Supplementary Data to the Manuscript Entitled

Supplementary Data to the manuscript entitled:

“Evaluating High Throughput Sequencing as a Method for Metagenomic Analysis of

Nematode Diversity”

Porazinska DL, Giblin-Davis RM, Farmerie W, Powers TO, Kanzaki N, Faller L, Morris

K, Sung W & Thomas KW

Resolving power of diagnostic regions

Prior to experimental work, we evaluated the resolving power of the selected diagnostic loci. To do this, we used a small subset of SSU and LSU sequences representing 3 genera: Bursaphelenchus (22 species, Ye et al. 2007a), Caenorhabditis (10 species, Kiontke and Fitch, 2005), and Fergusobia (17 species, Ye et al. 2007b) to predict errors associated with over- or underestimating nematode species using the chosen diagnostic loci (~ 400 bp long: NF1-18Sr2b and D3A-D3B) trimmed to ~200 bp

(an average read length generated by 454 GS FLX) along forward and reverse, SSU and

LSU primers. These three genera were chosen because they represented different lineages from different clades (Holterman et al. 2006) with different putative initiation times

(relative to C. elegans; but see Kiontke and Fitch, 2005) for each radiation. Taxa within each genus were selected to represent divergent species as well as closely related species or species within putative species complexes [ e.g., Bursaphelenchus mucronatus 163, B. mucronatus 165, B. xylophilus, and B. fraudulentus which are part of the xylophilus group (Ye et al. 2007a), Caenorhabditis briggsae 608043, C. elegans 3410584, and C. remanei 608045 of the Caenorhabditis elegans group (Sudhaus & Kiontke 2007), and 2

Fergusobia quinquenerviae mf 329 and F. quinquenerviae mq 421 which were described as a single species (Davies and Giblin-Davis, 2004), but are being considered different species based upon molecular and biological data (Ye et al. 2007b). Additionally in

Fergusobia, F. dealbata 352 and F. dealbata 18 (“Melaleuca dealbatae” complex) and

F. sp. ct 465, 327, and 444 (“Corymbia tessellaris” complex) are hypothesized to be possible members of cryptic species complexes (Ye et al. 2007b and Davies et al. unpubl. data)].

Using these four 200 bp long diagnostic sequences (SSU Forward, SSU Reverse,

LSU Forward, and LSU reverse) we created Neighbor-Joining trees (MEGA4, Tamura et al. 2007) using the Maximum Composite Likelihood (Tamura et al. 2004) and used them to estimate the resolving power of each sequence (the rationale for using NJ trees was to use the simplest, most robust, and familiar method to quickly determine the taxonomic resolution of the selected species). For instance, using a tree created for the 200 bp along

SSU forward primer NF1 (Figure SD1), we identified the number of resolved species (as a separate branch), the number of correctly resolved species (on the basis of prior knowledge of the phylogenetic relations), and the number of lumped species (not resolved as separate branches). The lumped category involved two subcategories: a cluster of closely related/sister species and a cluster of species that are not closely related

(should not cluster). Because the number of species among selected genera varied, we expressed the statistics per percent bases. To illustrate the procedure, we show only SSU

NF1 trees for the three genera, but we followed the same protocol for trees created along 3

18S r2b, and LSU D3A and D3B regions. Data are summarized in Table 1 of the manuscript. 4

Figure SD1. Neighbor Joining trees for selected species of A. Bursaphelenchus, B.

Caenorhabditis, and C. Fergusobia. Trees are based on 200 bp sequence length from the

SSU forward primer NF1. The percentage of replicate trees in which taxa clustered together in the bootstrap test (100 replicates) are shown next to branches. Bars indicate number of base substitutions per site.

A. Bursaphelenchus

Known Resolved Correctly Lumped Lumped (related spp) (unrelated spp) SSU NF1 Resolved 100 Fergusobia

Caenorhabditis 99 B. abruptus 136 1 1 1

B. kevini 361 1 1 1

94 B. anatolius 170 1 1 1

B. cocophilus 144 1 1 1

B. seani 175 1 1 1

99 74 B. fungivorus 153 1 1 1

B. platzeri 171 1 1 1

B. mucronatus 163 1 1

B. xylophilus 186 1 1 1 97 B. mucronatus 165 1 1 81 B. fraudulentus 150 1 1

B. tusciae 183 1 1 1

B. poligraphi 173 1 1 1 B. borealis 138 1 1

B. sexdentati 179 1 1 1

B. eggersi 146 1 1 1

1 1 1 77 B. hofmanni 155 B. paracorneolus 172 1 1 1

1 1 1 98 B. hellenicus 154 B. abietinus 137 1 1 1

B. hylobianum 160 1 1 1

71 B. gerberi 169 1 1 1

Total 22 18 16 6 0 0.005 Percent 100 82 73 27 0 5

B. Caenorhabditis

SSU NF1

Known Resolved Correctly Lumped Lumped Resolved (related spp) (unrelated spp)

