Multilocus phylogeny and species delimitation within the genus Glauconycteris (Chiroptera,Vespertilionidae), with the description of a new bat species from the Tshopo Province of the Democratic Republic of the Congo

The genus Glauconycteris Dobson, 1875 currently contains 12 species of butterfly bats, all endemic to sub‐Saharan Africa. Most species are rarely recorded, with half of the species known from less than six geographic localities. The taxonomic status of several species remains problematic. Here, we studied the systematics of butterfly bats using both morphological and molecular approaches. We examined 45 adult specimens for external anatomy and skull morphology, and investigated the phylogeny of Glauconycteris using DNA sequences from three mitochondrial genes and 116 individuals, which in addition to outgroup taxa, included nine of the twelve butterfly bat species currently recognized. Four additional nuclear genes were sequenced on a reduced sample of 69 individuals, covering the outgroup and Glauconycteris species. Our molecular results show that the genus Glauconycteris is monophyletic, and that it is the sister‐group of the Asian genus Hesperoptenus. Molecular dating estimates based on either Cytb or RAG2 data sets suggest that the ancestor of Glauconycteris migrated into Africa from Asia during the Tortonian age of the Late Miocene (11.6–7.2 Mya), while the basal diversification of the crown group occurred in Africa at around 6 ± 2 Mya. The species G. superba is found to be the sister‐group of G. variegata, questioning its placement in the recently described genus Niumbaha. The small species living in tropical rainforests constitute a robust clade, which contains three divergent lineages: (i) the “poensis” group, which is composed of G. poensis, G. alboguttata, G. argentata, and G. egeria; (ii) the “beatrix” group, which contains G. beatrix and G. curryae; and (iii) the “humeralis” group, which includes G. humeralis and a new species described herein. In the “poensis” group, G. egeria is found to be monophyletic in the nuclear tree, but polyphyletic in the mitochondrial tree. The reasons for this mito‐nuclear discordance are discussed.     

Molecular phylogeny and species delimitation in the section Longibrachiatum of Trichoderma

The phylogenetically most derived group of the genus Trichoderma - section Longibrachiatum, includes some of the most intensively studied species, such as the industrial cellulase producer T. reesei (teleomorph Hypocrea jecorina), or the facultative opportunistic human pathogens T. longibrachiatum and H. orientalis. At the same time, the phylogeny of this clade is only poorly understood. Here we used a collection of 112 strains representing all currently recognized species and isolates that were tentatively identified as members of the group, to analyze species diversity and molecular evolution. Bayesian phylogenetic analyses based on several unlinked loci in individual and concatenated datasets confirmed 13 previously described species and 3 previously recognized phylogenetic species all of which were not yet described formally. When the genealogical concordance criterion, the K/θ method and comparison of frequencies of pairwise nucleotide differences were applied to the data sample, 10 additional new phylogenetic species were recognized, seven of which consisted only of a single lineage. Our analysis thus identifies 26 putative species in section Longibrachiatum, what doubles the currently estimated taxonomic diversity of the group, and illustrates the power of combining genealogical concordance and population genetic analysis for dissecting species in a recently diverged group of fungal species.     

Integrating coalescent-based species delimitation with ecological niche modeling delimited two species within the Stewartia sinensis complex (Theaceae)

Accurate species delimitation is the key to precise estimation of species diversity and is fundamental to most branches of biology. Unclear species boundaries within species complexes could lead to the underestimation of species diversity. However, species delimitation of species complexes remains challenging due to the continuum of phenotypic variations. To robustly examine species boundaries within a species complex, integrative approaches in phylogeny, ecology, and morphology were applied to the Stewartia sinensis complex (Theaceae) endemic to China. Multispecies coalescent-based species delimitation using 572 nuclear ortholog sequences (anchored enrichment) supported reciprocal phylogenetic monophyly of the northern lineage (NL) and southern lineage (SL), which were not sister clades. Niche equivalency and similarity tests demonstrated significant climatic niche differentiation between NL and SL with observed Warren et al.'s I = 0.0073 and Schoener's D = 0.0021. Species distribution modeling also separated their potential distribution. Morphometric analyses suggested significant interlineage differentiation of multiple traits including the ratio of length and width, leaf width, and pedicel length, although overall similarity did not differ. Based on the integrative species concept, two distinct species were proposed with legitimate names of Stewartia gemmata for SL and S. sinensis for NL. Our empirical study of the S. sinensis complex highlights the importance of applying multiple species criteria, in particular the underappreciated niche differentiation, to species delimitation in species complexes pervasive in plants.     

