Adaptive divergence and post-zygotic barriers to gene flow between sympatric populations of a herbivorous mite.

Plant-herbivore interactions promote the generation and maintenance of both plant and herbivore biodiversity. The antagonistic interactions between plants and herbivores lead to host race formation: the evolution of herbivore types specializing on different plant species, with restricted gene flow between them. Understanding how ecological specialization promotes host race formation usually depends on artificial approaches, using laboratory experiments on populations associated with agricultural crops. However, evidence on how host races are formed and maintained in a natural setting remains scarce. Here, we take a multidisciplinary approach to understand whether populations of the generalist spider mite Tetranychus urticae form host races in nature. We demonstrate that a host race co-occurs among generalist conspecifics in the dune ecosystem of The Netherlands. Extensive field sampling and genotyping of individuals over three consecutive years showed a clear pattern of host associations. Genome-wide differences between the host race and generalist conspecifics were found using a dense set of SNPs on field-derived iso-female lines and previously sequenced genomes of T. urticae. Hybridization between lines of the host race and sympatric generalist lines is restricted by post-zygotic breakdown, and selection negatively impacts the survival of generalists on the native host of the host race. Our description of a host race among conspecifics with a larger diet breadth shows how ecological and reproductive isolation aid in maintaining intra-specific variation in sympatry, despite the opportunity for homogenization through gene flow. Our findings highlight the importance of explicitly considering the spatial and temporal scale on which plant-herbivore interactions occur in order to identify herbivore populations associated with different plant species in nature. This system can be used to study the underlying genetic architecture and mechanisms that facilitate the use of a large range of host plant taxa by extreme generalist herbivores. In addition, it offers the chance to investigate the prevalence and mechanisms of ecological specialization in nature.

Structural and molecular mechanisms for membrane protein biogenesis by the Oxa1 superfamily.

Members of the Oxa1 superfamily perform membrane protein insertion in bacteria, the eukaryotic endoplasmic reticulum (ER), and endosymbiotic organelles. Here, we review recent structures of the three ER-resident insertases and discuss the extent to which structure and function are conserved with their bacterial counterpart YidC.

First worldwide molecular phylogeny of the morphologically and ecologically hyperdiversified snapping shrimp genus Alpheus (Malacostraca: Decapoda).

Hyperdiverse animal groups raise intriguing questions regarding the factors that generate and maintain their diversity. The snapping shrimp genus Alpheus (with >300 described species) is a spectacularly diversified group of decapod crustaceans that serves as an exemplary system for addressing evolutionary questions regarding morphological adaptations, symbiosis, cryptic diversity and molecular divergence. A lack of information regarding evolutionary relationships among species has limited investigations into the mechanisms that drive the diversification of Alpheus. Previous phylogenetic studies of Alpheus have been restricted in scope, while molecular datasets used for phylogenetic reconstructions have been based solely on mitochondrial and a handful of nuclear markers. Here we use an anchored hybrid enrichment (AHE) approach to resolve phylogenetic relationships among species of Alpheus. The AHE method generated sequence data for 240 loci (>72,000 bp) for 65 terminal species that span the geographic, ecological and taxonomic diversity of Alpheus. Our resulting, well-supported phylogeny demonstrates a lack of monophyly for five out of seven morphologically defined species groups that have traditionally been used as a framework in Alpheus taxonomy. Our results also suggest that symbiotic associations with a variety of other animals have evolved independently in at least seven lineages in this genus. Our AHE phylogeny represents the most comprehensive phylogenetic treatment of Alpheus to date and will provide a useful evolutionary framework to further investigate questions, such as various modifications of the snapping claw and the role of habitat specialization and symbiosis in promoting speciation. Running head: PHYLOGENY OF THE SNAPPING SHRIMP GENUS ALPHEUS.

Ripe for reassessment: A synthesis of available molecular data for the speciose diatom family Bacillariaceae.

