PAUP*. Phylogenetic Analysis Using Parsimony (*and Other Methods). Version 4.0b10
Abstract
— We studied sequence variation in 16S rDNA in 204 individuals from 37 populations of the land snail Candidula unifasciata (Poiret 1801) across the core species range in France, Switzerland, and Germany. Phylogeographic, nested clade, and coalescence analyses were used to elucidate the species evolutionary history. The study revealed the presence of two major evolutionary lineages that evolved in separate refuges in southeast France as result of previous fragmentation during the Pleistocene. Applying a recent extension of the nested clade analysis (Templeton 2001), we inferred that range expansions along river valleys in independent corridors to the north led eventually to a secondary contact zone of the major clades around the Geneva Basin. There is evidence supporting the idea that the formation of the secondary contact zone and the colonization of Germany might be postglacial events. The phylogeographic history inferred for C. unifasciata differs from general biogeographic patterns of postglacial colonization previously identified for other taxa, and it might represent a common model for species with restricted dispersal.
... This method does not require gene tree estimations, and it can avoid gene tree estimation error when the locus has a low phylogenetic signal. For this method, we used SVDQuartets (Chifman & Kubatko, 2014) implemented within PAUP* (Swofford, 2003). ...
... Our analyses indicate that the gene concordance factor (UCEs: 34.9%; CDSs: 35.81%) is higher than the gene discordance factors (UCEs: 17.6%; CDSs: 11.46% and UCEs: 14.66%; CDSs: 15.89%) among the three genera. However, node-age and branch length estimations using quartet-based estimation (qAge) in PAUP* (Peng et al., 2022;Swofford, 2003) suggest that ILS may have influenced the mito-nuclear discordance, albeit not significantly and the short branch lengths indicate rapid speciation (Tables S8 and S9). Alternatively, the divergence observed in maternally inherited markers may arise from female-specific selection, which is reported in brood-parasitic birds (Spottiswoode et al., 2011), but is less concerned among bats. ...
The subfamily Murininae is renowned for its inherent taxonomic challenges associated with sampling difficulties and morphological similarities. At present, three genera are acknowledged within the subfamily; however, their phylogenetic interrelations and systematic classification remain debated. In this study, the separation of Harpiola at the genus level was robustly supported by our phylogenomic analyses based on the mitochondrial genome, coding sequences (CDSs) and ultraconserved elements (UCEs) from 12 individuals covering all three genera of Murininae. Notably, a distinctive mito‐nuclear discordance emerged, with the nuclear genealogy (( Harpiocephalus , Harpiola ), Murina ) contrasting with the mitochondrial genealogy ( Harpiocephalus , ( Harpiola , Murina )). The integration of these findings with inferences of demographic history reveals that dramatic environmental changes during the Pleistocene glacial and inter‐glacial cycles have shaped the current distribution of Murininae. Moreover, the detection of extensive gene flow between ancient lineages of Harpiola and Murina suggests that an ancestral ‘ghost’ Murina lineage may have contributed its mitochondrial DNA, along with a limited portion of nuclear DNA, to Harpiola in a bygone hybridization zone. In addition to molecular analyses, we employed traditional and geometric morphometric analyses of skulls to differentiate the three genera. Harpiocephalus is readily distinguishable, but Harpiola and certain species of Murina exhibit overlapping characteristics both morphometrically and geometrically which may be the outcome of ancient introgression events. This finding highlights the importance of fine‐scale morphological distinctions within the latter genera, which may be the outcome of ancient introgression events.
... b 2.0 (± 0.08) ab Disc floret diameter (mm) 1.5 (± 0.09) a 1.8 (± 0.05) b 2.8 (± 0.07) c Disc floret lobe length (mm) 0.5 (± 0.05) a 0.7 (± 0.03) b 1.3 (± 0.04) c Disc floret length (mm) 3.4 (± 0.09) a 3.9 (± 0.06) b 5.4 (± 0.08) c Disc floret tube width (mm) 0.7 (± 0.03) a 0.9 (± 0. resulting alignment by eye to ensure the comparison of orthologous subrepeats, as described by Timme et al. (2007). We determined the best-fit model of sequence evolution for the ETS dataset using PAUP* v4.0a Build 169 (Swofford 2002). The analysis selected the TVM+Γ model using the Bayesian Information Criterion (Schwarz 1978), and we set the starting model and prior parameters of subsequent phylogenetic analyses to be consistent with these results. ...
A new species of Helianthus (Asteraceae), Helianthus waccamawensis Ungberg, sp. nov., is described from the Cape Fear Arch Region of North and South Carolina. Most recently, it has been included in H. floridanus A. Gray ex Chapm. but differs in its narrower leaves, achene and floral morphology, and habitat preferences. It superficially resembles H. angustifolius L. but differs in its extensively rhizomatous habit, shorter leaves, and achene and floral morphologies. It is further differentiated from the diploids H. floridanus and H. angustifolius by being hexaploid, and molecular phylogenetic data do not indicate a close relation to either species. In situ photos, diagnostic morphological photos, molecular phylogenetic data, cytological data, a range map, a list of additional collections, and a revised key for relevant southeastern Helianthus species are provided.
... Combined and individual datasets were subjected to maximum likelihood (ML) and Bayesian inference (BI) analyses. The best-fit substitution models were evaluated using MrModeltest v. 2.3 [46] with the Akaike Information Criterion (AIC) as the selection criteria executed in PAUP v. 4.0b10 [47] . ML and BI analyses were performed on the CIPRES Science Gateway platform [48] . ...
... A maximum-likelihood (ML) tree was reconstructed using raxmlGUI 2.0 (Edler et al. 2021) with the GTR model and 1,000 bootstrap replicates. A model test was performed using the PAUP � plugin in Geneious Prime (Swofford 2002). ...
The complete mitochondrial genome sequence of Dendrodoris krusensternii (J. E. Gray, 1850) was determined using next-generation sequencing. The complete mitochondrial genome of D. krusensternii is 14,361 bp long, comprising 13 genes, including 13 protein-coding genes (PCGs), 22 transfer RNAs, and two ribosomal RNAs. The nucleotide composition was estimated: 28.7% A, 14.8% C, 19.6% G, and 36.9% T. Phylogenetic analysis was performed using the maximum likelihood method (including 13 PCGs). D. krusensternii is related to the Phyllidiidae (superfamily Phyllidioidea), suggesting a distinct phylogenetic placement within Nudibranchia. This study represents a genomic resource, contributing to molecular studies on the evolution of the Dendrodorididae.
... Minimum genetic distance based on uncorrected p-distance of COI was calculated using PAUP* (Swofford 2002) between the sampled specimens and the other 44 sequences from reported Osedax and non-Osedax species. These distances were calculated using the COI alignment employed in the phylogenetic analyses. ...
A new species of Osedax is described here using molecular and morphological data. It was found at the depth of 550 m off the Brazilian coast through experimental deployment of cow bones. Osedax nataliae sp. nov. is the second Osedax species from the Southwest Atlantic Ocean and had been previously reported as Osedax ‘BioSuOr-4’. Phylogenetic analysis of five concatenated genetic makers (28S rDNA, Histone H3, 18S rDNA, 16S rDNA, and cytochrome c oxidase I) placed Osedax nataliae sp. nov. within a well-supported Osedax Clade V, nested within a clade of Pacific Ocean Osedax though with poor support. The minimum interspecific COI distance between O. nataliae sp. nov. and another known Osedax was 13.92% (closest to O. ‘sagami-3’). The maximum intraspecific COI diversity (uncorrected) within O. nataliae sp. nov. sampled here was 2.44% and population structure was visualized via haplotype network analysis. Morphologically, O. nataliae sp. nov. is characterized by its reddish orange crown of palps and a ventral yellowish collar on the anterior trunk where it meets the base of the crown. Osedax nataliae sp. nov. shares features with other Clade V species, notably pinnules inserted on the outer margin of palps. Additionally, the presence of dwarf males within the tube lumen of females was documented. Further sampling and research in the Southern Hemisphere are needed to understand the diversity and biogeography of Osedax across the world’s oceans.
... The degree of saturation was investigated by plotting proportions of transitions and transversions against the pairwise divergence between the sequences using the Xia et al. (2003) test, implemented in the DAMBE 5.2.5 program (Xia and Xie, 2001). We examined the congruence of substitution rates between each of the data sets using the partition homogeneity test (Farris et al., 1995), as implemented in PAUP* (Swofford, 2003). The number of variable sites (S), haplotypes (h), haplotype (H d ), and nucleotide (π) diversity and mean number of pairwise differences (k) were calculated using DnaSP 6.0 (Rozas et al., 2017). ...
The brown howler, Alouatta guariba, endemic to the Atlantic Forest of Brazil and Argentina, is threatened by habitat loss and fragmentation, hunting, and its susceptibility to yellow fever. Two subspecies have been recognized, but their names, validity, and geographic ranges have been controversial. We obtained samples covering the species' entire distribution in Brazil and Argentina to clarify these issues by investigating their genetic diversity and structure and assessing their evolutionary history. We analyzed, for the first time, a set of ten microsatellite markers (N = 153), plus mitochondrial DNA (mtDNA) segments of the control region (N = 207) and cytochrome b gene (N = 116). The microsatellite data support two to three genetic clusters with biological significance. The southern populations (Argentina, Santa Catarina, and Rio Grande do Sul) presented a homogeneous genetic component, and populations from São Paulo (SP) to the north presented another component, although most presented~20% of the southern component. With K = 3, SP emerged as a third component while These authors have contributed equally to this work and share senior authorship ‡ These authors have contributed equally to this work and share last authorship sharing some ancestry with Rio de Janeiro and Argentina. The mtDNA phylogenies revealed three main clades that diverged almost simultaneously around 250 thousand years ago (kya). Clades A and B are from central SP to the north and east, while clade C is from SP to the south and southwest. Samples from SP presented haplotypes in all three clades, sometimes in the same population. The demographic history of the species estimated with the Bayesian skyline plot of the mtDNA showed a strong expansion~40-20 kya and a strong reduction over the last~4-2 kya. Although the genetic clusters identified here deserve appropriate management strategies as conservation units, the absence of (i) concordance between the mtDNA and microsatellite data, (ii) reciprocal monophyly in the mtDNA, and (iii) clear-cut non-genetic diagnostic characters advises against considering them as different taxonomic entities. None of the previous taxonomic proposals were corroborated by our data. Our results elucidate the taxonomy of the Atlantic Forest brown howler, indicating it should be considered a monotypic species, A. guariba. We also clarify the evolutionary history of the species regarding its intraspecific genetic diversity, which is crucial information for its conservation and population management.
... Eighteen gene sequences that consisted of 16 rice gene sequences [19] and two maize (Zea mays L.) gene sequences (outgroup) were aligned using Clustal W on UGENE software. The alignment results were saved as a Nexus file and then used to construct a phylogeny tree using the Neighbor-Joining method in PAUP4 software [20]. ...
Rice cv. Hawara Bunar is a local rice cultivar tolerant to aluminum (Al) and drought stress. However, the cultivar has inferior characteristics, such as a tall habitus and a small number of tillers, making the cultivar agronomically unattractive. Many genes control plant height and tiller number; one is the TAD1 gene. Analysis of gene expression in two contrasting rice cultivars for both characters is a prerequisite for selecting certain genes for gene editing targets. This study aimed to analyze the gene expression of the TAD1 in rice cv. IR64 and Hawara Bunar, and to construct a phylogenetic tree of genes that regulate rice plant height and tiller number. Gene expression analysis was conducted using the qRT-PCR technique, while the phylogenetic tree was constructed based on the Neighbor-Joining method using PAUP4 software. The results showed TAD1 gene expression in the tillering phase of rice cv. Hawara Bunar is higher than the cv. IR64. The gene expression level in both cultivars corresponds to the plant height and tiller number characters in both rice cultivars. Phylogenetic analysis showed that the TAD1 gene clustered with genes that cause the rice to have a tall habitus with few tillers. The results of a transcriptome meta-analysis reinforced the phylogenetic tree, which shows that the TAD1 gene was found in a group of downregulated genes based on the volcano plots. Therefore, the TAD1 gene can be selected as a target gene for editing in rice cv. Hawara Bunar to obtain superior characters.
