Advertisement

Organisms Diversity & Evolution

, Volume 18, Issue 2, pp 187–210 | Cite as

Rolling into the deep of the land planarian genus Choeradoplana (Tricladida, Continenticola, Geoplanidae) taxonomy

  • Fernando Carbayo
  • Marcos Santos Silva
  • Marta Riutort
  • Marta Álvarez-Presas
Original Article

Abstract

The land planarian genus Choeradoplana (Plathelminthes, Tricladida) is currently integrated by 13 species. In previous works, morphological variation in its type species, Choeradoplana iheringi, was reported, but no attempt to test whether it is just a single species has been made yet. In order to disentangle the taxonomy of this species and further members of the genus, we sampled new specimens and combined morphological and molecular data and also have evaluated the performance of diverse methods of molecular species delimitation. Our data point to the presence of two cryptic species named C. iheringi, plus two new species, all hidden under the same general appearance. An in-depth morphological study of the specimens allowed detection of diagnostic morphological traits in each species, for which we also propose a molecular diagnosis. This integrative taxonomic study demonstrates once again the usefulness of molecular tools to weigh minor morphological characteristics and thus reveal the existence of species that would otherwise remain cryptic. However, under certain parameters, the molecular methods may over-split species with a high genetic structure, maybe pointing to incipient speciation. This makes critical the use of these methods combined with a comprehensive morphological approach. We also present a comprehensive phylogenetic tree including most Choeradoplana species. The tree, well supported, allows making some preliminary inferences on the evolution of the group and its historical biogeography.

Keywords

ABGD bPTP BP&P Cryptic species Flatworms GMYC Histology Geoplaninae 

Abbreviations

cm

common muscular coat

CMI

cutaneous musculature thickness relative to body height at the pre-pharyngeal region

co

common glandular ovovitelline duct

dd

dorso-diagonal parenchymal muscles

e

eye

ES

State of Espírito Santo, Brazil

es

esophagus

f

fold

fa

female genital atrium

fg

female genital canal

g

gonopore

g1–g4

glandular sections of the prostatic vesicle

gl

glands

i

intestine

ls

normal longitudinal cutaneous muscles

m

muscle fiber

ma

male genital atrium

MG

State of Minas Gerais, Brazil

mo

mouth

MZU

Museu de Zoologia da Universidade do Vale do Rio dos Sinos

MZUSP

Museu de Zoologia da Universidade de São Paulo

ng

nervous ganglia

NHM

Natural History Museum Vienna

ov

ovoviteline duct

pg

sponge-like mass

ph

pharyngeal pocket

PR

State of Paraná, Brazil

pv

prostatic vesicle

RJ

State of Rio de Janeiro, Brazil

RS

State of Rio Grande do Sul, Brazil

SC

State of Santa Catarina, Brazil

sd

sperm duct

sg

shell glands

sk

sunken longitudinal cutaneous muscles

SMF

Senckenberg Museum Frankfurt

SP

State of São Paulo, Brazil

vi

vitellaria

vn

ventral nerve plate

Notes

Acknowledgements

We are grateful to the Instituto Chico Mendes de Conservação da Biodiversidade (ICMBio), Instituto Estadual do Ambiente do Governo do Rio de Janeiro (INEA), and Fundação de Amparo e Tecnologia do Meio Ambiente de Santa Catarina (FATMA) for licensing the fieldwork and Nivaldo Gularte and Juan R. E. García (Paulo Lopes, SC) for their kind field support and generous hospitality. We thank Fernanda Megiolaro for the picture of C. iheringi photographed in Parobé. We also thank Cláudia Olivares, Débora Redivo, Júlio Pedroni, Leonardo Zerbone, Marília Jucá, and Welton Araújo for the sampling help. Thanks to Geison Castro and Lucas Beltrami (EACH) for the histological processing. Dr. Dieter Fiege (SNM, Frankfurt) and Dr. Helmut Sattman (NHM, Vienna) are thanked for their kind assistance to FC during the museum visits. Ana Vasques (MZUSP) is thanked for the loan of specimens deposited in MZUSP. Sergio Vanin (MZUSP) is acknowledged for his advice on the zoological nomenclature. Two anonymous reviewers are kindly thanked for their valuable comments and suggestions. FC has financial support from FAPESP (proc. 2016/18295-5). M. R. and M. Á.-P. were supported by grant CGL2011-23466 of the Ministerio de Economía y Competitividad, Spain. M. Á.-P. acknowledges support from SEG, the Sociedad Española de Genética travel grant.

