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.     

A three-gene phylogeny of the Mycena pura complex reveals 11 phylogenetic species and shows ITS to be unreliable for species identification

Phylogenetic analyses of Mycena sect. Calodontes using ITS previously suggested ten cryptic monophyletic ITS lineages within the Mycena pura morphospecies. Here, we compare ITS data (645 bp incl. gaps) from 46 different fruit bodies that represent the previously described ITS diversity with partial tEF-1-α (423 bp) and RNA polymerase II (RPB1) (492 bp) sequence data to test the genealogical concordance.While neither of the markers were in complete topological agreement, the branches differing between the tEF and RPB1 trees had a low bootstrap (<50) support, and the partition homogeneity incongruence length difference (ILD) tests were not significant. ILD tests revealed significant discordances between ITS and the tEF and RPB1 markers in several lineages. And our analyses suggested recombination between ITS1 and ITS2, most pronounced in one phylospecies that was identical in tEF and RPB1. Based on the agreement between tEF and RPB1, we defined 11 mutually concordant terminal clades as phylospecies inside the M. pura morphospecies; most of them cryptic. While neither of the markers showed an unequivocal barcoding gap between inter- and intraspecific diversity, the overlap was most pronounced for ITS (intraspecific diversity 0-3.5 %, interspecific diversity 0.4 %-8.8 %). A clustering analysis on tEF separated at a 1.5 % level returned all phylogenetic species as Operational Taxonomic Units (OTUs), while ITS at both a 1.5 % level and at a 3 % threshold level not only underestimated diversity as found by the tEF and RPB1, but also identified an OTU which was not a phylogenetic species. Thus, our investigation does not support the universal suitability of ITS for species recognition in particular, and emphasises the general limitation of single gene analyses combined with single percentage separation values.     

Molecular systematics of the Philippine forest skinks (Squamata: Scincidae: Sphenomorphus): testing morphological hypotheses of interspecific relationships

Skinks of the genus Sphenomorphus are the most diverse clade of squamates in the Philippine Archipelago. Morphological examination of these species has defined six phenotypic groups that are commonly used in characterizations of taxonomic hypotheses. We used a molecular phylogeny based on four mitochondrial and two nuclear genes to assess the group's biogeographical history in the archipelago and examine the phylogenetic validity of the currently recognized Philippine species groups. We re‐examined traditional characters used to define species groups and used multivariate statistics to quantitatively evaluate group structure in morphometric space. Clustering analyses of phenotypic similarity indicate that some (but not all) members of previously defined species groups are phenotypically most similar to other members of the same group. However, when species group membership was mapped on our partitioned Bayesian phylogenetic hypothesis, only one species group corresponds to a clade; all other species group arrangements are strongly rejected by our phylogeny. Our results demonstrate that (1) previously recognized species group relationships were misled by phenotypic convergence; (2) Sphenomorphus is widely paraphyletic; and (3) multiple lineages have independently invaded the Philippines. Based on this new perspective on the phylogenetic relationships of Philippine Sphenomorphus, we revise the archipelago's diverse assemblage of species at the generic level, and resurrect and/or expand four previously recognized genera, and describe two new genera to accommodate the diversity of Philippine skinks of the Sphenomorphus group. © 2011 The Linnean Society of London, Zoological Journal of the Linnean Society, 2011, 163 , 1217–1243.     

Molecular data delineate four genera of "Thryothorus" wrens

Wrens of the genus Thryothorus comprise over a third of the species diversity in the family Troglodytidae. In addition to this species diversity, these wrens vary in a number of behavioral characteristics, in particular in the presence and structure of vocal duets, which makes them an interesting target for comparative evolutionary ecological and behavioral study. However, no phylogenetic hypothesis for this group-which would provide a sound basis for comparative analysis-is currently available. While previous molecular phylogenetic work established conclusively that the type of this genus, Thryothorus ludovicianus (Latham), was not part of a monophyletic group with other Thryothorus, the exact limits of the genus could not be established due to limited taxon sampling. Here, we present molecular data from all but four currently recognized species of Thryothorus. These data confirm that Thryothorus is paraphyletic, and that the type T. ludovicianus does not form a monophyletic group with any other member of the genus. Based on analyses of our data, we resurrect two previously recognized wren genera, Pheugopedius and Thryophilus, and erect a new genus-Cantorchilus-to house the remaining ex-Thryothorus species. Our hypothesis of relationships will provide a firm basis for future behavioral and morphological analyses of these species.     

