Rediscovery of the nemertean Prostoma graecense from Xochimilco, 75 years after its first and only record
Omar Lagunas-Calvo a, David Iván Hernández-Mena b, Gerardo Rivas a, Samantha Contreras-Mirón b, Berenit Mendoza-Garfias c, Olga M. Echeverría-Martínez d, Alejandro Oceguera-Figueroa b, *
a Departamento de Biología Comparada, Facultad de Ciencias, Universidad Nacional Autónoma de México, Apartado Postal 70-399, 04510 México City, Mexico
b Laboratorio de Helmintología, Instituto de Biología, Universidad Nacional Autónoma de México, Apartado postal 70-153, 04510 México City, Mexico
c Laboratorio de Microscopia Electrónica, Instituto de Biología, Universidad Nacional Autónoma de México, Apartado postal 70-153, 04510 México City, Mexico
d Laboratorio de Microscopia Electrónica, Facultad de Ciencias, Universidad Nacional Autónoma de México, Apartado Postal 70-181, 04510 México City, Mexico
*Corresponding author: aoceguera@ib.unam.mx (A. Oceguera-Figueroa).
Abstract
The identity of the freshwater nemertean Prostoma graecense (Böhming, 1892) from Lake Xochimilco is solved 75 years after its first recording. Identification of the specimens was accomplished via both morphological and molecular means, the latter employing the mitochondrial cytochrome c oxidase subunit I. Recent literature and our results suggest that this species has a cosmopolitan distribution and has, presumably, been recently introduced to areas well removed from its original distribution, which is unknown. The current study provides a background for continuing the work with freshwater and marine nemerteans and underscores the importance of including molecular data in future studies of the group.
Keywords:
Freshwater invertebrates; Nemertea; Tetrastemmatidae; Cox1; Taxonomy
© 2018 Universidad Nacional Autónoma de México, Instituto de Biología. Este es un artículo Open Access bajo la licencia CC BY-NC-ND
Redescubrimiento del nemertino Prostoma graecense de Xochimilco, 75 años después de su primer y único registro
Resumen
Se resuelve la identidad taxonómica del nemertino dulceacuícola Prostoma graecense (Böhming, 1892) del lago de Xochimilco 75 años después del primer registro. La identificación de los ejemplares se realizó con base en caracteres morfológicos y moleculares, esta última empleando el citocromo c oxidasa subunidad I mitocondrial. La literatura reciente y nuestros resultados sugieren que esta especie tiene una distribución cosmopolita y que ha sido introducida, presumiblemente, en áreas lejanas de su distribución original, la cual es desconocida. El presente estudio proporciona un contexto para continuar los estudios sobre nemertinos de agua marina y dulceacuícola y enfatiza la importancia de incluir datos moleculares en futuros estudios del grupo.
Palabras clave:
Invertebrados dulceacuícolas; Nemertea; Tetrastemmatidae; Cox1, Taxonomía
Introduction
Members of the phylum Nemertea (ribbon worms) are characterized by the presence of an eversible muscular proboscis housed within a cavity called a rhynchocoel; this structure is found in no other animal group. Nemerteans are unsegmented, bilaterian, vermiform animals with a closed circulatory system and protonephridia as excretory organs. The body size of these worms range in length from a few millimeters up to 30 meters (Sundberg & Strand, 2010; Strand & Sundberg, 2015), representing the longest metazoan on Earth. Members of Nemertea are mostly marine, with only 22 out of the 1,280 described species inhabiting freshwater (Kajihara et al., 2008; Sundberg & Gibson, 2008; Strand & Sundberg, 2015). Most of the species diversity within the group occurs in the Nearctic and Palearctic, but this may only reflect the intensity of sampling efforts and taxonomic work rather than a real biogeographical pattern (Strand & Sundberg, 2015). Because of the difficulty of identifying preserved specimens, several authors have suggested that morphological identification should always be accompanied by DNA sequence analyses (Chen et al., 2010; Sundberg & Strand, 2010; Sundberg et al., 2010, 2016).
