Codon usage patterns in Nematoda: analysis based on over 25 million codons in thirty-two species

gb-2006-7-8-r75 GBJ Research Codon usage patterns in Nematoda: analysis based on over 25 million codons in thirty-two species Mitreva Makedonka mmitreva@watson.wustl.edu Wendl C Michael mwendl@watson.wustl.edu Martin John jmartin@watson.wustl.edu Wylie Todd twylie@watson.wustl.edu Yin Yong yyin@watson.wustl.edu Larson Allan larsontl@biology.wustl.edu Parkinson John jparkin@sickkids.ca Waterston H Robert waterston@gs.washington.edu McCarter P James jmccarte@watson.wustl.edu

Genome Sequencing Center, Washington University School of Medicine, St Louis, Missouri 63108, USA

Department of Biology, Washington University, St. Louis, Missouri 63130, USA

Hospital for Sick Children, Toronto, and Departments of Biochemistry/Medical Genetics and Microbiology, University of Toronto, M5G 1X8, Canada

Department of Genome Sciences, University of Washington, Seattle, Washington 98195, USA

Divergence Inc., St Louis, Missouri 63141, USA

Genome Biology 1465-6906 2006 7 8 R75 http://genomebiology.com/2006/7/8/R75 16907990 10.1186/gb-2006-7-8-r75 20 4 2006 30 6 2006 14 8 2006 14 08 2006 2006 Mitreva et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Codon usage in worms

A codon usage table for 32 nematode species is presented and suggests that total genomic GC content drives codon usage.

Abstract

Background

Codon usage has direct utility in molecular characterization of species and is also a marker for molecular evolution. To understand codon usage within the diverse phylum Nematoda, we analyzed a total of 265,494 expressed sequence tags (ESTs) from 30 nematode species. The full genomes of Caenorhabditis elegans and C. briggsae were also examined. A total of 25,871,325 codons were analyzed and a comprehensive codon usage table for all species was generated. This is the first codon usage table available for 24 of these organisms.

Results

Codon usage similarity in Nematoda usually persists over the breadth of a genus but then rapidly diminishes even within each clade. Globodera, Meloidogyne, Pristionchus, and Strongyloides have the most highly derived patterns of codon usage. The major factor affecting differences in codon usage between species is the coding sequence GC content, which varies in nematodes from 32% to 51%. Coding GC content (measured as GC3) also explains much of the observed variation in the effective number of codons (R = 0.70), which is a measure of codon bias, and it even accounts for differences in amino acid frequency. Codon usage is also affected by neighboring nucleotides (N1 context). Coding GC content correlates strongly with estimated noncoding genomic GC content (R = 0.92). On examining abundant clusters in five species, candidate optimal codons were identified that may be preferred in highly expressed transcripts.

Conclusion

Evolutionary models indicate that total genomic GC content, probably the product of directional mutation pressure, drives codon usage rather than the converse, a conclusion that is supported by examination of nematode genomes.

Molecular biology Evolution Genetics

Background

Utilization of the degenerate triplet code for amino acid (AA) translation is neither uniform nor random. In particular, there are distinct patterns among different species and genes. Such patterns can readily be characterized by codon usage, namely the observed percentage occurrence with which each codon is used to encode a given AA. This measure has direct utility in molecular characterization of a species in that it enables efficient degenerate and nondegenerate primer design for cross-species gene cloning, open reading frame determination, and optimal protein expression 1. Such tools are particularly important with respect to species for which limited molecular information exists. Codon usage also serves as an indicator of molecular evolution 2. Codon usage bias, namely the degree to which usage departs from uniform use of all available codons for an AA, can be influenced by a number of evolutionary processes. The guanine and cytosine (GC) versus adenine and thymine (AT) composition of the species' genome, probably the product of directional mutation pressure 34, is a key driver of both codon usage and AA composition 56. Other factors that influence codon usage may include the relative abundance of isoaccepting tRNAs 789, especially for highly expressed mRNAs that require translational efficiency 1011, presence of mRNA secondary structure 1213, and facilitation of correct co-translational protein folding 14. Codon usage appears not to be optimized to minimize the impact of errors in translation and replication 15.

Nematodes are a highly abundant and diverse group of organisms that exploit niches from free-living microbivory to plant and animal parasitism. Molecular phylogenies divide nematodes into five major named and numbered clades within which parasitism has arisen multiple times 16: Dorylaimia (clade I), Enoplia (clade II), Spirurina (clade III), Tylenchina (clade IV), and Rhabditina (clade V). Following the sequencing of the complete genome of the model nematode Caenorhabditis elegans 17, we have begun to catalog the molecular diversity of nematode genomes through the generation of over 250,000 expressed sequence tags (ESTs) from more than 30 nematode species (including 28 parasites) in four clades. Gene expression analyses for several medically and economically important parasites such as filarial, hookworm, and root knot nematode species have been completed 181920212223 (for reviews 2425). Moreover, we recently conducted a meta-analysis of partial genomes across the whole phylum with a focus on the conservation and diversification of encoded protein families 26. Project information is maintained on several online resources 27282930.

Now, in the most extensive such study yet performed for any phylum, we extend the above analyses with a comprehensive survey of observed codon usage and bias based on nearly 26 million codons in 32 species of the Nematoda. Because of its completed genome, C. elegans has been the primary species utilized in nematode codon usage studies 31323334. Our findings provide more complete information for Caenorhabditis based on all 41,782 currently predicted proteins in C. elegans and C. briggsae 35. Studies for other nematode species have been more limited. Codon usage has been tabulated for a number of parasitic nematodes including filarial species Brugia malayi, Onchocerca volvulus, Wucheria bancrofti, Acanthocheilonema viteae, Dirofilaria immitis 36373839, Strongyloides stercoralis 40, Ascaris suum 41, Ancylostoma caninum, and Necator americanus 42. Although Fadiel and coworkers 39 used up to 60 genes per species, sample sizes in the other studies were quite small, typically fewer than 10 representative genes and 5,000 codons per species. In the present study we used an average of 2,350 genes and 270,000 codons per species for the 30 non-Caenorhabditis species. Our results provide the first codon usage tables for 24 of these organisms. Web available automated codon usage databases compiled from GenBank 43 lack almost all of this information because they rely only on full-length protein coding gene sequence submissions rather than the EST data used here.

In analyzing codon distribution in Nematoda, we describe how average usage varies between species and across the phylum. For instance, it has been shown that there is a level of conservation in codon distribution between 'closely' related nematodes such as Brugia malayi and B. pahangi 37 and Brugia and Onchocerca 38. These relationships do not appear to extend over greater evolutionary distances, for instance between Onchocerca and Caenorhabditis 36. The evolutionary distance at which conservation of codon usage diminishes has not previously been established 32. Here we show that codon usage similarity in Nematoda is a relatively short-range phenomenon, generally persisting over the breadth of a genus but then rapidly diminishing within each clade. We also show that the major factor affecting differences in mean codon usage between distantly related species is the coding sequence GC as compared with AT content. GC content also explains much of the observed variation in the effective number of codons, a measure of codon bias, and even differences in AA frequency.

Results

Determination of codon usage patterns and amino acid composition

Extensive nucleotide sequence data are now available for many nematode species, largely because of recent progress using genomic approaches 2544. To obtain a better understanding of codon usage and AA composition within the phylum Nematoda, we analyzed a total of 265,494 EST sequences originating from 30 nematode species. The ESTs define 93,645 clusters or putative genes, with 208-9,511 clusters per species (Table 1) 26. Table 1 also provides two letter codes for the nematode species used throughout the remainder of the report. We used prot4EST, a translation prediction pipeline optimized for EST datasets 45, to generate protein predictions. To reduce noise derived from poor translations, our analysis considered only the longest open reading frame (ORF) translations with strong supporting evidence in the form of similarity to known or predicted proteins (BLASTX cutoff 1 × e^-8) and retained only the polypeptide aligned portion of the nucleotide sequence. About 75% of the clusters met these criteria, yielding 8,080,057 codons originating from species other than Caenorhabditis, and 25,871,325 total codons from all 32 species including available predictions from C. elegans and C. briggsae. The 18 AA residues with redundant codons gave a total of (18) × C_32,2= 496 comparisons of codon usage between species. Comprehensive tables of AA composition (Tables 2 and 3) and codon usage (Table 4) for all 32 Nematoda species studied are provided. Below we use these tables to examine, first, variation in AA composition and its relationship to GC content and, second, codon usage and its relationship to GC content.

