Molecular phylogenetic analysis in ... - Prevenzione Oggi

Vol. 1, no. 3-4, 69-77 

Molecular phylogenetic analysis 

in ecogenotoxicological studies 

D. Davolos*,V. Iannilli**, E. De Matthaeis**, B. Pietrangeli* 

* ISPESL, Department of Production Plants and Environmental Interaction, Rome 

** University of Rome “La Sapienza”, Department of Animal and Human Biology 

ABSTRACT 

The usefulness of taxa belonging to the Amphipoda (Crustacea) in environmental toxicology is welldocumented 

in literature and a growing number of studies are addressing this line of research. However, as 

with most ecotoxicology studies, this research is rarely supported by analyses of the genetic structure and 

evolutionary history of the taxa under examination.The availability of such information becomes crucial 

when research is being carried out on the effects, such as genotoxic ones, of exposure to particular 

substances. In this work, two crustaceans (Orchestia garbinii and Gammarus aequicauda;Amphipoda) from 

aquatic habitats and with different ecological characteristics were chosen as potential candidates for 

ecogenotoxicological assessment for water, as well as for sediments in toto. Data are presented on the 

genetic structure and on the relationships among populations of these Crustaceans, inferred by using 

sequences of mitochondrial genes. Furthermore, various methods are discussed, which are helpful in 

assessing genotoxicity in tissues of organisms exposed in the laboratory and in populations taken from 

contaminated sites. 

Prevention Today July - December 2005 

69 

(Key words: ecogenotoxicology, molecular phylogeny,Amphipoda) 

BOW PO/base indexing: 

EUOSHA - OSH: Genetic toxicology (26601D), Environmental pollution (05481D), Measurement and assessment 

(12561D) 

CIS: Ecogenetics (Wopg), Ecotoxicology (Sepe), Crustacea (Fidu),Water pollution (Bubue), Sampling and analysis (Qe) 

Reviewed and accepted: 20/02/2006 by Giovanni Alfredo Zapponi; 17/03/2006 by Laura Mancini - National 

Institute of Health (ISS)

INTRODUCTION 


70 

The development of Community and national norms on the subject of water protection, geared initially 

towards the protection of drinking water, bathing water and of the consumption of edible aquatic 

organisms, is currently geared towards an approach of integrated protection, taking into account the 

necessity to safeguard the entire aquatic ecosystem. In fact, defence of the whole hydric environment from 

pollution caused by the introduction of dangerous substances from specific and widespread sources is 

necessary in order to ensure the protection of human health (e.g. from risks deriving from the transfer of 

contaminants through processes of bioaccumulation and biomagnification). 

Criteria for toxicity and ecotoxicity are therefore extremely useful for defining environmental quality 

standards that are necessary to achieve a good chemical state in bodies of water 1-2 . The usefulness of 

determined taxa in environmental toxicology is well-documented in scientific literature 3-5 . However, 

ecotoxicology studies are rarely supported by analyses of the genetic structure or the evolutionary history of 

the taxa under examination.The availability of this information 6-14 turns out to be indispensable when research 

is being carried out on the effects (e.g. the genotoxic effects) from exposure to particular substances 15-22 . 

Two Crustaceans belonging to Amphipoda and linked to aquatic environments were chosen for this study, 

as potential candidates for ecogenotoxicological assessment (direct, indirect and long-term effects) both for 

water and for sediments in toto.Analyses were carried out on the talitrid, Orchestia garbinii (Fig. 1), a semiterrestrial 

species found along the banks of lakes and rivers in Europe, and the gammarid, Gammarus 

aequicauda (Fig. 2), found in salt water and the lagoon systems of the Mediterranean. 

Figure 1 - Orchestia garbinii (Crustacea,Amphipoda,Talitridae): adult male, 20 mm 

Figure 2 - Gammarus aequicauda (Crustacea,Amphipoda, Gammaridae): adult male, 15 mm 

In this paper, we present phylogenetic results on those crustaceans with the focus on gene sequences of 

mitochondrial DNA (mtDNA). We will also discuss various methods that are helpful for evaluating 

genotoxicity and mutagenesis in tissues of organisms exposed in the laboratory and in populations taken 

from contaminated sites.

1. MATERIALS AND METHODS 

The sampling sites of O. garbinii were: Lake Garda (Verona ) and Lake Bracciano (Rome) (Fig. 3a,b).The 

samples of G. aequicauda were taken from the following sites: Lake Patria (Caserta); the mouth of the 

River Ombrone (Grosseto); the mouth of the River Mignone (Viterbo); the mouth of the River 

Candeloro, Manfredonia (Foggia). 