Molecular phylogenetics and biogeography of Neotropical tanagers in the genus Tangara

Species in the genus Tangara are distributed throughout the New World tropics and vary in their morphology, behavior, and ecology. We used data from the cytochrome b and ND 2 genes to provide the first phylogenetic perspective on the evolution of this diversity. Reconstructions based on parsimony, maximum likelihood, and Bayesian approaches were largely congruent. The genus is monophyletic and consists of two main clades. Within these clades, DNA sequence data confirm the monophyly of most previously recognized species groups within Tangara, indicating general concordance between molecular data and impressions based on geographic distribution, morphology, and behavior. Within some currently recognized species, levels of DNA sequence variation are larger than expected, suggesting multiple taxa may be involved. In contrast, some currently recognized species are only weakly differentiated from their sister species. Biogeographic analyses indicate that many early speciation events occurred in the Andes. More recently, dispersal events followed by subsequent speciation have occurred in other geographic areas of the Neotropics. Assuming a molecular clock, most speciation events occurred well before Pleistocene climatic cycles. The time frame of Tangara speciation corresponds more closely to a period of continued uplift in the Andes during the late Miocene and Pliocene.     

Lizards from the end of the world: phylogenetic relationships of the Liolaemus lineomaculatus section (Squamata: Iguania: Liolaemini)

The Liolaemus lineomaculatus section is a geographically widely distributed group of lizards from the Patagonian region of southern South America, and includes 18 described species representing the most southerly distributed Liolaemus taxa (the genus includes 228 species and extends from Tierra del Fuego north to south-central Peru). Despite high species diversity, the phylogenetic relationships of this section are unknown. In the present work we sampled all described species in the L. lineomaculatus section as well as currently undescribed candidate species to reconstruct the first complete phylogenetic hypothesis for the clade. Our data set included four anonymous nuclear loci, three nuclear protein-coding loci, and two mitochondrial genes. We compared results obtained with three different phylogenetic methods for the concatenated data set (Maximum Parsimony, Maximum Likelihood and Bayesian Inference) with a coalescent-based species tree approach (BEST), and recovered congruent, strongly-supported topological arrangements across all methods. We identified four main clades within the L. lineomaculatus section: the lineomaculatus, magellanicus, somuncurae, and kingii+archeforus groups, for which we estimated divergence times. We discuss the taxonomic implications of these results and how the future integration of phylogeographic, niche modeling and morphological approaches will allow testing biogeographical hypotheses in this clade.     

Phylogeny of North American amblemines (Bivalvia, Unionoida): prodigious polyphyly proves pervasive across genera

Abstract. The subfamily Ambleminae is the most diverse subfamily of fresh‐water mussels (order Unionoida), a globally diverse and ecologically prominent group of bivalves. About 250 amblemine species occur in North America; however, this diversity is highly imperiled, with the majority of species at risk. Assessing and protecting this diversity has been hampered by the uncertain systematics of this group. This study sought to provide an improved phylogenetic framework for the Ambleminae. Currently, 37 North American genera are recognized in Ambleminae. Previous phylogenetic studies of amblemines highlighted the need for more extensive sampling due to the uncertainties arising from polyphyly of many currently recognized taxa. The present study incorporated all amblemine genera occurring in North America north of the Rio Grande, with multiple species of most genera, including the type species for all but seven genera. A total of 192 new DNA sequences were obtained for three mitochondrial gene regions: COI, 16S, and ND1. In combination with published data, this produced a data matrix incorporating 357 gene sequences for 143 operational taxonomic units, representing 107 currently recognized species. Inclusion of published data provides additional taxa and a summary of present molecular evidence on amblemine phylogeny, if at the cost of increasing the amount of missing data. Parsimony and Bayesian analyses suggest that most amblemine genera, as currently defined, are polyphyletic. At higher taxonomic levels, the tribes Quadrulini, Lampsilini, and Pleurobemini were supported; the extent of Amblemini and the relationships of some genera previously assigned to that tribe remain unclear. The eastern North American amblemines appear monophyletic. Gonidea and some Eurasian taxa place as probable sister taxa for the eastern North American Ambleminae. The results also highlight problematic taxa of particular interest for further work.     

