Rosa Trobajo

Morphological, genetic and mating diversity within the widespread bioindicator Nitzschia palea (Bacillariophyceae)

Trobajo R., Clavero E., Chepurnov V.A., Sabbe K., Mann D.G., Ishihara S. and Cox E.J. 2009. Morphological, genetic and mating diversity within the widespread bioindicator Nitzschia palea (Bacillariophyceae). Phycologia 48: 443–459. DOI: 10.2216/08-69.1. Nitzschia palea (Kützing) W. Smith is believed to be a widely distributed diatom and is common in various lotic and lentic freshwater habitats. However, it is also taxonomically problematic. As part of a multidisciplinary study of this diatom, 25 clones identified morphologically as N. palea were isolated from different freshwater habitats around the world (Belgium, Brazil, Egypt, India, Japan, Paraguay, Spain, Sri Lanka and the United Kingdom). Morphological and genetic diversity (using the hypervariable D1–D2 domains of LSU rDNA) were investigated, and an almost complete set of interclonal crossing experiments was carried out. Results indicate that N. palea is not a simple, homogeneous taxon and that N. palea will probably have to be split into three or more species. Molecular and mating groups do not separate along the traditional morphological boundaries among N. palea varieties, in particular between vars palea and debilis, two taxa that are commonly used to discriminate between different degrees of water pollution. At least two of the putative species within the N. palea complex appear to be geographically widespread. Because of the complexity of variation revealed by the LSU, mating and morphometric data, it is clear that further work, using extra genetic markers and new isolates, will be needed to determine the full extent of cryptic and pseudocryptic speciation in N. palea and to investigate whether the segregate species are ecologically differentiated and have value as indicators.

TAXONOMY, LIFE CYCLE, AND AUXOSPORULATION OF NITZSCHIA FONTICOLA (BACILLARIOPHYTA)1

Nitzschia fonticola (Grunow) Grunow is a member of Nitzschia sect. Lanceolatae, a group of taxonomically intractable but ecologically important and widespread diatoms. We investigated the morphology and life cycle in three clones of N. fonticola and all exhibited reduced sexuality, with pedogamous production of auxospores in unpaired gametangia. The auxospores of all clones contained tangles of striplike elements that lay outside the perizonium and were distinct from it in structure and ontogeny. We introduce a new term, incunabula, to refer to such components of the auxospore wall. Semicryptic variation was detected: one clone differed from the other two in valve size and shape, stria density, and fibula density, as well as its nuclear large subunit ribosomal DNA (LSU rDNA) sequence. The implications of reduced sexuality for the taxonomy of sect. Lanceolatae are discussed. A lectotype is designated for N. fonticola from among original material of Grunow, and the application of the name is clarified further by designating illustrations and the LSU sequence AM182191 from one of our clones as epitypes.

The use of partial cox1, rbcL and LSU rDNA sequences for phylogenetics and species identification within the Nitzschia palea species complex (Bacillariophyceae)

Cox1 has been suggested as a barcode marker for diatoms but it has not been tested intensively in any group of closely related species outside Sellaphora. We evaluated the use of cox1, LSU and rbcL for phylogeny and identification in the taxonomically problematic but ecologically important freshwater diatom Nitzschia palea, for which LSU rDNA sequences, mating and morphological data had already been published, and for which DNA and/or clonal isolates were still available; some new isolates were added. The previous and new information concur in suggesting that N. palea is a complex of several or many species. Where cox1 sequences could be obtained, they were more variable than LSU and rbcL and discriminated between lineages that differed in their morphologies, mating compatibilities, LSU sequences or rbcL sequences. Repeated failures for some strains to recover cox1 sequences from DNA that yielded LSU and rbcL suggest that cox1 will be impractical as a universal barcode marker in diatoms until better primers are designed. LSU and rbcL, though less variable than cox1, can be sequenced reliably in N. palea and, together, seem to show sufficient discrimination to be worth further consideration for species recognition.