... Following the SCoT analyses, the DNA bands were scored by giving the value "1" in the presence of DNA, "0" in the absence of DNA and "?" or "9" for the missing cases. The UPGMA phylogenetic tree was drawn using the program PAUP 4.0b10 (Swofford, 2001). Pairwise distance was created with the same program. ...
In this study, 'Ak Sakı' and 'Kara Sakı' apple cultivars were collected from different locations in Erzincan province, Türkiye, and genetic diversity was determined using the Start Codon Targeted (SCoT) marker technique. The SCoT marker technique was chosen because its gene targeting, long primer, and high annealing temperature make it more effective than other marker techniques. Using ten SCoT primers, 60 bands were obtained, and 42 of them were polymorphic. The polymorphism rate was determined to be 70%. The UPGMA (Unweighted Pair Group Method with Arithmetic mean) dendrogram created using the PAUP 4.0b10 program consists of two clades. The genetic distance between apple cultivars varies between 0.13462 and 0.45614. Principal Component Analysis (PCA) results were compatible with the UPGMA dendrogram. With the SCoT marker technique, genetic diversity among apple cultivars can be determined in a shorter time and with more reliable results.
... Alignments were spliced using Mesquite v.3.2. The best-fit model of nucleotide evolution for the datasets was selected, respectively, with Akaike's information criterion (AIC) using MrModeltest 2.3 (Swofford, 2002;Guindon and Gascuel, 2003;Darriba et al., 2012). Phylogenetic analyses were conducted according to the previous study (Hu et al., 2022). ...
Members of the genera Gymnopus and Mycena are vital for litter decomposition in tropical and humid temperate forests. In this study, the difference in morphological features among Gymnopus gansuensis , Gymnopus subsepiiconicus , and Mycena glabera was confirmed by DNA data. Gymnopus gansuensis and Gymnopus subsepiiconicus showed separate relationships with other species in the ITS and nLSU combined dataset utilized for the phylogeny of Gymnopus sect. Impudicae . In addition, Gymnopus gansuensis is characterized by pileus honey yellow at the center, margin pinkish buff to buff, stipe pinkish buff to fuscous, and basidiospores elliptic to briolette. Gymnopus subsepiiconicus is characterized by pileus clay buff to grayish brown at the center, margin pinkish buff to fawn, stipe dark brown to fuscous, and basidiospores elliptic. Based on the combined dataset of the ITS and TEF-1α, Mycena glabera has been detected as a separate lineage in the phylogenetic studies of Mycena sect. Calodontes . The ecological behaviors of the new species are described with illustrations.
... Four Markov chains were run for 10,000,000 generations, with a burn-in fraction set to 0.25. Maximum parsimony (MP) phylogenies were calculated with heuristic searches in PAUP v. 4.0a133 (Swofford 2002). Bootstrap analysis of 1000 replicates was confirmed the support of the branches and shown next to the branches. ...
... A molecular phylogenetic tree was constructed using a Bayesian approach. MrModeltest version 2.4 (Nylander 2018) implemented in PAUP* version 4.0a (Swofford 2003) was used to select the model parameters. Within MrModeltest, the Akaike Information Criterion was used for model selection (Posada and Buckley 2004). ...
... The following software was used for data processing and phylogenetic analysis: BioEdit (Hall 1999), ClustalX (Thompson et al. 1997) and MAFFT (http:// mafft.cbrc.jp/alignment/server/, Katoh et al. 2017) for sequences and manually adjusted, PhyloSuite v.1.2.2 (Zhang et al. 2020) for concatenated the separate alignments, PAUP* 4.0b10 (Swofford 2002) for maximum parsimony (MP) analysis, raxmlGUI 1.2 (Silvestro and Michalak 2012) for maximum likelihood (ML) analysis and MrBayes 3.2.6 (Ronquist and Huelsenbeck 2003) for Besian Inference (BI), TreeView 1.5.0 and FigTree version 1.4.4 (Rambaut 2018) to show the phylogenetic tree. The best topologies from ML analyses are shown in this study and the final alignments and the retrieved topologies has been deposited at TreeBASE (http://treebase.org/treebase-web/home.html), study ID: 31700. ...
Fuscoporia is a polypore genus of Hymenochaetaceae that causes wood decay, although some species in the genus have medicinal values. Phylogenetic analyses of concatenated ITS1-5.8S-ITS2-nLSU sequence data and morphological features identified three new species, F. eucalypticola, F. resupinata and F. subtropica from Australia, China and Malaysia, and these new species derived from the Fuscoporia ferrea group. These three species are illustrated and described. A key to resupinate species of Fuscoporia without mycelial setae in the world is provided.
... under the accession number 29850. Phylogenetic analyses were performed by the maximum parsimony method in PAUP* 4.0b10 (Swofford, 2002) and the Bayesian inference method in MrBayes v.3.0b4 (Huelsenbeck & Ronquist, 2001). ...
Sclerotinia borealis is among the most psychrophilic snow mold pathogen, mainly attacking Poaceae crops such as winter wheat and forage grasses as well as non-Poaceae plants under snow. Isolates were collected through the extensive surveys from North Atlantic islands through European and Asian Russia to Japan. Morphological characterization as well as the effects of temperature and water potential on mycelial growth defined three varieties, i.e., vars. borealis, subarctica, and okhotskana. Phylogenetic analyses based on ITS and EF1-α regions of genomic DNA indicated the monophyly of S. borealis and supported varietal differentiation.
... Population distances based on haplotypes were measured as È ST linearized following Slatkin (1995), calculated in ARLEQUIN version 3.1 (Excoffier et al. 1992(Excoffier et al. , 2006. These È ST distances were used to generate a neighbour-joining (NJ) tree of population structure in PAUP* (Swofford 2002). The analysis was repeated on 100 bootstrap replicates generated by resampling the original data, and a 50%-majority-rule consensus tree was constructed using PAUP*. ...
In southern Alberta and north-central Montana, there is substantial mtDNA sequence divergence between two groups of the little brown bat, Myotis lucifugus (LeConte, 1831), previously thought to be subspecies (Myotis lucifugus lucifugus and Myotis lucifugus carissima) but recently hypothesized to be species. We tested this hypothesis using population genetic techniques. Using nuclear microsatellite markers (10 loci), we found a lack of differentiation between these two groups of bats (ascribed based on mitochondrial hypervariable region II sequence), suggesting interbreeding was sympatric. Our findings add to the recent discovery that M.l.lucifugus haplotypes are found throughout the range previously thought to be only M.l.carissima, suggesting widespread sympatry and extensively mixed gene pools, thus refuting the cryptic species hypothesis. Clinal morphology and individual variation demonstrated the impossibility to differentiate groups based on original subspecies definitions. The lack of geographic and morphological boundaries, in addition to the likelihood that the interbreeding observed in this study is occurring across western North America, suggests that no line can be drawn between these two groups. We thus suggest that the carissima subspecies designation be dropped. This study highlights the importance of investigating nuclear gene flow in widely sympatric animals suspected of being cryptic genetic species, and has important implications for applications of the DNA Barcoding Project.
... The generated partial sequences were assembled using Bioedit (Hall 1999). Phylogenetic analyses were conducted in PAUP* version 4.0b10 (Swofford 2002). Analyses were done under different optimality criteria as outlined in Cai et al. (2006). ...
A new species, Thozetella pinicola, was isolated from leaf litter of Pinus elliottii Engelm. in Hong Kong. This taxon is described and compared with existing species in the genus. It occurs on the substrate as creamy white sporodochia and has short black conidiophores. Morphological characters are typical of Thozetella and it most closely resembles Thozetella falcata, Thozetella gigantea and Thozetella nivea, but may be distinguished by its distinct microawns and different conidial size. To gain further taxonomic insight into the phylogenetic relationships of our new taxon and its allies, we sequenced and analysed 6 different regions of 3 genes (ribosomal DNA and protein coding genes: RNA polymerase II largest subunit (RBP2) and -tubulin). Resulting phylogenies are compared with existing morphological information. Molecular data support the relationship between Thozetella species and the Chaetosphaeriaceae (Chaetosphaeriales, Sordariomycetes). In addition, we recovered a new phylogenetic lineage (or group) within the existing phylogenetic framework of Thozetella as previously proposed. In particular, there is a close association between T. pinicola and T. nivea, which is strongly supported. The affinities of these 2 newly sequenced taxa are discussed in light of morphological and molecular characters.
... -Parsimony bootstrap analysis in PAUP v.4.0 (Swofford, 2003) of individual gene region alignments (ITS, ETS, trnL-trnF) was used to assess gene tree incongruence. Characters that were not parsimonyinformative were excluded, and 2000 bootstrap replicates were performed, each using 5 random taxon-addition starting trees, tree bisection-reconnection (TBR) branch swapping, and with heuristic search of a maximum of 500 trees. ...
Phylogenetic relationships in the South African daisy genus Dimorphotheca have long been uncertain, with the taxonomy of the genus relying on a few morphological traits, most prominently capitulum sexual system (e.g., cypsela type) and ray colour, which may not be evolutionarily conserved. Here we present the first well-sampled molecular phylogeny of Dimorphotheca, based on nuclear ribosomal (ITS, ETS) and plastid trnL-trnF region DNA sequences from multiple accessions per species. Although the relationships suggested by these markers are broadly congruent, we do find some instances of incongruence which we resolve using a combined decomposition and deletion approach. Using our best estimate of phylogenetic relationships, we reconstruct the evolution of capitulum fertility and ray colour to assess the evolutionary conservatism of these traits and their taxonomic utility. We find support for the monophyly of Dimorphotheca, excluding the recently segregated Osteospermum polypterum, and our data thus support the modern, enlarged circumscription of the genus incorporating the former genus Castalis and Osteospermum sect. Acanthotheca and O. sect. Blaxium. Major subclades within Dimorphotheca are largely cohesive in terms of geographic distribution and morphological traits such as growth form and cypsela structure. While many species are resolved as monophyletic, the polyphyly of a few species suggests a need for taxonomic re-evaluation. On the basis of both morphological and molecular data, we describe one new species, and elevate one variety to species level. A full taxonomic key to the enlarged genus is presented for the first time. Ancestral reconstructions show that capitulum sexual system and ray lamina colour are not evolutionarily conserved and that neither can therefore be used to delimit major lineages within Dimorphotheca. While our findings resolve some taxonomic problems, they also highlight the need for further species-level taxonomic work on Dimorphotheca.
... All six loci (ITS, LSU, act1, rpb2, tef1, tub2) were concatenated to a combined matrix using Phyutility v. 2.2 (Smith and Dunn 2008). The combined data matrix for phylogenetic analyses contained 4257 characters (528 nucleotides of ITS, 816 nucleotides of LSU, 839 nucleotides of act1, 879 nucleo- (Swofford 2002). A total of 1,000 bootstrap replicates were implemented using five rounds of heuristic search with random sequence addition, followed by tree-bisection-reconnection (TBR) branch swapping. ...
