Katharine M. Evans

Barcoding Diatoms: Exploring Alternatives to COI-5P

Diatoms are a diverse lineage with species that can be difficult to identify or cryptic, but DNA barcoding, a molecular technique, can assist identification and facilitate studies of speciation and biogeography. The most common region used for DNA barcoding, COI-5P, can distinguish diatom species, but has not displayed universality (i.e., successful PCR amplification from diverse taxa). Therefore, we have assessed the following alternative markers: ∼1400bp of rbcL; 748bp at the 3 end of rbcL (rbcL-3P); LSU D2/D3 and UPA. Sellaphora isolates were used to determine each markers ability to discriminate among closely related species and culture collection material was utilized to explore further marker universality. All of the alternative markers investigated have greater universality than COI-5P. Both full and partial (3P) rbcL regions had the power to discriminate between all species, but rbcL-3P can be sequenced more easily. LSU D2/D3 could distinguish between all but the most closely related species (96%), whereas UPA only distinguished 20% of species. Our observations suggest that rbcL-3P should be used as the primary marker for diatom barcoding, while LSU D2/D3 should be sequenced as a secondary marker to facilitate environmental surveys.

A MOLECULAR SYSTEMATIC APPROACH TO EXPLORE DIVERSITY WITHIN THE SELLAPHORA PUPULA SPECIES COMPLEX (BACILLARIOPHYTA)1

The common and cosmopolitan freshwater benthic diatom Sellaphora pupula (Kütz.) Mereschk. is a model system for studying the nature of species in microalgae; the biological significance of morphological variation in this species complex has been widely demonstrated. The aim of this study was to establish a two‐gene phylogeny (18S rDNA and rbcL) for 23 Sellaphora taxa, including 19 S. pupula aggregate (agg.) demes or species, S. bacillum (Ehrenb.) D. G. Mann, and S. laevissima (Kütz.) D. G. Mann. A range of analyses on separate and combined data sets indicated that Sellaphora is a monophyletic group containing four major clades. Of the traditionally recognized species, S. bacillum and S. laevissima are natural groups, but S. pupula is paraphyletic or polyphyletic because S. bacillum groups with S. pupula“small lanceolate” and S. lanceolata. Thickened bars at the poles of valves within the core “pupula–bacillum” group may be a morphological synapomorphy; the fossil record suggests that this group is at least 12 million years old. Otherwise, there was no clear pattern in the distribution of different morphologies among the major clades; each clade was also heterogeneous with respect to mating system. More intensive and extensive sampling will doubtless uncover even greater diversity; the challenge lies in its interpretation. Our results demonstrate the limitations of paleoecological, ecological, and biogeographical research based on morphospecies.

Highly differentiated populations of the freshwater diatom Sellaphora capitata suggest limited dispersal and opportunities for allopatric speciation

The diversities and distributions of diatoms are much more complex than was ever imagined. To understand the underlying mechanisms, research must focus on evolutionary processes occurring at a population level and employ sufficiently informative molecular markers. Using ten microsatellites and ITS rDNA sequence data, we investigated the genetic structure of populations of the benthic freshwater diatom Sellaphora capitata (until 2004 a cryptic entity within the S. pupula agg. species complex). This is the first time that microsatellites have been used to investigate the genetic structure of any freshwater or benthic microalga. Using an integrated approach (morphology, DNA barcoding and specificity of the microsatellite primers), we verified the identity of 70 S. capitata isolates obtained from lakes in the UK, Belgium and Australia. Standardized F(ST) values were very high (>0.4) and in Bayesian analyses, isolates clustered according to their country of origin, with limited evidence of admixture. However, selected isolates from all countries were sexually compatible, a result consistent with limited ITS divergence. Considering the apparent absence of desiccation-resistant resting stages in most diatoms, we conclude that such levels of differentiation are likely to be a consequence of limited dispersal. With restricted dispersal, previously unacknowledged opportunities for allopatric speciation exist, which may help to explain the huge extant diversity of diatoms.

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.

A proposed protocol for nomenclaturally effective DNA barcoding of microalgae

K.M. Evans and D.G. Mann. 2009. A proposed protocol for nomenclaturally effective DNA barcoding of microalgae. Phycologia 48: 70–74. DOI: 10.2216/08-70.1. A mechanism for giving DNA barcodes nomenclatural status in microalgae via culture-derived epitypes is demonstrated with reference to four species of the freshwater diatom Sellaphora. A fifth, Sellaphora caput, is described as a new species to illustrate application of barcoding via a holotype. Using cox1 barcodes, it is shown that S. capitata has a worldwide distribution, consistent with the ubiquitous dispersal hypothesis.

