Use of anatomical root markers for species identification in Catasetum (Orchidaceae) at the Portal da Amazônia region, MT, Brazil

Orchidaceae is one of the largest botanical families, with approximately 780 genera. Among the genera of this family, Catasetum currently comprises 166 species. The aim of this study was to characterize the root anatomy of eight Catasetum species, verifying adaptations related to epiphytic habit and looking for features that could contribute to the vegetative identification of such species. The species studied were collected at the Portal da Amazônia region, Mato Grosso state, Brazil. The roots were fixed in FAA 50, cut freehand, and stained with astra blue/fuchsin. Illustrations were obtained with a digital camera mounted on a photomicroscope. The roots of examined species shared most of the anatomical characteristics observed in other species of the Catasetum genus, and many of them have adaptations to the epiphytic habit, such as presence of secondary thickening in the velamen cell walls, exodermis, cortex, and medulla. Some specific features were recognized as having taxonomic application, such as composition of the thickening of velamen cell walls, ornamentation of absorbent root-hair walls, presence of tilosomes, composition and thickening of the cortical cell walls, presence of mycorrhizae, endodermal cell wall thickening, the number of protoxylem poles, and composition and thickening of the central area of the vascular cylinder. These traits are important anatomical markers to separate the species within the genus and to generate a dichotomous identification key for Catasetum. Thus, providing a useful tool for taxonomists of this group

Priming of a DNA metabarcoding approach for species identification and inventory in marine macrobenthic communities

Dissertação de mestrado em Molecular Genetics

Species identification in mammals from mixed biological samples based on mitochondrial DNA control region length polymorphism.

The ability to identify the species origin of an unknown biological sample is relevant in the fields of human and wildlife forensics. However, the detection of several species mixed in the same sample still remains a challenge. We developed and tested a new approach for mammal DNA identification in mixtures of two or three species, based on the analysis of mitochondrial DNA control region interspecific length polymorphism followed by direct sequencing. Contrary to other published methods dealing with species mixtures, our protocol requires a single universal primer pair and is not based on a pre-defined panel of species. Amplicons can be separated either on agarose gels or using CE. The advantages and limitations of the assay are discussed under different conditions, such as variable template concentration, amplicon sizes and size difference among the amplicons present in the mixture. For the first time, this protocol provides a simple, reliable and flexible method for simultaneous identification of multiple mammalian species from mixtures, without any prior knowledge of the species involved.

Molecular markers allow sibling species identification in red wood ants (Formica rufa group)

Red wood ants (Formica rufa group) constitute a group of species that are considered to be among the most promising bioindicators in forest ecosystems. However, because of their morphological similarity and intraspecific variability, morphological species identification can be difficult. Considerable expertise is necessary to discriminate between the sibling species F. lugubris and F. paralugubris, two species that often live in sympatry in the same Alpine forests. New taxonomic tools providing rapid and reliable species identification are needed. We present a simple and reliable molecular technique based on mtDNA (COI gene) and a restriction enzyme for discriminating between F. lugubris and F. paralugubris. We confirm the validity of this method with a Bayesian analysis based on microsatellites. This new molecular tool represents a clear breakthrough for discriminating between F. lugubris and F. paralugubris and is likely to be helpful in large-scale biomonitoring.

Species identification of two sympatric hakes by allozymic markers

Samples were collected from hake species Merluccius australis and M. hubbsi in the south west Atlantic Ocean. Enzyme electrophoretic analysis of the eye, liver and muscle revealed 5 out of 33 genetic loci with species-specific allelic frequencies. These five loci provide a set of genetic markers for individual classification

Comparing multiple criteria for species identification in two recently diverged seabirds

