Antonie van Leeuwenhoek DOI 10.1007/s10482-014-0148-x
INVITED REVIEW
Antonie van Leeuwenhoek 80th Anniversary Issue
Time to revisit polyphasic taxonomy Peter Vandamme • Charlotte Peeters
Received: 30 January 2014 / Accepted: 1 March 2014 Springer International Publishing Switzerland 2014
Abstract Although the International Code of Nomenclature of Bacteria does not specify a working strategy, editors and reviewers of taxonomic journals commonly request a polyphasic taxonomic approach that includes phenotypic, genotypic and chemotaxonomic information for the description of novel bacterial species. Whole genome sequences provide an insight into the genetic nature of microbial species, yield new and superior tools for delineating bacterial species and for studying their phylogeny, and provide a window on an organism’s metabolic potential. These new insights and tools are gradually introduced in the polyphasic taxonomic practice. The genus Burkholderia, a controversial group of bacteria with both benign and devastating characteristics, is used as an example to show that the modern practice of polyphasic taxonomy is counterproductive in light of the tremendous number of bacterial species that awaits formal description and naming. Bacterial taxonomists must urgently reconsider how to describe and name novel bacteria in the genomic era, and should consider using a full genome sequence and a minimal description of phenotypic characteristics as a basic, sufficient, cost-
P. Vandamme (&) C. Peeters Laboratory of Microbiology, K. L. Ledeganckstraat 35, 9000 Ghent, Belgium e-mail: [email protected]
effective and appropriate biological identity card for a species description. Keywords Bacterial taxonomy Polyphasic taxonomy Species description Whole genome sequence Burkholderia Genomics Introduction In this day and age of sustainable energy and green society every now and then there is a surge of interest in biodiversity. On such occasions bacterial taxonomists may be grinding their teeth. The staggering numbers of—for instance—insect and plant species render the list of a mere ca. 12,000 bacterial species confronting and embarrassing (Tamames and Rossello´-Mo´ra 2012; Oren and Garrity 2014). Yet each of these insect guts or plant rhizospheres very likely harbors several yet-to-be-described bacterial species. So what have bacterial taxonomists accomplished during the past centuries and why don’t we also have lists of hundreds of thousands of species? Why this is still the case is hard to explain to those who are not familiar with bacterial taxonomy, and if you seriously try to explain this, for instance to bachelor students in biotechnology, they shrug their shoulders. Can we mend this situation? Well, let’s step back in time. We know the historical roots of bacterial taxonomy very well (Oren and Garrity 2014). Early classifications were based on morphology and biochemical data. They were rendered more robust
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through the introduction of numerical taxonomy and the use of large numbers of characteristics, and were subsequently complemented and reinforced by chemotaxonomic and genotypic characterization. All of this culminated into polyphasic taxonomy, a term coined in 1970 (Colwell 1970) and a practice described in detail in an exhausting review paper of our research group, that has been highly cited though by the bacterial taxonomist community (Vandamme et al. 1996). Polyphasic taxonomy integrates all available genotypic, phenotypic, and phylogenetic information into a consensus type of general-purpose classification. It departs from the assumption that the overall biological diversity cannot be encoded in a single molecule and that the variability of characters is group dependent. The bacterial species was defined as an assemblage of isolates originating from a common ancestor population in which a steady generation of genetic diversity resulted in clones with different degrees of recombination and characterized by a certain degree of phenotypic consistency, a significant degree of DNA–DNA hybridization (DDH) and more than 97 % of 16S rRNA gene sequence similarity (Vandamme et al. 1996). This practice of polyphasic taxonomy is however also one of the main reasons why the field remained an exclusive playground for a happy few and why we have a ridiculously short list of a mere 12,000 bacterial species named today.
The Burkholderia example: pars pro toto, or our failure to truly report and name bacterial diversity Our research group has a strong interest in the biodiversity of the genus Burkholderia. When we started studying this group of bacteria in the early 1990s it was a rather obscure genus comprising a handful of species only (Yabuuchi et al. 1992). At present, the genus Burkholderia contains more than 80 validly named species (see http://www.bacterio.cict. fr/) and a large number of candidate species (Van Oevelen et al. 2004; Lemaire et al. 2011; Verstraete et al. 2011; Lemaire et al. 2012). Burkholderia species occupy extremely diverse ecological niches, including pristine and contaminated soils, plant rhizospheres and phytospheres, invertebrate intestinal tracts and the respiratory tract of humans (Coenye and Vandamme 2003; Compant et al. 2008; Sua´rez-Moreno et al. 2012). Their impressive metabolic potential has been
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exploited for biocontrol, bioremediation and plant growth promotion purposes, but safety issues regarding human infections, especially in cystic fibrosis patients, have not been solved. The latter has been the main driver behind recent proposals to split the genus into a cluster comprising the ‘bad’ species (i.e. the socalled pathogen group) and a cluster comprising the ‘good’ species (i.e. those with biotechnological potential, or the so-called plant beneficial or symbiotic group) (Gyaneshwar et al. 2011; Sua´rez-Moreno et al. 2012; Estrada-de Los Santos et al. 2013; Angus et al. 2014). While such oversimplification sends a clear message, the neutral reader should be aware that the largest body of literature addressing so-called beneficial or symbiotic Burkholderia strains concerns species belonging to the pathogen group (Parke and Gurian-Sherman 2001), and that strains of several species within the so-called plant beneficial group have been involved in human infections (Coenye et al. 2001; Goris et al. 2004; Gerrits et al. 2005; Deris et al. 2010). Of the latter, especially Burkholderia fungorum has been isolated from a wide range of human and veterinary samples including human blood, cerebrospinal fluid, vaginal secretions, sputum and lavage samples of cystic fibrosis patients, and the brain of a pig with neurological deficit, the brain stem of an injured deer, and the nose of several mice (Coenye et al. 2001, 2002; Gerrits et al. 2005) (unpublished data). Not a good pedigree for a plant beneficial organism clearly! This applied relevance of Burkholderia strains also catalyzed a fairly successful quest for research funding to keep the Burkholderia biodiversity study going for more than two decades, although the words ‘taxonomy’ and ‘biodiversity’ were always removed with rigorous scrutiny from early application drafts to increase the odds of success, which is another behavior many taxonomists are familiar with. We discovered that Burkholderia cepacia isolates from cystic fibrosis patients and other human clinical and environmental samples represented at least five genomic species, collectively referred to as the B. cepacia complex (Bcc) (Vandamme et al. 1997). During two decades of Burkholderia diversity research the list of Bcc species has grown to 18 validly named species (Peeters et al. 2013). Driven by the devastating effect of these infections in cystic fibrosis patients, microbiologists gained an interest in their environmental occurrence and many environmental Burkholderia species were