Table 1

Summary of sequences used by nematode species

Clade

Code

Species

ESTs

Total number of clusters

Clusters or genes used

Codons

GC content (%)

Necator americanus ^a

4,766

2,294

1,784

192,756

Ancylostoma caninum ^b

9,079

4,203

3,207

305,036

Ancylostoma ceylanicum ^b

10,544

3,485

2,814

387,372

Nippostrongylus brasiliensis ^b

1,234

742

630

75,934

Haemonchus contortus ^b

17,268

4,146

4,102

584,513

Ostertagia ostertagi ^b

6,670

2,355

1,961

222,616

Teladorsagia circumcincta ^b

4,313

1,655

1,616

194,351

Caenorhabditis elegans ^c

22,254

100

9,784,215

Caenorhabditis briggsae ^c

19,528

100

8,007,053

Pristionchus pacificus ^c

8,672

3,690

2,597

297,605

IVa

Strongyloides stercoralis ^a

11,236

3,635

2,803

367,308

Strongyloides ratti ^b

9,932

3,264

2,682

320,874

Parastrongyloides trichosuri ^b

7,712

3,086

2,457

284,785

IVb

Pratylenchus penetrans ^d

1,908

408

338

45,802

Globodera pallida ^d

1,317

977

479

65,699

Globodera rostochiensis ^d

5,905

2,851

2,192

290,614

Heterodera glycines ^d

18,524

7,198

5,564

742,990

Meloidogyne incognita ^d

12,394

4,408

3,214

366,435

Meloidogyne javanica ^d

5,282

2,609

2,086

203,135

Meloidogyne arenaria ^d

3,251

1,892

1,483

176,816

Meloidogyne hapla ^d

13,462

4,479

3,507

407,985

Meloidogyne chitwoodi ^d

7,036

2,409

1,906

205,612

Zeldia punctata ^c

388

208

102

16,723

III

Ascaris suum ^b

38,944

8,482

5,830

646,740

Ascaris lumbricoides ^a

1,822

853

508

42,919

Toxocara canis ^b

4,206

1,447

866

103,065

Brugia malayi ^a

25,067

9,511

6,483

561,296

Dirofiliaria immitis ^b

3,585

1,747

1,380

126,880

Onchocerca volvulus ^a

14,922

5,097

2,914

299,336

Trichinella spiralis ^a

10,384

3,680

2,693

290,794

Trichuris muris ^b

2,713

1,577

1,179

147,995

Trichuris vulpis ^b

2,958

1,257

1,000

106,071

^aHuman parasite, ^banimal parasite, ^cfree-living, and ^dplant parasite. EST, expressed sequence tag.

Table 2

Amino acid composition (%) of translations by nematode species

Clade

Species

Amino acid

Ala

Cys

Asp

Glu

Phe

Gly

His

Ile

Lys

Leu

Met

Asn

Pro

Gln

Arg

Ser

Thr

Val

Typ

Tyr

6.9

2.4

5.2

6.2

4.7

6.3

2.5

5.5

6.5

8.6

2.6

4.2

5.0

3.7

6.2

7.2

5.3

6.5

1.2

3.3

7.0

2.4

5.0

6.0

4.7

5.9

2.7

5.6

6.3

8.9

2.8

4.2

4.6

3.7

6.2

7.4

5.4

6.6

1.3

3.3

7.6

2.2

5.5

6.6

4.2

6.5

2.5

5.2

6.4

8.5

2.5

4.0

5.1

3.8

6.1

7.1

5.3

6.7

1.2

3.0

7.8

2.2

5.4

6.3

4.0

6.9

2.4

5.0

7.2

8.1

2.6

4.0

4.9

3.5

6.4

7.0

5.2

6.9

1.1

3.1

7.4

2.3

5.5

6.5

4.3

6.6

2.5

5.4

7.0

8.4

2.5

4.1

4.9

3.7

6.0

6.4

5.2

6.8

1.2

3.4

7.2

2.3

5.3

6.3

4.4

6.7

2.6

5.3

6.7

8.4

2.6

4.0

5.2

3.8

6.1

6.8

5.3

6.7

1.1

3.1

7.5

2.7

5.2

6.1

4.3

6.7

2.6

5.0

6.6

8.4

2.7

4.2

5.1

3.8

5.8

7.1

5.3

6.5

1.2

3.3

6.3

2.0

5.3

6.5

4.8

5.4

2.3

6.1

6.4

8.6

2.6

4.9

4.2

5.2

8.1

5.9

6.2

1.1

3.1

6.3

2.0

5.3

6.8

4.7

5.4

2.3

6.0

6.4

8.5

2.6

4.8

5.0

4.2

5.4

8.0

5.8

6.1

1.1

3.1

7.4

1.9

5.4

6.9

4.0

6.6

2.5

5.3

6.6

8.4

2.6

3.9

5.1

3.4

6.4

7.6

5.4

6.4

1.2

3.0

IVa

5.5

1.9

5.7

7.0

4.3

6.0

2.1

7.1

8.0

8.3

2.3

6.0

4.5

3.7

4.6

7.2

5.5

5.8

1.0

3.5

5.4

2.0

5.4

6.5

4.7

5.9

2.1

7.4

8.1

8.6

2.4

6.2

4.3

3.6

4.3

7.2

5.4

5.8

1.0

3.8

6.3

2.0

5.3

6.3

4.6

6.3

2.4

6.6

8.1

8.3

2.4

5.3

4.3

3.5

4.9

6.9

5.5

6.1

1.0

3.7

IVb

6.9

2.0

5.3

6.7

4.3

7.0

2.4

5.8

7.6

8.4

2.6

4.5

6.2

6.4

4.9

5.9

1.2

2.9

6.8

2.4

4.5

5.4

5.6

7.2

2.4

4.9

7.1

9.1

2.3

3.9

5.8

3.8

6.7

7.1

4.8

6.0

1.2

2.9

7.4

2.1

4.9

6.0

4.8

6.5

2.6

5.2

5.9

9.3

2.5

4.3

5.0

4.4

6.5

7.2

5.2

6.3

1.2

2.7

7.3

2.2

4.9

6.2

5.1

6.3

2.6

5.2

6.0

9.2

2.4

4.5

5.0

4.5

6.3

7.3

5.1

6.2

1.2

2.5

5.7

2.0

4.8

6.7

5.1

5.6

2.2

6.5

7.3

9.4

2.3

5.6

4.5

4.6

5.3

7.5

5.2

5.4

1.1

3.0

5.5

2.1

4.8

6.6

5.5

5.4

2.2

6.9

7.8

9.6

2.4

5.6

4.2

5.4

7.0

5.1

5.4

1.1

3.3

5.7

2.0

5.0

6.9

5.3

5.7

2.2

6.9

7.3

9.7

2.3

5.5

4.1

4.3

5.2

7.0

5.0

5.6

1.1

3.2

5.6

2.0

4.9

6.8

5.3

5.6

2.2

7.0

7.3

9.5

2.3

5.8

4.3

5.2

7.3

4.9

5.4

1.2

3.2

5.3

2.3

4.8

6.4

5.5

2.3

7.4

9.7

2.3

6.0

4.0

4.3

4.9

7.4

5.0

5.2

1.1

3.3

7.6

1.5

5.2

6.2

4.2

7.1

2.5

6.1

7.9

8.4

1.8

4.7

4.6

3.8

6.0

5.4

5.5

6.6

1.1

3.7

III

7.0

2.5

4.9

6.1

4.6

5.8

2.5

6.0

6.3

8.7

2.6

4.5

4.8

3.6

6.3

7.5

5.3

6.6

1.2

3.3

7.3

2.6

4.6

5.8

4.9

6.1

2.5

6.0

6.4

8.3

2.5

4.3

5.3

3.5

6.2

7.4

5.2

6.5

1.2

3.4

7.3

2.8

5.0

6.0

4.2

6.8

2.6

5.3

7.3

8.0

2.4

4.1

5.4

3.5

6.2

6.7

5.6

6.4

1.2

3.2

5.6

2.5

4.6

5.6

5.4

5.0

2.6

7.1

6.8

9.6

2.8

5.0

4.1

3.8

5.6

7.8

5.3

5.9

1.1

3.6

5.6

2.4

4.9

6.0

5.2

4.7

2.7

7.5

7.0

9.5

2.7

5.2

3.9

3.8

5.9

7.5

5.1

5.8

1.1

3.8

6.0

2.2

5.0

6.1

4.9

5.6

2.5

6.9

7.1

8.9

2.7

4.9

4.5

3.9

5.9

7.3

5.2

5.7

1.2

3.5

6.2

2.6

5.0

6.2

5.1

2.5

6.1

6.6

9.5

2.6

4.9

4.1

3.9

5.6

7.6

5.1

6.5

1.2

3.4

7.1

3.0

5.0

6.0

4.5

6.5

2.5

5.0

6.3

8.9

2.6

4.0

5.0

3.8

6.2

7.4

5.2

6.7

1.2

3.2

7.0

3.0

4.9

6.1

4.5

5.8

2.5

5.1

6.4

9.0

2.6

4.2

4.8

3.8

6.1

7.5

5.5

6.8

1.3

3.2

Definitions of species two letter codes are provided in Table 1.

