International Journal of Systematic Bacteriology (1 999), 49, 1255-1 262 Printed in Great Britain

The correlation between morphological and phylogenetic classification of

Cathrin Sproer,’ Hans Reichenbach’ and Erko Stackebrandtl

Author for correspondence: Erko Stackebrandt.Tel: +49 531 2616 352. Fax: +49 531 2616 418. e-mail : [email protected]

DSMZ-Deutsche Sammlung In order to determine whether morphological criteria are suitable to affiliate von Mikroorganismen und myxobacterial strains to species, a phylogenetic analysis of 16s rDNAs was Zellkulturen GmbH1 and G BF-Gesel Isc haft fur performed on 54 myxobacterial strains that represented morphologically 21 Biotechnologische species of the genera Angiococcus, Archangium, Chondromyces, , Forschung mbH*, 381 24 Melittangium, , Polyangium and , five invalid species Braunschweig, Germany and three unclassified isolates. The analysis included 12 previously published sequences. The branching pattern confirmed the deep trifurcation of the order Myxococcales. One lineage is defined by the genera Cystobacter, Angiococcus, Archangium, Melittangium, Myxococcus and Stigmatella. The study confirms the genus status of Corallococcus’, previously ‘Chondrococcus’,within the family . The second lineage contains the genus Chondromyces and the species Polyangium (‘Sorangium’) cellulosum, while the third lineage is comprised of Nannocystis and a strain identified as Polyangium vitellinum. With the exception of a small number of strains that did not cluster phylogenetically with members of the genus to which they were assigned by morphological criteria (‘Polyangium thaxteri’ PI t3, Polyangium cellulosum ATCC 25531T, Melittangium lichenicola ATCC 25947Tand Angiococcus disciformis An dl), the phenotypic classification should provide a sound basis for the description of neotype species in those cases where original strain material is not available or is listed as reference material.

Keywords : Myxococcales, 16s rDNA analysis, phylogeny

INTRODUCTION were described as one of the major eubacterial divisions (Woese et al., 1985). This phylum was subsequently The myxobacteria are among the few higher pro- described as the &subdivision of the division of purple karyotic taxa in which morphology and the presence of (Oyaizu & Woese, 1985) when its members a complex life cycle of its members can be considered a were found to form an individual branch within the reflection of their common ancestry. These organisms radiation of the division of purple and non-purple were first shown to form a phylogenetically coherent bacteria and their relatives, equivalent in phylogenetic cluster by partial 16s rRNA analysis (Ludwig et al., depth (i.e. in the range of 16s rRNA identity values 1983), as a result of which myxobacterial species from separating the most divergent species of this taxon) to five genera formed three major sublines of descent, the a-, p- and y-subdivisions. This division was later represented by Nannocystis exedens, ‘ Sorangium cellu- described as the class (Stackebrandt et losum’ and Myxococcus fulvus and its relatives of the al., 1988), with the subdivisions reclassified as sub- genera Stigmatella and Cystobacter. Later, when a classes. The most comprehensive phylogenetic analysis number of 16s rDNA oligonucleotide catalogues of of myxobacteria was published by Shimkets & Woese hitherto less well-represented genera became available, (1992), who determined the almost complete 16s the myxo bacteria, together with the bdellovibrio s rRNA sequences of 12 strains belonging to 11 species (Hespell et al., 1984) and the Gram-negative sulfate- of nine genera. The results of that study confirmed the and sulfur-reducing organisms (Fowler et al., 1986), partition of species as found in the original study by ...... Ludwig et al. (1983) but broadened the ‘Sorangium’ The EMBL accession numbers of the 165 rDNA sequences reported in this (this name has not been validated and ‘Sorangium paper are AJ233897-AJ233950. cellulosum ’ has been reclassified as Polyangium cellulo-