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isolated, described and named in polyphasic taxonomic studies. During these studies multilocus sequence analysis (MLSA) emerged as a most useful taxonomic tool. Although initially developed for strain genotyping below the species level and designated as multilocus sequence typing (MLST) (Maiden et al. 1998), MLSA employs phylogenetic procedures based on the nucleotide sequences of multiple gene fragments to reveal similarities between strains representing different species and genera (Gevers et al. 2006). Especially for depicting relationships within and between closely related species like the Bcc bacteria, this approach has a taxonomic resolution superior to that of the traditional 16S rRNA gene sequence analysis. Interestingly, an average concatenated allele sequence divergence of 3 % can be used as a threshold value to distinguish between Bcc species (Vanlaere et al. 2009; Peeters et al. 2013) and is therefore equivalent to the 70 % DDH threshold. This validation of the MLSA approach as a tool to replace DDH has speed up the description of novel species, albeit minimally. More recently, bacterial taxonomists have embraced whole-genome studies and introduced whole-genome sequence (WGS) based parameters that yielded thresholds equivalent to the magical 70 % DDH level (Goris et al. 2007; Richter and Rossello-Mora 2009). Although it can be argued that this introduction of WGS parameters is a convenient way to replace DDH without the need to revise existing classification schemes (most of our fellow microbiologists hate name changes), we should ponder on this practice. Remember that DDH was introduced as a tool to approach WGS derived information as close as possible (Wayne et al. 1987), and now that we have direct access to WGS information, we want it to mimic the results obtained through DDH experiments! Still, the introduction of WGS allows us to implement parameters like the calculation of the percentage of conserved DNA (pcDNA) (Goris et al. 2007) or MUM-indices (Deloger et al. 2009) which can be used as in silico imitations of DDH experiments. Complementary to these approaches, the average nucleotide identity (ANI) (Konstantinidis and Tiedje 2005; Richter and Rossello-Mora 2009) and core gene identity (CGI) methods (Vanlaere et al. 2009) are both natural extensions of MLSA in case complete or draft genome sequences are available. These WGS based approaches are superior compared to traditional 16S rRNA sequence analysis for studying phylogeny because they are based on a much larger part of the genome and
because they have a better resolution for discriminating both distantly and closely related bacteria. So we have new and superior taxonomy tools to delineate species and to study phylogeny, and no doubt there is a clear and bright future for bacterial taxonomy in the genomic era. While the old DDH species threshold is being translated into MLSA or WGS based thresholds (Gevers et al. 2005; Goris et al. 2007; Richter and Rossello-Mora 2009; Tindall et al. 2010), a fair reappraisal of bacterial taxonomy and the species definition will require more than a mere methodological translation of threshold levels (Achtman and Wagner 2008). Our study of the diversity of B. cepacia-like infections in cystic fibrosis patients triggered a long series of polyphasic taxonomic studies of clinical and environmental micro-organisms which primarily belong to various non-fermenting Gram-negative bacteria of the Betaproteobacteria, and which thus far entailed the description of several novel genera including Advenella, Inquilinus, Kerstersia and Pandoraea, and some 70 novel species belonging to the genera Achromobacter (10 species), Advenella (2 species), Alcaligenes (1 species), Bordetella (2 species), Burkholderia [both Bcc (14 species) and non-Bcc (23 species)], Cupriavidus (7 species), Inquilinus (1 species), Kerstersia (2 species), Pandoraea (5 species), and Ralstonia (1 species); it also revealed quite a few synonymies between validly published names. This list of novel taxa could only be produced through much efforts by devoted PhD students and with the help of numerous international collaborators. The above list may seem impressive at first sight but during these 20 years progress was being made painstakingly slow, not the least because we tend to describe novel species only when we have a set of multiple genetically distinct strains. The inclusion of multiple strains is considered old-fashioned but good taxonomic practice by many (Oren and Garrity 2014), but is counterproductive as it implies a lot of extra work, not the least to find differential biochemical characteristics. This search for differentiating biochemical reactions is a practice that originates from diagnostic medical microbiology, where in the meantime modern methods such as MALDI-TOF mass spectrometry have recently replaced biochemical identification algorithms that are intrinsically inadequate (Desai et al. 2012). The submission of these studies also provoked quite a few friendly (well… mostly friendly) wars with editors of taxonomic
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journals. Indeed, especially within the group of nonfermenting Gram-negative bacteria, it is notoriously difficult to find two (!) or more differential biochemical tests when one compares sets of multiple strains per species, while the corresponding type strains may differ simultaneously in a multitude of characteristics. Reducing such studies to type strains only and effectively removing all other strains and the corresponding data from the study would often have made life so much easier for the PhD students involved. The practice of polyphasic taxonomy is also discouraging when considering the overwhelming number of novel species that are available in public and private strain collections (let alone the natural environment). Two examples to illustrate and conclude this point: mining the Bcc PubMLST database (http://pubmlst.org/bcc/) using the 3 % threshold value of average concatenated allele sequence divergence for species delineation (Vanlaere et al. 2009; Peeters et al. 2013) revealed the presence of another 16 novel species (January 2014) within the Bcc that await description and formal naming (Table 1; Fig. 1). Or, a Table 1 Novel Bcc species, showing for each strain their ST, source and year of isolation (if known)
Bcc species Other Bcc A
Other Bcc B
Other Bcc C
Other Bcc D
Other Bcc E
Other Bcc F
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ST
recent search for Burkholderia caledonica in seven rhizosphere soil samples collected near Pretoria (South Africa) not only yielded the B. caledonica isolates that were aimed for (Verstraete et al. 2014), but also representatives of nearly a dozen novel Burkholderia species (unpublished data). These were added to a list of several dozens of novel Burkholderia species in our collection that await enthusiastic students and funding (unpublished data).