Only a single freshwater nemertean is known from Mexico, originally referred to as Stichostemma rubrum (Leydi, 1850) from Lake Xochimilco, México City (Rioja, 1941). Gibson and Moore (1976) listed S. rubrum under the genus Prostoma and concluded that no typically North American species of Prostoma can be recognized, thus the Mexican records of P. rubrum should be considered as P. graecense. In the most recent taxonomic account of freshwater nemerteans from North America, only 2 species were recognized: Prostoma canadiensis Gibson & Moore, 1978, which possesses a frontal organ (including previous records of P. graecense), and Prostoma asensoriatum (Montgomery, 1896), which lacks such an organ (Strand & Sundberg, 2015). To robustly infer the presence of a frontal organ, histological preparations are required, a procedure that has yet to be used to characterize nemerteans from Mexico.
More than 75 years have passed since the first and only record of the freshwater nemertean from Xochimilco, an area well known for its expansive biodiversity and number of endemics; arguably the most charismatic of these is Axolotl [Ambystoma mexicanum (Shaw & Nodder, 1798)] and the Montezuma leopard frog [Lithobates montezumae (Baird, 1854)]. During that time, Xochimilco has changed dramatically, due to a steep increase in human population numbers and the intentional and unintentional introduction of several exotic species that have reshaped the ecosystem, including the common carp Cyprinus carpio Linnaeus, 1758, tilapia Oreochromis niloticus (Linnaeus, 1758), and the water hyacinth Eichhornia crassipes (Mart.) Solms. (Cervantes-Sánchez & Rojas-Rabiela, 2000; Zambrano et al., 2010). The aim of the present study is to document the presence of the freshwater nemertean in Xochimilco, an area that has been severely impacted by human activities. In addition, this study aims to solidify the taxonomic and nomenclatural status of the nemertean and to generate the mitochondrial cytochrome c oxidase subunit I (cox1) DNA sequence, the preferred zoological barcoding region, which may serve as a framework for future studies of this charismatic taxon.
Materials and methods
In February, April, and October of 2016, 17 nemerteans were collected from “Embarcadero de Cuemanco”, Xochimilco, México City, Mexico (19º17’20.934” N, 99º06’06.585” W). Specimens were found within the roots of water lily Eichhornia crassipes that were collected from the edges of canals. The aquatic plants were placed in trays and, after about 10 minutes, ribbon worms were recovered from the bottom of the tray with the use of delicate paintbrushes. For subsequent morphological procedures, worms were relaxed in 7% MgCl2 and fixed in 4% formaldehyde. Two specimens were stained with Gomori trichrome and mounted on permanent slides with Canada balsam. For histological procedures, worms were fixed in 10% formaldehyde in phosphate-buffered saline (PBS) at pH 7.2, dehydrated with graded ethanol series and embedded in Epon epoxy resin. Semi-thin serial sections, obtained with a Leica Ultracut UTC Ultramicrotome, were stained with toluidine blue. In addition, 2 worms were dehydrated and critical point dried with CO2, mounted and coated with a mixture of gold-palladium and subsequently observed in a Hitachi model SUI510 scanning electron microscope (SEM) at the Laboratorio Nacional de Biodiversidad, Instituto de Biología, Universidad Nacional Autónoma de México (IB-UNAM). Voucher specimens were deposited in the National Invertebrates Collection at the IB-UNAM (accession number CNINV-002, 003). For molecular analyses, cox1 sequences from 3 specimens were obtained via procedures described elsewhere (Kvist et al., 2014; Oceguera-Figueroa et al., 2005). The newly generated cox1 sequences were aligned and compared with 15 sequences of members of the suborder Monostilifiera (Class Enopla) available from GenBank (Table 1) and selected based on BLASTn (Altschul et al., 1990) matches. Nucleotide sequences were aligned with ClustalW (Thompson et al., 1997), implemented in the web-version of the software at http://www.genome.jp/tools/clustalw/. The edges of the alignment were trimmed to include sequences
Table 1
Taxa used for the maximum likelihood analysis of nemerteans, including Prostoma graecense from Lake Xochimilco, Mexico with other specimens of the genus with localities and GenBank accession codes.
