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.

Molecular characteristics of Cryptosporidium spp. in human cases in five Finnish hospital districts during 2021: first findings of Cryptosporidium mortiferum (Cryptosporidium chipmunk genotype I) in Finland.

The aims of the study were to characterise the distribution of Cryptosporidium spp. and subtypes causing infections in Finland during 2021. This was carried out with 60 clinical samples from the hospital districts of Helsinki and Uusimaa, Vaasa, Kymenlaakso, South Karelia, and Central Finland, as well as with Finnish Infectious Diseases Register (FIDR) data. Additionally, the study aimed to explore the potential exposures related to Cryptosporidium mortiferum (Cryptosporidium chipmunk genotype I) infections via interview. Species identification was carried out with quantitative real-time PCR (qPCR) and 18S sequencing. Further typing was performed with gp60 subtyping. Over 70% of the samples were identified as Cryptosporidium parvum and 20% as C. mortiferum, which had not been identified in Finland before. Two cases of Cryptosporidium hominis were identified from patients reported to have travelled outside Europe. The C. parvum subtype IIaA15G2R1 and the C. mortiferum subtype XIVaA20G2T1 were the most common subtypes identified. The interviewed C. mortiferum cases did not report shared exposures such as contact with wild rodents. In conclusion, C. parvum and C. mortiferum were the major causes of cryptosporidiosis in the five studied Finnish hospital districts.

Population genomic structure of a widespread, urban-dwelling mammal: The eastern grey squirrel (Sciurus carolinensis).

Urbanization is a persistent and widespread driver of global environmental change, potentially shaping evolutionary processes due to genetic drift and reduced gene flow in cities induced by habitat fragmentation and small population sizes. We tested this prediction for the eastern grey squirrel (Sciurus carolinensis), a common and conspicuous forest-dwelling rodent, by obtaining 44K SNPs using reduced representation sequencing (ddRAD) for 403 individuals sampled across the species' native range in eastern North America. We observed moderate levels of genetic diversity, low levels of inbreeding, and only a modest signal of isolation-by-distance. Clustering and migration analyses show that estimated levels of migration and genetic connectivity were higher than expected across cities and forested areas, specifically within the eastern portion of the species' range dominated by urbanization, and genetic connectivity was less than expected within the western range where the landscape is fragmented by agriculture. Landscape genetic methods revealed greater gene flow among individual squirrels in forested regions, which likely provide abundant food and shelter for squirrels. Although gene flow appears to be higher in areas with more tree cover, only slight discontinuities in gene flow suggest eastern grey squirrels have maintained connected populations across urban areas in all but the most heavily fragmented agricultural landscapes. Our results suggest urbanization shapes biological evolution in wildlife species depending strongly on the composition and habitability of the landscape matrix surrounding urban areas.

Diversity and host specificity of Borrelia burgdorferi's outer surface protein C (ospC) alleles in synanthropic mammals, with a notable ospC allele U absence from mixed infections.

Interactions among pathogen genotypes that vary in host specificity may affect overall transmission dynamics in multi-host systems. Borrelia burgdorferi, a bacterium that causes Lyme disease, is typically transmitted among wildlife by Ixodes ticks. Despite the existence of many alleles of B. burgdorferi's sensu stricto outer surface protein C (ospC) gene, most human infections are caused by a small number of ospC alleles ["human infectious alleles" (HIAs)], suggesting variation in host specificity associated with ospC. To characterize the wildlife host association of B. burgdorferi's ospC alleles, we used metagenomics to sequence ospC alleles from 68 infected individuals belonging to eight mammalian species trapped at three sites in suburban New Brunswick, New Jersey (USA). We found that multiple allele ("mixed") infections were common. HIAs were most common in mice (Peromyscus spp.) and only one HIA was detected at a site where mice were rarely captured. ospC allele U was exclusively found in chipmunks (Tamias striatus), and although a significant number of different alleles were observed in chipmunks, including HIAs, allele U never co-occurred with other alleles in mixed infections. Our results suggest that allele U may be excluding other alleles, thereby reducing the capacity of chipmunks to act as reservoirs for HIAs.

Seasonal patterns of prolactin, prolactin receptor, and STAT5 expression in the ovaries of wild ground squirrels (Citellus dauricus Brandt).