Figure 3 - Sampling sites of Orchestia garbinii: (a) Lake Garda and (b) Lake Bracciano 

Lake Garda 

(a) 


71 

Lake Bolsena 

Lake Vico 

Lake Bracciano 

(b)

The methods used for amplification by Polymerase Chain Reaction (PCR) and the sequencing of 

mitochondrial genes of the subunits I and II of the cytochrome oxidase (COI and COII) 23-5 and of 16S 

ribosomal RNA (16S rRNA) are shown in 14,26-27 . Homologous sequences of the mtDNA of talitrids 14 (one 

sequence is deposited in GenBank, NCBI, with access number AY555730), of gammarids 28,29 and of other 

Crustacea (extracted from GenBank) were used to carry out phylogenetic analyses by the Neighbour- 

Joining and Maximum Parsimony methods 14 . Molecular phylogenetic inferences were carried out by using 

nucleotidic sequences (examining both transitions and transversions) and the deduced amino acid 

sequences (with Poisson correction) 14 . From 500 to 1000 bootstrap replications were calculated for each 

phylogenetic reconstruction 10-14 . 

2. RESULTS 


72 

Mitochondrial regions encoding protein and ribosomal RNA of O. garbinii and of G. aequicauda were 

amplified through the PCR method and then sequenced.The alignments with homologous sequences of 

Crustacea generally resulted as unambiguous.The gene tRNA LeuUUR was found between the genes for the 

subunits I and II of the cytochrome oxidase (COI and COII) in G. aequicauda, while COI-NC-COII 14 

rearrangement was found in O. garbinii. 

Inferences on the evolutionary relationships of O. garbinii and G. aequicauda (potential candidates for 

ecogenotoxicological assessment for water as well as for sediments) were obtained through phylogenetic 

reconstruction using mitochondrial sequences. Figs. 4 and 5 show some results of a phylogenetic analysis 

of the two Amphipods examined and of other Crustacea, based on sequences of nucleotides and amino 

acids encoded by the COI and COII genes. Fig. 6 shows a phylogenetic study of O. garbinii and G. aequicauda 

based on nucleotide sequences of ribosomal RNA of the large subunit (16S rRNA). 

Figure 4 - Molecular phylogeny of O. garbinii, talitrids and other Crustacea (sequences extracted from GenBank) 

obtained through (a) Neighbour-Joining on 121 amino acids (Poisson correction) encoded by a region of the COI 

gene and (b) Maximum Parsimony (consensus tree) on 367 nucleotides of the COI gene 

56 

91 

64 

80 61 

56 

90 

92 

99 

99 

Panulirus japonicus 

Pagurus longicarpus 

Penaeus monodon 

Orchestia gammarellus (I. Cumbrae) 

Orchestia gammarellus (I.Wight) 

Orchestia mediterranea (I.Wight) 

Talorchestia deshayesii (I.Wight) 

Talitrus saltator (I.Wight) 

Talitrus ssltator (I. Cumbrae) 

Orchestia stephenseni 

Orchestia garbinii (L. Garda) 

Orchestia garbinii (L. Bracciano) 

Parhjale hawaiiensis 

Triops cancriformis 

Daphnia pulex 

Artemia francescana 

Tigriopus japonicus 

Hyalidae 

Talitridae 

Talitroidea 

0,05 

(a)

100 

100 

100 

100 

100 

100 

100 

100 

66 

100 

100 

100 


Daphnia pulex 

Artemia francescana 

Panulirus japonicus 



Parhyale hawaiiensis Hyalidae 






Orchestia stephenseni (AY555730) 


Talitrus saltator (I. Cumbrae) 


Talitridae 

Talitroidea 

(b) 

Figure 5 - Molecular phylogeny of O. garbinii, of G. aequicauda and other Crustacea (sequences extracted from 

Genbank) obtained through Maximum Parsimony (consensus tree) on 114 amino acids encoded by regions of the 

COI and COII genes 

100 

65 

89 

100 

74 

62 

73 

67 

66 

100 

99 

94 

Daphnia pulex 




Gammarus aequicauda (Mignone) 

Gammarus aequicauda (L. Patria) 

Gammarus aequicauda (Ombrone) 

Gammarus aequicauda (Candeloro) 





Talitrus saltator (I. Cumbrae) 





Gammaridae 

Talitridae 

Talitroidea 


73 

Figure 6 - Molecular phylogeny of O. garbinii, of G. aequicauda and of other Crustacea (sequences extracted from 

GenBank) obtained through Maximum Parsimony (consensus tree) on 246 nucleotides of the 16S rRNA gene (500 

bootstrap replications) 

