Abstract
Despite the biological and economic importance of earthworms, the taxonomic status and evolutionary relationships of most lumbricid genera are still under debate. Further complications arise from the recognition that earthworms also show a high cryptic diversity. Past and current field studies of Iranian earthworm fauna have resulted in the identification of a total number of 28 earthworm species. However, many specimens do not fully fit into their original descriptions, making the species assignation very difficult. In this study, we evaluated the genetic diversity using mitochondrial markers as a tool to assess the species occurrence of some problematic species in Iran. Four species with high morphological variation were selected: Aporrectodea caliginosa (Savigny, 1826), Aporrectodea trapezoides (Dugès, 1828), Dendrobaena byblica (Rosa, 1893) and Dendrobaena veneta (Rosa, 1886). Morphological identification was contrasted with the molecular information generated through COI and 16S barcoding and the COI and 16S sequences stored in the Genbank. The results of this first integrative taxonomic analysis revealed that D. veneta consisted of two separated clades and that a number of species assigned to D. byblica showed very close relationships with those belonging to the genus Philomontanus. The lack of taxonomic expertise and identification characters providing a clear and unambiguous identification of earthworms highlights the urgent need of new tools to identify species unequivocally. Therefore, it is concluded that more taxonomical studies are needed to clarify the diagnostic characters and taxonomic status of the species belonging to two genera, Aporrectodea and Dendrobaena (Lumbricidae), in Iran.
References
Altschul, S.F., Madden, T.L., Schäffer, A.A., Zhang, J., Zhang, Z., Miller, W. & Lipman, D.J. (1997) Gapped BLASTvand PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Research, 25, 3389–3402.
https://doi.org/10.1093/nar/25.17.3389
Benson, D.A., Karsch-Mizrachi, I., Clark, K., Lipman, D.J., Ostell, J. & Sayers, E.W. (2012) Genbank. Nucleic Acids Research, 40 (D1), D36–D42.
https://doi.org/10.1093/nar/gkr1202
Blouin, M., Hodson, M.E., Delgado, E.A., Baker, G., Brussaard, L., Butt, K.R., Dai, J., Dendooven, L., Peres, G., Tondoh, J.E., Cluzeau, D. & Brun, J.J. (2013) A review of earthworm impact on soil function and ecosystem services. European Journal of Soil Science, 62 (4), 161–182.
https://doi.org/10.1111/ejss.12025
Bouché, M.B. (1972) Lombriciens de France: Écologie et Systématique. Institut national de la Recherche Agronomique—INRA, Paris, 671 pp.
Bozorgi, F., Seiedy, M., Malek, M., Aira, M., Pérez-Losada, M. & Domínguez, J. (2019) Multigene phylogeny reveals a new Iranian earthworm genus (Lumbricidae: Philomontanus) with three new species. PLOS ONE, 14 (1), e0208904.
https://doi.org/10.1371/journal.pone.0208904
Chang, C.H. & Chen, J.H. (2005) Taxonomic status and intraspecific phylogeography of two sibling species of Metaphire (Oligochaeta: Megascolecidae) in Taiwan. Pedobiologia, 49, 591–600.
https://doi.org/10.1016/j.pedobi.2005.07.002
Csuzdi, Cs. & Zicsi, A. (2003) Earthworms of Hungary (Annelida: Oligochaeta; Lumbricidae). In: Csuzdi, Cs. & Mahunka, S. (Eds.), Pedozoologica Hungarica. Vol. 1. Hungarian Natural History Museum, Budapest, 271 pp.
Csuzdi, Cs., Pop, A.A., Pop, VV., Zicsi, A. & Wink, M. (2005) Revision of the Dendrobaena alpina (Rosa, 1884) species group (Oligochaeta, Lumbricidae) by morphological and molecular methods. In: Pop, V.V. & Pop, A.A. (Eds.), Advances in earthworm taxonomy II. University Press Cluj, Cluj-Napoca, pp. 119–128.
Csuzdi, Cs. (2012) Earthworm species, a searchable database. Opuscula Zoologica, Budapest, 43 (1), 97–99.