Prolactin (PRL) is a hormone crucial for normal reproduction, functioning as an autocrine, paracrine, and endocrine factor. This study aimed to examine the immunolocalization and expression patterns of PRL, prolactin receptor (PRLR), and signal transducer and activator of transcription 5 (STAT5) in the ovaries of wild ground squirrels during both breeding and non-breeding periods. Significant seasonal variations were observed in ovarian weights, with higher values during the breeding season and relatively lower values during the nonbreeding season. PRL, PRLR, STAT5, and p-STAT5 were immunolocalized in granulosa cells and luteal cells during the breeding season, whereas they were exclusively found in granulosa cells during the non-breeding season. The mRNA expression levels of Prl, Prlr, and Stat5 were increased in ovarian tissues during the breeding season compared to the non-breeding season. Moreover, the mean mRNA levels of Prl, Prlr, and Stat5 exhibited a positive correlation with ovarian weights. Both circulating PRL and ovarian PRL concentrations were significantly elevated during the breeding season. Additionally, transcriptomic analysis of ovarian tissues revealed differentially expressed genes possibly associated with ovarian function and mammary gland development, including ovarian follicle development, steroid synthesis, and regulation of reproductive process. These findings suggest that PRL might play an essential endocrine, autocrine, or paracrine role in the regulation of seasonal changes in the ovarian functions in wild ground squirrels.

Integrated transcriptomics and metabolomics reveal protective effects on heart of hibernating Daurian ground squirrels.

Hibernating mammals are natural models of resistance to ischemia, hypoxia-reperfusion injury, and hypothermia. Daurian ground squirrels (spermophilus dauricus) can adapt to endure multiple torpor-arousal cycles without sustaining cardiac damage. However, the molecular regulatory mechanisms that underlie this adaptive response are not yet fully understood. This study investigates morphological, functional, genetic, and metabolic changes that occur in the heart of ground squirrels in three groups: summer active (SA), late torpor (LT), and interbout arousal (IBA). Morphological and functional changes in the heart were measured using hematoxylin-eosin (HE) staining, Masson staining, echocardiography, and enzyme-linked immunosorbent assay (ELISA). Results showed significant changes in cardiac function in the LT group as compared with SA or IBA groups, but no irreversible damage occurred. To understand the molecular mechanisms underlying these phenotypic changes, transcriptomic and metabolomic analyses were conducted to assess differential changes in gene expression and metabolite levels in the three groups of ground squirrels, with a focus on GO and KEGG pathway analysis. Transcriptomic analysis showed that differentially expressed genes were involved in the remodeling of cytoskeletal proteins, reduction in protein synthesis, and downregulation of the ubiquitin-proteasome pathway during hibernation (including LT and IBA groups), as compared with the SA group. Metabolomic analysis revealed increased free amino acids, activation of the glutathione antioxidant system, altered cardiac fatty acid metabolic preferences, and enhanced pentose phosphate pathway activity during hibernation as compared with the SA group. Combining the transcriptomic and metabolomic data, active mitochondrial oxidative phosphorylation and creatine-phosphocreatine energy shuttle systems were observed, as well as inhibition of ferroptosis signaling pathways during hibernation as compared with the SA group. In conclusion, these results provide new insights into cardio-protection in hibernators from the perspective of gene and metabolite changes and deepen our understanding of adaptive cardio-protection mechanisms in mammalian hibernators.

Toxoplasma gondii in small mammals in Romania: the influence of host, season and sampling location.

