Using directed phylogenetic networks to retrace species dispersal history

Methods designed for inferring phylogenetic trees have been widely applied to reconstruct biogeographic history. Because traditional phylogenetic methods used in biogeographic reconstruction are based on trees rather than networks, they follow the strict assumption in which dispersal among geographical units have occurred on the basis of single dispersal routes across regions and are, therefore, incapable of modelling multiple alternative dispersal scenarios. The goal of this study is to describe a new method that allows for retracing species dispersal by means of directed phylogenetic networks obtained using a horizontal gene transfer (HGT) detection method as well as to draw parallels between the processes of HGT and biogeographic reconstruction. In our case study, we reconstructed the biogeographic history of the postglacial dispersal of freshwater fishes in the Ontario province of Canada. This case study demonstrated the utility and robustness of the new method, indicating that the most important events were south-to-north dispersal patterns, as one would expect, with secondary faunal interchange among regions. Finally, we showed how our method can be used to explore additional questions regarding the commonalities in dispersal history patterns and phylogenetic similarities among species.     

A multilocus genealogical approach to phylogenetic species recognition in the model eukaryote Neurospora

To critically examine the relationship between species recognized by phylogenetic and reproductive compatibility criteria, we applied phylogenetic species recognition (PSR) to the fungus in which biological species recognition (BSR) has been most comprehensively applied, the well-studied genus Neurospora. Four independent anonymous nuclear loci were characterized and sequenced from 147 individuals that were representative of all described outbreeding species of Neurospora. We developed a consensus-tree approach that identified monophyletic genealogical groups that were concordantly supported by the majority of the loci, or were well supported by at least one locus but not contradicted by any other locus. We recognized a total of eight phylogenetic species, five of which corresponded with the five traditional biological species, and three of which were newly discovered. Not only were phylogenetic criteria superior to traditional reproductive compatibility criteria in revealing the full species diversity of Neurospora, but also significant phylogenetic subdivisions were detected within some species. Despite previous suggestions of hybridization between N. crassa and N. intermedia in nature, and the fact that several putative hybrid individuals were included in this study, no molecular evidence in support of recent interspecific gene flow or the existence of true hybrids was observed. The sequence data from the four loci were combined and used to clarify how the species discovered by PSR were related. Although species-level clades were strongly supported, the phylogenetic relationships among species remained difficult to resolve, perhaps due to conflicting signals resulting from differential lineage sorting.     

Genealogical concordance phylogenetic species recognition in the Fusarium oxysporum species complex

Fusarium oxysporum is an important plant and human pathogenic ascomycetous group, with near ubiquity in agricultural and non-cultivated ecosystems. Phylogenetic evidence suggests that F. oxysporum is a complex of multiple morphologically cryptic species. Species boundaries and limits of genetic exchange within this complex are poorly defined, largely due to the absence of a sexual state and the paucity of morphological characters. This study determined species boundaries within the F. oxysporum species complex using Genealogical Concordance Phylogenetic Species Recognition (GCPSR) with eight protein coding loci. GCPSR criteria were used firstly to identify independent evolutionary lineages (IEL), which were subsequently collapsed into phylogenetic species. Seventeen IELs were initially identified resulting in the recognition of two phylogenetic species. Further evidence supporting this delineation is discussed.     

Mitochondrial DNA sequences of six sturgeon species and phylogenetic relationships within Acipenseridae

Studies on mitochondrial DNA variability amongst six sturgeon species reared in Italian aquaculture plants are reported. Restriction analysis by Rsal of mitochondrial cytochrome b and D-loop fragments amplified by PCR permitted identification of interspecific variations that would be suitable as markers for species diagnosis. Data obtained by partial sequencing of the cytochrome b gene, analyzed through maximum parsimony, neighbour joining, and UPGMA distance methods, revealed species clusters that support previous morpho-meristic and geographical data for sturgeon classification into sub-genera. According to our cluster analysis, the genera Acipenser and Huso are monophyletic. Moreover, these and previous cytogenetic data suggest that three 120-chromosome species (H. huso, H. dauricus and A. ruthenus) are at the base of sturgeon differentiation, which occurred through two different events of polyploidization.     