Table 3

Amino acid composition (%) of translations from Nematoda and four reference species

Amino acid

Nematode

Mean

Ala

6.6

0.8

7.1

7.5

5.6

9.2

Cys

2.3

0.3

2.3

1.9

1.3

1.1

Asp

5.1

0.3

4.8

5.2

5.8

5.2

Glu

6.3

0.4

6.9

6.4

6.5

5.7

Phe

4.7

0.5

3.8

3.5

4.4

3.8

Gly

6.1

0.7

6.6

6.3

5.1

7.3

His

2.4

0.2

2.6

2.7

2.2

Ile

6.0

0.8

4.4

4.9

6.5

6.0

Lys

6.9

0.6

5.6

7.3

4.8

Leu

8.8

0.5

10.0

9.0

9.5

10.1

Met

2.5

0.2

2.2

2.4

2.1

2.6

Asn

4.7

0.7

3.6

4.7

6.1

4.3

Pro

4.7

0.5

6.1

5.5

4.4

4.2

Gln

3.9

0.3

4.7

5.2

4.0

4.3

Arg

5.8

0.6

5.7

5.5

4.4

5.5

Ser

7.2

0.5

8.1

8.3

8.9

6.4

Thr

5.3

0.2

5.3

5.7

5.9

5.7

Val

6.2

0.5

6.1

5.9

5.6

7.0

Typ

1.2

0.1

1.3

1.0

1.4

Tyr

3.2

0.3

2.8

2.9

3.4

3.0

DM, Drosophila melanogaster; EC, Escherichia coli; HS, Homo sapiens; SC, Saccharomyces cerevisiae.

Table 4

Codon usage of translations by nematode species

Species (codons [n])

Codon

NA (192,756)

AC (305,036)

AY (387,372)

NB (75,934)

HC (584,513)

OO (222,616)

TD (194,351)

CE (9,784,215)

CB (8,007,053)

PP (297,605)

SS (367,308)

SR (320,875)

PT (284,785)

PE (45,802)

GP (65,699)

GR (290,614)

