Maximum Parsimony Analyses Research Articles

Phylogenetic Relatedness Among Plasmids Harbored by Campylobacter jejuni and Campylobacter coli Isolated From Retail Meats.

Campylobacter jejuni and Campylobacter coli are two of the major causes of foodborne illness. In this study, 29 plasmids isolated from 20 retail meat isolates of Campylobacter jejuni and Campylobacter coli were fully-sequenced individually or as a part of a whole genome sequencing approach. The fully-sequenced plasmids ranged in size from 3 to 119 kb. Molecular characterization of the sequenced plasmids was based on pangenomic analysis and types of genes present on these plasmids and similar ones from GenBank. The plasmids were categorized into four different groups. These groups include type-1 that consisted mainly of pTet plasmids with the tetO gene, type-2 plasmids commonly found in C. coli strains, type-3 which has pVir plasmids, and type-4 that consisted mainly of smaller plasmids. The type-2 plasmids were unique, common among C. coli strains, and carried several conjugative transfer genes. The type-2 plasmids were most similar to a plasmid from Helicobacter pullorum. Maximum parsimony analysis and NeighborNet analysis were used to assess the phylogenetic relatedness among the 29 plasmid sequences presented in this study in addition to the other 104 plasmid sequences of Campylobacter species available in GenBank to date. Results from MP analysis revealed multiple lineages among Campylobacter plasmids which was supported by NeighborNet analysis. Clustering of plasmids did not conform to species-specific clades which suggested an intra-species dissemination of plasmids among Campylobacter species. To our knowledge, this is the first extensive phylogenetic analysis of Campylobacter plasmids sequenced to date.

Identification of ethnomedicinally important Kaempferia L. (Zingiberaceae) species based on morphological traits and suitable DNA region

Nuclear ribosomal internal transcribed spacer (ITS) sequences and morphological characteristics were used to identify medicinally important Kaempferia species cultivated in Peninsular Malaysia. Six species were evaluated for parameters such as nucleotide diversity (0.458) and estimated values of transition/transversion bias (0.89) using sequence data. Maximum parsimony (MP) analysis inferred divergence pattern in the Kaempferia genus. The resulting phylogenetic tree was compared to one formed using morphological traits. Morphological and molecular data both show 3 distinctive groupings within selected Kaempferia species. ITS 4 and 5 sequences are proposed as DNA barcodes for identification of Kaempferia species. In terms of morphological traits, plant habit, rhizome colour and leaf variation can be used for preliminary identification of this genus.

Saprobic Lophiostomataceae (Dothideomycetes): Pseudolophiostoma mangiferae sp. nov. and Neovaginatispora fuckelii, a new record from Mangifera indica

Pseudolophiostoma mangiferae, isolated from a dead stem of Mangifera indica, collected in Fanlu Township area, Dahu village in Chiayi, Taiwan (Elevation 634 m) and is introduced as a new species based on morphological data and DNA sequence analyses. Maximum parsimony, Maximum likelihood and Bayesian analyses of combined ITS, LSU, SSU and tef1-α sequence data were performed to clarify the phylogenetic affinities of the species. Pseudolophiostoma mangiferae is distinguished from the other Pseudolophiostoma species based on distinct size differences in ascomata, asci, ascospores and DNA sequence data. In addition, Neovaginatispora fuckelii is described as a new record from Taiwan. Both species are compared with other similar species and comprehensive descriptions and micrographs are provided.

Notes on Indian species of Calvatia and Langermannia including Calvatia natarajanii sp. nov.

A new species, Calvatia natarajanii, is presented as well as brief notes on the distribution of Calvatia sensu stricto and Langermannia in India. Calvatia natarajanii is distinguished by basidiomes having yellowish white to orange-white exoperidium, brownish orange to grayish red, persistent endoperidium, grayish orange to brownish orange cottony gleba, persistent cellular subgleba and globose to subglobose, echinulate basidiospores. It is described, illustrated and compared with the closely related taxa C. cyathiformis, C. fragilis, C. rugosa, C. aff. rugosa, C. pyriformis, C. vinosa and C. rosacea. Maximum Parsimony analysis based on ITS sequences confirmed that C. natarajanii is closely related to C. fragilis and distinct from other morphologically similar species. In addition, a key to the species of Calvatia and Langermannia recorded in India is presented. Four species recognized under Calvatia are excluded from the key since their transfer to Lycoperdon.

