Development of a DNA Barcoding System for Seagrasses: Successful but Not Simple

Seagrasses, a unique group of submerged flowering plants, profoundly influence the physical, chemical and biological environments of coastal waters through their high primary productivity and nutrient recycling ability. They provide habitat for aquatic life, alter water flow, stabilize the ground and mitigate the impact of nutrient pollution. at the coast region. Although on a global scale seagrasses represent less than 0.1% of the angiosperm taxa, the taxonomical ambiguity in delineating seagrass species is high. Thus, the taxonomy of several genera is unsolved. While seagrasses are capable of performing both, sexual and asexual reproduction, vegetative reproduction is common and sexual progenies are always short lived and epimeral in nature. This makes species differentiation often difficult, especially for non-taxonomists since the flower as a distinct morphological trait is missing. Our goal is to develop a DNA barcoding system assisting also non-taxonomists to identify regional seagrass species. The results will be corroborated by publicly available sequence data. The main focus is on the 14 described seagrass species of India, supplemented with seagrasses from temperate regions. According to the recommendations of the Consortium for the Barcoding of Life (CBOL) rbcL and matK were used in this study. After optimization of the DNA extraction method from preserved seagrass material, the respective sequences were amplified from all species analyzed. Tree- and character-based approaches demonstrate that the rbcL sequence fragment is capable of resolving up to family and genus level. Only matK sequences were reliable in resolving species and partially the ecotype level. Additionally, a plastidic gene spacer was included in the analysis to confirm the identification level. Although the analysis of these three loci solved several nodes, a few complexes remained unsolved, even when constructing a combined tree for all three loci. Our approaches contribute to the understanding of the morphological plasticity of seagrasses versus genetic differentiation

The phylogeography of Indoplanorbis exustus (Gastropoda: Planorbidae) in Asia

<p>Abstract</p> <p>Background</p> <p>The freshwater snail <it>Indoplanorbis exustus </it>is found across India, Southeast Asia, central Asia (Afghanistan), Arabia and Africa. <it>Indoplanorbis </it>is of economic importance in that it is responsible for the transmission of several species of the genus <it>Schistosoma </it>which infect cattle and cause reduced livestock productivity. The snail is also of medical importance as a source of cercarial dermatitis among rural workers, particularly in India. In spite of its long history and wide geographical range, it is thought that <it>Indoplanorbis </it>includes only a single species. The aims of the present study were to date the radiation of <it>Indoplanorbis </it>across Asia so that the factors involved in its dispersal in the region could be tested, to reveal potential historical biogeographical events shaping the phylogeny of the snail, and to look for signs that <it>I. exustus </it>might be polyphyletic.</p> <p>Results</p> <p>The results indicated a radiation beginning in the late Miocene with a divergence of an ancestral bulinine lineage into Assam and peninsular India clades. A Southeast Asian clade diverged from the peninsular India clade late-Pliocene; this clade then radiated at a much more rapid pace to colonize all of the sampled range of <it>Indoplanorbis </it>in the mid-Pleistocene.</p> <p>Conclusions</p> <p>The phylogenetic depth of divergences between the Indian clades and Southeast Asian clades, together with habitat and parasitological differences suggest that <it>I. exustus </it>may comprise more than one species. The timescale estimated for the radiation suggests that the dispersal to Arabia and to Southeast Asia was facilitated by palaeogeographical events and climate change, and did not require human involvement. Further samples from Afghanistan, Africa and western India are required to refine the phylogeographical hypothesis and to include the African Recent dispersal.</p

Maximize Resolution or Minimize Error? Using Genotyping-By-Sequencing to Investigate the Recent Diversification of Helianthemum (Cistaceae)

A robust phylogenetic framework, in terms of extensive geographical and taxonomic sampling, well-resolved species relationships and high certainty of tree topologies and branch length estimations, is critical in the study of macroevolutionary patterns. Whereas Sanger sequencing-based methods usually recover insufficient phylogenetic signal, especially in recently diversified lineages, reduced-representation sequencing methods tend to provide well-supported phylogenetic relationships, but usually entail remarkable bioinformatic challenges due to the inherent trade-off between the number of SNPs and the magnitude of associated error rates. The genus Helianthemum (Cistaceae) is a species-rich and taxonomically complex Palearctic group of plants that diversified mainly since the Upper Miocene. It is a challenging case study since previous attempts using Sanger sequencing were unable to resolve the intrageneric phylogenetic relationships. Aiming to obtain a robust phylogenetic reconstruction based on genotyping-by-sequencing (GBS), we established a rigorous methodological workflow in which we i) explored how variable settings during dataset assembly have an impact on error rates and on the degree of resolution under concatenation and coalescent approaches, ii) assessed the effect of two extreme parameter configurations (minimizing error rates vs. maximizing phylogenetic resolution) on tree topology and branch lengths, and iii) evaluated the effects of these two configurations on estimates of divergence times and diversification rates. Our analyses produced highly supported topologically congruent phylogenetic trees for both configurations. However, minimizing error rates did produce more reliable branch lengths, critically affecting the accuracy of downstream analyses (i.e. divergence times and diversification rates). In addition to recommending a revision of intrageneric systematics, our results enabled us to identify three highly diversified lineages in Helianthemum in contrasting geographical areas and ecological conditions, which started radiating in the Upper Miocene.España, MINECO grants CGL2014- 52459-P and CGL2017-82465-PEspaña, Ministerio de Economía, Industria y Competitividad, reference IJCI-2015-2345