|
Taxon |
Locality |
GenBankaccession number |
|
Gurjanovella littoralis |
Kandalaksha Bay, White sea, Russia |
AJ436904 |
|
Prosorhochmus nelsoni |
Coquimbo, Chile |
EF157586 |
|
Prosorhochmus belizeanus |
Carrie Bow Cay, Belize |
EF157591 |
|
Prosorhochmus cf. claparedii |
Armintza, Bizkaia, Spain |
EF157590 |
|
Prosorhochmus americanus |
Turbeville, Pawley’s Island, SC, USA |
EF157588 |
|
Prosorhochmus chafarinensis |
Savudrija, Adriatic Sea, Croatia |
EF157587 |
|
Prostoma sp. |
Los Angeles, USA |
HQ938796 |
|
Prostoma sp. |
Los Angeles, USA |
HQ939311 |
|
Prostoma graecense |
Ohrid Lake, Macedonia |
JX017298 |
|
Prostoma graecense |
West coast, Sweden |
EF208981 |
|
Prostoma graecense |
West coast, Sweden |
EU489490 |
|
Prostoma graecense 1 |
Xochimilco, Mexico |
KY523851 |
|
Prostoma graecense 2 |
Xochimilco, Mexico |
MF314116 |
|
Prostoma graecense 3 |
Xochimilco, Mexico |
MF314117 |
|
Nemertea sp. |
Italy |
KU840284 |
|
Prostoma cf. eilhardi |
Eastbrook Woods, USA |
HQ848594 |
|
Nemertopsis bivittata |
South Carolina, USA |
HQ848608 |
|
Psammamphiporus elongatus |
Coruña, Galicia, Spain |
HQ848609 |
of the same length. The final dataset included 18 terminals and 677 aligned characters. Phylogenetic analysis was run under the maximum likelihood (ML) criterion, employing a GTR+GAMMA+I model as suggested by jModelTest ver. 2 (Darriba et al., 2012). Likelihood inference (20 replicates), model parameters, and bootstrap (BS) support (500 pseudoreplicates) were implemented with RAxML v. 7.0.4 (Stamatakis, 2006). The resulting phylogenetic tree was visualized with the software FigTree ver. 1.4.2 (Rambaut, 2012). Based on the phylogenetic hypotheses of Andrade et al. (2012), Nemertopsis bivittata (Delle Chiaje, 1841) and Psammamphiporus elongatus (Stephenson, 1911) were selected as the outgroup. Genetic distances between selected terminals were calculated as uncorrected p-distances using MEGA ver. 5 (Tamura et al., 2011).
Results
Nemerteans small (8.45 ± 0.21 mm length by 0.67 ± 0.02 width), whitish-reddish, some specimens greenish when alive, number of eye spots variable, from 2-6 on the anterior end (Fig. 1A); proboscis large, with integument projections (Fig. 1B), with stylets at the mid-portion (Fig. 1C); rhynchocoel dorsal to the digestive tract, as large as proboscis (Fig. 1D). Histological analysis revealed a distinctive ciliated esophagus and no improvised ducts were observed (Fig. 1E). Frontal organ recovered with cephalic glands openings inside (Fig. 1F).
Maximum likelihood phylogenetic analysis (Fig. 2) recovered the 3 newly generated cox1 sequences from the Xochimilco specimens in a single group and nested within species of the genus Prostoma with 100% BS. Prostoma graecense from Sweden and Macedonia exhibit marginal genetic distances (> 0.2%) when compared to the Mexican samples. This group is sister to a moderately supported group (BS = 64%) formed by Prostoma cf. eilhardi from USA and a sample labeled as Nemertea sp. from Italy. The genetic distances between the members of these 2 latter groups are lower than 0.8%. Sister to this group are 2 identical sequences of unidentified Prostoma specimens from Los Angeles, California, USA with an average cox1 divergence of 3.2% with respect to the P. graecense group + the sample from Xochimilco.
