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Rivers are constantly in motion in a unidirectional flow; therefore it can be difficult assessing water quality measurements such as nutrient levels and other pollutants. It is estimated that there are hundreds of thousands of species of diatoms, with some researchers saying there could be up to one million species found in both freshwater and marine systems. Micro-algae, namely diatoms have been used as biological indicators in wetlands and watercourses for many years and can tell us about the ecosystem in which they live. Before one can use diatoms with confidence as biological indicators, however, diatom taxonomy must be reexamined for accuracy. Moreover, given that diatoms are extremely important ecological organisms, further studies should be undertaken to better understand micro-algae since they have not been studied extensively.
Because their taxonomy is in a state of flux, varying protocols regarding diatom identification have created confusion in their classification assessments. More accessible and newer technologies have lead to revisions of taxonomy, describing new genera and species, and providing ample evidence to query the accuracy of diatoms using morphology only. Specimens are often identified using morphological characteristics. These characteristics include shape, design and consistency and are often utilized for making comparisons of diatoms that have been described and identified using keys, photographs, drawings, microscope slides and other means such as on-line algae websites. This work is challenging and lengthy due to the enormous estimated number of diatom species. The similarities between species are difficult to differentiate because of their life cycle; asexual and sexual, and changes in structure because of phenotypic plasticity. Many researchers believe that using DNA barcoding would be a valuable tool to help identify diatoms and provide a consistent categorization methodology that can be used by diatomists, ecologists, limnologists, biologists, environmental researchers and anyone interested in the study of diatoms.
Introduction to Diatom Biology
Diatoms are protists belonging to the phylum Heterokontophyta. They are eukaryotes known as brown algae belonging to the class Bacillariophyceae. Researchers believe that diatoms emerged from secondary endosymbiosis in the early cretaceous period about 150 million years ago (mya) giving rise to centric diatoms and approximately 70 mya for pennate diatoms. Molecular studies have indicated that pennate diatoms, considered a monophyletic group, are derived from centric diatoms (Round and Crawford, 1990; Hoek et al., 1995; Delwiche et al., 1997). Diatoms are universally distributed and are ubiquitous in all types of aquatic environments. They can exist as single cells (5µm - 150µm) or form large chains or colonies, and can be branching, filamentous or tube forming.
Each diatom cell is surrounded by a cell wall known as a frustule, which consists of amorphous polymerized silica that resembles a Petri dish that overlaps a smaller Petri dish (Figure 1). The smaller frustule is known as the hypotheca and the larger frustule is called the epitheca. (Round, 1971; Hoek et al., 1997).
The frustules are elaborately ornamented, with a jewelry or artwork like appearance and can be quite beautiful. The wall structure is so consistent that diatom walls are sometimes used to calibrate high-precision instruments, including light microscopes.
There are two basic body shapes of diatoms based on symmetry, centric and pennate (Figure 2). Centric diatoms are typically round with radial symmetry but can be star shaped or can have three or four corners. The centric diatoms are from the order Centrales. Some pennate diatoms have bilateral symmetry and may be shaped like a pen, while other pennates have thicker midsections. All pennates have two mirror imaged valves with an oil filled vacuole called a raphe. The raphe helps the diatom slide on substrates. The pennate diatoms are from the order Pennales. They may be found as free floaters (i.e., plankton), but are more often attached to rocks, sticks, plants, larger diatoms and other substrates (Round et al., 1990).
The pennate diatoms are typically solitary cells, while the centric diatoms tend to be solitary or chain forming and are linked by their cell wall or membrane. The pennate and centric diatoms reflect a major ecological difference since the centrales are found more often in marine environments as phytoplankton and the pennates are typically found in freshwater attached to substrates or as plankton (Round, 1971).
Asexual reproduction in diatoms is the predominant mode. Diatoms are vegetatively diploid. In most species, when a diatom divides asexually the mother cell will produce two diploid daughter cells (Figure 3 and 7). One daughter cell inherits the larger half of the frustule (epitheca) while the second inherits the smaller half (hypotheca).
As a result of asexual division, the average size of diatom cells in the population gets smaller with time (Round et al., 1990). Once such cells reach a certain minimum size, they reverse this decline by means of sexual reproduction by forming an auxospore, a reproductive cell formed by the union of two cells that have diminished in size through repeated divisions. Sexual reproduction, therefore, is initiated predominantly in diatoms of small size (Round et al., 1990). Because of the alternative diatom life cycles, uncertainty and discrepancies in the accuracy of nomenclature is common.