Correct species identification is a crucial issue in systematics with key implications for prioritising conservation effort. However, it can be particularly challenging in recently diverged species due to their strong similarity and relatedness. In such cases, species identification requires multiple and integrative approaches. In this study we used multiple criteria, namely plumage colouration, biometric measurements, geometric morphometrics, stable isotopes analysis (SIA) and genetics (mtDNA), to identify the species of 107 bycatch birds from two closely related seabird species, the Balearic (Puffinus mauretanicus) and Yelkouan (P. yelkouan) shearwaters. Biometric measurements, stable isotopes and genetic data produced two stable clusters of bycatch birds matching the two study species, as indicated by reference birds of known origin. Geometric morphometrics was excluded as a species identification criterion since the two clusters were not stable. The combination of plumage colouration, linear biometrics, stable isotope and genetic criteria was crucial to infer the species of 103 of the bycatch specimens. In the present study, particularly SIA emerged as a powerful criterion for species identification, but temporal stability of the isotopic values is critical for this purpose. Indeed, we found some variability in stable isotope values over the years within each species, but species differences explained most of the variance in the isotopic data. Yet this result pinpoints the importance of examining sources of variability in the isotopic data in a case-by-case basis prior to the cross-application of the SIA approach to other species. Our findings illustrate how the integration of several methodological approaches can help to correctly identify individuals from recently diverged species, as each criterion measures different biological phenomena and species divergence is not expressed simultaneously in all biological traits.

Wood species identification by visual characterization of fibers

Kandidaatintyö tehtiin osana PulpVision-tutkimusprojektia, jonka tarkoituksena on kehittää kuvapohjaisia laskenta- ja luokittelumetodeja sellun laaduntarkkailuun paperin valmistuksessa. Tämän tutkimusprojektin osana on aiemmin kehitetty metodi, jolla etsittiin kaarevia rakenteita kuvista, ja tätä metodia hyödynnettiin kuitujen etsintään kuvista. Tätä metodia käytettiin lähtökohtana kandidaatintyölle. Työn tarkoituksena oli tutkia, voidaanko erilaisista kuitukuvista laskettujen piirteiden avulla tunnistaa kuvassa olevien kuitujen laji. Näissä kuitukuvissa oli kuituja neljästä eri puulajista ja yhdestä kasvista. Nämä lajit olivat akasia, koivu, mänty, eukalyptus ja vehnä. Jokaisesta lajista valittiin 100 kuitukuvaa ja nämä kuvat jaettiin kahteen ryhmään, joista ensimmäistä käytettiin opetusryhmänä ja toista testausryhmänä. Opetusryhmän avulla jokaiselle kuitulajille laskettiin näitä kuvaavia piirteitä, joiden avulla pyrittiin tunnistamaan testausryhmän kuvissa olevat kuitulajit. Nämä kuvat oli tuottanut CEMIS-Oulu (Center for Measurement and Information Systems), joka on mittaustekniikkaan keskittynyt yksikkö Oulun yliopistossa. Yksittäiselle opetusryhmän kuitukuvalle laskettiin keskiarvot ja keskihajonnat kolmesta eri piirteestä, jotka olivat pituus, leveys ja kaarevuus. Lisäksi laskettiin, kuinka monta kuitua kuvasta löydettiin. Näiden piirteiden eri yhdistelmien avulla testattiin tunnistamisen tarkkuutta käyttämällä k:n lähimmän naapurin menetelmää ja Naiivi Bayes -luokitinta testausryhmän kuville. Testeistä saatiin lupaavia tuloksia muun muassa pituuden ja leveyden keskiarvoja käytettäessä saavutettiin jopa noin 98 %:n tarkkuus molemmilla algoritmeilla. Tunnistuksessa kuitujen keskimäärinen pituus vaikutti olevan kuitukuvia parhaiten kuvaava piirre. Käytettyjen algoritmien välillä ei ollut suurta vaihtelua tarkkuudessa. Testeissä saatujen tulosten perusteella voidaan todeta, että kuitukuvien tunnistaminen on mahdollista. Testien perusteella kuitukuvista tarvitsee laskea vain kaksi piirrettä, joilla kuidut voidaan tunnistaa tarkasti. Käytetyt lajittelualgoritmit olivat hyvin yksinkertaisia, mutta ne toimivat testeissä hyvin.

