Comparative analysis of spatial genetic structures in sympatric populations of two riparian plants, Saxifraga acerifolia and Saxifraga fortunei.

PREMISE: The genetic structure between plant populations is facilitated by the spatial population arrangement and limited dispersal of seed and pollen. Saxifraga acerifolia, a local endemic species in Japan, is a habitat specialist that is confined to waterfalls in riparian environments. Its sister species, Saxifraga fortunei, is a generalist that is widely distributed along riverbanks. Here, we examined sympatric populations of the two Saxifraga species to test whether the differences in habitat preference and colonization process influenced regional and local genetic structures. METHODS: To reveal genetic structures, we examined chloroplast microsatellite variations and genome-wide nucleotide polymorphisms obtained by genotyping by sequencing. We also estimated the gene flow among and within populations and performed landscape genetic analyses to evaluate seed and pollen movement and the extent of genetic isolation related to geographic distance and/or habitat differences. RESULTS: We found strong genetic structure in the specialist S. acerifolia, even on a small spatial scale (<1 km part); each population on a different waterfall in one river system had a completely different predominant haplotype. By contrast, the generalist S. fortunei showed no clear genetic differentiation. CONCLUSIONS: Our findings suggest that the level of genetic isolation was increased in S. acerifolia by the spatially fragmented habitat and limited seed and pollen dispersal over waterfalls. Habitat differentiation between the sister taxa could have contributed to the different patterns of gene flow and then shaped the contrasting genetic structures.

A Comparative Test for Divergent Adaptation: Inferring Speciation Drivers from Functional Trait Divergence.

AbstractEcological differentiation between lineages is widely considered to be an important driver of speciation, but support for this hypothesis is mainly derived from the detailed study of a select set of model species pairs. Mounting evidence from nonmodel taxa, meanwhile, suggests that speciation often occurs with minimal differentiation in ecology or ecomorphology, calling into question the true contribution of divergent adaptation to species richness in nature. To better understand divergent ecological adaptation and its role in speciation generally, researchers require a comparative approach that can distinguish its signature from alternative processes, such as drift and parallel selection, in data sets containing many species pairs. Here we introduce new statistical models of divergent adaptation in the continuous traits of paired lineages. In these models, ecomorphological characters diverge as two lineages adapt toward alternative phenotypic optima following their departure from a common ancestor. The absolute distance between optima measures the extent of divergent selection and provides a basis for interpretation. We encode the models in the new R package diverge and extend them to allow the distance between optima to vary across continuous and categorical variables. We test model performance using simulation and demonstrate model application using published data sets of trait divergence in birds and mammals. Our framework provides the first explicit test for signatures of divergent selection in trait divergence data sets, and it will enable empiricists from a wide range of fields to better understand the dynamics of divergent adaptation and its prevalence in nature beyond just our best-studied model systems.

Phylogeographic evidence for two species of muriqui (genus Brachyteles).

The taxonomy of muriquis, the largest extant primates in the New World, is controversial. While some specialists argue for a monotypic genus (Brachyteles arachnoides), others favor a two-species classification, splitting northern muriquis (Brachyteles hypoxanthus) from southern muriquis (B. arachnoides). This uncertainty affects how we study the differences between these highly endangered and charismatic primates, as well as the design of more effective conservation programs. To address this issue, between 2003 and 2017 we collected over 230 muriqui fecal samples across the genus' distribution in the Brazilian Atlantic Forest, extracted DNA from these samples, and sequenced 423 base pairs of the mitochondrial DNA (mtDNA) control region. Phylogenetic and species delimitation analyses of our sequence dataset robustly support two reciprocally monophyletic groups corresponding to northern and southern muriquis separated by an average 12.7% genetic distance. The phylogeographic break between these lineages seems to be associated with the Paraíba do Sul River and coincides with the transition between the north and south Atlantic Forest biogeographic zones. Published divergence estimates from whole mitochondrial genomes and nuclear loci date the split between northern and southern muriquis to the Early Pleistocene (ca. 2.0 mya), and our new mtDNA dataset places the coalescence time for each of these two clades near the last interglacial (ca. 120-80 kya). Our results, together with both phenotypic and ecological differences, support recognizing northern and southern muriquis as sister species that should be managed as distinct evolutionarily significant units. Given that only a few thousand muriquis remain in nature, it is imperative that conservation strategies are tailored to protect both species from extinction.

