Highly resolved genome assembly and comparative transcriptome profiling reveal genes related to developmental stages of tapeworm Ligula intestinalis.

Ligula intestinalis (Cestoda: Diphyllobothriidae) is an emerging model organism for studies on parasite population biology and host-parasite interactions. However, a well-resolved genome and catalogue of its gene content has not been previously developed. Here, we present the first genome assembly of L. intestinalis, based on Oxford Nanopore Technologies, Illumina and Omni-C sequencing methodologies. We use transcriptome profiling to compare plerocercoid larvae and adult worms and identify differentially expressed genes (DEGs) associated with these life stages. The genome assembly is 775.3 mega (M)bp in size, with scaffold N50 value of 118 Mbp and encodes 27 256 predicted protein-coding sequences. Over 60% of the genome consists of repetitive sequences. Synteny analyses showed that the 10 largest scaffolds representing 75% of the genome display high correspondence to full chromosomes of cyclophyllidean tapeworms. Mapping RNA-seq data to the new reference genome, we identified 3922 differentially expressed genes in adults compared with plerocercoids. Gene ontology analyses revealed over-represented genes involved in reproductive development of the adult stage (e.g. sperm production) and significantly enriched DEGs associated with immune evasion of plerocercoids in their fish host. This study provides the first insights into the molecular biology of L. intestinalis and provides the most highly contiguous assembly to date of a diphyllobothriid tapeworm useful for population and comparative genomic investigations of parasitic flatworms.

Historical dispersal and host-switching formed the evolutionary history of a globally distributed multi-host parasite - The Ligula intestinalis species complex.

Studies on parasite biogeography and host spectrum provide insights into the processes driving parasite diversification. Global geographical distribution and a multi-host spectrum make the tapeworm Ligula intestinalis a promising model for studying both the vicariant and ecological modes of speciation in parasites. To understand the relative importance of host association and biogeography in the evolutionary history of this tapeworm, we analysed mtDNA and reduced-represented genomic SNP data for a total of 139 specimens collected from 18 fish-host genera across a distribution range representing 21 countries. Our results strongly supported the existence of at least 10 evolutionary lineages and estimated the deepest divergence at approximately 4.99-5.05 Mya, which is much younger than the diversification of the fish host genera and orders. Historical biogeography analyses revealed that the ancestor of the parasite diversified following multiple vicariance events and was widespread throughout the Palearctic, Afrotropical, and Nearctic between the late Miocene and early Pliocene. Cyprinoids were inferred as the ancestral hosts for the parasite. Later, from the late Pliocene to Pleistocene, new lineages emerged following a series of biogeographic dispersal and host-switching events. Although only a few of the current Ligula lineages show narrow host-specificity (to a single host genus), almost no host genera, even those that live in sympatry, overlapped between different Ligula lineages. Our analyses uncovered the impact of historical distribution shifts on host switching and the evolution of host specificity without parallel host-parasite co-speciation. Historical biogeography reconstructions also found that the parasite colonized several areas (Afrotropical and Australasian) much earlier than was suggested by only recent faunistic data.

The niches of nuthatches affect their lineage evolution differently across latitude.

Ecological niche evolution can promote or hinder the differentiation of taxa and determine their distribution. Niche-mediated evolution may differ among climatic regimes, and thus, species that occur across a wide latitudinal range offer a chance to test these heterogeneous evolutionary processes. In this study, we examine (a) how many lineages have evolved across the continent-wide range of the Eurasian nuthatch (Sitta europaea), (b) whether the lineages' niches are significantly divergent or conserved and (c) how their niche evolution explains their geographic distribution. Phylogenetic reconstruction and ecological niche models (ENMs) showed that the Eurasian nuthatch contained six parapatric lineages that diverged within 2 Myr and did not share identical climatic niches. However, the niche discrepancy between these distinct lineages was relatively conserved compared with the environmental differences between their ranges and thus was unlikely to drive lineage divergence. The ENMs of southern lineages tended to cross-predict with their neighbouring lineages whereas those of northern lineages generally matched with their abutting ranges. The coalescence-based analyses revealed more stable populations for the southern lineages than the northern ones during the last glaciation cycle. In contrast to the overlapping ENMs, the smaller parapatric distribution suggests that the southern lineages might have experienced competitive exclusion to prevent them from becoming sympatric. On the other hand, the northern lineages have expanded their ranges and their current abutting distribution might have resulted from lineages adapting to different climatic conditions in allopatry. This study suggests that niche evolution may affect lineage distribution in different ways across latitude.