Silica bodies in leaves of neotropical Podostemaceae: taxonomic and phylogenetic perspectives.

The presence, location and morphology of silica bodies are informative anatomical characters in angiosperms, mainly in Poales. In Podostemaceae, a strictly aquatic family, these structures are mentioned frequently, but there is limited insight into their location and morphological features. In the present study we focused on describing and analysing the morphological diversity of silica bodies in leaves of neotropical Podostemaceae at the intra- and interspecific levels to determine their taxonomic and phylogenetic relevance. We studied 103 specimens distributed across 40 species. Silica body morphological traits were analysed under light and scanning electron microscopy. Additionally, data from three species of Hypericaceae (sister group) were retrieved from the literature. A phylogenetic framework based on four molecular markers was built in order to reconstruct ancestral character states related to silica bodies in neotropical Podostemaceae. Silica bodies were detected in epidermal, subepidermal and perivascular cells, presenting different shapes and surface morphology. Presence and location were used for primary differentiation while surface morphology and lumen (presence and shape) were used for finer distinctions. Intraspecific comparisons among samples showed that the length and width of these structures were highly variable. Maximum parsimony and maximum likelihood analyses for ancestral character reconstruction were congruent. Three out of five characters showed a statistically strong phylogenetic signal. Silica bodies were reported for the first time for 19 taxa, and their morphological diversity is greater than reported in previous studies. Their presence can be considered an apomorphy in Podostemaceae. Although some significant differences were detected in length and width, qualitative characters are more informative at both specific and generic ranks.

Molecular and Morphological Phylogenetic Analysis of Naupactus Dejean (Curculionidae: Entiminae) and Allied Genera: The Dilemma of Classification

Naupactus (Curculionidae: Entiminae) is the most speciose weevil genus of the tribe Naupactini. The main objective of this work is to recognize species groups within Naupactus and to analyze the relationships between this and other Neotropical genera. For this purpose, we compiled a combined data matrix of 60 terminal units corresponding to 40 species for which we recorded 812 molecular and morphological characters (763 and 49 respectively), which were analyzed by Maximum Parsimony and Bayesian analyses. The single tree obtained from each analysis was rooted with Cyrtomon inhalatus. The species of Naupactus were recovered as different monophyletic groups, some of them closer to other genera of Naupactini (Lanterius, Teratopactus, Pantomorus and Parapantomorus) than to species of the same genus. We conclude that Naupactus is non-monophyletic, even though most species can be recognized based on a particular combination of morphological characters, which are probably symplesiomorphic. To be consistent with the cladistic principles, some genera diversified in marginal areas of the Pantomorus-Naupactus complex should be synonymized with Naupactus; however, these nomenclatural changes may not ensure a generic definition based on synapomorphies. We prefer to be conservative about the current classification until more evidence is available. The only nomenclatural amendments proposed herein are the transference of Naupactus inermis Hustache to Lanterius and of N. setarius to Symmathetes.

Morphological and molecular identification of three new species of Tomentella from Finland

