Biodiversity assessment of Eucyclops Claus, 1893 (Copepoda: Crustacea) in the Baikal region using genetic methods

Abstract

The genus Eucyclops is the most species-rich within the subfamily Eucyclopinae. Despite the significant research of Eucyclops in Russia, there are still vast areas including Siberia, the Baikal region, and the Far East with fragmentary study of this genus, particular with regard to molecular methods. In this work we have sequenced four molecular markers with different evolutionary rates and inheritance types (COI and 12S rRNA mtDNA, 18S rRNA and ITSn rDNA) to estimate the biodiversity of Eucyclops in the Baikal region. Five species E. serrulatus, E. speratus, E. macruroides, E. arcanus, and E. macrurus baicalocorrepus were identified through a combination of morphological and genetic methods in the Baikal region. The necessity for a taxonomic revision of endemic Baikal E. macrurus baicalocorrepus and E. macruroides baicalensis has been established. We assume that two forms of E. macrurus baicalocorrepus with short and long caudal rami are distinct endemic Baikal species. The genetic analysis of E. serrulatus, E. speratus, and E. macruroides revealed that the Baikal Cyclopoida, along with the Far Eastern and other Siberian representatives of the genus, form a distinct genetic lineage that differs from the majority of European representatives, with some exceptions.

Acta Biologica Sibirica 10: 1269–1291 (2024) doi: 10.5281/zenodo.14029856

Corresponding author: Tatyana Yu. Mayor (tatyanabfo@mail.ru)

Academic editor: R. Yakovlev | Received 9 September 2024 | Accepted 18 October 2024 | Published 5 November 2024

http://zoobank.org/3D180847-3D76-4E5E-B329-245EEB5ACDC3

Citation: Mayor TYu, Chaban OA, Kaskova KA, Sukhikh NM (2024) Biodiversity assessment of Eucyclops Claus, 1893 (Copepoda: Crustacea) in the Baikal region using genetic methods. Acta Biologica Sibirica 10: 1269–1291. https://doi.org/10.5281/zenodo.14029856

Keywords

Cyclopoida, Copepoda, endemic, molecular phylogenetics, Siberia, speciation, systematics, taxonomy

Introduction

The estimation of biodiversity is a fundamental element in the processes of its conservation and the understanding of evolutionary mechanisms in the context of a changing climate, biological invasions and anthropogenic impact on functional changes in ecosystems (Lynch et al. 2023). It has been predicted that biodiversity among Copepoda will increase by 37–126% by 2100, reaching approximately 5000 species (Macêdo et. al. 2024). In the study of microcrustaceans such as Copepoda, especially closely related species, integrative approaches involving microscopy, morphometrics and genetic methods are crucial (Karanovic and Bláha 2019).

The genus Eucyclops Claus, 1893 is the most species-rich within the subfamily Eucyclopinae, with approximately 100 species (Alekseev and Defaye 2011; Alekseev 2019). This genus is comparable to the most species-rich genera among freshwater Cyclopoida: Acanthocyclops Kiefer, 1927 and Diacyclops Kiefer, 1927. Representatives of Eucyclops are ubiquitous, inhabiting permanent and temporary water bodies, including lakes, ponds, rivers, estuaries and groundwater. The majority of species are freshwater, although brackish-water species are also found. Similar to Acanthocyclops and Diacyclops, the genus Eucyclops is taxonomically complex due to the similarity in morphology among species, as well as the vast expanse of their ranges. Previously, E. serrulatus (Fischer, 1851) was considered to be cosmopolitan (Monchenko 1974). Today, this species, like E. macrurus (G.O. Sars, 1863), E. macruroides Lilljeborg, 1901 and E. denticulatus (Graeter, 1903), has a Palearctic distribution (Alekseev 2019), and all finds of E. serrulatus outside the Palearctic are most likely relatively recent introductions. As shown by integrative revisions, employing molecular genetic methods, these widely distributed copepod species may represent species complexes. For example, three morphotypes and two genetic lineages of E. serrulatus were identified in a type locality in St. Petersburg (Alekseev et al. 2006; Sukhikh and Alekseev 2015). Additionally, eight genetic lineages were identified among E. serrulatus inhabiting European water bodies (Hamrova et al. 2012). Specimens of the two most widespread genetic lineages have been found in a single water body. Eurytemora affinis (Poppe, 1880), previously considered a Holarctic species, has now been split into at least three species (Lee 1999; Sukhikh et al. 2023). Furthermore, several species of Harpacticoida have also demonstated complex intraspecific structure in genetic studies (Kochanova et al. 2022; Kochanova et al. 2024). The Eucyclops complex taxonomy has resulted in a constant increase of the described and redescribed species number (Alekseev et al. 2006; Mercado-Salas and Suárez-Morales 2014, 2020; Gaponova and Hołyńska 2019, 2022). The most recent genus taxonomic revision was conducted by V.R. Alekseev (2019). This revision resulted in the separation of two new genera Isocyclops Kiefer, 1957 and Stygocyclops Pleša, 1971 from the genus Eucyclops, which were previously considered subgenera. The remaining species were assigned to nine subgenera.