Laurus nobilis is an important Mediterranean tree and shrub native to Italy that is also commercially grown as spice and ornamental plant. Field surveys conducted since 2021 in Sicily (Italy) revealed that bay laurel plants in urban and private gardens and nurseries were severely affected by symptoms of stem blight and internal necrosis, which were associated with ambrosia beetle entry holes in the bark and internal wood galleries. The occurring ambrosia beetle was identified as Xylosandrus compactus, an invasive wood-boring pest previously reported from Sicily. Investigation of fungi from symptomatic tissues primarily resulted in the isolation of Thyridium-like colonies. The main symbiont of X. compactus, Ambrosiella xylebori, was also isolated from infested plants. Phylogenetic analyses of a combined matrix of ITS, LSU, act1, rpb2, tef1, and tub2 gene regions revealed that the isolated Thyridium-like colonies represent a new fungal species within the genus Thyridium. Based on both phylogeny and morphology, the new isolated fungus is described as Thyridium laurisp. nov. Moreover, two recently described species, Phialemoniopsis hipposidericola and Phialemoniopsis xishuangbannaensis, are transferred to the genus Thyridium due to the confirmed synonymy of both genera, as supported by molecular phylogenies. Pathogenicity test conducted on potted plants demonstrated that T. lauri is pathogenic to bay laurel, causing internal necrosis and stem blight. The new species was consistently re-isolated from the symptomatic tissue beyond the inoculation point, thereby fulfilling Koch’s postulates. This study represents the first report of a new pathogenic fungus, T. lauri, causing stem blight and internal necrosis of bay laurel plants and associated with infestation of the invasive ambrosia beetle X. compactus.
... Coalescent phylogenetic reconstruction was conducted in SVDQUARTETS (Chifman and Kubatko 2014) as implemented in PAUP* 4.0 (Swofford 2019). We evaluated all possible quartets, and assessed branch support through 1000 replicates of standard bootstrap, resampling with replacement both loci and sites within loci (bootstrap = multilocus, loci = combined). ...
Large size, tropical climate, topographic complexity, and millions of years of isolation have turned the Greater Antillean islands into natural laboratories of evolution. Several groups have experienced explosive diversification across these islands, leading to highly diverse and endemic biotas and species experiencing unique adaptations. Here we describe a new tarantula species from the Viñales area, a biodiversity hotspot in western Cuba. Despite being a middle-sized species with large projected setae on the legs, reminiscent of those in arboreal tarantulas, phylogenomic analysis, diagnostic morphological features, and trap-door retreat building behaviour identifies it as the largest known representative of Trichopelma, and the only one with such hirsute legs. We provide mitochondrial barcoding data and a database of ultraconserved elements loci of the holotype of this species, which will help to include it in future phylogenetic studies of Theraphosidae. Given its limited distribution range, and its apparent absence in natural history collections despite its distinct appearance, this species might be scarce, positioning it as a potential conservation concern. http://www.zoobank.org/urn:lsid:zoobank.org:pub:2F9ED7A1-1345-4295-A876-6B617ABF7C75 ARTICLE HISTORY
... The phylogenetic analysis was conducted with PHYML v. 3.0 (Guindon et al., 2010), MRBAYES v. 3.1.2 (Ronquist & Huelsenbeck, 2003), and PAUP* 4.0b10 (Swofford, 2003) and trees were deposited into TreeBASEhttp:// purl. org/ phylo/ treeb ase/ phylo ws/ study/ TB2: S31487? ...
Fusarium diseases in forests, especially in naturally regenerated seedlings, are poorly recognized. Recently the rapid and severe decline of natural regeneration of European beech caused by Phytophthora spp. has been observed in Poland. The diseased beech seedlings were also co-infected by Fusarium species. The aim of this study was to isolate and identify the role of Fusarium species that may play a role in the decline of beech seedlings in Poland. A total of 487 Fusarium isolates were obtained from 450 symptomatic and asymptomatic beech seedlings: 466 from symptomatic plants and 21 from asymptomatic plants. Based on comparisons for two genes and phylogenetic analysis, all Fusarium strains were separated into 14 phylogenetically distinct species belonging to four Fusarium species complexes (SC), namely the F. incarnatum-equiseti species complex (FIESC), the F. nisikadoi species complex (FNSC), the F. sambucinum species complex (FSSC), and the F. tricinctum species complex (FTSC). Fusarium avenaceum and F. paeoniae were the most frequently isolated species from both above- and below-ground organs of seedlings affected by Phytophthora . In the pathogenicity trials, the most virulent species for beech seedlings were F. avenaceum and F. sporotrichioides . However, Fusarium isolates were significantly less aggressive compared to the P. × cambivora (positive control). Our results demonstrate that beech seedlings were frequently colonized by various Fusarium species. Fusarium species do not play an important role in the reduction of naturally regenerated European beech seedlings due to their low aggressiveness.
... Analysis of phylogeny based on Bayesian inference (BI) was performed using Mr-Bayes 3.2.7 (Huelsenbeck and Ronquist, 2001;Ronquist et al., 2012). Test of best nucleotide substitution model was performed using PAUP (Swofford, 2001) implemented in MrModeltest 2.3 (Nylander, 2004). For both 28S rRNA and cox1 genes, BI was run with two random starting trees and four Markov chains, three heated and one "cold" for 2 x 10 6 generations under the general time reversible model (GTR) with gamma distribution and invariable sites (G + I). ...
Salsola is one of the largest genera in the tribe Salsoleae within Amaranthaceae, with many species that share morphological characteristics, which makes it a challenging genus to study. In this study, 11 quantitative and eight qualitative morphological traits were evaluated and measured. Ward’s dendrogram showed two main clusters: one composed of S. brachiata and S. turcomanica , and the other composed of two subclusters, with populations of S. kali, S. incanescens, S. orientalis and S. dendroides . In addition, we performed molecular phylogenetic analyses conducted with sequence data from nrDNA (ITS and ETS) and cpDNA markers ( psb B- psb H and atp B- rbc L) under maximum parsimony, Bayesian inference and maximum likelihood approaches. All the members were clustered into a well-supported clade (PP = 1, ML/BS = 0.89) composed of two subclades: subclade A included S. turcomanica and S. brachiata (PP = 0.90, ML/BS = 73) and subclade B (PP = 0.95, ML/BS = 81) comprised S. incanescens, S. orientalis, S. dendroides and S. kali . The current study provides novel insights at morphological and molecular levels, as well as the results of molecular studies based on nr- and cpDNA sequence data that are congruent with morphological analyses.
The Canary Islands are an excellent natural laboratory for understanding ecological and evolutionary processes such as biogeographical colonisation. The morphology of the larva, puparium and adult of the endemic Canarian copper fly, Calliphora splendens , is described, illustrated and contrasted with those of the other species of Calliphora that occur in Africa, the Iberian Peninsula and Macaronesia. Partial cytochrome oxidase I sequences show a connection between C. splendens , Calliphora vicina , Calliphora loewi and Calliphora croceipalpis , but more distant relationship with Calliphora vomitoria . Calliphora splendens produced unisexual offspring in captivity. This work confirms the relict character of the Canarian copper fly associated with the endemic laurel forest habitat.
Coprophilous species of Coprinopsis sect. Niveae, commonly known as “snow inkcap”, are widespread in pastoral areas; however, wide sampling approaches are needed to discover new taxa and to clarify the taxonomic status of the so-called “snow inkcap”. Nationwide field work was conducted in China with a detailed record collected of the distribution and the animal origin of the dung. A four-loci phylogenetic study of Coprinopsis sect. Niveae was conducted based on the internal transcribed spacer regions (ITS), the ribosomal large subunit (LSU), and translation elongation factor 1-α (tef1-α)], and the mitochondria small ribosomal RNA subunit (mtSSU). Fourteen phylogenetic species were assigned to this section, including six novel species, namely Coprinopsis furfuracea, C. iliensis, C. khorqinensis, C. sericivia, C. subigarashii, and C. tenuipes. Macro-, micro-, and ultramicro-morphological observations of species collected from China were also conducted and the detailed descriptions and illustrations of the novel species are provided. Our studies revealed that the different origin of herbivore dung, the distribution, the color and thickness of the pileus, the shape of stipes, the shape and size of basidiospores, and the presence or absence of pleurocystidia can be used as characteristics for distinguishing species in sect. Niveae. The key to species belonging to this section is also provided.
Thaumetopoea is a genus of Lepidoptera referred to as the processionary moths species, that comprises famous defoliators of forest trees. They are also a threat to human and animal health due to the urticating setae carried by their larvae. Their phylogenetic relationships were studied so far using morphological data, mitochondrial DNA and only a few nuclear markers. Results remained controversial so far, notably for the winter Conifer‐feeding species complex encompassing T. pityocampa and T. wilkinsoni . In particular, since the first identification of divergent mitochondrial clades, several new species were described and incongruent phylogenies were proposed in this group. We here explored the potential of nuclear RAD‐seq data to resolve these inconsistencies and compare the resultant phylogeny with the information provided by the mitochondrial COI gene. It allows to propose a robust phylogeny and decipher cases of mitochondrial introgressions and nuclear‐mitochondrial discordance, which raises doubts about the validity of some recently described species. Hence, we recommend caution before describing new taxa in this group from mitochondrial data alone. Species delineation between geographically bounded and genetically differentiated lineages cannot be addressed pertinently without dedicated sampling and associated population genomic and ecological studies.
Context Lates calcarifer is a widespread Indo-Pacific fish that is important in aquaculture, recreational and commercial fisheries. Genetic divergences from different data sources and sampling schemes have been reported. Aims To conduct phylogenetic and population genetic analyses from a geographically and phylogenetically representative data set to identify hierarchical divisions within L. calcarifer. We further test the evolutionary significance of genetic units in terms of signatures of adaptation. Methods Using a whole-genome sequence data set of 61 fish, including an outgroup, we conducted phylogenetic and population genetic analyses. We also generated measures of fixation index (Fst), nucleotide diversity (π) and Tajima’s D (D). Key results We identified three main lineages of L. calcarifer, corresponding to the Indian subcontinent, South-east Asia and Australasia. Subdivision within each of the three main lineages was also identified and characterised. Adaptively significant differences are indicated within and among the three main lineages. Conclusions L. calcarifer exhibits genetic divergences at different levels that originate before and during the Pleistocene. These divergences are associated with adaptive divergence but unclear phenotypic changes. Implications This study has highlighted the need for comprehensive sampling and integrative study of genotypes and phenotypes across the range of L. calcarifer.
Recently, confirmed data on the identification of entomopathogenic fungi belonging to the genera Penicillium, Aspergillus, Fusarium, Beauveria, Cordyceps, Fusarium, Metarhizium, Purpureocillium and Beauveria have been gradually increasing. This study was conducted to evaluate the effect of Penicillium mallochii conidia and ethanol extract to control Cadra cautella. The study was carried out in Turkey, at Balıkesir University (BAUN), Faculty of Science and Literature, Department of Biology, under controlled laboratory conditions of 25 ± 1 °C temperature, 65 ± 5% relative humidity and a photoperiod of 12:12 h L/D. Topical application assays of ethanol extract gave an LD50 of 3.107 mg/mL (95% confidence limits, 1.339–15.421 mg/mL), and conidial suspensions gave an LD50 of 108.448 conidia/mL (95% confidence limits, (107.638− 1010.358 conidia/mL). The collected data in the current study compared to the control and a drastic reduction in the total number of eggs were observed more than 70% in all doses by P. mallochii conidial suspensions and over 80% in all doses by ethanol extract. As a result of our study, we found that the strong mortality rates of C. cautella larvae and significant reductions in the number of eggs depended on both the concentration of conidial suspensions and the concentration of the ethanol extract. Also, the cytotoxic effect of crude extracts was also tested on mouse cell lines found to be cytotoxic at higher concentrations than 0.708 mg/mL. Two important findings of the study are presented here for the first time; one of them is that P. mallochii is an entomopathogenic species that does not produce significant mycotoxins such as Aflatoxin B1, B2, G1 and G2, or Ochratoxin A. The second is that P. mallochii is effective on C. cautella larvae. Considering our findings P. mallochii can be recommended as a promising candidate entomopathogen for inclusion in pest control and use of ethanol extracts and conidial suspensions would demonstrate notable level of efficacy in biological control programs. Future research should explore the impacts of P. mallochii on other pest insects and focus on field trials in diverse environments to validate these laboratory findings.