Supplementary material

13127_2017_352_Fig11_ESM.gif (117 kb)
Supplementary Fig. 1

GMYC results represented on the ultrametric tree inferred with BEAST using the Cox1Del dataset. Orange vertical bar represents the threshold separating speciation and coalescence processes. (GIF 116 kb)

13127_2017_352_MOESM1_ESM.tif (50.9 mb)
High resolution image (TIFF 52075 kb)
13127_2017_352_Fig12_ESM.gif (131 kb)
Supplementary Fig. 2

DNA bar coding gap analysis of Cox1 gene sequence based on K2P distances. (GIF 131 kb)

13127_2017_352_MOESM2_ESM.tiff (1.4 mb)
High resolution image (TIFF 1392 kb)
13127_2017_352_Fig13_ESM.gif (99 kb)
Supplementary Fig. 3

BP&P results represented on the species tree inferred with *BEAST using the 5 datasets (18S, 28S, Cox1, EF and ITS). Numbers above nodes correspond to the PP values obtained in the first BP&P analysis (M1) and the numbers below the node correspond to the PP results of the second BP&P analysis (M2). The asterisks within the nodes denote the presence of a speciation event in BP&P analysis. The scale bar represents substitutions per site. (GIF 99 kb)

13127_2017_352_MOESM3_ESM.tif (77.8 mb)
High resolution image (TIFF 79621 kb)
13127_2017_352_MOESM4_ESM.docx (19 kb)
Supplementary Table 1 (DOCX 18 kb)
13127_2017_352_MOESM4_ESM.zip (10.3 mb)
Supplementary Figures 1-10 (ZIP 4.78 mb)

