New species of Kontrimavichusia Makarikov & Binkienė, 2022 (Eucestoda: Hymenolepididae) from arvicoline rodents (Rodentia: Cricetidae) from the North Caucasus

. Two previously unrecognized species attributable to the genus Kontrimavichusia Makarikov & Binkienė, 2022 in arvicoline rodents from the North Caucasus are described. Kontrimavichusia testiculata sp. nov. is described from Microtus majori (Thomas, 1906) from the northwestern Caucasus (Republic of Adygeya and Karachay-Cherkess Republic, Russia) and Kontrimavichusia hobergi sp. nov. is described from Microtus daghestanicus (Shidlovsky, 1919) from the central Caucasus (Republic of North Ossetia, Russia). Kontrimavichusia testiculata is readily distinguishable from K. asymmetrica (Janicki, 1904) and K. hobergi in having a larger number of testes (4–6 per proglottis), larger suckers and a longer cirrus and cirrus-sac. In addition, the new species differs from its congeners by the position of the cirrus-sac with regard to the poral osmoregulatory canals and position of distal end of the rostellar pouch relative to the posterior margins of the suckers. Kontrimavichusia hobergi can be readily distinguished from its congeners by the arrangement of the testes in a triangle and the position of the cirrus-sac with regard to the poral osmoregulatory canals. In addition, this previously unrecognized species differs from K. asymmetrica and K. testiculata by the smaller dimensions of the fully developed strobila and a narrower ovary. The cirrus-sac of K. hobergi is larger than that in K. asymmetrica but smaller than that in K. testiculata . We also used partial sequences of the nuclear ribosomal 28S rRNA gene and mitochondrial nad1 gen to justify the generic arrangement and independent status of these two new species which are characterized in the current manuscript.

Subsequent phylogenetic studies showed that K. asymmetrica could not be referred to any known genus among the hymenolepidids from mammals and thus had uncertain taxonomic affinities (Haukisalmi et al. 2010;Greiman & Tkach 2012;Makarikov et al. 2015;Neov et al. 2019).Recently, the generic allocation of this species was clarified based on the integration of a morphological criteria and molecular phylogenetic analysis.A monotypic genus, Kontrimavichusia Makarikov & Binkienė, 2022, was proposed for this cestode.
Concurrently, it was suggested that the genus may include additional uncharacterized species since interspecific lineages within the Kontrimavichusia clade were detected in molecular-based comparisons (Makarikov & Binkienė 2022).Following this suggestion, we continued to study the species diversity within this complex.Two previously unrecognized species attributable to the genus Kontrimavichusia, based on morphological criteria, among arvicoline rodents from the North Caucasus were discovered.One of them, Kontrimavichusia testiculata sp.nov., is described from Microtus majori (Thomas, 1906) from the Republic of Adygeya and the Karachay-Cherkess Republic of Russia.Originally reported as Rodentolepis sp.1 from the northwestern Caucasus, it was suggested this cestode may represent an undescribed species (Makarikov et al. 2017).Subsequently, a detailed redescription of K. asymmetrica (sensu stricto) provided a basis to differentiate this putative taxon (Makarikov & Binkienė 2022).The other species Kontrimavichusia hobergi sp.nov. is described from Microtus daghestanicus (Shidlovsky, 1919) from the Republic of North Ossetia, Russia.The descriptions of these two new species and their morphological differentiation from the type species of Kontrimavichusia are provided herein.We also used partial sequences of the nuclear ribosomal 28S rRNA gene and mitochondrial nad1 gen to analyze relationships among species of hymenolepidids and to justify the generic arrangement and independent status of the new species.