Branch dieback and general decline have been observed in pistachio orchards with trees of different ages in Iran. The aim of the study was to identify and characterize putative pathogens causing the symptoms. Isolations from rotted roots yielded predominantly Fusarium species. The causal agent was identified as Fusarium verticillioides based on morphological characteristics, sequencing of TEF1-α, and resultant symptoms and reisolation of the fungus in a pathogenicity test using 5-month-old pistachio seedlings (cv. Badami-Zarand). To the best of our knowledge, this is the first report of Fusarium verticillioides causing Fusarium wilt on Pistacia vera.
Integrative data from plastid and nuclear loci are increasingly utilized to resolve species boundaries and phylogenetic relationships within major angiosperm clades. Debregeasia (Urticaceae), an economically important genus, presents challenges in species delimitation due to its overlapping morphological traits and unstable taxonomic assignments. Here, we analyzed 14 morphological traits and generated 12 data matrices from the plastomes and nrDNA using genome skimming from the nine recognized morphospecies to clarify species boundaries and assess barcode performance in Debregeasia. We also used a universal set of 353 nuclear genes to explore reticulate evolution and biogeographic history of Debregeasia. Plastomes of Debregeasia exhibited the typical quadripartite structure with conserved gene content and marginal independent variations in the SC/IR boundary at inter- and intra-specific levels. Three Debregeasia species were non-monophyletic and could not be discerned by any barcode; however, ultra-barcodes identified the remaining six (67%), outperforming standard barcodes (56%). Our phylogenetic analyses placed Debregeasia wallichiana outside the genus and suggested six monophyletic clades in Debregeasia, although the placement between Debregeasia hekouensis and Debregeasia libera varied. There was extensive trait overlap in key morphologically diagnostic characters, with reticulation analysis showing potentially pervasive hybridization, likely influenced by speciation patterns and overlaps between species ranges. We inferred that Debregeasia crown diversification began at ca. 12.82 Ma (95% HPD: 11.54−14.63 Ma) in the mid-Miocene within Australia, followed by vicariance and later long-distance dispersal, mainly out of southern China. Our findings highlight the utility of genomic data with integrative lines of evidence to refine species delimitation and explore evolutionary relationships in complex plant lineages.
Living reptiles include more than 20,000 species with disparate ecologies. Direct anatomical evidence from Neodiapsida, which includes the reptile crown-group Sauria and its closest extinct relatives, shows that this diversity originates from a single common reptilian ancestor that lived some 255 million years ago in the Paleozoic. However, the evolutionary assembly of crown reptile traits is poorly understood due to the lack of anatomically close outgroups to Neodiapsida. We present a substantially revised phylogenetic hypothesis, informed by new anatomical data derived from high-resolution synchrotron tomography of Paleozoic reptiles. We find strong evidence placing the clade Millerettidae as a close sister to Neodiapsida, which uniquely share a suite of derived features among Paleozoic stem reptiles. This grouping, for which we name the new clade Parapleurota, replaces previous phylogenetic paradigms by rendering the group Parareptilia as a polyphyletic assemblage of stem reptiles, of which millerettids are the most crownward. Our analysis presents hypotheses that resolve long-standing issues in Paleozoic reptile evolution, including the placement of captorhinids on the amniote stem lineage and firm support for varanopids as synapsids, which taken together provide a greatly improved fit to the observed stratigraphic record. Optimizations of character evolution on our phylogenetic hypothesis reveals gradual assembly of crown reptile anatomy, including a Permian origin of tympanic hearing, the presence of a lower temporal fenestra in the amniote common ancestor, with subsequent modifications on the reptile stem lineage, leading to the loss of the lower temporal bar. This evolutionary framework provides a platform for investigating the subsequent radiations of the reptile crown group in the Early Triassic, including the lines leading to dinosaurs (including birds), crocodilians, lepidosaurs, and extinct marine reptiles.
Florida, USA is a hotspot of biological invasions with over 500 non-native species reported. Reptiles encompass most of the non-native wildlife with over 50 species established, many of which are sympatric and are identified as invasive due to their impacts to the environment, economy, and human health and safety. Reports of new non-native reptiles occur, and many established non-native reptiles continue to expand their ranges in Florida, increasing the need for multitaxa detection and monitoring capabilities. Invasive constrictor snakes are a primary focus of management efforts due to life history traits that favor successful establishment and dispersal in Florida as well as their impacts to native wildlife and Everglades restoration efforts. While traditional survey methods that rely on visual detections fail to reliably detect invasive constrictors, environmental DNA (eDNA) has proven to be a promising method for detection of cryptic and rare species across the landscape. To address emerging needs for multispecies detection and monitoring in Florida, we developed the first tetraplex dPCR assay designed for detection of four species of invasive constrictor snakes, including Burmese pythons (Python bivittatus), northern African pythons (P. sebae), boa constrictors (Boa constrictor), and rainbow boas (Epicrates cenchria). In this tetraplex assay, no cross-amplification across species was documented. This assay serves as a valuable tool for faster and more accurate monitoring efforts of these invasive species in South Florida. Additionally, eDNA samples comprised of soil and water both tested positive for Burmese python DNA under controlled and semicontrolled conditions with DNA being detectable up to 2-weeks post removal in soil samples. Water samples yielded positive detection as quickly as 5 min after exposure to the organism. These data highlight the utility and sensitivity of this protocol for eDNA monitoring.
Background: Genomic analysis is crucial for better understanding the evolutionary history of species and for their conservation. Shizhenia pinguicula is a rare medicinal plant endemic to China. However, the complete chloroplast genome of this species has not been reported to date. Insufficient genomic research on S. pinguicula has hindered effective conservation efforts for this valuable plant. Methods: In this study, we sequenced and assembled the first complete chloroplast genome of S. pinguicula using Illumina sequencing technology. We conducted a comparative analysis of its chloroplast genome with related species and reconstructed phylogenetic relationships. Results: The chloroplast genome of S. pinguicula exhibited a typical quadripartite structure with a length of 158,658 bp. A total of 123 genes, 118 simple sequence repeats, and 51 dispersed repetitive sequences were identified. The inverted repeat boundaries were significantly expanded, along with the pseudogenization and loss of multiple NDH genes. Codon usage bias is primarily influenced by natural selection and other factors, with the ycf3 gene under positive selection. Additionally, 10 hypervariable regions were detected for species identification and evolutionary studies. Phylogenetic analysis indicated that Ponerorchis gracilis and Hemipilia yajiangensis form a clade, with S. pinguicula as their sister species, located at the basal position of the Ponerorchis-Hemipilia alliance. Conclusions: The chloroplast genome structure and gene content of S. pinguicula exhibit certain degrees of variation compared to other species within the Orchidinae subtribe. This genome should be useful for further investigations into the biology of Shizhenia and the development of biodiversity conservation strategies.
Absidia is distributed worldwide and primarily isolated from soil, feces, and decaying plants. The genus was initially classified into Absidiaceae and then Mucoraceae, and currently belongs to Cunninghamellaceae and is further divided into Absidia s.s., Lichtheimia, and Lentamyces. Three new species of Absidia s.s. are identified and described from soil in Hainan Province of China based on morphological characteristics, molecular data, and maximum growth temperatures as well. They are named based on distinct shapes of projections on columellae: A. crystalloidessp. nov. (crystal-like), A. pacificasp. nov. (pacifier-like), A. pateriformissp. nov. (bowling-like). In SSU-ITS-LSU-TEF-Act phylogram, the A. crystalloides is closely related to A. oblongispora and A. heterospora, the A. pacifica is a sister group with A. edaphica, and the A. pateriformis has a close relationship with A. jiangxiensis. This study enriches the species diversity of Absidia in China.
During an ongoing investigation of bambusicolous ascomycetous fungi in southwestern China, a diverse range of asexual and sexual morphs of saprobic fungi were collected from freshwater and terrestrial habitats. Based on detailed morphological comparisons and multi-gene phylogenetic studies, we introduce one new family, Neodictyosporiaceae, along with seven new genera, viz. Sporidesmium chiangmaiense. Detailed, illustrated descriptions and notes for each taxon are provided, along with a brief note for each genus and family.
Colletotrichum species are the most common cause of maize anthracnose, which often manifests as leaf spots. However, field observations often reveal symptoms similar to those caused by other leaf spot pathogens, such as Curvularia and Bipolaris. In this study, 99 isolates were identified using tissue separation and single-spore isolation techniques. As preliminary measures of species diversity, all 99 isolates were identified morphologically, and the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene sequences were phylogenetically analyzed. Furthermore, 48 representative strains were selected for molecular identification using multi-locus phylogenetic analyses based on five gene loci (ITS, TUB, ACT, GAPDH, and CAL). Finally, 10 species of Colletotrichum isolated from maize leaf spots were identified. Colletotrichum cliviicola was the most dominant species (24.2%), followed by C. fructicola (18.2%), C. karstii (16.1%), C. siamense (13.1%), C. boninense (7.1%), C. kahawae (7.1%), C. brevisporum (6.1%), C. truncatum (5.1%), C. gigasporum (2.0%), and C. gloeosporioides (1.0%). For the first time, pathogenicity tests revealed that C. cliviicola, C. fructicola, C. siamense, C. karstii, and C. truncatum are the causative agents of maize anthracnose. Additionally, C. boninense was identified as an endophytic fungus on healthy maize. In conclusion, this study is the first to identify the pathogen of maize anthracnose in Sichuan Province. It provides valuable insights for accurately diagnosing and managing maize anthracnose.
Eriophyoid mites (Acari: Eriophyoidea) are among the smallest terrestrial arthropods possessing simplified morphological characters, which challenges the traditional morphology-based species delimitation. The Diptilomiopus spp. exhibit further morphological degeneration (e.g., lack of genu on both legs and some opisthosoma setae). Especially, they possess high similarity in the reticulate prodorsal shield patterns, leading to cryptic species complex and hampering our understanding of species diversity. In this study, we implemented an integrated approach, including genetic distance analysis, phylogenetic analysis, population genetic structure construction, introgression test, and morphometric analysis, to delimitate the Diptilomiopus ligustri complex. We obtained 52 mitochondrial COI gene sequences and 19 whole-genome sequences across 18 sample sites of this complex. All samples were collected from Ligustrum plants in China, which may cover their main distribution ranges. Our results demonstrate that the D. ligustri complex consists of at least four distinct species (i.e., D. ligustri, D. cf. ligustri sp1., D. cf. ligustri sp2., and D. cf. ligustri sp3.), which are genetically divergent but do not differ morphologically, assessed by principal component analysis of 27 morphological characteristics. No significant gene flow was observed among this complex. Divergence time estimates further showed that most Diptilomiopus species diverged in a short time interval at the Paleogene–Neogene boundary, indicating a rapid radiation. Our results highlight the integrated approaches in eriophyoid mite species complex delimitation.