A fourteen-species Eucyclops fauna is known to occur in Russia, including the E. serrulatus species complex. Four Palaearctic species E. macrurus, E. macruroides, E. denticulatus and E. speratus (Lilljeborg, 1901) are found in the European part of Russia. E. maritimus (Alekseev et Monchenko, 1991), E. persistens (Monchenko, 1978), and E. orthostylis Lindberg, 1952 were recorded in the Ponto-Caspian basin. E. delongi Alekseev, 2019 and E. roseus Ishida, 1997 were recorded in Siberia and the Far East, while E. roseus was also recorded in Baikal and Crimea. E. euacanthus (Sars, 1909) and Eucyclops cf. ohtakai Ishida, 1997 were found in the Far East. E. dumonti Alekseev, 2000 and E. arcanus Alekseev, 1990 have been observed in Siberia (Alekseev 2019, 2023; Hołyńska et al. 2021; Novichkova and Chertoprud 2022). Four of these species have subspecies. These are E. macrurus baicalocorrepus Mazepova, 1955 and E. macruroides baicalensis Mazepova, 1978 from Lake Baikal, E. arcanus arcticus Alekseev, 2022 from Arctic bogs of Bolshezemelskaya tundra, E. persistens persistens from the mouths of rivers in the Caucasus and E. persistens tauricus from wells in the Crimea (Mazepova 1978; Gaponova and Hołyńska 2019; Alekseev 2022). The systematic position of E. macrurus baicalocorrepus is taxonomically difficult, as it was originally described as subspecies of E. serrulatus. However, V.I. Monchenko proposed that it should be classified as a subspecies of E. macrurus (Monchenko 1974; Alekseev and Defaye 2011). Also E. roseus, 2023 was transferred to the rank of subspecies E. agiloides roseus (Alekseev 2023).

The Baikal region is situated within the territory of Eastern Siberia, comprising the Irkutsk region and Zabaykal'skij Kraj, wich is confined to Lake Baikal. Lake Baikal is a rift, the deepest and most ancient lake on Earth. It is a significant center of speciation for numerous animal groups, including Cyclopoida (Timoshkin 2009). The genera Diacyclops and Acanthocyclops, among the Baikal cyclopids, are the most species-rich with a high degree of endemism. Four species of Eucyclops have been recorded in Baikal and the surround region: E. macruroides, E. serrulatus, E. arcanus, and E. roseus, which occur in Siberia and two endemic subspecies E. macrurus baicalocorrepus and E. macruroides baicalensis (Mazepova 1978; Alekseev 1990; Alekseev et al. 2019; Sheveleva et al. 2020; Alekseev and Chaban 2021). The species E. serrulatus, E. arcanus, and E. macruroides baicalensis inhabit the littoral zone of the lake at depths up to 45 m. E. macrurus baicalocorrepus is widespread in the littoral zone and occurs rarely at depths greater than 50 m. E. macruroides baicalensis Mazepova, 1978 has been recorded from 20 m to the maximum depths of Baikal (Mazepova 1978). Furhermore, a fifth species E. dumonti has been recorded in the water bodies of the Baikal region (Alekseev 2019).

Despite the significant research of Eucyclops in Russia, there are still vast areas including Siberia, the Baikal region, and the Far East with fragmentary study of this genus, particular with regard to molecular methods. Novikov et al. (2022) have conducted studies on representatives of Eucyclops from the Lena River delta in the Arctic, which have shown that the species composition of the genus is likely to be significantly expanded. Already at the beginning of the Eucyclops revision in this region, five potentially new species for science were identified.