Evolutionary applications of phylogenetically-informed ecological niche modelling (ENM) to explore cryptic diversification over cryptic refugia.

We used the fat dormouse (Glis glis), a species from a monotypic genus of family Gliridae, as a model to promote the understanding of patterns of cryptic diversification along the ancient Hyrcanian Forests, one of the old-growth relicts of the temperate deciduous forests worldwide. Mitochondrial Cytb data was used to investigate the phylogenetic status of two geographically-different populations of G. glis along the Hyrcanian Forests among all the worldwide known lineages of the species. Regarding phylogenetically informed partitioning of occurrence data, we then used two analytically different ENMs (i.e. environmental-space and geographic-space) to address whether niche divergence conforms G. glis diversification over the study area. Phylogenetic reconstruction showed significant heterogeneity between other fat dormouse lineages and those belonging to the Hyrcanian Forests as well as within the two hypothesized cryptic groups in the study area. Quantifying niche differences using the two ENM frameworks additionally confirmed divergence between the two cryptic lineages by indicating niche conservatism. The integration of phylogeny and ENM in this study confirms the development of distinct cryptic species and suggests that the Hyrcanian Forests, a well-known Pleistocene refugium, might contain multiple cryptic refugia for small forest-dwelling species during paleontological oscillations.

Conservation Below the Species Level: Suitable Evolutionarily Significant Units among Mountain Vipers (the Montivipera raddei complex) in Iran.

Northern and western mountains of Iran are among the most important biodiversity and endemism hot spots for reptiles in the Middle East. Among herpetofauna, the montivipers represent an emblematic and fragmented endemic group for which estimating their level of genetic differentiation and defining conservation priorities is urgently needed. Here, we present the most comprehensive phylogenetic study on the Montivipera raddei species group comprising all 5 known taxa, among which 3 are endemic to Iran. Based on 2 mitochondrial genes, phylogenetic and phylogeographic analyses revealed 3 major lineages each presenting very contrasting distribution areas. The Iranian montivipers are highly structured in clades showing low genetic diversity and corresponding to high altitude summits. Molecular dating revealed the role of Quaternary paleo-climatic oscillations and altitudinal movements of montivipers in shaping genetic diversity and differentiation of these sky-island taxa. In addition, the best scenario of historical biogeography allowed identifying 3 possible refugial areas in Iran most likely arising by vicariance. Based on our mitochondrial results and pending additional data, we recognize 3 candidate species among the M. raddei complex: M. raddei, Montivipera latifii, and Montivipera kuhrangica that are coherent with their geographical distribution. We propose that the most appropriate evolutionary significant units for conservation of the montivipers are represented by 13 units among which 6 are recognized as high priority. Finally, we suggest some recommendations to the IUCN as well as to the Iranian conservation policies with respect to conservation prioritization.

Integrating phylogenetic, phylogeographic, and morphometric analyses to reveal cryptic lineages within the genus Asaccus (Reptilia: Squamata: Phyllodactylidae) in Iran.

The Middle Eastern endemic genus Asaccus comprises Southwest Asian leaf-toed geckos. To date, this genus includes 19 species of leaf-toed geckos (seven in Arabia and 12 in the Zagros Mountains). Despite a recent study on the taxonomy and phylogeny of Asaccus species in Iran, controversies still remain surrounding the phylogeny and phylogeography of the genus. Here, we used an integrative approach to determine the phylogeny and phylogeography of Asaccus species using two mitochondrial genes (12 S and Cyt b), and one nuclear gene (c-mos). Our results uncovered 22 distinct lineages, demonstrating a significant cryptic diversity that challenges the current morphological classifications of these species. Phylogenetic analyses reinforce the monophyly of the Asaccus group, positioning A. montanus as a basal lineage, which supports a deep evolutionary divergence dating back to the Late Oligocene, approximately 27.94 million years ago. This genetic diversity also highlights the impact of historical climatic and geographical changes on species diversification. The findings advocate for an integrative approach combining both molecular and morphological data to resolve species identities accurately, thereby enhancing conservation strategies to protect these genetically distinct lineages.