ABSTRACTThree new species of Tomentella (Thelephorales) from Finland, T. globosa, T. lammiensis, and T. longisterigmata, are described and illustrated with morphological characteristics and nuc rDNA ITS1-5.8S-ITS2 sequences. T. globosa is characterized by mucedinoid, pale to dark brown basidiocarps adherent to the substrate, generative hyphae with clamps and rarely with simple septa, and echinulate, globose basidiospores (echinuli up to 1.5 μm long). T. lammiensis is characterized by mucedinoid, oxide yellow to golden brown basidiocarps adherent to the substrate, generative hyphae with clamps and rarely with simple septa, and echinulate, ellipsoid, triangular, or lobbed basidiospores (echinuli up to 2 μm long). T. longisterigmata is characterized by mucedinoid, dark brown to chestnut basidiocarps separable from the substrate, generative hyphae clamped and rarely with simple septa, the long basidial sterigmata (7–11 μm long), and echinulate, globose basidiospores (echinuli up to 2 μm long). An absence of rhizomorphs and cystidia is their common morphological feature. Molecular analyses by maximum likelihood, maximum parsimony, and Bayesian analyses confirm the phylogenetic position of these three new species. The discriminating characters of these new species and their closely related species are discussed in this study, and a key to the species from Finland is provided.

Molecular phylogeny of Ischnocnema (Anura: Brachycephalidae) with the redefinition of its series and the description of two new species

We present a new phylogenetic hypothesis for Ischnocnema, a Neotropical brachycephaloid genus of ground-dwelling direct-developing frogs. We performed Bayesian inference, maximum likelihood, and maximum parsimony analyses using two nuclear (RAG1 and Tyr) and three mitochondrial genes (12S rRNA, tRNA-Val, and 16S rRNA) in a matrix comprising more than 80% of the described species. We recover Ischnocnema nanahallux outside the Ischnocnema parva series, and it is now unassigned to any species series, nor are Ischnocnema manezinho and Ischnocnema sambaqui. We propose the Ischnocnema venancioi species series to comprise Ischnocnema venancioi, Ischnocnema hoehnei, and two new species described herein (Ischnocnema parnaso sp. nov. and Ischnocnema colibri sp. nov.). Furthermore, we designate a lectotype for I. venancioi. The nuptial pad present in males is an important character in the genus, and having a large, conspicuous, and glandular-appearing nuptial pad seems to be a synapomorphy for the clade composed of the I. parva, Ischnocnema guentheri, and the newly proposed I. venancioi series.

Translucidithyrium thailandicum gen. et sp. nov.: a new genus in Phaeothecoidiellaceae

A sooty blotch and flyspeck fungus with semi-transparent ascomata was discovered in northern Thailand. Its multi-loculate-like ascomata arrangement is similar to species of Lecideopsella (Schizothyriaceae), but ascomata lack a network-like arrangement. The new genus also has ascospores with appendages at both ends. Maximum parsimony, maximum likelihood, and Bayesian inference analyses of a combined ITS and partial LSU sequence dataset revealed that this new taxon is a member of the family Phaeothecoidiellaceae, but it is distinct from any other genera. Therefore, Translucidithyrium thailandicum gen. et sp. nov. is introduced here with descriptions and illustrations. The discovery of this new genus with sexual characters in Capnodiales will make contributions to the further understanding of sooty blotch and flyspeck fungal group.

Fusarium species and moniliformin occurrence in sorghum grains used as ingredient for animal feed in Argentina.

A survey on Fusarium species and moniliformin (MON) occurrence in sorghum grains collected from one of the main sorghum-producing areas of Argentina was conducted. Also, growth of F. thapsinum, one of the main sorghum pathogens, and MON production under different water activity (aw ) conditions on a sorghum-based medium were determined. Infection of sorghum grains by Fusarium species ranged from 82.5 to 99%; closely related species F. verticillioides, F. thapsinum and F. andiyazi were the most frequently recovered, followed by F. proliferatum and F. subglutinans. By sequencing a portion of the translation elongation factor-1α (TEF-1α) gene and by maximum parsimony analysis, F. verticillioides and closely related species were identified as F. thapsinum, F. andiyazi and F. verticillioides. Species within the F. graminearum species complex (FGSC) were isolated in high frequency. Maximum growth rates of 12 F. thapsinum strains were obtained at 0.995 aw . All evaluated strains were able to produce MON at all aw values tested, but MON production was higher at 0.995-0.982 aw . MON was detected in 41% of the samples at levels ranging from 363.2 to 914.2 µg kg-1 . This study provides new data on the occurrence of Fusarium species in sorghum grains destined for animal consumption in Argentina. The production of MON at different aw values showed that the toxin can be produced under field conditions. The risk to livestock exposed to daily low levels of MON associated with the toxin occurrence in the sorghum grains analyzed is unknown. © 2018 Society of Chemical Industry.