In this study we have employed genetic methods to estimate the biodiversity of Eucyclops in the Baikal region.

Materials and methods

Sample collection

Samples were collected using a net and by scuba divers in 2009–2023 (Table 1). Copepods from the Baikal region, including Lake Baikal, water bodies on its shore, and water bodies from the Kuitun, Tulun, and Nizhneudinsk districts of the Irkutsk region, the vicinity of St. Petersburg, the Leningrad region, republic of Udmurtia, the Volgograd Reservoir, Sakhalin Island, and Kazakhstan were collected and subjected to analysis. The entire sample or only selected live copepods were fixed in 96% ethanol and stored at -20°C. Morphological analysis was performed using a Zeiss Imager A1 microscope (Zeiss, Germany), a MSP-1 (LOMO, Russia) and an Olympus CX 41 microscope (Olympus, Japan). Morphological features were measured using an Olympus CX 41 microscope (Olympus, Japan), a Levenhuk M 800 Plus digital camera and a LevenhukLite software (Levenhuk, Inc., USA). The mean error (m) was calculated according to the formula (1).

Figure 1.

Where SD is the standard deviation and N is the number of observations.

For confocal laser scanning microscopy (CLSM), specimens were stained with Congo Red overnight and mounted on a slide in a drop of 50% glycerol following the procedure described by Michels and Büntzow (2010). The material was scanned using a Carl Zeiss LSM 710 laser confocal microscope (Zeiss, Germany); lens: Plan-Apochromat 20 ×/0.8 and 63×/1.40 Oil DIC M27; filters: 570–670 nm; lasers: 561 nm: 6.0%. Spinules groups on the antenna coxobases are numbered according to Alexeev et al. (2006).

DNA extraction, PCR, and sequencing

DNA was extracted by two methods. In the first case, we used whole specimen or cephalothorax and the ExtractDNA Blood DNA extraction kit according to instructions (Evrogen, Russia). In the second case, the biological material (egg sac or cyclopids antennula) was incubated in a mixture of 2x Encyclo buffer for PCR (Evrogen, Russia) and 0.1 mg/ml Proteinase K for 1-3 hours at 56°C. The enzyme was then inactivated by heating for 5 min at 96°C. The resulting mixture containing the DNA was used as a matrix in PCR in 10-fold dilution and stored at -20°C. PCR was performed using universal primers (Table 2) in a 10-20 μl mixture of 1x Encyclo buffer (Evrogen, Russia), 3.5 mM magnesium, 0.5 μM of each primer, 0.2 mM of each dNTP, 0.5 units of Encyclo DNA polymerase (Evrogen, Russia) and 1-2 μl of DNA-containing solution in a 100TM thermocycler (Bio Rad, USA).

The PCR thermoprofile for all genes included a step of heating to 94°C for 4 min; 35–40 cycles consisting of the following steps: matrix melting at 94°C for 15 s, primer annealing at specific temperature (Table 2) for 20 s, DNA synthesis at 72°C for 1 min; elongation step at 72°C for 4 min. The amplicons were electrophoretically separated in a 0.6% agarose gel in 0.5x Tris-acetate buffer. A piece of gel containing the target DNA fragment was excised, followed by freezing at -20°C. The tube with gel then centrifuged for 10 min at 10,000 rpm, after which the resulting solution was used in sequencing as a matrix. Direct DNA sequencing was conducted using the ABI PRISM BigDye Terminator sequencing kit v. 3.1 in 8-capillary genetic analysers ABI 3500 (Thermo Fisher Scientific, USA) and Nanofor 05 (Syntol, Russia).