Two new species of Stillabothrium (Cestoda: Rhinebothriidea) from stingrays of the genus Fontitrygon from Senegal.

Morphological and molecular analyses of cestode specimens collected during survey work of batoid elasmobranchs and their parasites in Senegal revealed two new species of the rhinebothriidean cestode genus Stillabothrium Healy et Reyda 2016. Stillabothrium allisonae Dedrick et Reyda sp. n. and Stillabothrium charlotteae Iwanyckyj, Dedrick et Reyda sp. n. are both described from Fontitrygon margaritella (Compagno et Roberts) and Fontitrygon margarita (Günther). Both new cestode species overlap in geographic distribution, host use and proglottid morphology, but are distinguished from each other, and from the other seven described species of Stillabothrium, on the basis of their pattern of bothridial loculi. Phylogenetic analyses based on sequence data for 1,084 bp from the D1-D3 region of 28S rDNA that included multiple specimens of both new species and eight other species of Stillabothrium corroborated the morphologically-determined species boundaries. The phylogenetic analyses indicate that S. allisonae sp. n. and S. charlotteae sp. n. are sister species, a noteworthy pattern given that the two species of the stingray genus Fontitrygon they both parasitise, F. margaritella and F. margarita, are also sister species. Although species of Stillabothrium vary widely in their patterns of facial loculi, the variation does not appear to correlate with phylogeny. Most species of Stillabothrium parasitise myliobatiform elasmobranch genera of the Dasyatidae Jordan. This study brings the number of described species of Stillabothrium to nine, three of which occur in the eastern Atlantic, two of which occur off the northern coast of Australia, and four of which are from coastal Borneo.

Molecular evolution of Odorant-binding proteins gene family in two closely related Anastrepha fruit flies.

BACKGROUND: Odorant-binding proteins (OBPs) are of great importance for survival and reproduction since they participate in initial steps of the olfactory signal transduction cascade, solubilizing and transporting chemical signals to the olfactory receptors. A comparative analysis of OBPs between closely related species may help explain how these genes evolve and are maintained under natural selection and how differences in these proteins can affect olfactory responses. We studied OBP genes in the closely related species Anastrepha fraterculus and A. obliqua, which have different host preferences, using data from RNA-seq cDNA libraries of head and reproductive tissues from male and female adults, aiming to understand the speciation process occurred between them. RESULTS: We identified 23 different OBP sequences from Anastrepha fraterculus and 24 from A. obliqua, which correspond to 20 Drosophila melanogaster OBP genes. Phylogenetic analysis separated Anastrepha OBPs sequences in four branches that represent four subfamilies: classic, minus-C, plus-C and dimer. Both species showed five plus-C members, which is the biggest number found in tephritids until now. We found evidence of positive selection in four genes and at least one duplication event that preceded the speciation of these two species. Inferences on tertiary structures of putative proteins from these genes revealed that at least one positively selected change involves the binding cavity (the odorant binding region) in the plus-C OBP50a. CONCLUSIONS: A. fraterculus and A. obliqua have a bigger OBP repertoire than the other tephritids studied, though the total number of Anastrepha OBPs may be larger, since we studied only a limited number of tissues. The contrast of these closely related species reveals that there are several amino acid changes between the homologous genes, which might be related to their host preferences. The plus-C OBP that has one amino acid under positive selection located in the binding cavity may be under a selection pressure to recognize and bind a new odorant. The other positively selected sites found may be involved in important structural and functional changes, especially ones in which site-specific changes would radically change amino acid properties.

Comparison of neuromuscular development in two dinophilid species (Annelida) suggests progenetic origin of Dinophilus gyrociliatus.