Phylogeography of the parasitic mite Laelaps agilis in Western Palearctic shows lineages lacking host specificity but possessing different demographic histories.

BACKGROUND: Laelaps agilis C.L. Koch, 1836 is one the most abundant and widespread parasitic mite species in the Western Palearctic. It is a permanent ectoparasite associated with the Apodemus genus, which transmits Hepatozoon species via the host's blood. Phylogenetic relationships, genealogy and host specificity of the mite are uncertain in the Western Palearctic. Here, we investigated the population genetic structure of 132 individual mites across Europe from their Apodemus and Clethrionomys hosts. Phylogenetic relationships and genetic variation of the populations were analyzed using cytochrome c oxidase subunit I (COI) gene sequences. RESULTS: We recovered three main mtDNA lineages within L. agilis in the Western Palearctic, which differentiated between 1.02 and 1.79 million years ago during the Pleistocene period: (i) Lineage A, including structured populations from Western Europe and the Czech Republic, (ii) Lineage B, which included only a few individuals from Greece and the Czech Republic; and (iii) Lineage C, which comprised admixed populations from Western and Eastern Europe. Contrary to their population genetic differentiation, the lineages did not show signs of specificity to different hosts. Finally, we confirmed that the sympatric congener L. clethrionomydis is represented by a separated monophyletic lineage. CONCLUSION: Differences in the depth of population structure between L. agilis Lineages A and C, corroborated by the neutrality tests and demographic history analyses, suggested a stable population size in the structured Lineage A and a rapid range expansion for the geographically admixed Lineage C. We hypothesized that the two lineages were associated with hosts experiencing different glaciation histories. The lack of host specificity in L. agilis lineages was in contrast to the co-occurring highly host-specific lineages of Polyplax serrata lice, sharing Apodemus hosts. The incongruence was attributed to the differences in mobility between the parasites, allowing mites to switch hosts more often.

Different hosts in different lakes: prevalence and population genetic structure of plerocercoids of Ligula intestinalis (Cestoda) in Czech water bodies.

Ligula intestinalis (Linnaeus, 1758) is a tapeworm parasite with a worldwide distribution that uses a wide variety of fish species as its second intermediate host. In the present study, we investigated the prevalence and population genetic structure of plerocercoids of L. intestinalis in five common cyprinoid species, roach Rutilus rutilus (Linnaeus), freshwater bream Abramis brama (Linnaeus), white bream Blicca bjoerkna (Linnaeus), bleak Alburnus alburnus (Linnaeus), and rudd Scardinius erythrophthalmus (Linnaeus), collected in six water bodies of the Czech Republic (Milada, Most, Medard, Jordán, Rímov and Lipno). Of the six study sites, the highest frequency of parasitism was recorded in Lake Medard (15%). The overall prevalence rate among the species was as follows: roach > rudd ≥ freshwater bream > bleak > white bream. Two mitochondrial genes (cytb and COI) were used to compare the population genetic structure of parasite populations using selected samples from the five fish species. The results of the phylogenetic analysis indicated that all populations of L. intestinalis were placed in Clade A, previously identified as the most common in Europe. At a finer scale, haplotype network and PCoA analyses indicated the possible emergence of host specificity of several mtDNA haplotypes to the freshwater bream. Moreover, pairwise Fixation indices (FST) revealed a significant genetic structure between the parasite population in freshwater bream and other host species. Parasite populations in roach not only showed the highest rate of prevalence but also depicted a maximum number of shared haplotypes with populations from bleak and rudd. Our results suggest that recent ecological differentiation might have influenced tapeworm populations at a fine evolutionary scale. Thus, the differences in prevalence between fish host species in different lakes might be influenced not only by the parasite's ecology, but also by its genetic diversity.

The phylogeny, phylogeography, and diversification history of the westernmost Asian cobra (Serpentes: Elapidae: Naja oxiana) in the Trans-Caspian region.

