mythical far northern land) and ‘myzon’ (meaning sucking).Diagnosis. Medium-sized, elongate, sub-fusiform glossiphoniid leeches; body and posterior sucker densely covered by shallow, ‘fish-scale’-like papillae; somite V biannulate; somites XII–XXIII secondarily sexannulate dorsally and ventrally due to the presence of very deep, prominent furrows separating each annulus to two semi-annuli; six rows of prominent dorsal tubercle-like papillae at a2 (inner paramedian, inner paramarginal, and marginal series) from V to XXVI; two pairs of circular eyespots on II and Va1 at inner paramedian position; gonopores at the furrows XIa3/XIIa1 (male) and XIIa2/a3 (female) and separated by two annuli; male atrium spherical; proboscis pore opens in a thick velar fold in the anterior half of oral sucker; one pair of compact, massive, elongated, incurved salivary glands, each gland with a bunch of a few short processes apically; 9 crop caeca pairs. Comparison of the new genus with other genera in the family based on morphological and anatomical features is presented in
Supplementary Table S3. Sexannulate condition was also recorded in the genus Actinobdella Moore, 1901 from North America36, but it differs from Hyperboreomyzon gen. nov. by having one pair of eyespots, diffuse salivary glands, and an apical position of proboscis pore (Supplementary Table S3).Comments. This genus is established for a single species, which is described below.
Hyperboreomyzon polaris Bolotov, Eliseeva, Klass & Kondakov gen. & sp. nov.Figures 4J, 5e, 6a-j, 7c, Supplementary Figs. S21, S8, S9, S10, S11, Supplementary Table S2.LSID: https://zoobank.org/urn:lsid:zoobank.org:act:503A9A26-CEDE-4747-952D-8416AE4EF4EB.Holotype. RMBH Hir_0486-H (sequenced: COI sequence acc. No. ON810753; 18S rRNA sequence acc. No. ON819030), RUSSIA: small alpine lake on Putorana Plateau, 68.9008°N, 94.1599°E, July, 2021, E. S. Chertoprud leg.Paratypes (N = 2). RUSSIA: 1 specimen RMBH Hir_0689 (dissected and placed on 60 permanent slides as a series of slices), small alpine lake on Putorana Plateau, 68.6659° N, 93.1365° E, August 11, 2021, E. S. Chertoprud leg.; 1 specimen RMBH Hir_0216 (sequenced and dissected; COI sequence acc. No. ON810677; 18S rRNA sequence acc. No. ON819005), water puddle on Kolguev Island, 68.9300° N, 49.0303° E, August 12, 2018, O. V. Travina & V. M. Spitsyn leg.Etymology. The name of the new species reflects its occurrences in polar (Arctic) areas of Eurasia.Differential diagnosis. As for the genus.Molecular diagnosis. None of congeneric species is known. Based on uncorrected pairwise COI p-distances between a haplotype of the new taxon and selected species-level haplotypes in each genus (Supplementary Table S1), Hyperboreomyzon seems to be more closely related to members of Hemiclepsis (mean distance ± s.e.m. = 11.62 ± 0.15%, range = 9.75–14.08%, N = 9) and Theromyzon (mean distance ± s.e.m. = 11.37 ± 0.07%, range = 10.47–12.64%, N = 9) without significant differences between distances from these two genera (Mann–Whitney test: p = 0.72). Other Glossiphoniidae genera are more distantly related, with a mean pairwise uncorrected COI p-distance of > 13.0% (Mann–Whitney test: p More