01048 0 1999 IUMS 1255 C. Sproer, H. Reichenbach and E. Stackebrandt sum) subline by the inclusion of Chondromyces and phology and structure, important characteristics in myxo- Polyangium and the Myxococcus subline by the ad- bacterial , are often substantially modified or dition of Angiococcus, Archangiurn, Melittangiurn and disappear completely after several transfers, which may lead ' Corallococcus'. to misidentification. The availability of type strains for species of the order 165 rDNA sequence determination and analysis. Genomic Myxococcales DNA was extracted from the strains and used for the PCR- is a point of contention. The Approved mediated amplification of the 16s rDNA (Rainey et al., Lists of Bacterial Names (Skerman et al., 1980) and 1996). The purified PCR products were sequenced as subsequent Notification and Validation Lists contain described previously (Rainey et al., 1996). The sequences 33 species and nine genera. However, only 12 species of were determined by electrophoresis by using a model 373A seven genera are available from culture collections. automatic DNA sequencer (Applied Biosystems). The majority of species (1 5)are deposited as herbarium material, many of which have lost the ability to grow The phylogenetic position of the myxobacterial strains was determined by using the ae2 editor (Maidak et al., 1996). and to form fruiting bodies, which is the primary basis Evolutionary distances were calculated by the method of of species classification. For some species, no type Jukes & Cantor (1969). Phylogenetic dendrograms were material has been designated at all (e.g. Polyangium reconstructed by using the neighbour-joining treeing algor- aureum, Polyangiurn luteum, Polyangium parasiticurn, ithms contained in the PHYLIP package (Felsenstein, 1993) Polyangium sorediatum, Polyangium spumosum and as well as the algorithm of DeSoete (1983). Bootstrap Melittangium boletus). The situation is also confused values were determined by using the PHYLIP package by the fact that several species are listed with a (Felsenstein, 1993). The accession numbers of the 16s reference strain in the chapter ' Fruiting Gliding rDNAs of reference strains were : , Bacteria : The Myxobacteria' in Bergey 's Manual of M34 1 14; Myxococcus coralloides, M94278 ; Melittangium Systematic Bacteriology (McCurdy, 1989), a taxon lichenicola, M94277 ; Angiococcus discifomis, M94280 ; that has no standing in nomenclature according to Archangium gephyra, M94273 ; Cystobacter fuscus, M94276 ; Rule 19 of the Bacteriological Code (Lapage et al., , M9428 1 ; Polyangium (' Sorangium ') cellulosum, M94280 ; Chondromyces crocatus, M94275 ; 1975). Many type strains are deposited as non- Chondromyces apiculatus, M94274 ; Polyangium sp. P14943, culturable, dried herbarium material (accepted by Rule M94280 ; and Nannocystis exedens, M94279. 18a of the Bacteriological Code), which excludes the possibility of using these strains in comparative phylo- genetic and biochemical studies. Morphology and RESULTS AND DISCUSSION habitats are then the only characteristics that can be used to verify the affiliation of new isolates to known The almost complete primary structure of the 16s species. rDNA, consisting of a region between positions 44 and 1465 ( nomenclature ; Brosius et al., In this study, most of the 54 myxobacterial strains 1978) was determined for 54 myxobacterial strains investigated by comparative 16s rDNA analysis were isolated at various times from different locations. The tentatively assigned to described species and non- 16s rDNA sequences have been deposited in the validated species on the basis of morphological charac- EMBL database under the accession numbers in- teristics. Unfortunately, reference material of many dicated in Table 1. These sequences, representing 21 type strains was not available, which limited the species of nine genera and some invalid taxa and non- present investigation to some extent. In the light of the classified isolates, were aligned against the 16s rDNA future effort to replace non-living herbarium material database of 12 myxobacterial strains, including eight with culturable neotype strains, it was the goal of this type species of seven genera (Oyaizu & Woese, 1985; study to broaden the phylogenetic basis of species Shimkets & Woese, 1992), and phylogenetic trees were definitions within the order Myxococcales and to test generated. The 16s rDNA of Nannocystis exedens Na the importance of morphology in the classification of el, sequenced in this study, was 99-8YO identical to the these organisms. 16s rRNA sequence of the same strain published by Shimkets & Woese (1992). The branching patterns of METHODS the trees generated were highly similar with the two algorithms used and small differences occurred only in Bacterial strains. Most isolates analysed in this study were the order of branches among highly related strains. new isolates (collection Hans Reichenbach, GBF). All High bootstrap values (>80 YO)supported the tri- strains were cultivated aerobically on yeast agar (VY/2 agar) plates at 30 "C (Reichenbach & Dworkin, 1992). The furcation of the suborders and many other branching strains were classified by morphological criteria : shape, size points (Fig. 1). The three major lineages were separated and structure of vegetative cells, myxospores, fruiting bodies from each other by 16s rDNA identity values ranging and swarm colonies. Myxococcus stipitatus strains were from 73 to 85 YO. tentatively identified by the bright yellow fluorescence of their swarm colonies under UV irradiation at 366 nm All strains identified as members of the order Myxo- (Lampky & Brockman, 1977). Myxococcus xanthus and coccales on the basis of morphological criteria were Myxococcus virescens were distinguished from each other by also found to be phylogenetically related to members the greenish-yellow pigmentation of the latter which, how- of this order by analysis of their 16s rDNA. The ever, is easily lost during cultivation. Fruiting-body mor- inclusion of the isolates did not change the topology of