References

  1. Álvarez-Presas, M., & Riutort, M. (2014). Planarian (Platyhelminthes, Tricladida) diversity and molecular markers: a new view of an old group. Diversity, 6, 323–338.CrossRefGoogle Scholar
  2. Álvarez-Presas, M., Baguñà, J., & Riutort, M. (2008). Molecular phylogeny of land and freshwater planarians (Tricladida, Platyhelminthes): from freshwater to land and back. Molecular Phylogenetics and Evolution, 47, 555–568.CrossRefPubMedGoogle Scholar
  3. Álvarez-Presas, M., Carbayo, F., Rozas, J., & Riutort, M. (2011). Land planarians (Platyhelminthes) as a model organism for fine-scale phylogeographic studies: understanding patterns of biodiversity in the Brazilian Atlantic forest hotspot. Journal of Evolutionary Biology, 24, 887–896.CrossRefPubMedGoogle Scholar
  4. Álvarez-Presas, M., Sánchez-Gracia, A., Carbayo, F., Rozas, J., & Riutort, M. (2014). Insights into the origin and distribution of biodiversity in the Brazilian Atlantic forest hot spot: a statistical phylogeographic study using a low-dispersal organism. Heredity, 112, 656–665.CrossRefPubMedPubMedCentralGoogle Scholar
  5. Álvarez-Presas, M., Amaral, S. V., Carbayo, F., Leal-Zanchet, A. M., & Riutort, M. (2015). Focus on the details: morphological evidence supports new cryptic land flatworm (Platyhelminthes) species revealed with molecules. Organisms Diversity & Evolution, 15, 379–403.CrossRefGoogle Scholar
  6. Bálint, M., Domisch, S., Engelhardt, C. H. M., Haase, P., Lehrian, S., Sauer, J., Theissinger, K., Pauls, S. U., & Nowak, C. (2011). Cryptic biodiversity loss linked to global climate change. Nature Climate Change, 1, 313–318.CrossRefGoogle Scholar
  7. Ball, I. A. (1981). The phyletic status of the Paludicola. Hydrobiologia, 84, 7–12.CrossRefGoogle Scholar
  8. Batalha-Filho, H., & Miyaki, C. Y. (2016). Late Pleistocene divergence and postglacial expansion in the Brazilian Atlantic forest: multilocus phylogeography of Rhopias gularis (Aves: Passeriformes). Journal of Zoological Systematics and Evolutionary Research, 54(2), 137–147.CrossRefGoogle Scholar
  9. Bickford, D., Lohman, D. J., Sodhi, N. S., Ng, P. K. L., Meier, R., Winker, K., Ingram, K. K., & Das, I. (2007). Cryptic species as a window on diversity and conservation. Trends in Ecology & Evolution, 22, 148–155.CrossRefGoogle Scholar
  10. Bois-Reymond, M. (1951). On South American geoplanids. Boletim da Faculdade de Filosofia, Ciências e Letras da Universidade de São Paulo, Série Zoologia, 16, 217–255.Google Scholar
  11. Carbayo, F., & Almeida, A. L. (2015). Anatomical deviation of male organs of land planarians from Rio de Janeiro, Brazil, with description of two new species of Cratera (Platyhelminthes, Tricladida). Zootaxa, 3931, 27–40.CrossRefPubMedGoogle Scholar
  12. Carbayo, F., & Froehlich, E. M. (2012). Three new Brazilian species of the land planarian Choeradoplana (Platyhelminthes: Tricladida: Geoplaninae), and an emendation of the genus. Journal of Natural History, 46(19–20), 1153–1177.CrossRefGoogle Scholar
  13. Carbayo, F., & Leal-Zanchet, A. M. (2003). Two new genera of geoplaninid land planarians (Platyhelminthes: Tricladida: Terricola) of Brazil in the light of cephalic specialisations. Invertebrate Systematics, 17, 449–468.CrossRefGoogle Scholar
  14. Carbayo, F., Leal-Zanchet, A. M., & Vieira, E. M. (2002). Terrestrial flatworm (Platyhelminthes: Tricladida: Terricola) diversity versus man-induced disturbance in an ombrophilous forest in southern Brazil. Biodiversity and Conservation, 11, 1091–1104.CrossRefGoogle Scholar
  15. Carbayo, F., Álvarez-Presas, M., Olivares, C. T., Marques, F. P. L., Froehlich, E. M., & Riutort, M. (2013). Molecular phylogeny of Geoplaninae (Platyhelminthes) challenges current classification: proposal of taxonomic actions. Zoologica Scripta, 42, 508–528.CrossRefGoogle Scholar