DECIPHERING THE EVOLUTIONARY HISTORY OF THE MONTANE NEW GUINEA AVIFAUNA: COMPARATIVE PHYLOGEOGRAPHY AND INSIGHTS FROM PALEODISTRIBUTIONAL MODELING IN A DYNAMIC LANDSCAPE

Integrating comparative phylogeographic methods with taxon-specific paleodistributional modeling provides a powerful approach for assessing historical environmental factors that have contributed to patterns of population genetic structure and species formation. Herein, I reconcile spatial analyses of genetic diversity with contemporary and paleoecological niche reconstructions in four co-distributed montane passerines to examine how Pleistocene climate change and topographic relief have influenced avian diversification across the New Guinea highlands. Phylogeographic analyses revealed substantial disparity in the distribution of genetic diversity among focal taxa, with Peneothello cyanus and Crateroscelis robusta exhibiting deep divergences along the Strickland River Valley, whereas Rhipidura atra and Amblyornis macgregoriae displayed evidence of gene flow and shallow genetic structure across this biogeographic boundary. Patterns of population genetic structure in P. cyanus and C. robusta were largely congruent with the distribution of contemporary sky-islands and historical population connectivity inferred from Last Glacial Maximum ecological niche reconstructions; however, Mantel tests indicate an isolation-by-distance effect has also impacted the distribution of genetic diversity in each of these taxa. By contrast, R. atra and A. macgregoriae exhibited weak geographic structure and indications of admixture or ancestral polymorphism among most sky-island populations, yet have maintained highly divergent lineages in the Vogelkop and Huon Peninsula, respectively. Signatures of demographic expansion were observed across each species complex, corroborating elevational shifts and range expansion predicted by Last Glacial Maximum ecological niche models. Although differences in dispersal capacity may have contributed to the discordant evolutionary histories among these taxa, limitations of the mtDNA data set preclude assessing the impact of stochastic or selective processes with confidence. This investigation yields novel insight into the evolutionary dynamics that have shaped patterns of avian diversification and historical demography across the New Guinea highlands. Moreover, the phylogenetic relationships recovered within these geographically structured lineages have important implications for understanding the evolution of phenotypic traits, redefining species limits, and clarifying areas of endemism--knowledge critical to guiding future biodiversity investigation and developing informed conservation policies across the region

Choosing and Using Introns in Molecular Phylogenetics

Introns are now commonly used in molecular phylogenetics in an attempt to recover gene trees that are concordant with species trees, but there are a range of genomic, logistical and analytical considerations that are infrequently discussed in empirical studies that utilize intron data. This review outlines expedient approaches for locus selection, overcoming paralogy problems, recombination detection methods and the identification and incorporation of LVHs in molecular systematics. A range of parsimony and Bayesian analytical approaches are also described in order to highlight the methods that can currently be employed to align sequences and treat indels in subsequent analyses. By covering the main points associated with the generation and analysis of intron data, this review aims to provide a comprehensive introduction to using introns (or any non-coding nuclear data partition) in contemporary phylogenetics

Assessing the potential of RAD-sequencing to resolve phylogenetic relationships within species radiations: The fly genus Chiastocheta (Diptera: Anthomyiidae) as a case study