The Bacillariaceae is a very species-rich family of raphid diatoms and includes the large and taxonomically difficult genus Nitzschia, whose species are often small-celled and finely structured and have few discrete morphological characters visible in the light microscope. The classification of Nitzschia is still mostly based on one developed in the second half of the 19th century by Grunow, who separated the genus into a series of sections largely on cell shape and symmetry, the position of the raphe, transverse extension of the fibulae, and folding of the valve. We assembled and analysed single-gene and concatenated alignments of nSSU, nLSU, rbcL, psbC and cox1 to test Grunow's and subsequent classifications and to examine selected morphological characters for their potential to help define monophyletic groups. The maximum likelihood trees were equivocal as to monophyly of the family itself but showed good support for each of eight main clades of Bacillariaceae, three of which corresponded more or less to existing genera (Hantzschia, Cylindrotheca and Bacillaria). The other five main clades and some subclades comprised groups of Nitzschia species or assemblies of Nitzschia species with other genera (Pseudo-nitzschia, Fragilariopsis, Neodenticula, Tryblionella, Psammodictyon). Relationships between most of the eight main clades were not resolved robustly but all analyses recovered Nitzschia as non-monophyletic. The Grunowian classification of Nitzschia into sections was not supported, though in some respects (e.g. treatment of sigmoid species) it is better than subsequent reclassifications. Several of the main clades and subclades are cryptic (lacking morphological synapomorphies) and homoplasy is common in both light microscopical and ultrastructural characters (to the extent that organisms initially assigned to the same species sometimes prove to belong to a different main clade). Nevertheless, some characters, including the structure of the raphe canal and girdle, seem to be sufficiently conservative evolutionarily to give a provisional estimate of relationships if molecular data are unavailable. No new formal classifications are proposed but various options are explored and research needs identified.

Genomic fingerprints of palaeogeographic history: The tempo and mode of rift tectonics across tropical Africa has shaped the diversification of the killifish genus Nothobranchius (Teleostei: Cyprinodontiformes).

This paper reports a phylogeny of the African killifishes (Genus Nothobranchius, Order Cyprinodontiformes) informed by five genetic markers (three nuclear, two mitochondrial) of 80 taxa (seven undescribed and 73 of the 92 recognized species). These short-lived annual fishes occupy seasonally wet habitats in central and eastern Africa, and their distribution coincides largely with the East African Rift System (EARS). The fossil dates of sister clades used to constrain a chronometric tree of all sampled Nothobranchius recovered the origin of the genus at ~13.27 Mya. It was followed by the radiations of six principal clades through the Neogene. An ancestral area estimation tested competing biogeographical hypotheses to constrain the ancestral origin of the genus to the Nilo-Sudan Ecoregion, which seeded a mid-Miocene dispersal event into the Coastal ecoregion, followed closely (~10 Mya) by dispersals southward across the Mozambique coastal plain into the Limpopo Ecoregion. Extending westwards across the Tanzanian plateau, a pulse of radiations through the Pliocene were associated with dispersals and fragmentation of wetlands across the Kalahari and Uganda Ecoregions. We interpret this congruence of drainage rearrangements with dispersals and cladogenic events of Nothobranchius to reflect congruent responses to recurrent uplift and rifting. The coevolution of these freshwater fishes and wetlands is attributed to ultimate control by tectonics, as the EARS extended southwards during the Neogene. Geobiological consilience of the combined evidence supports a tectonic hypothesis for the evolution of Nothobranchius.

A five-gene molecular phylogeny reveals Parapanteles Ashmead (Hymenoptera: Braconidae) to be polyphyletic as currently composed.