Conclusion Where is this field going? We will never succeed in formally naming all Burkholderia species if the taxonomic community continues to describe and name novel species like it does now, and we will never be able to generate more than the proverbial drop of water on a hot plate in our efforts to formally describe prokaryotic diversity. The issue was recently raised by Sutcliffe et al. (2012), who called for a significant reappraisal of the procedures used to describe novel Strain
Source
Country
Year
112
T21
ENV
USA
113
LMG 21824
CF
USA
114
BC003
ENV
USA
519
AU7314
NON
USA
527
HI3541
ENV
USA
690
NII
787
NII
789 47
NII BCC0276
ENV
USA
48
MRL-10
ENV
USA
49
BCC0517
ENV
USA
481
AU7004
NON
USA
510
AU0278
CF
USA
522
AU9897
CF
USA
775
QLD039
CF
Australia
130
MDII-110p
ENV
Italy
1996
144
MVP-C1-49
ENV
Italy
1996
284
MW10-3
ENV
USA
357
BCC0430
ENV
Italy
445
MVPCII 48
ENV
Italy
588
HT15
ENV
China
735
B3
ENV
USA
471
NII
1999 2004
Antonie van Leeuwenhoek Table 1 continued
Bcc species
ST
Strain
Source
Country
Year
Other Bcc G
535
AU2654
CF
USA
2001
654
R117
CF
Germany
2010
533
AU0141
CF
USA
1997
346
NII
115
MD II 367
ENV
Italy
147
MVP-C2-84
ENV
Italy
156
MDIII-T224
ENV
Italy
2002
401 774
MDIII-P-292 QLD028
ENV CF
Italy Australia
2002 2008
116
C1714
CF
UK
206
CEP0178
CF
UK
252
AU2242
CF
USA
730
CC-AI74
ENV
Taiwan
791
NII
792
NII
794
NII
333
LMG 13014
IND
Belgium
334
BCC1300
IND
N America
597
JW13.3a
IND
UK
2007
812
SBL13/154
NON
New Zealand
2013
335
NII
Other Bcc N
810
BURK106
ENV
Australia
2007
Other Bcc O
797 763
NII R-50211
ENV
Argentina
2011
Other Bcc P
764
R-50214
ENV
Argentina
2011
Other Bcc H Other Bcc I
Other Bcc J Other Bcc K Other Bcc L
All data available in the Bcc PubMLST database (http:// pubmlst.org/bcc) CF isolated from a cystic fibrosis patient, NON isolated from a non-CF patient, ENV environmental isolate, IND isolated from an industrial product, NII no isolate information available, only sequence information
Other Bcc M
prokaryotic taxa, including the introduction of new publication formats, but the message appeared unheard in a field that may be too conservative. Sutcliffe et al. rightfully pointed out that although progress in the description of new microbial taxa is being made at accelerating rates, there is an enormous backlog of work. Even if progress in describing new taxa can be accelerated considerably over current rates, the challenge of adequately describing prokaryotic diversity could take several centuries (Sutcliffe et al. 2012; Tamames and Rossello´-Mo´ra 2012)! Conflicting with this need to dramatically change our throughput capacity, comprehensive guidelines for the description of novel species have been published by leading scientists of the systematics community (Tindall et al. 2010) which safeguard bacterial taxonomy as the playground of a few privileged with full access to a battery of phenotypic, genotypic and
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chemotaxonomic tools, and the essential funding to pay for all analyses. In light of the vast unnamed microbial diversity this is counterproductive and we should indeed assess how much characterization is enough (Sutcliffe et al. 2012). Another practical consideration in this respect is that the number of bacterial taxonomists especially in Western countries is in sharp decline and that the extent of new species descriptions is primarily constrained by journal capacities, rather than taxonomists’ activities (Tamames and Rossello´-Mo´ra 2012). Polyphasic taxonomy has been so valuable for several decades because it provided a multifaceted view on an organism’s biology in an era where we did not have access to WGS information; not because we felt that one particular data type should be part of every organism’s identity card. The attempts to predict a dozen biochemical characteristics (Amaral et al. 2014) or to reconstruct an organism’s
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Antonie van Leeuwenhoek b Fig. 1 Phylogenetic tree based on the concatenated sequences
(2,773 bp) of seven housekeeping gene fragments [atpD (443 bp), gltB (400 bp), gyrB (454 bp), recA (393 bp), lepA (397 bp), phaC (385 bp) and trpB (301 bp)] of the 18 established Bcc species and novel Bcc species (labeled ‘other Bcc A to P’). For each of the 18 established Bcc species a maximum of 5 strains were selected: LMG 1222T, LMG 2161, LMG 17997, LMG 18821 and R-49849 for B. cepacia; LMG 13010T, LMG 18822, LMG 18824, R-18885 and LMG 16665 for B. multivorans; LMG 16656T, LMG 18826, LMG 18828, LMG 6986, MSMB554, LMG 18830, HI-2629, LMG 18829, PC28, LMG 18832, LMG 19230, LMG 19238, LMG 19240, LMG 19246, LMG 21462 and R-922 for B. cenocepacia (recA groups IIIA, IIIB, IIIC and IIID); LMG 14294T, LMG 7000, 5702, AU6735 and J5 for B. stabilis; LMG 10929T, LMG 18835, LMG 18836, LMG 6999 and G4 for B. vietnamiensis; LMG 18943T, LMG 19468, LMG 24508, IST4507 and AU3960 for B. dolosa; LMG 19182T, LMG 19467, LMG 17828, BCC0399 and BCC1222 for B. ambifaria; LMG 20980T, LMG 20983, LMG 16670, LMG 21821 and AU3904 for B. anthina; LMG 14191T, LMG 21822, LMG 21823, AU5468 and SBL12/065 for B. pyrrocinia; LMG 20358T, MSMB056, MSMB1396-A, LMG 24263 and R-52253 for B. ubonensis; LMG 24064T, LMG 24264, QLD058, MSMB101 and BURK108 for B. latens; LMG 24065T, LMG 24266, LMG 24267, HI3672 and R-9913 for B. diffusa; LMG 24066T, J1757, CEP0823, R-16920 and AU1125 for B. arboris; LMG 24067T, LMG 24271, LMG 19587, AU2877 and AU2283 for B. seminalis; LMG 24068T, R-2712 and AU7680 for B. metallica; LMG 23361T, LMG 16227, CEP0964, R-18428 and AU0059 for B. contaminans; LMG 22485T, LMG 14095, R-50215, BCC1406 and R-3211 for B. lata; LMG 26883T, LMG 16669, LMG 26914, LMG 26915 and TJI49 for B. pseudomultivorans. The sequences of LMG 16225T (B. fungorum), ATCC 23344T (B. mallei), LMG 19076T (B. caledonica) and LMG 14190T (B. glathei) were used as outgroup. Concatenated allele sequences of all strains were exported from the Bcc PubMLST database (http://pubmlst.org/ bcc) (Jolley and Maiden 2010) and a phylogenetic tree was constructed using the maximum likelihood method in RAxML version 7.4.2 (Stamatakis 2006). Rapid bootstrapping and ML search were performed using the general time reversible model with CAT approximation (GTRCAT) and the best scoring ML tree was exported in Newick format. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1,000 replicates) are shown next to the branches if greater than 70 %
chemotaxonomic components (Sutcliffe et al. 2012) illustrate the versatile exploitation of WGS information but should not pave the way for adding more guidelines to the characterization process of novel bacteria (Tindall et al. 2010). In a similar manner Ramasamy et al. (2014) presented a modernized schema for polyphasic taxonomy but although their approach accepted the utility of WGS and other modern data, they advocated adding these to the
conventional polyphasic approach rather than using their power to replace old methods and reduce the labor of describing new taxa. Together, all of this should indeed call us to arms and we must revitalize the practice of polyphasic taxonomy if we want to uncover more than the tip of the iceberg of microbial diversity in the next few decades. Urgent action from International Committee on Systematics of Prokaryotes is indeed badly needed (Sutcliffe et al. 2013) or the Committee may receive a wake-up call from rebelling taxonomists which recently proved very effective in moving the field of fungal taxonomy to a more modern practice (Hibbett and Taylor 2013). In the present day and age, a full genome sequence and a minimal description of phenotypic characteristics would provide a basic biological identity card that could be considered sufficient, cost-effective and appropriate for a species description. Acknowledgments We thank Iain Sutcliffe, Editor-in-Chief of Antonie van Leeuwenhoek, for his kind invitation to contribute to this festive issue of the journal and for giving us the opportunity to express some of our concerns. C. P. is indebted to the Special Research Council of Ghent University. The Burkholderia cepacia complex National Reference Center is supported by the Belgian Ministry of Social Affairs through a fund within the Health Insurance System.