Ecology of Diatoms
Ecologically diatoms are diverse. They can be planktonic and can move with the current and are found in the water column. They may be epiphytic, attaching to other submerged plants including macrophytes and larger diatoms, or epilithic, attaching to the aquatic substrate, such as pebbles, rocks, and other hard surfaces and as epipelons which are found in sediments (Stevenson and Pan, 1999). Planktonic diatoms in freshwater and marine environments can exhibit a bloom when conditions, such as light and nutrients in the upper mixed layer, are favorable. Blooms are most notable during the start of spring and warmer summer months when they can quickly dominate phytoplankton communities (Furnas, 1990).
iatoms as Biological Indicators
Diatoms have been widely used as biological indicators of water quality and can be utilized to detect chemical conditions in aquatic environments (McCormick and Cairns, 1994; Stoermer and Smol, 1999). Diatoms reproduce and respond rapidly to changes in their environment and are therefore good indicator organisms. They can be used as a measure of environmental health and change, such as a pollution event, and can therefore provide a better understanding and important information on wetland, stream, and other habitat changes as well as aquatic restoration projects (Rott, 1991).
Because diatom walls are made of silica, the preparation process allows diatom samples to be preserved and kept in storage for future analyses, making them an indispensable tool for further investigation (Stevenson and Pan, 1999). Researchers have shown that changes in diatom communities are frequently related to poor water quality, such as high or low pH, pollution from mining, contamination from stormwater runoff, thermal pollution, and other anthropogenic activities that influence diatom populations and communities (Stevenson and Pan, 1999; Mann, 1996). Diatoms are sensitive to the aforementioned and can be used to help us make better land-use decisions, and incorporate beneficial management practices when managing drinking water and fragile water systems, and surface and ground water management planning. This can be accomplished by utilizing diatoms as biological indicators for water quality assessments, provided they are accurately identified.
Since diatom frustules slides are easy to keep for long periods of time, they can be used for future reference (Stevenson and Pan, 1999). This is one of the features that can make diatoms useful as environmental tools in a number of fields, including paleoclimatology, ecology, geology, anthropology, biomonitoring, and paleontology. They are of significant ecological importance as they are the major food source for both marine and freshwater organisms; the base of the food chain within the ecological community. Diatoms are important as well for the global carbon and silica cycles (Smetacek, 1999; Kemp et al., 2000). Interestingly micro-algae and macro-algae are often found in food products, drinks, shampoo, cosmetics, are used as insect pesticides and are found in other products (Round et al, 1996).
Diatoms are major primary producers and contribute 25-40% of the total production on earth (Van Den Hoek et al., 1997). Bacillariophyceae and other algae are important contributors of energy flow. They are essential for the cycling of nutrients in surface waters and other water bodies. Since they are the base of the food chain, they create a balance within aquatic ecosystems and are important to all life on Earth (Sgro and Johansen, 1995; Van Den Hoek et al., 1997).
Since the eighteenth century the study of diatom taxonomy has been ongoing though it wasn 't until Diatom taxonomy ha s been studied since the late eighteenth century; however, the first real advances in the field came in the the early nineteenth century when researchers started using diatoms to help make improvements in microscope resolution and the first real advances began to occur. In 1847, Joseph Hooker confirmed Ehrenberg's discovery of Diatomaceae and viewed diatoms under the microscope extensively when on board the H.M.S. Challenger. Hooker made many drawings of diatom morphology and their life cycle, though at the time unaware that some of the life cycle changes he was viewing were of the same diatom species (Blinderman and Joyce, 1998).
Diatoms were once identified solely by morphology using light microscopes. Until the scanning electron microscope (SEM) and transmission electron microscope (TEM) became more widely available, it was nearly impossible to distinguish the ultrastructures of these microscopic algae. With the use of DNA sequencing, molecular data, in conjunction with SEM and TEM for morphological identification, better assurance of the correct identification and classification of diatoms should occur. They should be used, however, with some degree of discretion since a genomic region hasn't been found that works consistently for all diatoms (Silva, 2007).
