Chasing a Unicorn for Model Host-Microbiome-Systems
phages manuscript HHMI (1)
1. Dramatic variation in phage genome structures revealed by whole genome comparisons
Welkin Pope1
, Charles Bowman1
, SEA-PHAGES2
, PHIRE3
, K-RITH MGC4
, Deborah Jacobs-
Sera1
, Daniel A. Russell1
, Steven Cresawn5
, William R. Jacobs Jr.6
, Jeffrey G. Lawrence1
,
Roger W. Hendrix1
, and Graham F. Hatfull1
*.
1
Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260
2
Science Education Alliance Phage Hunters Advancing Genomics and Evolutionary Science
3
Phage Hunters Integrating Research and Education
4
KwaZulu-Natal Institute for TB and HIV research Mycobacterial Genetics Course
5
Department of Biology, James Madison University, Harrisonburg, VA
6
Department of Microbiology and Immunology, Albert Einstein College of Medicine, NY
*Corresponding Author
2. 2
Bacteriophages are the dark matter of the biological universe1
, forming a vast, dynamic,
old, and genetically diverse population2
. Horizontal exchange generates pervasive
genome mosaicism, with different genome segments having distinct evolutionary
histories3
. Phages of phylogenetically distant hosts typically share low nucleic acid
sequence similarity, and few share genes with amino acid sequence similarity2
. Phages
of a single common host can also span considerable sequence diversity even though
they are in direct genetic contact1
. Comparative genomics of a large collection of phages
isolated on Mycobacterium smegmatis provides insights into the size and diversity of
groups of related phages and the extent to which the groups are discrete and genetically
isolated from other phages. We show that both the diversity and genetic isolation of
phage groups varies enormously. Some are discrete and share few genes with other
phages, whereas others are genetically connected to many other phages. The phage
population thus spans a continuum of relationships, but with phages of different types
varying enormously in prevalence. The reticulate relationships resulting from pervasively
mosaic architectures confound hierarchical taxonomic phage classification or
application of simple numerical values to distinguish among phage genomic types.
Bacteriophages are the most abundant organisms in the biosphere, and the ~1031
tailed phage
particles participate in ~1023
infections per second on a global scale, with the entire population
turning over every few days4
. Virion structures suggest the population is also extremely old5
and
thus the great genetic diversity of phages is not surprising2
. Phages likely evolved with common
ancestry and access to a large common gene pool3
, although rates of horizontal exchange are
heterogeneous, being influenced by host range, varying phage migration rates across the
microbial landscape, and lifestyle (temperate or virulent)6
. Multiple processes determine this
including local host diversity and mutation rates, as well as resistance mechanisms such as
receptor availability, restriction, CRISPRs, and abortive infection systems6,7
. Constraints on
3. 3
gene acquisition may also be imposed by synteny – particularly among virion structural genes –
and by size limits of DNA packaging2,8
.
Genomic comparison of phages infecting a common host provides insights into evolutionary
mechanisms and the structure of their genetic diversity9
. Relatively small numbers of phage
genomes have been sequenced for hosts such as Escherichia coli, Salmonella,
Staphylococcus, Pseudomonas, and Propionibacterium10-13
revealing varying degrees of genetic
diversity. Mycobacteriophages isolated from environmental samples using Mycobacterium
smegmatis mc2
155 as a host are architecturally mosaic1
and span considerable diversity, but
can be grouped into ‘clusters’ of related phages that share little or no nucleotide sequence
similarity with other phages1,14-18
. Some clusters are heterogeneous and can be readily divided
into subclusters by their nucleotide similarities. Recent analysis of phages adsorbed to
Synechococcus revealed 26 discrete ‘populations’, although they were obtained from a single
sample and are predominantly morphologically myoviral (T4-like)9
. However, these populations
likely represent only a small portion Synechococcus phages because the genomes of 17 fully
sequenced phages infecting Synechoccocus or closely-related hosts fail to associate with these
“populations”9
. These populations may thus reflect sampling bias of the single environment
examined, and extensive genomic mosaicism found in phages of Synechococcus and other
hosts1,3,19
warrants caution in extrapolation of the concept of discrete phage populations in the
absence of complete genome sequences.