Molecular phylogenetic analysis

The study included 38 specimens of Eucyclops, for which 66 sequences of one to four molecular markers were obtained. All sequences were deposited in GenBank and their NCBI accession numbers are provided in Table 3: for 18SrRNA PQ164692- PQ164696, for nITS PQ165826-PQ165851, PQ319846-PQ319849 for 12SrRNA-PQ218851.1-PQ218871.1, for COI PQ216000-PQ216007. The sequences were aligned using MegaX (Kumar et al. 2018) and CLC Sequence Viewer 8.0.0 (URL: https://resources.qiagenbioinformatics.com/manuals/clcsequenceviewer/current/index.php?manual=Introduction_CLC_Sequence_Viewer.html, accessed on August 20, 2024). The model-corrected genetic distances and p-distances were calculated using MegaX. The saturation of nucleotide substitutions was evaluated using the DAMBE (Xia et al. 2003). Nucleotide substitution model selection based on the Bayesian information criterion and phylogenetic tree construction by maximum likelihood (ML) method were performed using IQ-TREE2 software (Minh et al. 2020). Bootstrap algorithm was employed to evaluate the branching node support, with 1000 replications. Eucyclops and other cyclopid species sequences available in the GenBank were included in the analyses as outgroups. The GenBank accession numbers are provided on the phylogenetic trees. The trees were visualised and edited using the Interactive Tree Of Life (iTOL) version 6.8.1 (URL: https://itol.embl.de, accessed 07 May 2024) (Letunic, Bork, 2021) and Inkscape 1.3.2.

Results

The 12S rRNA sequences were obtained for 22 specimens of Eucyclops (182-435 bp). Of these, 15 specimens were collected in Lake Baikal and in water bodies on its shore, and one specimen was collected in the Irkutsk region. We identified E. serrulatus, E. speratus, E. macruroides, E. arcanus, and two forms of E. macrurus baicalocorrepus inhabiting Lake Baikal using a combination microscopic examination and molecular genetic analysis.

The morphological differences between specimens of E. m. baicalocorrepus form 1 (F192-5 to F192-7) and E. m. baicalocorrepus form 3 (F10-1, F10-6, F10-7) for which molecular data were obtained include shorter caudal rami (Lf/Wf), smaller proportion of the length and width of the third endopodite segment of the fourth pair of swimming legs (LenpP4/WenpP4), longer inner apical spine relative to the outer apical spine of the same segment (IAS/EAS), greater proportion of inner apical spine and length of the third endopodite segment of the fourth pair of swimming legs (IAS/LenpP4) (Table 4), larger spinules of the caudal rami lateral row, longer row of spinules near to the lateral seta (Fig. 1A, B), additional row of small spinules on the caudal side of the coxopodite of the fourth pair of swimming legs (P4) (Fig. 1D) and one row of large spinuless in group 18 on the antenna coxobase on the caudal side instead of two rows of small spinules.

Note: *– our measurements; **– data of G.F. Mazepova (1978).

A saturation analysis of nucleotide substitutions in the 12S dataset using fully resolved sites showed Iss = 0.3073, significantly less than Iss.c = 0.6539 for the symmetric topology and less than Iss.c = 0.7267 for the asymmetric topology. The sequences for the symmetric topology have a small saturation of nucleotide substitutions, rendering them suitable for phylogenetic analysis. The intraspecific p-distances are 15.3% among E. macruroides, 14.5% among E. serrulatus, and 0.2% among E. arcanus. The interspecific p-distances ranged from 4.1 to 33.7%. E. arcanus is the most distant from all others species (30.1-33.7%). E. speratus and V genetic line of E. serrulatus according to Hamrova et al. (2012) are the closest (4.1%). The p-distance between the Baikalian E. m. baicalocorrepus forms 1 and 3 is 14.7%. One haplotype was selected from the population for inclusion in the 12S phylogenetic tree data set for each species. The E. macrurus baicalocorrepus forms 1 and 3 from Lake Baikal are sister taxons (Fig. 2). Specimens of E. macruroides from the eastern and western sides of the Baikal coast have the same haplotype, and form a cluster with E. macruroides from the vicinity of St. Petersburg and the VII and VIII genetic lineages of E. cf. serrulatus as defined by Hamrova et al. (2012). However, Baikalian E. macruroides are genetically isolated from E. macruroides from the vicinity of St. Petersburg. Specimens of E. serrulatus from water bodies on the shores of Baikal, Udmurtia and Kazakhstan belong to a single genetic lineage. This lineage was identified as the I lineage of E. cf. serrulatus by Hamrova et al. (2012), and is genetically close to E. cf. serrulatus from Great Britain. A single pond in St. Petersburg (the type locality for the species) contains individuals of E. serrulatus that are presented by not two, as previously thought (Sukhikh and Alekseev 2015), but three genetically distant lineages. These belong to lineages I, III and possibly a new, closest VI lineage of E. cf. serrulatus according to Hamrova et al. (2012) (specimen 468). The specimen 468 was identified as E. cf. serrultus based on morphology and together with the specimen from United Kindom forms a separate cluster VI.