We conducted a comprehensive analysis of the phylogenetic, phylogeographic, and demographic relationships of Caspian cobra (Naja oxiana; Eichwald, 1831) populations based on a concatenated dataset of two mtDNA genes (cyt b and ND4) across the species' range in Iran, Afghanistan, and Turkmenistan, along with other members of Asian cobras (i.e., subgenus Naja Laurenti, 1768). Our results robustly supported that the Asiatic Naja are monophyletic, as previously suggested by other studies. Furthermore, N. kaouthia and N. sagittifera were recovered as sister taxa to each other, and in turn sister clades to N. oxiana. Our results also highlighted the existence of a single major evolutionary lineage for populations of N. oxiana in the Trans-Caspian region, suggesting a rapid expansion of this cobra from eastern to western Asia, coupled with a rapid range expansion from east of Iran toward the northeast. However, across the Iranian range of N. oxiana, subdivision of populations was not supported, and thus, a single evolutionary significant unit is proposed for inclusion in future conservation plans in this region.

The legacy of Eastern Mediterranean mountain uplifts: rapid disparity of phylogenetic niche conservatism and divergence in mountain vipers.

BACKGROUND: The orogeny of the eastern Mediterranean region has substantially affected ecological speciation patterns, particularly of mountain-dwelling species. Mountain vipers of the genus Montivipera are among the paramount examples of Mediterranean neo-endemism, with restricted ranges in the mountains of Anatolia, the Levant, Caucasus, Alborz, and Zagros. Here we explore the phylogenetic and ecological diversification of Montivipera to reconstruct its ecological niche evolution and biogeographic history. Using 177 sequences of three mitochondrial genes, a dated molecular phylogeny of mountain vipers was reconstructed. Based on 320 occurrence points within the entire range of the genus and six climatic variables, ecological niches were modelled and used to infer ancestral niche occupancy. In addition, the biogeographic history and ancestral states of the species were reconstructed across climate gradients. RESULTS: Dated phylogenetic reconstruction revealed that the ancestor of mountain vipers split into two major clades at around 12.18 Mya followed by multiple vicariance events due to rapid orogeny. Montivipera colonised coastal regions from a mountain-dwelling ancestor. We detected a highly complex ecological niche evolution of mountain vipers to temperature seasonality, a variable that also showed a strong phylogenetic signal and high contribution in niche occupation. CONCLUSION: Raising mountain belts in the Eastern Mediterranean region and subsequent remarkable changes in temperature seasonality have led to the formation of important centres of diversification and endemism in this biodiversity hotspot. High rates of niche conservatism, low genetic diversity, and segregation of ranges into the endemic distribution negatively influenced the adaptive capacity of mountain vipers. We suggest that these species should be considered as evolutionary significant units and priority species for conservation in Mediterranean mountain ecosystems.

Evolutionary history and postglacial colonization of an Asian pit viper (Gloydius halys caucasicus) into Transcaucasia revealed by phylogenetic and phylogeographic analyses.

It has been generally acknowledged that glacial climates at the time of the Pleistocene altered the patterns of species distributions, prompting latitudinal and altitudinal distribution shifts in several species, including poikilothermic species commonly known for their thermal sensitivity. However, the historical phylogeographic patterns of such species have remained largely unknown. Here, we present the historical biogeographic, phylogenetic, and phylogeographic relationships of the Caucasian pit viper, G. h. caucasicus, based on two mtDNA (cyt b and ND4) and one nDNA (c-mos) genes. This pit viper represents the westernmost member of the Crotalinae subfamily in the Palearctic and occurs in a variety of habitats, from 30 m to 3,000 m above sea level. In Iran, it is distributed on the northern and southern slopes of the Alborz Mountains, rendering it a target for phylogenetic and phylogeographic studies of a terrestrial poikilothermic animal. Our study identified four Iranian lineages of G. h. caucasicus along the northeastern to northwestern slopes of the Alborz Mountains and southern Azerbaijan (Talysh Mountains). Diversification of the Iranian lineages highlights population expansion and subsequent isolation into four plausible refugial areas during the Quaternary paleo-climatic oscillations, confirmed by our molecular dating and historical biogeographic analyses. The results of coalescence-based simulations support the incursion of the species from northeastern Iran to the western end of the Alborz, and then toward Transcaucasia via two directions: northern and southern slopes of the Alborz Mountains. Furthermore, our results clearly implied that G. h. caucasicus should be elevated to species rank and further referred to as G. caucasicus (Nikolsky, 1916).