Japanese species of the Longibrachiatum Clade of Trichoderma

The Longibrachiatum Clade of the genus Trichoderma in Japan was examined, among which two new species and three new records are herewith reported. The new species, T. tsugarense and T. kunigamense were isolated from a bed log (cultivation of Lentinula edodes) and volcanic ash soil, respectively. These species are distinguished from closely related species by growth and morphological characteristics and in phylogenetic analysis. Additional species new to Japan were T. ghanense, T. parareesei and T. sinense. The significance of their distribution is discussed. Most species of the Longibrachiatum Clade are tropical rather than temperate in distribution. Their in vitro optimum growth tends to be >35 °C but the optimum temperature for some Japanese species was lower. Some species are endophytes of temperate plant species, some of which are endemic in Japan.     

Molecular systematics of threadfin breams and relatives (Teleostei,Nemipteridae)

Threadfin breams and relatives of the family Nemipteridae comprise 69 currently recognized species in five genera. They are found in the tropical and subtropical Indo‐West Pacific and most are commercially important. Using recently developed molecule‐based approaches exploiting DNA sequence variation among species/specimens, this study reconstructed a comprehensive phylogeny of the Nemipteridae, examined the validity of species and explored the cryptic diversity of the family, and tested previous phylogenetic hypotheses. A combined data set (105 taxa from 41 morphospecies) with newly determined sequences from two nuclear genes (RAG1 and RH) and one mitochondrial gene (COI), and a data set with only COI gene sequences (329 newly obtained plus 328 from public databases from a total of 53 morphospecies) were used in the phylogenetic analysis. The latter was further used for species delimitation analyses with two different tools to explore species diversity. Our phylogenetic results showed that all the currently recognized genera were monophyletic. The monotypic genus Scaevius is the sister group of Pentapodus and they together are sister to Nemipterus. These three genera combined to form the sister group of the clade comprising Parascolopsis and Scolopsis. The validity of most of the examined species was confirmed except in some cases. The combined evidence from the results of different analyses revealed a gap in our existing knowledge of species diversity in the Nemipteridae. We found several currently recognized species contain multiple separately evolving metapopulation lineages within species; some lineages should be considered as new species for further assignment. Finally, some problematic sequences deposited in public databases (probably due to misidentification) were also revised in this study to improve the accuracy for prospective DNA barcoding work on nemipterid fishes.     

Reproductive isolation and phylogenetic divergence in Neurospora: comparing methods of species recognition in a model eukaryote

We critically examined methods for recognizing species in the model filamentous fungal genus Neurospora by comparing traditional biological species recognition (BSR) with more comprehensive applications of both BSR and phylogenetic species recognition (PSR). Comprehensive BSR was applied to a set of 73 individuals by performing extensive crossing experiments and delineating biological species based on patterns of reproductive success. Within what were originally considered two species, N. crassa and N. intermedia, we recognized four reproductively isolated biological species. In a concurrent study (Dettman et al. 2003), we used genealogical concordance of four independent nuclear loci to recognize phylogenetic species in Neurospora. Overall, the groups of individuals identified as species were similar whether recognized by reproductive success or by phylogenetic criteria, and increased genetic distance between parents was associated with decreased reproductive success of crosses, suggesting that PSR using genealogical concordance can be used to reliably recognize species in organisms that are not candidates for BSR. In one case, two phylogenetic species were recognized as a single biological species, indicating that significant phylogenetic divergence preceded the development of reproductive isolation. However, multiple biological species were never recognized as a single phylogenetic species. Each of the putative N. crassa x N. intermedia hybrids included in this study was confidently assigned to a single species, using both PSR and BSR. As such, no evidence for a history of hybridization in nature among Neurospora species was observed. By performing reciprocal mating tests, we found that mating type, parental role, and species identity of parental individuals could all influence the reproductive success of matings. We also observed sympatry-associated sexual dysfunction in interspecific crosses, which was consistent with the existence of reinforcement mechanisms.     