98 

74 

99 

95 

100 

98 

77 

100 

88 

76 



Gammarus locusta 

Gammarus aequicauda (Black Sea) 

Gammarus aequicauda (L. Patria) 

Gammarus aequicauda (Mignone) 

Gammarus balcanicus 

Gammarus fasciatus 

Gammarus mucronatus 

Gammarus annulatus 

Gammarus oceanicus 

Gammarus elvirae 

Gammarus lacustris (L. Hovsgol) 

Chaetogammarus marinus 



Orchestia “cavimana” (AY744911) 


Gammaridae 

Talitridae 

Talitroidea

3. DISCUSSION 


74 

Many human activities (in the industrial, urban, agricultural sectors, etc.) have caused an increase in the 

environmental concentration of particular pollutants including heavy metals, mutagenic substances, etc.The 

negative effects (direct and indirect) of xenobiotic agents can be significant in natural populations 12-30 , with 

a potential risk of exposure and repercussions on human health 31 . 

In recent years, many research projects have focussed attention on the identification of species that are 

useful in ecogenotoxicological assessments on different types of matrices. However, the lack of 

phylogenetic analyses of the taxa under examination may entail a margin of error in the interpretation of 

the research results, e.g. environmental-genotoxicological 16,17,21,22 . 

Low levels of genetic divergence found among the populations of O. garbinii 33-34 validate this organism as a 

suitable subject for ecotoxicological investigation on various geographical scales 21-22-35 . It should be noted 

that our research group is involved in further molecular studies to examine populations of O. garbinii and 

G. aequicauda sampled in other geographical areas, analysing especially the control region of mtDNA 

which generally presents hypervariable portions 36 and non-coding mitochondrial segments 14,37,38 . 

Furthermore, studies into genotoxicology 39 and mutagenesis (Fig. 7) are underway on organisms exposed 

in the laboratory. 

Figure 7 - Nucleotidic mutation in the COI gene highlighted by sequencing of mtDNA. (Davolos, study pending).The 

chromatograms are visualized with Chromas software version 1.41 

870 880 890 900 910 

950 960 970 980

4. CONCLUSIONS 

Due to their biological characteristics and the ease with which they may be grown in the laboratory 22,40 , 

the O. garbinii and G. aequicauda species analyzed here are particularly suited to experiments on 

genotoxicological exposures. 

In literature, we can find various molecular protocols that are used to assess the levels of genotoxicity 18,31,41 . 

Among these, the electrophoretic analysis of DNA samples in agarose gel is useful for the identification and 

quantification of the kind of damage caused at DNA level 39,42-45 . In addition, using in vivo and in vitro methods 

followed in DNA sequencing analysis, it is possible to identify any nucleotidic mutations induced 21,22,45 . 

Nevertheless, the information on the genetic structure of the populations examined is absolutely 

necessary for the interpretation of the results of environmental-genotoxicological studies 15-22 . 

REFERENCES 

1. Italia. Decreto Legislativo 11 maggio 1999, n. 152.Testo aggiornato del decreto legislativo 11 maggio 

1999, n. 152, recante:“Disposizioni sulla tutela delle acque dall’inquinamento e recepimento della 

direttiva 91/271/CEE concernente il trattamento delle acque reflue urbane e della direttiva 

91/676/CEE relativa alla protezione delle acque dall’inquinamento provocato dai nitrati provenienti da 

fonti agricole”, a seguito delle disposizioni correttive ed integrative di cui al decreto legislativo18 

agosto 2000, n. 258”. Gazzetta Ufficiale n. 246, Supplemento Ordinario n. 172, 20 ottobre 2000. 

2. Unione Europea. Direttiva 2000/60/CE del Parlamento Europeo e del Consiglio del 23 ottobre 2000 

che istituisce un quadro per l’azione comunitaria in materia di acque. Gazzetta ufficiale delle Comunità 

europee L327/1-72, 22 dicembre 2000. 

3. Schulz R. Using a freshwater amphipod in situ bioassay as a sensitive tool to detect pesticide effects in 

the field. Environ Toxicol Chem 2003;22:1172-6. 

4. Schill RO, Kohler HR. Does the environment or the source of the population define stress status and 

energy supply in the freshwater amphipod Gammarus fossarum? Ecotoxicology 2004;13:683-95. 

5. Borgmann U, Couillard Y, Doyle P, Dixon DG.Toxicity of sixty-three metals and metalloids to Hyalella 

azteca at two levels of water hardness. Environ Toxicol Chem 2005;24:641-52. 