  16. Carbayo, F., Álvarez-Presas, M., Jones, H. D., & Riutort, M. (2016). The true identity of Obama (Platyhelminthes: Geoplanidae) flatworm spreading across Europe. Zoological Journal of the Linnean Society, 177, 5–28.CrossRefGoogle Scholar
  17. Carnaval, A. C., Hickerson, M. J., Haddad, C. F. B., Rodrigues, M. T., & Moritz, C. (2009). Stability predicts genetic diversity in the Brazilian Atlantic forest hotspot. Science, 323, 785–789.CrossRefPubMedGoogle Scholar
  18. Carranza, S., Ruiz-Trillo, I., Littlewood, D. T. J., Riutort, M., & Baguñà, J. (1998). A reappraisal of the phylogenetic and taxonomic position of land planarians (Platyhelminthes, Turbellaria, Tricladida) inferred from 18S rDNA sequences. Pedobiologia, 42, 433–440.Google Scholar
  19. Carranza, J., Salinas, M., de Andrés, D., & Pérez-González, J. (2016). Iberian red deer: paraphyletic nature at mtDNA but nuclear markers support its genetic identity. Ecology and Evolution, 6(4), 905–922.CrossRefPubMedPubMedCentralGoogle Scholar
  20. Cason, J. E. (1950). A rapid one-step Mallory-Heidenhain stain for connective tissue. Stain Technology, 25(4), 225–226.CrossRefPubMedGoogle Scholar
  21. Darriba, D., Taboada, G. L., Doallo, R., & Posada, D. (2012). jModelTest 2: more models, new heuristics and parallel computing. Nature Methods, 9, 772.CrossRefPubMedPubMedCentralGoogle Scholar
  22. Drummond, A. J., & Rambaut, A. (2007). BEAST: Bayesian Evolutionary Analysis by Sampling Trees. BMC Evolutionary Biology, 7, 214.CrossRefPubMedPubMedCentralGoogle Scholar
  23. Drummond, A. J., Suchard, M. A., Xie, D., & Rambaut, A. (2012). Bayesian phylogenetics with BEAUti and the BEAST 1.7. Molecular Biology and Evolution, 29, 1969–1973.CrossRefPubMedPubMedCentralGoogle Scholar
  24. Felsenstein, J. (1985). Phylogenies and the comparative method. American Naturalist, 125, 1–15.CrossRefGoogle Scholar
  25. Flot, J. F., Blanchot, J., Charpy, L., Cruaud, C., Licuanan, W. Y., Nakano, Y., Payri, C., & Tillier, S. (2011). Incongruence between morphotypes and genetically delimited species in the coral genus Stylophora: phenotypic plasticity, morphological convergence, morphological stasis or interspecific hybridization? BMC Ecology, 11(22), 925–931.Google Scholar
  26. Fontaneto, D., Flot, J.-F., & Tang, C. Q. (2015). Guidelines for DNA taxonomy, with a focus on the meiofauna. Marine Biodiversity, 45(3), 433–451.CrossRefGoogle Scholar
  27. Froehlich, C. G. (1955a). On the biology of land planarians. Boletim da Faculdade de Filosofia, Ciências e Letras da Universidade de São Paulo, Série Zoologia, 20, 263–271.CrossRefGoogle Scholar
  28. Froehlich, C. G. (1955b). Sobre morfologia e taxonomia das Geoplanidae. Boletim da Faculdade de Filosofia, Ciências e Letras da Universidade de São Paulo, Série Zoologia, 19, 195–279.CrossRefGoogle Scholar
  29. Froehlich, C. G. (1956). Planárias terrestres do Paraná. Dusenia, 7(4), 173–196.Google Scholar
  30. Froehlich, E. M. (1978). On a collection of Chilean land planarians. Boletim de Zoologia, Universidade de São Paulo, 3, 7–80.Google Scholar
  31. Fujisawa, T., & Barraclough, T. G. (2013). Delimiting species using single-locus data and the generalized mixed yule coalescent approach: a revised method and evaluation on simulated data sets. Systematic Biology, 62, 707–724.CrossRefPubMedPubMedCentralGoogle Scholar
  32. Graff, L.V. (1894). Viaggio del Dott. A. Borelli nella Republica Argentina e nel Paraguay. V. Landplanarien. Bollettino dei musei di zoologia ed anatomia comparata della R. Università di Torino 9 (182): 1–4.Google Scholar
  33. Graff, L. V. (1896). Über das System und die geographische Verbreitung der Landplanarien. Verhandlungen der deutschen zoologischen Gesellschaft, 6, 61–75.Google Scholar