Ala

GCA

28.8

25.0

23.9

19.7

26.0

24.9

25.4

31.5

26.1

20.7

33.2

31.4

24.2

31.4

20.4

22.1

Ala

GCC

18.5

23.0

24.2

30.0

22.8

23.4

24.0

19.9

22.7

32.4

9.1

9.7

23.4

29.5

31.9

32.6

Ala

GCG

16.0

17.7

17.3

17.6

13.6

16.3

16.6

13.1

15.2

16.7

2.0

1.6

7.1

13.2

26.7

27.3

Ala

GCT

36.7

34.2

34.6

32.7

37.6

35.4

34.0

35.5

35.9

30.2

55.7

57.4

45.3

25.9

20.9

18.1

Codons per AA

13,237

21,208

29,600

5,916

43,104

16,126

14,513

618,499

502,187

22,118

20,285

17,201

17,930

3,173

4,494

21,522

Cys

TGT

54.0

46.9

44.4

39.5

46.7

49.0

44.3

55.3

56.9

39.2

84.4

85.3

63.9

42.6

42.1

43.7

Cys

TGC

46.0

53.1

55.6

60.5

53.3

51.0

55.7

44.7

43.1

60.8

15.6

14.7

36.1

57.4

57.9

56.3

Codons per AA

4,538

7,295

8,510

1,702

13,213

5,109

5,154

196,660

159,737

5,789

6,996

6,285

5,682

925

1,584

6,219

Asp

GAC

40.5

46.8

47.4

48.5

40.8

45.1

45.4

32.4

35.6

43.0

13.5

12.7

29.1

39.1

61.8

63.8

Asp

GAT

59.5

53.2

52.6

51.5

59.2

54.9

54.6

67.6

64.4

57.0

86.5

87.3

70.9

60.9

38.2

36.2

Codons per AA

9,934

15,229

21,124

4,117

32,318

11,797

10,014

520,465

423,125

16,048

20,825

17476

15,182

2,448

2,930

14,122

Glu

GAA

58.8

52.0

49.5

46.3

57.9

56.8

55.3

62.5

59.1

37.7

80.4

84.6

65.3

60.4

50.2

49.7

Glu

GAG

41.2

48.0

50.5

53.7

42.1

43.2

44.7

37.5

40.9

62.3

19.6

15.4

34.7

39.6

49.8

50.3

Codons per AA

11,865

18,355

25,474

4,774

38,175

14,008

11,873

638,649

543,774

20,537

25,838

20,812

18,057

3,075

3,570

17,574

Phe

TTC

56.4

61.0

67.7

72.3

63.7

63.5

63.9

50.5

58.6

81.9

17.8

17.7

41.5

52.8

43.3

50.0

Phe

TTT

43.6

39.0

32.3

27.7

36.3

36.5

36.1

49.5

41.4

18.1

82.2

82.3

58.5

47.2

56.7

50.0

Codons per AA

8,977

14,376

16,102

3,031

24,881

9,726

8,311

464,354

373,697

12,020

15,752

15,138

13,032

1,966

3,650

14,036

Gly

GGA

42.7

39.5

39.7

41.4

40.4

40.0

40.9

58.7

60.7

55.6

50.1

51.2

52.3

35.1

26.9

24.8

Gly

GGC

17.8

23.3

23.5

24.1

20.9

22.5

12.5

12.1

20.6

4.3

3.1

10.5

29.7

34.9

41.8

Gly

GGG

10.1

10.0

9.5

5.9

9.7

8.8

8.4

8.3

8.4

6.9

5.9

3.9

6.8

11.7

20.6

17.5

Gly

GGT

29.4

27.2

27.3

28.6

29.0

28.8

28.2

20.5

18.8

16.9

39.8

41.8

30.4

23.5

17.6

15.9

Codons per AA

12,228

18,073

25,292

5,264

38,407

14,914

13,068

524,163

433,832

19,759

22,207

18,910

18,057

3,189

4,741

18,802

His

CAC

43.1

48.8

51.9

55.5

43.5

46.8

45.9

39.5

39.7

53.5

15.7

16.2

35.7

37.2

50.5

52.6

His

CAT

56.9

51.2

48.1

44.5

56.5

53.2

54.1

60.5

60.3

46.5

84.3

83.8

64.3

62.8

49.5

47.4

Codons per AA

4,853

8,224

9,726

1,835

14,460

5,779

4,965

226,949

183,283

7,363

7,664

6,679

6,789

1,086

1,589

7,500

Ile

ATA

21.9

20.1

16.9

14.4

19.6

19.5

18.5

15.6

13.4

10.5

30.2

30.3

24.3

16.0

15.0

11.5

Ile

ATC

35.1

41.2

46.0

49.6

39.9

41.4

42.2

31.2

39.2

57.4

9.1

9.2

27.0

33.1

36.9

37.1

Ile

ATT

43.0

38.7

37.2

36.0

40.5

39.2

39.3

53.3

47.4

32.2

60.7

60.6

48.7

50.9

48.1

51.4

Codons per AA

10,621

17,171

20,026

3,808

31,585

11,807

9,787

596,151

477,819

15,856

26,077

23,850

18,935

2,659

3,216

15,144

Lys

AAA

50.2

44.1

39.5

36.1

47.5

48.5

45.0

59.3

57.9

24.2

80.4

82.4

56.7

55.5

58.9

53.3

Lys

AAG

49.8

55.9

60.5

63.9

52.5

51.5

55.0

40.7

42.1

75.8

19.6

17.6

43.3

44.5

41.1

46.7

Codons per AA

12,606

19,080

24,922

5,451

41,187

14,926

12,874

622,428

511,710

19,693

29,246

25,932

23,023

3,465

4,650

17,224

Leu

CTA

11.8

10.7

9.7

9.0

10.2

10.9

9.8

9.2

9.9

7.3

6.2

5.5

7.4

4.4

3.6

Leu

CTC

17.2

19.5

21.1

22.8

18.6

20.0

20.7

17.3

18.8

36.9

3.4

3.5

15.2

15.0

18.4

16.0

Leu

CTG

16.1

21.6

23.5

24.6

18.8

19.5

20.5

14.1

16.0

20.1

1.7

1.5

9.3

17.4

21.8

25.2

Leu

CTT

23.1

19.6

20.2

18.9

22.7

21.2

21.8

24.6

21.1

18.4

30.9

30.5

24.8

20.6

19.3

17.4

Leu

TTA

12.5

9.6

7.2

6.2

9.9

8.8

7.7

11.4

9.0

4.8

45.8

46.6

29.4

11.3

8.6

6.1

Leu

TTG

19.3

19.0

18.3

18.4

19.9

19.7

19.5

23.3

25.2

12.5

12.0

12.3

15.8

28.3

27.5

31.7

Codons per AA

16,664

27,074

32,761

6,176

49,075

18,693

16,399

841,631

680,113

25,061

30,422

27,556

23,764

3,828

6,003

27,096

Met

ATG

100.0

Codons per AA

5,102

8,525

9,696

1,943

14,531

5,794

5,165

255,677

209,897

7,598

8,490

7,569

6,725

1,189

1,489

7,131

Asn

AAC

48.0

52.3

53.9

58.0

46.9

48.9

50.7

37.8

42.2

49.7

13.3

13.7

31.5

35.9

53.0

53.7

Asn

AAT

52.0

47.7

46.1

42.0

53.1

51.1

49.3

62.2

57.8

50.3

86.7

86.3

68.5

64.1

47.0

46.3

Codons per AA

8,173

12,784

15,647

3,003

24,165

9,013

8,111

477,965

383,675

11,638

21,928

19,815

15,025

2,073

2,589

12,490

Pro

CCA

35.0

33.6

31.2

34.6

35.7

35.1

52.8

53.1

15.3

66.2

66.5

48.2

44.0

20.0

22.5

Pro

CCC

14.0

15.9

16.5

16.7

15.8

15.0

14.5

9.1

10.3

38.3

3.7

3.2

17.3

16.2

28.1

23.4

Pro

CCG

20.0

21.8

23.1

28.4

19.5

20.9

22.0

19.8

19.1

17.7

2.4

2.1

8.5

19.9

33.0

39.1

Pro

CCT

31.0

27.3

26.8

23.7

30.0

28.5

28.4

18.2

17.5

28.6

27.7

28.2

26.0

19.9

18.9

15.1

Codons per AA

9,552

14,020

19,732

3,711

28,449

11,512

9,972

481,470

403,504

15,120

16,634

13,870

12,379

2,068

3,838

14,519

Gln

CAA

56.3

48.9

45.4

42.6

52.6

52.7

52.3

65.6

63.3

37.3

89.1

88.4

69.6

62.9

52.8

52.7

Gln

CAG

43.7

51.1

54.6

57.4

47.4

47.3

47.7

34.4

36.7

62.7

10.9

11.6

30.4

37.1

47.2

47.3

Codons per AA

7,217

11,341

14,843

2,691

21,339

8,424

7,339

405,452

332,326

10,116

13,651

11,696

10,098

2,058

2,515

12,900

Arg

AGA

20.8

18.7

17.1

19.3

18.6

18.4

19.8

29.4

31.8

30.6

50.6

52.2

39.1

13.9

12.8

10.7

Arg

CGA

23.0

21.9

21.5

20.7

21.3

23.2

21.4

22.9

22.0

16.4

6.8

6.3

8.3

20.4

14.3

17.9

Arg

AGG

11.7

13.3

13.8

10.5

12.5

12.0

12.4

7.4

8.1

9.8

6.9

9.3

10.2

10.3

8.9

Arg

CGC

13.9

16.3

17.3

18.2

14.0

15.0

14.7

9.9

10.0

16.2

3.3

3.5

12.3

19.9

26.3

25.1

Arg

CGG

8.0

9.1

8.9

7.9

8.5

9.0

8.4

8.9

8.3

5.9

1.7

1.3

3.5

10.9

17.2

17.3

Arg

CGT

22.6

20.7

21.3

23.5

25.0

22.4

23.2

21.6

19.8

21.1

27.8

29.8

27.5

24.7

19.1

20.2

Codons per AA

11,856

19,042

23,683

4,857

35,121

13,588

11,363

511,021

432,791

19,008

17,062

13,744

14,051

2,840

4,378

18,826

Ser

AGC

14.0

16.4

16.6

18.0

14.6

14.7

16.2

10.3

9.8

3.1

2.6

7.7

15.5

17.8

17.5

Ser

TCA

21.3

19.6

18.1

16.4

21.1

21.4

19.7

25.5

20.4

14.8

36.6

37.6

27.8

24.1

13.5

13.8

Ser

TCC

13.6

15.6

16.2

16.3

14.0

14.6

14.7

13.2

16.0

20.7

5.2

4.1

13.2

16.9

20.7

21.0

Ser

AGT

14.8

13.4

12.6

11.8

14.3

13.4

14.0

15.0

14.4

10.4

23.8

21.7

17.5

12.9

11.6

11.9

Ser

TCG

16.6

16.8

18.6

22.2

16.9

18.2

17.6

15.1

16.7

21.4

2.2

2.0

8.0

14.3

20.3

22.7

Ser

TCT

19.7

18.1

18.0

15.5

19.1

17.7

17.8

20.8

22.7

22.9

29.2

31.9

25.8

16.1

13.1

Codons per AA

13,892

22,627

27,519

5,278

37,542

15,230

13,768

787,872

641,565

22,591

26,438

22,992

19,598

2,921

4,637

21,063

Thr

ACA

30.6

28.0

24.8

21.6

27.1

28.4

28.3

34.2

29.6

16.3

51.5

50.9

38.6

35.0

21.9

22.6

Thr

ACC

20.4

22.9

25.5

30.8

22.8

23.2

22.4

17.9

21.8

25.7

7.6

7.5

20.8

22.9

27.4

26.8

Thr

ACG

18.9

21.8

22.6

19.9

21.5

22.1

15.2

17.2

24.9

3.9

3.2

9.2

16.3

27.9

28.8

Thr

ACT

30.1

27.3

27.2

25.0

30.2

26.9

27.2

32.7

31.4

33.2

36.9

38.3

31.4

25.7

22.7

21.7

Codons per AA

10,197

16,333

20,529

3,959

30,547

11,909

10,364

571,606

461,093

15,952

20,070

17,360

15,532

2,255

3,164

14,970

Val

GTA

18.7

16.9

14.6

12.9

18.1

16.2

15.8

15.9

13.7

11.9

26.5

19.0

12.8

8.3

7.3

Val

GTC

21.2

24.7

25.1

28.6

24.9

24.3

25.4

21.8

26.3

34.1

10.0

8.5

20.3

24.5

26.3

28.2

Val

GTG

26.8

29.3

30.8

31.8

26.3

29.5

29.1

23.4

25.5

30.6

5.1

3.8

15.8

28.0

37.3

39.2

Val

GTT

33.2

29.1

29.5

26.7

30.8

30.0

29.7

38.9

34.6

23.5

58.4

61.2

44.9

34.7

28.1

25.3

Codons per AA

12,606

20,139

25,863

5,243

39,506

14,814

12,547

605,528

491,117

18,939

21,158

18,523

17,487

2,707

3,946

18,216

Typ

TGG

100.0

Codons per AA

2,289

3,952

4,517

833

7,229

2,494

2,424

107,642

90,785

3,498

3,508

3,090

2,830

531

791

3,496

Tyr

TAC

51.6

56.5

59.8

62.8

52.7

54.6

55.7

44.0

47.1

64.9

18.5

18.0

36.6

39.4

57.1

61.6

Tyr

TAT

48.4

43.5

40.2

37.2

47.3

45.4

44.3

56.0

52.9

35.1

81.5

82.0

63.4

60.6

42.9

38.4

Codons per AA

6,322

10,100

11,774

2,335

19,614

6,915

6,328

307,728

250,436

8,842

12,998

12,250

10,470

1,346

1,894

7,748

Species (codons [n])

Codon

HG (742,990)

Mi (366,435)

Mj (203,135)

Ma (176,816)

Mh (407,985)

Mc (205,612)

ZP (16,723)

AS (646,740)