Csuzdi, Cs., Chang, CH., Pavlíček, T., Szederjesi, T., Esopi, D. & Szlávecz, K. (2017) Molecular phylogeny and systematics of native North American lumbricid earthworms (Clitellata: Megadrili). PLoS ONE, 12 (8), e0181504.
https://doi.org/10.1371/journal.pone.0181504
Edgar, R.C. (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Research, 32 (5), 1792–1797.
https://doi.org/10.1093/nar/gkh340
Fernández, R., Almodóvar, A., Novo, M., Gutiérrez, M. & Díaz Cosín, D.J. (2011) A vagrant clone in a peregrine species: Phylogeography, high clonal diversity and geographical distribution in the earthworm Aporrectodea trapezoides (Dugès, 1828). Soil Biology and Biochemistry, 43 (10), 2085–2093.
https://doi.org/10.1016/j.soilbio.2011.06.007Fernández, R., Almodóvar, A., Novo, M., Simancas, B. & Díaz Cosín, D.J. (2012) Adding complexity to the complex: new insights into the phylogeny, diversification and origin of parthenogenesis in the Aporrectodea caliginosa species complex (Oligochaeta, Lumbricidae). Molecular Phylogenetics and Evolution, 64 (2), 368–379.
https://doi.org/10.1016/j.ympev.2012.04.011
Fernández, R., Almodóvar, A., Novo, M., Gutiérrez, M. & Díaz Cosín, D.J. (2013) Earthworms, good indicators for palaeogeographical studies? Testing the genetic structure and demographic history in the peregrine earthworm Aporrectodea trapezoides (Dugès, 1828) in southern Europe. Soil Biology and Biochemistry, 58, 127–135. https://doi.org/10.1016/j.soilbio.2012.10.021
Hall, T.A. (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. In Nucleic Acids Symposium series, 41, 95–98.
Hebert, P.D.N., Cywinska, A., Ball, S.L. & deWaard, J.R. (2003) Biological identifications through DNA barcodes. Proceedings of the Royal Society of London, Series B, Biological Sciences, 270, 313–321.
https://doi.org/10.1098/rspb.2002.2218
Heethoff, M., Etzold, K. & Scheu, S. (2004) Mitochondrial COII sequences indicate that the parthenogenetic earthworm Octolasion tyrtaeum (Savigny 1826) constitutes of two lineages differing in body size and genotype. Pedobiologia, 48 (1), 9–13. https://doi.org/10.1016/j.pedobi.2003.04.001
Huang, J., Xu, Q., Sun, Z.J., Tang, G.L. & Su, Z.Y. (2007) Identifying earthworms through DNA barcodes. Pedobiologia, 51, 301–309.
https://doi.org/10.1016/j.pedobi.2007.05.003James, S., Porco, D., Decaëns, T., Richard, B., Rougerie, R. & Erséus, C. (2010) DNA barcoding reveals cryptic diversity in Lumbricus terrestris L., 1758 (Clitellata): resurrection of L. herculeus (Savigny, 1826). PLoS One, 5 (12), e15629.
https://doi.org/10.1371/journal.pone.0015629
King, R.A., Tibble, A.L. & Symondson, W.O.C. (2008) Opening a can of worms: unprecedented sympatric cryptic diversity within British lumbricid earthworms. Molecular Ecology, 17, 4684–4698.
https://doi.org/10.1111/j.1365-294X.2008.03931.xKing, R.A., Tibble, A.L. & Symondson, W.O.C. (2008) Opening a can of worms: unprecedented sympatric cryptic diversity within British lumbricid earthworms. Molecular Ecology, 17, 4684–4698.
https://doi.org/10.1111/j.1365-294X.2008.03931.xLavelle, P., Decaëns, T., Aubertb M., Barot, S., Blouin, M., Bureau, F., Margerie, P., Mora, P. & Rossi, J.P. (2006) Soil invertebrates and ecosystem services. European Journal of Soil Biology, 42, 3–15.
https://doi.org/10.1016/j.ejsobi.2006.10.002Marchán, D.F., Fernández, R., Domínguez, J. & Díaz Cosin, D.J. (2020) Genome-informed integrative taxonomic description of three cryptic species in the earthworm genus Carpetania (Oligochaeta, Hormogastridae). Systematics and Biodiversity, 18 (3), 203–215.
https://doi.org/10.1080/14772000.2020.1730474
Novo, M., Almodóvar, A. & Díaz Cosín, D.J. (2009) High genetic divergence of hormogastrid earthworms (Annelida, Oligochaeta) in the central Iberian Peninsula: evolutionary and demographic implications. Zoologica Scripta, 38 (5), 537–552. https://doi.org/10.1111/j.1463-6409.2009.00389.x
Novo, M., Almodóvar, A., Fernández, R., Trigo, D. & Díaz Cosín, D.J. (2010) Cryptic speciation of hormogastrid earthworms revealed by mitochondrial and nuclear data. Molecular Phylogenetics and Evolution, 56, 507–512.
https://doi.org/10.1016/j.ympev.2010.04.010Nylander, J.A.A. (2004) MrModeltest. Version 2. Program Distributed by the Author. Evolutionary Biology Centre, Uppsala University, Uppsala.