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Antonie van Leeuwenhoek Coenye T, Goris J, Spilker T, Lipuma JJ, Vandamme P (2002) Characterization of unusual bacteria isolated from respiratory secretions of cystic fibrosis patients and description of Inquilinus limosus gen. nov. J Clin Microbiol 40:2062–2069. doi:10.1128/JCM.40.6.2062 Colwell RR (1970) Polyphasic taxonomy of the genus vibrio: numerical taxonomy of Vibrio cholerae, Vibrio parahaemolyticus, and related Vibrio species. J Bacteriol 104:410–433 Compant S, Nowak J, Coenye T, Cle´ment C, Ait Barka E, Clement C, Barka EA (2008) Diversity and occurrence of Burkholderia spp. in the natural environment. FEMS Microbiol Rev 32:607–626. doi:10.1111/j.1574-6976. 2008.00113.x Deloger M, El Karoui M, Petit MA (2009) A genomic distance based on MUM indicates discontinuity between most bacterial species and genera. J Bacteriol 191:91–99. doi:10. 1128/Jb.01202-08 Deris ZZ, Van Rostenberghe H, Habsah H, Noraida R, Tan GC, Chan YY, Rosliza AR, Ravichandran M (2010) First isolation of Burkholderia tropica from a neonatal patient successfully treated with imipenem. Int J Infect Dis 14:e73–e74. doi:10.1016/j.ijid.2009.03.005 Desai AP, Stanley T, Atuan M, McKey J, Lipuma JJ, Rogers B, Jerris R (2012) Use of matrix assisted laser desorption ionisation-time of flight mass spectrometry in a paediatric clinical laboratory for identification of bacteria commonly isolated from cystic fibrosis patients. J Clin Pathol 65:835–838. doi:10.1136/jclinpath-2012-200772 Estrada-de Los Santos P, Vinuesa P, Martı´nez-Aguilar L, Hirsch AM, Caballero-Mellado J (2013) Phylogenetic analysis of Burkholderia species by multilocus sequence analysis. Curr Microbiol 67:51–60. doi:10.1007/s00284-013-0330-9 Gerrits G, Klaassen C, Coenye T, Vandamme P, Meis JF (2005) Burkholderia fungorum septicemia. Emerg Infect Dis 11:1115–1117 Gevers D, Cohan FM, Lawrence JG, Spratt BG, Coenye T, Feil EJ, Stackebrandt E, Van de Peer Y, Vandamme P, Thompson FL, Swings J (2005) Re-evaluating prokaryotic species. Nat Rev Microbiol 3:733–739. doi:10.1038/ Nrmicro1236 Gevers D, Dawyndt P, Vandamme P, Willems A, Vancanneyt M, Swings J, De Vos P (2006) Stepping stones towards a new prokaryotic taxonomy. Philos Trans R Soc B-Biological Sci 361:1911–1916. doi:10.1098/Rstb.2006.1915 Goris J, De Vos P, Caballero-Mellado J, Park J, Falsen E, Quensen JF, Tiedje JM, Vandamme P (2004) Classification of the biphenyl and polychlorinated biphenyl-degrading strain LB400T and relatives as Burkholderia xenovorans sp. nov. Int J Syst Evol Microbiol 54:1677–1681. doi:10. 1099/ijs.0.63101-0 Goris J, Konstantinidis KT, Klappenbach JA, Coenye T, Vandamme P, Tiedje JM (2007) DNA–DNA hybridization values and their relationship to whole-genome sequence similarities. Int J Syst Evol Microbiol 57:81–91. doi:10. 1099/ijs.0.64483-0 Gyaneshwar P, Hirsch AM, Moulin L, Chen W-M, Elliott GN, Bontemps C, Estrada-de Los Santos P, Gross E, Dos Reis FB, Sprent JI, Young JPW, James EK (2011) Legumenodulating betaproteobacteria: diversity, host range, and
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future prospects. Mol plantmicrobe Interact MPMI 24:1276–1288. doi:10.1094/MPMI-06-11-0172 Hibbett DS, Taylor JW (2013) Fungal systematics: is a new age of enlightenment at hand? Nat Rev Microbiol 11:129–133. doi:10.1038/nrmicro2963 Jolley KA, Maiden MC (2010) BIGSdb: scalable analysis of bacterial genome variation at the population level. BMC Bioinformatics 11:595. doi:10.1186/1471-2105-11-595 Konstantinidis KT, Tiedje JM (2005) Genomic insights that advance the species definition for prokaryotes. Proc Natl Acad Sci USA 102:2567–2572. doi:10.1073/pnas. 0409727102 Lemaire B, Robbrecht E, Wyk B, Oevelen S, Verstraete B, Prinsen E, Smets E, Dessein S (2011) Identification, origin, and evolution of leaf nodulating symbionts of Sericanthe (Rubiaceae). J Microbiol 6:935–941. doi:10.1007/s12275011-1163-5 Lemaire B, Van Oevelen S, De Block P, Verstraete B, Smets E, Prinsen E, Dessein S (2012) Identification of the bacterial endosymbionts in leaf nodules of Pavetta (Rubiaceae). Int J Syst Evol Microbiol 62:202–209. doi:10.1099/ijs.0. 028019-0 Maiden MCJ, Bygraves JA, Feil E, Morelli G, Russell JE, Urwin R, Zhang Q, Zhou JJ, Zurth K, Caugant DA, Feavers IM, Achtman M, Spratt BG (1998) Multilocus sequence typing: a portable approach to the identification of clones within populations of pathogenic microorganisms. Proc Natl Acad Sci USA 95:3140–3145. doi:10.1073/pnas.95.6.3140 Oren A, Garrity GM (2014) Then and now: a systematic review of the systematics of prokaryotes in the last 80 years. Antonie Van Leeuwenhoek. doi:10.1007/s10482-013-0084-1 Parke JL, Gurian-Sherman D (2001) Diversity of the Burkholderia cepacia complex and implications for risk assessment of biological control strains. Annu Rev Phytopathol 39:225–258. doi:10.1146/annurev.phyto.39.1.225 Peeters C, Zlosnik JEA, Spilker T, Hird TJ, Lipuma JJ, Vandamme P (2013) Burkholderia pseudomultivorans sp. nov., a novel Burkholderia cepacia complex species from human respiratory samples and the rhizosphere. Syst Appl Microbiol 36:483–489. doi:10.1016/j.syapm.2013.06.003 Ramasamy D, Mishra AK, Lagier J-C, Padhmanabhan R, Rossi M, Sentausa E, Raoult D, Fournier P-E (2014) A polyphasic strategy incorporating genomic data for the taxonomic description of novel bacterial species. Int J Syst Evol Microbiol 64:384–391. doi:10.1099/ijs.0.057091-0 Richter M, Rossello-Mora R (2009) Shifting the genomic gold standard for the prokaryotic species definition. Proc Natl Acad Sci USA 106:19126–19131. doi:10.1073/pnas. 0906412106 Stamatakis A (2006) RAxML-VI-HPC: maximum likelihoodbased phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics 22:2688–2690 Sua´rez-Moreno ZR, Caballero-Mellado J, Coutinho BG, Mendonc¸a-Previato L, James EK, Venturi V (2012) Common features of environmental and potentially beneficial plantassociated Burkholderia. Microb Ecol 63:249–266. doi:10. 1007/s00248-011-9929-1 Sutcliffe IC, Trujillo ME, Goodfellow M (2012) A call to arms for systematists: revitalising the purpose and practises underpinning the description of novel microbial taxa.