Discussion
The placement of the 3 samples of nemerteans from Xochimilco, Mexico within a group of samples from
http://rev.mex.biodivers.unam.mx/wp-content/uploads/2018/vol-89/89-1-mar-2018/html/05-Fig1.tif
Figure 1. Prostoma graecense from Xochimilco, Mexico. Living specimen: A) eyes spots (black arrow). Microphotography with SEM: B) details of integument projections of the proboscis. Specimens stained with Gomori trichrome: C) stylet in the middle portion of proboscis (black arrow); D) rhynchocoel (white arrow). Sagittal section through the body: E) ciliated epithelium of the esophagus (black arrow) at middle body region; F) cup-shaped frontal organ (black arrow) at the cephalic region.
disparate areas including Macedonia, Italy, Sweden, and USA, and with genetic distances lower than 1%, strongly suggests that all of these specimens belong to the same species. This result contrast with the most recent taxonomic treatment of the group by Sundberg and Gibson (2008) and Strand and Sundberg (2015) who applied the name Prostoma canadiensis for the North American freshwater nemerteans in possession of a frontal organ and maintained the name Prostoma graecense for specimens from other major biogeographic areas in possession of the
http://rev.mex.biodivers.unam.mx/wp-content/uploads/2018/vol-89/89-1-mar-2018/html/05-Fig2.tif
Figure 2. Phylogenetic tree obtained from the analysis of maximum likelihood including 3 samples of Prostoma graecense from Xochimico, Mexico in bold. Dotted lines show the genetic distances between the groups of Prostoma.
same organ. The presence of a frontal organ in the samples from Mexico and the negligible genetic divergence found in the 3 Mexican specimens and between the samples labeled as P. graecense from North America and Europe, allowed us to suggest that all of them should be considered the same species for which the name P. graecense should be used. The unusual disjunct biogeographic distribution accompanied with low genetic divergences found by Strand and Sundberg (2005) in their analysis of partial 18S rRNA sequences of 3 P. graecense samples from Sweden and New Zealand and a sample labeled as P. eilhardi, was interpreted as evidence of a recent introduction, and suggested that these taxa should potentially be synonymized. The cox1 locus has been suggested as a reliable marker for species delineation in nemerteans under the assumption that 4-5% divergence indicates a difference in species-level identity (Sundberg et al., 2016). In our results, the low genetic divergence in cox1 (less than 1%) when comparing the samples from Xochimilco and other localities is also interpreted as a relatively recent introduction. The fact that international efforts to introduce the South American water hyacinth, at least in Europe and North America were conducted intensively in the second half of the 19th Century, may explain the current distribution of P. graecense, a species that might be transported inadvertently when introducing this aquatic plant. This phenomenon also has been recorded in other freshwater invertebrates such as leeches (Garduño-Montes de Oca et al., 2016; Reyes-Prieto et al., 2013) and the red crayfish Procambarus clarkii (Hernández et al., 2008). Prostoma graecense has, based mainly on morphological data to be confirmed by molecular studies, a cosmopolitan distribution: in the Americas (from USA to Argentina), Europe, Asia, Africa, Australia, and Oceania (Cordero, 1943; Corrêa, 1986; Pennak, 1989; Tamburi & Cazzaniga, 2006; Weidenbach,
1995).
Phylogenetic analyses by Kvist et al. (2015) with 158 terminals and 4 molecular markers, recovered a monophyletic group with Prostoma cf. eilhardi and brackish and marine sacconemertids (Sacconemertidae), suggesting that at least in this part of the tree, the transition from marine to brackish/freshwater habitat may be the result of a single transitional event. Unfortunately, this hypothesis cannot be tested with our dataset given that our taxon selection, based on BLASTn matches, only recovered species of Prosorhochmus as the most similar sequences to those of Prostoma, leaving the transitional hypothesis to be tested in future studies after the inclusion of more species of Prostoma and additional markers.
Enrique Rioja Lo Bianco, who first reported the nemertean from Xochimilco, also contributed to the characterization of Mexican fauna of annelids and crustaceans (Caso, 1964; Dosil-Mancilla & Cremades-Ugarte, 2004). In addition, he studied freshwater sponges, hydrozoans, and bryozoans (Rioja, 1940a, b, c; 1953a, b). These taxa, like Nemertea, have not been studied in the last 75 years in Mexico. The omission of taxonomic studies in several phyla of invertebrates is a serious problem for the current studies of comparative biology and biodiversity.