Pérez Onteniente, A. & Rodríguez Babío, C. (2002) Three new species of earthworm, Annelida: Oligochaeta: Lumbricidae, from the Valencian Community, Spain. Journal of Natural History, 36, 515–530.
https://doi.org/10.1080/00222930010020252
Pérez-Losada, M., Ricoy, M., Marshall, J.C., Domínguez, J. (2009) Phylogenetic assessment of the earthworm Aporrectodea caliginosa species complex (Oligochaeta: Lumbricidae) based on mitochondrial and nuclear DNA sequences. Molecular Phylogenetics and Evolution, 52 (2), 293–302.
https://doi.org/10.1016/j.ympev.2009.04.003Perel, T.S. (1997) The earthworms of the fauna of Russia: cadaster and key. Nauka, Moscow, 102 pp. [in Russian]
Posada, D. & Buckley, T.R. (2004) Model Selection and Model Averaging in Phylogenetics: Advantages of Akaike Information Criterion and Bayesian Approaches Over Likelihood Ratio Tests. Systematic Biology, 53 (5), 793–808.
https://doi.org/10.1080/10635150490522304Rambaut, A. (2012) FigTree version 1.4.0, A Graphical Viewer of Phylogenetic Trees. Available from http://tree.bio.ed.ac.uk/software/figtree/ (accessed 5 December 2012)
Ronquist, F., Teslenko, M., van der Mark, P., Ayres, D.L., Darling, A., Höhna, S., Larget, B., Liu, L., Suchard, M.A. & Huelsenbeck, J.P. (2012) MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Systematic Biology, 61 (3), 539–542.
https://doi.org/10.1093/sysbio/sys029
Rougerie, R., Decaëns, T., Deharveng, L., Porco, D., James, S.W., Chang, C. H., Richard, B., Potapov,M., Suhardjono,Y. & Hebert, P.D.N. (2009) DNA barcodes for soil animal taxonomy. Pesquisa Agropecuaria Brasileira, 44, 789–801.
https://doi.org/10.1590/S0100-204X2009000800002
Sambrook, J., Fritsch, E.F. & Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual. 2nd Edition. Cold Spring Harbor Press, Cold Spring Harbor, New York, 412 pp.
Szederjesi, T., Pop, V.V., Pavlíček, T., Márton, O., Krízsik, V. & Csuzdi, Cs. (2018) Integrated taxonomy reveals multiple species in the Dendrobaena byblica (Rosa, 1893) complex (Oligochaeta: Lumbricidae). Zoological Journal of the Linnean Society, 182 (3), 500–516.
https://doi.org/10.1093/zoolinnean/zlx049Szederjesi, T., Pavlíček, T., Márton, O., Krízsik, V. & Csuzdi, Cs. (2019) Integrative taxonomic revision of Dendrobaena veneta (Rosa, 1886) sensu lato with description of a new species and resurrection of Dendrobaena succinta (Rosa, 1905) (Megadrili: Lumbricidae). Journal of Natural History, 53, 301–314.
https://doi.org/10.1080/00222933.2019.1593537
Sims, R.W. & Gerard, B.M. (1999) Earthworms: Synopses of the British Fauna (New Series). No. 31. Earthworms. Linnean Society of London and the Estuarine and Coastal Sciences Association, London, 52 line drawings.
Taheri, S., James, S., Roy,V., Decaëns, T., Williams, B.W., Anderson, F., Rougerie, R., Chang, C.-H., Brown, G.G., Cunha, L., Stanton, D.W.G., Silva, E. Da, Chen, J.-H., Lemmon, A., Bartz, M., Baretta, D., Barois, I., Lapied, E., Coulis, M. & Dupont, L. (2018) Complex taxonomy of the“brush tail”peregrine earthworm Pontoscolex corethrurus. Molecular Phylogenetics and Evolution, 124, 60–70.
https://doi.org/10.1016/j.ympev.2018.02.021
Tamura, K., Stecher, G., Peterson, D., Filipski, A. & Kumar, S. (2013) MEGA6: molecular evolutionary genetics analysis version 6.0. Molecular biology and evolution, 30 (12), 2725–2729.
https://doi.org/10.1093/molbev/mst197
Trifinopoulos, J., Nguyen, L.T., von Haeseler, A. & Minh, B.Q. (2016) W-IQ-TREE: a fast online phylogenetic tool for maximum likelihood analysis. Nucleic Acids Research, 44 (W1), W232–W235.