Antonie van Leeuwenhoek Antonie Van Leeuwenhoek 101:13–20. doi:10.1007/ s10482-011-9664-0 Sutcliffe IC, Trujillo ME, Whitman WB, Goodfellow M (2013) A call to action for the international committee on systematics of prokaryotes. Trends Microbiol 21:51–52. doi:10.1016/j.tim.2012.11.004 Tamames J, Rossello´-Mo´ra R (2012) On the fitness of microbial taxonomy. Trends Microbiol 20:514–516. doi:10.1016/j. tim.2012.08.012 Tindall BJ, Rossello-Mora R, Busse HJ, Ludwig W, Kampfer P (2010) Notes on the characterization of prokaryote strains for taxonomic purposes. Int J Syst Evol Microbiol 60:249–266. doi:10.1099/Ijs.0.016949-0 Van Oevelen S, De Wachter R, Vandamme P, Robbrecht E, Prinsen E (2004) ‘‘Candidatus Burkholderia calva’’ and ‘‘Candidatus Burkholderia nigropunctata’’ as leaf gall endosymbionts of African Psychotria. Int J Syst Evol Microbiol 54:2237–2239. doi:10.1099/ijs.0.63188-0 Vandamme P, Pot B, Gillis M, DeVos P, Kersters K, Swings J (1996) Polyphasic taxonomy, a consensus approach to bacterial systematics. Microbiol Rev 60:407–438 Vandamme P, Holmes B, Vancanneyt M, Coenye T, Hoste B, Coopman R, Revets H, Lauwers S, Gillis M, Kersters K, Govan JRW (1997) Occurrence of multiple genomovars of Burkholderia cepacia in cystic fibrosis patients and proposal of Burkholderia multivorans sp. nov. Int J Syst Bacteriol 47:1188–1200 Vanlaere E, Baldwin A, Gevers D, Henry D, De Brandt E, LiPuma JJ, Mahenthiralingam E, Speert DP, Dowson C,
Vandamme P (2009) Taxon K, a complex within the Burkholderia cepacia complex, comprises at least two novel species, Burkholderia contaminans sp. nov. and Burkholderia lata sp. nov. Int J Syst Evol Microbiol 59:102–111. doi:10.1099/Ijs.0.001123-0 Verstraete B, Van Elst D, Steyn H, Van Wyk B, Lemaire B, Smets E, Dessein S (2011) Endophytic bacteria in toxic South African plants: identification, phylogeny and possible involvement in gousiekte. PLoS ONE. doi:10.1371/ annotation/e7fa3cdf-c7bc-439f-83a2-36da63ef6c08 Verstraete B, Peeters C, van Wyk B, Smets E, Dessein S, Vandamme P (2014) Intraspecific variation in Burkholderia caledonica: Europe vs. Africa and soil vs. endophytic isolates. Syst Appl Microbiol in press: 1–6. doi: 10.1016/j. syapm.2013.12.001 Wayne LG, Brenner DJ, Colwell RR, Grimont PAD, Kandler O, Krichevsky MI, Moore LH, Moore WEC, Murray RGE, Stackebrandt E, Starr MP, Truper HG (1987) Report of the ad hoc committee on reconciliation of approaches to bacterial systematics. Int J Syst Bacteriol 37:463–464 Yabuuchi E, Kosako Y, Oyaizu H, Yano I, Hotta H, Hashimoto Y, Ezaki T, Arakawa M (1992) Proposal of Burkholderia gen. nov. and transfer of seven species of the genus Pseudomonas homology group II to the new genus, with the type species Burkholderia cepacia (Palleroni and Holmes 1981) comb. nov. Microbiol Immunol 36:1251–1275
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INVITED REVIEW
Antonie van Leeuwenhoek 80th Anniversary Issue
Time to revisit polyphasic taxonomy Peter Vandamme • Charlotte Peeters
Received: 30 January 2014 / Accepted: 1 March 2014 Springer International Publishing Switzerland 2014
Abstract Although the International Code of Nomenclature of Bacteria does not specify a working strategy, editors and reviewers of taxonomic journals commonly request a polyphasic taxonomic approach that includes phenotypic, genotypic and chemotaxonomic information for the description of novel bacterial species. Whole genome sequences provide an insight into the genetic nature of microbial species, yield new and superior tools for delineating bacterial species and for studying their phylogeny, and provide a window on an organism’s metabolic potential. These new insights and tools are gradually introduced in the polyphasic taxonomic practice. The genus Burkholderia, a controversial group of bacteria with both benign and devastating characteristics, is used as an example to show that the modern practice of polyphasic taxonomy is counterproductive in light of the tremendous number of bacterial species that awaits formal description and naming. Bacterial taxonomists must urgently reconsider how to describe and name novel bacteria in the genomic era, and should consider using a full genome sequence and a minimal description of phenotypic characteristics as a basic, sufficient, cost-
P. Vandamme (&) C. Peeters Laboratory of Microbiology, K. L. Ledeganckstraat 35, 9000 Ghent, Belgium e-mail: [email protected]
effective and appropriate biological identity card for a species description. Keywords Bacterial taxonomy Polyphasic taxonomy Species description Whole genome sequence Burkholderia Genomics Introduction In this day and age of sustainable energy and green society every now and then there is a surge of interest in biodiversity. On such occasions bacterial taxonomists may be grinding their teeth. The staggering numbers of—for instance—insect and plant species render the list of a mere ca. 12,000 bacterial species confronting and embarrassing (Tamames and Rossello´-Mo´ra 2012; Oren and Garrity 2014). Yet each of these insect guts or plant rhizospheres very likely harbors several yet-to-be-described bacterial species. So what have bacterial taxonomists accomplished during the past centuries and why don’t we also have lists of hundreds of thousands of species? Why this is still the case is hard to explain to those who are not familiar with bacterial taxonomy, and if you seriously try to explain this, for instance to bachelor students in biotechnology, they shrug their shoulders. Can we mend this situation? Well, let’s step back in time. We know the historical roots of bacterial taxonomy very well (Oren and Garrity 2014). Early classifications were based on morphology and biochemical data. They were rendered more robust
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through the introduction of numerical taxonomy and the use of large numbers of characteristics, and were subsequently complemented and reinforced by chemotaxonomic and genotypic characterization. All of this culminated into polyphasic taxonomy, a term coined in 1970 (Colwell 1970) and a practice described in detail in an exhausting review paper of our research group, that has been highly cited though by the bacterial taxonomist community (Vandamme et al. 1996). Polyphasic taxonomy integrates all available genotypic, phenotypic, and phylogenetic information into a consensus type of general-purpose classification. It departs from the assumption that the overall biological diversity cannot be encoded in a single molecule and that the variability of characters is group dependent. The bacterial species was defined as an assemblage of isolates originating from a common ancestor population in which a steady generation of genetic diversity resulted in clones with different degrees of recombination and characterized by a certain degree of phenotypic consistency, a significant degree of DNA–DNA hybridization (DDH) and more than 97 % of 16S rRNA gene sequence similarity (Vandamme et al. 1996). This practice of polyphasic taxonomy is however also one of the main reasons why the field remained an exclusive playground for a happy few and why we have a ridiculously short list of a mere 12,000 bacterial species named today.