6. Meyran JC, Monnerot M,Taberlet P.Taxonomic status and phylogenetic relationships of some species 

of the genus Gammarus (Amphipoda) from mtDNA sequences. Mol Phylogenet Evol 1997;8:1-10. 

7. Meyran JC, Gielly L,Taberlet P. Environmental Ca and mtDNA polymorphism among populations of 

Gammarus fossarum (Amphipoda). Mol Ecol 1998;7:1391-400. 

8. Meyran JC, Taberlet P. mtDNA polymorphism among alpine populations of Gammarus lacustris 

(Amphipoda). Freshw Biol 1998;39:259-65. 

9. Müller J. Mitochondrial DNA variation and the evolutionary history of cryptic Gammarus fossarum 

types. Mol Phylogenet Evol 2000;15:260-8. 

10. De Matthaeis E, Davolos D, Cobolli M 1998. Genetic divergence between populations and species of 

talitrids from Aegean islands. J Hered 2000;89:37-43. 

11. De Matthaeis E, Davolos D, Cobolli M, Ketmaier V. Isolation by distance in equilibrium and non 

equilibrium populations of four talitrid amphipod species in the Mediterranean sea. Evolution 

2000;54:1606-13. 

12. Davolos D, Ketmaier V, Cobolli M, De Matthaeis E. Struttura genetica e livelli di differenziamento tra 

popolazioni e specie di Orchestia (Amphipoda, Talitridae) del Mediterraneo. Biogeographia 

2002;23:23-34. 


75


76 

13. Iannilli V, Ketmaier V, Ruffo S, De Matthaeis E. Multidisciplinary approach to the systematics of the Italian 

freshwater Gammarus (Amphipoda). In: Proceedings of the 4th European Crustacean Conference. 

Lodz (Poland); 2002.Abstracts, p. 46. 

14. Davolos D, Maclean N. Mitochondrial COI-NC-COII sequences of talitrid amphipods (Crustacea). 

Heredity 2005;94:81-6. 

15. Hogg ID, Larose C, de Lafontaine Y, Doe KG. Genetic evidence for a Hyalella species complex within 

the Great Lakes - St. Lawrence River drainage basin: implications for ecotoxicology and conservation 

biology. Can J Zool 1998;76:1134-40. 

16. Stanton JL, Schizas NV, Chandler GT, Coull BC, Quattro JM. Ecotoxicology and population genetics: the 

emergence of phylogepgraphic and evolutionary ecotoxicology. Ecotoxicology 2001;10:217-22. 

17. Theodorakis CW. Integration of genotoxic and population genetic endpoints in biomonitoring and 

risk assessment. Ecotoxicology 2001;10:245-56. 

18. Perkins EJ, Lotufo GR. Playing in the mud-using gene expression to assess contaminant effects on 

sediment dwelling invertebrates. Ecotoxicology 2003;12:453-6. 

19. Whitehead A, Anderson SL, Kuivila KM, Roach JL, May B. Genetic variation among interconnected 

populations of Catostomus occidentalis: implications for distinguishing impacts of contaminants from 

biogeographical structuring. Mol Ecol 2003;12:2817-33. 

20. Snape JR, Maund SJ, Pickford DB, Hutchinson TH. Ecotoxicogenomics: the challenge of integrating 

genomics into aquatic and terrestrial ecotoxicology.Aquat Toxicol 2004;67:143-54. 

21. Davolos D, Pietrangeli B, Maclean N. Mitochondrial DNA sequence analysis and molecular phylogeny 

of Orchestia cavimana (Crustacea) in freshwater genotoxicological studies. In: Proceedings of the 34th 

Annual Meeting of the European Environmental Mutagen society (EEMS 2004). Maastricht, the 

Netherlands: University of Maastricht; 2004.Abstracts PW2016. 

22. Davolos D, Maclean N, Pietrangeli B. A molecular phylogenetic study of the freshwater Orchestia 

cavimana (Crustacea). Riv Biol 2004;97:161-8. 

23. Capaldi RA.The complexity of a respiratory complex. Nature Struct Biol 1996;3:570-4. 

24. Saraste M. Oxidative Phosphorylation at the fin de siècle. Science 1999;283:1488-93. 

25. Tsukihara T,Aoyama H,Yamashita E,Tomizaki T,Yamaguchi H, Shinzawa-Itoh, et al. Structures of metal 

sites of oxidized bovine heart cytochrome c oxidase at 2.8 Å. Science 1995;269:1069-74. 

26. Davolos D, Maclean N. Evolutionary relationships among supralittoral talitrid amphipods (Crustacea) 

inferred from mitochondrial sequence analysis. In: Proceedings of the “XXI Giornata dell’Ambiente. 

