Traditional multilocus phylogeny fails to fully resolve Palearctic ground squirrels (Spermophilus) relationships but reveals a new species endemic to West Siberia.

Previous efforts to reconstruct evolutionary history of Palearctic ground squirrels within the genus Spermophilus have primarily relied on a single mitochondrial marker for phylogenetic data. In this study, we present the first phylogeny with comprehensive taxon sampling of Spermophilus via a conventional multilocus approach utilizing five mitochondrial and five nuclear markers. Through application of the multispecies coalescent model, we constructed a species tree revealing four distinct clades that diverged during the Late Miocene. These clades are 1) S. alaschanicus and S. dauricus from East Asia; 2) S. musicus and S. pygmaeus from East Europe and northwestern Central Asia; 3) the subgenus Colobotis found across Central Asia and its adjacent regions and encompassing S. brevicauda, S. erythrogenys, S. fulvus, S. major, S. pallidicauda, S. ralli, S. relictus, S. selevini, and S. vorontsovi sp. nov.; and 4) a Central/Eastern Europe and Asia Minor clade comprising S. citellus, S. taurensis, S. xanthoprymnus, S. suslicus, and S. odessanus. The latter clade lacked strong support owing to uncertainty of taxonomic placement of S. odessanus and S. suslicus. Resolving relationships within the subgenus Colobotis, which radiated rapidly, remains challenging likely because of incomplete lineage sorting and introgressive hybridization. Most of modern Spermophilus species diversified during the Early-Middle Pleistocene (2.2-1.0 million years ago). We propose a revised taxonomic classification for the genus Spermophilus by recognizing 18 species including a newly identified one (S. vorontsovi sp. nov.), which is found only in a limited area in the southeast of West Siberia. Employing genome-wide single-nucleotide polymorphism genotyping, we substantiated the role of the Ob River as a major barrier ensuring robust isolation of this taxon from S. erythrogenys. Despite its inherent limitations, the traditional multilocus approach remains a valuable tool for resolving relationships and can provide important insights into otherwise poorly understood groups. It is imperative to recognize that additional efforts are needed to definitively determine phylogenetic relationships between certain species of Palearctic ground squirrels.

Diversity of trematodes from the amphibian anomaly P hotspot: Role of planorbid snails.

Trematode infection of the second intermediate hosts can lead to changes in their fitness and, as a result, a change in the invasion rate of animal communities. It is especially pronounced during the invasion of parasite species that reduce activity due to the manipulation of hosts through the changes of their morphology and physiology. One of these cases is an anomaly P syndrome hotspot found in some populations of water frogs and toads in Europe caused by the trematode Strigea robusta metacercariae. The occurrence of pathogen and their participation in ecosystems are intrigues questions in the anomaly P phenomenon, as well as the role of planorbid snails that serve as the first intermediate hosts for many trematode species. Herein, we focused on trematodes spectra from planorbid snails and amphibians from the anomaly P hosts with the aim to undetected interactions between the pathways of parasites. Emerging cercariae of 6802 planorbid snails of dominant species (Planorbarius corneus, Planorbis planorbis, and Anisus spp.) were detected by both morphological and molecular methods in seven waterbodies in Privolzhskaya Lesostep Nature Reserve (Russia). A total of 95 sequences of 18 species were received, and 48 sequences were unique and did not present in any genetic databases. The 18 species of trematodes from snails and 14 species of trematodes from amphibian hosts (Pelophylax ridibundus; Ranidae; Anura) were detected. Three species (Echinostoma nasincovae, Tylodelphys circibuteonis and Australapatemon burti) was new for the trematode fauna of the Middle Volga River region and Russia as a whole. Eleven species of parasitic flatworms have amphibians in their life cycles and nine species used amphibians as metacercariae hosts: Echinostoma nasincovae, E. miyagawai, Echinoparyphium recurvatum, Tylodelphys circibuteonis, Neodiplostomum spathula, Paralepoderma cloacicola, Macrodera longicollis, Strigea robusta, and Strigea strigis. The occurrence of trematode species from planorbid mollusks and frogs were compared.

Strigea robusta (Digenea: Strigeidae) infection effects on the gonadal structure and limb malformation in toad early development.