BACKGROUND: Toxoplasma gondii is a protozoan parasite that infects a large spectrum of warm-blooded animals, including humans. Small rodents and insectivores play an important role in the epidemiology of T. gondii and may serve as a source of infection for both, domestic and wild definitive felid hosts. Factors influencing the occurrence of T. gondii in wild small mammals are unknown, despite the fact that many intermediate host species are identified. We have used small mammals (Rodentia and Lipotyphla) captured over two years in various habitats, both in urbanised and in natural landscapes. We assessed the importance of land-use, season and host ecology on T. gondii infection. RESULTS: We examined 471 individuals belonging to 20 small mammal species, collected at 63 locations spread over wide altitude, habitat and land-use ranges from Romania. Heart tissue samples were individually analysed by PCR targeting the 529 bp repetitive DNA fragment of T. gondii. The overall prevalence of infection was 7.3%, with nine species of rodents and two species of shrews being found to carry T. gondii DNA. Five species showed high frequency of infection, with the highest prevalence found in Myodes glareolus (35.5%), followed by Spermophilus citellus (33.3%), Sorex minutus (23.1%), S. araneus (21.7%) and Micromys minutus (11.1%). Adults seemed more often infected than young, however when controlling for season, the difference was not significant, as in spring both adults and young showed higher infection rates, but more adults were sampled. Contrary to our expectations, urban/rural areas (with their implicit high density of domestic feline presence) had no effect on infection prevalence. In addition, neither habitat, nor land-use at sampling sites was important as only geographical location and host species were contributing factors to the infection risk. CONCLUSIONS: High prevalence of T. gondii infection showed a highly localised, patchy occurrence, with long living and higher mobility host species being the most common carriers, especially during autumn.

Seasonal changes of vitamin D3 and ovarian steroidogenesis in the wild ground squirrels (Citellus dauricus Brandt).

There is mounting evidence that vitamin D3 regulates female reproductive function critically, while little is known about the function of seasonally variable vitamin D3 in regulating ovarian steroidogenesis. This study examined the seasonal expressions of vitamin D receptor (VDR), vitamin D metabolic molecules (CYP2R1, CYP27B1, and CYP24A1), and steroidogenic enzymes (P450scc, 3ß-HSD, P450c17, and P450arom) in the ovaries of the wild ground squirrels (Citellus dauricus Brandt) during the different breeding seasons. VDR, CYP2R1, CYP27B1, and CYP24A1 were shown to be localized in different types of ovarian cells in the wild ground squirrels during the breeding and non-breeding seasons. Meanwhile, the mRNA levels of VDR, CYP2R1, CYP27B1, CYP11A1, HSD3B1, CYP17A1, and CYP19A1 in the ovaries were remarkably higher in the breeding season. Furthermore, RNA-seq data of ovaries revealed that 6036 genes were differentially expressed genes (DEGs); further analysis revealed that several DEGs known to be involved in ovarian steroidogenesis pathway and cellular response to vitamin D pathway were identified. In addition, during the breeding season, the concentrations of luteinizing hormone (LH), follicle-stimulating hormone (FSH), progesterone, and 17ß-estradiol were greater in the serum of the wild female ground squirrels. This observation was positively correlated with seasonal changes in the concentration of 25(OH)D3, supporting the fact that the 25(OH)D3 content in the ovaries was significantly higher in the breeding season. These findings suggested that seasonal changes in vitamin D3 might regulate the ovarian steroidogenesis of the wild female ground squirrels.

Shifts in immune responses of an invasive alien species: A test of the evolution of increased competitive ability hypothesis using American Eastern gray squirrels in Italy.

Based on the Evolution of Increased Competitive Ability (EICA) hypothesis, a reduced investment in immunity, consequent to parasite loss, could partly explain the success of invasive alien species. We investigated variation in parasite load and immune responses of alien Eastern gray squirrels (Sciurus carolinensis) along the invasion wave of an expanding population. We first verified by fecal analyses that 1) parasite abundance decreased moving from the core towards the invasion front. Next, we used multiple measures of immunity to investigate whether, in response to the lower parasite pressure, individuals at the invasion front 2) dampened their costly inflammatory response, and 3) increased their investment in less expensive acquired immunity. We first explored variation in hematological variables related either to the inflammatory or the acquired response. On a subset of individuals, we carried out ex vivo cell cultures to analyse the basal expression of MHC class II genes and the expression of TNF-α genes in response to an immune challenge. Platelet counts and TNF-α expression suggested higher inflammation in individuals living at the invasion core, whereas parameters associated with an acquired response (lymphocyte counts and MHC II expression by spleen cells), conversely, were higher in squirrels at the front. Overall, our results suggest a shift between different immune strategies along the invasion wave, supporting a reduced investment in costly inflammatory responses and an increased investment in acquired immunity in individuals at the expanding edge of the range, which are subjected to high selective pressures for dispersal and reproduction.

Development of Genetic Markers for Sex and Individual Identification of the Japanese Giant Flying Squirrel (Petaurista leucogenys) by an Efficient Method Using High-Throughput DNA Sequencing.

