Using morphological characters of subfossil daphniid postabdominal claws to improve taxonomic resolution within species complexes

Daphnia subfossils from lake sediments are useful for exploring the impacts of environmental stressors on aquatic ecosystems. Unfortunately, taxonomic resolution of Daphnia remains is coarse, as only a small portion of the animal is preserved, and so the identification of daphniid subfossils typically relies upon postabdominal claws. Daphniid claws can be assigned to one of two species complexes: D. longispina or D. pulex. Both complexes contain species with differing environmental optima, and therefore improved taxonomic resolution of subfossil daphniid claws would aid paleolimnological analyses. To identify morphological features that may be used to help differentiate between species within complexes, we used species presence/absence data from net tows to select lakes in central Ontario (Canada) containing only a single species from a particular complex, then used remains preserved in surface sediments of these lakes to isolate four Daphnia species: D. ambigua and D. mendotae from the D. longispina complex, and D. pulicaria and D. catawba from the D. pulex complex. Our analyses demonstrate that, within the D. longispina complex, postabdominal claw length (PCL) and spinule length can be used to distinguish D. mendotae from D. ambigua. In addition, within the D. pulex complex, there are differences between D. pulicaria and D. catawba in the relative lengths of the proximal and middle combs on the postabdominal claw. However, the number of stout spines on the middle comb is an unreliable character for differentiating species. Overall, our data demonstrate that greater resolution within Daphnia species complexes is possible using postabdominal claws; however, the process is arduous, and applicability will likely decrease with the number of taxa present.     

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

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

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

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

Structure and function of a mating-type gene from the homothallic species Neurospora africana

The homothallic Neurospora species, N. africana, contains sequences that hybridize to the A but not to a mating-type sequences of the heterothallic species N. crassa. In this study, the N. africana mating-type gene, mt A-1, was cloned, sequenced and its function analyzed in N. crassa. Although N. africana does not mate in a heterothallic manner, its mt A-1 gene functions as a mating activator in N. crassa. In addition, the N. africana mt A-1 gene confers mating type-associated vegetative incompatibility in N. crassa. DNA sequence analysis shows that the N. africana mt A-1 open reading frame (ORF) is 93% identical to that of N. crassa mt A-1. The mt A-1 ORF of N. africana contains no stop codons and was detected as a cDNA which is processed in a similar manner to mt A-1 of N. crassa. By DNA blot and orthogonal field agarose gel electrophoretic analysis, it is shown that the composition and location of the mating-type locus and the organization of the mating-type chromosome of N. africana are similar to that of N. crassa.     

九种绢丝昆虫线粒体12S rRNA基因的序列特征和系统发育分析

为探讨鳞翅目中绢丝昆虫之间的系统发育关系和分子进化特征,本研究测定了中国柞蚕Antheraea pernyi野生型和放养型的线粒体12S rRNA基因的部分序列,结合来自GenBank数据库的17条序列,对总共9种绢丝昆虫（2科3属）的12S rRNA基因序列进行了分析。利用软件MEGA 3.1进行碱基组成、变异位点的统计和分子进化分析,分别用类平均聚类法（UPGMA）、邻接法（NJ）、最小进化法（ME）、最大简约法（MP）重建系统发生树。测定的中国柞蚕野生型的12S rRNA基因序列（427 bp）与放养型“豫早1号”的序列完全一致。序列对齐后共鉴定80个变异位点,50个简约信息位点。碱基组成分析显示在科属间具有明显差异,AT含量蚕蛾科高于大蚕蛾科;在A和T碱基的使用上,大蚕蛾科偏好使用T,而蚕蛾科则偏好使用A。与动物中常见的以转换为主的碱基替换模式不同,所分析的9种昆虫中除桑蚕属内部为转换与颠换基本一致外,其余物种间均是颠换多于转换。进化分析支持柞蚕属、樗蚕属和桑蚕属的单系。基于UPGMA法的进化树支持琥珀蚕是柞蚕属的较原始类型,而NJ、ME和MP法则支持印度柞蚕是较原始的类型,因此,柞蚕属种间的进化关系尚需进一步研究。     

PCR-based phylogenetic walking: Isolation of para-homologous sodium channel gene sequences from seven insect species and an arachnid