Material and methods
Specimens of Kontrimavichusia testiculata sp.nov.were found in 7 out of 25 Microtus majori, prevalence 28%, collected in July 2014 from the suburbs of Nikel (44º10′31″ N, 40º09′24″ E), a village located in the Maykopsky District, Republic of Adygeya, Russia.Also, specimens attributable to this species were found in one out of 11 Microtus majori collected in September 2016 from the Djamagat River, suburbs of Teberda (43º27′21″ N, 41º47′44″ E), a town located in the Khabezsky District, Karachay-Cherkess Republic, Russia.Specimens of Kontrimavichusia hobergi sp.nov.were found in 15 out of 26 Microtus daghestanicus, prevalence 57.7%, collected in July 2017 from the suburbs of Verkhniy Tsey (42º48′02″ N, 43º56′03″ E), a village located in the Alagirsky District, Republic of North Ossetia-Alania, Russia.
Host specimens were dissected fresh.Cestodes were isolated, rinsed and relaxed in water, and preserved in 70% ethanol.Specimens were stained with Ehrlich's haematoxylin, dehydrated in an ethanol series, cleared in clove oil and mounted in Canada balsam.Some scoleces and fragments of strobilae were mounted in Berlese's medium to facilitate detailed examination of the rostellar hooks, suckers, cirrus armature and structure of the eggs.Additional tissue was subsampled from some strobila and stored in 96% ethanol for molecular analyses.Specimens were studied using standard light and differential interference contrast microscopy.In the descriptions, measurements are given in micrometers except where otherwise stated; they are presented as the range followed by the mean and the number of the measurements (n) in parentheses.
The type material and voucher specimens of the new species have been deposited in the collection of the Institute of Systematics and Ecology of Animals, Novosibirsk, Russia (ISEA).Mammalian taxonomy follows Musser & Carelton (2005).
Voucher specimens of K. asymmetrica deposited in the helminthological collections of the Geneva Museum of Natural History, Switzerland (MHNG) and ISEA were studied for comparison purposes.A list of examined collection material is outlined in Makarikov & Binkienė (2022).
Genomic DNA for the molecular phylogenetic analysis was extracted from fragments (1.5-2 mm long) of holotype, paratype and voucher specimens of Kontrimavichusia testiculata sp.nov.and Kontrimavichusia hobergi sp.nov.from the type locality and from voucher specimens of K. asymmetrica from Lithuania and the Republic of Bashkortostan, Russia, following the protocol of Tkach & Pawlowski (1999).Scoleces and the remaining strobila have been mounted on slides.DNA fragments approximately 1090 base pairs long at the 5' end of the nuclear large ribosomal subunit (28S) gene and approximately 755 base pairs long fragment of the mitochondrial NAD(P)H dehydrogenase 1 gene (nad1) were amplified by PCR and sequenced for inter-and intraspecific molecular comparisons.PCRs were run on an Eppendorf Mastercycler ep Gradient thermal cycler using OneTaq Quick-load Mastermix from New England Biolabs (Ipswich, MA) according to the manufacturer's instructions.All PCR protocols included 40 cycles.Forward primer 28S-5' (5'-TAC CCG CTG AAC TTA AGC ATA T-3') and reverse primer 28S-3' (5'-CTC CTT GGT CCG TGT TTC AAG AC-3') designed by Zehnder & Mariaux (1999) were used for amplification; annealing temperature 53ºC.Degenerate forward primer nad1f (5'-GGNTATTSTCARTNTCGTAAGGG-3') and degenerate reverse primer trnNR (5'-TTCYTGAAGTTAACAGCATCA-3') from Littlewood et al. (2008) were used for nad1 amplification; annealing temperature for these reactions was set at 45ºC.The same primers were used for sequencing both genes.Sequences were aligned using BioEdit software ver.7.0.1 (Hall 1999).Pairwise comparisons of sequences of Kontrimavichusia spp.were calculated using MEGA X (Kumar et al. 2018).To build phylogenetic tree and reconstruct relationships between Kontrimavichusia testiculata sp.nov., Kontrimavichusia hobergi and K. asymmetrica, we used maximum likelihood (ML) with a general time reversible model as distance substitution.For phylogenetic analyses, we used newly obtained nucleotide sequences of 28S and nad1 genes of the two new species which were submitted to GenBank; Kontrimavichusia testiculata (5 and 7 respectively), accession numbers OR992632-OR992636 and PP133258-PP133264 and Kontrimavichusia hobergi (7 and 6 respectively), accession numbers OR992638-OR992642 and PP133265-PP133270.We also sequenced nad1 gene of K. asymmetrica (sensu stricto) from two relatively remote localities (Republic of Bashkortostan, Russia, and Lithuania), with GenBank accession numbers PP133271-PP133272 and PP133273.Nucleotide sequences of 28S of K. asymmetrica (sensu lato) and species of Hymenolepis were downloaded from the GenBank for comparison purposes (Lockyer et al. 2003;Haukisalmi et al. 2010;Nkouawa et al. 2016;Binkienė et al. 2019;Makarikov & Binkienė 2022).Rodentolepis microstoma (Dujardin, 1845) was used as an outgroup.Bootstrap values were calculated using MEGA as the percentage of 1000 replicates.

Etymology
This specific epithet refers to the very distinctive morphological feature of the species, namely its relatively great number of testes.