Morphology and identity of some ecologically important small Nitzschia species

The taxonomy of several small-celled, ecologically significant Nitzschia species, which are frequently confused with each other or whose names are misapplied, is clarified. Following an examination of type material and modern samples by light and electron microscopy, it was concluded that N. frustulum (Kützing) Grunow, N. inconspicua Grunow, N. soratensis E. Morales & Vis and N. invisitata Hustedt are independent species. No morphological basis was found for separating N. frustulum var. subsalina Hustedt or N. boliviana E. Morales & Vis from N. inconspicua and they are therefore placed in synonymy with N. inconspicua. Nitzschia soratensis, described recently from Bolivia, has previously been misidentified in Europe, either as N. inconspicua (from which it differs most obviously in having more bluntly rounded poles, striae within the raphe canal that are composed of triplets, and fibulae that can be seen in light microscopy to widen at their bases) or as N. abbreviata Hustedt ex Simonsen (from which it differs in pore ultrastructure). Nitzschia frustulum resembles N. inconspicua in every morphological feature examined, but with wider valves and consistently higher maximum length.

Effects of salinity on growth and on valve morphology of five estuarine diatoms

The effects of salinity on the growth and valve morphology of five benthic estuarine diatoms (Nitzschia pusilla, N. frustulum, N. palea, N. filiformis var. conferta and Eolimna subminuscula), isolated from both freshwater and brackish/marine habitats, were investigated. The four Nitzschia strains grew well over a broad salinity range, though some (N. pusilla, N. frustulum) showed a broader salinity range tolerance (from fully saline down to at least 9.5 ppt) than others (N. palea, N. filiformis var. conferta had reduced growth at salinities of 16 ppt and above). Salinity significantly affected the valve morphology of the five strains studied. However, there was no consistent pattern in either the morphological characters affected or the direction of the effects. Although significant, the effects of salinity on valve morphology were very small and therefore it seems that the taxonomic usefulness of some of the classical taxonomical characters is not undermined.

The use of diatom assemblages as ecological indicators in highly stratified estuaries and evaluation of existing diatom indices.

Diatom indices are used to evaluate the ecological status of rivers but they have been rarely applied in estuaries. This study aimed to identify the diatom species indicating the main environmental gradients and pressures in a highly stratified estuary; and to evaluate the applicability of existing freshwater diatom indices. Marine influence due to salt-wedge intrusion and sea water mixing appeared as the main factor affecting diatom community. Three diatom assemblages were identified: indicators of riverine conditions (without marine influence), indicators of estuarine conditions (heterogeneous conditions with higher conductivities due to marine influence) and those specifically indicating well-established salt-wedge situations. Nowadays, the main human pressure affecting diatom community in the Ebro Estuary is the hydrological alteration resulting from flow regulation and abstraction. Several limitations were encountered in the application of diatom indices (e.g. inverse response with nutrients; ecologically important species not considered). Therefore, their use in estuaries should be done cautiously.

When is Sampling Complete? The Effects of Geographical Range and Marker Choice on Perceived Diversity in Nitzschia palea (Bacillariophyta)

DNA barcoding, being developed for biomonitoring, requires a database of reference sequences and knowledge of how much sequences can deviate before they are assigned to separate species. The molecular hunt for hidden species also raises the question of species definitions. We examined whether there are objective criteria for sequence-based species delimitation in diatoms, using Nitzschia palea, an important monophyletic indicator species already known to contain cryptic diversity. Strains from a wide geographical range were sequenced for 28S rRNA, COI and rbcL. Homogeneity indices and the Chao index failed to objectively select a precise number of species existing in N. palea as well as an evolutionary method based on coalescence theory. COI always gave higher diversity estimations than 28S rRNA or rbcL. Mating data did not provide a precise calibration of molecular species thresholds. Rarefaction curves indicated that further MOTUs would be detected with more isolates than we sampled (81 clones, 42 localities). Although some genotypes had intercontinental distributions, there was a positive relationship between genetic and geographical distance, suggesting even higher richness than we assessed, given that many regions were not sampled. Overall, no objective criteria were found for species separation; instead barcoding will need a consensual approach to molecular species limits.