Taxonomic and phylogenetic revision of three rare irpicoid species within the Meruliaceae

We have carried out a morphological and molecular study of Mycoacia nothofagi and Steccherinum lusitanicum, two rare irpicoid species collected in Spain. From Steccherinum lusitanicum, only the type of the species, collected in Portugal, was known to date. This has been compared with type material of Hydnum setulosum (≡Hyphodontia setulosa) collected in the USA, confirming that both species are synonymous. We contribute with microphotographs of the most representative characters of the specimens. The molecular data based on ITS and partial 28S rRNA gene sequences show that both taxa should be accommodated within genus Phlebia. Our data suggest a close relationship between S. lusitanicum and the core of Phlebia sensu stricto (including the type species P. radiata), whereas M. nothofagi is apparently more closely related with another group of species that would include M. aurea, P. subserialis and P. livida, among others. The molecular phylogenetic analysis confirms that the genera Mycoacia and Mycoaciella, as well as Merulius, should be considered as synonyms of Phlebia.     

Using multi-locus sequence data for addressing species boundaries in commonly accepted lichen-forming fungal species

Accurate species delimitations are of great importance for effectively characterizing biological diversity. Our criteria for delimiting species have changed dramatically over the last decades with the increasing availability of molecular data and improvement of analytical methods to evaluate these data. Whereas reciprocal monophyly is often seen as an indicator for the presence of distinct lineages, recently diverged species often fail to form monophyletic groups. At the same time, cryptic species have repeatedly been detected in numerous organismal groups. In this study, we addressed the species delimitation in the crustose lichen-forming fungal genus Diploschistes using multilocus sequence data from specimens representing 16 currently accepted species. Our results indicate the presence of previously undetected, cryptic species-level lineages in the subgenus Limborina. In the subgenus Limborina, samples from different continents currently classified under the same species were shown to be only distantly related. At the same time, in parts of subgen. Diploschistes characterized by short branches, none of the currently accepted species formed monophyletic groups. In spite of the lack of monophyly in phylogenetic reconstructions, a multispecies coalescent method provided support for eight of the nine accepted species in subgen. Diploschistes as distinct lineages. We propose to reduce D. neutrophilus to synonymy with D. diacapsis and point out that additional sampling will be necessary before accepting additional species in subgen. Limborina.     

Conflicting preferences within females: sexual selection versus species recognition

Preferences for mates within and between species are often harmonious, as traits that females prefer are usually more developed in conspecifics than heterospecifics. This need not be the case, however. When it is not, conflict between these arenas of mate choice can be resolved if females attend to different cues for each task. But this raises the potential for correlations among preferences to limit the opportunity for these two processes to operate independently. Here, we show that, within individual female pygmy swordtails (Xiphophorus pygmaeus), directional preferences for conspicuous ornamentation are inversely associated with discrimination against a sympatric heterospecific, Xiphophorus cortezi. Thus, mate choice among and within species need not be separate, independent processes; instead, they can be mechanistically intertwined. As a consequence, different arenas of mate choice can constrain one another, even when females assess multiple cues.     

Using micropylar ultrastructure for species identification and phylogenetic inference among four species of Sparidae

Among Sparus sarba, Acanthopagrus latus, A. schlegeli and Pagrus major, P. major had the largest egg size, with the biggest micropyle funnel and the most numerous accessory openings. The reinforcement in the micropyle canals was species specific with eight spiral clockwise, five two-spiral clockwise, seven two-spiral clockwise, and 10 triangular ridges in S. sarba, A. latus, A. schlegeli , and P. major , respectively. A key to identify these four species based on micropyle characters is proposed for future applications. Cladistic analysis by using different parsimonious methods on the morphological character of the micropyle suggested that the generic interrelationship between Sparus and Acanthopagrus was closer than to the genus Pagrus . Furthermore, the congeneric species of A. latus and A. schlegeli were the most closely related, with S. sarba the second, and P. major the last. This result agreed with conclusions obtained from other character suites including morphological, biochemical and molecular data.     