Parapanteles Ashmead (Braconidae: Microgastrinae) is a medium-sized genus of microgastrine wasps that was erected over a century ago and lacks a unique synapomorphic character, and its monophyly has not been tested by any means. Parapanteles usually are parasitoids of large, unconcealed caterpillars (macrolepidoptera) and have been reared from an unusually large diversity of hosts for a relatively small microgastrine genus. We used Cytochrome Oxidase I sequences ("DNA barcodes") available for Parapanteles and other microgastrines to sample the generic diversity of described and undescribed species currently placed in Parapanteles, and then sequenced four additional genes for this subsample (wingless, elongation factor 1-alpha, ribosomal subunit 28s, and NADH dehydrogenase subunit 1). We constructed individual gene trees and concatenated Bayesian and maximum-likelihood phylogenies for this 5-gene subsample. In these phylogenies, most Parapanteles species formed a monophyletic clade within another genus, Dolichogenidea, while the remaining Parapanteles species were recovered polyphyletically within several other genera. The latter likely represent misidentified members of other morphologically similar genera. Species in the monophyletic clade containing most Parapanteles parasitized caterpillars from only five families - Erebidae (Arctiinae), Geometridae, Saturniidae, Notodontidae, and Crambidae. We do not make any formal taxonomic decisions here because we were not able to include representatives of type species for Parapanteles or other relevant genera, and because we feel such decisions should be reserved until a comprehensive morphological analysis of the boundaries of these genera is accomplished.

Unraveling the systematics and evolution of the 'Geophagus' brasiliensis (Cichliformes: Cichlidae) species complex.

The 'Geophagus' brasiliensis complex is one of the most abundant groups of cichlids from eastern coastal basins in South America. Traditionally, this fish group has been recognized as incertae sedis because of phylogenetic uncertainties and unclear taxonomy. In addition, the remarkable morphological, chromosomal, and DNA variation reported over recent years in several populations of these cichlids has increased the debate about their species richness and their distributional range. Here, we tested the presence of independent evolutionary lineages within the 'G.' brasiliensis complex, addressing their taxonomic status and evolutionary relationships, including a comparative analysis of genetic and morphological patterns, based on an extensive dataset, comprising 172 sampling sites along most of their known range using a mitochondrial marker, RADseq data and geometric morphometrics. The number of putative species in the present study varied from 9 to 11 depending on the molecular species delimitation methods used. Our results revealed at least two putative new taxa ('Geophagus' sp. Doce and 'Geophagus' sp. Upper Contas). Morphometric analyses, particularly those based on Canonical Variate Analysis (CVA), revealed significant morphological differentiation between species within the main clades. On the other hand, analyses of morphological phylogenetic signal and phylomorphospace provided no evidence of adaptive differentiation among these species. Thus, diversification in the 'G.' brasiliensis complex seems to have been influenced by hydrogeological events that promoted allopatry, such as the presence of paleodrainages and distributional reconfiguration through river captures. We propose major changes in the known distribution of some species within the complex and conservatively suggest the recognition of 10 species within the 'Geophagus' brasiliensis complex, with the potential for further dividing 'G.' rufomarginatus after additional taxonomic evaluation.

Phylogenetic relationships between fungus-associated Neotropical species of the genera Hirtodrosophila, Mycodrosophila and Zygothrica (Diptera, Drosophilidae), with insights into the evolution of breeding sites usage.

The Neotropical region harbors an astonishing diversity of species, but still encompasses the least studied biogeographic region of the world. These properties apply for different taxonomic groups, and can be exemplified by drosophilids. In fact, high levels of cryptic diversity have recently been discovered for Neotropical species of the Zygothrica genus group, but relationships among these species, or them and other Drosophilidae species still remains to be addressed. Therefore, the aim of this study was to evaluate the phylogenetic relationships between fungus-associated Neotropical species of the genera Hirtodrosophila, Mycodrosophila and Zygothrica, which together with Paramycodrosophila and Paraliodrosophila compose the Zygothrica genus group. For this, fragments of the mitochondrial cytochrome oxidase subunits I (COI) and II (COII) genes, and the nuclear alpha methyldopa (Amd) and dopa decarboxylase (Ddc) genes were newly characterized for 43 Neotropical specimens of fungus-associated drosophilids, and analyzed in the context of 51 additional Drosophilinae sequences plus one Steganinae outgroup. Based on the resulting phylogeny, the evolution of breeding sites usage was also evaluated through ancestral character reconstructions. Our results revealed the Zygothrica genus group as a monophyletic lineage of Drosophila that branches after the subgenera Sophophora and Drosophila. Within this lineage, Mycodrosophila species seem to encompass the early offshoot, followed by a grade of Hirtodrosophila species, with derived branches mostly occupied by representatives of Zygothrica. This genus, in particular, was subdivided into five major clades, two of which include species of Hirtodrosophila, whose generic status needs to be reevatuated. According to our results, the use of fungi as breeding sites encompasses a symplesiomorphy for the Zygothrica genus group, since one of the recovered clades is currently specialized in using flowers as breeding sites whereas a sole species presents a reversal to the use of fruits of a plant of Gentianales. So, in general, this study supports the paraphyly of Drosophila in relation to fungus-associated Neotropical species of Drosophilidae, providing the first molecular insights into the phylogenetic patterns related to the evolution of this diverse group of species and some of its characteristic traits.