AL (42,919)

TC (103,065)

BM (561,296)

DI (126,880)

OV (299,336)

TS (290,794)

TM (147,995)

TV (106,071)

Ala

GCA

22.3

32.2

32.3

32.8

34.9

36.9

19.5

32.7

31.7

29.3

39.1

40.0

37.5

31.7

24.1

26.7

Ala

GCC

33.0

13.3

12.8

11.6

11.4

11.2

24.9

18.1

20.9

20.6

12.3

11.7

13.8

16.4

27.7

25.9

Ala

GCG

25.9

8.1

8.0

7.0

7.3

6.8

7.4

22.8

23.9

23.7

12.3

12.8

14.3

18.4

22.7

22.1

Ala

GCT

18.7

46.3

46.9

48.6

46.4

45.2

48.2

26.4

23.5

26.4

36.3

35.5

34.4

33.5

25.5

25.3

Codons per AA

54,049

21,050

11,150

10,069

22,749

10,844

1,264

45,388

3,142

7,531

31,439

7,087

17,813

18,017

10,515

7,439.0

Cys

TGT

44.5

71.4

72.8

73.7

74.3

72.9

56.3

48.8

42.8

61.2

62.9

60.0

51.8

31.8

33.8

Cys

TGC

55.5

28.6

27.2

26.3

25.7

27.1

43.8

51.2

51.3

57.2

38.8

37.1

40.0

48.2

68.2

66.2

Codons per AA

16,227

7,328

4,217

3,502

8,174

4,656

256

16,446

1,120

2,901

14,026

2,998

6,689

7,559

4,428

3,174.0

Asp

GAC

65.5

26.5

27.1

24.8

24.2

22.2

32.1

35.2

41.0

40.5

24.0

20.5

20.6

33.2

56.3

56.1

Asp

GAT

34.5

73.5

72.9

75.2

75.8

77.8

67.9

64.8

59.0

59.5

76.0

79.5

79.4

66.8

43.7

43.9

Codons per AA

36,495

17,627

9,747

8,876

19,863

9,894

873

31,446

1,970

5,114

26,014

6,223

15,056

14,599

7,378

5,214.0

Glu

GAA

55.7

75.3

74.6

76.2

76.6

78.4

77.9

56.5

55.4

52.8

73.9

77.0

77.1

79.0

61.4

61.8

Glu

GAG

44.3

24.7

25.4

23.8

23.4

21.6

22.1

43.5

44.6

47.2

26.1

23.0

22.9

21.0

38.6

38.2

Codons per AA

45,714

24,640

13,289

12,192

27,589

13,117

1,041

39,335

2,469

6,214

31,136

7,577

18,333

18,001

8,907

6,459.0

Phe

TTC

48.9

24.3

21.8

20.9

21.3

18.1

55.3

54.5

56.6

60.0

35.0

37.3

36.9

34.3

53.0

52.6

Phe

TTT

51.1

75.7

78.2

79.1

78.7

81.9

44.7

45.5

43.4

40.0

65.0

62.7

63.1

65.7

47.0

47.4

Codons per AA

37,855

18,687

11,103

9,412

21,612

11,345

704

29,754

2,102

4,296

30,333

6,568

14,714

14,907

6,593

4,737.0

Gly

GGA

25.5

44.5

44.7

45.8

46.2

33.3

31.2

31.0

32.5

32.7

35.4

35.0

31.1

29.9

31.7

Gly

GGC

41.8

14.8

14.9

13.7

14.0

13.0

19.2

25.0

28.1

26.4

16.8

16.9

17.2

24.6

33.8

33.2

Gly

GGG

15.7

13.7

13.6

13.0

10.0

8.6

4.9

10.6

11.1

10.6

10.2

7.9

9.2

9.1

9.5

11.0

Gly

GGT

17.0

27.1

26.7

27.5

30.2

32.1

42.6

33.2

29.8

30.5

40.2

39.8

38.6

35.3

26.9

24.0

Codons per AA

46,667

20,413

10,992

10,064

22,693

11,194

1,186

37,251

2,633

7,031

27,963

5,944

16,849

14,863

9,577

6,196.0

His

CAC

51.6

28.1

27.8

24.8

25.4

22.3

33.7

38.8

41.3

43.8

29.2

22.2

25.2

39.0

51.0

49.7

His

CAT

48.4

71.9

72.2

75.2

74.6

77.7

66.3

61.2

58.7

56.2

70.8

77.8

74.8

61.0

49.0

50.3

Codons per AA

19,477

7,978

4,459

3,819

9,003

4,628

421

16,467

1,060

2,710

14,787

3,389

7,427

7,245

3,626

2,601.0

Ile

ATA

9.7

23.0

23.1

22.9

23.5

25.3

13.8

23.2

21.9

19.6

29.5

29.2

27.5

25.0

26.6

27.0

Ile

ATC

35.7

12.5

11.7

11.2

11.0

9.8

36.5

36.2

39.7

40.7

22.3

23.0

24.7

21.5

29.7

27.9

Ile

ATT

54.7

64.5

65.1

65.9

65.5

64.8

49.7

40.6

38.3

39.7

48.3

47.8

47.7

53.5

43.6

45.2

Codons per AA

38,860

23,849

13,986

12,183

28,528

15,200

1,018

38,551

2,582

5,486

39,971

9,443

20,692

17,746

7,330

5,428.0

Lys

AAA

60.2

72.5

72.6

73.2

74.7

76.6

61.2

53.8

53.4

69.0

69.4

69.5

69.4

44.6

47.3

Lys

AAG

39.8

27.5

27.4

26.8

25.3

23.4

38.8

46.2

46.6

31.0

30.6

30.5

30.6

55.4

52.7

Codons per AA

44,829

26,575

15,780

12,963

29,876

15,288

1,324

40,639

2,742

7,563

37,793

8,913

21,167

19,233

9,334

6,794.0

Leu

CTA

3.1

6.7

6.4

6.3

6.9

8.1

10.4

9.9

10.5

10.3

9.9

9.5

5.8

7.9

8.7

Leu

CTC

17.1

7.1

6.6

6.0

6.4

5.7

15.9

18.9

19.3

19.1

7.8

7.2

7.6

6.9

10.9

11.1

Leu

CTG

21.5

5.0

4.7

4.8

4.5

14.8

16.1

18.0

13.0

10.9

13.8

16.0

24.7

23.4

Leu

CTT

17.9

24.7

25.0

25.2

25.3

26.0

25.1

21.2

20.7

20.5

19.7

19.8

19.6

17.3

18.0

18.6

Leu

TTA

6.6

30.9

31.4

32.3

33.4

35.3

17.5

13.5

12.8

10.1

26.1

27.8

24.6

17.3

9.2

9.7

Leu

TTG

33.8

25.6

25.9

25.5

23.8

21.7

29.0

21.3

21.2

21.7

23.1

24.5

24.9

36.8

29.4

28.5

Codons per AA

68,607

34,549

19,485

17,118

38,589

20,003

1,397

56,369

3,564

8,233

53,591

12,048

26,576

27,491

13,110

9,558

Met

ATG

100.0

Codons per AA

18,014

8,593

4,930

4,095

9,532

4,730

306

16,510

1,091

2,477

15,883

3,373

7,990

7,625

3,836

2,785

Asn

AAC

48.6

22.3

20.9

19.9

18.8

17.6

46.6

43.2

44.1

49.1

27.2

23.8

25.1

34.9

53.1

Asn

AAT

51.4

77.7

79.1

80.1

81.2

82.4

53.4

56.8

55.9

50.9

72.8

76.2

74.9

65.1

46.9

Codons per AA

33,133

20,562

11,306

9,756

23,509

12,351

779

28,917

1,836

4,201

28,196

6,554

14,572

14,343

5,930

4,424

Pro

CCA

24.2

40.6

39.9

41.3

44.8

45.0

46.5

36.9

32.5

34.9

45.0

45.5

44.2

38.3

30.0

30.3

Pro

CCC

24.5

10.6

9.6

8.8

7.9

7.2

14.2

14.9

18.4

17.1

10.7

8.5

9.7

10.9

17.7

17.1

Pro

CCG

34.9

10.5

11.2

10.7

8.5

11.7

24.7

24.5

24.6

19.1

19.2

22.9

24.4

28.4

28.7

Pro

CCT

16.4

38.3

39.3

39.1

38.9

39.3

27.6

23.5

24.7

23.4

25.2

26.8

23.2

26.3

23.8

23.9

Codons per AA

36,929

16,614

8,537

7,305

17,440

8,307

768

30,784

2,276

5,519

23,108

4,915

13,464

11,909

7,466

5,066

Gln

CAA

57.1

79.4

79.6

80.2

80.9

80.3

82.0

57.4

57.1

50.2

61.4

63.6

62.8

59.9

50.4

50.3

Gln

CAG

42.9

20.6

20.4

19.8

19.1

19.7

18.0

42.6

42.9

49.8

38.6

36.4

37.2

40.1

49.6

49.7

Codons per AA

33,107

16,926

8,552

7,532

17,739

8,805

640

23,271

1,514

3,612

21,428

4,769

11,797

11,420

5,644

4,047

Arg

AGA

11.6

28.8

28.1

29.2

30.0

16.4

17.7

16.3

17.2

22.3

21.9

21.2

21.8

16.3

17.6

Arg

CGA

18.0

16.8

17.3

16.7

17.2

17.3

21.3

18.5

19.7

24.0

26.3

25.4

22.7

18.1

17.7

Arg

AGG

9.0

9.7

9.4

8.8

8.9

8.6

2.9

12.3

11.8

12.4

10.9

8.8

9.5

7.1

12.4

13.2

Arg

CGC

25.0

9.4

9.2

8.5

8.8

7.9

15.3

15.8

20.2

17.3

9.6

9.4

10.4

13.7

22.9

21.8

Arg

CGG

15.8

5.6

5.4

4.9

4.5

1.2

8.3

7.9

7.2

9.2

8.4

8.8

8.5