Genetic diversity in a collection of Italian long storage tomato landraces as revealed by SNP markers array

Tomato (Solanum lycopersicum L.) is one of the most important crops worldwide. In this study, we used 7720 genome-wide SNPs to characterize the genetic diversity within a tomato germplasm collection enriched with 64 accessions from southern Italy of the so called “da serbo” type i.e. drought-tolerant and long storage landraces. Notwithstanding the relatively small collection area, 1575 (20.4%) polymorphic SNPs, mostly on Chr11, detected considerable levels of genetic variation. Maximum parsimony analysis of genetic distance revealed four main clusters and clearly separated most “da serbo” landraces from the outgroups. One of the clusters grouped the landraces from the Mount Vesuvius area, though no further indications of a geographic-specific structure were found. STRUCTURE analysis confirmed the presence of four genetic groups within the collection, with admixture between them. A survey of non-synonymous SNPs highlighted several mutations in genes related to stress tolerance and fruit maturation/quality. Overall, our results suggest possible exchange of “da serbo” genetic material between southern Italy regions and indicate that the long storage “da serbo” germplasm could be a promising reservoir of peculiar alleles for traits of interest.

Molecular Phylogenetic Taxonomy of Some Parrotfish Species (Perciformes, Scaridae) From the Red Sea Using -Actin Gene

a--actin gene (ACTA 1) was studied as a potential phylogenetic marker for selected members of Subfamily Scarinea (Scaridae, Perciformes). The samples collected from the Red Sea. The nucleotide sequences of six parrotfish (Scarus niger, Scarus fuscopurpureus, Scarus ferrugineus, Scarus psittacus, Chlorurus gibbus and Hipposcarus harid)were analyzed with respect to their molecular evolution and phylogenetic relationships among themselves and other related percoid species depending on available sequence data. a-skeletal muscle actin gene segments isolated from the skeletal muscle of the six species that were sequenced and recorded in gene bank with the Accession number for the first time. The six-nucleotide sequences compared to fourteen other percoid sequences from Gene Bank/NCBI, altogether comprising 20 percoid sequences and 3 outgroup sequences (Order Scoraeniformes). The scores of p-distance and sequence divergence of the alpha-skeletal muscle actin gene among the tested species were calculated. Studied A+T of the six sequence rates were variant between 44.4 and 52.4 % for all species. The phylogenetic trees for 23 species (6 parrotfish and 14 sequences of other percoid families from GeneBank together with 3 fishes as outgroup) were developed using actin gene and 5 different analytical approaches: Neighbour Joining (NJ), Minimum Evolution (ME), Maximum Parsimony (MP), Maximum Likelihood (ML) and Bayesian Inference method (BI). The analysis revealed a monophyletic origin for the five tested species of the scarinea, which was the principal subfamily investigated (87, 92, 100, 88 and 100% support in our NJ, ME, MP, ML and BI analyses, respectively). While the sixth species Scarus fuscopurpureus of the tested fishes formed a complete separate clade that indicates this species more related to genus labrus than genus scarus .The phylogenetic implications of actin gene or other phylogenetic markers in the family Scaridae or even all families of Order Perciformes until now were shortly discussed.