Integrative description of bisexual Paramacrobiotus experimentalis sp. nov. (Macrobiotidae) from republic of Madagascar (Africa) with microbiome analysis.

In a moss samples collected on Madagascar two populations of Paramacrobiotus experimentalis sp. nov. were found. Paramacrobiotus experimentalis sp. nov. with the presence of a microplacoid and areolatus type of eggs is similar to Pam. danielae, Pam. garynahi, Pam. hapukuensis, Pam. peteri, Pam. rioplatensis and Pam. savai, but it differs from them by some morphological and morphometric characters of the eggs. The p-distance between two COI haplotypes of Pam. experimentalis sp. nov. was 0.17%. In turn, the ranges of uncorrected genetic p-distances of all Paramacrobiotus species available in GenBank was from 18.27% (for Pam. lachowskae) to 25.26% (for Pam. arduus) with an average distance of 20.67%. We also found that Pam. experimentalis sp. nov. is bisexual. This observation was congruent on three levels: (i) morphological - specimen size dimorphism; (ii) structural (primary sexual characteristics) - females have an unpaired ovary while males have an unpaired testis and (iii) molecular - heterozygous and homozygous strains of the ITS-2 marker. Although symbiotic associations of hosts with bacteria (including endosymbiotic bacteria) are common in nature and these interactions exert various effects on the evolution, biology and reproductive ecology of hosts, there is still very little information on the bacterial community associated with tardigrades. To fill this gap and characterise the bacterial community of Pam. experimentalis sp. nov. populations and microbiome of its microhabitat, high throughput sequencing of the V3-V4 hypervariable regions in the bacterial 16S rRNA gene fragment was performed. The obtained 16S rRNA gene sequences ranged from 92,665 to 131,163. In total, 135 operational taxonomic units (OTUs) were identified across the rarefied dataset. Overall, both Pam. experimentalis sp. nov. populations were dominated by OTUs ascribed to the phylum Proteobacteria (89-92%) and Firmicutes (6-7%). In the case of samples from tardigrades' laboratory habitat, the most abundant bacterial phylum was Proteobacteria (51-90%) and Bacteroides (9-48%). In all compared microbiome profiles, only 16 of 137 OTUs were shared. We found also significant differences in beta diversity between the partly species-specific microbiome of Pam. experimentalis sp. nov. and its culturing environment. Two OTUs belonging to a putative bacterial endosymbiont were identified - Rickettsiales and Polynucleobacter. We also demonstrated that each bacterial community was rich in genes involved in membrane transport, amino acid metabolism, and carbohydrate metabolism.

Genetic variation, pseudocryptic diversity, and phylogeny of Erpobdella (Annelida: Hirudinida: Erpobdelliformes), with emphasis on Canadian species.

Leeches of the family Erpobdellidae are important members of benthic freshwater environments, where they are voracious predators of other invertebrates and an important source of nutrition for several species of vertebrates. Beset by a lack of reliable diagnostic morphological characters and destructive identification processes, molecular approaches have, in recent years, been employed to illuminate the relationships within this family, and DNA barcoding has been employed for identification purposes. However, an understanding of the levels of genetic variation across the geographic distributions of members of the genus is still lacking. Herein, we sequence the mitochondrial COI locus for 249 newly collected North American individuals, representing 5 species, as well as mitochondrial 12S rDNA, nuclear 18S rDNA, and nuclear 28S rDNA for a select subset of these. Our COI dataset was leveraged to detect potential cryptic species, and to calculate genetic distances as a proxy for the degree of gene flow between populations. Augmented by numerous sequences from GenBank, the multilocus dataset was used to reconstruct a phylogenetic hypothesis for worldwide members of the genus. Beyond corroborating previous overarching phylogenetic frameworks, our results show that an undescribed species that is morphologically and genetically similar to Erpobdella punctata exists in sympatry with this species - the new species has likely been overlooked in previous studies due to its morphological similarity with Erpobdella punctata. Erpobdella bucera is reported from Canada for the first time; and Erpobdella microstoma is newly reported from Saskatchewan and placed in a phylogeny for the first time. Finally, we find evidence for genetic structure in both E. cf. punctata and Erpobdella obscura that is correlated with major river drainage basin boundaries in North America.