Genome size is an adaptive trait, and its variations influence the organismal phenotype and fitness. In this study, we propose a hypothesis linking variations in genome size within Scolopendra to ecological factors. To test this hypothesis, we employed flow cytometry to estimate genome size in seven Scolopendra species from Chinese mainland. Subsequently, we reconstructed the phylogenetic relationship of these species using the cytochrome c oxidase subunit 1 gene and conducted phylogenetic comparative analysis to assess the relationships between genome size and niche breadth or 19 bioclimatic variables. Our findings indicate the following: (1) genome size in Scolopendra can be categorized into three groups, similar to the phylogenetic clades; (2) there is a negative correlation between genome size in Scolopendra species and the precipitation niche breadth of species; (3) the estimated divergence time of Scolopendra dates back 153 Mya, during the Jurassic period. We assume that consistent aridity geological periods may promote the evolution of Scolopendra species with a large genome size, whereas rapidly fluctuating humidity geological periods may have the opposite effect.
Worldwide, humans have strongly altered river networks. Key changes resulted in modified hydromorphology, poor habitat quality and availability, migration barriers, and pollution. Restoration measures aim at mitigating anthropogenic stressors and at restoring connectivity, but the biological success of these measures is not guaranteed. Analyzing genetic diversity and metapopulation structure of target species in the river network with genetic markers can help to understand recolonization processes and to identify persisting gene flow barriers. Here, we studied the population genetic structure of the two pollution‐tolerant detritivorous isopod species, Asellus aquaticus and Proasellus coxalis, in the former heavily degraded and polluted, but now mostly restored Emscher catchment in Germany. For both species, we analyzed mitochondrial cytochrome c oxidase I (COI) gene sequences and nuclear genome‐wide single nucleotide polymorphism (SNP) data. Surprisingly, we found a strong metapopulation structure for both species with several isolated populations on a small‐scale of few kilometers, but a still high genetic diversity, especially in the COI gene. For both taxa, potentially cryptic species are known, but our SNP data showed that the mitochondrial lineages represent only one species, each, in the study area. This highlights the importance of integrating high‐resolution nuclear markers into species identification because species diversity may otherwise be overestimated. While we could identify some migration barriers and find indications for passive dispersal by birds or humans, these factors could not fully explain the local metapopulation structure, suggesting that also other drivers, such as isolation by adaptation, priority effects, or biotic interactions, play a role in shaping the population genetic structure.
Using the example of multiple species groups and species complexes, it is empirically shown that the resolving power of a set of 10–20 nuclear loci is insufficient for the study of their genetic differentiation. For the adequate analysis of phylogenetic relationships (including reticulation events) and assessment of divergence levels within such groups of closely related species, it is necessary to multiply the number of nuclear loci and transit to high-throughput next-generation deep sequencing. However, the question of the genomic coverage required for the purposes of such study remains open. In other words, exactly how many genes in a set are needed to measure the genetic distance resolving the relationships between branches on the phylogenetic tree reconstruction of a complex of closely related species? We chose protein-coding sequences as a standard set of markers. Distances for 160–180 genes with a combined length of 270 000–300 000 bp were used to estimate the divergence levels of closely related mammalian species. In most cases, the interspecific distances are within the 0.15–0.75% range (median of 0.33%). The range of distances between semi-species is 0.12–0.28% (median of 0.14%). Intraspecific distances are always lower than 0.11%. Rodents show higher values of interspecific distances, 0.25–2.3% (median of 0.72%); distances between half-species range from 0.1 to 0.35%. To determine the number of nuclear loci and their combined length sufficient for the calculation of a genetic distance, we use simulations based on a model that included the following parameters: the average rate of gene evolution, its dispersion, and the level of polymorphism in the modern and ancestral populations. We performed a preliminary analysis of the distribution of loci evolution rates among mammalian taxa based on the data on ~50 thousand nuclear markers. It is shown that a relative error of 10–15%, comparable to the same value for mitochondrial distances between close species based on individual genes (approximately 1000 bp in length), is achieved using approximately 100 loci of 300 bp in length. Based on these data, we propose the following working hypothesis: the threshold of interspecific/intraspecific genetic distance calculated on the basis of the exons most frequently used in mammalian molecular phylogenetics is approximately 0.15%. This hypothesis assumes a species rank for the forms in statu nascendi. It should be noted that not all “good” species have significant genetic distances, since in some cases reproductive barriers form faster than substitutions accumulate in the genome.
Fusarium wilt of tobacco (FWT), caused by Fusarium spp., has emerged as a severe threat to tobacco production in China. In all, 132 isolates of Fusarium were isolated from tobacco and pathogenic to tobacco-causing FWT in Jilin Province, China. In this study, we identified 7 of 132 isolates as a novel species Fusarium tobaccum sp. nov. Zhao Xie & Jie Gao, using multi-gene phylogenetic analyses of translation elongation factor (tef1), β-tubulin (tub2), calmodulin (cmdA), and RNA polymerase II second largest subunit (rpb2) genes, along with subtle morphological differences. Isolates of F. tobaccum sp. nov. were clustered in a distinct branch in the maximum parsimony phylogenetic tree generated from the sequences of tef1-rpb2-tub2-cmdA and can be distinguished from closely related species F. cugenangense, F. callistephi, and F. elaeidis. The morphological characteristics of F. tobaccum sp. nov. are distinct from other Fusarium species. F. tobaccum sp. nov. exhibits abundant aerial mycelia and pigment production on potato dextrose agar (PDA), microconidia with 0–1 septa, and macroconidia with 2–5 septa on carnation leaf-piece agar (CLA), and produces abundant chlamydospores on Spezieller Nährstoffarmer agar (SNA) and CLA. The mycelia of F. tobaccum exhibited optimal growth at a pH of 7.1 and a temperature of 23.6°C. Sucrose and NaNO3 significantly promoted the mycelial growth of F. tobaccum. PD medium was optimal for total sporulation. However, the sporulation ratios of the macrospores of F. tobaccum in PD, SN, and CMC were relatively low (0.48%, 2.51%, and 2.16%, respectively). These findings provided valuable insights into the morphological and biological characteristics of F. tobaccum.
IMPORTANCE
Fusarium wilt of tobacco (FWT) is a prevalent issue in tobacco-growing regions globally, leading to significant losses in yield and quality. This study identified F. tobaccum sp. nov., a novel species of Fusarium causing FWT in China. The identification was based on multi-gene phylogenetic analyses and morphological characteristics. The effects of temperature, pH, carbon source, nitrogen source, medium, and light on the mycelial growth of F. tobaccum sp. nov. were determined. These findings might contribute to future research on the pathogenic mechanisms of this novel species and the development of strategies to control FWT.
Macrobenthic communities in a lake are affected by the type of bottom cover such as macrophytes or algae. In the southern basin of Lake Biwa, mats of the benthic cyanobacteria (BC) Microseira wollei widely cover the lake bottom and are interspersed with submerged macrophytes (SMs). Because different macrobenthos species appear to occur at those bottoms, we investigated the composition of the communities. Furthermore, as M. wollei is supposed to be inedible to consumer organisms owing to its hard sheath and toxins, the food possibility of the cyanobacteria and macrophytes for macrobenthos was analyzed. In this study, macrobenthos were collected, identified molecularly, counted in the BC and SM zones, and analyzed for carbon and nitrogen stable‐isotopic compositions. In the BC zone, asellids and amphipods dominated the macrobenthic community, while chironomids dominated the SM zone. The stable‐isotope analysis revealed that M. wollei was a minor food source for macrobenthos and other higher‐level consumers, with some macrophytes, particulate organic matter and bottom sediment potentially being a major source. The dominance of crustacean macrobenthos in M. wollei mats suggested that they provided a refuge from predation for crustaceans, whereas SMs were not sufficiently abundant to achieve this. Although different macrobenthic communities in the BC and SM zones were likely supported by common food sources, with the exeption of M. wollei, the present study was conducted over a short period and lacked advanced methods for gut content analysis. Therefore, further monitoring and food web analysis are required.
While investigating macrofungi diversity in Gansu province, northwestern China, five fresh and fleshy specimens were collected, which are characterized by nearly white to buff hemispherical pileus with waved margins, a disc depressed with coral to brownish red fibrillose scales, adnate to sub-decurrent lamellae with four relatively regular rows of lamellulae, a stipe that is central, hollow, frequently straight to curved; basidiospores that are globose to subglobose, 5.0-6.0 × (3.5−) 4.0-5.0 (−5.5) µm, narrowly clavate cheilocystidia predominantly, pleurocystidia and caulocystidia not observed; and a cutis pileipellis, with hyphae slightly inflated in the KOH. The results of phylogeny analysis indicated that the species forms an independent lineage in Phyllotopsidaceae based on the ITS (ITS5/ITS4) and nLSU (LR0R/LR7) dataset. Molecular clock analyses suggested the common ancestor of Neotricholomopsis emerged later than upper Cretaceous with a mean crown age of 229.36 Mya (95% highest posterior density of 129.63-343.08 Mya). These five specimens were described as an unreported taxon based on the phylogeny analysis combined with morphological examination and ecological and geographical distribution. Detailed descriptions, illustrations, and phylogenetic trees to demonstrate the placement of this species and discussions with its related species are provided.
Cover crops, typically planted during off-seasons and requiring less agronomic manipulation, may provide abundant fungal resources. Certain species of Chaetomiaceae could serve as potential agents for controlling plant diseases and developing bioorganic fertilizers. Eight species from five genera of Chaetomiaceae were identified from healthy Astragalus sinicus and Vicia villosa, two major cover crops, through multigene phylogenetic analysis, morphological identification, and pairwise homoplasy index testing. The identified species comprise three new species: Achaetomium astragali, Subramaniula henanensis, and S. sichuanensis, as well as five known but new host record species: Botryotrichum murorum, Chaetomium coarctatum, C. pseudocochliodes, C. pseudoglobosum, and Collariella pachypodioides. Dual culture tests revealed that isolates of all eight Chaetomiaceae species exhibited antagonistic effects on multiple phytopathogens. Among the identified fungi, the NSJA2 isolate, belonging to C. coarctatum, exhibited significant relative inhibition effects on 14 out of 15 phytopathogens tested in this study, indicating its broad-spectrum antagonistic effects. Additionally, NSJA2 exhibited excellent salt tolerance. Overall, our study has identified multiple fungi with anti-phytopathogens potential, among which NSJA2 exhibits high potential for practical application. This finding paves the way for further exploration and exploitation of NSJA2 as a promising biocontrol agent.
With populations of land snails of very small size like Vertiginidae, questions have arisen as to whether populations of relatively distant islands in archipelagos are really isolated from each other. Apart from other flight agencies, airborne transport of loose specimens is not improbable in stormy weather conditions. Currently, mechanisms of wind-borne transport of sand particles over short and long distances have been intensively studied. The results are available in the literature on sediments, allowing the calculation of probable flight distances for particles in suspension.
For living snails of the Aegean species Truncatel-lina rothi, an average fall velocity of 2.6–2.7 m s⁻¹ has been determined in experiments under laboratory conditions. Applying these results, Truncatellina living on an island at 100 m altitude and close to the coast could be transported up to several kilometers in heavy storms, which are not uncommon in the Aegean archipelago (Greece). This would imply that many of the Aegean islands are not effectively isolated for minute snail species, and that genetic interchange between island populations is probably frequent.
A new statistical method for estimating divergence dates of species from DNA sequence data by a molecular clock approach is developed. This method takes into account effectively the information contained in a set of DNA sequence data. The molecular clock of mitochondrial DNA (mtDNA) was calibrated by setting the date of divergence between primates and ungulates at the Cretaceous-Tertiary boundary (65 million years ago), when the extinction of dinosaurs occurred. A generalized leastsquares method was applied in fitting a model to mtDNA sequence data, and the clock gave dates of 92.311.7, 13.31.5, 10.91.2, 3.70.6, and 2.70.6 million years ago (where the second of each pair of numbers is the standard deviation) for the separation of mouse, gibbon, orangutan, gorilla, and chimpanzee, respectively, from the line leading to humans. Although there is some uncertainty in the clock, this dating may pose a problem for the widely believed hypothesis that the bipedal creatureAustralopithecus afarensis, which lived some 3.7 million years ago at Laetoli in Tanzania and at Hadar in Ethiopia, was ancestral to man and evolved after the human-ape splitting. Another likelier possibility is that mtDNA was transferred through hybridization between a proto-human and a protochimpanzee after the former had developed bipedalism.