The Burkholderia example: pars pro toto, or our failure to truly report and name bacterial diversity Our research group has a strong interest in the biodiversity of the genus Burkholderia. When we started studying this group of bacteria in the early 1990s it was a rather obscure genus comprising a handful of species only (Yabuuchi et al. 1992). At present, the genus Burkholderia contains more than 80 validly named species (see http://www.bacterio.cict. fr/) and a large number of candidate species (Van Oevelen et al. 2004; Lemaire et al. 2011; Verstraete et al. 2011; Lemaire et al. 2012). Burkholderia species occupy extremely diverse ecological niches, including pristine and contaminated soils, plant rhizospheres and phytospheres, invertebrate intestinal tracts and the respiratory tract of humans (Coenye and Vandamme 2003; Compant et al. 2008; Sua´rez-Moreno et al. 2012). Their impressive metabolic potential has been
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exploited for biocontrol, bioremediation and plant growth promotion purposes, but safety issues regarding human infections, especially in cystic fibrosis patients, have not been solved. The latter has been the main driver behind recent proposals to split the genus into a cluster comprising the ‘bad’ species (i.e. the socalled pathogen group) and a cluster comprising the ‘good’ species (i.e. those with biotechnological potential, or the so-called plant beneficial or symbiotic group) (Gyaneshwar et al. 2011; Sua´rez-Moreno et al. 2012; Estrada-de Los Santos et al. 2013; Angus et al. 2014). While such oversimplification sends a clear message, the neutral reader should be aware that the largest body of literature addressing so-called beneficial or symbiotic Burkholderia strains concerns species belonging to the pathogen group (Parke and Gurian-Sherman 2001), and that strains of several species within the so-called plant beneficial group have been involved in human infections (Coenye et al. 2001; Goris et al. 2004; Gerrits et al. 2005; Deris et al. 2010). Of the latter, especially Burkholderia fungorum has been isolated from a wide range of human and veterinary samples including human blood, cerebrospinal fluid, vaginal secretions, sputum and lavage samples of cystic fibrosis patients, and the brain of a pig with neurological deficit, the brain stem of an injured deer, and the nose of several mice (Coenye et al. 2001, 2002; Gerrits et al. 2005) (unpublished data). Not a good pedigree for a plant beneficial organism clearly! This applied relevance of Burkholderia strains also catalyzed a fairly successful quest for research funding to keep the Burkholderia biodiversity study going for more than two decades, although the words ‘taxonomy’ and ‘biodiversity’ were always removed with rigorous scrutiny from early application drafts to increase the odds of success, which is another behavior many taxonomists are familiar with. We discovered that Burkholderia cepacia isolates from cystic fibrosis patients and other human clinical and environmental samples represented at least five genomic species, collectively referred to as the B. cepacia complex (Bcc) (Vandamme et al. 1997). During two decades of Burkholderia diversity research the list of Bcc species has grown to 18 validly named species (Peeters et al. 2013). Driven by the devastating effect of these infections in cystic fibrosis patients, microbiologists gained an interest in their environmental occurrence and many environmental Burkholderia species were
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isolated, described and named in polyphasic taxonomic studies. During these studies multilocus sequence analysis (MLSA) emerged as a most useful taxonomic tool. Although initially developed for strain genotyping below the species level and designated as multilocus sequence typing (MLST) (Maiden et al. 1998), MLSA employs phylogenetic procedures based on the nucleotide sequences of multiple gene fragments to reveal similarities between strains representing different species and genera (Gevers et al. 2006). Especially for depicting relationships within and between closely related species like the Bcc bacteria, this approach has a taxonomic resolution superior to that of the traditional 16S rRNA gene sequence analysis. Interestingly, an average concatenated allele sequence divergence of 3 % can be used as a threshold value to distinguish between Bcc species (Vanlaere et al. 2009; Peeters et al. 2013) and is therefore equivalent to the 70 % DDH threshold. This validation of the MLSA approach as a tool to replace DDH has speed up the description of novel species, albeit minimally. More recently, bacterial taxonomists have embraced whole-genome studies and introduced whole-genome sequence (WGS) based parameters that yielded thresholds equivalent to the magical 70 % DDH level (Goris et al. 2007; Richter and Rossello-Mora 2009). Although it can be argued that this introduction of WGS parameters is a convenient way to replace DDH without the need to revise existing classification schemes (most of our fellow microbiologists hate name changes), we should ponder on this practice. Remember that DDH was introduced as a tool to approach WGS derived information as close as possible (Wayne et al. 1987), and now that we have direct access to WGS information, we want it to mimic the results obtained through DDH experiments! Still, the introduction of WGS allows us to implement parameters like the calculation of the percentage of conserved DNA (pcDNA) (Goris et al. 2007) or MUM-indices (Deloger et al. 2009) which can be used as in silico imitations of DDH experiments. Complementary to these approaches, the average nucleotide identity (ANI) (Konstantinidis and Tiedje 2005; Richter and Rossello-Mora 2009) and core gene identity (CGI) methods (Vanlaere et al. 2009) are both natural extensions of MLSA in case complete or draft genome sequences are available. These WGS based approaches are superior compared to traditional 16S rRNA sequence analysis for studying phylogeny because they are based on a much larger part of the genome and