Reconstructing the phylogenetic history of the tribe Leucocoryneae (Allioideae): Reticulate evolution and diversification in South America

At present, the Allioideae is included within the Amaryllidaceae, which is an economically important bulb crop subfamily that includes onion, garlic, and ornamental species worldwide. The Allioideae includes four tribes geographically disjunct namely: Allieae, widespread in the northern hemisphere, tribe Tulbaghieae distributed in South Africa, and tribes Leucocoryneae and Gilliesieae are endemic to South America. Although we agree with the current tribal circumscription of the Leucocoryneae including Beauverdia, Ipheion, Latace, Leucocoryne, Nothoscordum, and Tristagma, there are still taxonomic and phylogenetic uncertainties regarding the monophyly, phylogenetic relationships, and divergence time of several lineages in a biogeographic context. In this study, a comprehensive molecular phylogeny of the tribe Leucocoryneae was inferred based on nuclear ribosomal ITS and plastid (ndhF and matK) sequences. We used Bayesian inference and maximum parsimony analyses to predict ancestor-descendant relationships. Our results confirmed the monophyly of the four tribes of subfamily Allioideae. Similarly, within the Leucocoryneae, Ipheion, Leucocoryne, and Nothosocordum Sect. Inodorum were also monophyletic; Tristagma and Nothoscordum would be monophyletic if including Ipheion and Beauverdia, respectively. Network analyses were implemented to reveal putative scenarios of reticulate evolution. Both, current and ancestral hybridization events have presumably occurred among species of Nothoscordum Sect. Nothoscordum and Beauverdia favored by spatial overlapping of populations, flowering synchrony and a puzzling pattern of cytogenetic attributes. The estimation of divergence time indicates that the tribe Leucocoryneae originated in the Late Oligocene in southern South America with possible ancestors in Africa. Most crown lineages within the tribe diversified in conjunction with biogeographical events during the Late Miocene to Pliocene. We posit that new suitable environments available after the Andean uplift and during the Age of the Southern Plains provided the favorable geographic setting for the major lineages of Leucocoryneae in southern Pampas, extra-Andean Patagonia, Andean mountains, and in Chile. Hybridization, polyploidization, and Robertsonian translocations of chromosomes have been the driving forces and major sources of speciation in the evolution of tribe Leucocoryneae.

Resolving the systematic positions of enigmatic taxa: Manipulating the chloroplast genome data of Saxifragales

Accurately resolving the phylogeny of enigmatic taxa is always a challenge in phylogenetic inference. Such uncertainties could be due to systematic errors or model violations. Here, we provide an example demonstrating how these factors affect the positioning of Paeoniaceae within Saxifragales based on chloroplast genome data.We newly assembled 14 chloroplast genomes from Saxifragales, and by combining these genomes with those of 63 other angiosperms, three datasets were assembled to test different hypotheses proposed by recent studies. These datasets were subjected to maximum parsimony, maximum likelihood and Bayesian analyses with site-homogeneous/heterogeneous models, different data partitioning strategies, and the inclusion/exclusion of weak phylogenetic signals.Three datasets exhibited remarkable heterogeneity among sites and among taxa of Saxifragales. Phylogenetic analyses under homogeneous models or maximum parsimony showed a closer relationship of Paeoniaceae with herbaceous families in the order. Data partitioning strategies did not change the general tree topology. However, PhyloBayes analysis under the CAT+GTR model resulted in a relationship closer to woody families.We conclude that although genomic data significantly increase the phylogenetic resolution of enigmatic taxa with high support, the phylogenetic results inferred from such data might be analysis or signal dependent. The analytical pipeline outlined here combines phylogenomic inference methods with evaluation of lineage-specific rates of substitution, model selection, and assessment of systematic error. These methods would be applicable to resolve similar difficult questions in the tree of life.

Epitypification, characterization and phylogenetic positioning of Pseudobeltrania cedrelae, the causal agent of pseudobeltrania spot on Cedrela fissilis