Biogeography and early diversification of Tapinotaspidini oil-bees support presence of Paleocene savannas in South America.

Worldwide distributed tropical savannas were established only in the Miocene, with climatic cooling and rise of C4 grasses. However, there is evidence for an earlier presence of savanna-like vegetation in southern parts of South America. Here we investigated the biogeographic history of a clade of solitary bees which have endemic groups in areas covered by savannas and other types of open vegetation as well as forested areas. We hypothesized that these bees originated in savanna-like biomes and that shifts to forested areas and floral host shifts increased species diversification along their evolutionary history. We reconstructed a comprehensive phylogeny for Tapinotaspidini bees based majorly on original DNA sequences. We then used macroevolutionary tools to estimate ancestral range area and reconstructed ancestral habitat (open versus forested) and host plant association to analyze the effects of shifts in vegetation type and flower hosts on their diversification. Tapinotaspidini bees originated in the Paleocene and in a savanna-type, Cerrado-like, which is reinforced by reconstruction of open vegetation as the most probable ancestral area, thus bringing additional evidence to a much earlier origin of this vegetation type in South America. Shifts to forested areas occurred at least three times in a period of 30 Ma and were responsible for slight increases in diversification rates. Malpighiaceae is the ancestral floral host; host broadening occurred only in the Miocene and at least in three occasions. Host shifts, i.e. from Malpighiaceae to other oil families, occurred in the Eocene and Miocene. Both host broadening and host shifts did not significantly alter diversification rates, however exploitation of other oil sources were important in occupying new habitats. The link between biomes and host plant shifts and changes in diversification rate brings us additional insights into the evolution of bees and associated flora in South America.

From taxonomic deflation to newly detected cryptic species: Hidden diversity in a widespread African squeaker catfish.

Cryptic genetic diversity and erroneous morphological species determination represent frequent problems in biodiversity research. Here, examination of 138 specimens of Synodontis (Mochokidae, Siluriformes) from the Nile River and Lake Turkana revealed the presence of both S. schall-like and S. frontosus-like morphotypes, with a phenotypic gradient between them. We concluded phylogenetic and population genetic analyses based on two mitochondrial and one nuclear marker including 131 coxI (565 bp), 96 cytb (973 bp) and 19 RAG2 (896 bp) sequences from the Nile-Turkana population, plus additional GenBank data of Synodontis spp. Whilst nuclear data were inconclusive, mitochondrial sequences suggested that both morphotypes and intermediate forms are conspecific. The results imply probable synonymy of S. frontosus with S. schall. Conversely, a strong biogeographical signal was revealed among widely distributed and supposedly conspecific S. schall-like catfish of the Nilo-Sudanian ichthyological province. Synodontis schall sensu stricto (=Eastern clade), as defined by type locality in the Nile, is apparently restricted to the eastern part of the Nilo-Sudanian ichthyological province (e.g. Nile, Turkana, Chad). Synodontis schall Western clade (Senegambia, Niger, Chad) most probably represents a cryptic taxon, unrecognized thus far due to the absence of distinctive morphological differences.

Identification of closely related Ixodes species by protein profiling with MALDI-TOF mass spectrometry.