DNA barcoding of Cyclamen species: another approach to species identification

Cyclamen is a diverse genus with some well defined and some intransigent species complexes. Here we report on an attempt to use DNA barcoding techniques to help evaluate species boundaries. DNA barcoding uses multiple samples, each from a different individual of a species, to generate a series of DNA sequences that can be compared for similarity.

Plant species identification using multi-scale fractal dimension applied to images of adaxial surface epidermis

This paper presents the study of computational methods applied to histological texture analysis in order to identify plant species, a very difficult task due to the great similarity among some species and presence of irregularities in a given species. Experiments were performed considering 300 ×300 texture windows extracted from adaxial surface epidermis from eight species. Different texture methods were evaluated using Linear Discriminant Analysis (LDA). Results showed that methods based on complexity analysis perform a better texture discrimination, so conducting to a more accurate identification of plant species. © 2009 Springer Berlin Heidelberg.

Measuring and analyzing color and texture information in anatomical leaf cross sections: An approach using computer vision to aid plant species identification

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Analysis of inteins in the Candida parapsilosis complex for simple and accurate species identification

Inteins are coding sequences that are transcribed and translated with flanking sequences and then are excised by an autocatalytic process. There are two types of inteins in fungi, mini-inteins and full-length inteins, both of which present a splicing domain containing well-conserved amino acid sequences. Full-length inteins also present a homing endonuclease domain that makes the intein a mobile genetic element. These parasitic genetic elements are located in highly conserved genes and may allow for the differentiation of closely related species of the Candida parapsilosis (psilosis) complex. The correct identification of the three psilosis complex species C. parapsilosis, Candida metapsilosis, and Candida orthopsilosis is very important in the clinical setting for improving antifungal therapy and patient care. In this work, we analyzed inteins that are present in the vacuolar ATPase gene VMA and in the threonyl-tRNA synthetase gene ThrRS in 85 strains of the Candida psilosis complex (46 C. parapsilosis, 17 C. metapsilosis, and 22 C. orthopsilosis). Here, we describe an accessible and accurate technique based on a single PCR that is able to differentiate the psilosis complex based on the VMA intein. Although the ThrRS intein does not distinguish the three species of the psilosis complex by PCR product size, it can differentiate them by sequencing and phylogenetic analysis. Furthermore, this intein is unusually present as both mini- and full-length forms in C. orthopsilosis. Additional population studies should be performed to address whether this represents a common intraspecific variability or the presence of subspecies within C. orthopsilosis. Copyright © 2013, American Society for Microbiology. All Rights Reserved.

Species identification, distributioin and reproductive interactions of common smoot-hound and blackspotted smooth-hound shark (Genus mustelus) in the Adriatic Sea

Longstanding taxonomic ambiguity and uncertainty exist in the identification of the common (M. mustelus) and blackspotted (M. punctulatus) smooth-hound in the Adriatic Sea. The lack of a clear and accurate method of morphological identification, leading to frequent misidentification, prevents the collation of species-specific landings and survey data for these fishes and hampers the delineation of the distribution ranges and stock boundaries of the species. In this context, adequate species-specific conservation and management strategies can not be applied without risks of population declining and local extinction. In this thesis work I investigated the molecular ecology of the two smooth-hound sharks which are abundant in the demersal trawl surveys carried out in the NC Adriatic Sea to monitor and assess the fishery resources. Ecological and evolutionary relationships were assessed by two molecular tests: a DNA barcoding analysis to improve species identification (and consequently the knowledge of their spatial ecology and taxonomy) and a hybridization assay based on the nuclear codominant marker ITS2 to evaluate reproductive interactions (hybridization or gene introgression). The smooth-hound sharks (N=208) were collected during the MEDITS 2008 and 2010 campaigns along the Italian and Croatian coasts of the Adriatic Sea, in the Sicilian Channel and in the Algerian fisheries. Since the identification based on morphological characters is not strongly reliable, I performed a molecular identification of the specimens producing for each one the cytochrome oxidase subunit 1 (COI) gene sequence (ca. 640 bp long) and compared them with reference sequences from different databases (GenBank and BOLD). From these molecular ID data I inferred the distribution of the two target species in the NC Adriatic Sea. In almost the totality of the MEDITS hauls I found no evidence of species sympatry. The data collected during the MEDITS survey showed an almost different distribution of M. mustelus (confined along the Italian coasts) and M. punctulatus (confined along the Croatian coasts); just one sample (Gulf of Venice, where probably the ranges of the species overlap) was found to have catches of both the species. Despite these data results suggested no interaction occurred between my two target species at least during the summertime (the period in which MEDITS survey is carried out), I still wanted to know if there were inter-species reproductive interactions so I developed a simple molecular genetic method to detect hybridization. This method is based on DNA sequence polymorphism among species in the nuclear ribosomal Internal Transcribed Spacer 2 locus (ITS2). Its application to the 208 specimens collected raised important questions regarding the ecology of this two species in the Adriatic Sea. In fact results showed signs of hybridization and/or gene introgression in two sharks collected during the trawl survey of 2008 and one collected during the 2010 one along the Italian and Croatian coasts. In the case that it will be confirmed the hybrid nature of these individuals, a spatiotemporal overlapping of the mating behaviour and ecology must occur. At the spatial level, the northern part of the Adriatic Sea (an area where the two species occur with high frequency of immature individuals) could likely play the role of a common nursery area for both species.