Multilocus species delimitation in Mesoamerican Scaptotrigona stingless bees (Apidae: Meliponini) supports the existence of cryptic species

Accelerating taxonomic knowledge and making accurate species identifications are critically important given the current biodiversity crisis, particularly in biodiversity hotspots such as Mesoamerica. Objective species delimitation that reduces investigator‐driven bias is fundamental to the establishment of appropriate conservation strategies, above all in managed species. Previous morphological and molecular studies on three managed stingless bee species of the genus Scaptotrigona distributed in Mexico (S. mexicana, S. pectoralis and S. hellwegeri) suggested that both S. mexicana and S. hellwegeri are cryptic species complexes. Herein we tested species delimitation by analysing sequence information of five markers (two mitochondrial: cox1 and 16S, and three nuclear: ITS1, EF1‐α, ArgK) within a Bayesian coalescent framework to test the putative species. We obtained two different hypotheses using a Generalized Mixed Yule Coalescent (GMYC) model: four (cox1) and six (16S) species. After the species validation step with the Bayesian species‐delimitation analysis (BPP), we suggest that only S. mexicana is a complex of two species with different distribution (along the Pacific and the Atlantic coasts, respectively). We highly recommend avoiding colony exchange between geographical regions in order to conserve the genetic integrity of both taxa.     

A molecular phylogeny of the Sylvia cantillans complex: cryptic species within the Mediterranean basin

The subalpine warbler Sylvia cantillans is formally considered a polytypic species, with four subspecies, European S. c. cantillans, albistriata, moltonii (recently resumed name: subalpina) and North African S. c. inornata. They are very similar in external morphology but clearly differ in their vocalizations. We evaluated their uncertain taxonomic status reconstructing the phylogenetic and phylogeographic relationships among populations sampled across major biogeographical areas in the European species’ range, using nucleotide sequences of the mitochondrial cytochrome b gene (mtDNA cyt b). A variety of phylogenetic analyses concordantly led to identify four major groups, only partially corresponding to the three European nominal subspecies. Phylogenetic trees showed a monophyletic group including all moltonii individuals, well diverged from all other taxa. Populations taxonomically assigned to cantillans were polyphyletic being split into two distinct clades (western and southern cantillans), with monophyletic albistriata closely related to southern cantillans. Individuals of moltonii and southern cantillans sampled in sites of sympatry in central Italy were assigned to their respective groups, with perfect concordance between phenotypic and genetic identifications. All findings indicate that moltonii should be ranked as a distinct species. Former subspecies cantillans is polyphyletic, but additional data are needed to define the taxonomic status of its two clades. Albistriata is phylogenetically related to southern cantillans and should be provisionally kept as a subspecies of S. cantillans. The cantillans complex thus provides an interesting case-study illustrating geographical structuring across small geographical ranges, and it exemplifies speciation through differentiation in allopatry leading to reproductive isolation after a secondary contact.     

Size diversity and species diversity relationships in fish assemblages of Western Palearctic lakes

Body size, coupled with abundance and taxonomy, may help to understand the mechanisms shaping community structure. Since the body size of fish is closely related to their trophic niche, size diversity (based on individual body size) of fish communities may capture intraspecific variations in fish trophic niches that are not detected by species diversity. Thus, the relationship between size diversity and species diversity may help to integrate variation at both intraspecific and interspecific levels. We studied the relationship between species diversity and size diversity as a measure of the degree of overlap in size among species and thereby the potential overlap in niches in a community. We hypothesized that the relationship between size diversity and species would be different across the European continent due to different levels of size overlap in fish communities. The data were derived from samplings of fish communities using standardised benthic gill nets in 363 lakes. At the continental scale, size diversity increased with species diversity; at the ecoregion scale, the slope of the relation changed across the continent, with the greatest mismatch occurring in northern Europe where communities comprised only one or a few species, but each of which exhibited a great range in size. There was an increase in slope towards the south with significant relations for four out of six ecoregions. The steeper size diversity‐species diversity slope at lower latitudes is attributable to a lower overlap in fish size and thus likely to finer niche separation. Our results also suggest that size diversity is not a strong surrogate for species diversity in European lake fish communities. Thus, particularly in fish communities composed of few species, measuring size diversity may help to detect potential functional variation which may be neglected by measuring species diversity alone.     

A species delimitation approach in the Trochulus sericeus/hispidus complex reveals two cryptic species within a sharp contact zone

Background  

Mitochondrial DNA sequencing increasingly results in the recognition of genetically divergent, but morphologically cryptic lineages. Species delimitation approaches that rely on multiple lines of evidence in areas of co-occurrence are particularly powerful to infer their specific status. We investigated the species boundaries of two cryptic lineages of the land snail genus Trochulus in a contact zone, using mitochondrial and nuclear DNA marker as well as shell morphometrics.     

