The Future of Taxonomy
AMANDA LOUSIE JONES
Department of Biology, Food and Nutritional Sciences, School of Life
Sciences, Northumbria University, Newcastle upon Tyne, NE1 8ST United Kingdom
I.
Abstract
Microbial systematics has always been a misunderstood scientific discipline. It is readily
assumed that systematists use antiquated techniques to examine the molecular,
morphological, physiological and biochemical properties of microorganisms. It is also
believed that the circumscription of novel taxa is not essential let alone a requirement and it
is due to this that systematics has become a dying art. It is rarely appreciated that
systematics is a discipline that is essential to all sciences and that without the use of current
techniques, descriptions of novel species or higher taxa cannot be correctly published. Since
Woese and colleagues first publicised the use of the small subunit ribosomal RNA as a
molecular tool, phylogenetic analysis of 16S rRNA gene sequences has become an essential
step in the polyphasic approach of microbial systematics. However this molecular technique
has limitations which have become apparent and therefore it is evident that full genome
comparisons are soon going to be a requirement for the full circumscription of novel taxa.
The next generation of sequencing technology has enabled more information to be
incorporated into the full systematic picture and that is immense as it is only the start of the
genomic era. It is hoped that high throughput sequencing will compliment polyphasic data
rather than throwing a different light on it and thus soon become an essential minimal
standard for taxonomic descriptions.
II.
Introduction
Systematics is a fundamental discipline to the field of microbiology. It has an impact in all
aspects of microbiological sciences, from medical microbiology, environmental microbial
studies to the investigations in the biotechnological field. This immense impact is due to the
ability of systematics to achieve three goals introduced by Cowan (1965), namely:
classification, identification and nomenclature. Classification is the ordering of
microorganisms into taxonomic groups, this tends to be based on their numerous
differences and similarities, with reliance upon the differences for most of the analytical
techniques (Zhi et al., 2012). Identification can be established by comparison with key
characteristics of previously described taxa and can result in unknown microorganisms
being identified as members of known species or subsequently requiring classification as a
novel taxa (Zhi et al., 2012). Nomenclature is the third goal of systematics (Sutcliffe et al.,
2012) and this is also an essential part of systematics and is directed by the International
Code of Nomenclature of Bacteria (Sneath, 1992) to ensure microorganisms are given
internationally recognised names, as well as denoting the taxonomic hierarchy, such as
species, genus, family and order (Zhi et al., 2012).
A fourth goal of systematics has been recently suggested by Staley (2010), which is that
comprehending microbial diversity is also a benefit. The use of 16S rRNA gene sequence
analysis has enabled microbial taxonomists to understand microbial communities,
particularly by ‘deep’ sequencing, which has resulted in a greater understanding of the
microbial diversity within these environments, in particular highlighting the number of taxa
present in microbial communities and what has not been cultured at present. The process of
microbial description has improved, with greater than 9,000 prokaryotic species described
so far, and over 50% in the last decade. However it is still thought that the currently
described species only account for less than 1% of the microbial diversity present (Sutcliffe
et al., 2012). It is thought that current methods are dated and new approaches such as
genomics are required. Iverson and colleagues (2012) have recently documented the
development of novel computer software that enables full genomes obtained from a
metagenomic analysis of a microbial community. Using next generation sequencing, the
software analysed the data obtained, thus enabling the production of individual genomes of
all microbes present (Iverson et al., 2012). This technology would enable the genomes of
previous uncultured microorganisms to be obtained and provide the opportunity for
cultivation strategies for these organisms to be devised.
These techniques are the future of microbial taxonomy and ecology, and there are
misconceptions that microbial taxonomists do not exploit these modern techniques and
instead prefer to utilise well established methodology. However this concept is not true
with the work of Peter Sneath bringing the advent of numerical taxonomy in 1957, an early
adoption of computational techniques to be exploited by systematists (Sneath and Sokal,
1973) and then Rita Colwell established the use of polyphasic taxonomy to improve
taxonomic descriptions (Colwell, 1970). Finally the addition of the phylogenetic analysis of
the 16S rRNA gene sequence by Carl Woese and colleagues enabled a better understanding
of the microbial world (Woese and Fox, 1977). To better appreciate the future applications
that will be available to systematists we need to look at the previous studies and the
resources available, these then need to be compared with the current techniques, the
protocols used and the standards required, to aid the understanding of the applications that
are required and give a better indication of the microbial taxonomic needs for the not too
distant future.
III.