100 Fergusobia

97 C. sp. SB341 C. sp. SB341 1 1 1 C. sonorae 8515065

C. plicata 4886908 1 1 1 99 C. sp. PS1010 1 1 1

1 1 96 C. sp. DF5070 1 C. drosophilae Caenorhabditis1 1

C. sp. CB5161 1 1 1

C. japonica 488690 1 1 1 1 1 91 C. briggsae 608043 1 C. elegans 3410584 1 1

C. vulgarisremanei 608045608045 1 1

Bursaphelenchus 99

Total 10 7 5 5 0

0.005 Percent 100 70 50 50 0 6

C. Fergusobia

Known Resolved Correctly Lumped Lumped Resolved (unresolved spp) SSU NF1 (resolved spp)

F. dealbatae 352 1 1 1 F. dealbatae 18 1 1

F. sp. csp. 39 1 1 1 1 F. quinquenerviae mf 329 1 1 1 1 1 F. sp. mq 357 1 1 F. quinquenerviae mq 421

F. sp. af 346 1 1 1 1 F. sp. ma 469 1 1

F. sp. ec 54 1 1 1

F. sp. esp. 339 1 1 1 Fergusobia F. sp. sl 739 1 1 1

F. sp. ef 330 1 1 1 1 1 1 87 F. sp. esp. 446 F. sp. ec 56 1 1 1

F. sp. ct 465 1 1 1

F. sp. ct 327 1 1 1

71 F. sp. ct 444 1 1 1 1 1 1 100 F.F. pukutukawasp. me 359 F. leucadendrae 451 1 1 1

F. sp. ed 281 1 1 1

Caenorhabditis 99

Bursaphelenchus Total 20 13 10 10 5 99 Percent 100 65 50 50 25

0.005

Primer analysis

The general protocol for evaluating primers within the phylum Nematoda is described in the manuscript along with the percentage of base substitutions. Here, we detail the type of base substitutions with the 3’-end 10bp of each primer.

Figure SD2. Types of base substitutions for A. 3’ end 10bp of the SSU forward

(NF1) and reverse (18Sr2b) primers (X-axis) and B. 3’ end 10bp of the LSU forward

(D3A) and reverse (D3B) primers 7

A T G C Y M N A T G C Y W M N 100% 100% s n o i

t 80%

n 80% u o t i i t t u s t i b t 60% u

s 60% s b

u e s p

y e t

40% 40% p e y t s

a e s B

a 20% 20% B

0% 0% G T T C T T A G T T A T T A C G T C C C NF1 3' 10bp 18Sr2b 3' 10bp

A T G C N A T G C K 100% 100% n o i n

80% t 80% o i u t t i u t t i s s

60% b 60% b u u s s

e e p p 40% 40% y y t t

e e s s a 20% a 20% B B

0% 0% G A A A C A C G G A T A G T A G C T G G D3A 3' 10bp D3B 3' 10bp

To define the taxonomic scope of the mismatches, we focused on the distribution of mismatching primer sequences within orders, families, and genera. Of the 19 orders in the database, 9 contained sequences with mismatches in either of the SSU primers

(Figure SD3 A, B). Only in the case of Ascaridida, did the percentage of sequences with mismatches represent greater than 10% of the sequences. Of the 139 families represented in the database, only 18 had sequences with mismatches to either SSU primer (Figure

SD4 A, B). The order Ascaridida had the most families (5) with mismatches. In contrast, 8 the mismatches in the order Tylenchida came from a single species, Rotylenchus reniformis.

Among the LSU sequences, the only obvious taxonomic bias for D3A mismatches occurred within the order Tylenchida (Figure SD3 C, D), primarily within the family

Meloidogynidae and the genus Meloidogyne (Figure SD4 C, D). In contrast, the D3B primer showed mismatches in the orders Dorylaimida, Rhabditida, and Tylenchida. For the Dorylaimida and Tylenchida, the mismatches were found in at least 3 families. In the

Rhabditida, the mismatches were concentrated in the Cephalobidae.

Figure SD3. Percent of mismatches in the 3’ end 10 bp and 4 bp of the four priming sites for sequences of all nematode orders available at GenBank. The number in parenthesis e.g. (1) indicates the total number of sequences with both priming regions retrieved from GenBank.