Ticks are vectors of infectious diseases of major importance in human and veterinary medicine. For epidemiological studies, accurate identification of ticks is crucial to define their potential role as vectors and to develop control and prevention strategies. Although morphological and molecular methods are widely used to identify ticks, an innovative approach using MALDI-TOF MS technology recently emerged as an alternative tool. Previous works showed that MALDI-TOF MS was highly effective in identifying ticks, but these works mainly tested tick specimens of different genera. To confirm the accuracy of this new tool for tick identification, nine closely related tick species belonging to the Ixodes genus were analysed, specimens of the Dermacentor reticulatus species were also included in the analysis as an outer group. Three of the species used for the present study belonged to the I. ricinus species complex, which are known to transmit Borrelia burgdorferi sensu lato, the causative agent of Lyme borreliosis. A total of 246 tick specimens were submitted to MALDI-TOF MS analysis, and two body parts (half-idiosoma and four legs) were individually investigated. For each body part, intraspecies reproducibility and interspecies specificity of the MS profiles were determined. The profile analysis revealed that the main determinant for spectra clustering was the tick species for both legs and half-idiosoma. For each body part, a reference database of spectra was set up including 2 to 5 specimens per species randomly selected, and genotyped using 16s rDNA and COI genes to confirm their morphological identification. Both created spectral databases were individually blind tested with their respective body part using the remaining specimens, which were correctly identified in 98.5% of the cases. MALDI-TOF MS is a reliable tool for tick identification, including specimens belonging to closely related species and hardly distinguishable using morphology. The 4-legs as well as the half-idiosoma of ticks can now be applied for specimen identification using two different databases. The combined use of these two body parts improves the rate of tick identification and their confidence level.

Further analyses on the phylogeny of the subclass Scuticociliatia (Protozoa, Ciliophora) based on both nuclear and mitochondrial data.

So far, the phylogenetic studies on ciliated protists have mainly based on single locus, the nuclear ribosomal DNA (rDNA). In order to avoid the limitations of single gene/genome trees and to add more data to systematic analyses, information from mitochondrial DNA sequence has been increasingly used in different lineages of ciliates. The systematic relationships in the subclass Scuticociliatia are extremely confused and largely unresolved based on nuclear genes. In the present study, we have characterized 72 new sequences, including 40 mitochondrial cytochrome oxidase c subunit I (COI) sequences, 29 mitochondrial small subunit ribosomal DNA (mtSSU-rDNA) sequences and three nuclear small subunit ribosomal DNA (nSSU-rDNA) sequences from 47 isolates of 44 morphospecies. Phylogenetic analyses based on single gene as well as concatenated data were performed and revealed: (1) compared to mtSSU-rDNA, COI gene reveals more consistent relationships with those of nSSU-rDNA; (2) the secondary structures of mtSSU-rRNA V4 region are predicted and compared in scuticociliates, which can contribute to discrimination of closely related species; (3) neither nuclear nor mitochondrial data support the monophyly of the order Loxocephalida, which may represent some divergent and intermediate lineages between the subclass Scuticociliatia and Hymenostomatia; (4) the assignments of thigmotrichids to the order Pleuronematida and the confused taxon Sulcigera comosa to the genus Histiobalantium are confirmed by mitochondrial genes; (5) both nuclear and mitochondrial data reveal that the species in the family Peniculistomatidae always group in the genus Pleuronema, suggesting that peniculistomatids are more likely evolved from Pleuronema-like ancestors; (6) mitochondrial genes support the monophyly of the order Philasterida, but the relationships among families of the order Philasterida remain controversial due to the discrepancies between their morphological and molecular data.

Molecular phylogeny and biogeography of the land snail subfamily Leptaxinae (Gastropoda: Hygromiidae).