Representatives of E. arcanus from distant populations in the Baikal region and the Far East are genetically close. Three individuals of E. arcanus have two haplotypes, one of which is represented in both the Irkutsk region and on Sakhalin Island.

The COI sequences were obtained from nine specimens of Eucyclops (219-645 bp), five of which were collected in the Baikal region. The species E. serrulatus, E. speratus, E. arcanus, and E. macruroides were identified by the integral method. The COI dataset, which included GenBank sequences, was saturated at the third codon position. The value of Iss = 0.6104 for symmetric trees is slightly less than Iss.c = 0.7407, while for asymmetric trees it is slightly greater than Iss.c = 0.4949. A minor effect is observed at the first and second codon positions (Iss = 0.4430 is significantly smaller than Iss.c = 0.7003). The intraspecies genetic model-corrected distances (K2P+G), calculated on the basis of the1st and 2nd codon positions ranged from 0 to 3.3%. The most genetically diverse species were E. serrulatus (3.6%), E. arcanus (2.7%), and E. speratus (2%). The interspecies, genetic model-corrected distances considering the 1st and 2nd codon positions ranged from 3.1 to 10.1%. The most closely related species are E. taiwanensis and E. speratus (3.1%). The p-distance values were close to the model-corrected distances, ranging from 0-3.2% within species and 2.9-8.7% between species. The phylogenetic tree was constructed using only the 1st and 2nd codon positions (Fig. 3). The nucleotide sequences of E. speratus and E. taiwanensis from Taiwan form a distinct cluster. Specimens of E. speratus from the Baikal and Far Eastern populations are genetically close and separated from a pair of other genetically similar specimens from the Volgograd reservoir and the Orlovsky pond in St. Petersburg.

Figure 2.Figure 1. CLSM microphotographs of E. m. baicalocorrepus forms 1, 3. A-caudal rami; arrows indicate lateral row of spinules. B-abdomen. C-4th pair of swimming legs (P4), caudal. D-intercoxal sclerite and coxa of P4, caudal. E-antenna coxobase, caudal. Scale: A, C-50 µm; B-100 µm; D, E-20 µm.

Figure 3.Figure 2. Phylogenetic tree based on 12S rRNA (TPM2u+F+I+G4) and sampling map. The number in the node is the bootstrap support value. Sequences obtained in this study are marked in bold. Sequences of specimens from the Baikal region are marked in blue. Sequences from GenBank are preceded by their accession numbers. Roman numerals indicate genetic lineages.

Figure 4.Figure 3. Phylogenetic tree based on the 1st, 2nd codon positions of COI (TN+F+I+G4) and sampling map. The number in the node is the bootstrap support value. Obtained sequences are marked in bold. Sequences of individuals from the Baikal region are marked in blue. Sequences from GenBank are preceded by their access numbers.

The СО1 E. arcanus sequences from the Baikal region and Sakhalin Island form a separate cluster, comprising three genetic lineages. The first lineage is represented by a specimen from Sakhalin Island, the second by a specimen from the eastern coast of Lake Baikal, and the third by specimens from the western side of the Baikal coast and the Irkutsk region. The Baikal region representative of E. serrulatus is genetically close to a specimen from New Zealand and a representative of clade II (eastern lineage) of E. serrulatus from St. Petersburg (Sukhikh and Alekseev 2015; Kochanova et al. 2021). The sequences of E. serrulatus collected in the same pond in the vicinity of St. Petersburg exhibit a significant degree of genetic divergence. Specimen 409, as observed in the 12S tree, represent a descrete lineage from the eastern clade. The sequences of E. macruroides form a separate cluster with two genetic lineages found in the vicinity of St. Petersburg. One of the lineages includs also Eucyclops from India.

The ITSn sequences, comprising the first and second transcribed spacers of rDNA and the 5.8S rRNA gene, were determined for 30 individuals belonging to five species: E. speratus, E. macrurus baicalocorrepus (forms 1 and 3), E. arcanus, E. serrulatus, and E. macruroides (206-681 bp). The intraspecific model-corrected (JC+G) genetic distances ranged from 0 to 10.3%. The maximum values were observed in the distances among E. macruroides (10.3%), E. serrulatus (2.5%), and E. speratus (2.1%). The interspecific model-corrected genetic distances ranged from 3.9 to 25.1%. E. macrurus baicalocorrepus form 1 and E. macrurus baicalocorrepus form 3 from Baikal genetically are the closest (3.9%), while and the greatest divergence was observed between E. arcanus and other species (22.1–25.1%). The intra- and interspecific p-distances were similar to the model-corrected distances and were 0-8.6% and 3.7-18.6%, respectively. The mitochondrial genes and nuclear ITSn tree topologies are consistent in general (Fig. 4).