Acknowledgments
We thank Rosario Ortiz Hernández for helping with the histological techniques. Brenda Atziri García García, Mauricio Ramírez Carmona, Jossué Jiménez Armenta, and Jorge Damián López Caballero assisted in the collection of specimens. Thanks to Ofelia Delgado Hernández, Andrea Jiménez Marín, and Laura Márquez Valdelamar for the assistance in the molecular laboratory. We also acknowledge Luis García Prieto for incorporating the specimens into the Colección de Invertebrados, Instituto de Biología, Universidad Nacional Autónoma de México, México City. This project was partially founded by PAPIIT (IA202016) to AO-F.
References
Altschul, S. F., Gish, W., Miller, W., Myers, E. W., & Lipman D. J. (1990). Basic local alignment search tool. Journal of Molecular Biology, 215, 403–410.
Andrade, S. C. S., Strand, M., Schwartz, M., Chen, H., Kajihara, H., von Döhren, J. et al. (2012). Disentangling ribbon worm relationships: multi-locus analysis supports traditional classification of the phylum Nemertea. Cladistics, 28, 141–159.
Caso, M. E. (1964). La labor de don Enrique Rioja como investigador, maestro y amigo de México. Revista Mexicana de Historia Natural, 25, 77–96.
Cervantes-Sánchez, J. M., & Rojas-Rabiela, T. (2000). Introducción del lirio acuático (Eichhornia crassipes) a México durante el porfiriato. Quipu, 13, 177–190.
Chen, H. X., Strand, M., Norenburg J. L., Sun S. C., Kajihara, H., Chernyshev A. V. et al. (2010). Statistical parsimony networks and species assemblages in cephalotrichid nemerteans (Nemertea). Plos One, 5, e12885.
Cordero, E. H. (1943). Hallazgos en diversos países de Sud América de Nemertinos de agua dulce del género Prostoma. Anais da Academia Brasileira de Ciências, 15, 125–134.
Corrêa, D. D. (1986). Nemertini. In R. Schaden (Eds.) Manual de identificação de invertebrados límnicos do Brasil 9 (pp. 1–20). Brasilia: CNPq Assessoria Editorial.
Darriba, D., Taboada, G. L., Doallo, R., & Posada, D. (2012). jModelTest 2: more models, new heuristcs and parallel computing. Nature Methods, 9, 772.
Dosil-Mancilla, F. J., & Cremades-Ugarte, J. (2004). El zoólogo Enrique Rioja (1895-1963). Datos sobre su vida y su contribución a la ciencia y a la cultura en España y en México. Actas VIII Congreso de la Sociedad Española de Historia de las Ciencias y de las Técnica, 497–517.
Garduño-Montes de Oca, U., Martínez-Flores, G., Contreras-Mirón, S., Jiménez-Armenta, J., & Oceguera-Figueroa, A. (2016). Primer registro de la sanguijuela introducida Barbronia weberi (Annelida: Clitellata) en Nuevo León, México. Revista Mexicana de Biodiversidad, 87, 1124–1126.
Gibson, R., & Moore, J. (1976). Freshwater nemerteans. Zoological Journal of the Linnean Society, 58, 177–218.
Hernández, L., Maeda-Martínez, A. M., Ruiz-Campos, G., Rodríguez-Almaraz, G., Alonzo-Rojo, F., & Sainz, J. C. (2008). Geographic expansion of the invasive red crayfish Procambarus clarkii (Girard, 1852) (Crustacea: Decapoda) in Mexico. Biological Invasions, 10, 977–984.
Kajihara, H., Chernyshev, A. V., Sun, S., Sundberg, P., & Crandall, F. B. (2008). Checklist of nemertean genera and species published between 1995 and 2007. Species Diversity, 13, 245–274.
Kvist, S., Chernyshev, A. V., & Giribet, G. (2015). Phylogeny of Nemertea with special interest in the placement of diversity from far East Russia and Northeast Asia. Hydrobiologia, 760, 105–119.
Kvist, S., Laumer, C. E., Junoy, J., & Giribet, G. (2014). New insights into the phylogeny, systematics and DNA barcoding of Nemertea. Invertebrate Systematics, 28, 287–309.
Oceguera-Figueroa, A., León-Règagnon, V., & Siddall, M. E. (2005). Phylogeny and revision of Erpobdelliformes (Annelida, Arhynchobdellida) from Mexico based on nuclear and mitochondrial gene sequences. Revista Mexicana de Biodiversidad, 72, 191–198.