  34. Graff, L. V. (1899). Monographie der Turbellarien II. Tricladida Terricola (Landplanarien). Atlas von Achtundfünfzig Tafeln zur Monographie der Turbellarien II. Tricladida Terricola (Landplanarien). Leipzig: W. Engelmann.Google Scholar
  35. Hall, T. A. (1999). BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series, 41, 95–98.Google Scholar
  36. Hamilton, C. A., Hendrixson, B. E., Brewer, M. S., & Bond, J. E. (2014). An evaluation of sampling effects on multiple DNA barcoding methods leads to an integrative approach for delimiting species: a case study of the North American tarantula genus Aphonopelma (Araneae, Mygalomorphae, Theraphosidae). Molecular Phylogenetics and Evolution, 71, 79–93.CrossRefPubMedGoogle Scholar
  37. Hebert, P., Cywinska, A., Ball, S., & Dewaard, J. (2003). Biological identifications through DNA barcodes. Proceedings of the Royal Society B: Biological Sciences, 270, 313–321.CrossRefPubMedPubMedCentralGoogle Scholar
  38. Katoh, K., & Standley, D. M. (2013). MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Molecular Biology and Evolution, 30, 772–780.CrossRefPubMedPubMedCentralGoogle Scholar
  39. Kearse, M., Moir, R., Wilson, A., Stones-Havas, S., Cheung, M., Sturrock, S., Buxton, S., Cooper, A., Markowitz, S., Duran, C., Thierer, T., Ashton, B., Meintjes, P., & Drummond, A. (2012). Geneious basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics, 28(12), 1647–1649.CrossRefPubMedPubMedCentralGoogle Scholar
  40. Knowles, L. L., & Carstens, B. C. (2007). Delimiting species without monophyletic gene trees. Systematic Biology, 56(6), 887–895.CrossRefPubMedGoogle Scholar
  41. Kumar, S., Stecher, G., & Tamura, K. (2016). MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets. Molecular Biology and Evolution, 33(7), 1870–1874.CrossRefPubMedGoogle Scholar
  42. Leal-Zanchet, A. M., & de Souza, S. A. (2003). Redescrição de Choeradoplana iheringi Graff (Platyhelminthes, Tricladida, Terricola). Revista Brasileira de Zoologia, 20(3), 523–530.CrossRefGoogle Scholar
  43. Leal-Zanchet, A. M., Rossi, I., Seitenfus, A. R. L., & Alvarenga, J. (2012). Two new species of land flatworms and comments on the genus Pasipha Ogren & Kawakatsu, 1990 (Platyhelminthes: Continenticola). Zootaxa, 3583, 1–21.Google Scholar
  44. Lemos, V. S. A., Cauduro, G. P. B., Valiati, H. V., & Leal-Zanchet, A. M. (2014). Phylogenetic relationships within the flatworm genus Choeradoplana Graff (Platyhelminthes: Tricladida) inferred from molecular data with the description of two new sympatric species from Araucaria moist forests. Invertebrate Systematics, 28, 605–627.CrossRefGoogle Scholar
  45. Marcus, E. (1951). Turbellaria brasileiros (9). Boletim da Faculdade de Filosofia, Ciências e Letras da Universidade de São Paulo, Série Zoologia, 16, 5–215.Google Scholar
  46. Mateos, E., Sluys, R., Riutort, M., & Álvarez-Presas, M. (2017). Species richness in the genus Microplana (Platyhelminthes, Tricladida, Microplaninae) in Europe: as yet no asymptote in sight. Invertebrate Systematics, 31, 269–301.CrossRefGoogle Scholar
  47. Negrete, L., & Brusa, F. (2012). Choeradoplana crassiphalla sp. nov. (Platyhelminthes: Tricladida: Geoplanidae): a new species of land planarian from the Atlantic Forest of Argentina. Studies on Neotropical Fauna and Environment, 47(3), 227–237.Google Scholar
  48. Ogren, R. E., & Darlington, J. T. (1991). Geoplana arkalabamensis n. sp., a land planarian from the southern United States (Turbellaria: Tricladida: Geoplanidae). Transactions of the American Microscopical Society, 110(3), 226–236.Google Scholar
  49. Prévot, V., Jordaens, K., Sonet, G., & Backeljau, T. (2013). Exploring species level taxonomy and species delimitation methods in the facultatively self-fertilizing land snail genus Rumina (Gastropoda: Pulmonata). PLoS One, 8(4), e60736.CrossRefPubMedPubMedCentralGoogle Scholar