The Past
Since Ferdinand Cohn, in the 1870’s, the ability to classify microorganisms was based on
microbial physiology, pathogenicity, products they produce and their morphological
appearance. This later improved with the Society of American Bacteriologists’ Committee on
Bacterial Classification and Nomenclature using additional criteria such as growth conditions
to aid genus descriptions (Logan, 1994). In 1923 Bergey’s Manual of Determinative
Bacteriology was published in a bid to provide an identification scheme based on phenetic
traits, biochemical profiles, morphological traits and nutritional requirements. This became
the first out of eight editions that were subsequently published (Logan, 1994). However
after the eighth edition in 1974, it was found that the system created was not appropriate,
leading to unrealistic groupings of microorganisms (Logan, 1994). This led to the decision for
proposals of new bacterial names to be validly published or their descriptive publication to
be approved in the International Journal of Systematic Bacteriology (IJSB), now known as
the International Journal of Systematic and Evolutionary Microbiology (IJSEM). This journal is
the ‘cornerstone’ of microbial systematics and adheres to the strict guidelines of the
Bacteriological Code (www.ijs.sgmjournals.org).
During this time of change and uncertainty, Peter Sneath brought to light the use of
computers for taxonomic analyses. The numerical taxonomic techniques that were originally
proposed and later established were referred to as Adansonian taxonomy, following the
principles of Adanson, of studying phenetic differences and traits. Numerical taxonomy
involved grouping microorganisms, referred to as operational taxonomic units (OTUs;
Sneath and Sokal, 1973), by numerical methods into taxonomic units based upon the
organisms characteristics. The important characteristic of this technique was that each
character is of equal importance and therefore in the analysis has equal weighting (Priest
and Austin, 1993). Once the computer analysis has been performed certain traits could then
be deemed of higher importance and used as differential characteristics. The types of traits
that are taken into consideration are morphological data, growth requirements, biochemical
analysis, antibiotic inhibitory analyses and carbon source utilisation data.
In 1970 it was proposed by Rita Colwell that polyphasic taxonomy was the next progressive
step for improving bacterial classification (Colwell, 1970). This approach enabled traits of
phenotypic and genotypic nature to be analysed and therefore classifications to be based on
a consensus of high quality data. The characteristics that were analysed were phenetic traits
such as those used in numerical taxonomy, chemical analysis of the cell wall
(chemotaxonomy) and genotypic data such as DNA G+C content (mol %). The extensive
incorporation of the polyphasic approach into taxonomy has led to an improved
classification system, resulting in improved identifications which is evident in the recent
edition of Bergey’s Manual of Systematic Bacteriology (de Vos et al., 2009) and is now seen
to be the ‘gold standard approach’ for taxonomic descriptions.
The start of the molecular era saw the use of the small subunit ribosomal RNA (16S rRNA),
with Carl Woese and colleagues demonstrating the application of phylogenetic analysis to
determine that Archaea form their own kingdom, which enabled a better understanding of
the microbial world (Woese and Fox, 1977). At the beginning of the use of this molecular
marker, only small sections of the gene were able to be amplified and analysed and Sab
values calculated (Woese, 1987). In addition to the sequencing data, DNA:DNA hybridization
of genomic DNA was becoming more prevalent since it was first reported by Johnson and
colleagues (1968). Wayne and colleagues (1987) subsequently illustrated how a cut-off point
of 97% of the 16S rRNA gene sequence similarity is indicative of a novel species, which was
found to be comparable to a 70% threshold using the DNA:DNA hybridization technique
(Wayne et al., 1987). This has subsequently led to DNA:DNA hybridization as being
considered as the ‘gold standard’ in microbial classification.
IV.
The Present
Since the advent of PCR based 16S rRNA gene sequence analysis, microbial taxonomy has
not changed dramatically, however the different aspects of taxonomy have been clarified
for better understanding by scientists. It has been suggested that there are different stages
of taxonomy, and these should be referred to as alpha, beta and gamma. The alpha
taxonomic stage is the classification of species, the naming and the identification (Zhi et al.,
2012). The beta taxonomy is the assignment of species to a natural classification, based on
their phylogeny but also chemotaxonomy, phenotypic and additional genotypic information.
Natural classifications are based on the phenetic approach, which is based on the whole
organisms attributes, phenotype and genotype, with natural phylogenetic classifications
based on evolution (Priest and Austin, 1993). The final stage known as gamma taxonomy is
based on intraspecific categories, such as subspecies and ecotypes, although most of these
investigations are carried out by ecologists (Zhi et al., 2012).