BACKGROUND: Several independent meiofaunal lineages are suggested to have originated through progenesis, however, morphological support for this heterochronous process is still lacking. Progenesis is defined as an arrest of somatic development (synchronously in various organ systems) due to early maturation, resulting in adults resembling larvae or juveniles of the ancestors. Accordingly, we established a detailed neuromuscular developmental atlas of two closely related Dinophilidae using immunohistochemistry and CLSM. This allows us to test for progenesis, questioning whether i) the adult smaller, dimorphic Dinophilus gyrociliatus resembles a younger developmental stage of the larger, monomorphic D. taeniatus and whether ii) dwarf males of D. gyrociliatus resemble an early developmental stage of D. gyrociliatus females. RESULTS: Both species form longitudinal muscle bundles first, followed by circular muscles, creating a grid of body wall musculature, which is the densest in adult D. taeniatus, while the architecture in adult female D. gyrociliatus resembles that of prehatching D. taeniatus. Both species display a subepidermal ganglionated nervous system with an anterior dorsal brain and five longitudinal ventral nerve bundles with six sets of segmental commissures (associated with paired ganglia). Neural differentiation of D. taeniatus and female D. gyrociliatus commissures occurs before hatching: both species start out forming one transverse neurite bundle per segment, which are thereafter joined by additional thin bundles. Whereas D. gyrociliatus arrests its development at this stage, adult D. taeniatus condenses the thin commissures again into one thick commissural bundle per segment. Generally, D. taeniatus adults demonstrate a seemingly more organized (= segmental) pattern of serotonin-like and FMRFamide-like immunoreactive elements. The dwarf male of D. gyrociliatus displays a highly aberrant neuromuscular system, showing no close resemblance to any early developmental stage of female Dinophilus, although the onset of muscular development mirrors the early myogenesis in females. CONCLUSION: The apparent synchronous arrest of nervous and muscular development in adult female D. gyrociliatus, resembling the prehatching stage of D. taeniatus, suggests that D. gyrociliatus have originated through progenesis. The synchrony in arrest of three organ systems, which show opposing reduction and addition of elements, presents one of the morphologically best-argued cases of progenesis within Spiralia.

The geography and ecology of plant speciation: range overlap and niche divergence in sister species.

A goal of evolutionary biology is to understand the roles of geography and ecology in speciation. The recent shared ancestry of sister species can leave a major imprint on their geographical and ecological attributes, possibly revealing processes involved in speciation. We examined how ecological similarity, range overlap and range asymmetry are related to time since divergence of 71 sister species pairs in the California Floristic Province (CFP). We found that plants exhibit strikingly different age-range correlation patterns from those found for animals; the latter broadly support allopatric speciation as the primary mode of speciation. By contrast, plant sisters in the CFP were sympatric in 80% of cases and range sizes of sisters differed by a mean of 10-fold. Range overlap and range asymmetry were greatest in younger sisters. These results suggest that speciation mechanisms broadly grouped under 'budding' speciation, in which a larger ranged progenitor gives rise to a smaller ranged derivative species, are probably common. The ecological and reproductive similarity of sisters was significantly greater than that of sister-non-sister congeners for every trait assessed. However, shifts in at least one trait were present in 93% of the sister pairs; habitat and soil shifts were especially common. Ecological divergence did not increase with range overlap contrary to expectations under character displacement in sympatry. Our results suggest that vicariant speciation is more ubiquitous in animals than plants, perhaps owing to the sensitivity of plants to fine-scale environmental heterogeneity. Despite high levels of range overlap, ecological shifts in the process of budding speciation may result in low rates of fine-scale spatial co-occurrence. These results have implications for ecological studies of trait evolution and community assembly; despite high levels of sympatry, sister taxa and potentially other close relatives, may be missing from local communities.

Not so cryptic-differences between mating calls of Hyla arborea and Hyla orientalis from Bulgaria.