The Howards Hughes Medical Institute (HHMI) Science Education Alliance Phage Hunters
Advancing Genomics and Evolutionary Science (SEA-PHAGES) program has facilitated
expansion of the number of sequenced mycobacteriophage genomes to 627 (Table S1) by
engaging large numbers of undergraduates in phage discovery and genomics20
. The size of this
collection now provides sufficient resolution to offer insights into the diversity and genetic
4. 4
isolation of phage genome types. Here we address the question of whether the groups of
related phages represent primarily discrete populations or genetically intermixed groups.
Although the collection excludes viruses that don’t form plaques under laboratory conditions, the
phages were isolated from widely dispersed geographical locations, including nine countries
and 36 of the continental United States (Fig. S1), over a dozen or more years. All are dsDNA
tailed phages (Caudovirales), and are morphologically siphoviral, except cluster C myoviruses.
Most have isometric heads except for singleton MooMoo and the Cluster I and O phages, which
have prolate heads21
.
Using previously reported parameters15
the 627 genomes were assembled into 20 clusters (A –
T) and 8 singletons (with no close relatives) with large variations in Cluster sizes (Table 1, Fig.
S2); 11 clusters can be subdivided into 2 to 11 subclusters (Table 1). Clustered phages typically
share genome architectures; for example, Cluster A phages are similar in size, transcriptional
organization, and share an unusual immunity system16,22
. A different set of clustering
parameters would generate different profiles, but not alter the core observation that there are
large variations among the different phage types. Cluster designation is simple for some phage
types because of extensive nucleotide similarity (e.g. Cluster C; Fig. S2), and if all clusters
resembled Cluster C, our data would be congruent with the Synechococcus populations 9
. But
many do not, revealing more complex relationships.
To compare mycobacteriophage gene contents we grouped related genes into phamilies using
Phamerator23
, modified to use kclust24
. The 69,633 genes assembled into 5,205 phams of which
1,613 (31%) are orphams14
(single-gene phamilies), and the gene content relationships are
represented as a network phylogeny in Fig. 1. In general, branch lengths provide strong support
for cluster and subcluster designations (Table 1, Fig. S2); the proportions of orphams per
genome provide additional support, which as expected is highest for singletons and single-
5. 5
genome subclusters (Fig. S3). Determination of the proportions of shared genes by pairwise
comparisons reveals the complexity of the genetic relationships (Fig. 2), and three major
features are apparent.
First, the overall phage relationships closely mirror the cluster and subcluster designations
derived by DNA similarities (Fig. S2). Secondly, the intra-cluster and intra-subcluster diversity
varies enormously, and this is quantified as the Cluster Cohesion Index (CCI, average number
of genes/genome divided by the total number of phamilies in the cluster; Table 1, Fig. 3). Thus
in clusters such as Cluster A (CCI, 0.08), the total number of phamilies is vastly greater than the
average number of genes per genome, indicating high diversity. The diversity of the A
subclusters is also highly varied with CCI values ranging from 0.22 to 0.91 (Table S1). In
contrast, Clusters G and O have low diversity (high CCI values) and closely related genomes
(Table 1; Fig. 3).
Thirdly, the degree to which clusters are genetically connected to other phages varies greatly,
and is quantified as the Cluster Isolation Index (CII, the percentage of phamilies not present in
genomes outside of the cluster; Table 1, Fig. 3). Some clusters such as Clusters A, B, C, and Q
share relatively few genes (<25%) with other phages and have high CCI values (Fig. 3). Other
groups, such as Clusters I and P, share >60% of their genes with other phages (Table 1),
reflecting the DNA relationships (Fig. S4). There are therefore no universally applicable values
of either diversity or isolation for different phage groups, and the most striking picture emerging
is one of great diversity with unequal representation of different types (Fig. 3). This is in marked
contrast to the discreet populations reported for Synechococcus phages9
.