Genetic diversity of a Botrytis cinerea cryptic species complex in Hungary

Botrytis cinerea has been described as a species complex containing two cryptic species, referred to as groups I and II. The first B. cinerea group I strains outside of Western Europe were collected in Hungary in 2008 from strawberry and rape plants. Sympatric B. cinerea cryptic species were analyzed using a population genetic approach and phenotypic markers. Statistically significant, but moderate population differentiation was found between the two groups in Hungary. Group I was originally typified by the lack of the transposable elements Boty and Flipper. However, all the Hungarian group I isolates carried the Boty element and one isolate additionally contained Flipper, indicating a much wider genetic variation than previously believed. Vegetative compatibility analyses showed that twelve of the thirteen B. cinerea group I isolates studied belonged to a unique vegetative compatibility group (VCG), but VCGs overlapped between groups. Phenotypic markers such as fenhexamid resistance or asexual spore size were found unsuitable to differentiate between the cryptic species. The results did not confirm the complete separation of the two cryptic species, previously determined with genealogical concordance of the phylogenetic species recognition using multiple gene sequences, and suggest instead the possibility of information exchange between them.     

Phylogeny of the genus Arenavirus

The family Arenaviridae consists of a unique genus (Arenavirus) that currently comprises 22 viral species, as recognized by the International Committee for Taxonomy of Viruses. Seven newly discovered represent putative new species. Here, our aims were to provide the most comprehensive phylogenetic analysis of members and putative members of the family Arenaviridae to date, and to investigate the genetic diversity observed within and between recognized species of New world arenaviruses to determine whether the genetic criteria previously proposed to define arenavirus species for Old world arenaviruses should be retained or are more widely applicable to the whole genus.     

Phylogenetic diversity of termite gut spirochaetes

A molecular phylogenetic analysis was done of not-yet-cultured spirochaetes inhabiting the gut of the termite, Reticulitermes flavipes (Kollar). Ninety-eight clones of near-full-length spirochaetal 16S rDNA genes were classified by ARDRA pattern and by partial sequencing. All clones grouped within the genus Treponema , and at least 21 new species of Treponema were recognized within R. flavipes alone. Analysis of 190 additional clones from guts of Coptotermes formosanus Shiraki and Zootermopsis angusticollis (Hagen), as well as published data on clones from Cryptotermes domesticus (Haviland), Mastotermes darwiniensis Froggatt, Nasutitermes lujae (Wasmann) and Reticulitermes speratus (Kolbe), revealed a similar level of novel treponemal phylogenetic diversity in these representatives of five of the seven termite families. None of the clones was closely related (i.e. all bore ≤ 91% sequence similarity) to any previously recognized treponeme. The data also revealed the existence of two major phylogenetic groups of treponemes: one containing all of the currently known isolates of Treponema and a large number of phylotypes from the human gingival crevice, but only a minority of the termite gut spirochaete clones; another containing the majority of termite spirochaete clones and two Spirochaeta ( S. caldaria and S. stenostrepta ), which, although free living, group within the genus Treponema on the basis of 16S rRNA sequence. Signature nucleotides that almost perfectly distinguished the latter group, herein referred to as the 'termite cluster', occurred at the following ( E. coli numbering) positions: 289-G · C-311; A at 812; and an inserted nucleotide at 1273. The emerging picture is that the long-recognized and striking morphological diversity of termite gut spirochaetes is paralleled by their phylogenetic diversity and may reflect substantial physiological diversity as well.     