1256 International Journal of Systematic Bacteriology 49 Phylogeny of myxobacteria

Table 1. Strains investigated in this study, their morphology-based classification and 165 rDNA accession numbers

Isolate Morphology-based classification 16s rDNA accession no.

An dl Angiococcus discformis A5233909 An d4 Angiococcus disc formis A5233910 An d6 Angiococcus disc formis A523391 1 Ar gl Archangium gephyra A52339 12 Ar 82 Archangium gephyra A52339 13 Cm a14 Chondromyces apiculatus A5233938 sy t2 Chondromyces lanuginosus A5233939 Cm p17 Chondromyces pediculatus AJ23 3940 Cm a13 ‘ Chondromyces robustus’ A5233942 Cm a22 ‘ Chondromyces robustus’ A523394 1 Cc e12b ‘ Corallococcus exiguus’ A5233931 Cc el00 ‘ Corallococcus exiguus ’ A5233932 Cb f6 Cystobacterfuscus A5233897 Cb f10 Cystobacter fuscus A5233898 Cb f39 Cystobacter fuscus A5233899 Cb fe13 Cystobacter ferrugineus A5233900 Cb fe18 Cystobacter ferrugineus A523390 1 Cb fe27 Cystobacter ferrugineus A5233902 Cb m2 Cystobacter minor A5233903 Cb m6 Cystobacter minor A5233904 Cb vi29 ‘ Cystobacter violaceus’ A5233905 Cb vi34 ‘ Cystobacter violaceus’ A5233906 Me b7 Melittangium alboraceum A5233907 Me b8 Melittangium boletus AJ23 3908 cc c494 Myxococcus (‘ Corallococcus ’) coralloides A5233914 Cc c806 Myxococcus (‘ Corallococcus ’) coralloides A5233915 Mx fll Myxococcus jlavescens A52339 16 Mx f2 Myxococcus fulvus A52339 17 Mx f421 Myxococcus fulvus A5233918 Mx f428 Myxococcus fulvus A52339 19 Cc m7 Myxococcus macrosporus AJ23 3920 Cc m8 Myxococcus macrosporus A5233921 Mx s33 Myxococcus stipitatus A5233922 Mx s42 Myxococcus stipitatus A5233923 Mx s52 Myxococcus stipitatus A5233924 Mx v2 Myxococcus virescens A5233925 Mx v76 Myxococcus virescens A5233926 Mx vl Myxococcus virescens A5233927 Mx v144 Myxococcus virescens A5233928 DSM 435; Mx xl Myxococcus xanthus A5233929 Mx x132 Myxococcus xanthus A5233930 Na a1 ‘ Nannocystis aggregans’ A5233945 Na el Nannocystis exedens A5233946 Na e571 Nannocystis exedens A5233947 P1 t3 ‘ Polyangium thaxteri’ A5233943 P1 vtl Polyangium vitellinum A5233944 Pd e3 Stigmatella erecta A 523 3933 Pd el9 Stigmatella erecta A5233934 Sg a1 Stigmatella aurantiaca A5233935 Sg a15 Stigmatella aurantiaca A5233936 Sg a28 Stigmatella aurantiaca AJ23 3937 NOSO- 1 Unidentified A5233948 NOCB-2 Unidentified A5233949 NOCB-4 Unidentified A5233950

International Journal of Systematic Bacteriology 49 1257 C. Sproer, H. Reichenbach and E. Stackebrandt

100

cyenObecter~#rrsCb Rg Cystobactw~~CbblB cysdDb.ctsrhsATCC 25104~ Mellttangiumboletus Me MI Sfigmtella emda Pd d stigmdda aumdaca ATCC 25191 Stigmatallaerecia Pd el0 Si&na- aumnllaca Sg a28 1 1UI StigmaWa aurenlyBcB Sg a1