The ITSn sequences of E. speratus form a separate cluster, in which sequences of the Far Eastern and Baikal populations are genetically isolated from those of the St. Petersburg population. The two forms of E. macrurus baicalocorrepus are closely related and sister to E. macruroides, which is genetically subdivided into two lineages from geographically distant populations: the Baikal region and St. Petersburg. The individuals of E. arcanus from the Baikal region and the Far Eastern populations are genetically close according to this fragment and form a distinct cluster. The E. serrulatus specimens from the four water bodies in the Baikal region belong to the same genetic lineage and are closest to E. serrulatus from St. Petersburg and Udmurtia. The representatives of E. serrulatus from St. Petersburg form three lineages, with one being the most distant and represented by a specimen of E. cf. serrulatus (468).

An 18S rRNA gene fragment (326 to 565 bp) was obtained from five specimens sourced from the Baikal region. The interspecific genetic model-corrected distances ranged from 0 to 1.7%, while p-distances ranged from 0 to 1.9%. The maximum values were observed between E. agilis and other species. The phylogenetic tree, based on 18S rRNA, with a high degree of support reveals two clusters. E. agilis from the USA, and E. ensifer from Brazil are sister species. E. macruroides from a water body on the shore of Lake Baikal, the VII and VIII genetic lineages of E. cf. serrulatus, as delineated by Hamrova et al. (2012) from Europe, form another cluster (Fig. 5). E. macruroides from the Baikal region and St. Petersburg reservoir (GenBank data) form distant genetic lines and the reason for this is unknown.

Figure 5.Figure 4. Phylogenetic tree based on ITSn rDNA (TNe+G4) and sampling map. The number in the node is the bootstrap support value. Obtained sequences are marked in bold. Sequences of specimens from the Baikal region are marked in blue. Sequences from GenBank are preceded by their access numbers.

Discussion

In the present study, four molecular markers with different evolutionary rates and inheritance types were sequenced: COI and 12S rRNA mtDNA, 18S rRNA and ITSn rDNA. The study permitted a considerable expansion of the genetic database of Eucyclops species, as well as estimation of its diversity and phylogeny within the Baikal region. Of the four molecular markers, the ITSn rDNA fragment, comprising two internal transcribed spacers and the 5.8S rRNA gene, was amplified with the highest efficiency using universal primers. For the COI data set, a saturation effect of nucleotide substitutions at the third codon position was detected, which appears to be characteristic phenomen among Copepoda. This phenomen has been shown in the genus Diacyclops, which is species-rich among freshwater Cyclopoida and the widespread species Eurytemora affinis (Lee 1999; Mayor et al. 2010; Novikov et al. 2024). For another mtDNA fragment, 12S rRNA, our analysis revealed extremely high interspecific genetic distances. In the case of E. arcanus and the other Eucyclops species, p-distances were approximately 30-35%. The high genetic diversity among Eucyclops may be related to both an ancient evolutionary age and an accelerated rate of mtDNA evolution. In light of the considerable taxonomic diversity observed in copepods, Boxshall and Jaume (2000), suggested that they represent the earliest colonisers of freshwater habitats, with an evolutionary history extending back to the Palaeozoic era. This hypothesis is confirmed by the fossil record of crustaceans. Harpacticoids of the extant family Canthocamptidae, were found in a bitumen clast of a late Carboniferous age (ca. 303 Ma) from a glacial diamictite deposit in eastern Oman (Selden et al. 2010). Conversely, Copepoda fossils are rare and therefore there are no estimates of specific evolutionary rates. The evolutionary dating of Copepoda uses estimates of the overall mtDNA evolutionary rate for Crustacea, which ranges from 0.9 to 2.6% per MY (Miracle et al. 2013; Marrone et al. 2013; Yong et al. 2014; Cornils and Held 2014). The latest estimates of evolutionary rates for aquatic invertebrates, including arthropods, indicate that the level of nucleotide substitutions of mtDNA can be significantly higher than the commonly used evolutionary rates for Copepoda (Calvo et al. 2015; Loeza-Quintana et al. 2019). Kochanova et al. (2024) observed an unexpectedly high level of mtDNA genetic diversity in Harpacticella inopinata Sars, 1908 from Lake Baikal, which they attributed to a possible accelerated evolutionary rate. A similar hypothesis was proposed by Sukhikh et al. (2023) in their study of the E. affinis group.