A. SSU NF1

25 10 bp 20 4 pb

t 15 n e c r e

P 10

0 ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) 5 0 3 3 5 2 1 5 1 6 9 3 5 2 8 7 4 1 1 0 ( ( ( ( 1 2 9 2 ( 5 6 5 4 9 1 2 6 7 5 4 ( ( ( ( ( ( ( ( ( 3 ( ( a 2 a 5 ( a

a a

( ( ( a a d a d d a a a a a d a i i a d i a n i i t d d a d i d d d a d d r a d i i i d c d i i i i i i i i l m a w r r d s i d r r u d i h r e h i h i l d p t o o o t u i a m e i e y i m u i l c c h f o t t r m o n d d r i x d e i a o n n c e s m s c a l n k a o a r r b n p s o o n l a y O s o c l n E I e a o T m m n S e y g h o e s p l u r i h g o s o o n i a M r y A l r o m e r o R h M T T p h s i D A D R M e C D D 9

B. SSU 18S r2b

30 10 bp 25 4 bp

t 15 n e c r e

P 10

0 ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) 0 0 1 5 3 3 5 2 5 2 8 6 5 1 9 3 7 4 1 1 4 ( ( ( ( 1 2 9 ( 2 2 1 5 6 5 4 9 ( 6 7 5 ( ( ( ( ( ( ( ( ( 3 (

( a a 2 a 5

a ( ( ( N a a d d d a a a a a d a a i a i i d a i i t d a d d i d d d r a d d a d i i c d i d i i i i i W i i i l m a r r d s r u d r i d e i r h h i h i l d p t o o t u O m e i e a i y i u m i f l c c h t o t m r o d r d i x d e i N a o n e n c s c s m l a n o a a r b n p r l s o o n K y s O a o c l E I a T e o m m y S n e h o g e s N p l r i h g s o o n o i a M r y A l r m o U r e o R h p M T T s h i D A D R M e C D D

C. LSU D3A

12 10 bp 10 4 bp

8 t n e

c 6 r e P

0 ) ) ) ) ) ) ) ) 2 2 2 6 4 2 2 2 ( ( ( ( 3 6 8 2 ( ( ( a 1 a a a

( a d a d d d i n i i i l d d a r i h i h w t p t i d e i i c o t m o i d h n m n s n a r b l c o k y l E e a y n n h p r i h e n M l r U o R o y T D T M 10

D. LSU D3B.

100

80 10 bp 4 bp

t 60 n e c r e

P 40

0 ) ) ) ) ) ) ) ) 2 2 2 2 1 4 6 3 ( ( ( ( 3 6 8

2 ( ( ( a 1 a a a

( a d d d a d i n i i i l d r d a i h i h w p t t d i e i i c t o m o i d h n m s n n a r b l c o y k l E e a y n h n p r i h e n M l r U o o R y T D T M

Figure SD4. Percent of mismatches in the 3’ end 10 bp and 4 bp of the four priming sites for the phylum Nematoda at the family level.

A. SSU NF1

100 10 bp 80 4 bp

t 60 n e c r e

P 40

0 ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) 0 0 2 0 6 7 0 8 6 8 2 1 8 6 0 1 6 0 1 ( ( 1 3 0 4 3 ( 2 2 1 3 7 1 9 3 1 5 6 ( ( e ( e ( ( ( ( ( ( ( 1 ( ( 1 e ( (

e e ( a e ( a e e e e e e e a e e n e a e d a a d a e a a a a i a a i a a d a w i d d a d a i d d d d d i k d i i d d d i d i i i i i i i i d l i o d i d i d a i r i i c h b k r d m m t y n n r m r m i i i r i r i i n a c o a a r a k o p l e l a a u a i e a a y t c l l n l s l a d r n a i u c i c g m e s o y g o o r o c u h l n T s g r o n e a h o n e n H s p p t A n o h n A A d d o a o i i e o A a l c o a D i h o h y M e l L C n H s p p e B C d O a S u M R Q 11

B. SSU 18Sr2b

80 10 bp 4 bp 60 t n e

c 40 r e P

0 ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) 4 0 5 6 9 0 4 2 1 7 6 8 4 3 8 2 5 2 6 7 9 0 6 7 0 ( ( ( 2 0 1 1 ( ( 4 3 3 7 2 3 5 2 8 1 4 9 3 7 5 6 e ( ( e ( ( e ( ( ( ( ( ( ( ( ( ( 1 e e ( ( ( 1 ( e ( a e a e a e e e e e e e ( e e e e a a e e e n a e d a a d a d a a e a a a a a a a i a d d i a i a a i i t w d a d d d l d d d d d d d d a d i d i i d i d d i i i i i i i i i i i m a i i o m d t a t i i m r r d i r i i i h b c i h r m t m n d m m n m c y a i i i i i i a r o e i o m d l a o n c l c a a l l k e l a o a a X e d u d a a l d e b l l l o l o n y n l i a n A u h o g o r c m n a o P d o o o o g e o g r h r l l u n c c o h e o h g b h o h i n l n n e p e p y i t n h c n A t a y c o i d o o r R a e o i l a M T e r c a e l h n i L h N o e y M D C H n l T e e s H p R l B e C h d O y S p u M T A Q