The voltage-sensitive sodium channel is the site of action of two important classes of insecticides, DDT and pyrethroids. We recently used the polymerase chain reaction (PCR) to amplify sodium channel gene sequences in the house fly genome and showed the direct use of the amplification product as a conspecific hybridization probe. This report describes the use of this method to isolate sodium channel gene sequences from seven insect species (representing four orders) and an arachnid, thereby demonstrating its general utility for quickly and efficaciously isolating homologous sequences from distantly related species. DNA sequence analysis of the amplified products revealed that all but a few were homologous to the IS5-6 region of the para gene of Drosophila melanogaster, the region upon which the design of the target primers was based. Although unique nucleotide sequences were obtained for each species (with some species having more than one sequence variant), the inferred amino acid sequences of the 15 residue stretch between the amino acid target sequences were found to be completely conserved or to contain a single conservative replacement of serine with threonine. We suggest that this methodology now permits specific knowledge obtained from molecular genetic analysis of D. melanogaster to be applied straightforwardly to the characterization of many genes and the primary products of their expression in other insect specs.     

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

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

Use of rpoB gene sequence analysis for phylogenetic identification of Cronobacter species

Here, we report the RNA polymerase beta-subunit gene (rpoB) as a new molecular marker for the identification of the Cronobacter species. The results indicated that members of the Cronobacter genus are more easily discriminated by rpoB sequencing than 16S rRNA sequencing, and reliable identification could be achieved by rpoB gene sequence comparison.     

Development of high‐resolution DNA barcodes for Dioscorea species discrimination and phylogenetic analysis

The genus Dioscorea is widely distributed in tropical and subtropical regions, and is economically important in terms of food supply and pharmaceutical applications. However, DNA barcodes are relatively unsuccessful in discriminating between Dioscorea species, with the highest discrimination rate (23.26%) derived from matK sequences. In this study, we compared genic and intergenic regions of three Dioscorea chloroplast genomes and found that the density of SNPs and indels in intergenic sites was about twice and seven times higher than that of SNPs and indels in the genic regions, respectively. A total of 52 primer pairs covering highly variable regions were designed and seven pairs of primers had 80%–100% PCR success rate. PCR amplicons of 73 Dioscorea individuals and assembled sequences of 47 Dioscorea SRAs were used for estimating intraspecific and interspecific divergence for the seven loci: The rpoB‐trnC locus had the highest interspecific divergence. Automatic barcoding gap discovery (ABGD), Poisson tree processes (PTP), and generalized mixed Yule coalescence (GMYC) analysis were applied for species delimitation based on the seven loci and successfully identified the majority of species, except for species in the Enantiophyllum section. Phylogenetic analysis of 51 Dioscorea individuals (28 species) showed that most individuals belonging to the same species tended to cluster in the same group. Our results suggest that the variable loci derived from comparative analysis of plastid genome sequences could be good DNA barcode candidates for taxonomic analysis and species delimitation.     

Using directed phylogenetic networks to retrace species dispersal history

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

TreeGeneBrowser: phylogenetic data mining of gene sequences from public databases

MOTIVATION: Sequence databases represent an enormous resource of phylogenetic information, but there is a lack of tools for accessing that information in order to assess the amount of evolutionary information in these databases that may be suitable for phylogenetic reconstruction and for identifying areas of the taxonomy that are under-represented for specific gene sequences. RESULTS: We have developed TreeGeneBrowser which allows inspection and evaluation of gene sequence data for phylogenetic reconstruction. This program improves the efficiency of identification of genes that may be useful for particular phylogenetic studies and identifies taxa and taxonomic branches that are under-represented in sequence databases.     

Analysis of phylogenetic relationships of Brassicaceae species based on Chs sequences

Sequences of nuclear chalcone synthase gene (Chs) were analyzed for species of the Brassicaceae family to reconstruct phylogenetic relationships. The phylogeny for 106 species of 60 genera was reconstructed, and assigned to 24 tribes, using maximum parsimony, maximum likelihood, and neighbor-joining methods. Most of the tribes can be assigned to the major lineages (Lineages I–III) suggested by Beilstein et al. (2006). The tribe Camelineae was not monophyletic. Conringia planisiliqua together with Orychophragmus violaceus would not be recognized as a new tribe proposed by the previous studies, and C. planisiliqua should be a member of tribe Isatideae. The genera delimitation and monophyly of the expanded Solms-laubachia were also confirmed by our data. Furthermore, one parent of inter-tribal allopolyploid Pachycladon appeared to be most closely associated with Crucihimalaya, Transberingia and tribes Boechereae and Halimolobeae, another parent was proved to be in tribe Smelowskieae.     