Remarks
Until the present study, K. asymmetrica was the only known representative of the genus Kontrimavichusia.Kontrimavichusia testiculata sp.nov. is readily distinguishable from the type species by the number of testes; the former species has 4-6 testes per proglottis while in K. asymmetrica those are usually 3.The cirrus-sac in K. testiculata substantially crosses the poral osmoregulatory canals, whereas the cirrus-sac of K. asymmetrica overlaps or rarely crosses the ventral longitudinal canal.Further, the distal end of the rostellar pouch does not attain the level of the posterior margins of suckers; in K. asymmetrica the rostellar pouch reaches to or slightly extends beyond the level of the posterior margins of the suckers.In addition, specimens of K. testiculata are characterized by larger suckers and a longer cirrus and cirrussac relative to K. asymmetrica (Table 1).Kontrimavichusia hobergi sp.nov.urn:lsid:zoobank.org:act:8EBE9219-C6BA-4B85-8740-3A3E05F51581Figs 3-6, Tables 1-2 Diagnosis Kontrimavichusia hobergi sp.nov.has morphological characters typical of the genus Kontrimavichusia, namely rhynchus armed with cricetoid-like hooks, apex of rostellum invaginable and blades of retracted hooks directed anteriorly, suckers armed with minute spines, ventral canals connected by irregularly spaced transverse anastomoses, copulatory part of vagina surrounded by circular musculature and covered externally by dense layer of intensely-stained cells, labyrinthine uterus extending beyond osmoregulatory canals into both lateral fields, situated dorsally to osmoregulatory canals and genital ducts and embryophore without polar filaments (Makarikov & Binkienė 2022).

Etymology
This species has been named in honour of the outstanding parasitologist Dr Eric P. Hoberg in recognition of his seminal and critical studies of parasites of vertebrates, helminth systematics, biogeography, ecology, phylogeny and evolution.It is also recognition of his significant contribution to the advancement of parasitology and the preservation of important archival materials on helminths during his tenure as chief curator of the U.S. National Parasite Collection from 1990 to 2014.

Remarks
Kontrimavichusia hobergi sp.nov. is readily distinguishable from K. asymmetrica by the triangular arrangement of the testes; those in the type species most often are situated in one row.The number of testes distinguishes the new species from K. testiculata sp.nov.: the former has three 3 per proglottis, the latter has 4-6 per proglottis.The cirrus-sac in K. hobergi substantially crosses the poral osmoregulatory canals; in contrast, the cirrus-sac of K. asymmetrica overlaps or rarely crosses the ventral longitudinal canal.The distal end of the rostellar pouch does not attain the level of the posterior margins of the suckers in K. hobergi, directly contrasting with the condition in K. asymmetrica.In addition, specimens of K. hobergi differ from K. asymmetrica and K. testiculata by distinctly smaller dimensions of the fully developed strobila.Further, the cirrus-sac is larger than in K. asymmetrica but smaller than in K. testiculata; the cirrus of the new species is smaller than in K. testiculata.The ovary of K. hobergi is narrower than in both congeners (Table 1).

Molecular phylogenetic analysis
Phylogeny was based on the nuclear ribosomal 28S rRNA gene.The length of the alignment after trimming was 1042 nucleotides.Consistent with a previous study, the monophyly of each of the two clades corresponding to Kontrimavichusia and Hymenolepis is well supported (0.97 and 1.0 posterior probability, respectively) (Makarikov & Binkienė 2022).Further, we demonstrated at least four independent phylogenetic lineages within the Kontrimavichusia clade including the two species from the North Caucasus described herein, with differences between all available sequences up to 1-11 bp (Fig. 5).Current analyses strongly support the generic allocation of those species to the genus.One of those lineages is represented by specimens of K. asymmetrica (sensu stricto) from Europe from Microtus agrestis (Linnaeus, 1761) (GenBank: GU166232, HM138528, ON562540 and ON562542-ON562544) and M. arvalis (Pallas, 1778) (GenBank: ON562541).The second lineage consists of species from the North Caucasus, namely K. testiculata sp.nov.and K. hobergi sp.nov.including the sequence from M. majori from Turkey (GU166234) attributed to K. asymmetrica (sensu lato) by Haukisalmi et al. (2010).It distinctly differs from the K. asymmetrica lineage by 8-9 bp.However, interspecific differences based on 28S gene in the Caucasian cluster are not pronounced.The two remaining lineages represented by specimens from Chionomys nivalis (Martins, 1842) from France (GU166231) and Dinaromys bogdanovi (Martino, 1922) from Bosnia (GU166233).Those differ from Caucasian linage by up to 11 bp and 7 bp respectively.
Phylogeny was based on the mitochondrial nad1 gen.The length of the alignment after trimming was 731 nucleotides.Until present, sequences of nad1 gen of K. asymmetrica were missing in GenBank.We used for analysis the sequences of K. asymmetrica (sensu stricto) from Eastern Europe (Lithuania and the Republic of Bashkortostan, Russia) and two new species from the North Caucasus K. testiculata sp.nov.and K. hobergi sp.nov.that we obtained.Three well-supported lineages within Kontrimavichusia corresponded to morphologically recognized species (Fig. 6).Unlike the 28S rRNA gene sequences, mitochondrial gene nad1 provided strong evidence for the description of K. testiculata and K. hobergi.The interspecific pairwise distances between lineages of K. testiculata and K. hobergi vary within 4.76-5.81%(33-40 bp).Also, both species distinctly differ from K. asymmetrica (sensu stricto) by 13.36-14.35%(84-92 bp) and 14.51-15.32%(91-98 bp) respectively (Table 2).
Among these, the level of intraspecific differences of the lineages based on nad1 gen has the following values: in the lineage of K. asymmetrica (sensu stricto) the two specimens from the same locality (Republic of Bashkortostan, Russsia) have no intraspecific variability, while the sequence originated from the relatively remote population (more than 2000 km) in Lithuania differs from these by 0.69% (5 bp) (Table 2); in K. testiculata sp.nov.from the Republic of Adygeya the intraspecific differences reached up to 0.97% (7 bp), while the sequences from Karachay-Cherkess Republic showed slightly higher values of intraspecific variability 1.11%-1.81%(8-13 bp); all sequences of K. hobergi sp.nov.originating from the same locality showed up to 0.14%-0.41%(1-3 bp) intraspecific variability.