  50. Puillandre, N., Lambert, A., Brouillet, S., & Achaz, G. (2012). ABGD, Automatic Barcode Gap Discovery for primary species delimitation. Molecular Ecology, 21, 1864–1877.CrossRefPubMedGoogle Scholar
  51. Rambaut, A. (2007–2017). FigTree. Tree figure drawing tool. Available from http://tree.bio.ed.ac.uk/.
  52. Rambaut, A., Suchard, M., Xie, D. & Drummond, A. (2014). Tracer v1.6. Available from http://beast.bio.ed.ac.uk/Tracer.
  53. Riester, A. (1938). Beiträge zur Geoplaniden-Fauna Brasiliens. Abhandlungen der senkenbergischen naturforschenden Gesellschaft, 441, 1–88.Google Scholar
  54. 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, 539–542.CrossRefPubMedPubMedCentralGoogle Scholar
  55. Schwarzfeld, M. D., & Sperling, F. A. H. (2015). Comparison of five methods for delimitating species in Ophion Fabricius, a diverse genus of parasitoid wasps (Hymenoptera, Ichneumonidae). Molecular Phylogenetics and Evolution, 93, 234–248.CrossRefPubMedGoogle Scholar
  56. Sluys, R. (1989). Phylogenetic relationships of the triclads (Platyhelminthes, Seriata, Tricladida). Bijdragen tot de Dierkunde, 59, 3–25.Google Scholar
  57. Stamatakis, A. (2014). RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics, 30, 1312–1313.CrossRefPubMedPubMedCentralGoogle Scholar
  58. Sukumaran, J., & Knowles, L. L. (2017). Multispecies coalescent delimits structure, not species. Proceedings of the National Academy of Sciences, 114(7), 201607921.CrossRefGoogle Scholar
  59. Talavera, G., & Castresana, J. (2007). Improvement of phylogenies after removing divergent and ambiguously aligned blocks from protein sequence alignments. Systematic Biology, 56, 564–577.CrossRefPubMedGoogle Scholar
  60. Waeschenbach, A., Porter, J., & Hughes, R. (2012). Molecular variability in the Celleporella hyalina (Bryozoa; Cheilostomata) species complex: evidence for cryptic speciation from complete mitochondrial genomes. Molecular Biology Reports, 39, 8601–8614.CrossRefPubMedGoogle Scholar
  61. Winsor, L. (2006). New and revised terrestrial flatworm taxa (Platyhelminthes: Tricladida: Terricola) of Australia and the Subantarctic Islands of New Zealand. Tuhinga, 17, 81–104.Google Scholar
  62. Winsor, L. (2009). A new subfamily, new genus and new species of terrestrial flatworm (Platyhelminthes: Tricladida: Geoplanidae) from Stewart Island, New Zealand. Tuhinga, 20, 23–32.Google Scholar
  63. Winsor, L. (2011). Some terrestrial flatworm taxa (Platyhelminthes: Tricladida: Continenticola) of the Subantarctic Islands of New Zealand. Tuhinga, 22, 161–169.Google Scholar
  64. Yang, Z. (2015). The BPP program for species tree estimation and species delimitation. Current Zoology, 61(5), 854–865.CrossRefGoogle Scholar
  65. Yang, Z., & Rannala, B. (2010). Bayesian species delimitation using multilocus sequence data. Proceedings of the National Academy of Sciences, 107, 9264–9269.CrossRefGoogle Scholar
  66. Yang, Z., & Rannala, B. (2014). Unguided species delimitation using DNA sequence data from multiple loci. Molecular Biology and Evolution, 31, 3125–3135.CrossRefPubMedPubMedCentralGoogle Scholar
  67. Zhang, J., Kapli, P., Pavlidis, P., & Stamatakis, A. (2013). A general species delimitation method with applications to phylogenetic placements. Bioinformatics, 29, 2869–2876.CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Gesellschaft für Biologische Systematik 2017

Authors and Affiliations

  1. 1.Laboratório de Ecologia e Evolução, Escola de Artes, Ciências e HumanidadesUniversidade de São Paulo (USP)São PauloBrazil
  2. 2.Departament de Genètica, Microbiologia i Estadística, Facultat de Biologia and Institut de Recerca de la Biodiversitat (IRBio)Universitat de BarcelonaBarcelonaSpain

Personalised recommendations