Angkxuis disdbnis ATCC 33172 Cystobder minor Cb m2

- -Sv8@m7 lLhruS Mx f2

hfyxococcuswanthus DSM 435, Mx xi

hiyxococcus flevsooensMX ni Myxocmx~swanthus ATCC 25232T Myxcnaxcus vkescsns Mx vl Mywococcxrs vhescens Mx v144 M~ococcuswanthus Mx x132

lO9b

...... ,...... ,...... Fig. 1. Neighbour-joining tree of 16s rDNAs showing the phylogenetic position of the type strains of different genera of the order Myxococcales and isolates that were assigned to myxobacterial species on the basis of morphological characteristics (e.g. fruiting bodies, myxospores, colour). The sequences of Gram-negative, sulfate-reducing bacteria were used to root the dendrogram. Numbers within the dendrogram indicate the percentages of occurrence of the branching order in 100 bootstrapped trees. The bar represents 10 nucleotide substitutions per 100 nucleotides.

the phylogenetic tree significantly, with three major The morphology-based affiliation of isolates to genera branches as originally defined on the basis of the 16s agreed for most strains, while a higher degree of rDNA cataloguing approach (Ludwig et al., 1983). discrepancies could be observed at the species level. Some of the families have been described, i.e. Myxo- However, for most genera, the intrageneric differences coccaceae Jahn 1924, Polyangiaceae Jahn 1924 emend. between the 16s rDNA primary structures of members Brockman 1989, Archangiaceae Jahn 1924 and Cysto- were very small, in some cases below 1 %. In these bacteraceae McCurdy 1970, while ' Sorangiaceae ' has cases, the branching pattern was based on sequence been proposed by Reichenbach & Dworkin (1992), differences within highly variable regions of the mol- although the genus name ' Sorangiurn' has not yet been ecule, which do not necessarily reflect phylogenetic validated. relationships (Stackebrandt & Goebel, 1994) and