Paedogamy and auxosporulation in Nitzschia sect. Lanceolatae (Bacillariophyta)

Mann D.G., Sato S., Rovira L. and Trobajo R. 2013. Paedogamy and auxosporulation in Nitzschia sect. Lanceolatae (Bacillariophyta). Phycologia 52: 204–220. DOI: 10.2216/12-077.1 Paedogamy (fusion of gametes produced within the same gametangium following meiosis) has rarely been reported in diatoms, with fewer than 10 confirmed examples. One of these, reported by L. Geitler, was in a diatom from Illmitz, Lake Neusiedl (Austria), identified as ‘Nitzschia frustulum var. perpusilla’. We observed uniparental auxosporulation in two Nitzschia clones isolated from the lower Ebro River (Catalonia, Spain), morphologically similar to Geitlers material and belonging to the N. inconspicua species complex. We established that the auxospores were formed paedogamously by Feulgen staining of the nuclei and time-lapse microscopy of living cells. However, reinvestigation of Geitlers original cytological preparations revealed differences between the Illmitz and Ebro material with respect to the length of the initial cells, the structure of the perizonium, and the timing of degeneration of superfluous haploid nuclei during gametogenesis, indicating a genetic and possibly a taxonomic separation. Scanning electron microscopic studies of Ebro auxospores revealed a novel form of longitudinal perizonium with bilateral asymmetry, and also scaly incunabula surrounding the unexpanded zygote, which contrast with the strip incunabula of another paedogamous Nitzschia species, N. fonticola. Molecular phylogenies, based on rbcL and partial LSU rDNA sequences, and evaluation of trees constrained to make the paedogamous species monophyletic, indicate that paedogamy probably evolved at least twice independently in Nitzschia sect. Lanceolatae, in the N. inconspicua and N. fonticola lineages.

Holocene palaeoenvironmental evolution of the Ebro Delta (Western Mediterranean Sea): Evidence for an early construction based on the benthic foraminiferal record

Major Mediterranean deltas began to develop during a period between 8000 and 6000 yr BP when the rate of fluvial sediment input overtook the declining rate of sea-level rise. However, different authors have argued that the Ebro Delta primarily formed during the late Middle Ages as a consequence of increased anthropogenic pressure on its river basin and these arguments are supported by the scarcity of previous geological studies and available radiocarbon dates. To reconstruct the environmental evolution of the Ebro Delta during the Holocene, we used micropalaeontological analysis of continuous boreholes drilled in two different locations (Carlet and Sant Jaume) on the central delta plain. Different lithofacies distributions and associated environments of deposition were defined based on diagnostic foraminiferal assemblages and the application of a palaeowater-depth transfer function. The more landward Carlet sequence shows an older and more proximal progradational delta with a sedimentary record composed of inner bay, lagoonal and beach materials deposited between 7600 and >2000 yr BP under rising sea-level and highstand conditions. This phase was followed by a series of delta plain environments reflected in part by the Carlet deposits that formed before 2000 yr BP. The Sant Jaume borehole is located closer to the present coastline and contains a much younger sequence that accumulated in the last 2.0 ka during the development of three different deltaic lobes under highstand sea-level conditions. The results of this study reinforce the idea that the Ebro Delta dates to the early Holocene, similar to other large Mediterranean deltas.

Genetic and Physiological Diversity in the Diatom Nitzschia inconspicua.

Nitzschia inconspicua is an ecologically important diatom species, which is believed to have a widespread distribution and to be tolerant to salinity and to organic or nutrient pollution. However, its identification is not straightforward and there is no information on genetic and ecophysiological diversity within the species. We used morphological, molecular (rbcL and LSU D1–D3), ecophysiological and reproductive data to investigate whether N. inconspicua constitutes a single species with a broad ecological tolerance or two or more cryptic species with shared or different ecological preferences. Molecular genetic data for clones from upstream and deltaic sites in the Ebro River basin (Catalonia, Spain) revealed seven N. inconspicua rbcL + LSU genotypes grouped into three major clades. Two of the clades were related to other Nitzschia and Denticula species, making N. inconspicua paraphyletic and suggesting the need for taxonomic revision. Most clones were observed to be automictic, exhibiting paedogamy, and so the biological species concept cannot be used to establish species boundaries. Although there were morphological differences among clones, we found no consistent differences among genotypes belonging to different clades, which are definable only through sequence data. Nevertheless, separating the genotypes could be important for ecological purposes because two different ecophysiological responses were encountered among them.