Anurans are among the most vocally active vertebrate animals and emit calls with different functions. In order to attract a mate, during the breeding season male frogs produce mating calls which have species-specific structure and parameters, and have been successfully used to resolve issues in taxonomy and phylogenetic relations. This is particularly useful when closely related taxa are concerned, as many species are morphologically almost identical, but still their status is well-supported by molecular and genetic data, suggesting the existence of mechanisms for reproductive isolation. Such is the case for treefrogs from the Hyla arborea group, which are now recognized as several distinct species. The present study aims to establish differences in call parameters between the European tree frog, Hyla arborea, and the Eastern tree frog, Hyla orientalis, which both occur on the territory of Bulgaria. Using autonomous audio loggers, calls from six sites (three in the range of H. arborea and three in the range of H. orientalis) were recorded between 7 p.m. and 12 a.m. during the breeding season in 2020-2023. The following parameters in a total of 390 mating calls were analyzed: call count, pulse count, call series duration, call period, peak (dominant) frequency, entropy. Results indicated that sites formed two distinct groups, which corresponded to the known distribution ranges of H. arborea and H. orientalis. The first two components of the PCA explained 71% of the total variance, with variables call count, call series duration, peak frequency and entropy being most important for differentiation between the sites. This study presents the first attempt to differentiate between the calls of these two sister taxa, which both fall within the "short-call treefrogs" group, and results are discussed in terms of known data for mating calls in Hyla sp., as well as limitations and future perspectives.

Sexual selection accelerates signal evolution during speciation in birds.

Sexual selection is proposed to be an important driver of diversification in animal systems, yet previous tests of this hypothesis have produced mixed results and the mechanisms involved remain unclear. Here, we use a novel phylogenetic approach to assess the influence of sexual selection on patterns of evolutionary change during 84 recent speciation events across 23 passerine bird families. We show that elevated levels of sexual selection are associated with more rapid phenotypic divergence between related lineages, and that this effect is restricted to male plumage traits proposed to function in mate choice and species recognition. Conversely, we found no evidence that sexual selection promoted divergence in female plumage traits, or in male traits related to foraging and locomotion. These results provide strong evidence that female choice and male-male competition are dominant mechanisms driving divergence during speciation in birds, potentially linking sexual selection to the accelerated evolution of pre-mating reproductive isolation.

Evolutionary Genomics of Sister Species Differing in Effective Population Sizes and Recombination Rates.

Studies of closely related species with known ecological differences provide exceptional opportunities for understanding the genetic mechanisms of evolution. In this study, we compared population-genomics data between Daphnia pulex and Daphnia pulicaria, two reproductively compatible sister species experiencing ecological speciation, the first largely confined to intermittent ponds and the second to permanent lakes in the same geographic region. Daphnia pulicaria has lower genome-wide nucleotide diversity, a smaller effective population size, a higher incidence of private alleles, and a substantially more linkage disequilibrium than D. pulex. Positively selected genes in D. pulicaria are enriched in potentially aging-related categories such as cellular homeostasis, which may explain the extended life span in D. pulicaria. We also found that opsin-related genes, which may mediate photoperiodic responses, are under different selection pressures in these two species. Genes involved in mitochondrial functions, ribosomes, and responses to environmental stimuli are found to be under positive selection in both species. Additionally, we found that the two species have similar average evolutionary rates at the DNA-sequence level, although approximately 160 genes have significantly different rates in the two lineages. Our results provide insights into the physiological traits that differ within this regionally sympatric sister-species pair that occupies unique microhabitats.

Analysis of autapomorphic point mutations provides a key for the tangled taxonomic distinction of the closely related species, Euplotes crassus, E. minuta and E. vannus (Ciliophora, Euplotida).

A well-defined clade of the Euplotes phylogenetic tree is represented by marine species characterized by a single-type dargyrome and ten fronto-ventral cirri. Three of them, namely Euplotes crassus, E. minuta and E. vannus, form a complex of closely related species of large use in experimental ciliatology. Despite morphometric and genetic analyses having substantiated their taxonomic separation, ambiguities still persist in strain assignments to one or another species. In addition to objective reasons intrinsic to significant overlapping of most morphological parameters, ambiguities also result from divergences (inherited from past literature) in deciding which of the two morphotypes, E. crassus or E. vannus, is characterized by a larger or a medium cell body size (E. minuta being clearly distinct by a smaller morphotype). By analysing nuclear SSU-rRNA gene and ITS region sequences from 37 strains, previously assigned to E. crassus, E. minuta and E. vannus based on conventional taxonomic parameters, we identified and used ITS autapomorphic point mutations to design three species-specific primers. In combination with an Euplotes-generic primer, they proved to be very effective in running polymerase chain reactions that produce amplicons of species-specific size that reliably resolve ambiguities in assigning strains to E. crassus, E. minuta or E. vannus.