Discussion
The number of testes in the family Hymenolepididae is traditionally considered as generic level character (Skrjabin & Matevosyan 1945;Mas-Coma & Galan-Puchades 1991;Czaplinski & Vaucher 1994).In the following genera of hymenolepidids from rodents the presence of numerous testes (more than three per proglottis) was used to discriminate among genera: Chitinolepis Baylis, 1926;Hymenandrya Smith, 1954;Paraoligorchis Wason & Johnson, 1977;Pseudandrya Fuhrmann, 1943;Pseudanoplocephala Baylis, 1927and Sudarikovina Spassky, 1951(Czaplinski & Vaucher 1994;Gulyaev & Chechulin 1996).Although specimens of K. testiculata sp.nov., unlike other representatives of the genus, have more than three testes per proglottis there is no evidence to separate this species in a distinct genus; all morphological characters are typical for Kontrimavichusia.Molecular analyses also clearly support the placement of K. testiculata in this genus.Similarly, it was discovered that the number of testes apparently does not have generic significance for other hymenolepidids of the genus Arostrilepis Mas-Coma & Tenora, 1997.Currently the genus includes 16 nominal species all having three testes per proglottis (e.g., Makarikov et al. 2013Makarikov et al. , 2020)).However, this genus had been shown to be paraphyletic with respect to Hymenandrya thomomyis Smith, 1954 having 7-15 testes per proglottis (Haas et al. 2020;Galbreath et al. 2023).Also, recently it was shown that Pseudanoplocephala crawfordi Baylis, 1927, with numerous testes, is clustered in a subclade of Hymenolepis (all cestodes characterized by three testes), based on molecular phylogenetic data (Jia et al. 2014).
Among this assemblage Arostrilepis-Hymenandrya, Hymenolepis-Pseudanoplocephala and Kontrimavichusia, it was shown that the number of testes does not appear to be a generic feature.The present data do not support revision of supraspecific taxa based on the number of testes.In any case, a detailed study of the phylogenetic relationships of these hymenolepidids should be carried out, since it may be quite obvious that morphological characters, including the number of testes, may have different taxonomic significance in different groups of cestodes.It seems more likely that a secondary increase in the number of testes occurred in different groups of hymenolepidids of small mammals independently and at different times.This issue needs further study.
The shape of the spines or microtriches and their pattern of distribution on suckers are usually used as distinctive characters for differentiation among species and genera (Mas-Coma & Galan-Puchades 1991; Czaplinski & Vaucher 1994).However, no visible differences among K. asymmetrica and the two new species from the North Caucasus were detected by light microscopy.
Of interest are the two phylogenetic lineages based on the 28S gene published in GenBank and attributed to K. asymmetrica (Haukisalmi et al. 2010).Those sequences significantly differ both from the type species and from K. testiculata sp.nov.and K. hobergi sp.nov.These are the following: cestode specimens collected from C. nivalis from France (GU166231) and from D. bogdanovi from Bosnia (GU166233).Also, specimens collected from M. majori from Turkey (GU166234) distinctly differ from K. asymmetrica but do not show interspecific differences from K. testiculata or K. hobergi in this region of DNA.It is possible that these specimens of K. asymmetrica (sensu lato) may be conspecific to one of the described species from the Caucasus (i.e., GU166234), or represent yet undiscovered species (i.e., GU166231, GU166233 and GU166234).Unfortunately, there are no morphological vouchers for these sequences (Haukisalmi et al. 2010;Makarikov & Binkienė 2022); their status requires further study.The present phylogenetic analysis based on the 28S gene has shown that interspecific distances within the genus Kontrimavichusia can reach up to 11 base pairs.However, this gene has apparent limitations for differentiating among species, as the two new species from the North Caucasus, which clearly differ in morphological features, are indistinguishable in this region of DNA.For a reliable differentiation between species of these hymenolepidids it is necessary to use more variable genes.Further, the presence of a species complex among specimens attributed to Hymenolepis hibernia Montgomery, Montgomery & Dunn, 1986 cannot be excluded.As even based on the 28S gene the 3 sequences deposited in GenBank (KT148842, KT148843 and KT148845) differed up to 5 positions and that exceeds the limits of intraspecific variability in this relatively conserved region of DNA (Tkach et al. 2013).