Nucleotide sequence analysis is still open for debate and interpretation as to whether mitochondrial cox1 (a protein found in the mitochondria) is adequate for all diatom species. Even with the cox1 debate, DNA barcoding can be an important means for proper identification and should be considered for diatom taxonomy purposes (Silva, 2007; Evans and Mann, 2009).
Although ecologists believe that diatoms make good indicators of water quality and can be used for geological, ecological, and limnological studies, others believe that unless the taxonomy is accurate the data is subjective and likely incorrect. Without knowing the diversity of diatoms, the concerns with phenotypic plasticity, life cycle, cryptic species, along with environmental factors, can create inadequate taxonomy of diatoms and may be difficult to resolve (Mann and Droop, 1996; Kooistra et al., 2008; Vyverman et al., 1998; 2007; Darling et al., 2004).
There has been much debate as to whether morphology alone can accurately identify diatoms. Archibald (1984) highlighted that because of incorrect taxonomy of diatoms by inexperienced taxonomists many water quality assessments and other studies resulted in scientific and technical inaccuracies (Moniz and Kaczmarska, 2009). Perhaps most importantly, until a genome region is found that possesses enough variation that is informative and able to differentiate between divergent diatom lineages, having the generic taxonomy accurate may be sufficient enough to differentiate diatom attributes. The problem remains, however, with renaming already named species. Diatom taxonomy is currently in a state of significant transformation and will continue to evolve for the unforeseeable future as genera continues to be divided and new genera and species are created (John, 1993b; John, 1998).
As noted above, diatom taxa have been characterized and identified by valve morphology, but taxonomy of diatoms is tenuous due to phenotypic polymorphisms (Sarno et al., 2005; Amato et al., 2007; Vanormelingen et al., 2008, Evans and Mann, 2009). When exposed to different environmental factors, such as pH, chemical conditions, and ecological changes, phenotypic plasticity, a property by which a genotype may produce different phenotypes (physical characteristics of an organism), can occur among diatoms. Diatom valve shape and size, and consistency can be affected by photosynthetic potential and physical absorption of nutrients, which can affect their rate of growth (Bartual, 2008).
Figure 8. Different morphs of Fragilaria construens from Avery Pond, CT (USA) as shown by SEM. First column: typical morphs of F. contruens, F. construens aff. var. subsalina and F. construens var. venter. Second column: morphs with wider areolae arranged in shortened striae. Third column: morphs with wider areolae arranged in longer striae. Scale bars (from top to bottom): first column: 2, 5, and 2 µm; second column: 2, 2, and 2 µm; third column: 2, 2, and 2 µm.
Phaeodactylum tricornutum have been extensively studied because of its phenotypic expression when changes in temperature, photosynthetic rate, and other environmental factors occur. They produce oval, fusiform, and triradiate morphs during these changes (Figure 9). Researchers have extensive knowledge regarding Phaeodactylum tricornutum plasticity features and have written various papers noting morphological alterations attributable to environmental fluctuations (Gutenbrunner et al., 1994, Morales and Trainor, 2002).
Figure 9: Phenotypic response of the diatom Phaeodactylum tricornutum Bohlin to experimental changes in their organic carbon system
Cryptic species are those that are reproductively isolated from each other and therefore genetically divergent; however their morphology is very similar and in some cases nearly identical. One of the better-studied cryptic species complexes of diatoms are in Sellaphora pupula agg, a widespread and cosmopolitan benthic freshwater group of species. There has been an ample amount of life cycle and morphological data generated on this species (Behnke et al., 2004; Mann and Droop, 1996; Mann, 1999). Because of these studies, the data have verified a large number of pseudo, cryptic, and semi-cryptic species.
When examining Figure 10 (Amato, 2007), one can see the slight morphological differences between micrographs A and B (light microscopy), and C and D (TEM). The diatoms were thought to be the same species until further diagnostic methods were employed. Only then were those slight variances observed resulting in the formal recognition of two species.
Figure 10: Cryptic species
Micrographs from light (A,B) and transmission electron microscopy (C,D) of Pseudo-nitzschia calliantha pse4 (A-C) and Pseudo-nitzschia delicatissima del1 (B-D). Solid circles indicate the overlapping region between two adjacent cells in a chain, dotted arrows the fibulae, solid arrows the wider separation of the two central fibulae, solid ellipses the striae, dotted ellipses the interstriae, and arrowheads the poroids. Scale bars: 20 mm (A,B), 1mm (C,D). Amato et. al 2007
Amato (2007) suggests that identifying diatoms by their morphological features alone are not suitable for accurate identification because of cryptic characteristics and are difficult if not impossible to distinguish (Amato et al., 2007).