because they have a better resolution for discriminating both distantly and closely related bacteria. So we have new and superior taxonomy tools to delineate species and to study phylogeny, and no doubt there is a clear and bright future for bacterial taxonomy in the genomic era. While the old DDH species threshold is being translated into MLSA or WGS based thresholds (Gevers et al. 2005; Goris et al. 2007; Richter and Rossello-Mora 2009; Tindall et al. 2010), a fair reappraisal of bacterial taxonomy and the species definition will require more than a mere methodological translation of threshold levels (Achtman and Wagner 2008). Our study of the diversity of B. cepacia-like infections in cystic fibrosis patients triggered a long series of polyphasic taxonomic studies of clinical and environmental micro-organisms which primarily belong to various non-fermenting Gram-negative bacteria of the Betaproteobacteria, and which thus far entailed the description of several novel genera including Advenella, Inquilinus, Kerstersia and Pandoraea, and some 70 novel species belonging to the genera Achromobacter (10 species), Advenella (2 species), Alcaligenes (1 species), Bordetella (2 species), Burkholderia [both Bcc (14 species) and non-Bcc (23 species)], Cupriavidus (7 species), Inquilinus (1 species), Kerstersia (2 species), Pandoraea (5 species), and Ralstonia (1 species); it also revealed quite a few synonymies between validly published names. This list of novel taxa could only be produced through much efforts by devoted PhD students and with the help of numerous international collaborators. The above list may seem impressive at first sight but during these 20 years progress was being made painstakingly slow, not the least because we tend to describe novel species only when we have a set of multiple genetically distinct strains. The inclusion of multiple strains is considered old-fashioned but good taxonomic practice by many (Oren and Garrity 2014), but is counterproductive as it implies a lot of extra work, not the least to find differential biochemical characteristics. This search for differentiating biochemical reactions is a practice that originates from diagnostic medical microbiology, where in the meantime modern methods such as MALDI-TOF mass spectrometry have recently replaced biochemical identification algorithms that are intrinsically inadequate (Desai et al. 2012). The submission of these studies also provoked quite a few friendly (well… mostly friendly) wars with editors of taxonomic
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journals. Indeed, especially within the group of nonfermenting Gram-negative bacteria, it is notoriously difficult to find two (!) or more differential biochemical tests when one compares sets of multiple strains per species, while the corresponding type strains may differ simultaneously in a multitude of characteristics. Reducing such studies to type strains only and effectively removing all other strains and the corresponding data from the study would often have made life so much easier for the PhD students involved. The practice of polyphasic taxonomy is also discouraging when considering the overwhelming number of novel species that are available in public and private strain collections (let alone the natural environment). Two examples to illustrate and conclude this point: mining the Bcc PubMLST database (http://pubmlst.org/bcc/) using the 3 % threshold value of average concatenated allele sequence divergence for species delineation (Vanlaere et al. 2009; Peeters et al. 2013) revealed the presence of another 16 novel species (January 2014) within the Bcc that await description and formal naming (Table 1; Fig. 1). Or, a Table 1 Novel Bcc species, showing for each strain their ST, source and year of isolation (if known)
Bcc species Other Bcc A
Other Bcc B
Other Bcc C
Other Bcc D
Other Bcc E
Other Bcc F
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ST
recent search for Burkholderia caledonica in seven rhizosphere soil samples collected near Pretoria (South Africa) not only yielded the B. caledonica isolates that were aimed for (Verstraete et al. 2014), but also representatives of nearly a dozen novel Burkholderia species (unpublished data). These were added to a list of several dozens of novel Burkholderia species in our collection that await enthusiastic students and funding (unpublished data).
Conclusion Where is this field going? We will never succeed in formally naming all Burkholderia species if the taxonomic community continues to describe and name novel species like it does now, and we will never be able to generate more than the proverbial drop of water on a hot plate in our efforts to formally describe prokaryotic diversity. The issue was recently raised by Sutcliffe et al. (2012), who called for a significant reappraisal of the procedures used to describe novel Strain
Source
Country
Year
112
T21
ENV
USA
113
LMG 21824
CF
USA
114
BC003
ENV
USA
519
AU7314
NON
USA
527
HI3541
ENV
USA
690
NII
787
NII
789 47
NII BCC0276
ENV
USA
48
MRL-10
ENV
USA
49
BCC0517
ENV
USA
481
AU7004
NON
USA
510
AU0278
CF
USA
522
AU9897
CF
USA
775
QLD039
CF
Australia
130
MDII-110p
ENV
Italy
1996
144
MVP-C1-49
ENV
Italy
1996
284
MW10-3
ENV
USA
357
BCC0430
ENV
Italy
445
MVPCII 48
ENV
Italy
588
HT15
ENV
China
735
B3
ENV
USA
471
NII
1999 2004
Antonie van Leeuwenhoek Table 1 continued
Bcc species
ST
Strain
Source
Country
Year
Other Bcc G
535
AU2654
CF
USA
2001
654
R117
CF
Germany
2010
533
AU0141
CF
USA
1997
346
NII
115
MD II 367
ENV
Italy
147
MVP-C2-84
ENV
Italy
156
MDIII-T224
ENV
Italy
2002
401 774
MDIII-P-292 QLD028
ENV CF
Italy Australia
2002 2008
116
C1714
CF
UK
206
CEP0178
CF
UK
252
AU2242
CF
USA
730
CC-AI74
ENV
Taiwan
791
NII
792
NII
794
NII
333
LMG 13014
IND
Belgium
334
BCC1300
IND
N America
597
JW13.3a
IND
UK
2007
812
SBL13/154
NON
New Zealand
2013
335
NII
Other Bcc N
810
BURK106
ENV
Australia
2007
Other Bcc O
797 763
NII R-50211
ENV
Argentina
2011
Other Bcc P
764
R-50214
ENV
Argentina
2011
Other Bcc H Other Bcc I
Other Bcc J Other Bcc K Other Bcc L