Aree costiere”. Roma, Italy:Accademia Nazionale dei Lincei. [2004];205:233-42. 

27. Müller JC, Schramm S, Seitz A. Genetic and morphological differentiation of Dikerogammarus invaders 

and their invasion history in Central Europe. Fresh Biol 2002;47:2039-48. 

28. Macdonald KS III,Yampolsky L Duffy JE. Molecular and morphological evolution of the amphipod 

radiation of Lake Baikal. Mol Phylogenet Evol 2005;35:323-43. 

29. Iannilli V, De Matthaeis E. Genetic divergence within Gammarus aequicauda (Martynov, 1931) based 

on 16S mitochondrial DNA. In: Proceedings of The Sixth International Crustacean Congress. 

Glasgow, Scotland UK: University of Glasgow; 2005. 

30. Wurgler FE, Kramers PG. Environmental effects of genotoxins (eco-genotoxicology). Mutagenesis 

1992;7:321-7. 

31. Jha AN. Genotoxicological studies in aquatic organisms: an overview. Mutat Res 2004;552:1-17. 

32. Sato H, Aoki Y. Mutagenesis by environmental pollutants and bio-monitoring of environmental 

mutagens. Curr Drug Metab 2002;3:311-9.

33. De Matthaeis E, Ketmaier V, Latella L, Ruffo S, Scapini F,Tarocco M. Multidisciplinary approach to the 

study of a terrestrial Amphipod: the case of Orchestia cavimana. In: Proceedings of the IV European 

Crustacean Conference. Lodz, Poland; 2002.Abstracts 36. 

34. Ketmaier V,Amendola D, Scapini F, De Matthaeis E. Large scale phylogeography of the landhopper 

Orchestia cavimana: combining allozymes and mtDNA. In: Proceedings of the XIth International 

Colloquium on Amphipoda.Tunis; 2003. 

35. Fialkowski W, Rainbow PS, Smith BD, Zmudzinski L. Seasonal variation in trace metal concentrations in 

three talitrid amphipods from the Gulf of Gdansk, Poland. J Exp Mar Biol Ecol 2003;288:81-93. 

36. Chu KH, Li CP,Tam YK, Lavery S.Application of mitochondrial control region in population genetic 

studies of the shrimp Penaeus. Mol Ecol Notes 2003;3:120-2. 

37. Davolos D, Iannilli V, De Matthaeis E.Analysis on the mitochondrial region between the COI and COII 

genes in Crustacea. In: Proceedings of The Sixth International Crustacean Congress. Glasgow, 

Scotland UK: University of Glasgow; 2005. 

38. Davolos D, Iannilli V, De Matthaeis E. Molecular analysis on the mitochondrial COI-tRNALeuUUR- 

COII region in Gammarus aequicauda (Amphipoda, Gammaridae). In: Proceedings of the First 

Congress of Italian Evolutionary Biologists. Ferrara, Italy: University of Ferrara; 2005. 

39. Setini A, Iannilli V. DNA strand breakage come biomarker di esposizione ai metalli. In: Proceedings of 

the 66th Annual Congress of the U.Z.I. 19-22 Sept 2005. Roma, Italy: 2005. 

40. Hecker A, Quennedey B,Testeniere O, Quennedey A, Graf F, Luquet G. Orchestin, a calcium-binding 

phosphoprotein, is a matrix component of two successive transitory calcified biomineralizations 

elaborated by a terrestrial crustacean. J Struct Biol 2004;146:310-24. 

41. Lee RF, Steinert S. Use of the single cell gel electrophoresis/comet assay for detecting DNA damage in 

aquatic (marine and freshwater) animals. Mutat Res 2003;544:43-64. 

42. Costa FO, Neuparth T, Costa MH,Theodorakis CW, Shugart LR. Detection of DNA strand breakage 

in a marine amphipod by agarose gel electrophoresis: exposure to X-rays and copper. Biomarkers 

2002;7:451-63. 

43. Dixon DR, Pruski AM, Dixon LR, Jha AN. Marine invertebrate eco-genotoxicology: a methodological 

overview. Mutagenesis 2002;17:495-507. 

44. Park S, Imlay JA. High levels of intracellular cysteine promote oxidative DNA damage by driving the 

fenton reaction. J Bacteriol 2003;185:1942-50. 

45. Braman J (ed.). In Vitro Mutagenesis Protocols. 2nd Edn.Totowa NJ: Humana Press; 2002. 


77

Molecular phylogenetic analysis in ... - Prevenzione Oggi

You also want an ePaper? Increase the reach of your titles

Delete template?

Save as template?