Determining phylogenetic relationships among recently diverged species has long been a challenge in evolutionary biology. Cytoplasmic DNA markers, which have been widely used, notably in the context of molecular barcoding, have not always proved successful in resolving such phylogenies. However, with the advent of next-generation-sequencing technologies and associated techniques of reduced genome representation, phylogenies of closely related species have been resolved at a much higher detail in the last couple of years. Here we examine the potential and limitations of one of such techniques—Restriction-site Associated DNA (RAD) sequencing, a method that produces thousands of (mostly) anonymous nuclear markers, in disentangling the phylogeny of the fly genus Chiastocheta (Diptera: Anthomyiidae). In Europe, this genus encompasses seven species of seed predators, which have been widely studied in the context of their ecological and evolutionary interactions with the plant Trollius europaeus (Ranunculaceae). So far, phylogenetic analyses using mitochondrial markers failed to resolve monophyly of most of the species from this recently diversified genus, suggesting that their taxonomy may need a revision. However, relying on a single, non-recombining marker and ignoring potential incongruences between mitochondrial and nuclear loci may provide an incomplete account of the lineage history. In this study, we applied both classical Sanger sequencing of three mtDNA regions and RAD-sequencing, for reconstructing the phylogeny of the genus. Contrasting with results based on mitochondrial markers, RAD-sequencing analyses retrieved the monophyly of all seven species, in agreement with the morphological species assignment. We found robust nuclear-based species assignment of individual samples, and low levels of estimated contemporary gene flow among them. However, despite recovering species’ monophyly, interspecific relationships varied depending on the set of RAD loci considered, producing contradictory topologies. Moreover, coalescence-based phylogenetic analyses revealed low supports for most of the interspecific relationships. Our results indicate that despite the higher performance of RAD-sequencing in terms of species trees resolution compared to cytoplasmic markers, reconstructing inter-specific relationships among recently-diverged lineages may lie beyond the possibilities offered by large sets of RAD-sequencing markers in cases of strong gene tree incongruence

The development of nuclear protein coding genes as phylogenetic markers in bark and ambrosia beetles (Coleoptera: Curculionidae)

Bark and ambrosia beetles are grouped into two different subfamilies (Scolytinae and Platypodinae), within the superfamily Curculionoidea (more than 60,000 described species). These insects constitute a large part (circa 8,000 species) of the advanced weevils (family Curculionidae). The subfamilies Scolytinae and Platypodinae were traditionally considered closely related, due to anatomical affinities and similar ecological behavior of their members. Indeed, these beetles present morphological modifications which allow them to spend almost all the entire lifecycle in tunnels constructed mainly in dead wood, though showing extraordinary variation in ecological adaptations to thrive in different niches. Despite the large interest focused on Scolytinae and Platypodinae which include economically important pests, the evolutionary history of these two groups is largely unclear (especially for Scolytinae) as well as their precise placement in the weevil tree. Due to the high number of species and the lack of molecular markers, obtaining high phylogenetic resolution for framing the timing and ecological circumstances under which each of the largest radiations originated still represent a great challenge. Even though this is one of the beetle taxa where more efforts were concentrated in collecting molecular data, the low phylogenetic resolution at deeper nodes has not been markedly improved adding only a few protein coding genes. Morphological characters in larvae, pupae and adults together with few mitochondrial and nuclear molecular markers clarified only a limited number of important evolutionary issues in Scolytinae, while Platypodinae phylogeny is significantly more resolved. This PhD research project focused on the development and standardization of nuclear protein coding genes as phylogenetic markers for weevils. One hundred genes were tentatively PCR amplified and sequenced with &lsquo;classic&rsquo; Sanger technology for different species of Scolytinae, Platypodinae and other weevils. After this preliminary screening, unsuitable genes were discarded and the most promising ones were further tested in their capacity to recover monophyly for well-supported tribes. A total of sixteen protein coding genes emerged as first choice markers for reconstructing the phylogeny of Scolytinae, a subset of them were tested in other members of Curculionoidea and additional eighteen markers were shown to present different degree of utility for shallow level phylogenetics in weevils (e.g. tribes, genera and at population level). In the first section of this study (paper I), the procedure of development and optimization of each selected marker was described. Information on the intron length and number were reported for all the sixteen nuclear genes. Problems of unspecific amplification or primer failure in particular taxa were also emphasized. Finally, the novel genes were tested under different methods of phylogeny reconstruction (NJ, maximum parsimony and Bayesian inference), for their ability to recover well-established relationships among closely related species. The integrative knowledge provided by comparison among the different analyses allowed ranking the selected markers according to their utility for higher level phylogenetics in Scolytinae. In the second section of this work, a total of 18 markers (five previously defined and 13 out of the 16 developed in this study) were used to reconstruct the phylogeny of the subfamily Scolytinae applying two different phylogenetic methods: maximum parsimony and Bayesian inference. Among the major findings, the tribe Scolytini and the genus Microborus were confirmed to be early divergent lineages. However, their placement at the base of the Scolytinae tree or close to other subfamilies in the weevil tree remains to be clarified. The tribe Hypoborini was recovered as the sister lineage to a group containing the species-rich Dryocoetini and Ipini. Better resolution was achieved within different tribes and the placement of a few enigmatic species was unambiguously solved, but the relationships among older tribes remained elusive (paper II). Finally, ten genes (five developed in this study) were used to reconstruct the phylogeny of different weevil families and subfamilies (paper III). All the analyses placed the subfamily Platypodinae as the sister lineage to Dryophthorinae with high node support, therefore more distantly related to Scolytinae