SummaryThe fungus Pseudobeltrania cedrelae, the type species of the genus, is the causal agent of an important leaf spot in seedlings and adult plant of cedar (Cedrela fissilis). Due to the contradictory phylogenetic position of the genus Pseudobeltrania, epitypification of P. cedrelae was carried out based on a culture obtained from the same locality and host of the original type. Samples were collected, and 10 isolates of P. cedrelae associated with lesions on cedar leaflets were obtained. For morphological characterization, conidia, conidiophores, conidiogeneous cells, conidiogeneous loci and basal cells were taken both from the fungus obtained from leaf lesions and from that obtained in slide culture. Mycelial growth rates and sporulation were evaluated in six different culture media. For molecular phylogeny, maximum parsimony analyses were performed from the ITS and 28S sequences of the isolates. Both the morphological characteristics of the fungal structures obtained from symptomatic leaves and the slide culture technique presented variations. In foliar lesions, isolates presented the same morphological characteristics as the type material. Mycelial growth rate and sporulation of P. cedrelae were greatest on malt extract agar and V8 juice agar. Pseudobeltrania cedrelae was pathogenic when inoculated into healthy cedar plants. According to the phylogenetic tree, isolates grouped in the same clade, but in a distinct clade of Pseudobeltrania ocoteae. The results suggested that P. ocoteae belongs to the genus Hemibeltrania. This paper presents new information on P. cedrelae that contributes to clarifying the phylogeny of the Beltraniaceae.

Chloroplast variation is incongruent with classification of the Australian bloodwood eucalypts (genus Corymbia, family Myrtaceae).

Previous molecular phylogenetic analyses have resolved the Australian bloodwood eucalypt genus Corymbia (~100 species) as either monophyletic or paraphyletic with respect to Angophora (9–10 species). Here we assess relationships of Corymbia and Angophora using a large dataset of chloroplast DNA sequences (121,016 base pairs; from 90 accessions representing 55 Corymbia and 8 Angophora species, plus 33 accessions of related genera), skimmed from high throughput sequencing of genomic DNA, and compare results with new analyses of nuclear ITS sequences (119 accessions) from previous studies. Maximum likelihood and maximum parsimony analyses of cpDNA resolve well supported trees with most nodes having >95% bootstrap support. These trees strongly reject monophyly of Corymbia, its two subgenera (Corymbia and Blakella), most taxonomic sections (Abbreviatae, Maculatae, Naviculares, Septentrionales), and several species. ITS trees weakly indicate paraphyly of Corymbia (bootstrap support <50% for maximum likelihood, and 71% for parsimony), but are highly incongruent with the cpDNA analyses, in that they support monophyly of both subgenera and some taxonomic sections of Corymbia. The striking incongruence between cpDNA trees and both morphological taxonomy and ITS trees is attributed largely to chloroplast introgression between taxa, because of geographic sharing of chloroplast clades across taxonomic groups. Such introgression has been widely inferred in studies of the related genus Eucalyptus. This is the first report of its likely prevalence in Corymbia and Angophora, but this is consistent with previous morphological inferences of hybridisation between species. Our findings (based on continent-wide sampling) highlight a need for more focussed studies to assess the extent of hybridisation and introgression in the evolutionary history of these genera, and that critical testing of the classification of Corymbia and Angophora requires additional sequence data from nuclear genomes.

Evolutionary relationships of the Tehuelche scallop Aequipecten tehuelchus (Bivalvia: Pectinidae) from the south-western Atlantic Ocean

This study addresses aspects of the phylogenetic relationships of the commercial Tehuelche scallop, Aequipecten tehuelchus s.l. (Bivalvia: Pectinidae), from southern South America using molecular techniques. The Tehuelche scallop presents two different putative subspecies, A. t. tehuelchus and A. t. madrynensis, and a potentially related sympatric species, Flexopecten felipponei. The Tehuelche scallop is a very important component of ecosystems and is the target of artisanal fisheries in the northern Patagonian gulfs of Argentina. Despite its importance, the systematic relationships of these taxa have not been fully addressed. The main goal of this study is to place the Tehuelche scallop within a partial phylogenetic framework of the family Pectinidae. Scallops were sampled at 10 localities distributed along the south-western Atlantic Ocean. Phylogenetic reconstructions were carried out from two mitochondrial (12S rRNA and 16S rRNA) and two nuclear markers (28S rRNA and H3) using Bayesian, maximum likelihood and maximum parsimony analyses. Our phylogenetic analysis indicates that the two putative subspecies of the Tehuelche scallop together with F. felipponei form a monophyletic clade, without differentiating at the specific level. Observed differences would be the result of phenotypic plasticity, probably caused by environmental factors. However, further analysis using genes with faster evolution rate are needed to corroborate it. Our phylogenetic analysis resolved to Aequipecten as polyphyletic. The Tehuelche scallop has a basal position within the Argopecten group and we recommend that it should be transferred to this genus. The relationship between the hypotheses about the origin of the Tehuelche scallop implicit in the literature and our results are discussed.