The anomaly P is a mass morphological anomaly reported in some water frog populations across Europe. It was found that polydactyly is only a mild attenuated form of heavy cases of the anomaly P syndrome, which have strong deformations of the hindlimbs and, partly, forelimbs. It was shown that the anomaly P is caused by the trematode Strigea robusta and this syndrome can be considered as a special case of strigeosis in amphibians. The anomaly P for a long time considered to be specific for water frogs of the genus Pelophylax. Herein, we describe polydactyly and heavy forms of the anomaly P syndrome in toads of the genera Bufo and Bufotes, as a result of exposure to S. robusta cercariae. A total of 150 tadpoles of Bufo bufo, 60 tadpoles of Bufotes viridis, and 60 tadpoles of Bufotes baturae were divided into five experimental and four control groups (30 tadpoles in each group). All anomalies in the toads were similar to those observed in water frogs. The survival of tadpoles in the experimental groups was 76%. The anomaly P was observed in 57.9% of toad tadpoles (51.8% of mild forms and 6.1% of heavy forms). The occurrence of the anomaly P varied among groups from 19% to 78%. Heavy forms of the anomaly P were found in all experimental groups. We described rare asymmetrical cases of the anomaly P. According to severe modification of limb morphology, we supposed changes of gonadal morphology (any modifications of the germ and somatic cells). The gonadal development of infected tadpoles was however the same as in uninfected toad tadpoles, and heterochromatin distribution within gonocytes had no differences as well. It seems like the parasite doesn't have any effect on the gonadal development of the toads. The lack of heavy forms in natural populations of toads, as well as a development of gonads were discussed.

A new record of odd-scaled snake (Serpentes, Xenodermidae) from Vietnam: expanded description of Parafimbrios vietnamensis based on integrative taxonomy.

Based on the combination of molecular and morphological data, we herein report the second known finding of the xenodermid snake species Parafimbrios vietnamensis Ziegler, Ngo, Pham, Nguyen, Le & Nguyen, 2018. The male individual was found in the Yen Bai Province of northwestern Vietnam, more than 200 km from the type locality in Lai Chau Province. Genetic divergence between the newly-collected male and the holotype was low (1.7%), and is in agreement with morphological data that supports that they are conspecific. We give a detailed description of the morphological characters and coloration of the new record and provide an expanded diagnosis of P. vietnamensis. Parafimbrios is a poorly-understood genus, and our recent discovery brings the total number of known specimens of the genus to nine, 1/3 of them having been found in Vietnam (one specimen of P. lao and now two specimens of P. vietnamensis).

Strigea robusta causes polydactyly and severe forms of Rostand's anomaly P in water frogs.

BACKGROUND: Cases of polydactyly in natural populations of amphibians have attracted great interest from biologists. At the end of the 1940s, the French biologist Jean Rostand discovered a polymorphic syndrome in some water frog (Anura: Pelophylax) populations that included polydactyly and some severe morphological anomalies (he called it 'anomaly P'). The cause of this anomaly remains unknown for 70 years. In a previous study, we obtained anomaly P in the laboratory in tadpoles of water frogs that developed together with molluscs Planorbarius corneus (Mollusca: Gastropoda) collected in the field. We thus proposed the 'trematode hypothesis', according to which the infectious agent responsible for anomaly P is a trematode species. METHODS: Metacercariae from tadpoles with anomaly P were identified using ITS2 gene sequencing as Strigea robusta (Trematoda: Strigeidae). To verify teratogenic features of the species, cercariae of S. robusta were tested for the possibility to cause anomalies. Identification of cercariae species was made using morphological and molecular methods (sequencing of ITS2 and 28S rRNA). The tadpoles were exposed to parasites at four doses of cercariae (control, low, medium and high) and divided into two groups: "early" (at 25-27 Gosner stages) and "late" (at 29-34 Gosner stages) exposure. RESULTS: A total of 58 (72.5%) tadpoles survived until metamorphosis under the dose-dependent experiment with the trematode S. robusta. Differences in the survival rates were observed between the exposed and unexposed tadpoles both in the group of "early" tadpoles and "late" tadpoles. The exposure of tadpoles to the cercariae of S. robusta induced anomaly P in 82% of surviving tadpoles. The severe forms developed only in "early" stages under all doses of cercariae exposure. Polydactyly predominantly developed in the "late" stages; under a light exposure dose, polydactyly also developed in "early" tadpoles. Laboratory-hatched tadpoles reared together with infected snails had different rates of survival and complexity of deformations associated with the period of coexistence. CONCLUSIONS: The experiments with direct cercariae exposure provide compelling evidence that S. robusta leads to anomaly P in tadpoles of water frogs. The manifestation of anomaly P turned out to be dependent on the stage of development, cercariae dose, and the location of the cysts.