Viewed from a geological perspective, present-day animal and plant communities in many parts of the world have a remarkably short history. The environmental revolution at the end of the Pleistocene, a mere 10 000 years ago, triggered major shifts in the ranges of species and hence composition of communities. Present-day communities in the boreal and temperate zones assembled at this time by combining species that survived the northern environment of the Last Cold Stage with those returning from more temperate refugia. Increasing evidence suggests that the well-studied European southern and eastern refugia for thermophilous animal and plant taxa were supplemented by cryptic refugia in northern Europe during the Late Pleistocene. These northern refugia would have been in areas of sheltered topography that provided suitable stable microclimates, and could partially explain the ‘nonanalogue’ mammalian assemblages of the Late Pleistocene. They also have implications for phylogeography and speciation.
Indirect methods using genetic markers are the primary measure of gene flow levels among interbreeding populations. Results from studies employing these methods are often ambiguous and open to multiple interpretation. This is primarily due to low resolution of molecular markers and low precision of model-based estimates. Studies of paternity, kinship and phylogeography generate the most reliable results. Future studies should employ more powerful analytical methods, analyse loci independently, and attempt to distinguish confounding contributions of vicariance to isolation-by-distance studies. Moreover, direct assessment of movement remains the most valid approach to many key issues in population biology.
Although several decades of study have revealed the ubiquity of variation of evolutionary rates among sites, reliable methods for studying rate variation were not developed until very recently. Early methods fit theoretical distributions to the numbers of changes at sites inferred by parsimony and substantially underestimate the rate variation. Recent analyses show that failure to account for rate variation can have drastic effects, leading to biased dating of speciation events, biased estimation of the transition:transversion rate ratio, and incorrect reconstruction of phylogenies.
We previously developed a cladistic approach to identify subsets of haplotypes defined by restriction endonuclease mapping or DNA sequencing that are associated with significant phenotypic deviations. Our approach was limited to segments of DNA in which little recombination occurs. In such cases, a cladogram can be constructed from the restriction site or sequence data that represents the evolutionary steps that interrelate the observed haplotypes. The cladogram is used to define a nested statistical design to identify mutational steps associated with significant phenotypic deviations. The central assumption behind this strategy is that any undetected mutation causing a phenotypic effect is embedded within the same evolutionary history that is represented by the cladogram. The power of this approach depends upon the confidence one has in the particular cladogram used to draw inferences. In this paper, we present a strategy for estimating the set of cladograms that are consistent with a particular sample of either restriction site or nucleotide sequence data and that includes the possibility of recombination. We first evaluate the limits of parsimony in constructing cladograms. Once these limits have been determined, we construct the set of parsimonious and nonparsimonious cladograms that is consistent with these limits. Our estimation procedure also identifies haplotypes that are candidates for being products of recombination. If recombination is extensive, our algorithm subdivides the DNA region into two or more subsections, each having little or no internal recombination. We apply this estimation procedure to three data sets to illustrate varying degrees of cladogram ambiguity and recombination.
Nonrandom associations of alleles or haplotypes with geographical location can arise from restricted gene flow, historical events (fragmentation, range expansion, colonization), or any mixture of these factors. In this paper, we show how a nested cladistic analysis of geographical distances can be used to test the null hypothesis of no geographical association of haplotypes, test the hypothesis that significant associations are due to restricted gene flow, and identify patterns of significant association that are due to historical events. In this last case, criteria are given to discriminate among contiguous range expansion, long-distance colonization, and population fragmentation. The ability to make these discriminations depends critically upon an adequate geographical sampling design. These points are illustrated with a worked example: mitochondrial DNA haplotypes in the salamander Ambystoma tigrinum. For this example, prior information exists about restricted gene flow and likely historical events, and the nested cladistic analyses were completely concordant with this prior information. This concordance establishes the plausibility of this nested cladistic approach, but much future work will be necessary to demonstrate robustness and to explore the power and accuracy of this procedure.
Empirical data sets of intraspecific restriction site polymorphism in Drosophila have been gathered in order to test hypotheses derived from coalescent theory. Three main ideas are tested: (1) haplotype frequency in the sample contains information on the topological position of a given haplotype in a cladogram, (2) the frequency of a haplotype is related to the number of mutational connections to other haplotypes in the cladogram and (3) geographic location can be used to infer topological positioning of haplotypes in a cladogram. These relationships can then be used to better estimate intraspecific phylogenies in two ways: (1) rooting the phylogeny and (2) resolving ambiguities in a cladogram. This information will allow one to reduce the number of alternative phylogenies and incorporate the uncertainties involved in reconstructing intraspecific phylogenies into subsequent analyses that depend heavily on the topology of the tree.
Using two sets of nucleotide sequences of the human and simian T-cell leukemia/lymphoma virus type I (HTLV-I/STLV-I), one consisting of 522 bp of the env gene from 70 viral strains and the other a 140-bp segment from the pol gene of 52 viral strains, I estimated cladograms based on a statistical parsimony procedure that was developed specifically to estimate within-species gene trees. An extension of a nesting procedure is offered for sequence data that forms nested clades used in hypothesis testing. The nested clades were used to test three hypotheses relating to transmission of HTLV/STLV sequences: (1) Have cross-species transmissions occurred and, if so, how many? (2) In what direction have they occurred? (3) What are the geographic relationships of these transmission events? The analyses support a range of 11-16 cross-species transmissions throughout the history of these sequences. Additionally, outgroup weights were assigned to haplotypes using arguments from coalescence theory to infer directionality of transmission events. Conclusions on geographic origins of transmission events and particular viral strains are inconclusive due to small samples and inadequate sampling design. Finally, this approach is compared directly to results obtained from a traditional maximum parsimony approach and found to be superior at establishing relationships and identifying instances of transmission.
Mitochondrial DNA, inherited predominantly through the female line, has been exceptionally useful for reconstructing phylogenies (Avise, in Molecular markers, natural history and evolution. New York: Chapman and Hall (1994)). However, at the lowest taxonomic level, if there are polymorphisms within species the lineages of mitochondria need not correspond to the lineages of the species (Avise, in Phil. Trans. R. Soc. Lond. B 312, 325-342 (1986)). We find that a classic organism in ecological genetics, Cepaea nemoralis, has the most extreme intraspecific variation and polymorphism so far recorded, and that at least one other pulmonate land mollusc also has very high levels of mitochondrial diversity. Making the simplest assumptions, the data suggest times of divergence as long ago as 20 million years between haplotypes now coexisting within a single population. There are four overlapping explanations of the diversity: (i) that mitochondrial evolution in pulmonates is exceptionally fast; (ii) that the morphs have differentiated in isolated 'refuges' and then come together; (iii) that natural selection has acted to preserve the variation; and (iv) that the population structure of pulmonates favours the persistence of ancient haplotypes. We argue for the importance of the last explanation.
The program MODELTEST uses log likelihood scores to establish the model of DNA evolution that best fits the data.
AVAILABILITY: The MODELTEST package, including the source code and some documentation is available at http://bioag.byu. edu/zoology/crandall_lab/modeltest.html.
Keywords:cladogram estimation;gene genealogy;phylogeography;population genetics;software
Phylogenetic reconstruction--the method by which biologists examine the relationship between living and extinct organisms in an effort to identify evolutionary pathways--has seen radical changes in the last ten years. But as rapid advances in mathematical, molecular, developmental, and cladistic techniques have greatly improved reconstruction efforts, they have also sparked controversy, especially with regard to the assumptions and data underlying the cladistic method. This book brings together contributions from a wide range of practitioners in order to find common ground. By examining numerous models from a variety of fields, the book serves as a guide to the latest methods of classification and phylogeny reconstruction and provides insights into the relative merits that each approach has to offer. In particular, developmental studies emerge as a significant factor in cladogram construction. Splendidly written and wide-ranging in scope, Models in Phylogeny Reconstruction will be welcomed by students and researchers in systematic and evolutionary biology.
A new coalescent is introduced to study the genealogy of a sample from the infinite-alleles model of population genetics. This coalescent also records the age ordering of alleles in the sample. The distribution of this process is found explicitly for the Moran model, and is shown to be robust for a wide class of reproductive schemes.
Properties of the ages themselves and the relationship between ages and class sizes then follow readily.
The possibility that a high rate of selectively neutral mutation can account for both genetic polymorphism and gene substitution in evolution is investigated theoretically. A species divided into partially isolated local populations is considered, and relations found between the local population size, the number of populations, the migration and mutation rates, and the degree of polymorphism within and between populations. It is shown that if selectively neutral mutation rates are high enough (perhaps 10-6 per generation per cistron) to account for observed rates of evolution, then either the total population size of a species, now or in the geologically recent past, must be small (⩽106), or hybrids between local populations will be heterozygous at almost all loci.
Fossil land snail shells constitute a valuable source of paleoenvironmental information for the Quaternary. They can be dated by a variety of methods, including radiocarbon, amino acid racemization and epimerization, and perhaps also and ESR. The vast majority of paleoenvironmental studies based on land snail shells have examined the faunal composition of fossil assemblages, from which a variety of paleoenvironmental characteristics such as biome, temperature, and moisture conditions have been reconstructed. Still, there are a number of problems involved in using this approach and these are discussed. Shell morphology has occasionally been used to reconstruct such factors as rainfall and temperature. Stable isotope studies on Quaternary land snails include: analysis of δ18O values of organic matter in the shells, to reconstruct C4 plant distributions from which rainfall amounts can be inferred, and analysis of δ18O values of shell carbonate, from which trends in the oxygen isotope composition of rainfall can be reconstructed. Stable carbon isotopes of shell carbonate have also been studied but their interpretation is not clear. Amino acid epimerization analysis ( ratios) of land snail shells has been used for estimation of paleotemperatures. Some potential uses of land snail shells for paleoenvironmental reconstruction include the study of stable isotopes of H and N, periodic growth lines, and deposits of pedogenic carbonates on the shells.
When a population splits in two and the resulting populations evolve independently, genetic divergence may occur due to mutation and genetic drift. This paper considers the influence these factors have on the genetic composition of two samples drawn from the respective populations at some time after the split. Analytical formulas and numerical examples are given of (i) the probability distribution and moments of the allele frequencies, (ii) the number of alleles in common between the samples, and (iii) the probability that the samples are monomorphic.
Studies of population structure were undertaken at six sites in the Tenby (South Wales) colony of the Mediterranean coastal land snail Theba pisana (Müller). Over four years, the density of adult (two-year-old) snails was estimated as ranging from 39 to 202 m-
2 during the summer active season; half-grown juvenile (one-year-old) density was estimated during the same time of year as between 13 and 436 m-
2; no estimates were made of the density of younger snails. Significant aggregation was shown for both adults and juveniles. Estimates of variance of dispersal over 100 days (0489 to 1601 m2) suggest that T. pisana is rather sedentary when compared with the few other snail species for which comparable information is available. Combining the data on density and dispersal with an estimate of variance of family size, yields estimates of effective neighbourhood size ranging from 115 to 4130. These results are compared with data for other organisms, especially other species of land snail.