These comparisons reveal additional complexities arising from highly mosaic genomes (Figs.
S5-S8). For example, Dori is clearly related to Cluster B phages (Fig. 1) with which it shares 20-
6. 6
26% of its genes and limited DNA similarity (Fig. S5), but also has nucleotide similarity and
shares genes with Cluster N and I2 phages, among others (Fig. S5, S7A), as reflected in its low
CII (Table 1, Fig. 3). Likewise, the singleton MooMoo has segments of DNA similarity and
shares ~20% of its genes with Cluster F phages (Fig. 1, S6, S7B), but also has similarity to
Clusters N and I; it also has a low CII (Table 1, Fig. 3). It has low DNA similarity to Cluster O
(Fig. S6), but shares several genes and has the same unusual prolate morphology (Fig. 1).
Complex relationships are also seen in the singletons Gaia and Sparky (Fig. S8).
Bacteriophage taxonomic classification reflecting phylogeny presents substantial challenges
because of genome mosaicism25
. Classification by viral morphology is well established, but may
not accurately report the genetic relationships, as observed for the prolate-headed MooMoo
(Fig. 1). We also note that the mycobacteriophage myoviruses have a high CII and form a
discrete group (Table 1) as for the Synechococcus phages9
, perhaps reflecting a virulent
lifestyle that constrains productive gene exchange; host range mutability may also differ in
phages with different morphotypes, limiting access to the gene pool. Although grouping phages
into clusters and subclusters provides analytical advantages because of the wide range in
prevalence of the different types (Table 1), it is not suitable as a broadly applicable hierarchical
taxonomic system. Reticulate taxonomies more accurately reflect the phylogenetic
complexities25,26
.
Given the sampling ranges of these phages, it seems unlikely that the population profile
reported here is specific for M. smegmatis mc2
155 phages and we predict that related profiles
will be found for phages isolated from similar environments using different hosts. However,
phage types occurring rarely in M. smegmatis may be abundant in phylogenetically proximal
hosts, and we predict that phage populations at large – regardless of host – represent a
continuum of complex reticulate relationships. Finally, we predict that the overall diversity of the
7. 7
phage population is in large part a consequence of narrow but mutable viral host ranges, which
promotes local genetic isolation and constrains access to the common gene pool.
METHODS
In addition to extant GenBank sequence information, mycobacteriophages were isolated,
sequenced, and annotated in the Phage Hunters Integrating Research and Education (PHIRE)
or Science Education Alliance Phage Hunters Advancing Genomics and Evolutionary Science
(SEA-PHAGES) programs. All genome sequences are publically available at phagesDB.org or
in GenBank. Nucleotide comparisons used BlastN or Gepard27
. To create database
Mykobacteriophage_627, phamilies were constructed by first clustering to an equivalent of 70%
amino acid sequence identity and a 25% size threshold, followed by multiple sequence
alignment using kAlign28
. Consensus sequences were extracted using hhmake and
hhconsensus29
, and passed through a second iteration of kClust, clustering proteins above a
threshold e-value of 10-4
. CCI values were calculated as the average number of genes/genome
divided by the total number of phams in that cluster. Thus if all genomes in a cluster are
identical (and if phamilies occur only once in a genome), CCI would be one; the CCI for two sets
of five randomly chosen genomes is ~0.02. CII is the percentage of phams present within a
cluster that are not present in other mycobacteriophage genomes. Students, faculty and their
contributions to authorship are listed in Table S3.
ACKNOWLEDGEMENTS
This work was supported in part by the Howard Hughes Medical Institute SEA-PHAGES
program, by the Howard Hughes Medical Institute through its Professorship grant to GFH, and
by NIH grant GM51975 to GFH.
8. 8
Author Contributions
Authors and contributions are listed in Table S3.