The subfamily Leptaxinae is included within the highly diverse land snail family Hygromiidae. In the absence of clear diagnostic morphological differences, the subfamily status is currently based solely on molecular information and includes three disjunctly distributed tribes, Leptaxini, Cryptosaccini and Metafruticicolini. However, the phylogenetic relationships among these tribes are not fully resolved and the clustering of some of the genera to the tribes is not statistically supported. To resolve the relationships within Leptaxinae and their position within Hygromiidae, we reconstructed their phylogeny using a multi-locus approach with two mitochondrial genes and eight nuclear markers. The phylogeny was further calibrated and an analysis of ancestral area estimation was carried out to infer the biogeographic history of the group. We elevated Metafruticicolini to subfamily level (Metafruticicolinae) and we restricted Leptaxinae to Cryptosaccini and Leptaxini. The Lusitanian genus Portugala was moved to Leptaxini, previously containing only the Macaronesian genus Leptaxis. Within Cryptosaccini, a new genus strictly confined to the Sierra de la Cabrera (Spain) is described, Fractanella gen. nov. According to our results, Leptaxinae originated in the Early Miocene in the Iberian Peninsula, from which the Macaronesian Islands were colonized. Due to the old split recovered for the divergence between Macaronesian and Iberian lineages, we hypothesize that this colonization may have occurred via the once emerged seamounts located between the archipelagos and the European and African continents, although this could also have occurred through the oldest now emerged islands of Macaronesia. In the Iberian Peninsula, the climatic shift that began during the Middle Miocene, changing progressively from subtropical climate towards the present-day Mediterranean climate, was identified as an important factor shaping the subfamily's diversification, along with Pleistocene climatic fluctuations.

Pheromones and Barcoding Delimit Boundaries between Cryptic Species in the Primitive Moth Genus Eriocrania (Lepidoptera: Eriocraniidae).

Animal classification is primarily based on morphological characters, even though these may not be the first to diverge during speciation. In many cases, closely related taxa are actually difficult to distinguish based on morphological characters alone, especially when there is no substantial niche separation. As a consequence, the diversity of certain groups is likely to be underestimated. Lepidoptera -moths and butterflies- represent the largest group of herbivorous insects. The extensive diversification in the group is generally assumed to have its origin in the spectacular radiation of flowering plants and the resulting abundance of ecological niches. However, speciation can also occur without strong ecological divergence. For example, reproductive isolation can evolve as the result of divergence in mate preference and the associated pheromone communication system. We combined pheromone trapping and genetic analysis to elucidate the evolutionary relationships within a complex of primitive moth species (Lepidoptera: Eriocraniidae). Mitochondrial and nuclear DNA markers provided evidence that Eriocrania semipurpurella, as currently defined by morphological characters, includes three cryptic species in Northern and Western Europe. Male moths of these cryptic species, as well as of the closely related E. sangii, exhibited relative specificity in terms of their attraction to specific ratios of two major pheromone components, (2S,6Z)-nonen-2-ol and (2R,6Z)-nonen-2-ol. Our data suggest strong assortative mating in these species in the absence of apparent niche separation, indicating that Eriocrania moths may represent an example of non-ecological speciation. Finally, our study argues in favour of combining pheromone investigations and DNA barcoding as powerful tools for identifying and delimitating species boundaries.

Cytochrome C Oxidase Subunit 1, Internal Transcribed Spacer 1, Nicotinamide Adenine Dinucleotide Hydrogen Dehydrogenase Subunits 2 and 5 of Clonorchis sinensis Ancient DNA Retrieved from Joseon Dynasty Mummy Specimens.

We analyzed Clonorchis sinensis ancient DNA (aDNA) acquired from the specimens of the Joseon mummies. The target regions were cytochrome C oxidase subunit 1 (CO1), internal transcribed spacer 1 (ITS1), nicotinamide adenine dinucleotide hydrogen (NADH) dehydrogenase subunits 2 (NAD2) and 5 (NAD5). The sequences of C. sinensis aDNA was completely or almost identical to modern C. sinensis sequences in GenBank. We also found that ITS1, NAD2 and NAD5 could be good markers for molecular diagnosis between C. sinensis and the other trematode parasite species. The current result could improve our knowledge about genetic history of C. sinensis.

Integrating multilocus DNA data and 3D geometric morphometrics to elucidate species boundaries in the case of Pyrenaearia (Pulmonata: Hygromiidae).