Phylogenetic analysis of Aspergillus species using DNA sequences from four loci

DNA sequences were determined for beta tubulin (BT2), calmodulin (CF), ITS and lsu rDNA (ID) and RNA polymerase II (RPB2) from ca. 460 Aspergillus isolates. RPB2 and rDNA sequences were combined and analyzed to determine relationships in the genus and in the family Trichocomaceae. Eupenicillium species form a statistically supported clade with origins among the Aspergillus clades. A. crystallinus, A. malodoratus and H. paradoxus are members of the Eupenicillium clade. A. zonatus, A. clavatoflvus and W. spinulosa occur in a clade along with Hamigera sp. Other than these exceptional species, Aspergillus species and sections occur on three strongly supported clades that descend from a polytomy. Section Versicolores as a monophyletic group includes only A. versicolor and A. sydowii and is superfluous. The other sections were retained but modified. All four loci were used in genealogical concordance analysis of species boundaries. Fennellia flavipes and F. nivea are not conspecific with their supposed anamorphs A. flavipes and A. nivea. Synonymies were found for some species and more than 20 undescribed taxa were identified in genealogical concordance analysis. Newly discovered taxa will be described elsewhere. Possibly paralogous gene fragments were amplified with the BT2 primers in sections Nidulantes, Usti and Nigri. Use of nonhomologous sequences in genealogical concordance analysis could lead to false conclusions and so BT2 sequences were not used in analysis of those sections.     

Utility of the DNA barcoding gene fragment for parasitic wasp phylogeny (Hymenoptera: Ichneumonoidea): data release and new measure of taxonomic congruence

The enormous cytochrome oxidase subunit I (COI) sequence database being assembled from the various DNA barcoding projects as well as from independent phylogenetic studies constitutes an almost unprecedented amount of data for molecular systematics, in addition to its role in species identification and discovery. As part of a study of the potential of this gene fragment to improve the accuracy of phylogenetic reconstructions, and in particular, exploring the effects of dense taxon sampling, we have assembled a data set for the hyperdiverse, cosmopolitan parasitic wasp superfamily Ichneumonoidea, including the release of 1793 unpublished sequences. Of approximately 84 currently recognized Ichneumonoidea subfamilies, 2500 genera and 41,000 described species, barcoding 5'-COI data were assembled for 4168 putative species-level terminals (many undescribed), representing 671 genera and all but ten of the currently recognized subfamilies. After the removal of identical and near-identical sequences, the 4174 initial sequences were reduced to 3278. We show that when subjected to phylogenetic analysis using both maximum likelihood and parsimony, there is a broad correlation between taxonomic congruence and number of included sequences. We additionally present a new measure of taxonomic congruence based upon the Simpson diversity index, the Simpson dominance index, which gives greater weight to morphologically recognized taxonomic groups (subfamilies) recovered with most representatives in one or a few contiguous groups or subclusters.     

CHLOROPLAST DNA EVOLUTION AND PHYLOGENETIC RELATIONSHIPS IN CLARKIA SECT. PERIPETASMA (ONAGRACEAE)

Restriction-site analysis of chloroplast DNA in Clarkia sect. Peripetasma (Onagraceae) was done to test previously proposed phylogenetic models. One hundred nineteen restriction-site mutations were identified among the nine species using 29 restriction enzymes, and these were used to construct rooted most parsimonious trees (Wagner and Dollo). A chloroplast DNA evolutionary clock could not be statistically rejected. Branch points of this tree were statistically tested by Felsenstein's bootstrap method. This tree 1) provided an unambiguous and detailed genealogical history for the section, 2) verified a previous partial phylogenetic model for the section based on gene duplications and differential silencing, 3) provided details of the phylogenetic model not inferred or expected based on morphology and reproductive isolation, and 4) indicated that morphology evolves at markedly different rates within and between lineages in the section.     