The current approach to the description of a novel taxon would be to initially perform a
phylogenetic analysis of the isolate with an almost complete 16S rRNA gene sequence in
comparison with from the genus the isolate was most similar to from the nBLAST search
(Altschul et al., 1990), in addition to representative type species residing within the same
order. This analysis enables the visualisation of the phylogenetic positioning of the isolate in
question within the order, but a further analysis of the nucleotide differences and
percentage similarity between the strains analysed, gives a better indication of the novelty
of the organism. If the isolate is deemed presumptively novel, additional phylogenetic
algorithms should be performed to ensure parity in the analysis presented. The algorithms
that tend to be chosen are least-squares likelihood (Fitch and Margoliash, 1967), neighbourjoining (Saitou and Nei, 1987), maximum likelihood (Felsenstein, 1981) and maximumparsimony (Kluge and Farris, 1969) methods along with evolutionary distance matrices
prepared after Jukes and Cantor (Jukes and Cantor, 1969). In addition the unrooted tree
topologies are further evaluated with the use of a bootstrap analysis (Felsenstein, 1985)
based on 1,000 resamplings. In addition to the phylogenetic analysis the DNA G+C content
(mol %) should be ascertained, along with DNA:DNA hybridization depending on the
percentage 16S rRNA gene similarity of the isolate and its closest neighbour.
The delineation of a novel species is currently a tricky situation, with systematists differing
in the choice of tree algorithms and with the multiple versions of 16S rRNA operon, which
can have differences from 2-5% (Kampfer, 2012). Moreover, the 16S rRNA gene has become
limited in its use for taxonomic characterisation of many taxa. For example members of the
genus Tsukamurella are not able to be defined with the phylogenetic analysis of 16S rRNA
gene sequence and therefore more reliance is placed on DNA:DNA hybridization
(Goodfellow and Kumar, 2012). Unfortunately the previously stated 70% cut off for
DNA:DNA hybridization data, equating to ~97 % 16S rRNA gene sequence similarity, can also
be debateable with a more realistic value of >70%, that is readily seen particularly within the
genus Rhodococcus, with evidence that the cut-off point for members of the genus
Rhodococcus needs to be set at 80% (Goodfellow et al., 2002). Additional limitations of the
16S rRNA gene sequence are also seen with the lack of resolution of higher taxa; however
there are other limitations in the classification of higher taxa with the lack of phenotypic
data and even further molecular data.
The next stage in the descriptive process is to examine the novel isolate along with its
nearest neighbours, which would have been determined from the phylogenetic analysis, by
phenotypic characterisation. The types of analysis that would be performed are the
morphological appearance of the isolate after growth on specific nutritious media for a
specified time and temperature. Micro-morphological characteristics will also be examined
such as spore forming, motility and it is also essential to establish the morphological life
cycle of the isolate. The nutritional requirements of the novel isolate are the next criteria to
establish. These analyses tend to take the form of utilization of carbohydrates and organic
acids (Shirling and Gottlieb, 1966), in addition the degradation of a number of compounds is
analysed (Goodfellow and Pirouz, 1982; Gordon et al., 1974) along with antimicrobial
susceptibility testing (Groth et al., 2004) and enzyme activities, which tends to be
ascertained by the use of API ZYM test kits (bioMérieux). Finally the isolate would be
subjected to tolerance testing for temperature, pH and NaCl concentrations. The data from
all of the phenotypic assessment, which can amount to over 100 tests, will be incorporated
into the taxonomic description; however a minimum of 20 characteristics will need to be
documented, to establish differences between the related taxa tested.
The final stage for a novel species description, is to ascertain the isolates cell wall
chemotype (Lechevalier and Lechevalier, 1970). This is determined by examining the
diagnostic isomers of diaminopimelic acid (A2pm; Staneck and Roberts, 1974) and wholeorganism sugars (Hasegawa et al., 1983). Additional key chemotaxonomic markers should
also be established, including fatty acid methyl ester analysis (Komagata and Suzuki, 1987),
determination of the predominant isoprenoid quinones (Collins, 1994), muramic acid type
(Uchida et al., 1999), the presence and length of mycolic acids (Minnikin et al., 1975) and
the polar lipid profile (Minnikin et al., 1984). Until recently whole cell protein analysis was
performed by Curie point mass spectrometry (Ferguson et al., 1997; Sanglier et al., 1992).
Pyrolysis mass spectrometry data was examined using combined principal component and
canonical variate analyses (Windig et al., 1983), thus enabling the clustering of pyrogroups,
groups that tended to differentiate between the environments isolates are cultivated from.