Genetic and phylogenetic evidence for misidentification of Vibrio species within the Harveyi clade

Aim: This report describes the use of a six‐gene multi‐locus sequence analysis (MLSA) to correctly identify Vibrio strains of the Harveyi clade. Methods and Results: Vibrio isolates were characterized using a six housekeeping gene MLSA. The study provided evidence supporting: (i) a substantial number of reference strains maintained within commercial culture collections are misidentified taxonomically at the species level; (ii) two V. alginolyticus subclades retain species‐level divergence; and (iii) V. communis and V. owensii likely are the same species. Conclusion: A significant number (n = 10) of Harveyi clade Vibrio strains have been inaccurately identified, including evidence that V. communis and V. owensii strains, two recently discovered species assigned to the Harveyi clade, comprise a single species. Significance and Impact of the study: As Harveyi clade vibrios have an enormous impact on human and aquatic animal health, it is of paramount importance to identify members of the Harveyi clade correctly.     

Using multiple sources of characters to delimit species in the genus Crataegus (Rosaceae): the case of the Crataegus rosei complex

The aim of this study was to delimit the taxa of the Crataegus rosei complex using an integrative approach that incorporates a suite of molecular (cpDNA and nuclear microsatellite markers), morphological, and geometric morphometric characters. One hundred and ten plants from 19 populations that encompass the entire distribution range of the species complex were collected and examined along with herbarium specimens. Parsimony and Bayesian inference analyses were run using morphological, molecular, and both the morphological and molecular data sets combined. Analyses to determine genetic structure based on microsatellite data and multivariate analyses incorporating geometric morphometrics were also done to identify differences in leaf shape. The results supported the recognition of two taxa: C. rosei with high levels of gene flow among its populations, remarkable morphological variation and a wide distribution range and C. rosei var. amoena, composed of a few isolated populations in the high elevation location of Cerro Potosí; a new specific epithet will be decided for the latter in accordance with the International Code of Nomenclature for algae, fungi, and plants.     

Phylogeny, biogeography, and species boundaries within the Alexandrium minutum group

The geographic range and bloom frequency of the toxic dinoflagellate Alexandrium minutum and other members of the A. minutum group have been increasing over the past few decades. Some of these species are responsible for paralytic shellfish poisoning (PSP) outbreaks throughout the world. The origins of new toxic populations found in previously unaffected areas are typically not known due to a lack of reliable plankton records with sound species identifications and to the lack of a global genetic database. This paper provides the first comprehensive study of minutum-group morphology and phylogeny on a global scale, including 45 isolates from northern Europe, the Mediterranean, Asia, Australia and New Zealand.Neither the morphospecies Alexandrium lusitanicum nor A. angustitabulatum was recoverable morphologically, due to large variation within and among all minutum-group clonal strains in characters previously used to distinguish these species: the length:width of the anterior sulcal plate, shape of the 1′ plate, connection between the 1′ plate and the apical pore complex, and the presence of a ventral pore. DNA sequence data from the D1 to D2 region of the LSU rDNA also fail to recognize these species. Therefore, we recommend that all isolates previously designated as A. lusitanicum or A. angustitabulatum be redesignated as A. minutum. A. tamutum, A. insuetum, and A. andersonii are clearly different from A. minutum on the basis of both genetic and morphological data.A. minutum strains from Europe and Australia are closely related to one another, which may indicate an introduction from Europe to Australia given the long history of PSP in Europe and its recent occurrence in Australia. A minutum from New Zealand and Taiwan form a separate phylogenetic group. Most strains of A. minutum fit into one of these two groups, although there are a few outlying strains that merit further study and may represent new species. The results of this paper have greatly improved our ability to track the spread of A. minutum species and to understand the evolutionary relationships within the A. minutum group by correcting inaccurate taxonomy and providing a global genetic database.     

Multiple nuclear gene sequences identify phylogenetic species boundaries in the rapidly radiating clade of Mexican ambystomatid salamanders

Delimiting the boundaries of species involved in radiations is critical to understanding the tempo and mode of lineage formation. Single locus gene trees may or may not reflect the underlying pattern of population divergence and lineage formation, yet they constitute the vast majority of the empirical data in species radiations. In this study we make use of an expressed sequence tag (EST) database to perform nuclear (nDNA) and mitochondrial (mtDNA) genealogical tests of species boundaries in Ambystoma ordinarium, a member of an adaptive radiation of metamorphic and paedomorphic salamanders (the Ambystoma tigrinum complex) that have diversified across terrestrial and aquatic environments. Gene tree comparisons demonstrate extensive nonmonophyly in the mtDNA genealogy of A. ordinarium, while seven of eight independent nuclear loci resolve the species as monophyletic or nearly so, and diagnose it as a well-resolved genealogical species. A differential introgression hypothesis is supported by the observation that western A. ordinarium localities contain mtDNA haplotypes that are identical or minimally diverged from haplotypes sampled from a nearby paedomorphic species, Ambystoma dumerilii, while most nDNA trees place these species in distant phylogenetic positions. These results provide a strong example of how historical introgression can lead to radical differences between gene trees and species histories, even among currently allopatric species with divergent life history adaptations and morphologies. They also demonstrate how EST-based nuclear resources can be used to more fully resolve the phylogenetic history of species radiations.     