The mitochondrial gene nad1 has been used in different groups of hymenolepidids from small mammals and apparently this marker is a reliable one for distinguishing among species.For instance, the proposed interspecific differences among species of Staphylocystis Villot, 1877 based on nad1 gen vary within 33-49 bases (Tkach et al. 2013;Greiman et al. 2013).Results of pairwise comparisons of the two new species from the North Caucasus showed the close values of interspecific differences in 33-40 bases.While specimens of K. testiculata sp.nov.and K. hobergi sp.nov.even more distanced from K. asymmetrica in 84-92 and 91-98 bases, respectively.At the same time, intraspecific variability in species of Kontrimavichusia can reach up to 13 bases and is found in specimens from remote or orographically isolated populations.Thus, molecular analysis of nad1 sequences together with morphological data provides compelling evidence for the description of the two new species from the North Caucasus belonging to the genus Kontrimavichusia.
The diversity of relief and climate in different parts of the Greater Caucasus leads to extraordinary diversity and heterogeneity of its nature in general and the animals in particular.These factors are closely related to the fact that the North Caucasus represents one of the important centers of speciation in the Western Palaearctic which includes a large number of endemics.For instance, among small mammals of the Caucasus a significant portion of the diversity is represented by endemics (Sokolov & Tembotov 1989).In this regard, it can be assumed that the fauna of their helminths also has features of uniqueness and includes species endemic to this region.Thus, it was noted that 5 out of 27 species of cestodes from shrews reported in the North Caucasus are endemic to this region (Kornienko et al. 2021).Although the fauna of cestodes from rodents from the Northern Caucasus has not yet been sufficiently studied, it has been suggested that at least four currently undescribed putative species of cestodes from the northwestern Caucasus may be endemic to this region (Makarikov et al. 2017).One of these species was later described from the fat dormouse Glis glis (Linnaeus, 1766) as Armadolepis longisoma Makarikov, Stakheev & Tkach, 2018, the second species is described herein as K. testiculata sp.nov.(Makarikov et al. 2018;present study).An additional potentially endemic species was found in the central Caucasus and described in this paper as K. hobergi sp.nov.Thus, the expanding knowledge on the diversity of helminths in small mammals of the Northern Caucasus confirms the presence of a unique cestode fauna in this region which remains to be evaluated in detail.

Fig. 5 .
Fig. 5. Maximum likelihood phylogenetic tree of Kontrimavichusia Makarikov & Binkienė, 2022 and Hymenolepis Weinland, 1858 based on analysis of partial sequences of the 28S rRNA gene.Bootstrap support given for maximum likelihood analysis based on 1000 replicates.Bootstrap support values lower than 70% are not shown.

Fig. 6 .
Fig. 6.Maximum likelihood phylogenetic tree of species of Kontrimavichusia Makarikov & Binkienė, 2022 based on analysis of partial sequences of the nad1 gen.Bootstrap support given for maximum likelihood analysis based on 1000 replicates.Bootstrap support values lower than 70% are not shown.

Table 1 .
Makarikov & Binkienė, 2022ta sp.nov.andK.hobergi sp.nov.highlighted in bold show the most remarkable differences between the described and new species.Comparative morphometric data of species of KontrimavichusiaMakarikov & Binkienė, 2022(measurements in micrometres except where otherwise stated).