C. LSU D3A

100 10 bp 80 4 bp t

n 60 e c r

e 40 P 20

0 ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) 2 1 6 4 2 6 1 2 2 4 2 0 3 3 4 ( ( ( ( ( ( ( ( 3 4 1 8 2 7 2 e e e e e e ( e e ( ( ( ( ( ( a a a a a a a e a e e e e e n d d d d d d d d a a a a i i i a i i i a i i t l t w r d r d d d d h d i i i i i a m i m y a o t t i o i e i r i b n h t p d n a d i a i m o m d y l o l c s m r l k o o e e d b r o g y n i y a n T r r h h n n e a o e h g h u l g c o c a o h i d i n l n i M p y i a r n t D s o r R o e o n e l T a l d L D r M C a e e u P P h M Q p A 12

D. LSU D3B

100 10 bp 80 4 bp t

n 60 e c r

e 40 P 20

0 ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) 2 4 1 6 2 1 2 2 4 2 0 6 3 3 4 ( ( ( ( ( ( ( ( 3 4 1 8 2 7 2 e e e e e ( ( e e ( ( e ( ( ( a a a a a e e a a e e a e e n d d d d d a d a a a d d a i i i i i i i a i t l t w d d d d r r d h i i i d i i y t m i o m a a t i e i i o r i n b h t d n p i i a a d o d m m o y l c l m s l k r o o d r e e b g n y o i y a n T h r r h n e n a o e g h h l u c o g c o h a d i n i n l i M p y i a n r t D o o r R s o e l e n a T l L d D r M C e a e u P P h M Q p A

We also assayed the ability of the chosen primer sets to amplify the target loci from all known other eukaryotic SSU and LSU sequences (Figure SM5 A, B, C, D).

Figure SD5. Percent of mismatches in the 3’ end 10 bp and 4 bp of the four priming sites for all eukaryotic sequences available at GenBank at the level of phylum.

A. SSU NF1

60 10 bp 50 4 pb 40 t n e

c 30 r e P 20

0 ) ( ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) 0 a 3 5 0 0 1 3 2 1 3 0 9 2 2 9 1 7 5 2 4 1 2 3 4 1 0 3 t 0 6 8 2 1 8 6 1 8 4 0 ( ( ( ( ( ( 8 ( ( ( ( 5 2 6 6 ( ( ( 9 1 ( 9 4 1 1 0 2 7 7 6 6 4 o 9 5 5 3 3 1 a ( ( ( ( a ( a a ( 3 a s a ( ( a a a ( 2 a a ( a e 0 3 1 1 i t 3 4 c 5 9 t l 8 ( r 2 r 9 7 r ( a a e d a a ( h ( a a h ( e e a ( ( ( d i ( o a a 1 ( t i d a t o t y t u 1 o r a i o 2 u 7 a e s d r ( a c d a a r i a a a a t ( o z a e r t i a ( o a i d ( c a t y t c t o m h l h t e e o e a r m d a r r c e h o x a p o c d c o t f d a t n y y a t h n y h i t p o p i u y h m c o l d o e c a c s c e o t y y t y p e d i a r o h l g d o p u i r h p o p r o o h i i r y m h g e u y r y s l r E h f i n o n s l o c o p m p h e i p i p o i n g c n t l a p t m h m i t i n o B c h i i o o p i l o i p y o e c d b P c o m y C e s l o r o o s o p e m i e p S d n r m h i t z t o d c o o l a o e r a o g r p N a P o m m a o r o o y A m r d M N p h a y r t l e C i C n c p a l a o a t i e i l o h c e h o r G e i e m i l B a h h s t i i c t C M n P r T h H l P a H r c d C l p t y n c i o y a C m c m t h s t a A A S u s a h o g X a E l s i P A c l a t B C a o P l G s B e B u N E 13