One hypothesis of determining molecular alpha-taxonomy is based on the divergence of a small DNA fragment that is variable enough to determine interspecific and even intraspecific genetic differentiation. A DNA database specifically for micro-algae, similar to the rapidly growing animal phyla found in the Canadian Barcode of Life or GenBank, may be employed as a tool to distinguish between genera and species. It may also be useful to determine how diatoms can be utilized effectively for research, including water quality biological assessments, as well as phylogenetic, and biogeographic studies (Hajibabaei et al., 2007; Moniz and Kaczmarska et al., 2009).
Molecular data has the potential to enhance the identification of diatoms. DNA sequencing may improve diatom nomenclature. Further methods need to be employed to evaluate diatom identity and to develop an improved approach for water quality characterization, facilitating the conservation of surface and groundwater. To accomplish this, proper identification must be employed. Workers have traditionally utilized morphological features for identifying diatoms. However, in recent times nucleotide sequence analysis has been proposed as a tool for diatom taxonomy (Jahn et al., 2007; Evans et al., 2007; Kaczmarska et al., 2007).
Recently many questions regarding the accurate nomenclature of sequenced species by molecular biologists have emerged. There has been concern associated with the large number of sequences in the public databases that have discrepancies or are inaccurate. However some researchers believe that diatom taxonomy can be built solely on a framework based on DNA sequence data (McManus et al., 2009). Presently, DNA barcoding appears to be a useful addition in the toolbox of techniques utilized to identify diatoms.
If DNA was the only tool for identification purposes, it is thought that the already described characteristics with other pertinent information regarding diatoms (i.e., ecology, morphology, and plastic features) would be lost and no longer available (McManus et al., 2009). Another issue that is of concern, is that currently the accessions to public genetic databases are not regulated by a governing body thus creating confusion and inaccuracies between nucleotide sequence data and incorrectly identified organisms. A regulatory or policy making committee to ensure appropriate accuracy should be considered (McManus et al., 2009).
Several researchers have tried to locate a specific site in the genome region to amplify, which includes the mitochondrial site cox1. Other sites include plastid-encoded rbcL and nuclear encoded 18S rDNA (SSU), and internal transcribed spacer (ITS-2). These regions do not, however, consistently work for all diatom species. The list of successful taxa that have used cox1 as a DNA barcode is short (Moniz and Kaczmarska, 2009). There have been studies performed in one genus where the cox1 region has worked successfully, however specific primers had to be developed (Moniz and Kaczmarska, 2009; Evans et al., 2007). Evans et al. (2007) revealed that finding an appropriate genome region that can be used for all diatoms was difficult. They found that trying to find a region that was conserved enough to design universal primers for diatoms was complicated and challenging. Moniz and Kaczmarska (2009) found that DNA from other organisms (e.g., bacteria) was amplified rather than that of the diatom.
None of the three markers (SSU, cox1, and ITS-2 DNA) combined with the 5.8S gene tested by Moniz and Kaczmarska (2009) fulfilled the role that cox1 seemed to provide for animals. The animal phylum has a 95 percent rate of success distinguishing between species using cox1 as a genetic marker (Moniz and Kaczmarska, 2009).
There has been some recent development that presently indicates the potential use of ITS-2, the 5.8S + ITS-2 fragment as a diatom DNA barcode (Moniz and Kaczmarska, 2009). Once a genome region that satisfies all diatoms is found and primers can be developed, using DNA would be an added feature in the toolbox to help make identification more accurate, reliable, and accessible.
More research is needed before DNA barcoding will be accurate enough to use in distinguishing between diatom species. First, an informative genetic marker that can be used across a majority if not all diatom lineages will have to be found. Specific diatom primers will need to be designed if molecular techniques are to be a successful method to support morphological identification and taxonomy. In my opinion, using a combined approach of both DNA barcoding and morphological characters to identify diatoms is the best approach. The toolbox approach, i.e. having more information and more tools to work with, is better than using just one method to identify distinct evolutionary units of microscopic algae.
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