Analysis of the diet of Natterer's bat Myotis nattereri and the common long-eared bat Plecotus auritus in the West of Ireland

The diets of Natterer's bat Myotis nattereri and the common long-eared bat Plecotus auritus were investigated at a nursery colony of each species by analysis of droppings collected monthly from May to September. Respectively, 68% and 42% of the diets comprised items presumed to have been gleaned from foliage or other surfaces: diurnal insects, insects which rarely fly, and non-flying arthropods. Such surfaces included the ground: centipedes were eaten by both bats, and the muscid, Scatophaga stercoraria , usually associated with cattle dung, was a common prey. Indeed, the prevalence of cattle-farming around both of the bat roosts almost certainly contributed indirectly, in the form of S. stercoraria , to a significant part of the Diptera consumed. The chief food of M. nattereri was the larger Diptera, but Trichoptera, Hymenoptera and Arachnida were also important. Lepidoptera, Coleoptera and Hemiptera were minor prey, with Dermaptera and Chilopoda taken occasionally. Diptera, closely followed by Lepidoptera, together accounted for nearly two-thirds of the diet of P. auritus. Also taken, in descending order of importance, were Trichoptera, Arachnida, Chilopoda, Coleoptera, Dermaptera, Hymenoptera and Hemiptera. Plecotus auritus , but not M. nattereri , was evidently able to take a few small insects such as aphids and lesser nematoceran Diptera.     

Multilocus sequence analysis reveals multiple symbiovars within Mesorhizobium species

The genus Mesorhizobium includes species nodulating several legumes, such as chickpea, which has a high agronomic importance. Chickpea rhizobia were originally described as either Mesorhizobium ciceri or M. mediterraneum. However, rhizobia able to nodulate chickpea have been shown to belong to several different species within the genus Mesorhizobium. The present study used a multilocus sequence analysis approach to infer a high resolution phylogeny of the genus Mesorhizobium and to confirm the existence of a new chickpea nodulating genospecies. The phylogenetic structure of the Mesorhizobium clade was evaluated by sequence analysis of the 16S rRNA gene, ITS region and the five core genes atpD, dnaJ, glnA, gyrB, and recA. Phylogenies obtained with the different genes are in overall good agreement and a well-supported, almost fully resolved, phylogenetic tree was obtained using the combined data. Our phylogenetic analyses of core genes sequences and their comparison with the symbiosis gene nodC, corroborate the existence of one new chickpea Mesorhizobium genospecies and one new symbiovar, M. opportunistum sv. ciceri. Furthermore, our results show that symbiovar ciceri spreads over six species of mesorhizobia. To our knowledge this study shows the most complete Mesorhizobium multilocus phylogeny to date and contributes to the understanding of how a symbiovar may be present in different species.     

Revisiting species delimitation within the genus Oxystele using DNA barcoding approach

The genus Oxystele, a member of the highly diverse marine gastropod superfamily Trochoidea, is endemic to southern Africa. Members of the genus include some of the most abundant molluscs on southern African shores and are important components of littoral biodiversity in rocky intertidal habitats. Species delimitation within the genus is still controversial, especially regarding the complex O. impervia / O. variegata. Here, we assessed species boundaries within the genus using DNA barcoding and phylogenetic tree reconstruction. We analysed 56 specimens using the mitochondrial gene COI. Our analysis delimits five molecular operational taxonomic units (MOTUs), and distinguishes O. impervia from O. variegata. However, we reveal important discrepancies between MOTUs and morphology-based species identification and discuss alternative hypotheses that can account for this. Finally, we indicate the need for future study that includes additional genes, and the combination of both morphology and genetic techniques (e.g. AFLP or microsatellites) to get deeper insight into species delimitation within the genus.     

Species delimitation and hybridization history of a hazel species complex

Background and AimsHybridization increases species adaptation and biodiversity but also obscures species boundaries. In this study, species delimitation and hybridization history were examined within one Chinese hazel species complex (Corylus chinensis–Corylus fargesii). Two species including four varieties have already been described for this complex, with overlapping distributions.MethodsA total of 322 trees from 44 populations of these four varieties across their ranges were sampled for morphological and molecular analyses. Climatic datasets based on 108 geographical locations were used to evaluate their niche differentiations. Flowering phenology was also observed for two co-occurring species or varieties.Key ResultsFour statistically different phenotypic clusters were revealed, but these clusters were highly inconsistent with the traditional taxonomic groups. All the clusters showed statistically distinct niches, with complete or partial geographical isolation. Only two clusters displayed a distributional overlap, but they had distinct flowering phenologies at the site where they co-occurred. Population-level evidence based on the genotypes of ten simple sequence repeat loci supported four phenotypic clusters. In addition, one cluster was shown to have an admixed genetic composition derived from the other three clusters through repeated historical hybridizations.ConclusionsBased on our new evidence, it is better to treat the four clusters identified here as four independent species. One of them was shown to have an admixed genetic composition derived from the other three through repeated historical hybridizations. This study highlights the importance of applying integrative and statistical methods to infer species delimitations and hybridization history. Such a protocol should be adopted widely for future taxonomic studies.     