B. SSU 18S r2b

100 10 bp 80 4 bp t

n 60 e c r

e 40 P

0 ( ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) a 0 0 1 3 2 3 5 0 1 7 0 3 9 2 0 2 9 1 2 4 1 2 3 4 1 5 0 3 t 6 8 8 2 6 1 1 8 0 4 ( ( ( ( ( ( ( ( 8 ( ( ( 2 1 6 ( ( ( 5 9 6 1 7 9 4 2 7 1 0 6 6 4 o 9 5 5 3 3 1 ( ( ( a a ( ( ( ( a s a a a 3 ( a ( a a e a a 2 ( a 3 0 3 1 i t 1 4 t c 5 9 l 8 2 7 r 9 r ( r e a d a ( a a a a ( h ( ( e e a ( h ( ( d i a ( a o ( 1 t i d a t y 1 o t t u 2 r u 7 o a i o a d s e r a ( a a r i a d a c a a ( o t a e z a ( r d ( o a t i i c a t t y c t o h m h l t e o e e c a a h r m d r r e x a p o c o d a o t d c f t y n y a h n t y h i t o p i u y p c m h s l d c o o a e c e o c t y y d i p a e y t r l o g h d r p i o u h p o o p r i o h y i r e m h u g y r E y s l h f r o n l i s n o p c o p i i h e m p p o c l i n g n t a p h m t m i o t i c i n B h i o o p l p i o i y b c c o d e P o y C m l o o e s r o p e s o m i e d n p S r h i z m o t t c o d e a o o l r o p r a g o a m N P o r m a o A m o M y N h r d p y t a r p l C i C n c e a l a a t o i e i l o h c h e o r G e l a i i B e m t s h h i c C M i t n P T h r l H P a r H c p d C l t y n c i o y a m C m c t h t s A a A S u a s h g o X E a i s l P A c l t a B C a o l P s G B e u B N E

C. LSU D3A

100

80 10 bp t 4 bp n

e 60 c r e

P 40

0 ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) 4 6 7 2 3 4 8 8 1 3 7 1 1 2 6 1 2 1 1 2 3 ( 3 4 3 ( ( ( ( ( ( ( 1 ( 5 ( 4 ( 0 6 7 0 1 4 1 a 5 9 3 ( ( a ( s ( a a a a ( a ( t a a ( a ( ( 9 1 t t 2 r r 3 a a d a e r a ( o a a o o h a a ( a t i t 2 t o a l o 6 u o e h r e a z r i ( d c z c d d o t t t ( a f o c d u i h i e c r l a h o a o y o r n t c a n e a f c y c t i p i i m s e e d c c p f y o y o y d i r y i r n o m o r m m u n a o h E h m l o n l o i c e m l R u o c l n m r e m o n B r i p h e C d e P e P p y o C o o o i o A i N i d e o c p o h g n N r S m d i M d a a i y s n i l o m i t h o r r K n t s t c o P h a c l r B l u a y o c s t A P h G B E s A C a l B 14

D. LSU D3B

100

80 10 bp

t 4 bp n

e 60 c r e

P 40

0 ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) 4 4 6 7 2 3 8 8 1 1 2 7 1 3 1 6 1 1 2 3 2 4 ( 3 3 ( ( ( ( ( ( ( 1 5 ( 4 ( ( 6 7 0 0 1 4 1 9 a 5 3 s ( ( a ( ( a a ( a a a ( t a ( a ( a ( 9 t 1 t r 2 3 r e a a d a r a a ( o a o h a a o a i ( t t o 2 t a l o 6 u h o e r e r a i z ( d c c z d t t d ( o a t f c o d u h i i e c r l a h o a o r y n t o n f e a c a c y c t i p i i m s e e d c c p o o f y y r d y i y r n i o m o m r m u n a m h E o h l o o n l e m l R u c i l c o n m e r m o n B i r p C d e P e P p e h o y C o o i o o A N i d o i e h p c o g N n r S m M d i a d y s a i n i l o m i t h o r r K n t s c t o P h a c l r l B u a o y c s t A P h G B E s A C a l B

Sequence Diversity

Table SD1. Sequence diversity (Pi) for 5 potentially least divergent species pairs for SSU and LSU loci. Within – indicates sequence diversity among copies within a species, and

Between – indicates sequence diversity between selected species pairs.

SSU LSU Within Between Within Between Overall 0.031 0.061 0.022 0.069

B. borealis 0.019 0.053 B. sexdentati 0.041 0.034 0.018 0.044

B. fungivorus 0.071 0.021 B. seani 0.023 0.049 0.018 0.049

Koerneria n. sp. 1 0.024 0.014 Koerneria n. sp. 2 0.019 0.026 0.021 0.042

D. metamasius 0.035 0.022 Rhabditidoides n. sp. 0.023 0.114 0.015 0.084

Poikololaimus n. sp. 0.013 0.018 References C. elegans 0.022 0.082 0.017 0.125 15

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