Structure and function of a mating-type gene from the homothallic species Neurospora africana

The homothallic Neurospora species, N. africana, contains sequences that hybridize to the A but not to a mating-type sequences of the heterothallic species N. crassa. In this study, the N. africana mating-type gene, mt A-1, was cloned, sequenced and its function analyzed in N. crassa. Although N. africana does not mate in a heterothallic manner, its mt A-1 gene functions as a mating activator in N. crassa. In addition, the N. africana mt A-1 gene confers mating type-associated vegetative incompatibility in N. crassa. DNA sequence analysis shows that the N. africana mt A-1 open reading frame (ORF) is 93% identical to that of N. crassa mt A-1. The mt A-1 ORF of N. africana contains no stop codons and was detected as a cDNA which is processed in a similar manner to mt A-1 of N. crassa. By DNA blot and orthogonal field agarose gel electrophoretic analysis, it is shown that the composition and location of the mating-type locus and the organization of the mating-type chromosome of N. africana are similar to that of N. crassa.     

The dnaJ gene as a novel phylogenetic marker for identification of Vibrio species

The utility of the dnaJ gene for identifying Vibrio species was investigated by analyzing dnaJ sequences of 57 type strains and 22 clinical strains and comparing sequence homologies with those of the 16S rDNA gene and other housekeeping genes (recA, rpoA, hsp60). Among the 57 Vibrio species, the mean sequence similarity of the dnaJ gene (77.9%) was significantly less than that of the 16S rDNA gene (97.2%), indicating a high discriminatory power of the dnaJ gene. Most Vibrio species were, therefore, differentiated well by dnaJ sequence analysis. Compared to other housekeeping genes, the dnaJ gene showed better resolution than recA or rpoA for differentiating Vibrio coralliilyticus from Vibrio neptunius and Vibrio harveyi from Vibrio rotiferianus. Among the clinical strains, all 22 human pathogenic strains, including an atypical strain, were correctly identified by the dnaJ sequence. Our findings suggest that analysis of the dnaJ gene sequence can be used as a new tool for the identification of Vibrio species.     

High resolution analysis and phylogenetic network construction using complete mtDNA sequences in sardinian genetic isolates

For mitochondrial phylogenetic analysis, the best result comes from complete sequences. We therefore decided to sequence the entire mitochondrial DNA (mtDNA) (coding and D-loop regions) of 63 individuals selected in 3 small Ogliastra villages, an isolated area of eastern Sardinia: Talana, Urzulei, and Perdasdefogu. We studied at least one individual for each of the most frequent maternal genealogical lineages belonging to haplogroups H, V, J, K, T, U, and X. We found in our 63 samples, 172 and 69 sequence changes in the coding and in the D-loop region, respectively. Thirteen out of 172 sequence changes in the coding region are novel. It is our hypothesis that some of them are characteristic of the Ogliastra region and/or Sardinia. We reconstructed the phylogenetic network of the 63 complete mtDNA sequences for the 3 villages. We also drew a network including a large number of European sequences and calculated various indices of genetic diversity in Ogliastra. It appears that these small populations remained extremely isolated and genetically differentiated compared with other European populations. We also identified in our samples a never previously described subhaplogroup, U5b3, which seems peculiar to the Ogliastra region.     

Genetic distinctness and phylogenetic relationships among Undaria species (Laminariales, Phaeophyceae) based on mitochondrial cox3 gene sequences

Genetic relationships among Undaria species and among populations of each species were studied based on DNA sequences of the mitochondrial cox3 gene. Although three Undaria species, U. peterseniana (Kjellman) Okamura, U. pinnatifida (Harvey) Suringar and U. undarioides (Yendo) Okamura, have been described based mostly on blade morphology, plants with intermediate morphologies have also been found. Multiple plants from several populations in Japan were collected. Morphological characters could identify most of the samples unambiguously. A few samples with intermediate morphologies were also collected. Mitochondrial haplotypes found in each population were different for each identified species, and each species had multiple haplotypes. In the cox3 haplotype network analysis, the numbers of steps between haplotypes within and between species were similar, and haplotypes of each species did not group together. The close genetic relationships among species strongly suggest that these species are conspecific. Alternatively, recent speciation could be possible with maintenance of ancestral polymorphisms within the species (i.e. incomplete lineage sorting). Haplotypes of samples with intermediate morphologies were different for each sample and the same as ones found in the local population, suggesting interspecific hybridizations among species.