Higher level phylogeny for the passion-vine butterflies (Nymphalidae, Heliconiinae) based on early stage and adult morphology

A higher level phylogeny for the passion-vine butterflies (Nymphalidae, Heliconiinae) was generated by cladistic analysis of 146 morphological characters from all life stages. The 24 species studied were selected representatives of the ten currently accepted genera of the sub-tribe Heliconiiti. Analyses of only characters from larvae and pupae did not produce well resolved trees. However, some characters of the immature stages provided critical support for the monophyly of two clades. Analysis of only adult characters yielded a tree that closely resembled that obtained from all data combined. The phylogeny here derived from the combined analysis of early stage and adult characters differed in topology from all previously proposed hypotheses, and supported the monophyly of all currently recognized genera. Characters supporting each clade are described and illustrated, and various hypotheses of phylogenetic relatedness of passion-vine butterfly taxa are discussed.     

An analysis of species boundaries and biogeographic patterns in a cryptic species complex: the rotifer--Brachionus plicatilis

Since the advent of molecular phylogenetics, there is increasing evidence that many small aquatic and marine invertebrates--once believed to be single, cosmopolitan species--are in fact cryptic species complexes. Although the application of the biological species concept is central to the identification of species boundaries in these cryptic complexes, tests of reproductive isolation do not frequently accompany phylogenetic studies. Because different species concepts generally identify different boundaries in cryptic complexes, studies that apply multiple species concepts are needed to gain a more detailed understanding of patterns of diversification in these taxa. Here we explore different methods of empirically delimiting species boundaries in the salt water rotifer Brachionus plicatilis by comparing reproductive data (i.e., the traditional biological species concept) to phylogenetic data (the genealogical species concept). Based on a high degree of molecular sequence divergence and largely concordant genetic patterns in COI and ITS1, the genealogical species hypothesis indicates the existence of at least 14 species--the highest estimate for the group thus far. A test of the genealogical species concept with biological crosses shows a fairly high level of concordance, depending on the degree of reproductive success used to draw boundaries. The convergence of species concepts in this group suggests that many of the species within the group may be old. Although the diversity of the group is higher than previously understood, geographic distributions remain broad. Efficient passive dispersal has resulted in global distributions for many species with some evidence of isolation by distance over large geographic scales. These patterns concur with expectations that micro-meiofauna (0.1-1mm) have biogeographies intermediate to microbial organisms and large vertebrates. Sympatry of genetically distant strains is common.     

Phylogeny of all recognized species of ammonia oxidizers based on comparative 16S rRNA and amoA sequence analysis: implications for molecular diversity surveys

The current perception of evolutionary relationships and the natural diversity of ammonia-oxidizing bacteria (AOB) is mainly based on comparative sequence analyses of their genes encoding the 16S rRNA and the active site polypeptide of the ammonia monooxygenase (AmoA). However, only partial 16S rRNA sequences are available for many AOB species and most AOB have not yet been analyzed on the amoA level. In this study, the 16S rDNA sequence data of 10 Nitrosomonas species and Nitrosococcus mobilis were completed. Furthermore, previously unavailable 16S rRNA sequences were determined for three Nitrosomonas sp. isolates and for the gamma-subclass proteobacterium Nitrosococcus halophilus. These data were used to revaluate the specificities of published oligonucleotide primers and probes for AOB. In addition, partial amoA sequences of 17 AOB, including the above-mentioned 15 AOB, were obtained. Comparative phylogenetic analyses suggested similar but not identical evolutionary relationships of AOB by using 16S rRNA and AmoA as marker molecules, respectively. The presented 16S rRNA and amoA and AmoA sequence data from all recognized AOB species significantly extend the currently used molecular classification schemes for AOB and now provide a more robust phylogenetic framework for molecular diversity inventories of AOB. For 16S rRNA-independent evaluation of AOB species-level diversity in environmental samples, amoA and AmoA sequence similarity threshold values were determined which can be used to tentatively identify novel species based on cloned amoA sequences. Subsequently, 122 amoA sequences were obtained from 11 nitrifying wastewater treatment plants. Phylogenetic analyses of the molecular isolates showed that in all but two plants only nitrosomonads could be detected. Although several of the obtained amoA sequences were only relatively distantly related to known AOB, none of these sequences unequivocally suggested the existence of previously unrecognized species in the wastewater treatment environments examined.