DNA markers that detect differences in the number of microsatellite repeats can be highly effective for genotyping individuals that lack differences in external morphology. However, isolation of sequences with different microsatellite repeat numbers between individuals has been a time-consuming process in the development of DNA markers. Individual identification of Japanese giant flying squirrels (Petaurista leucogenys) has been challenging because this species is arboreal and nocturnal and exhibits little to no morphological variation between individuals. In this study, we developed DNA markers for sex and individual identification of this species by an efficient method using high-throughput DNA sequence data. Paired-end 5 Gb (2 × 250 bp) and 15 Gb (2 × 150 bp) genome sequences were determined from a female and a male Japanese giant flying squirrel, respectively. We searched SRY and XIST genes located on Y and X chromosomes, respectively, from high-throughput sequence data and designed primers to amplify these genes. Using these primer sets, we succeeded to identify the sex of individuals. In addition, we selected 12 loci containing microsatellites with different numbers of repeats between two individuals from the same data set, and designed primers to amplify these sequences. Twenty individuals from nine different locations were discriminated using these primer sets. Furthermore, both sex and microsatellite markers were amplified from DNA extracted non-invasively from single fecal pellet samples. Based on our results for flying squirrels, we expect our efficient method for developing non-invasive high-resolution individual- and sex-specific genotyping to be applicable to a diversity of mammalian species.

Seasonal change of circulating leptin associated with testicular activities of the wild ground squirrels (Citellus dauricus).

The purpose of this study was to explore the variations in the circulating leptin concentrations of the wild ground squirrels in relation to seasonal changes in testicular activities. Hematoxylin-eosin staining showed all types of elongated spermatids and spermatogenic cells existed in the testis in April, while the primary spermatocytes and spermatogonia were most advanced stages of germ cells in June. In addition, the primary spermatocytes, secondary spermatocytes, and spermatogonia were most advanced stages of germ cells in September. The highest circulating leptin concentration was consistent with the maximum body weight results from accumulation of adipose tissue in September. The mRNA expression level of leptin receptor (Ob-R) and STAT3 was lowest in June, raised in September, and remained increased in April. Ob-R and STAT3 were stronger staining in the Leydig cells in July. Moreover, the concentrations of testosterone (T) showed the maximum values in April, the minimum values in June, and significant increases in September. Furthermore, it is worth noting that the levels of T increased with the mRNA levels of Ob-R, STAT3, StAR, and testicular steroidogenic enzymes (3ß-HSD, P450c17, and P450scc). Moreover, RNA-seq analyses of testis during the different periods showed that a total of 4209 genes were differentially expressed genes (DEGs); further analysis revealed that DEGs related with the Jak/STAT pathways and reproduction were altered. Taken together, the results suggested that the leptin regulated testicular function through the Jak/STAT pathways and testicular steroidogenic factor expressions.

Genetic characterization of potential venom resistance proteins in California ground squirrels (Otospermophilus beecheyi) using transcriptome analyses.

Understanding the molecular basis of adaptations in coevolving species requires identifying the genes that underlie reciprocally selected phenotypes, such as those involved in venom in snakes and resistance to the venom in their prey. In this regard, California ground squirrels (CGS; Otospermophilus beecheyi) are eaten by northern Pacific rattlesnakes (Crotalus oreganus oreganus), but individual squirrels may still show substantial resistance to venom and survive bites. A recent study using proteomics identified venom interactive proteins (VIPs) in the blood serum of CGS. These VIPs represent possible resistance proteins, but the sequences of genes encoding them are unknown despite the value of such data to molecular studies of coevolution. To address this issue, we analyzed a de novo assembled transcriptome from CGS liver tissue-where many plasma proteins are synthesized-and other tissues from this species. We then examined VIP sequences in terms of three characteristics that identify them as possible resistance proteins: evidence for positive selection, high liver expression, and nonsynonymous variation across CGS populations. Based on these characteristics, we identified five VIPs (i.e., α-2-macroglobulin, α-1-antitrypsin-like protein GS55-LT, apolipoprotein A-II, hibernation-associated plasma protein HP-20, and hibernation-associated plasma protein HP-27) as the most likely candidates for resistance proteins among VIPs identified to date. Four of these proteins have been previously implicated in conferring resistance to the venom in mammals, validating our approach. When combined with the detailed information available for rattlesnake venom proteins, these results set the stage for future work focused on understanding coevolutionary interactions at the molecular level between these species.