9. 9
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20 Jordan, T. C. et al. A broadly implementable research course in phage discovery and
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23 Cresawn, S. G. et al. Phamerator: a bioinformatic tool for comparative bacteriophage
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12. 12
Figure Legends
Figure 1. Network phylogeny of 627 mycobacteriophages based on gene content.
Genomes of 627 mycobacteriophages were compared according to shared gene content using
the Phamerator23
database mykobacteriophage_627, and displayed using Splitstree30
. Colored
circles indicate grouping of phages labeled according to their cluster designations generated by
nucleotide sequence comparison (Fig. S2); singleton genomes with no close relatives are
labeled but not circled. Micrographs show morphotypes of the singleton MooMoo, the Cluster F
phage Mozy, and the Cluster O phage Corndog. With the exception of DS6A, all of the phages
infect M. smegmatis mc2
155.
Figure 2. Heat map representation of shared gene content among 627
mycobacteriophages. The percentages of pairwise shared genes was determined using a
database (mykobacteriophage_627) generated by Phamerator23
populated with 627 completely
sequenced phage genomes. The 69,574 genes were assembled into 5,205 phamilies (phams)
of related sequences using kclust, and the average percentages of shared phams calculated.
Genomes are ordered on both axes according to their cluster and subcluster designations
determined by nucleotide sequence similarities (Fig. S2). The values are colored as indicated.
Figure 3. Relationships between Cluster Cohesion and Cluster Isolation Indexes of
Mycobacteriophage groups. Mycobacteriophage clusters and singletons are plotted
according to their Cluster Isolation Index and Cluster Cohesion Index. Groups are colored
according to the numbers of phages in that group; scale is shown above. There is enormous
variation in both cluster isolation and cluster diversity among the different groups.
13. Table 1. Diversity and genetic isolation of mycobacteriophage genome clusters
Cluster # Subclusters # Genomes Avg # genes
1
Ave length (bp) Total phams
2
Total genes Cluster Cohesion
3
Cluster Isolation
4
A 11 232 90 51514 1085 20880 0.08 80.2
B 5 109 100.4 68653 421 10944 0.24 81.0
C 2 45 231 155504 486 10395 0.48 84.6
D 2 10 89.3 64965 147 893 0.61 71.4
E 1 35 141.9 75526 236 4967 0.60 59.3
F 3 66 105.3 57416 658 6950 0.16 55.8
G 1 14 61.5 41845 72 861 0.85 55.6
H 2 5 98.4 69469 207 492 0.48 67.6
I 2 4 78 49954 147 312 0.53 23.8
J 1 16 239.8 110332 530 3776 0.45 58.5
K 5 32 95.7 59720 411 3069 0.23 73.5
L 3 13 127.9 75177 246 1663 0.52 72.4
M 2 3 141 81636 201 423 0.70 69.2
N 1 7 69.1 42888 152 484 0.45 40.8
O 1 5 124.2 70651 151 621 0.82 64.2
P 2 9 78.8 47668 159 709 0.50 34.0
Q 1 5 85.2 53755 90 426 0.95 73.3
R 1 4 101.5 71348 117 406 0.87 71.8
S 1 2 109 65172 117 218 0.93 70.9
T 1 3 66.7 42833 83 200 0.80 62.7
Dori 1 1 94 64613 94 94 1.00 35.8
DS6A 1 1 97 60588 96 97 1.01 58.3
Gaia 1 1 194 90460 193 194 1.01 58.0
MooMoo 1 1 98 55178 98 98 1.00 31.6
Muddy 1 1 71 48228 70 71 1.01 71.4
Patience 1 1 109 70506 109 109 1.00 57.8
Sparky 1 1 93 63334 93 93 1.00 48.4
Wildcat 1 1 148 78296 148 148 1.00 69.6
1
Average number of protein-coding genes per genome
2
Total phams is the sum of all phamilies (groups of homologous mycobacteriophage genes) in that cluster
3
Cluster Cohesion Index (CCI) is generated by dividing the average number of genes per genome by the total number of phamilies (phams) in
that cluster. For singleton phages (bottom eight rows) the number of phams is equivalent to the number of genes (.e. CCI is one), except
where phams are represented by two or more genes in the same genome.