All data available in the Bcc PubMLST database (http:// pubmlst.org/bcc) CF isolated from a cystic fibrosis patient, NON isolated from a non-CF patient, ENV environmental isolate, IND isolated from an industrial product, NII no isolate information available, only sequence information
Other Bcc M
prokaryotic taxa, including the introduction of new publication formats, but the message appeared unheard in a field that may be too conservative. Sutcliffe et al. rightfully pointed out that although progress in the description of new microbial taxa is being made at accelerating rates, there is an enormous backlog of work. Even if progress in describing new taxa can be accelerated considerably over current rates, the challenge of adequately describing prokaryotic diversity could take several centuries (Sutcliffe et al. 2012; Tamames and Rossello´-Mo´ra 2012)! Conflicting with this need to dramatically change our throughput capacity, comprehensive guidelines for the description of novel species have been published by leading scientists of the systematics community (Tindall et al. 2010) which safeguard bacterial taxonomy as the playground of a few privileged with full access to a battery of phenotypic, genotypic and
2000
1991
chemotaxonomic tools, and the essential funding to pay for all analyses. In light of the vast unnamed microbial diversity this is counterproductive and we should indeed assess how much characterization is enough (Sutcliffe et al. 2012). Another practical consideration in this respect is that the number of bacterial taxonomists especially in Western countries is in sharp decline and that the extent of new species descriptions is primarily constrained by journal capacities, rather than taxonomists’ activities (Tamames and Rossello´-Mo´ra 2012). Polyphasic taxonomy has been so valuable for several decades because it provided a multifaceted view on an organism’s biology in an era where we did not have access to WGS information; not because we felt that one particular data type should be part of every organism’s identity card. The attempts to predict a dozen biochemical characteristics (Amaral et al. 2014) or to reconstruct an organism’s
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Antonie van Leeuwenhoek b Fig. 1 Phylogenetic tree based on the concatenated sequences
(2,773 bp) of seven housekeeping gene fragments [atpD (443 bp), gltB (400 bp), gyrB (454 bp), recA (393 bp), lepA (397 bp), phaC (385 bp) and trpB (301 bp)] of the 18 established Bcc species and novel Bcc species (labeled ‘other Bcc A to P’). For each of the 18 established Bcc species a maximum of 5 strains were selected: LMG 1222T, LMG 2161, LMG 17997, LMG 18821 and R-49849 for B. cepacia; LMG 13010T, LMG 18822, LMG 18824, R-18885 and LMG 16665 for B. multivorans; LMG 16656T, LMG 18826, LMG 18828, LMG 6986, MSMB554, LMG 18830, HI-2629, LMG 18829, PC28, LMG 18832, LMG 19230, LMG 19238, LMG 19240, LMG 19246, LMG 21462 and R-922 for B. cenocepacia (recA groups IIIA, IIIB, IIIC and IIID); LMG 14294T, LMG 7000, 5702, AU6735 and J5 for B. stabilis; LMG 10929T, LMG 18835, LMG 18836, LMG 6999 and G4 for B. vietnamiensis; LMG 18943T, LMG 19468, LMG 24508, IST4507 and AU3960 for B. dolosa; LMG 19182T, LMG 19467, LMG 17828, BCC0399 and BCC1222 for B. ambifaria; LMG 20980T, LMG 20983, LMG 16670, LMG 21821 and AU3904 for B. anthina; LMG 14191T, LMG 21822, LMG 21823, AU5468 and SBL12/065 for B. pyrrocinia; LMG 20358T, MSMB056, MSMB1396-A, LMG 24263 and R-52253 for B. ubonensis; LMG 24064T, LMG 24264, QLD058, MSMB101 and BURK108 for B. latens; LMG 24065T, LMG 24266, LMG 24267, HI3672 and R-9913 for B. diffusa; LMG 24066T, J1757, CEP0823, R-16920 and AU1125 for B. arboris; LMG 24067T, LMG 24271, LMG 19587, AU2877 and AU2283 for B. seminalis; LMG 24068T, R-2712 and AU7680 for B. metallica; LMG 23361T, LMG 16227, CEP0964, R-18428 and AU0059 for B. contaminans; LMG 22485T, LMG 14095, R-50215, BCC1406 and R-3211 for B. lata; LMG 26883T, LMG 16669, LMG 26914, LMG 26915 and TJI49 for B. pseudomultivorans. The sequences of LMG 16225T (B. fungorum), ATCC 23344T (B. mallei), LMG 19076T (B. caledonica) and LMG 14190T (B. glathei) were used as outgroup. Concatenated allele sequences of all strains were exported from the Bcc PubMLST database (http://pubmlst.org/ bcc) (Jolley and Maiden 2010) and a phylogenetic tree was constructed using the maximum likelihood method in RAxML version 7.4.2 (Stamatakis 2006). Rapid bootstrapping and ML search were performed using the general time reversible model with CAT approximation (GTRCAT) and the best scoring ML tree was exported in Newick format. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1,000 replicates) are shown next to the branches if greater than 70 %
chemotaxonomic components (Sutcliffe et al. 2012) illustrate the versatile exploitation of WGS information but should not pave the way for adding more guidelines to the characterization process of novel bacteria (Tindall et al. 2010). In a similar manner Ramasamy et al. (2014) presented a modernized schema for polyphasic taxonomy but although their approach accepted the utility of WGS and other modern data, they advocated adding these to the
conventional polyphasic approach rather than using their power to replace old methods and reduce the labor of describing new taxa. Together, all of this should indeed call us to arms and we must revitalize the practice of polyphasic taxonomy if we want to uncover more than the tip of the iceberg of microbial diversity in the next few decades. Urgent action from International Committee on Systematics of Prokaryotes is indeed badly needed (Sutcliffe et al. 2013) or the Committee may receive a wake-up call from rebelling taxonomists which recently proved very effective in moving the field of fungal taxonomy to a more modern practice (Hibbett and Taylor 2013). In the present day and age, a full genome sequence and a minimal description of phenotypic characteristics would provide a basic biological identity card that could be considered sufficient, cost-effective and appropriate for a species description. Acknowledgments We thank Iain Sutcliffe, Editor-in-Chief of Antonie van Leeuwenhoek, for his kind invitation to contribute to this festive issue of the journal and for giving us the opportunity to express some of our concerns. C. P. is indebted to the Special Research Council of Ghent University. The Burkholderia cepacia complex National Reference Center is supported by the Belgian Ministry of Social Affairs through a fund within the Health Insurance System.
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