Power of Bayesian and Heuristic Tests to Detect Cross-Species Introgression with Reference to Gene Flow in the Tamias quadrivittatus Group of North American Chipmunks.

In the past two decades, genomic data have been widely used to detect historical gene flow between species in a variety of plants and animals. The Tamias quadrivittatus group of North America chipmunks, which originated through a series of rapid speciation events, are known to undergo massive amounts of mitochondrial introgression. Yet in a recent analysis of targeted nuclear loci from the group, no evidence for cross-species introgression was detected, indicating widespread cytonuclear discordance. The study used the heuristic method HYDE to detect gene flow, which may suffer from low power. Here we use the Bayesian method implemented in the program BPP to re-analyze these data. We develop a Bayesian test of introgression, calculating the Bayes factor via the Savage-Dickey density ratio using the Markov chain Monte Carlo (MCMC) sample under the model of introgression. We take a stepwise approach to constructing an introgression model by adding introgression events onto a well-supported binary species tree. The analysis detected robust evidence for multiple ancient introgression events affecting the nuclear genome, with introgression probabilities reaching 63%. We estimate population parameters and highlight the fact that species divergence times may be seriously underestimated if ancient cross-species gene flow is ignored in the analysis. We examine the assumptions and performance of HYDE and demonstrate that it lacks power if gene flow occurs between sister lineages or if the mode of gene flow does not match the assumed hybrid-speciation model with symmetrical population sizes. Our analyses highlight the power of likelihood-based inference of cross-species gene flow using genomic sequence data. [Bayesian test; BPP; chipmunks; introgression; MSci; multispecies coalescent; Savage-Dickey density ratio.].

Chromosome-level genome assembly of the Siberian chipmunk (Tamias sibiricus).

Tamias sibiricus is regarded as one predominant scatter-hoarder that stores their food items both in small scattered caches and underground larder-hoards. This unique behavior, though providing essential seed dispersal services for many plant species worldwide, relies highly on accurate spatial memory and acute sense of olfaction. Here, we assembled a chromosome-scale genome of T. sibiricus using Illumina sequencing, PacBio sequencing and chromosome structure capture technique. The genome was 2.64 Gb in size with scaffold N50 length of 172.61 Mb. A total of 2.59 Gb genome data was anchored and orientated onto 19 chromosomes (ranging from 28.70 to 222.90 Mb) with a mounting rate of up to 98.03%. Meanwhile, 25,311 protein-coding genes were predicted with an average gene length of 32,936 bp, and 94.73% of these genes were functionally annotated. This reference genome will be a valuable resource for in-depth studies on basic biological possess and environmental adaptation of the Siberian chipmunk, as well as promoting comparative genomic analyses with other species within Rodentia.

Differential bone metabolism and protein expression in mice fed a high-fat diet versus Daurian ground squirrels following natural pre-hibernation fattening.

This study compared the effects on bone metabolism and morphology of pathological obesity induced by excessive fat intake in a non-hibernator (mice) versus healthy obesity due to pre-hibernation fattening in a hibernator (ground squirrels). Kunming mice were fed a high-fat diet to provide a model of pathological obesity (OB group). Daurian ground squirrels fattened naturally in their pre-hibernation season (PRE group) were used as a healthy obesity model. Micro-computed tomography (micro-CT) and three-point bending tests were used to determine the microstructure and mechanical properties of bone. Western blots were used to analyze protein expression levels related to bone metabolism (Runt-related transcription factor 2 (RunX2), osteocalcin (OCN), alkaline phosphatase (ALP), osteoprotegerin (OPG), receptor activator of nuclear factor-|κB ligand (RANKL), cathepsin K, matrix metallopeptidase 9 (MMP9), patched protein homolog 1 (Ptch1), phosphorylated ß|-|catenin (P|-|ß|-|catenin), and glycogen synthase kinase-3ß (GSK-3ß)). Compared with controls, there was no obvious bone loss in the OB mice, and the stiffness of the femur was increased significantly. Compared with summer active squirrels, bone formation was enhanced but the mechanical properties did not change in the PRE group squirrels. In OB mice, western blots showed significantly increased expression levels of all proteins except RunX2, OPG, and Ptch1. PRE ground squirrels showed significantly increased expression of most proteins except OCN and Ptch1, which decreased significantly, and P|-|ß|-|catenin and OPG, which did not change. In conclusion, for non-hibernating mice, moderate obesity had a certain protective effect on bones, demonstrating two-way regulation, increasing both bone loss and bone formation. For pre-hibernating ground squirrels, the healthy obesity acquired before hibernation had a positive effect on the microstructure of bones, and also enhanced the expression levels of proteins related to bone formation, bone resorption, and Wnt signaling.