1258 International Journal of Systematic Bacteriology 49 Phylogeny of myxobacteria should not be taken as evidence that species affiliation of members of the taxa. The most unrelated strain was is erroneous. ‘Cystobacter violaceus’ Cb vi29, showing as little as 97.4% identity to other members of the cluster. Genus Cystobacter The phylogenetic separateness of Cystobacter minor and ‘ Cystobacter violaceus ’ from the type species, This genus contains three species, Cystobacter fuscus together with unique morphological properties of these (the type species), Cystobacter ferrugineus and Cysto- strains, supports the proposal to elevate this cluster to bacter minor. The epithet minus is incorrect and should genus status. be corrected to minor, L. neut. Only reference but no type material is available for these species. While strains of Cystobacter minor were phylogenetically well Genus Myxococcus separated from the other two species and must prob- ably be considered members of a new genus (see All of the 13 strains identified as members of Myxo- below), strains of Cystobacter fuscus and Cystobacter coccus clustered with the type strain of Myxococcus ferrugineus were phylogenetically intermixed at a 16s xanthus, ATCC 19368T(Oyaizu & Woese, 1985) [listed rDNA identity level above 99.4 YO. as ATCC 25232T in the latest edition of the ATCC catalogue of strains (1994)l. The intrageneric structure recognized five subclusters, which were separated by Genus Me/ittangium 16s rDNA identity values of about 98-2% and showed bootstrap values above 55%. The first subcluster The genus Melittangium is a closely related sister taxon contained two isolates identified as members of Myxo- of the genus Cystobacter sensu stricto, from which it coccus xanthus, strains Mx x132 and Mx xl (=DSM was separated by 98.2% 16s rDNA identity. The 435). They shared more than 99% identity with the branching point was supported by a bootstrap value of type strain, ATCC 25232T. Other strains that were 90 YO.Melittangium contains three species but type highly related to Myxococcus xanthus were identified material is not available for the type species of the as Myxococcus virescens (four strains), a strain of genus, Melittangium boletus. The Melittangium cluster Myxococcus flavescens and a single strain of Myxo- contained two strains, which on the basis of mor- coccus (‘ Corallococcus ’) macrosporus (strain Cc m8). phological characteristics most closely matched Melit- The genus name ‘ Corallococcus ’ has been proposed by tangium boletus (strain Me b8) and Melittangium Reichenbach & Dworkin (1992) for former ‘ Chon- alboraceum (Me b7). The type strain of Melittangium drococcus’ species, but neither genus name has been lichenicola clustered with strains of ‘ Corallococcus ’ validated so far. The second subcluster consisted of a and was probably not correctly identified. group of three strains, two of which were identified as Myxococcus stipitatus (Mx s42 and Mx s52) and a Genera Archangium and Angiococcus strain of Myxococcus fulvus (Mx f2). Two strains of Myxococcus fulvus (Mx f421 and Mx f428) and a strain This cluster embraced the type species of Archangium of Myxococcus stipitatus (Mx s33) formed subcluster as well as strains assigned to the genera Archangiurn, 3, while the other two subclusters were defined by Cystobacter and Angiococcus. Closely related to this single strains of Myxococcus (‘ Corallococcus ’) macro- cluster was a neighbouring taxon, the strains of which sporus (Cc m7) and Angiococcus disciformis strain An are related the neotype strain of Angiococcus disci- dl. The data may indicate that the subtle differences in formis, ATCC 33 172? Angiococcus disciformis strain 16s rDNA sequences do not allow a reliable species An dl clearly does not belong to the same species as differentiation. They may also suggest that Myxo- the neotype strain of the species, strain ATCC 33 172T. coccus xanthus and Myxococcus virescens can not be The swarm and morphology of the latter cor- distinguished reliably by pigmentation alone, viz. respond to those of the genus Cystobacter, to which orange versus greenish-yellow, a colour difference that the species has been transferred in the latest edition of may disappear during cultivation anyway. DNA- Bergey’s Manual of Systematic Bacteriology. Since in DNA reassociation should establish whether the two our hands the neotype strain no longer produced species should be united. Myxococcus flavescens may fruiting bodies and myxospores, comparison of fruit- belong to the same complex. The Myxococcus fulvus ing bodies was not possible. The description of the and Myxococcus stipitatus strains used in this study neotype strain (Hook et al., 1980) suggests that this appeared to be typical representatives of each species, strain indeed represents the species originally described which are clearly distinguished by smaller, often oval by Thaxter (1904). The strain was a phylogenetic myxospores and the yellow fluorescence mentioned neighbour of Cystobacter ferrugineus Cb fe 13 and two above for Myxococcus stipitatus. Yet, strains of either moderately related strains of a tentative morpho- species were placed at different locations in the species ‘ Cystobacter violaceus ’. Strains of the invalid dendrogram. The position of the two Myxococcus species ‘ Cystobacter violaceus ’ grouped with strains of macrosporus strains is less of a surprise. Both produced Cystobacter minor. The Cystobacter minor strains are only degenerated fruiting bodies and therefore could morphologically similar to Angiococcus disciformis really represent Myxococcus virescens strains. As both ATCC 33 172T, supporting the phylogenetic clustering strains have large myxospores, 2-2.5 pm in diameter, hternational lo~irnalof Systematic Bacteriology 49 1259 