What about Cryptangieae (Cyperaceae)—a molecular hypothesis about its tribal status and circumscription

We endorse the tribal status of Cryptangieae and discuss its most probable sister-group based on phylogenetic analyses using rbcL, trnL-trnL-trnF and ITS sequences. Cryptangieae were poorly sampled in previous molecular phylogenies of Cyperaceae: only three of about 50 species were sampled and all of them are currently included in the genus Lagenocarpus. We gathered more data about the tribe to verify its monophyletic status and to clarify the relationships among the tribes of subfamily Cyperoideae. Here we present a phylogeny with 18 species of Cryptangieae including all three currently recognized genera. Cryptangieae appears as monophyletic with great support in our Maximum Parsimony and Bayesian Inference analyses based on single and combined matrices. We emphasize the need for morphological, molecular and biogeographical data to achieve a broadly supported interpretation of the early divergent taxa of subfamily Cyperoideae.

Filogenetik Jenis-jenis Annonaceae dari Jawa Timur Koleksi Kebun Raya Purwodadi Berdasarkan Coding dan Non-coding sekuen DNA

Annonaceae species of Purwodadi Botanic Garden collections from East Java encountered problems in species identification based on morphological characters. In addition, the unavailability of molecular data to support identification based on morphological characters hinders accurate identification of species. The aim of this research is to discern phylogenetic of Annonaceae species from East Java collections of Purwodadi Botanic Garden based on coding and non-coding sequence DNA. Annonaceae species used in this study are 30 species, includes outgroup from family of Magnoliaceae. Materials for DNA analysis were young leaves which were processed through DNA extraction, DNA amplification through PCR technique, DNA sequencing and data analysis with Maximum Likelihood (ML), Maximum Parsimony (MP) and Neighbour-Joining (NJ) method analysis. The results show that phylogenetic tree is divided into two subfamilies, i.e. Annonoideae and Malmeoideae. The topology of phylogenetic tree from three DNA molecular marker shows that non-coding sequence DNA (trnL-F molecular marker) has the best grouping of relationship and be able to explain the relationship between species of Annonaceae than rbcL and matK molecular marker as coding sequence DNA. Bootstrap value of trnL-F tree is very weak to high (36-100%) while rbcL is very weak to weak (17-63%) and matK tree is high (98-99%).

Phylogenetic and morphological resolution of the Helobdella stagnalisspecies-complex (Annelida: Clitellata: Hirudinea).

The glossiphoniid freshwater leech, Helobdella stagnalis, was described by Linnaeus 1758 based on common European specimens. The presence of a brown, chitinous scute on the dorsal-anterior surface, as observed on leeches elsewhere in the world, has generally led to the classification of all scute-bearing members of the genus as H. stagnalis. Here we describe the morphology and behavior of the type species from Europe, and analyze H. stagnalis-like specimens collected worldwide. We present evidence for at least four distinct scute-bearing Helobdella species that can be morphologically resolved. Maximum Parsimony (MP) and Bayesian Inference (BI) analyses at the mitochondrial cytochrome c oxidase subunit I (COI) locus further supported this notion, with divergence values suggesting a mid-Miocene ancestor of this successful group of cosmopolitan hirudineans. New species, Helobdella echoensis nov. sp., Helobdella eriensis nov. sp., and Helobdella serendipitious nov. sp., are described, based on morphological, anatomical and molecular data. Current distribution patterns of Helobdella spp. suggest a robust, global dispersal mechanism, as well as local pockets of endemism.