11.6

11.5

Arg

CGT

20.6

29.7

31.0

31.4

31.1

31.8

47.0

24.6

25.3

26.2

24.0

25.2

24.8

26.1

18.7

18.2

Codons per AA

47,178

19,486

11,042

9,171

21,409

9,973

1,001

40,766

2,650

6,429

31,420

7,457

17,634

16,384

9,203

6,468

Ser

AGC

15.5

9.1

9.4

8.4

7.9

7.6

11.8

16.4

16.6

17.1

11.0

9.7

11.1

15.1

23.2

22.5

Ser

TCA

14.4

24.5

24.3

24.1

27.0

28.6

18.1

22.3

20.8

19.6

28.4

29.5

27.7

21.0

14.4

15.5

Ser

TCC

20.8

9.0

8.6

7.9

6.8

6.6

15.7

9.9

12.0

11.0

10.4

9.3

10.7

10.3

17.7

17.3

Ser

AGT

12.5

17.6

17.2

18.5

18.1

18.4

13.6

15.8

13.8

14.4

19.2

18.7

19.1

13.3

13.8

Ser

TCG

22.4

7.8

8.3

7.9

6.2

13.3

21.4

22.3

24.8

12.2

12.8

14.0

14.4

19.1

18.3

Ser

TCT

14.3

31.9

32.1

33.3

34.0

32.5

27.5

14.3

14.4

13.1

18.9

20.0

17.5

20.1

12.2

12.6

Codons per AA

54,466

27,606

14,239

12,353

29,702

15,207

899

48,736

3,157

6,860

43,504

9,483

21,762

22,175

10,980

7,907

Thr

ACA

23.2

40.6

39.2

41.1

42.3

42.7

19.5

30.2

30.3

27.5

39.0

40.1

38.1

31.8

21.3

25.1

Thr

ACC

25.6

11.1

9.9

9.0

9.1

7.9

28.6

19.2

22.4

20.0

15.6

12.9

15.6

16.3

24.9

23.1

Thr

ACG

26.6

8.4

8.8

8.6

8.5

7.7

12.5

26.8

24.9

29.2

15.4

16.3

17.4

19.8

30.9

30.3

Thr

ACT

24.6

39.8

42.1

41.4

40.1

41.7

39.4

23.8

22.4

23.2

30.0

30.7

28.9

32.1

22.9

21.5

Codons per AA

37,607

19,046

10,254

8,837

20,194

10,213

919

34,207

2,219

5,814

29,901

6,460

15,488

14,901

7,680

5,829

Val

GTA

6.0

17.6

16.9

17.5

17.0

20.7

14.1

16.4

16.6

16.4

25.4

26.2

26.3

19.4

16.6

17.7

Val

GTC

29.6

13.2

12.3

11.9

11.4

10.7

29.2

20.6

21.7

21.3

14.3

14.0

14.4

16.7

22.8

21.9

Val

GTG

38.3

13.5

13.2

11.7

12.9

11.7

12.9

29.7

31.1

30.7

22.9

21.2

21.6

26.3

27.6

28.3

Val

GTT

26.1

55.8

57.6

58.9

58.6

56.9

43.8

33.2

30.6

31.5

37.5

38.7

37.7

37.6

33.0

32.1

Codons per AA

45,942

19,706

10,925

9,899

21,917

10,703

1,109

42,490

2,780

6,580

33,067

7,389

17,037

18,805

9,977

7,169

Typ

TGG

100.0

Codons per AA

9,033

4,055

2,294

2,002

4,746

2,298

188

7,902

532

1,213

6,380

1,377

3,584

3,486

1,777

1,350

Tyr

TAC

58.9

23.2

22.2

20.9

21.4

19.6

34.6

37.3

41.2

46.0

30.3

26.0

28.4

38.7

61.8

62.7

Tyr

TAT

41.1

76.8

77.8

79.1

78.6

80.4

65.4

62.7

58.8

54.0

69.7

74.0

71.6

61.3

38.2

37.3

Codons per AA

18,687

11,019

6,656

5,582

12,940

6,716

627

21,299

1,453

3,267

20,353

4,785

10,337

9,939

4,691

3,401

Values are given as % per AA, or as numbers for Codons per AA. Definitions of species two letter codes are provided in Table 1. AA, amino acid.

To examine these variables independent of species relatedness, correlations were calculated using phylogenetically independent contrasts (see Materials and methods, below). The variances of the contrasts were computed for each character as a measure of the variance accumulating per unit branch length. The branch lengths were estimated from the maximum likelihood phylogeny assuming a molecular clock (Figure 1); by this criterion, the tips of the tree are all equidistant in branch length from its root. Computed contrasts were plotted in all figures representing pair-wise comparisons, and the correlation coefficients were calculated from the paired contrasts. This method is robust to changes in molecular clock assumptions. (Trees calculated without the assumption of a molecular clock are similar in topology but differ in rooting, and branch lengths vary according to amount of base substitution in the 18S rRNA; the clock-based tree provides branch lengths that should estimate most closely the relative durations of branches in evolutionary time. Because independent contrasts are influenced mainly by relative branch lengths, our results should be robust to alternative placements of the root.)

Figure 1

Maximum likelihood (ML) analysis of 18S ribosomal RNA from 25 nematode species

Maximum likelihood (ML) analysis of 18S ribosomal RNA from 25 nematode species. The ML calculation assumes a molecular clock; thus, the tips of the tree are all equidistant, in branch length, from its root. This model of base substitution allows the expected frequencies of the four bases to be unequal, and different rates of evolution at different sites are allowed. The numbers indicate reconstruction of percentage changes in overall codon usage on this phylogenetic topology (see Codon usage patterns and relationships to sampling method, nematode phylogeny, and GC content [under Results]). A distance matrix of D values corrected for non-additivity [1 - antilog(-D)] × 100 was partitioned on the topology using the cyclic neighbor-joining algorithm, as illustrated by Avise [82]. Approximate percentage change in overall codon usage is indicated for five branches inferred to have undergone 5% or more divergence from an ancestral nematode pattern. This analysis identified genera Globodera, Meloidogyne, Pristionchus, and Strongyloides as having the most highly derived patterns of codon usage, and the remaining species as having relatively little net divergence from an ancestral nematode pattern. Definitions of species two letter codes are provided in Table 1; GenBank accession numbers are listed on right. Clades V are shown in red, IVa in blue, IVb in green, III in yellow, and I in brown.

Amino acid composition of nematode proteins and relationship to GC content

AA composition of predicted proteins in nematodes varies among species within a narrow window and is similar to that observed in other organisms (Tables 2 and 3). (Standard deviations in AA usage among nematodes range from 5% to 15% of mean usage, and mean nematode AA usage differs from the mean of four representative organisms by an average of 8%.) Across nematodes, Leu is the most common AA (8.8% of all codons) and Trp the least common (1.1%). Eight AAs contribute an average of more than 6% each to AA content (Ile, Gly, Val, Glu, Ala, Lys, Ser, and Leu); these AAs are also among the most common in the proteomes of other representative species, including humans (Table 3). As in other taxa 46, nematodes show a correlation between AA usage and the degree of codon degeneracy (R = 0.72).