C. Sproer, H. Reichenbach and E. Stackebrandt they should cluster together and with Myxococcus genetically coherent cluster. The phylogenetic distance virescens. Again, DNA-DNA reassociation studies among certain species, however, indicates that the should resolve the problem of species affiliation. genus is genomically more diverse than the other Angiococcus disciformis strain An dl has a typical genera of the Myxococcales. While some species myxobacterial swarm with soft, snaking veins and exhibited 16s rDNA identities above 98.5 % (‘ Chon- spherical myxospores but, in contrast to all other drornyces robustus ’, Chondromyces apiculatus and members of the family, produces fruiting bodies with Chondromyces lanuginosus), the species Chondromyces sporangioles. Strain An dl does not belong to the same crocatus. and Chondromyces pediculatus showed species and genus represented by the neotype strain of around 96.5 70 sequence identity to each other and to Angiococcus disciformis, ATCC 33 172? other species of the genus. A more detailed analysis of Chondromyces crocatus strains has recently demon- strated the phylogenetic identity of most of the The ‘Corallococcus‘ cluster analysed strains. Three strains were slightly less re- This cluster is a sister taxon of the genus Myxococcus, lated, showing around 98.5% 16s rDNA identity to containing the type strain of Myxococcus (‘ Corallo- the other strains of the species (Jacobi et al., 1996). coccus ’) coralloides ATCC 25202T (previously ‘ Chon- Also included in the Chondromyces cluster is a strain drococcus’) and, unexpectedly, the type strain of classified as ‘ Polyangium thaxteri’ on the basis of its Melittangium lichenicola, ATCC 25946T. Two strains unique morphology (see Reichenbach & Dworkin, that were identified as typical members of the emerging 1992). This organism produces clusters or chains of genus ‘ Corallococcus ’, ‘ Corallococcus exiguus ’ Cc large, spherical, orange-brown sporangioles, which e 100 and Cc e2 16, were also found to be placed within may be borne on a short slime pedicle. Thus, the this cluster, while two strains of Myxococcus (‘ Coral- fruiting body has little resemblance to a Chondromyces lococcus’) coralloides, Cc c494 and Cc c806, branched fruiting body but is also clearly different from those somewhat deeper. The ‘ Corallococcus ’ strain cluster described for Polyangium species. was separated from members of Myxococcus by 16s rDNA identity values between 97 and 98 YO.A boot- Genus Polyangium. Ten species of this genus have been strap value of 100% strongly supported the bifur- described, but with the exception of Polyangium cation point of these two neighbouring taxa. The (‘ Sorangiurn ’) cellulosum, culturable reference material separate position of ‘ Corallococcus’ species, together is not available for any of them. For many species, not with their distinct morphological characteristics and even a reference strain is available. While Polyangium physiological differences, indicate that this taxon (‘ Sorangium ’) cellulosum subsp. ‘ferrugineum ’ ATCC merits genus status. 2553 1 and Polyangium sp. PI 4943 (Shimkets & Woese, 1992) clustered distantly with members of Chondro- rnyces, Polyangium vitellinum PI vt 1 defined a separate Genus Stigmatella line of descent. The morphology of strain P1 vtl most closely matches that of the type species, ‘Polyangium This genus, containing two species for which type vitellinum, in that the organism produces greenish- material is available, formed a phylogenetically well- yellow swarms and large, spherical, yellow sporan- defined taxon. Strains of the two species showed gioles within and on the substrate. Obviously, the greater than 99.4 YO16s rDNA sequence identity and phylogenetic position of Polyangium vitellinum is the branching pattern of strains did not allow clear neither close to Polyangium (‘ Sorangium ’) cellulosum discrimination between the two species, Stigmatella ATCC 25531 nor to any species of Chondromyces. erecta and Stigmatella aurantiaca. The two are very Apparently, the genus-specific morphological charac- difficult to distinguish if fruiting body morphology has teristics of Polyangium do not bear phylogenetic somewhat degenerated. A clear difference in significance, as strains identified as members of this size appears to support the species differentiation genus were placed at three different positions within (Neumann et al., 1992). the suborder Sorangineae (see below). In fact, the fruiting bodies of the Polyangiurn species are simply Suborder Sorangineae clusters and piles of smaller and larger sporangioles, sometime closely packed and polyhedral, and they Genus Chondromyces. The genus Chondromyces consists resemble the fruiting bodies of Cystobacter and Angio- of five species, all of which have been deposited as coccus. Cystobacter and Polyangiurn species have herbarium material and hence are not available for therefore previously been classified together, but can phylogenetic analysis. However, Chondromyces strains easily be separated by the shape of the vegetative cells. are the most differentiated morphologically among the From this it follows that Polyangium cellulosum and myxobacteria, meaning that strains can be allocated to possibly ‘ Polyangium thaxteri’ probably represent the described species with considerable confidence. Com- nuclei of new genera. The genus name Sorangium parison of 16s rDNA sequences revealed that all should be revived for the cellulose degraders of the isolates affiliated to the species Chondromyces lanugin- Polyangium cellulosum complex. A new genus will be oms, Chondromyces apiculatus, Chondromyces cro- defined for the ‘Polyangium’ species of the PI 4943 catus and Chondromyces pediculatus formed a phylo- type, which are very common myxobacteria in soil.