Compensatory Base Changes and Varying Phylogenetic Effects on Angiosperm ITS2 Genetic Distances.

A compensatory base change (CBC) that coevolves in the secondary structure of ribosomal internal transcribed spacer 2 (ITS2) influences the estimation of genetic distance and thus challenges the phylogenetic use of this most popular genetic marker. To date, however, the CBC effect on ITS2 genetic distance is still unclear. Here, ITS2 sequences of 46 more recent angiosperm lineages were screened from 5677 genera and phylogenetically analyzed in sequence-structure format, including secondary structure prediction, structure-based alignment and sequence partition of paired and unpaired regions. ITS2 genetic distances were estimated comparatively by using both conventional DNA substitution models and RNA-specific models, which were performed in the PHASE package. Our results showed that the existence of the CBC substitution inflated the ITS2 genetic distances to different extents, and the deviation could be 180% higher if the relative ratio of substitution rate in ITS2 secondary structure stems was threefold higher than that in the loops. However, the CBC effect was minor if that ratio was below two, indicating that the DNA model is still applicable in recent lineages in which few CBCs occur. We thus provide a general empirical threshold to take account of CBC before ITS2 phylogenetic analyses.

Ancient Tethyan Vicariance and Long-Distance Dispersal Drive Global Diversification and Cryptic Speciation in the Red Seaweed Pterocladiella.

We investigated the globally distributed red algal genus Pterocladiella, comprising 24 described species, many of which are economically important sources of agar and agarose. We used DNA-based species delimitation approaches, phylogenetic, and historical biogeographical analyses to uncover cryptic diversity and infer the drivers of biogeographic patterns. We delimited 43 species in Pterocladiella, of which 19 are undescribed. Our multigene time-calibrated phylogeny and ancestral area reconstruction indicated that Pterocladiella most likely originated during the Early Cretaceous in the Tethys Sea. Ancient Tethyan vicariance and long-distance dispersal have shaped current distribution patterns. The ancestor of Eastern Pacific species likely arose before the formation of the formidable Eastern Pacific Barrier-a first confirmation using molecular data in red algae. Divergences of Northeast and Southeast Pacific species have been driven by the Central American Seaway barrier, which, paradoxically, served as a dispersal pathway for Atlantic species. Both long- and short-distance dispersal scenarios are supported by genetic relationships within cosmopolitan species based on haplotype analysis. Asymmetrical distributions and the predominance of peripatry and sympatry between sister species suggest the importance of budding speciation in Pterocladiella. Our study highlights the underestimation of global diversity in these crucial components of coastal ecosystems and provides evidence for the complex evolution of current species distributions.

Comparative reproductive ecology of two sister Asarum species (Aristolochiaceae) in relation to the evolution of elongated floral appendage.

Genus Asarum (Aristolochiaceae) shows diverse floral morphology and is hypothesized to have diversified as a result of pollinator-mediated selection. Yet most aspects of their reproductive ecology, including pollinators, remain unclear. This study focuses on A. costatum and A. minamitanianum in Japan, a sister species pair having remarkable differences in calyx lobe length (10-20 mm and 70-180 mm, respectively). The objectives of this study are to elucidate multiple aspects of reproductive ecology of these two species and obtain evolutionary insights into floral organ elongation. We adopted combined approaches, including field observations, molecular analyses and cultivation experiments, such as pollinator observation for 3 years, fine-scale spatial genetic analysis of 769 individuals, paternity analysis based on 566 seeds over 4 years, and control pollination experiments. Both Asarum species had strong spatial genetic structures, indicating limited seed dispersal. Pollinator observation revealed that flies and ground-dwelling insects visited flowers of both species, but that the pollinator fauna differed between the species. The visitation rate of flies was extremely low but was more than twice as high in the species with an elongated floral appendage. Paternity analysis revealed A. minamitanianum was predominantly outcrossing, while A. costatum showed a wide range of selfing rates among fruits. These two Asarum species are likely adapted to fly pollination in the shady forest understorey, where available pollinator fauna is limited. In addition, although its function remains unclear, the elongated calyx lobe of A. minamitanianum could have evolved for effective pollen dispersal by attracting fly visitors.