In nematodes, coding sequence GC content, derived from our EST clusters, varies from 32% to 51% (Table 1) among species, with a mean of 43.6 ± 5.9%. The distribution is biphasic, with a peak at 36% GC and a second peak at 48%. Strongyloides (SS and SR), Meloidogyne (MI, MJ, and so on), and filarial parasites (BM, DI, and OV) are the most AT rich (low GC); and NB, PP, and cyst nematodes (GP, GR, and HG) are the most GC rich (approximately 50%). The variation observed in AA composition among species shows a clear relationship to the species' coding sequence GC content. The frequency of AAs encoded by WWN codons (AA, AT, TA, or TT in the first and second nucleotide positions; Asn, Ile, Lys, Try, Phe, and Met) decreases with increasing coding sequence GC content (Figure 2a), whereas the proportion of AAs encoded by SSN codons (GG, GC, CG, and CC; Ala, Arg, Pro, and Gly) increases with higher coding sequence GC content (Figure 2b), and these relationships remain even after removing the effect of evolutionary relationships using phylogenetically independent contrasts. Among AAs, the most uniform and precipitous decrease with increasing GC content was seen with Ile and Tyr whereas the most uniform and rapid increase with higher GC content was seen with Ala and Arg. The trend is less pronounced for other AAs (flatter slope, lower R value). Thr, encoded by four GC/AT 'balanced' codons (ACN), exhibits no change in its frequency with changing GC content (data not shown).

Figure 2

Correlation between phylogenetically independent contrasts of coding sequence GC3 content and AA usage for 25 nematode species

Correlation between phylogenetically independent contrasts of coding sequence GC3 content and AA usage for 25 nematode species. (a) AAs lysine (Lys), isoleucine (Ile), asparagine (Asn), and tyrosine (Tyr) are used less frequently as the species' coding sequence GC3 content increases. (b) AAs alanine (Ala), glycine (Gly), arginine (Arg), and proline (Pro) are used more frequently as the coding sequence GC3 content increases. AA, amino acid.

Base composition by codon position in nematode transcripts and relationship to GC content

Codon usage in nematode species was examined by several methods, including comparison of base usage by position (1-3) over all AAs and comparison of codon usage within each AA. Over all AAs, purine (AG) and pyrimidine (TC) usage in positions 1, 2, and 3 is remarkably uniform between species, favoring purines in position 1 (AG 59.6 ± 1.5%), near equal usage in position 2 (AG 50.0 ± 0.8%), and pyrimidines in position 3 (AG 47.9 ± 1.5%; Additional data file 1). Similar values were observed in Schistosoma mansoni (AG 61%, 53%, and 48% in positions 1, 2, and 3, respectively) 1. GC versus AT usage also varies by position but with much greater variance, with near equal usage in position 1 (50.3% GC) and lower GC usage in positions 2 and 3 (39.1 and 41.4%, respectively), mainly due to greater use of G in position 1 and T in positions 2 and 3 4.

Additional file 1

Click here for file

The variation observed in GC usage by codon position among species exhibits a clear relationship to the species' overall coding sequence GC content. Not surprisingly, both GC1 and GC2 composition increase with higher coding sequence GC3 content (Figure 3). Specifically, species with high AT content like root-knot Meloidogyne species (MI, MJ, and so on) and filarial worms (BM, DI, and OV) 3839 are biased toward codons terminating in A or T, whereas species with higher GC content such as NB, PP, cyst nematodes, and whipworms (TM and TV) prefer codons ending with G or C. Differences in calculated GC composition by codon position (1-3) between species are determined both by the species' AA usage (as described above) and the codons used for each AA. For example, Cys was encoded by TG

T as much as 85% of the time for the AT-rich Strongyloides genomes, whereas TG

C was used up to 60% of the time in GC-rich genomes such as NB, PP, and HG. To compare codon usage more systematically for individual AAs between species, we employed a statistical approach (described in Materials and Methods and in the following section).

Figure 3

Correlation between phylogenetically independent contrasts of the third position GC content (GC3) and that of the first (GC1) and second (GC2) codon positions for 25 nematode species

Correlation between phylogenetically independent contrasts of the third position GC content (GC3) and that of the first (GC1) and second (GC2) codon positions for 25 nematode species.

Codon usage patterns and relationships to sampling method, nematode phylogeny, and GC content

Similarity in codon usage was quantified and reported as D₁₀₀values for each species and AA compared 4748 (matrix of D₁₀₀values for each species and AA compared is available in Additional data file 2).

Additional file 2

Click here for file

Because analyses of all but two of the nematode species were based on EST-derived partial genomes 26, comparisons were performed to estimate the differences in codon usage pattern that could be expected using EST collections versus gene predictions derived from a fully assembled and annotated genome. Using C. elegans, parallel analyses were performed using either all 22,254 predicted gene products or two EST datasets (CE-A and CE-B) each comprising 10,000 ESTs. Clustering and peptide predictions were performed using the same algorithms as for the other 30 species. The average D₁₀₀value for the comparison of codon usage pattern between the CE-A and CE-B datasets was 0.18, which was not statistically different at the P < 0.05 threshold and less than the D₁₀₀value of the C. elegans to C. briggsae comparison (0.40). Comparing the CE-A and CE-B datasets to the genome-derived full gene set for C. elegans yielded average D₁₀₀values of 0.67 and 0.26, respectively. At a practical level, the calculated use of the average codon in C. elegans based on CE-A and CE-B differs from that based on prediction from the whole genome by just 3.4 ± 2.3% and 2.0 ± 1.5%, respectively. Therefore, although differences in calculated codon usage using partial versus whole genome data are modest enough to make EST-derived codon usage data highly informative, care must be taken not to over-interpret minor differences in D₁₀₀values because such differences are probably within the range of sampling error (see Discussion, below). However, such uncertainty around small differences in D₁₀₀values does not alter the major trends that we describe.

The 16 intragenus comparisons of species sharing the same genus name (Ancylostoma, Caenorhabditis, Strongyloides, Globodera, Meloidogyne, Ascaris, and Trichuris) all have low D₁₀₀values, with a mean of 0.14 ± 0.11 (median 0.09, range 0.02-0.40), indicating very similar patterns of codon usage among species within the same genera. By contrast, the 480 comparisons beyond named genera vary greatly, with a mean D₁₀₀value of 8.10 ± 7.46 (median 5.26, range 0.08-40.56). Low D₁₀₀values do sometimes extend to comparisons among genera. For instance, relatively low D₁₀₀values (0.08-1.94) are observed within the following: order Haemonchidae (HC, OO, and TD); subfamily Heteroderinae (GP, GR, and HG); superfamily Ascaridoidea (AS, AL, and TC); and superfamily Filarioidea (BM, DI, and OV). However, low D₁₀₀values are not maintained across family Ancylostomatidae (NA, AC, and AY), family Strongyloididae (SS, SR, and PT), superfamily Tylenchoidea (PE-MC), and order Trichocephalida (TS, TM, and TV). Similarity in codon usage, as indicated by low D₁₀₀values, does not extend to the level of the major clades (I, III, IVb, IVa, and V).

Furthermore, species with very similar GC content, although distantly related, can exhibit extremely similar codon usage (for instance Ancylostoma caninum versus Toxocara canis, GC = 48%, D₁₀₀= 0.79). Species with the lowest average D₁₀₀values in one-versus-all comparisons are those closest to the median species GC content, such as PE (GC = 46%). Taxa with the highest AT content, such as Strongyloides and Meloidogyne species, have among the most extreme differences in codon usage when compared with species beyond their genus (median D₁₀₀values are 15.3 and 9.4, respectively).

Phylogenetic analysis of changes in codon usage using (1 - antilog [-D]) × 100, interpretable as percentage divergence in overall codon usage (Figure 1), identifies five branches that have accumulated more than 5% change in codon usage. These branches are as follows: the most recent common ancestor of clades III, IVa, and IVb (5.2%); the most recent common ancestor of clade IVa (11.2%); the most recent common ancestor of genus Meloidogyne (6.7%); the most recent common ancestor of genus Globodera (7.3%); and the lineage represented by PP (8.3%). Genera Globodera, Meloidogyne, Pristionchus, and Strongyloides therefore represent the most highly derived patterns of codon usage in nematodes, with the remaining species exhibiting less relatively divergence from an ancestral nematode pattern.