4
Cluster Isolation Index (CII) is the percentage of phams that are present only in that cluster, and not present in other mycobacteriophages
16. A B
C
K
F
N
P
I
J
H
L D
M
E
O
T
R S
Q
G
ClusterIsolationIndexMoreIsolatedLessIsolated
Cluster Cohesion Index
Less DiverseMore Diverse
0 0.2 0.4 0.6 0.8 1.0
20
30
40
50
60
70
80
90
Wildcat
Muddy
MooMoo
Dori
Sparky
Gaia
DS6A
Patience
>200 100-200 50-100 10-50 5-10 2-5 Singleton
Figure 3
17. SUPPLEMENTARY DATA
Supplementary Tables
Table S1. Phages used in this study and their cluster designation
Table S2. Genometrics and Cluster Cohesion Index of mycobacteriophages.
Supplementary Figures
Figure S1. Geographical distribution of sequenced mycobacteriophages. (A) Locations of
sequenced mycobacteriophages across the globe. (B) Locations of sequenced
mycobacteriophages across the United States. Data from www.phagesDB.org.
Figure S2. Nucleotide sequence comparison of 627 mycobacteriophages displayed as a
dotplot. Complete genome sequences of 627 mycobacteriophages were concatenated into a
single file and compared with itself using Gepard1
and displayed as a dotplot. The order of the
genomes is as listed in Table S1. Nucleotide similarity is a primary component in assembling
phages into Clusters, which typically requires evident DNA similarity spanning more than 50% of
the genome lengths.
Figure S3. Proportions of orphams in mycobacteriophage genomes. The proportions of
genes that are orphams (i.e. single-gene phamilies with no homologues within the
mycobacteriophage dataset) are shown for each phage. The order of the phages is as shown in
Table S1. All of the singleton genomes have >30% orphams, and most of the other genomes
with relatively high proportions of orphams are the single-genome subclusters (see Table S2)
including Hawkeye (D2), Myrna (C2), Squirty (F3), Barnyard (H2), Che9c (I2), Whirlwind (L3),
Rey (M2), and Purky (P2). Three phages shown in red type are not singletons or single-
genome subclusters but have relatively high proportion of orphams. Predator and Menkokysei
18. are members of the diverse and small clusters (5 or fewer genomes) H, and T respectively;
KayaCho is a member of Subcluster B4 but has a sufficiently high proportion of orphams to
arguably warrant formation of a new subcluster, B6.
Figure S4. Dotplot of phages in Clusters I, N, P and the singleton Sparky. Dotplot was
generated using a concatenated file of genome sequences using Gepard1
. The complexity of
the genome relationships is illustrated by the Cluster I phages which share varying degrees of
similarity to phages in Clusters N and P, as well as the singleton Sparky. Because inclusion of
a phage in a cluster typically requires sharing a span of similarity over half of the genome
lengths, these phages are not assembled into a single larger cluster.
Figure S5. Dotplot of Carcharodon, Che9c, Kheth and Dori. The dotplot of concatenated
genome sequences illustrates the ambiguity of whether the singleton Dori warrants inclusion in
Cluster B. Dori shares DNA sequence similarity with its closest relative Kheth (Subcluster B2),
but it does not span 50% of the genome lengths. Dori also share DNA sequence similarity with
Che9c (Cluster I2) and Carcharodon (Cluster N).
Figure S6. Dotplot of Corndog, Brujita, SG4, Yoshi, and MooMoo. The dotplot of
concatenated genome sequences illustrates the complex relationships between the singleton
MooMoo and other phages. MooMoo shares DNA sequence similarity with SG4 (Subcluster F1)
and Yoshi (Subcluster F2), but also with Brujita (Subcluster I1). MooMoo has barely detectable
DNA sequence similarity with Corndog (Cluster O), but has a similar prolate virion morphology.