This technique has since become redundant with differing mass spectrometry techniques
taking its place.
It needs to be kept in mind that the examination of and the subsequent description of novel
taxa should conform to the minimal standards for the taxon, the strains. The aim of the
minimal standards is to provide a guidance on the description of novel taxa and to direct the
appropriate testing required to establish the taxonomic positioning of the isolate in
question (Whitman, 2012). However the number of available minimal standards covers only
a tiny fraction of known microbial diversity. A number of members within the taxonomic
community think the polyphasic approach and the current battery tests required is costly in
time and money. Moreover, in many cases the taxonomic utility of these tests is
undermined by the current situation whereby 70% or more novel taxa are described based
on single strains (Sutcliffe et al., 2012). With the start of the genomic era it can be decided
which tests should be used to create a new ‘gold standard’ along with novel up and coming
genomic techniques.
V.
The Future
Genomics is the future of systematics; however its full potential has yet to be fully realised
and exploited. Since the start of the genomic era, scientists have started to use genomes to
assess the phylogenetic relationships between organisms, with a variety of techniques used
including the order of the genes being examined, analysis of the shared genes and finally the
construction of super trees (Beiko et al., 2005; Belda et al., 2005; Ciccarelli et al., 2006; Ding
et al., 2008; Gao and Gupta, 2012; Lathe et al., 2000; Snel et al., 1999). The results from
these differing analyses do not match the current phylogenetic relationships established by
phylogenetic analysis of the 16S rRNA gene sequence, as the genomes are dynamic as well
as much more diverse than the phenotypes, genotypes and chemotypes illustrated (Gao and
Gupta, 2012; Gogarten et al., 2002; Gogarten and Townsend, 2005; Lawrence and
Hendrickson, 2005; Snel et al., 2005).
It is thought bacterial genomes can have between 0 and 20% genes obtained by horizontal
gene transfer (HGT), which can be seen in some instances as a process of evolution such as
the transfer of antibiotic resistance which does not occur in conserved regions of the
genome. Definite HGT is usually when genes are found only in a particular genome which is
phylogenetically diverse from other genomes containing the specific genes. However with
genomes containing homologues it is harder to unravel where the HGT started and if it
happened, although HGT is not readily seen in genes involved in transcription and
translation. HGT has already thrown questions on using single genes for phylogenetic trees,
such as 16S rRNA gene, and therefore it is thought core genes in plural should be used for
better differentiation (Gao and Gupta, 2012).
It is seen that genomes of related taxa can be very different. Current phylogenetic analysis
of genomes illustrates strains within species can be very different and may indicate that the
taxonomic positioning is too rigid and strains are too wide a term and further differentiation
is required. This issue with phylogenetic analysis of genes may lead to modification of the
methods required for species description (Gao and Gupta, 2012). One suggested method is
the use of conserved inserts and deletions (indels) in gene/proteins sequences which are
classed as rare genetic elements (RGCs), which could be used for the phylogeny of
microorganisms. This is due to indels being flanked by conserved regions and therefore
possess a reliability insurance, also the rarity and highly specific nature of these types of
changes makes their characteristics good candidates, as well as the difference in
evolutionary rates should not affect the indel or the interpretation of it. In addition genetic
elements could be incorporated at any time during evolution and therefore the
identifications of indels at different phylogenetic depths becomes possible (Gao and Gupta,
2012).
Another analysis that can be derived from with genomic information is conserved signature
proteins or lineage specific proteins. This is usually a number of specific proteins which are
unique to certain strains, species or higher taxa (Gao and Gupta, 2012). Alternatively
systematists can analyses single nucleotide polymorphisms (SNPs) which can be exploited as
tools for genome relatedness studies as well at chromosomal and gene levels (Zhi et al.,
2012). In 2009, Jim Staley (2009) suggested a phylogenomic species concept which involves
more information from the genomic data via a multilocus sequence analysis (MLSA), an in
depth approach involving the use of housekeeping genes as they evolve slowly and at a
constant rate, which should give a good understanding of true phylogenetic relationships.
This technique is thought to be a possible replacement of DNA:DNA hybridization, however
whole genome comparisons using average nucleotide identity (ANI; Konstantinidis and
Tiedje, 2005), which compares the shared genes between two genomes, has shown to give
the best correlation with DNA:DNA hybridization, with 95-96% ANI correlating with the 70%
cut off for DNA:DNA hybridization data (Goris et al., 2007; Konstantinidis and Tiedje, 2005).