The recently-developed statistical method known as the "bootstrap" can be used to place confidence intervals on phylogenies. It involves resampling points from one's own data, with replacement, to create a series of bootstrap samples of the same size as the original data. Each of these is analyzed, and the variation among the resulting estimates taken to indicate the size of the error involved in making estimates from the original data, In the case of phylogenies, it is argued that the proper method of resampling is to keep all of the original species while sampling characters with replacement, under the assumption that the characters have been independently drawn by the systematist and have evolved independently. Majority-rule consensus trees can be used to construct a phylogeny showing all of the inferred monophyletic groups that occurred in a majority of the bootstrap samples. If a group shows up 95% of the time or more, the evidence for it is taken to be statistically significant. Existing computer programs can be used to analyze different bootstrap samples by using weights on the characters, the weight of a character being how many times it was drawn in bootstrap sampling. When all characters are perfectly compatible, as envisioned by Hennig, bootstrap sampling becomes unnecessary; the bootstrap method would show significant evidence for a group if it is defined by three or more characters.
Helicid snails are suitable organisms for research addressing both ecological and evolutionary questions. They are easy to collect and mark, and because of their limited power of dispersal and their generally patchy distribution, snails exhibit extensive demographic and genetic population structure even on a microgeographic scale (Baur 1993). As one major group of land snails, several species of the family Helicidae are adapted to grassland ecosystems. Small land snails of the sub-family Helicellinae especially prefer habitats of dry open grassland with sparse vegetation. In Germany, habitat of this type is generated and maintained mainly by agricultural use, especially sheep grazing and viticulture. In the course of structural changes in agriculture during the last decades, sheep grazing has nearly vanished, and former grasslands and vineyards have either turned into intensively used monocultures or have been abandoned. This has led to a substantial loss of habitat due to natural succession and has resulted in progressive fragmentation and isolation (Henle & Kaule 1991). Thus, several snail species are endangered in Germany (Jungbluth 1986, Bogon 1990, Körnig 1992). We therefore focussed our research on two Helicellinae snails, Trochoidea geyeri (SOOS, 1926) and Candidula unifasciata (POIRET, 1801). Both species are rare today in Germany and are considered to be extinct in several states (Bundesländer). In the present case study, we analyse the population structure of T. geyeri and C. unifasciata in different grassland ecosystems: firstly, in the agricultural landscape near Halle (Bliss et al. this volume: 161–168), and secondly, in an abandoned vineyard complex on a limestone hill in the upper Kinzig valley (Hesse, Germany) with characteristic dry grassland on calciferous soil.
Population structure was estimated in a continuous population of a small land snail (Trochoidea geyeri). Mark-recapture experiments and randomly amplified polymorphic DNA analyses indicate that the population structure can be described by the isolation by distance model of Wright (1946). Estimates of density and dispersal suggest a neighbourhood size of 70-208 individuals on an area of 13-21 m(2). A principal component analysis of the randomly amplified polymorphic DNA data reveals clinal variation of genetic composition across the population, as predicted by the neighbourhood concept. An analysis of molecular variance indicates substantial genetic structuring. Comparisons of the genetic distances, expressed as euclidean distances among individuals, versus the geographic distance between sampling sites yield a highly significant positive correlation (Mantel test: r = 0.567, p < 0.0001). The revealed pattern of populational subdivision on a microgeographic scale seems to be one of the principal processes generating and maintaining genetic diversity within populations of small land gastropods.
The Quaternary cold periods in Europe are thought to have heavily influenced the amount and distribution of intraspecific genetic variation in both animals and plants. The phylogeographies of 10 taxa, including mammals (Ursus arctos, Sorex spp., Crocidura suaveolens, Arvicola spp.), amphibians (Triturus spp.), arthropods (Chorthippus parallelus), and plants (Abies alba, Picea abies, Fagus sylvatica, Quercus spp.), were analysed to elucidate general trends across Europe. Only a small degree of congruence was found amongst the phylogeographies of the 10 taxa, but the likely postglacial colonization routes exhibit some similarities. A Brooks parsimony analysis produced an unrooted area phylogram, showing that: (i) the northern regions were colonized generally from the Iberic and Balkanic refugia; and (ii) the Italian lineages were often isolated due to the presence of the Alpine barrier. The comparison of colonization routes highlighted four main suture-zones where lineages from the different refugia meet. Some of the intraspecific genetic distances among lineages indicated a prequaternary divergence that cannot be connected to any particular cold period, but are probably related mainly to the date of arrival of each taxon in the European continent. As a consequence, molecular genetics so far appears to be of limited use in dating Quaternary events.
The n-coalescent is a continuous-time Markov chain on a finite set of states, which describes the family relationships among a sample of n members drawn from a large haploid population. Its transition probabilities can be calculated from a factorization of the chain into two independent components, a pure death process and a discrete-time jump chain. For a deeper study, it is useful to construct a more complicated Markov process in which n-coalescents for all values of n are embedded in a natural way.
The application of principles from coalescence theory to genealogical relationships within species can provide insights into the process of diversification and the influence of biogeography on distributional patterns. There are several features that make some organisms more suitable for detailed studies of historical processes; in particular, limited dispersal, which serves to conserve the patterns of genetic variation that developed during colonization. We describe the potential benefits of studies that integrate analyses of genetic variation with information from the fossil pollen record and present recent examples of the application of quantitative methods of phylogeographic analysis.
Spectacular progress has been made recently in the study of evolution at the molecular level, primarily due to new biochemical techniques such as gene cloning and DNA sequencing. In this book, the author summarizes new developments and seeks to unify studies of evolutionary histories of organisms and the mechanisms of evolution into a single science - molecular evolutionary genetics.
A population of constant size is subjected to mutation, such that each mutant is of a new allelic type. For the particular population model studied in this paper, the age of an allele, whose present frequency is known, is a random variable with distribution independent of the frequencies of other alleles. As a consequence of reversibility of the population process, the age of an allele, from the past to the present, has the same distribution as its time to extinction, from the present into the future. This verifies, and re-interprets, certain diffusion approximations found by Kimura and Ohta [Genetics75, 199–212 (1973)] and Maruyama [Genet. Res. Cambridge23, 137–143 (1974)].
The n0 coalescent of Kingman (1982a, b) describes the family relationships among a sample of n0 individuals drawn from a panmictic species. It is a stochastic process resulting from n0 − 1 independent random events (coalescences) at each of which n (2 ≤ n ≤ n0) ancestral lineages of a sample are descended from n − 1 distinct ancestors for the first time. Here a similar genealogical process is studied for a species consisting of two populations with migration between them. The main interest is with the probability density of the time length between two successive coalescences and the spatial distribution of n − 1 ancestral lineages over two populations when n to n − 1 coalescence takes place. These are formulated based on a non-linear birth and death process with killing, and are used to derive several explicit formulae in selectively neutral population genetics models. To confirm and supplement the analytical results, a simulation method is proposed based on the underlying bivariate Markov chain. This method provides a general way for solving the present problem even when an analytical approach appears very difficult. It becomes clear that the effects of the present population structure are most conspicuous on 2 to 1 coalescence, with lesser extents on n to n − 1 (3 ≤ n) coalescence.This implies that in a more general model of population structure, the number of populations and the way in which a sample is drawn are important factors which determine the n0 coalescent.
The widespread use of restriction endonucleases and DNA sequencing provides a wealth of data on the genetic structure of natural populations. From such data, detailed phylogenies can be constructed and qualitatively different kinds of mutational and substitutional processes can be studied. A neutral model can be constructed to describe the frequencies of sequence haplotypes according to the haplotypes from which they arose and the types of substitution that distinguish them. One feature of such a model is that it examines the ancestors of various sequences. Deleterious selection against a character has a distinct effect on descendant sequences. Individuals containing many deleterious characters leave few or no descendants because these individuals are quickly eliminated by selection. Hence, such a model lends itself to the study of deleterious selection. It is possible to determine if selection is required by searching for any set of mutation rates that can explain an observed set of data. Simulations of artificial populations without selection suggest that this method seldom indicates selection when none is present. Furthermore, recent recombination events between the sequences do not induce false indications of deleterious selection. The method may, however, require relatively large simple sizes in order to accurately reflect the true nature of populations. The method is often very conservative and may not indicate selection when it is, in fact, present.
The application of maximum likelihood techniques to the estimation of evolutionary trees from nucleic acid sequence data is discussed. A computationally feasible method for finding such maximum likelihood estimates is developed, and a computer program is available. This method has advantages over the traditional parsimony algorithms, which can give misleading results if rates of evolution differ in different lineages. It also allows the testing of hypotheses about the constancy of evolutionary rates by likelihood ratio tests, and gives rough indication of the error of ;the estimate of the tree.
The sensitivity of the commonly used progressive multiple sequence alignment method has been greatly improved for the alignment
of divergent protein sequences. Firstly, individual weights are assigned to each sequence in a partial alignment in order
to downweight near-duplicate sequences and up-weight the most divergent ones. Secondly, amino acid substitution matrices are
varied at different alignment stages according to the divergence of the sequences to be aligned. Thirdly, residue-specific
gap penalties and locally reduced gap penalties in hydrophilic regions encourage new gaps in potential loop regions rather
than regular secondary structure. Fourthly, positions in early alignments where gaps have been opened receive locally reduced
gap penalties to encourage the opening up of new gaps at these positions. These modifications are incorporated into a new
program, CLUSTAL W which is freely available.
The rooting of intraspecific gene or haplotype trees has proven to be difficult using the traditional techniques of rooting species trees such as outgroups. As an alternative, we apply neutral coalescent theory to the problems of determining the root of an intraspecific gene tree and the relative ages of the haplotypes. By using a recursive equation, exact root probabilities can be calculated for small cladograms and sample sizes. These exact probabilities indicate that root probabilities are not very sensitive to the parameter theta, which is four times the mutation rate times the inbreeding effective size. The exact probabilities also indicate that root probabilities are not very sensitive to the absolute numbers of each haplotype (allele) class, only their relative proportions. The exact method, unfortunately, is not feasible for implementation with larger data sets with present algorithms and computers. However, the exact results suggested a simple heuristic for determining outgroup weights; that is, finding the haplotype that is the oldest in the sample and that can serve as an outgroup for the remainder of the haplotype tree. Computer simulations revealed that these outgroup weights are strongly correlated with actual age and are much better indicators of haplotype age than is the haplotype frequency, another commonly used indicator of relative age.
We previously developed an analytical strategy based on cladistic theory to identify subsets of haplotypes that are associated with significant phenotypic deviations. Our initial approach was limited to segments of DNA in which little recombination occurs. In such cases, a cladogram can be constructed from the restriction site data to estimate the evolutionary steps that interrelate the observed haplotypes to one another. The cladogram is then used to define a nested statistical design for identifying mutational steps associated with significant phenotypic deviations. The central assumption behind this strategy is that a mutation responsible for a particular phenotypic effect is embedded within the evolutionary history that is represented by the cladogram. The power of this approach depends on the accuracy of the cladogram in portraying the evolutionary history of the DNA region. This accuracy can be diminished both by recombination and by uncertainty in the estimated cladogram topology. In a previous paper, we presented an algorithm for estimating the set of likely claodgrams and recombination events. In this paper we present an algorithm for defining a nested statistical design under cladogram uncertainty and recombination. Given the nested design, phenotypic associations can be examined using either a nested analysis of variance (for haploids or homozygous strains) or permutation testing (for outcrossed, diploid gene regions). In this paper we also extend this analytical strategy to include categorical phenotypes in addition to quantitative phenotypes. Some worked examples are presented using Drosophila data sets. These examples illustrate that having some recombination may actually enhance the biological inferences that may derived from a cladistic analysis. In particular, recombination can be used to assign a physical localization to a given subregion for mutations responsible for significant phenotypic effects.