Codon bias in nematode transcripts and relationship to GC content

We used the effective number of codons (ENC) to measure the degree of codon bias for a gene 49. ENC is a general measure of non-uniformity of codon usage and ranges from 20 if only one codon is used for each AA to 61 if all synonymous codons are used equally. The mean ENC across all sampled nematode species is 46.7 ± 5.1, and many nematodes have ENC values similar to those obtained for various bacteria, yeast, and Drosophila species (ENCs of 45-48) 50. Outliers with low ENC values include SS and SR, for which transcripts on average utilize only about 35 of 61 available codons. The variation observed in ENC values among species exhibits a clear relationship to the species' overall coding sequence GC3 content (R = 0.70 following phylogenetic correction; Figure 4). The correlation confirms that species with lower GC3 content in coding sequence have greater codon usage bias than those with higher GC3. ENC values for nematodes peak at 47-49% GC (data not shown). In addition to comparing species' mean ENC values, we also examined the distribution of ENC values across all transcripts within each species. Although all species have examples of transcripts across nearly the full range of possible ENC values, in species with low GC3 content, such as SR, the distribution is shifted toward a lower ENC peak (Additional data file 3).

Additional file 3

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Figure 4

Correlation between phylogenetically independent contrasts of each species' %GC3 and its mean ENC for 25 nematode species

Correlation between phylogenetically independent contrasts of each species' %GC3 and its mean ENC for 25 nematode species. ENC, effective number of codons.

To ensure that differences in our available data for each species (for instance, cluster number and cluster length) were not creating artifacts in ENC values, quality checks were performed. Unlike measures such as codon bias index, scaled ×2, and intrinsic codon bias index, ENC values should be independent of translated polypeptide length and sample size 4951, and our analysis confirmed this. No correlation with ENC was observed with either average translated polypeptide length or number of clusters for a species. In fact, SS and SR with the lowest ENC values had above average cluster length and number. As additional confirmation, we randomly selected 2,400 C. elegans genes (the average number of clusters for species other than CE and CB) and calculated ENC based on either full-length genes or genes trimmed to 121 AAs (the average length cluster translation for species other than CE and CB). Differences in the average ENC numbers for these datasets were not statistically significantly different from zero (P > 0.05).

In addition to codon bias, neighboring nucleotides influence the codon observed at a position relative to synonymous codons. The most important nucleotide determining such context dependent codon bias 525354 is the first one following the codon (N1 context) 5556. An analysis using the complete genesets of Homo sapiens, Drosophila melanogaster, C. elegans, and Arabidopsis thaliana revealed that 90% of codons have a statistically significant N1 context-dependent codon bias 57. Using the same method we calculated that, for the 30 nematode species represented by EST-derived codon data, an average of 63% of codons with N1 context have a statistically significant bias (because the R values differed from 1 by more than 3 standard deviations). Fedorov and colleagues 57 showed that their results were not considerably affected by gene sampling. However, for our dataset the calculated CE-A and CE-B N1 context with statistically significant bias was 75% and 83% of the codons, respectively, as compared with 96% when the complete C. elegans gene set was used. Therefore, the extent of significant N1 context-dependent codon bias determined from EST-based codon usage data may change as more complete nematode genomes become available. The complete list of relative abundance of all nematode species with N1 context, R values, and standard deviations are available in Additional data file 4.

Additional file 4

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Coding sequence GC content versus total genome GC content

Because of the clear relationships of AA composition, codon usage pattern, and codon bias to the GC content of coding sequences and the interest in the underlying cause of these correlations (see Discussion, below), we examined the relationship between coding sequence GC3 content and genomic GC content in nematodes. Total genomic GC content was calculated for the six nematode species for which significant genome sequence data were available as unassembled sequences (TS and HC), partial assemblies (BM and AC), or finished assemblies (CE and CB). Noncoding genomic GC content was calculated for CB and CE based on published estimates of the percentage of each genome that is composed of noncoding sequence, namely 74.5% and 77.1%, respectively 35. Extrapolations were made for other species using the CE percentage noncoding estimate. Although GC content varies across the genome for some organisms (for example, isochors in vertebrates 58), GC content is fairly uniform across the C. elegans genome 17; furthermore, as yet there is no evidence of non-uniformity in other nematode genomes. A positive correlation was observed between coding GC3 content and both total GC content and extrapolated noncoding GC content (R = 0.92; Figure 5). Noncoding genomic sequences varied across a wider span of GC values than did coding sequences. In all six nematodes, coding sequences were somewhat more GC rich than were noncoding sequences (2-10%).

Figure 5

Correlation between coding sequence (transcriptome) %GC3 and genome %GC for six nematode species with extensive available genomic sequence

Correlation between coding sequence (transcriptome) %GC3 and genome %GC for six nematode species with extensive available genomic sequence. The green line indicates the coding sequence %GC versus the full genomic %GC. In this case, coding sequence %GC3 is a contributor to the full genomic %GC such that X and Y are not independent variables. The red line indicates the coding sequence %GC3 versus noncoding genomic %GC. In this case, the coding sequence contribution has been removed from genomic totals such that X and Y are independent variables. For BM, TS, HC, and AC, the calculation of noncoding genomic %GC relies on the assumption that the species will have a similar breakdown of coding and noncoding sequence as CE. Assembly and gene calling for the BM, HC, TS, and AC sequences are needed to test this assumption. Definitions of species two letter codes are provided in Table 1.

Comparison of coding sequence GC versus 3'-untranslated region (UTR) GC also supports this conclusion. Calculated 3'-UTR GC for the 30 species in our EST dataset ranges from 28.6% to 46.1%. Correlation between phylogenetically independent contrasts of coding GC content (Table 1) and 3'-UTR has an R value of 0.81 (data not shown).

Codon usage patterns in abundantly expressed genes and candidate optimal codons

Representation in cDNA library generally correlates with abundance in the original biologic sample 59 although artifacts occur 6061. To investigate the difference in the codon usage patterns in highly abundant transcripts as compared with less abundantly expressed genes, as determined by ESTs, we selected five species, each of which is a member of a different clade. The selected species (AY, MI, OV, SR, and TS) were represented by approximately 3,000 clusters each (range 2,693-3,214), and codon usage tables were generated for subsets of genes from each species: the 20 most abundant clusters versus all remaining clusters, and the 50 most abundant clusters versus all remaining clusters. Results of both comparisons were similar, and for simplicity we discuss only the results based on the comparison of the 50 most abundant versus all remaining clusters. Clusters 51 to about can be described as containing mainly genes with low to moderate expression because transcripts of extremely low abundance are less likely to be represented in EST collections (for instance, neuronal 7-transmembrane receptors). Codon usage tables, AA frequencies, and relative differences between AA usage of the most abundant and less abundant genes are available in Additional data file 5.

Additional file 5

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D values were calculated across all AAs and the codon usage in each species was generally similar for genes represented by abundant EST clusters and genes represented by low to moderate expression EST clusters. SR exhibited the greatest difference between the two usage patterns (D₁₀₀= 6.15). Additionally, for all the species at least seven AAs were used significantly more frequently in the abundant genes than in the remainder of the genes. For example, although the abundant OV clusters had a Pro composition of 10.5% of all AAs, the rest of the clusters were only 4.4% Pro.

Examining the codon usage frequencies within an AA, an increase in usage has been noted with higher gene expression for specific so-called 'optimal' codons 6263. Using the codon usage tables for the top 50 and remaining clusters, we have defined a list of potentially optimal codons with usage that is higher in abundant transcripts by a statistically significant measure. Out of the 59 synonymous codons there were 24, 28, 25, 27, and 23 candidate optimal codons (Table 5) in AY, MI, OV, SR, and TS, respectively. For example, Tyr is encoded by two codons (TAC and TAT); in AY TAC is used 75% of the time in the abundant clusters and 59% of the time in the less abundant clusters. Similar analysis documented about 21 candidate optimal codons in C. elegans for which usage differed significantly when comparing high and low expressed genes 313364. Confirmation of these candidate codons as truly 'optimal' will require additional investigations, including other means of verifying relative expression levels (for example, microarrays and reverse transcription [RT]-polymerase chain reaction [PCR]).

Table 5

Candidate optimal codons in five species, determined as frequency increase by increased expression level^a

Species

Codon

Ala

GCA

Asn

AAC

Ala

GCC

Asn

AAT

Ala

GCT

Pro

CCA

Cys

TGC

Pro

CCC

Pro

CCG

Asp

GAC