SCALY INCUNABULA, AUXOSPORE DEVELOPMENT, AND GIRDLE POLYMORPHISM IN SELLAPHORA MARVANII SP. NOV. (BACILLARIOPHYCEAE)1

Uniparental auxosporulation was observed in a monoclonal culture of a Sellaphora clone isolated from the epipelon of a fishpond in the Czech Republic. The cox1 sequence for the clone confirmed that it belonged to the Sellaphora pupula–bacillum species complex but showed significant differences from all previously characterized Sellaphora species, and it is therefore described as S. marvanii sp. nov. Protoplast, valve, and girdle structure resembled those of other Sellaphora species, but a novel finding for all diatoms was a change in girdle structure during the life cycle: the most advalvar girdle band (valvocopula) bore a single line of pores in enlarged postauxospore cells but was entirely plain in small cells and gametangia. The young auxospores were covered by incunabula containing large, delicate, ± circular scales, resembling those of centric diatom auxospores; similar scales have been reported in a few other raphid diatoms (Pseudo‐nitzschia multiseries, Diploneis sp.) but contrast with the strip incunabula of some Nitzschia and Pinnularia and the helmet‐like caps of Neidium. The scales persisted during auxospore expansion, mostly as two caps over the auxospore poles. The transverse perizonium comprised a very wide, closed primary band, flanked by numerous secondary bands whose open ends were strongly incurved toward the center. Initial valves were differentiated from their immediate descendants by the very strong external demarcation of the raphe sternum, irregular shape, and curved transapical profile.

A new integrated approach to taxonomy: the fusion of molecular and morphological systematics with type material in benthic foraminifera

A robust and consistent taxonomy underpins the use of fossil material in palaeoenvironmental research and long-term assessment of biodiversity. This study presents a new integrated taxonomic protocol for benthic foraminifera by unequivocally reconciling the traditional taxonomic name to a specific genetic type. To implement this protocol, a fragment of the small subunit ribosomal RNA (SSU rRNA) gene is used in combination with 16 quantitative morphometric variables to fully characterise the benthic foraminiferal species concept of Elphidium williamsoni Haynes, 1973. A combination of live contemporary topotypic specimens, original type specimens and specimens of genetic outliers were utilised in this study. Through a series of multivariate statistical tests we illustrate that genetically characterised topotype specimens are morphologically congruent with both the holotype and paratype specimens of E. williamsoni Haynes, 1973. We present the first clear link between morphologically characterised type material and the unique SSU rRNA genetic type of E. williamsoni. This example provides a standard framework for the benthic foraminifera which bridges the current discontinuity between molecular and morphological lines of evidence, allowing integration with the traditional Linnaean roots of nomenclature to offer a new prospect for taxonomic stability.

Genotypic diversity and differentiation among populations of two benthic freshwater diatoms as revealed by microsatellites

Given their large population sizes and presumed high dispersal capacity, protists are expected to exhibit homogeneous population structure over large spatial scales. On the other hand, the fragmented and short‐lived nature of the lentic freshwater habitats that many protists inhabit promotes strong population differentiation. We used microsatellites in two benthic freshwater diatoms, Eunotia bilunaris ‘robust’ and Sellaphora capitata, sampled from within a pond and connected ponds, through isolated ponds from the same region to western Europe to determine the spatial scale at which differentiation appears. Because periods of low genotypic diversity contribute to population differentiation, we also assessed genotypic diversity. While genotypic diversity was very high to maximal in most samples of both species, some had a markedly lower diversity, with up to half (Eunotia) and over 90% (Sellaphora) of the strains having the same multilocus genotype. Population differentiation showed an isolation‐by‐distance pattern with very low standardized FST values between samples from the same or connected ponds but high values between isolated ponds, even when situated in the same region. Partial rbcL sequences in Eunotia were consistent with this pattern as isolated ponds in the same region could differ widely in haplotype composition. Populations identified by Structure corresponded to the source ponds, confirming that ‘pond’ is the main factor structuring these populations. We conclude that freshwater benthic diatom populations are highly fragmented on a regional scale, reflecting either less dispersal than is often assumed or reduced establishment success of immigrants, so that dispersal does not translate into gene flow.

Repeated evolution of uniparental reproduction in Sellaphora (Bacillariophyceae)

Diatoms possess a remarkable life cycle in which cell size decreases slowly during vegetative cell division and then increases rapidly via special expanding cells called ‘auxospores’, which are usually formed as a result of biparental sexual reproduction. However, auxospores are sometimes produced by single unpaired cells, i.e. uniparentally. We examined the nature of uniparental auxosporulation in Sellaphora and used a two-gene dataset to study phylogenetic relationships between uniparental and biparental Sellaphora demes and species; we tested whether uniparental reproduction has evolved once or repeatedly in the genus. In at least two of the uniparental demes auxosporulation occurred through autogamy (i.e. intra-tetrad mating within an undivided cell). Maximum likelihood phylogenies indicated four lineages of uniparental Sellaphora and significance tests of alternative topologies, in which combinations of uniparental Sellaphora were constrained to be monophyletic, coupled with likelihood reconstruction of ancestral character states, led to rejection of the hypothesis that uniparental auxosporulation evolved only once in the genus. Uniparentally reproducing lineages appear to arise not infrequently in diatoms but do not persist. Two small extranuclear bodies, apparently containing DNA and lying outside the chloroplast (one close to each pole of the cell), were revealed by DAPI staining.

Ten microsatellite markers for the freshwater diatom Sellaphora capitata.

Ten polymorphic microsatellite markers were developed for the benthic freshwater diatom Sellaphora capitata and tested on 40 isolates from a Belgian pond. Genotyping was very successful (95%). The number of alleles per locus ranged from three to 12 (mean 6.6) and expected heterozygosities from 0.2 to 0.86 (mean 0.67). This is the first time that microsatellite markers have been developed for a freshwater or benthic diatom.