Figure S7. Shared gene content between Dori, MooMoo, and other mycobacteriophages.
A. Average percentages of genes shared between Dori and other mycobacteriophages. B.
Average percentages of genes shared between MooMoo and other mycobacteriophages.
19. Genomes on the x axis are listed in the same order as in Table S1 and the cluster designations
are indicated.
Figure S8. Shared gene content between Gaia, Sparky, and other mycobacteriophages.
A. Average percentages of genes shared between Gaia and other mycobacteriophages. B.
Average percentages of genes shared between Sparky and other mycobacteriophages.
Genomes on the x axis are listed in the same order as in Table S1 and the cluster designations
are indicated.
20. References
1 Krumsiek, J., Arnold, R. & Rattei, T. Gepard: a rapid and sensitive tool for creating dotplots
on genome scale. Bioinformatics 23, 1026-1028 (2007).
21. Table S1. Phages used in this study and their cluster designation
Phage Name Clus
Abrogate A1
Aeneas A1
Alsfro A1
Anglerfish A1
Arcanine A1
BPBiebs31 A1
BeesKnees A1
Bethlehem A1
BillKnuckles A1
Bob3 A1
Bruns A1
Bxb1 A1
ConceptII A1
Corvo A1
DD5 A1
Doom A1
Dreamboat A1
Dynamix A1
Edtherson A1
Euphoria A1
Fascinus A1
Forsytheast A1
Fushigi A1
GageAP A1
Hope4ever A1
Ichabod A1
JC27 A1
Jasper A1
KBG A1
KSSJEB A1
Kugel A1
Kykar A1
Lamina13 A1
Lesedi A1
Lockley A1
MPlant7149 A1
Magnito A1
Manatee A1
Marcell A1
McGuire A1
MetalQZJ A1
MrGordo A1
Museum A1
Papez A1
Pari A1
PattyP A1
Pepe A1
Perseus A1
Petp2012 A1
PhrostyMug A1
Pinto A1
RidgeCB A1
Ringer A1
Rufus A1
Ruotula A1
Rutherferd A1
Sarfire A1
Scowl A1
SkiPole A1
Solon A1
Switzer A1
Target A1
Thor A1
Treddle A1
Tripl3t A1
Trouble A1
Turj99 A1
U2 A1
Violet A1
Wheeler A1
Zephyr A1
Zeuska A1
ADZZY A2
Bugsy A2
Changeling A2
Che12 A2
ChipMunk A2
D29 A2
EagleEye A2
Echild A2
Equemioh13 A2
EvilGenius A2
Heffalump A2
IronMan A2
Jerm A2
Jsquared A2
L5 A2
Larenn A2
Loser A2
Odin A2
Piro94 A2
Power A2
Pukovnik A2
RedRock A2
SemperFi A2
Serenity A2
SweetiePie A2
Trixie A2
Turbido A2
Whabigail7 A2
Aglet A3
Bxz2 A3
DaHudson A3
EpicPhail A3
Farber A3
GingkoMaracino A3
Grum1 A3
Hercules11 A3
JHC117 A3
Jobu08 A3
Lilith A3
Mainiac A3
MarQuardt A3
Marie A3
Methuselah A3
Microwolf A3
Misomonster A3
Ollie A3
P28Green A3
Phoxy A3
PotatoSplit A3
PurpleHaze A3
Sabia A3
Spike509 A3
Taurus A3
Tiffany A3
Vix A3
Zetzy A3
BabyRay A31
HelDan A31
Norbert A31
Phantastic A31
Pocahontas A31
Popcicle A31