However prokaryotic species concepts should be based on more than one characteristic,
such a phenotypic or genotypic, and also be based on more than one type of analysis i.e. the
phylo-phenetic species is based on DNA:DNA hybridization, 16S rRNA gene sequence
analysis and phenotypic descriptions to establish a taxonomic species (Zhi et al., 2012).
Previous chemotaxonomic, phenotypic and genotypic data, classed in this instance as
phenotypes can be correlated with molecular phenotypes, with reference to genomic data.
However a full understanding of metabolic and biosynthetic processes will be needed when
using genomic data for taxonomic comparisons. An example of this is the analysis of
isoprenyl diphosphate synthase, an enzyme which is involved in the determination of the
menaquinone chain length in actinomycetes, yet it will be uncertain when the change length
is terminated (Zhi et al., 2012). However the menaquinone biosynthetic pathway has shown
differing genes involved and therefore has been shown as a useful aid in speciation. Care
needs to be taken when looking at genomic information. It also needs to be considered that
when using genomic data it will have to be fully analysed as the core genome may indicate
essential function, although still can be dispensable which may not aid classification of
species but may aid higher taxa to be classified, however the peripheral genome which
relates to survival in niches environments, such as biochemical pathways which are essential
for bacterial growth will enable more accurate classification of taxa (Zhi et al., 2012).
The recommendation of Zhi and colleagues (2012) is that the phylogenomic backbone of
microbial systematics should be merged with biochemical traits, physiological traits,
morphological traits, and the biology of the organism. This will enable a full understanding
of the evolution and taxonomic relationships between taxa in regard to both their
taxonomic positioning and relationships between microbes residing in the same
environmental niche. Also until an agreed approach to establishing the phylogenetic
positioning of taxa by using genomic data, DNA G+C content (mol %) and DNA:DNA
hybridisation, via ANI or multilocus sequence typing (MLST), can be achieved by using the
genomic data alone. Therefore it is becoming apparent that draft genomes are soon going
to be required with each species description, especially as the cost of analysing genomes is
coming down especially with next generation sequencing (Sutcliffe et al., 2012). Genomic
analyses would enable ecological and physiological insights along with the taxonomy of
microorganisms through their diverse metabolic systems. Since ‘deep’ sequencing of
microbial communities can be achieved, culturable and non-culturable, niche adaptation
can be assessed, including defining the human microbiome, the pathogenic potential and
disease states in the human body.
For a species description in the future, more than one isolate should be analysed although
this is in an ideal situation and a rare occurrence, with a genome of the type strain, along
with a selection of phenotypic characters to be analysed which could take the form of
phenotypic microarray panels. Phenotypic microarray is a rapid method to ascertain
phenotypic characteristics and if the same protocol was used then possibly a database could
be created and utilised so that only a handful of panels would need to be performed per
species description. In addition the phenotypic microarray can also give comparison with
the genomic information on metabolic pathways (Bochner, 2009). An additional analysis
that could be incorporated in the future descriptive studies is mass spectral analysis by
MALDI-TOF-Mass spectrometry. This analysis has been readily incorporated in clinical
microbiological laboratories. Standardized protocols of a selected few chemotaxonomic
markers would also enable the storage of data online and therefore preventing the
numerous tests being repeated. The absences of online databases indicate that taxonomy is
currently behind the times, with the lack of databases hindering the development of rapid
and cost effective methods.
It is evident that a new era of systematics is rapidly approaching and hopefully it will be a
high through-put era, enabling the descriptions of numerous taxa but in a cost and time
effective format. It will hopefully enable systematists to concentrate on microorganisms of
importance in the biotechnological and clinical field. However this will only be of benefit if
the sources of information are frequently updated and widely available and therefore it
would be appropriate for the next and subsequent editions of Bergey’s Manual of
Systematic Microbiology to become an online version, with access to databases and
genomic information, and therefore usable by all microbiologists. An example of a
information resource that can be combined with additional resources the Genomic
Encyclopaedia of Bacteria and Archaea (GEBA; Wu et al., 2009). Microbial taxonomy is an art
form, and possibly a dying one, as emphasised in reports from the UK House of Lords
Science and Technology Committee (2007-2008) and the Review Team at the Natural
History Museum (Boxshall and Self, 2010).Therefore building a sustainable future for
taxonomy is paramount to the survival of systematists and a fundamental discipline.
ACKNOWLEDGMENTS
The author is grateful to I.C. Sutcliffe for a critical reading of the manuscript and comments.
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