Multilocus genotyping and molecular phylogenetics resolve a novel head blight pathogen within the Fusarium graminearum species complex from Ethiopia

A survey of Fusarium head blight (FHB)-contaminated wheat in Ethiopia recovered 31 isolates resembling members of the Fusarium graminearum species complex. Results of a multilocus genotyping (MLGT) assay for FHB species and trichothecene chemotype determination suggested that 22 of these isolates might represent a new species within the Fg complex. Phylogenetic analyses of multilocus DNA sequence data resolved the 22 Ethiopian isolates as a novel, phylogenetically distinct species. The new species also appears to be novel in that MLGT probe data and sequence analysis of both ends of the TRI-cluster identified 15ADON and NIV recombination blocks, documenting inter-chemotype recombination involving the chemotype-determining genes near the ends of the TRI-cluster. Results of pathogenicity experiments and analyses of trichothecene mycotoxins demonstrated that this novel Fg complex species could induce FHB on wheat and elaborate 15ADON in planta. Herein the FHB pathogen from Ethiopia is formally described as a novel species.     

Multilocus phylogenetic structure within the Trichoderma harzianum/Hypocrea lixii complex

Trichoderma harzianum is a ubiquitous species in the environment and is effective in the biological control of plant-pathogenic fungi. T. harzianum has not been linked unequivocally to its sexual state nor has its phylogeny been studied in detail. It has been suggested that T. harzianum is a species complex based on the phenotypic and genotypic variability encountered. On the basis of morphological and cultural characters and DNA sequence data analysis of four genes (ITS rDNA, translation elongation factor 1-alpha, calmodulin, and alpha-actin), Hypocrea lixii was found to be the sexual state of T. harzianum. Both the asexual and sexual states of this species have wide geographic distributions. Phylogenetic analysis of four genes showed that T. harzianum/H. lixii is a cohesive group that is supported by bootstrap values higher than 95%. Principles of genealogical concordance indicated that T. harzianum/H. lixii is a complex of independent monophyletic lineages, but no diagnostic morphological distinctions were identified that justify formal taxonomic recognition for the different lineages.     

Generic delimitation and phylogeny of theCarduncellus-Carthamus complex (Asteraceae) based on ITS sequences

Within the Mediterranean complexCarduncellus-Carthamus, taxonomic classification has proven problematic. Numerous attempts to clarify the relative systematic boundaries have included splittingCarduncellus andCarthamus into several genera, but none of these proposed classifications have been generally accepted. For a comprehensive resolution of the relationships within this group, we used sequences of the Internal Transcribed Spacers (ITS) of nuclear ribosomal DNA. The results indicate that the complex should be classified into four genera:Carduncellus, Carthamus, Femeniasia andPhonus. The relationship between the western group (Carduncellus, Femeniasia andPhonus) and the eastern genusCarthamus are not resolved by analysis of ITS sequences, but the two groups are probably not close relatives. The ITS classifications corresponded with biogeography and less with morphological characters, which have also been the main source of confusion in traditional classifications. Most of the unusual morphological features in theCarduncellus-Carthamus complex appear to be reversals to ancestral character states.     

Molecular phylogeny of the Sphaerophorus globosus species complex

The Sphaerophorus globosus complex (Lecanorales, lichenized Ascomycota) shows a large morphological variation, and three relatively distinct morphotypes can be distinguished in parts of the distribution area. Here, we utilize a multigene‐based maximum‐parsimony approach (nITS+ LSU rDNA, mtSSU rDNA, β‐tubulin, and actin) to investigate whether these morphotypes constitute distinct species. The results show that there are at least two well‐supported monophyletic groups that we interpret as phylogenetic species within the S. globosus complex. These species do not completely correspond to the predefined morphotypes. One group, an apparently undescribed species, contains noncoralloid specimens from the North American Pacific Northwest. The other group, S. globosus, consists of two well‐supported monophyletic groups: one contains coralloid epiphytic specimens from the North American Pacific Northwest that are morphologically indistinguishable from epiphytic specimens from Europe and are presently interpreted as belonging to the same species and the other is morphologically variable and contains terrestrial specimens from Europe, North America, and southernmost South America and coralloid epiphytic and epilithic specimens from Europe. The results suggest that the population in southernmost South America originated by long‐distance dispersal from arctic populations in the Northern Hemisphere.     