To accurately delimit species the use of multiple character types is essential as all speciation processes are not equally reflected in different data (e.g. morphological, molecular or ecological characters). With the introduction of geometric morphometrics methods and advances in 3D technology, a comprehensive combination of molecular and morphological data has been enabled in groups where exhaustively quantifying and measuring morphological shape change was not possible before such as gastropod shells. In this study, we combined multilocus coalescent species delimitation methods with 3D geometric morphometrics of shell shape to delimit species within the land snail genus Pyrenaearia. A new taxonomic scheme was constructed for the genus identifying ten species. Two nominal species were synonymized and a hitherto unrecognized cryptic species was identified. Our findings support the importance of combining multiple lines of evidence as molecular and morphological data on their own do not yield the same information. Further, the integration of morphological and molecular data shows the importance of allometry in shell shape and suggests a combined effect of population history and selection in different environments on shells morphological variation. Our new taxonomy and phylogenetic reconstruction suggest that, besides the glacial cycles of the Pleistocene, passive dispersal and rock substrate complexity could also have been involved in the speciation of the genus.

When is a "cryptic" species not a cryptic species: A consideration from the Holarctic micro-landsnail genus Euconulus (Gastropoda: Stylommatophora).

Naive use of molecular data may lead to ambiguous conclusions, especially within the context of "cryptic" species. Here, we integrated molecular and morphometric data to evaluate phylogenetic relationships in the widespread terrestrial micro-snail genus, Euconulus. We analyzed mitochondrial (16S + COII) and nuclear (ITS1 + ITS2) sequence across 94 populations from Europe, Asia and North America within the nominate species E. alderi, E. fulvus and E. polygyratus, and used the southeastern USA E. chersinus, E. dentatus, and E. trochulus as comparative outgroups. Phylogeny was reconstructed using four different reconstruction methods to identify robust, well-supported topological features. We then performed discriminant analysis on shell measurements between these genetically-identified species-level clades. These analyses provided evidence for a biologically valid North American "cryptic" species within E. alderi. However, while highly supported polyphyletic structure was also observed within E. fulvus, disagreement in placement of individuals between mtDNA and nDNA clades, lack of morphological differences, and presence of potential hybrids imply that these lineages do not rise to the threshold as biologically valid cryptic species, and rather appear to simply represent a complex of geographically structured populations within a single species. These results caution that entering into a cryptic species hypothesis should not be undertaken lightly, and should be optimally supported along multiple lines of evidence. Generally, post-hoc analyses of macro-scale features should be conducted to attempt identification of previously ignored diagnostic traits. If such traits cannot be found, i.e. in the case of potentially "fully cryptic" species, additional criteria should be met to propound a cryptic species hypothesis, including the agreement in tree topology among both mtDNA and nDNA, and little (or no) evidence of hybridization based on a critical analysis of sequence chromatograms. Even when the above conditions are satisfied, it only implies that the cryptic species hypothesis is plausible, but should optimally be subjected to further careful examination.

Cryptic lineages in the Wolf Cardinalfish living in sympatry on remote coral atolls.

Coral reef health and biodiversity is under threat worldwide due to rapid climate change. However, much of the inter- and intra-specific diversity of coral reefs are undescribed even in well studied taxa such as fish. Delimiting previously unrecognised diversity is important for understanding the processes that generate and sustain biodiversity in coral reef ecosystems and informing strategies for their conservation and management. Many taxa that inhabit geographically isolated coral reefs rely on self-recruitment for population persistence, providing the opportunity for the evolution of unique genetic lineages through divergent selection and reproductive isolation. Many such lineages in corals and fish are morphologically similar or indistinguishable. Here, we report the discovery and characterisation of cryptic lineages of the Wolf Cardinalfish, Cheilodipterus artus, from the coral atolls of northwest Australia using multiple molecular markers from mitochondrial (CO1 and D-loop) and nuclear (microsatellites) DNA. Concordant results from all markers identified two highly divergent lineages that are morphologically cryptic and reproductively isolated. These lineages co-occurred at daytime resting sites, but the relative abundance of each lineage was strongly correlated with wave exposure. It appears, therefore, that fish from each lineage are better adapted to different microhabitats. Such cryptic and ecologically based diversity appears to be common in these atolls and may well aid resilience of these systems. Our results also highlight that underwater surveys based on visual identification clearly underestimate biodiversity, and that a taxonomic revision of the Cheilodipterus genus is necessary.