~ ~~ 1260 International Journal of Systematic Bacteriology 49 Phylogeny of myxobacteria

Genus Nannocystis. The separate position of the genus neotype strains. As myxobacteria have a highly de- Nannocystis has already been noted in the first phylo- veloped morphology, it should be possible to find genetic study on myxobacteria (Ludwig et al., 1983) strains that correspond to the original descriptions. and was later explained by rapidly evolving 16s rDNA There is, however, a basic difficulty. Fruiting-body (Shimkets & Woese, 1992). The presence of the morphology often degenerates or is lost completely sequence idiosyncrasies in the strains of Nannocystis during cultivation, with the consequence that the type investigated in this study was confirmed. The two or neotype strain no longer shows the characteristics of strains of Nannocystis exedens (Nannocystis exedens the species. In fact, this observation has made myxo- Na el was sequenced twice, see above) shared more bacterial taxonomists hesitate to deposit neotypes. than 99.4 % sequence identity, and this species is highly There are, however, two ways to circumvent the related to the new species ‘ Nannocystis aggregans ’ problem. Firstly, the use of preservation methods that (99 % sequence identity). allow the strains to produce fruiting bodies, e.g. drying fruiting bodies on filter paper at an early stage of Unassigned isolates. Three isolates could not be assigned to any of the acknowledged myxobacterial species on cultivation. Secondly, the deposition of type strains the basis of morphological criteria. The phylogenetic together with careful and comprehensive photographic analysis confirmed their morphological uniqueness. documentation of fruiting body and myxospore mor- phology, both being made available to interested While isolate NOSO- 1, showing some morphological : resemblance to species of Polyangium, branched deeply investigators. Some difficulties will still remain fruit- within the suborder Sorangineae, the isolates NOCB-2 ing bodies often change their shape and structure and NOCB-4, resembling Cystobacter species, are during cultivation. Thus, members of several genera, Stigmatella. e.g. Stigmatella, Cystobacter and Melittangium, may distantly related to members of These Archangium-type two new groups of isolates probably represent novel produce fruiting bodies. This may lead to erroneous classification, even by experienced genera. specialists. The search for differentiating physiological properties has not been particularly successful so far. Conclusions Eventually, systematic monitoring for differentiating characteristics at the chemotaxonomic level, e.g. pro- The present study confirms not only that the myxo- tein patterns and lipids, and at the molecular level, e.g. bacteria are a phylogenetically coherent group, but, to species-specific oligonucleotide probes, may help to a large extent, the phylogenetic significance of mor- overcome the presently unsatisfactory taxonomic situ- phological characteristics of myxobacteria. Strains ation within the Myxococcales. classified on morphological grounds, such as Arch- angium gephyra, Cystobacter fuscus, Nannocystis exe- dens and Stigmatella aurantiaca, indeed clustered next REFERENCES to the respective neotype strains in the phylogenetic Brosius, J., Palmer, M. L., Kennedy, P. J. & Noller, H. F. (1978). analysis. In two cases, no match was observed. The Complete nucleotide sequence of a 16s ribosomal RNA gene type strain of Angiococcus disciformis, ATCC 33 127T, from Escherichia coli. Proc Nut1 Acud Sci USA 75, 4801-4805. clearly does not belong to the family Myxococcaceae. DeSoete, G. (1983). A least square algorithm for fitting additive On the basis of cell and swarm morphology, it seems trees to proximity data. Psychometrika 48, 621-626. justified that the species has been transferred to the genus Cystobacter. This means that strain An dl, Felsenstein, J. (1993). PHYLIP (phylogenetic inference package) Myxococcaceae, version 3.5.1. Department of Genetics, University of Wash- which belongs to the represents a new ington, Seattle, USA. genus. The second case refers to the neotype strain of Melittangium lichenicola, ATCC 25946T, which clus- Fowler, V. J., Widdel, F., Pfennig, N., Woese, C. R. & Stackebrandt, ‘ Corallococcus’ E. (1986). Phylogenetic relationship of sulfate- and sulfur- tered within the radiation of species reducing eubacteria. Syst Appl Microbiol6, 32-41. and may have been misclassified. There are many fruiting-body-forming strains that conform to the Hespell, R. B., Paster, J., Macke, T. & Woese, C. R. (1984). The origin and phylogeny of the bdellovibrios. Syst Appl Microbiol description of the species and those strains certainly do 5, not belong to the family Myxococcaceae. 196-203. Hook, L. A., Larkin, J. M. & Brockman, E. R. (1980). Isolation, The main problem identified in this study refers to the characterization, and emendation of description of Angiococcus availability of type strains for myxobacterial species. disciformis (Thaxter 1904) Jahn 1924 and proposal of a neotype The original descriptions of most species are based on strain. Int J Syst Bucteriol30, 135-142. fruiting bodies developing in crude cultures and many Jacobi, C. A., Reichenbach, H., Tindall, B. 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~ International Journal of Systematic Bacteriology 49 1261 C. Sproer, H. Reichenbach and E. Stackebrandt

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