Multilocus phylogeny and MALDI-TOF analysis of the plant pathogenic species Alternaria dauci and relatives

The genus Alternaria includes numerous phytopathogenic species, many of which are economically relevant. Traditionally, identification has been based on morphology, but is often hampered by the tendency of some strains to become sterile in culture and by the existence of species-complexes of morphologically similar taxa. This study aimed to assess if strains of four closely-related plant pathogens, i.e., accurately Alternaria dauci (ten strains), Alternaria porri (six), Alternaria solani (ten), and Alternaria tomatophila (ten) could be identified using multilocus phylogenetic analysis and Matrix-Assisted Laser Desorption Ionisation Time of Flight (MALDI-TOF) profiling of proteins. Phylogenetic analyses were performed on three loci, i.e., the internal transcribed spacer (ITS) region of rRNA, and the glyceraldehyde-3-phosphate dehydrogenase (gpd) and Alternaria major antigen (Alt a 1) genes. Phylogenetic trees based on ITS sequences did not differentiate strains of A. solani, A. tomatophila, and A. porri, but these three species formed a clade separate from strains of A. dauci. The resolution improved in trees based on gpd and Alt a 1, which distinguished strains of the four species as separate clades. However, none provided significant bootstrap support for all four species, which could only be achieved when results for the three loci were combined. MALDI-TOF-based dendrograms showed three major clusters. The first comprised all A. dauci strains, the second included five strains of A. porri and one of A. solani, and the third included all strains of A. tomatophila, as well as all but one strain of A. solani, and one strain of A. porri. Thus, this study shows the usefulness of MALDI-TOF mass spectrometry as a promising tool for identification of these four species of Alternaria which are closely-related plant pathogens.     

Integrating morphology and mitochondrial DNA for species delimitation within the spruce budworm (Choristoneura fumiferana) cryptic species complex (Lepidoptera: Tortricidae)

Species in cryptic complexes tend to be very difficult, if not impossible, to identify using morphological characters. One such complex is the spruce budworm (Choristoneura fumiferana Clemens, 1865) species group, an economically important group of Nearctic forest pests. Morphological, ecological, behavioural and genetic characters have been studied to try to understand the taxonomy of this group better, but diagnostic character states differ in frequency rather than being complete replacements between each species. We used mitochondrial DNA (mtDNA), together with a new morphology‐based character system that focuses on forewing colour components, to determine if one or a combination of character sources can be used for species diagnoses within the spruce budworm complex. We characterized 47 forewing morphometric measurements and sequenced a 470 bp region of cytochrome c oxidase I mtDNA for 111 ingroup individuals comprising five taxa within the complex. Larval host association and coloration or adult pheromone attraction were used as the prior method for grouping individuals. Our results showed that linear discriminant analysis of morphometric wing characters gave unique clusters for all species on the first and second canonical axes, except for a partial overlap between C. fumiferana and C. biennis, which are not sympatric in nature. In contrast, mtDNA distinguished C. fumiferana, C. pinus pinus Freeman, 1953 and a group of western species, but the three western species (C. occidentalis Freeman, 1967 , C. biennis Freeman, 1967 and C. lambertiana Busck, 1915) shared mtDNA haplotypes. On the basis of the linear discriminant analysis of the combined character set, this study supports the application of both morphology and mtDNA within a framework of integrative taxonomy as the most accurate method for species identification. Furthermore, it demonstrates the utility of quantitative colour analysis, which may be particularly helpful for groups in which colour characters are difficult to divide into discrete units due to intergrading hues.     

Morphological and molecular species delimitation within the Holarctic Ilybius angustior complex with a focus on Beringia (Coleoptera: Dytiscidae)

A fourth species of the Holarctic Ilybius angustior complex, Ilybius minakawai n. sp., is described from the Island of Sakhalin in the Russian far east. Male genitalia are diagnostic within this species complex, although from body size and shape the new species cannot be separated from I. churchillensis Wallis from north Alaska and the Canadian low arctic. The small and narrow body characteristic of these two species represents an extreme of the cold climate form of the widespread I. angustior (Gyllenhal). Low variation within the studied large ribosomal and cytochrome c oxidase subunit I mitochondrial genes suggest that speciation events within the I. angustior-complex are fairly recent. In combination with known geographical ranges, the low genetic variation within this species complex suggest speciation within Pleistocene refugia including Beringia.