Species identification of archived fish bones collected from the Mediterranean Sea 100 years ago using molecular techniques

With the discovery that DNA can be successfully recovered from museum collections, a new source of genetic information has been provided to extend our comprehension of the evolutionary history of species. However, historical specimens are often mislabeled or report incorrect information of origin, thus accurate identification of specimens is essential. Due to the highly damaged nature of ancient DNA many pitfalls exist and particular precautions need to be considered in order to perform genetic analysis. In this study we analyze 208 historical remains of pelagic fishes collected in the beginning of the 20th century. Through the adaptation of existing protocols, usually applied to human remains, we manage to successfully retrieve valuable genetic material from almost all of the examined samples using a guanidine and silica column-based approach. The combined use of two mitochondrial markers cytochrome-oxidase-1(mtDNA COI) and Control Region (mtDNA CR), and the nuclear marker first internal transcriber space (ITS1) allowed us to identify the majority of the examined specimens using traditional PCR and Sanger sequencing techniques. The creation of primers capable of amplifying heavily degraded DNA have great potential for future uses, both in ancient and in modern investigation. The methodologies developed in this study can in fact be applied for other ancient fish specimens as well as cooked or canned samples.

Real-time quantitative broad-range PCR assay for detection of the 16S rRNA gene followed by sequencing for species identification

Here we determined the analytical sensitivities of broad-range real-time PCR-based assays employing one of three different genomic DNA extraction protocols in combination with one of three different primer pairs targeting the 16S rRNA gene to detect a panel of 22 bacterial species. DNA extraction protocol III, using lysozyme, lysostaphin, and proteinase K, followed by PCR with the primer pair Bak11W/Bak2, giving amplicons of 796 bp in length, showed the best overall sensitivity, detecting DNA of 82% of the strains investigated at concentrations of < or =10(2) CFU in water per reaction. DNA extraction protocols I and II, using less enzyme treatment, combined with other primer pairs giving shorter amplicons of 466 bp and 342 or 346 bp, respectively, were slightly more sensitive for the detection of gram-negative but less sensitive for the detection of gram-positive bacteria. The obstacle of detecting background DNA in blood samples spiked with bacteria was circumvented by introducing a broad-range hybridization probe, and this preserved the minimal detection limits observed in samples devoid of blood. Finally, sequencing of the amplicons generated using the primer pair Bak11W/Bak2 allowed species identification of the detected bacterial DNA. Thus, broad-spectrum PCR targeting the 16S rRNA gene in the quantitative real-time format can achieve an analytical sensitivity of 1 to 10 CFU per reaction in water, avoid detection of background DNA with the introduction of a broad-range probe, and generate amplicons that allow species identification of the detected bacterial DNA by sequencing. These prerequisites are important for its application to blood-containing patient samples.