Seasonal Change in Adiponectin Associated with Ovarian Morphology and Function in Wild Ground Squirrels (Citellus dauricus Brandt).

The goal of this study is to explore the relationship between altered circulating adiponectin concentration, ovarian tissue morphology, ovarian steroidogenesis, and sex hormone production in ovaries of wild ground squirrels. The ovarian mass differed significantly during the breeding and non-breeding seasons, and the circulating estradiol and progesterone concentrations were significantly higher in the breeding season, while the circulating adiponectin level was significantly lower. The expression levels of gonadotropin receptors (FSHR and LHR) and steroidogenic enzymes (StAR, P450scc, P450arom, and 3ß-HSD) were significantly higher during the breeding season. Comparing the ovarian transcriptome data of wild ground squirrels between the two periods, we found that some differentially expressed genes were enriched for ovarian steroidogenesis and the adipocytokine signaling pathway, which correlated with our present results. Notably, the MAPK signaling pathway was also enriched and its related genes (Erk1, p38 Mapk, Jnk) were up-regulated by qPCR during the non-breeding season. These findings suggested that adiponectin may be involved in the regulation of seasonal changes in the ovarian function of wild ground squirrels, possibly by acting on the MAPK signaling pathway to regulate sex steroidogenesis in the ovaries.

Six new species of Pristimantis (Anura: Strabomantidae) from Llanganates National Park and Sangay National Park in Amazonian cloud forests of Ecuador.

We describe six new species of rainfrogs of the genus Pristimantis (Strabomantidae) from Amazonian cloud forests in Ecuador. We also present a phylogeny showing the relationships of the new species. The phylogeny is based on mitochondrial genes 16S rRNA (16S), 12 rRNA (12S), NADH-ubiquinone oxidoreductase chain 1 (ND1) and the nuclear gene recombination-activating 1 (RAG1). We also describe the osteology of two of the new species using high-resolution x-ray computed tomography. The new species belong to two clades. The first clade is sister to the subgenus Huicundomantis and includes P. tamia sp. nov., P. miktos, and P. mallii. Pristimantis tamia sp. nov. is morphologically similar to P. miktos, P. mallii, P. martiae, and P. incomptus, but differs from them by lacking vocal slits and tympanic membrane and by having light greenish blue iris. Based in our results we expand the subgenus Huicundomantis to include the P. miktos species group. The second clade is remarkable by being highly divergent and consisting exclusively of new species: P. anaiae sp. nov., P. glendae sp. nov., P. kunam sp. nov., P. resistencia sp. nov., and P. venegasi sp. nov. The new species resemble P. roni, P. yanezi, P. llanganati, P. katoptroides, P. verecundus, and P. mutabilis but can be distinguished from them by lacking vocal slits and tympanic membrane and by having large dark round areas with thin clear borders in the sacral region. All six new species occur in the eastern slopes of the Ecuadorian Andes and are known from a single locality in Llanganates or Sangay National Park. We recommend assigning all of them to the Data Deficient (DD) Red List category. Based in our high-resolution x-ray tomographies, we report the presence of structures that appear to be intercalary elements. This would be the first report of such structures in Terrarana.

Complete mitogenomes of Turkish tree squirrels, Sciurus anomalus and S. vulgaris, (Sciuridae: Rodentia: Mammalia) and their phylogenetic status within the tribe Sciurini.