Phylogenetic relationship and variation of alarm call traits of populations of red-cheeked ground squirrels (Spermophilus erythrogenys sensu lato) suggest taxonomic delineation.

Distribution area and taxonomic borders within the species complex Spermophilus erythrogenys sensu lato remain questionable. Early evidence suggests that red-cheeked ground squirrels of Southeast Kazakhstan are remarkably different in terms of the acoustic structure of their alarm calls from the red-cheeked ground squirrels of the Kurgan region in Russia. In this study, we analyzed the differences in the acoustic structure of the alarm call and mitochondrial DNA (complete control region, 1005-1006 bp and complete cytochrome b gene, 1140 bp) in 3 populations of red-cheeked ground squirrels (Tara, Altyn-Emel and Balkhash), all located within areas isolated by geographical barriers in Southeast Kazakhstan. We found that the alarm call variables were similar between the 3 study populations and differed by the maximum fundamental frequency (8.46 ± 0.75 kHz) from the values (5.62 ± 0.06 kHz) reported for the red-cheeked ground squirrels from the Kurgan region of Russia. Variation in mtDNA control region was only 3% and variation in cytochrome b gene was only 2.5%. Phylogenetic trees based on cytochrome b gene polymorphism of 44 individuals from the study area and adjacent territories indicated 3 clades with high (98-100%) bootstrap support: "intermedius," "brevicauda" and "iliensis"). We conclude that the 3 study populations in Southeast Kazakhstan belong to the clade intermedius and suggest a taxonomical revision of the species complex Spermophilus erythrogenys sensu lato, including analyses of nuclear DNA and alarm calls for populations of the brevicauda and iliensis clades.

DNA analysis of a 30,000-year-old Urocitellus glacialis from northeastern Siberia reveals phylogenetic relationships between ancient and present-day arctic ground squirrels.

In contrast to the abundant fossil record of arctic ground squirrels, Urocitellus parryii, from eastern Beringia, only a limited number of fossils is known from its western part. In 1946, unnamed GULAG prisoners discovered a nest with three mummified carcasses of arctic ground squirrels in the permafrost sediments of the El'ga river, Yakutia, Russia, that were later attributed to a new species, Citellus (Urocitellus) glacialis Vinogr. To verify this assignment and to explore phylogenetic relationships between ancient and present-day arctic ground squirrels, we performed 14C dating and ancient DNA analyses of one of the El'ga mummies and four contemporaneous fossils from Duvanny Yar, northeastern Yakutia. Phylogenetic reconstructions, based on complete cytochrome b gene sequences of five Late Pleistocene arctic ground squirrels and those of modern U. parryii from 21 locations across western Beringia, provided no support for earlier proposals that ancient arctic ground squirrels from Siberia constitute a distinct species. In fact, we observed genetic continuity of the glacialis mitochondrial DNA lineage in modern U. parryii of the Kamchatka peninsula. When viewed in a broader geographic perspective, our findings provide new insights into the genetic history of U. parryii in Late Pleistocene Beringia.

Implications of hybridization, NUMTs, and overlooked diversity for DNA Barcoding of Eurasian ground squirrels.

The utility of DNA Barcoding for species identification and discovery has catalyzed a concerted effort to build the global reference library; however, many animal groups of economical or conservational importance remain poorly represented. This study aims to contribute DNA barcode records for all ground squirrel species (Xerinae, Sciuridae, Rodentia) inhabiting Eurasia and to test efficiency of this approach for species discrimination. Cytochrome c oxidase subunit 1 (COI) gene sequences were obtained for 97 individuals representing 16 ground squirrel species of which 12 were correctly identified. Taxonomic allocation of some specimens within four species was complicated by geographically restricted mtDNA introgression. Exclusion of individuals with introgressed mtDNA allowed reaching a 91.6% identification success rate. Significant COI divergence (3.5-4.4%) was observed within the most widespread ground squirrel species (Spermophilus erythrogenys, S. pygmaeus, S. suslicus, Urocitellus undulatus), suggesting the presence of cryptic species. A single putative NUMT (nuclear mitochondrial pseudogene) sequence was recovered during molecular analysis; mitochondrial COI from this sample was amplified following re-extraction of DNA. Our data show high discrimination ability of 100 bp COI fragments for Eurasian ground squirrels (84.3%) with no incorrect assessments, underscoring the potential utility of the existing reference librariy for the development of diagnostic 'mini-barcodes'.