A number of methods have already been described for the isolation of genomic DNA. Some methods of DNA isolation, for example CsCl-gradients, require large amounts of tissue, and are not suitable for the isolation of DNA from small tissue samples. Protocols that yield DNA in quality from small amounts of tissue frequently involve detergents (cetyltrimethylammonium bromide, SDS) or enzyme treatment (e.g. proteinases) in specialized buffer systems, whose preparation is often time consuming and expensive. We therefore developed a rapid method of DNA isolation using commercial biological laundry detergent concentrate (Persil Mega Perls( and Frosch( ) as detergent/buffer-system. Tissue (500 mg) was ground with sea sand in a 1.7 ml Eppendorf tube containing one spatula point of washing powder (40 mg) dissolved in 1.5 ml deionized water. The tissue/laundry detergent-mixture was then incubated overnight at 37C, and half of the resulting homogenate (750 ml) was extracted once with phenol-chloroform-isoamylalcohol (25:24:1), followed by RNAse treatment for 1 h at 37C (0.2 mg/ml), and one extraction with chloroform-isoamylalcohol. The DNA was precipitated overnight at -20C by adding 0.1 vol 3 M sodium acetate and 2 vol ice-cold 98% ethanol and centrifugated for 15 min
Since the 1920s, population geneticists have had measures that describe how genetic variation is distributed spatially within a species' geographical range. Modern genetic survey techniques frequently yield information on the evolutionary relationships among the alleles or haplotypes as well as information on allele frequencies and their spatial distributions. This evolutionary information is often expressed in the form of an estimated haplotype or allele tree. Traditional statistics of population structure, such as F statistics, do not make use of evolutionary genealogical information, so it is necessary to develop new statistical estimators and tests that explicitly incorporate information from the haplotype tree. One such technique is to use the haplotype tree to define a nested series of branches (clades), thereby allowing an evolutionary nested analysis of the spatial distribution of genetic variation. Such a nested analysis can be performed regarding the geographical sampling locations either as categorical or continuous variables (i.e. some measure of spatial distance). It is shown that such nested phylogeographical analyses have more power to detect geographical associations than traditional, nonhistorical analyses and, as a consequence, allow a broader range of gene-flow parameters to be estimated in a precise fashion. More importantly, such nested analyses can discriminate between phylogeographical associations due to recurrent but restricted gene flow vs. historical events operating at the population level (e.g. past fragmentation, colonization, or range expansion events). Restricted gene flow and historical events can be intertwined, and the cladistic analyses can reconstruct their temporal juxtapositions, thereby yielding great insight into both the evolutionary history and population structure of the species. Examples are given that illustrate these properties, concentrating on the detection of range expansion events.
A new method for the estimation of migration rates and effective population sizes is described. It uses a maximum-likelihood framework based on coalescence theory. The parameters are estimated by Metropolis-Hastings importance sampling. In a two-population model this method estimates four parameters: the effective population size and the immigration rate for each population relative to the mutation rate. Summarizing over loci can be done by assuming either that the mutation rate is the same for all loci or that the mutation rates are gamma distributed among loci but the same for all sites of a locus. The estimates are as good as or better than those from an optimized FST-based measure. The program is available on the World Wide Web at http://evolution.genetics. washington.edu/lamarc.html/.
Coalescent theory represents the most significant progress in theoretical population genetics in the past two decades of this century. It is now widely recognized as a cornerstone for rigorous statistical analyses of molecular data from populations. In the future, challenges from the rapidly expanding body of molecular data will continue to inject fresh blood into the development of coalescent theory. As the 21st century looms, we review recent progress in coalescent theory and discuss some prospects for future developments, particularly with regard to human population genetics.
The Iowa Pleistocene snail, Discus macclintocki, is an endangered species that survives only in relictual populations on algific (cold-air) talus slopes in northeast Iowa and northwest Illinois in the central region of the USA. These populations are believed to have been isolated since the temperatures began to warm at the end of the last glacial period around 16 500 years ago. DNA sequencing of the 16s rRNA gene of the mitochondria was used to determine the genetic relationship among 10 populations and the genetic diversity within these populations. Genetic diversity is extremely high within this species with 40 haplotypes spread across the 10 populations sampled within a 4000 km2 region. Phylogenetic analyses showed that haplotypes formed monophyletic groups by the watershed on which they were found, suggesting that watersheds were important historical avenues of gene flow. Genetic distances were strongly related to the geographical distance among all populations, but this relationship was dependent on the scale being considered.
Keywords:cladistic nested analysis;historical processes;phylogeography;population genetics;software
Mollusks are an extraordinarily diverse group of animals with an estimated 200,000 species, second only to the phylum Arthropoda. We conducted a comparative analysis of complete mitochondrial ribosomal large subunit sequences (LSU) of a chiton, two bivalves, six gastropods, and a cephalopod. In addition, we determined secondary structure models for each of them. Comparative analyses of nucleotide variation revealed substantial length variation among the taxa, with stylommatophoran gastropods possessing the shortest lengths. Phylogenetic analyses of the nucleotide sequence data supported the monophyly of Albinaria, Euhadra herklotsi + Cepaea nemoralis, Stylommatophora, Cerithioidea, and when only transversions are included, the Bivalvia. The phylogenetic limits of the mitochondrial LSU rRNA gene within mollusks appear to be up to 400 million years, although this estimate will have to be tested further with additional taxa. Our most novel finding was the discovery of phylogenetic signal in the secondary structure of rRNA of mollusks. The absence of entire stem/loop structures in Domains II, III, and V can be viewed as three shared derived characters uniting the stylommatophoran gastropods. The absence of the aforementioned stem/loop structure explains much of the observed length variation of the mitochondrial LSU rRNA found within mollusks. The distribution of these unique secondary structure characters within mollusks should be examined.
Global climate has fluctuated greatly during the past three million years, leading to the recent major ice ages. An inescapable consequence for most living organisms is great changes in their distribution, which are expressed differently in boreal, temperate and tropical zones. Such range changes can be expected to have genetic consequences, and the advent of DNA technology provides most suitable markers to examine these. Several good data sets are now available, which provide tests of expectations, insights into species colonization and unexpected genetic subdivision and mixture of species. The genetic structure of human populations may be viewed in the same context. The present genetic structure of populations, species and communities has been mainly formed by Quaternary ice ages, and genetic, fossil and physical data combined can greatly help our understanding of how organisms were so affected.
Intraspecific gene evolution cannot always be represented by a bifurcating tree. Rather, population genealogies are often multifurcated, descendant genes coexist with persistent ancestors and recombination events produce reticulate relationships. Whereas traditional phylogenetic methods assume bifurcating trees, several networking approaches have recently been developed to estimate intraspecific genealogies that take into account these population-level phenomena.
Molecular methods as applied to the biogeography of single species (phylogeography) or multiple codistributed species (comparative phylogeography) have been productively and extensively used to elucidate common historical features in the diversification of the Earth's biota. However, only recently have methods for estimating population divergence times or their confidence limits while taking into account the critical effects of genetic polymorphism in ancestral species become available, and earlier methods for doing so are underutilized. We review models that address the crucial distinction between the gene divergence, the parameter that is typically recovered in molecular phylogeographic studies, and the population divergence, which is in most cases the parameter of interest and will almost always postdate the gene divergence. Assuming that population sizes of ancestral species are distributed similarly to those of extant species, we show that phylogeographic studies in vertebrates suggest that divergence of alleles in ancestral species can comprise from less than 10% to over 50% of the total divergence between sister species, suggesting that the problem of ancestral polymorphism in dating population divergence can be substantial. The variance in the number of substitutions (among loci for a given species or among species for a given gene) resulting from the stochastic nature of DNA change is generally smaller than the variance due to substitutions along allelic lines whose coalescence times vary due to genetic drift in the ancestral population. Whereas the former variance can be reduced by further DNA sequencing at a single locus, the latter cannot. Contrary to phylogeographic intuition, dating population divergence times when allelic lines have achieved reciprocal monophyly is in some ways more challenging than when allelic lines have not achieved monophyly, because in the former case critical data on ancestral population size provided by residual ancestral polymorphism is lost. In the former case differences in coalescence time between species pairs can in principle be explained entirely by differences in ancestral population size without resorting to explanations involving differences in divergence time. Furthermore, the confidence limits on population divergence times are severely underestimated when those for number of substitutions per site in the DNA sequences examined are used as a proxy. This uncertainty highlights the importance of multilocus data in estimating population divergence times; multilocus data can in principle distinguish differences in coalescence time (T) resulting from differences in population divergence time and differences in T due to differences in ancestral population sizes and will reduce the confidence limits on the estimates. We analyze the contribution of ancestral population size (theta) to T and the effect of uncertainty in theta on estimates of population divergence (tau) for single loci under reciprocal monophyly using a simple Bayesian extension of Takahata and Satta's and Yang's recent coalescent methods. The confidence limits on tau decrease when the range over which ancestral population size theta is assumed to be distributed decreases and when tau increases; they generally exclude zero when tau/(4Ne) > 1. We also apply a maximum-likelihood method to several single and multilocus data sets. With multilocus data, the criterion for excluding tau = 0 is roughly that l tau/(4Ne) > 1, where l is the number of loci. Our analyses corroborate recent suggestions that increasing the number of loci is critical to decreasing the uncertainty in estimates of population divergence time.
Intraspecific phylogeographic methods provide a means of examining the history of genetic exchange among populations. As part of a study of the history of Helix aspersa in the Western Mediterranean, we performed a phylogenetic analysis based on partial sequences of the mitochondrial large ribosomal subunit (16S) gene. Our samples include 31 H. a. aspersa populations from North Africa previously investigated for anatomical and biochemical characters. To clarify subspecific relationships, three individuals of the subspecies H. a. maxima were also studied. The molecular phylogeny inferred agrees largely with previous results, in splitting H. a. aspersa haplotypes into an eastern and a western group. H. a. maxima haplotypes form a third lineage arising before the H. a. aspersa groups. Divergence times estimated between the lineages suggest that dispersal during Pleistocene glaciation and vicariance events due to Pliocene geological changes in the western Mediterranean may both have played a significant part in the establishment of the present range of H. aspersa.
A maximum likelihood estimator based on the coalescent for unequal migration rates and different subpopulation sizes is developed. The method uses a Markov chain Monte Carlo approach to investigate possible genealogies with branch lengths and with migration events. Properties of the new method are shown by using simulated data from a four-population n-island model and a source-sink population model. Our estimation method as coded in migrate is tested against genetree; both programs deliver a very similar likelihood surface. The algorithm converges to the estimates fairly quickly, even when the Markov chain is started from unfavorable parameters. The method was used to estimate gene flow in the Nile valley by using mtDNA data from three human populations.
In an effort to link quantitative morphometric information with molecular data on the population level, we have analysed 19 populations of the conchologically variable land snail Candidula unifasciata from across the species range for variation in quantitative shell traits and at the mitochondrial 16S ribosomal (r)DNA locus. In genetic analysis, including 21 additional populations, we observed two fundamental haplotype clades with an average pairwise sequence divergence of 0.209 +/- 0.009 between clades compared to 0.017 +/- 0.012 within clades, suggesting the presence of two different evolutionary lineages. Integrating additional shell material from the Senckenberg Malacological Collection, a highly significant discriminant analysis on the morphological shell traits with fundamental haplotype clades as grouping variable suggested that the less frequent haplotype corresponds to the described subspecies C. u. rugosiuscula, which we propose to regard as a distinct species. Both taxa were highly subdivided genetically (FST = 0.648 and 0.777 P < 0.001). This was contrasted by the partition of morphological variance, where only 29.6% and 21.9% of the variance were distributed among populations, respectively. In C. unifasciata, no significant association between population pairwise FST estimates and corresponding morphological fixation indices could be detected, indicating independent evolution of the two character sets. Partial least square analysis of environmental factors against shell trait variables in C. u. unifasciata revealed significant correlations between environmental factors and certain quantitative shell traits, whose potential adaptational values are discussed.