QuinnKiro A31
Rockstar A31
Veracruz A31
Abdiel A4
Achebe A4
Arturo A4
Backyardigan A4
BellusTerra A4
Broseidon A4
Bruiser A4
BubbleTrouble A4
Burger A4
Caelakin A4
Camperdownii A4
Clarenza A4
Dhanush A4
Eagle A4
Eris A4
Flux A4
Funston A4
Gadost A4
HamSlice A4
Holli A4
ICleared A4
KFPoly A4
Kampy A4
Kratark A4
LHTSCC A4
Lemur A4
LittleGuy A4
Maverick A4
Medusa A4
MeeZee A4
Melvin A4
Millski A4
Morpher26 A4
Mundrea A4
Nyxis A4
Obama12 A4
Peaches A4
Phighter1804 A4
Pipcraft A4
Sabertooth A4
Shaka A4
TinaFeyge A4
TiroTheta9 A4
TygerBlood A4
Wander A4
Wile A4
Airmid A5
Aragog A5
Archetta A5
Benedict A5
Chadwick A5
Cuco A5
ElTiger69 A5
ForGetIt A5
George A5
LittleCherry A5
Naca A5
Phlorence A5
Swirley A5
Theia A5
Tiger A5
UnionJack A5
Blue7 A6
DaVinci A6
EricB A6
Gladiator A6
Hammer A6
Jeffabunny A6
JewelBug A6
Kazan A6
McFly A6
SuperAwesome A6
VohminGhazi A6
HINdeR A7
Sheen A7
Timshel A7
Astro A8
Expelliarmus A8
Saintus A8
Smeadley A8
Alma A9
Catalina A9
Myxus A9
PackMan A9
Goose A10
KittenMittens A10
Rebeuca A10
RhynO A10
Severus A10
Trike A10
Twister A10
Bachome A11
Et2Brutus A11
Fibonacci A11
Mulciber A11
Adjutor D1
BigMama D1
Butterscotch D1
Gumball D1
Nova D1
PBI1 D1
PLot D1
SirHarley D1
Troll4 D1
Hawkeye D2
244 E
ABCat E
Bask21 E
Cactus E
Cjw1 E
Contagion E
Czyszczon1 E
DrDrey E
Dumbo E
Dusk E
Elph10 E
Eureka E
Goku E
Henry E
Hopey E
Kostya E
Lilac E
MadamMonkfish E
Murphy E
NelitzaMV E
NoSleep E
Pharsalus E
Phaux E
Phrux E
Porky E
Pumpkin E
Rakim E
RiverMonster E
Simpliphy E
SirDuracell E
Stark E
TeardropMSU E
Toto E
Tuco E
Ukulele E
Ardmore F1
Batiatus F1
Bipolar F1
Bobi F1
Boomer F1
Brocalys F1
Bubbles123 F1
BuzzLyseyear F1
Cabrinians F1
CaptainTrips F1
Cerasum F1
Che8 F1
DLane F1
Daenerys F1
Dante F1
DeadP F1
Dorothy F1
DotProduct F1
Drago F1
Empress F1
Estave1 F1
Fruitloop F1
GUmbie F1
Girr F1
Hades F1
Hamulus F1
Hegedechwinu F1
Ibhubesi F1
Inventum F1
Job42 F1
Krakatau F1
Llama F1
Llij F1
Mantra F1
MilleniumForce F1
Minnie F1
MisterCuddles F1
Mozy F1
Mutaforma13 F1
Ogopogo F1
Ovechkin F1
PMC F1
Pacc40 F1
Pippy F1
Ramsey F1
RockyHorror F1
Ruby F1
SG4 F1
Saal F1
Shauna1 F1
ShiLan F1
SiSi F1
Spartacus F1
Spoonbill F1
SuperGrey F1
Taj F1
Tweety F1
Velveteen F1
Wee F1
dirtMcgirt F1
Avani F2
Che9d F2
Jabbawokkie F2
Yoshi F2
Zapner F2
Squirty F3
Angel G
Annihilator G
Avrafan G
BPs G
BQuat G
BruceB G
Cherrybomb426 G
Frosty24 G
Gomashi G
Halo G
Hope G
Liefie G
Phreak G
Zombie G
Damien H1
Konstantine H1