Phylogeny and Classification of the Trapdoor Spider Genus Myrmekiaphila: An Integrative Approach to Evaluating Taxonomic Hypotheses

Background: Revised by Bond and Platnick in 2007, the trapdoor spider genus Myrmekiaphila comprises 11 species. Species delimitation and placement within one of three species groups was based on modifications of the male copulatory device. Because a phylogeny of the group was not available these species groups might not represent monophyletic lineages; species definitions likewise were untested hypotheses. The purpose of this study is to reconstruct the phylogeny of Myrmekiaphila species using molecular data to formally test the delimitation of species and species-groups. We seek to refine a set of established systematic hypotheses by integrating across molecular and morphological data sets. Methods and Findings: Phylogenetic analyses comprising Bayesian searches were conducted for a mtDNA matrix composed of contiguous 12S rRNA, tRNA-val, and 16S rRNA genes and a nuclear DNA matrix comprising the glutamyl and prolyl tRNA synthetase gene each consisting of 1348 and 481 bp, respectively. Separate analyses of the mitochondrial and nuclear genome data and a concatenated data set yield M. torreya and M. millerae paraphyletic with respect to M. coreyi and M. howelli and polyphyletic fluviatilis and foliata species groups. Conclusions: Despite the perception that molecular data present a solution to a crisis in taxonomy, studies like this demonstrate the efficacy of an approach that considers data from multiple sources. A DNA barcoding approach during the species discovery process would fail to recognize at least two species (M. coreyi and M. howelli) whereas a combine

Pleistocene glacial refugia across the Appalachian Mountains and coastal plain in the millipede genus Narceus: Evidence from population genetic, phylogeographic, and paleoclimatic data

Background: Species that are widespread throughout historically glaciated and currently nonglaciated areas provide excellent opportunities to investigate the role of Pleistocene climatic change on the distribution of North American biodiversity. Many studies indicate that northern animal populations exhibit low levels of genetic diversity over geographically widespread areas whereas southern populations exhibit relatively high levels. Recently, paleoclimatic data have been combined with niche-based distribution modeling to locate possible refugia during the Last Glacial Maximum. Using phylogeographic, population, and paleoclimatic data, we show that the distribution and mitochondrial data for the millipede genus Narceus are consistent with classical examples of Pleistocene refugia and subsequent post-glacial population expansion seen in other organismal groups. Results: The phylogeographic structure of Narceus reveals a complex evolutionary history with signatures of multiple refugia in southeastern North America followed by two major northern expansions. Evidence for refugial populations were found in the southern Appalachian Mountains and in the coastal plain. The northern expansions appear to have radiated from two separate refugia, one from the Gulf Coastal Plain area and the other from the mid-Atlantic coastal region. Distributional models of Narceus during the Last Glacial Maximum show a dramatic reduction from the current distribution, with suitable ecological zones concentrated along the Gulf and Atlantic coastal plain. We found a strong correlation between these zones of ecological suitability inferred from our paleo-model with levels of genetic diversity derived from phylogenetic and population estimates of genetic structuring. Conclusion: The signature of climatic change, during and after the Pleistocene, on the distribution of the millipede genus Narceus is evident in the genetic data presented. Niche-based historical distribution modeling strengthens the conclusions drawn from the genetic data and proves useful in identifying probable refugia. Such interdisciplinary biogeographic studies provide a comprehensive approach to understanding these processes that generate and maintain biodiversity as well as the framework necessary to explore questions regarding evolutionary diversification of taxa. Originally published BMC Evolutionary Biology, Vol. 9, No. 25, Jan 200

SYSTEMATICS AND EVOLUTION OF ANTENNARIA (ASTERACEAE, GNAPHALIEAE) AND NEW INSIGHTS INTO THE ANTENNARIA ROSEA POLYPLOID AGAMIC COMPLEX

SYSTEMATICS AND EVOLUTION OF ANTENNARIA (ASTERACEAE, GNAPHALIEAE) AND NEW INSIGHTS INTO THE ANTENNARIA ROSEA POLYPLOID AGAMIC COMPLE