The genus Sciurus, a member of the family Sciuridae, is widely distributed in the Holarctic region. To better understand mitogenomic characteristics and to reveal internal phylogenetic relationships of the genus, 20 complete mitogenomes of Turkish tree squirrels were successfully sequenced for the first time, including 19 for S. anomalus (from 16,505 bp to 16,510 bp) and one for S. vulgaris (16,511 bp). The mitogenomes of two species were AT-biased. All tRNAs for two species displayed a typical clover-leaf structure, except for tRNASer(AGY). The tRNA Serine1 (S1)-GCT structure lacked the dihydrouridine (DHU) loop and stem. Based on mitogenomic dataset for phylogeny of Sciurinae, phylogenetic analyses (Bayesian Inference and Maximum Likelihood) did not support monophyly of Sciurus and proposed that S. anomalus, the most basal taxa in the Sciurini tribe, had at least five mitogenome lineages, which were also supported by network analysis. The dissimilarities among the five lineages of S. anomalus ranged from 0.0042 (0.42%) to 0.0062 (0.62%) using K2P sequence pairwise distances. In addition to this mitogenomic analysis result, phylogenetic analyses using the CYTB + D-loop dataset proposed the existence of at least nine lineages for S. anomalus, which was different than those of the previous studies. The current study proposed that the use of mitogenomic data for reconstructing the phylogeny of Turkey's Sciurus holds an important value for revealing evolutionary relationships.

Genomic resolution of cryptic species diversity in chipmunks.

Discovery of cryptic species is essential to understand the process of speciation and assessing the impacts of anthropogenic stressors. Here, we used genomic data to test for cryptic species diversity within an ecologically well-known radiation of North American rodents, western chipmunks (Tamias). We assembled a de novo reference genome for a single species (Tamias minimus) combined with new and published targeted sequence-capture data for 21,551 autosomal and 493 X-linked loci sampled from 121 individuals spanning 22 species. We identified at least two cryptic lineages corresponding with an isolated subspecies of least chipmunk (T. minimus grisescens) and with a restricted subspecies of the yellow-pine chipmunk (Tamias amoenus cratericus) known only from around the extensive Craters of the Moon lava flow. Additional population-level sequence data revealed that the so-called Crater chipmunk is a distinct species that is abundant throughout the coniferous forests of southern Idaho. This cryptic lineage does not appear to be most closely related to the ecologically and phenotypically similar yellow-pine chipmunk but does show evidence for recurrent hybridization with this and other species.

Old specimens for old branches: Assessing effects of sample age in resolving a rapid Neotropical radiation of squirrels.

Ultraconserved Elements (UCEs) have been useful to resolve challenging phylogenies of non-model clades, unpuzzling long-conflicted relationships in key branches of the Tree of Life at both deep and shallow levels. UCEs are often reliably recovered from historical samples, unlocking a vast number of preserved natural history specimens for analysis. However, the extent to which sample age and preservation method impact UCE recovery as well as downstream inferences remains unclear. Furthermore, there is an ongoing debate on how to curate, filter, and properly analyze UCE data when locus recovery is uneven across sample age and quality. In the present study we address these questions with an empirical dataset composed of over 3800 UCE loci from 219 historical and modern samples of Sciuridae, a globally distributed and ecologically important family of rodents. We provide a genome-scale phylogeny of two squirrel subfamilies (Sciurillinae and Sciurinae: Sciurini) and investigate their placement within Sciuridae. For historical specimens, recovery of UCE loci and mean length per locus were inversely related to sample age; deeper sequencing improved the number of UCE loci recovered but not locus length. Most of our phylogenetic inferences-performed on six datasets with alternative data-filtering strategies, and using three distinct optimality criteria-resulted in distinct topologies. Datasets containing more loci (40% and 50% taxa representativeness matrices) yielded more concordant topologies and higher support values than strictly filtered datasets (60% matrices) particularly with IQ-Tree and SVDquartets, while filtering based on information content provided better topological resolution for inferences with the coalescent gene-tree based approach in ASTRAL-III. We resolved deep relationships in Sciuridae (including among the five currently recognized subfamilies) and relationships among the deepest branches of Sciurini, but conflicting relationships remain at both genus- and species-levels for the rapid Neotropical tree squirrel radiation. Our results suggest that phylogenomic consensus can be difficult and heavily influenced by the age of available samples and the filtering steps used to optimize dataset properties.