Using mating-type gene sequences for improved phylogenetic resolution of Collectotrichum species complexes

Colletotrichum species are defined primarily on the basis of host preference and morphology of the organism in planta and in culture. However the genus contains several species complexes that encompass such a broad range of morphological and pathological variation that the species name is of relatively little use either to the taxonomist or plant pathologist. Phylogenetic analyses, primarily based on variable regions of the ribosomal DNA (rDNA) sequences, have indicated that these species complexes comprise a variable number of identifiable monophyletic clades. However rDNA sequences often are insufficiently diverse to fully resolve such closely related lineages. A group of isolates representing three species complexes (C. graminicola, C. gloeosporioides and C. acutatum) were analyzed by using the high mobility group (HMG)-encoding sequence of the MAT1-2 mating type sequence, which has been shown in other fungi to be especially suitable for distinguishing relationships among closely related groups. Results were compared with those obtained from analysis of variable regions of the rDNA as well as from standard morphological classification methods. Results achieved through analysis of MAT1-2 sequences correlated well with those obtained by analysis of rDNA sequences but provided significantly better resolution among the various lineages. Morphological traits, including hyphopodia size, colony appearance, spore size, appresorial shape and size and host preference, frequently were unreliable as indicators of phylogenetic association. Spore shape and hyphopodia shape more often were useful for this purpose.     

Species boundaries and phylogenetic relationships within the green algal genus Codium (Bryopsidales) based on plastid DNA sequences

Despite the potential model role of the green algal genus Codium for studies of marine speciation and evolution, there have been difficulties with species delimitation and a molecular phylogenetic framework was lacking. In the present study, 74 evolutionarily significant units (ESUs) are delimited using 227 rbcL exon 1 sequences obtained from specimens collected throughout the genus' range. Several morpho-species were shown to be poorly defined, with some clearly in need of lumping and others containing pseudo-cryptic diversity. A phylogenetic hypothesis of 72 Codium ESUs is inferred from rbcL exon 1 and rps3-rpl16 sequence data using a conventional nucleotide substitution model (GTR+Gamma+I), a codon position model and a covariotide (covarion) model, and the fit of a multitude of substitution models and alignment partitioning strategies to the sequence data is reported. Molecular clock tree rooting was carried out because outgroup rooting was probably affected by phylogenetic bias. Several aspects of the evolution of morphological features of Codium are discussed and the inferred phylogenetic hypothesis is used as a framework to study the biogeography of the genus, both at a global scale and within the Indian Ocean.     

Using average nucleotide identity (ANI) to evaluate microsporidia species boundaries based on their genetic relatedness

Microsporidia are obligatory intracellular parasites related to fungi and since their discovery their classification and origin has been controversial due to their unique morphology. Early taxonomic studies of microsporidia were based on ultrastructural spore features, characteristics of their life cycle and transmission modes. However, taxonomy and phylogeny based solely on these characteristics can be misleading. SSU rRNA is a traditional marker used in taxonomical classifications, but the power of SSU rRNA to resolve phylogenetic relationships between microsporidia is considered weak at the species level, as it may not show enough variation to distinguish closely related species. Overall genome relatedness indices (OGRI), such as average nucleotide identity (ANI), allows fast and easy-to-implement comparative measurements between genomes to assess species boundaries in prokaryotes, with a 95% cutoff value for grouping genomes of the same species. Due to the increasing availability of complete genomes, metrics of genome relatedness have been applied for eukaryotic microbes taxonomy such as microsporidia. However, the distribution of ANI values and cutoff values for species delimitation have not yet been fully tested in microsporidia. In this study we examined the distribution of ANI values for 65 publicly available microsporidian genomes and tested whether the 95% cutoff value is a good estimation for circumscribing species based on their genetic relatedness.