Elucidation of the speciation history of three sister species of crown-of-thorns starfish (Acanthaster spp.) based on genomic analysis.

The crown-of-thorns starfish (COTS) is a coral predator that is widely distributed in Indo-Pacific Oceans. A previous phylogenetic study using partial mitochondrial sequences suggested that COTS had diverged into four distinct species, but a nuclear genome-based analysis to confirm this was not conducted. To address this, COTS species nuclear genome sequences were analysed here, sequencing Northern Indian Ocean (NIO) and Red Sea (RS) species genomes for the first time, followed by a comparative analysis with the Pacific Ocean (PO) species. Phylogenetic analysis and ADMIXTURE analysis revealed clear divergences between the three COTS species. Furthermore, within the PO species, the phylogenetic position of the Hawaiian sample was further away from the other Pacific-derived samples than expected based on the mitochondrial data, suggesting that it may be a PO subspecies. The pairwise sequentially Markovian coalescent model showed that the trajectories of the population size diverged by region during the Mid-Pleistocene transition when the sea-level was dramatically decreased, strongly suggesting that the three COTS species experienced allopatric speciation. Analysis of the orthologues indicated that there were remarkable genes with species-specific positive selection in the genomes of the PO and RS species, which suggested that there may be local adaptations in the COTS species.

Chloroplast genomic comparison of two sister species Allium macranthum and A. fasciculatum provides valuable insights into adaptive evolution.

BACKGROUND: Allium macranthum and Allium fasciculatum are two sister species and their natural populations are separated by high mountains and deep valleys with exact opposite habitat. The chloroplast genome in angiosperms has showed useful for investigating plant evolution and systematic studies. OBJECTIVE: Comparative analysis of these genomes revealed potential markers and phylogenetic analysis, and discuss the influence of positive selected sites on adaptive evolution. METHODS: Here, we sequenced the complete chloroplast genomes of these two species and analyzed the repeat sequences components, nucleotide diversity, selection pressure and the phylogeny relationships with related species. RESULTS: A typical quadripartite structure was detected with a genome size changed from 152,148 to 152,931 bp. We identified 67 and 79 simple sequence repeats in A. macranthum and A. fasciculatum, in which the mono-nucleotide repeats A/T possess the highest percentage. Three mutational hotspots (rpl32, rps16 and matK) at the SSC and LSC regions were observed, which showed remarkably higher Pi value (> 0.03). Additionally, eight genes (rpoA, atpF, cemA, rps4, ccsA, rpoC2, rpl14 and clpP) exhibited elevated pairwise Ka/Ks ratios in alpine species. Phylogenetic analyses based on the CDS sequences and the whole complete genomes showed same topologies with high support, and A. macranthum was closely clustered with A. fasciculatum within the fourteen Amaryllidaceae species. CONCLUSION: Their coding proteins of these genes often functioned in chloroplast protein synthesis, gene transcription, energy transformation and regulation and photosynthesis. These results provide valuable insights into the alpine species adaptation and evolution.

Welcome back Mr. Rudkin: differentiating Papilio zelicaon and Papilio polyxenes in Southern California (Lepidoptera: Papilionidae).