Pennak, R. W. (1989). Fresh-water invertebrates of the United States, 3 ed. John Wiley and Sons, New York, 628 pp.
Rambaut, A. (2012). FigTree v1.4. Available from http://tree.bio.ed.ac.uk/software/figtree/
Reyes-Prieto, M., Oceguera-Figueroa, A., Snell, S., Negredo, A., Barba, E., Fernández, L. et al. (2013). DNA barcodes reveal the presence of the introduced freshwater leech Helobdella europaea in Spain. Mitochondrial DNA, 25, 1–7.
Rioja, E. (1940a). Esponjas, Hidrozoarios y Briozoos del Lago de Pátzcuaro. Anales del Instituto de Biología, Universidad Nacional Autónoma de México, 11, 443–443.
Rioja, E. (1940b). Contribución al conocimiento de los briozoarios del Lago de Xochimilco. Anales del Instituto de Biología, Universidad Nacional Autónoma de México, 11, 585–592.
Rioja, E. (1940c). Estudios Hidrobiológicos. I. Estudio crítico sobre las esponjas del Lago de Xochimilco. Anales del Instituto de Biología, Universidad Nacional Autónoma de México, 11, 173–189.
Rioja, E. (1941). Hallazgo en Xochimilco de Stichostemma rubrum (Leidy), nemerte de aguadulce. Anales del Instituto de Biología, Universidad Nacional Autónoma de México, 12, 663–668.
Rioja, E. (1953a). Estudios Hidrobiológicos. XI. Contribución al estudio de las esponjas de agua dulce de México. Anales del Instituto de Biología, Universidad Nacional Autónoma de México, 24, 425–433.
Rioja, E. (1953b). Datos históricos acerca de las esponjas de agua dulce de México. Revista de la Sociedad Mexicana de Historia Natural, 14, 51.
Stamatakis, A. (2006). RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics, 22, 2688–90.
Strand, M., & Sundberg, P. (2005). Delimiting species in the hoplonemertean genus Tetrastemma (phylum Nemertea): morphology is not concordant with phylogeny as evidenced from mtDNA sequences. Biological Journal of the Linnean Society, 86, 201–212.
Strand, M., & Sundberg, P. (2015). Phylum Nemertea. In J. H. Thorp & D. C. Rogers (Eds.) Thorp and Covich’s freshwater invertebrates: ecology and general biology, Fourth Edition (pp. 205–209). London, United Kingdom: Academic
Press.
Sundberg, P., Andrade, S. C. S., Bartolomaeus, T., Beckers, P., von Döhren, J., Krämer, D. et al. (2016). The future of nemertean taxonomy (phylum Nemertea) ̶ a proposal. Zoologica Scripta, 45, 579–582.
Sundberg, P., & Gibson, R. (2008). Global diversity of nemerteans (Nemertea) in freshwater. Hydrobiologia, 595, 61–66.
Sundberg, P., & Strand, M. (2010). Nemertean taxonomy—time to change lane? Journal of Zoological Systematics and Evolutionary Research, 48, 283–284.
Sundberg, P., Vodoti, E. T., & Strand, M. (2010). DNA barcoding should accompany taxonomy – the case of Cerebratulus spp (Nemertea). Molecular Ecology Resources, 10, 274–281.
Tamburi, N. E., & Cazzaniga, N. J. (2006). The freshwater ribbon-worm Prostoma graecense (Hoplonemertea: Monostilifera) in South America (Argentina). Zootaxa, 1248, 27–34.
Tamura K., Peterson, D., Peterson, N., Stecher, G., Nei, M., & Kumar, S. (2011). MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular Biological Evolution, 28, 2731–2379.
Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F., & Higgins, D. G. (1997). The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research, 25, 4876–4882.
Weidenbach, R. P. (1995). Freshwater pond life, a guide to the animals and plants of Hawaii’s freshwater aquaculture ponds. Hawaii: Aquaculture Development Program, DLNR.
Zambrano, L., Valiente, E., & Vander-Zanden, M. J. (2010). Stable isotope variation of a highly heterogeneous shallow freshwater system. Hydrobiologia, 646, 627–336.