The integrative method employed in our study enabled the identification of five Eucyclops species from the Baikal region: E. speratus, E. serrulatus, E. macruroides, E. arcanus, and E. macrurus baicalocorrepus. E. speratus was detected for the first time in the area. All other species had previously been recorded in the area by microscopy methods (Mazepova 1978; Alekseev 1990; Sheveleva et al. 2020; Alekseev 2022). Interestingly that only E. macrurus baicalocorrepus, which presents two forms by Mazepova (1955), was found in Lake Baikal. The remaining species were found in the various water bodies of the Baikal region. The available data indicate that, the fauna of the Baikal region is similar to that of Siberia, reflecting their common evolutionary history. However, the fauna of Lake Baikal is the most specific, it is although influenced by the Siberian fauna. The lake is notable for its high endemic species richness. We did not find the same species in Lake Baikal and the Baikal region. The sole exception is the species E. macruroides, which was found by us on the Baikal coast and is quite different from its European relatives. E. macruroides with the subspecies E. macruroides baicalensis was also described from Lake Baikal. The subspecies differs from E. macruroides in morphology, including in the shape and armament of the caudal rami, the structure of P5, and so forth. Moreover, the drawings provided for this taxon (Mazepova 1978) are more correspond to E. macrurus and the long-furcal form of E. m. baicalocorrepus that was previously described by the same author. The specimens of E. m. baicalocorrepus with long caudal rami, designated as form 3, differ from E. m. baicalensis by the cylindrical shape of the caudal rami without thickening downwards and converge with it towards the side of the outer terminal seta of the caudal rami. The structure of P5 makes it challenging to distinguish between these two taxa, as both have similar ratios of the inner spine to the length of the segment: 1.1-1.6 for E. m. baicalensis and 1.0-1.8 for E. m. baicalocorrepus.

Figure 6.Figure 5. Phylogenetic tree based on 18S rRNA (JC+I) and sampling map. The number in the node is the bootstrap support value. Obtained sequences are marked in bold. Sequences of individuals from the Baikal region are marked in blue. Sequences from GenBank are preceded by their access numbers.

The taxonomic status of E. macrurus baicalocorrepus, initially described as E. serrulatus baicalocorrepus (Mazepova, 1955), requires further verification using genetic methods and the inclusion of E. macrurus from the type locality in the analysis. The results of morphological analysis (Monchenko, 1974) and our genetic study of the two forms demonstrate that E. macrurus baicalocorrepus is genetically distinct from any genetic lineage of E. serrulatus, not confirming its original description as E. serrulatus baicalocorrepus. It is evident that the considerable range of morphological variability (length of caudal rami from 3.4 to 8.2 and serra from a few spinules to the entire length of caudal rami, etc.) described for this subspecies cannot be attributed to a single taxon. It seems probably that we are dealing with several species or at least subspecies at once, which require accurate taxonomic revision and redescription. Similar species complexes were revealed for three endemic Baikal species of Diacyclops (Mayor et al. 2024). In this study, at least two of the three forms of E. m. baicalocorrepus identified by the caudal rami index were genotyped. The two forms of E. m. baicalocorrepus form a separate clade based on mitochondrial and nuclear DNA fragments. At the same time, the p-distances between two forms are 3.7% for ITSn and 14.7% for 12S rRNA, which corresponds to the species level among Copepoda (Zagoskin et al. 2014; Krajicek et al. 2016; Sukhikh et al. 2023). The forms of E. m. baicalocorrepus differ both in quantitative (index of caudal rami, indices of the third endopodite of the fourth pair of swimming legs) and qualitative characters (ornamentation of the P4 coxa and antenna coxobase). The level of genetic and morphological differences observed between the forms corresponds to the species level. Baikal is the deepest and most ancient lake on Earth, and is one of the most significant centers for hydrobiont speciation. The fauna of Baikal Cyclopoida includes 46 species and subspecies, of which 64% are endemic (Sheveleva et al. 2012). It is probable that both forms of E. macrurus baicalocorrepus diverged in the recent past in Baikal and are endemic. Genetic and morphological comparative analysis of the type material as well as other population of the species will allow to solve this question.