We studied wing pattern characters to distinguish closely related sympatric species Papilio zelicaon Lucas, 1852 and Papilio polyxenes Fabricius, 1775 in Southern California, and developed a morphometric method based on the ventral black postmedian band. Application of this method to the holotype of Papilio [Zolicaon variety] Coloro W. G. Wright, 1905, the name currently applied to the P. polyxenes populations, revealed that it is a P. zelicaon specimen. The name for western US polyxenes subspecies thus becomes Papilio polyxenes rudkini (F. R. Chermock, 1981), reinstated status, and we place coloro as a junior subjective synonym of P. zelicaon. Furthermore, we sequenced mitochondrial DNA COI barcodes of rudkini and coloro holotypes and compared them with those of polyxenes and zelicaon specimens, confirming rudkini as polyxenes and coloro as zelicaon.

Molecular Characterization of the Myoliquefactive Fish Parasite Kudoa mirabilis (Cnidaria, Kudoidae) from SE Indian Ocean and Its Phylogenetic Relationship with the Kudoa thyrsites Species Complex.

Myxosporean parasites of the genus Kudoa are fish parasites of great economic importance, as some species can affect the fish fillet quality by producing macroscopic cysts or generating post mortem myoliquefaction, commonly referred to as 'soft flesh'. Kudoa mirabilis is a 'soft flesh'-inducing species originally described based on morphology in the musculature of Trichiurus lepturus from the Indian Ocean. An integrative morphological and genetic characterization of K. mirabilis from the type host caught off the coast of Tanzania is here provided. The spores were stellate with four unequal polar capsules, showing similarities to Kudoa thyrsites. For comparative and validation purpose, K. mirabilis was compared morphologically and genetically with K. thyrsites reference isolates, including new obtained samples from the type host Thyrsites atun caught in the SE Atlantic Ocean. Morphological analyses of spores revealed key diagnostic characters clearly distinguishing the two Kudoa species. Phylogenetic analyses based on SSU and LSU rRNA genes demonstrated that K. mirabilis is a distinct and valid species, representing a sister group to a K. thyrsites subclade that comprises several isolates from Japan and one single isolate from South Africa. This finding raises questions about the true diversity likely hidden in the K. thyrsites complex.

Genus Pempeliella Caradja, 1916 and P.bayassensis Leraut, 2001 (Lepidoptera, Pyralidae) in Italy.

Pempeliellabayassensis has been reported for the first time in Italy. This species has been confused with P.ornatella for a long time. Our study of the historical collections of Carlo Prola and Federico Hartig, and also newly collected materials from central Italy, allowed us to verify the presence of P.bayassensis in Italy. At present, this species is known only in central Italy (Lazio), where it coexists with P.ornatella and P.sororiella. We also provide information on the geographical distribution of the genus Pempeliella in Italy. In northern Italy we found P.ornatella and P.sororiella, and in the south (Puglia), P.sororiella. In Sardinia, P.matilella, which has been confused with Delplanqueiacortella in the past, coexists with P.sororiella.

Alternative treatments of genetic distances for species delimitation in Callosciurinae and Sciurinae (Rodentia: Sciuridae).

There are many DNA taxonomy methods available, but single-locus data coupled with distance-based analyses still dominate species identification in practice. Genetic distance thresholds are often used for assigning genetic diversity into entities corresponding to species but are criticized for non-universality or arbitrary. This study investigated an alternative approach for determining genetic thresholds that used iteratively sister-clade clustering for sister species boundary detection within a phylogenetic framework. This method was separately applied to two close subfamilies of Callosciurinae and Sciurinae in Rodentia using the cyt b gene. Our results showed that genetic thresholds for Callosciurinae and Sciurinae were 4.0% and 6.0%, respectively, indicating that the optimized thresholds could be lineage-specific. The use of these thresholds for taxa partitioning yielded a very similar putative species number as the prevailing ABGD method, and increased species diversity by 74.2% and 20.7% in Callosciurinae and Sciurinae, respectively. This suggested that additional cryptic species were present that warrant further investigation. We further tested the performance limitation of our method by simulating the impacts of tree construction and sampling limitation. The results showed that it preformed equally well for different trees but failed to work when inter- or intraspecific sampling is insufficient. These findings support the feasibility of this approach as an alternative tool for species delineation when only single-locus information is available for large datasets.