The tree topologies obtained by mitochondrial and nuclear markers are generally in agreement. Forms 1 and 3 of E. macrurus baicalocorrepus formed a monophyletic group with E. macruroides, indicating their common ancestral form. Nevertheless, the bootstrap support for this node is relatively low (57 and 65%), and it is conceivable that constructing a single tree based on several genetic fragments could enhance the reliability of this node. However, in the present study, we did not employ this method, as the data on four genetic fragments were obtained for different individuals due to varying PCR efficiency. The close relationship between E. dumonti and E. macruroides was previously demonstrated on the basis of the initial molecular genetic studies of Eucyclops using 18S rRNA (Alekseev et al. 2006). The latest revision of the genus places E. arcanus and E. dumonti in the subgenus Speratocyclops, E. macruroides in the subgenus Denticyclops, and E. macrurus in the subgenus Macrurocyclops (Alekseev 2019). However, the molecular phylogeny data do not fully agree with this systematics for this group of species.

In our analysis, E. macruroides is represented by a number of sister genetic lines. The isolation of the lines from the Baikal region and St. Petersburg was confirmed by ITSn and 12S rRNA analysis. Furthermore, two lines from the vicinity of St. Petersburg were isolated according to COI. The intraspecific genetic heterogeneity is shown for E. speratus by 12S rRNA and ITSn fragments. Similarly, to E. macruroides genetic lines from the Baikal region/Far East and St. Petersburg are distinguished from one another. Probably these lineages may represent new species, the status of which requires further study. All E. serrulatus specimens from the Baikal region belong to a single genetic lineage. This lineage has been identified as one of two in the study conducted by Sukhikh and Alekseev (2015) and as one of eight lineages within the species according to Hamrova et al. (2012). Specimens of this lineage are distributed across a vast area, including Siberia, Central Asia, and even New Zealand, where the species is apparently introduced. In the vicinity of St. Petersburg, three genetically distinct lineages were found in one pond, wich is the type locality of E. serrulatus. These lineages are consistent in 12S rRNA with clades 1–3 published by Hamrova et al. (2012). Three more clades out of eight under the name E. cf. serrulatus in the cited work reliably belong to other species (12S rRNA tree).

Conclusion

Five species of Eucyclops were identified through a combination of morphological and genetic methods in the Baikal region: E. serrulatus, E. speratus, E. macruroides, E. arcanus, and E. macrurus baicalocorrepus. E. speratus is included in the list of Copepoda fauna of the region for the first time. The necessity for a taxonomic revision of the species E. macrurus baicalocorrepus and E. macruroides baicalensis, which are endemic to Baikal, has been established. The results of the analysis of the description, in conjunction with the findings of the morphological and genetic studies, indicate that at least two species are under the common subspecies name E. macrurus baicalocorrepus. Additionally E. macruroides baicalensis is actually E. macrurus and most likely corresponds to one of the form of E. m. baicalocorrepus. Each analysed species was genotyped by four mitochondrial and nuclear genes: 12S rRNA, COI, ITSn, and 18S rRNA. The genetic analysis of E. serrulatus, E. speratus, and E. macruroides revealed that the near Baikal Cyclopoida, along with the Far Eastern and other Siberian representatives of the genus, form a distinct genetic lineage that differs from the majority of European representatives, with some exceptions.

Acknowledgements

We are grateful to V.I. Lazareva, V.R. Alexeev, I.A. Nyapshaev, A.P. Fedotov and E.V. Dzyuba for helping us collect the material. Genetic studies were done at the Taxon Centre of ZIN and LIN SB RAS. Sequencing was done at Ultramicroanalysis, Evrogen and Syntol (Moscow). Confocal laser scanning microscopy was done at the Instrumentation Center “Electronic Microscopy” of the Collective Instrumental Center "Ultramicroanalysis" (LIN SB RAS). Lake Baikal material and CLSM were funded in the framework of The State Assignment No. 0279-2021-0005 (121032300224-8). The molecular genetic analysis was supported by RSF grant 23-24-00296 (N.S.).

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