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DNA barcoding supports identification of Malacobdella species (Nemertea: Hoplonemertea)

  • 1, 2,
  • 1, 2,
  • 3,
  • 4,
  • 5 and
  • 6Email author
Zoological Studies201554:10

  • Received: 16 March 2014
  • Accepted: 16 December 2014
  • Published:



Nemerteans of the genus Malacobdella live inside of the mantle cavity of marine bivalves. The genus currently contains only six species, five of which are host-specific and usually found in a single host species, while the sixth species, M. grossa, has a wide host range and has been found in 27 different bivalve species to date. The main challenge of Malacobdella species identification resides in the similarity of the external morphology between species (terminal sucker, gut undulations number, anus position and gonad colouration), and thus, the illustrations provided in the original descriptions do not allow reliable identification. In this article, we analyse the relationships among three species of Malacobdella: M. arrokeana, M. japonica and M. grossa, adding new data for the M. grossa and reporting the first for M. japonica, analysing 658 base pairs of the mitochondrial cytochrome c oxidase subunit I gene (COI). Based on these analyses, we present and discuss the potential of DNA barcoding for Malacobdella species identification.


Sixty-four DNA barcoding fragments of the mitochondrial COI gene from three different Malacobdella species (M. arrokeana, M. japonica and M. grossa) are analysed (24 of them newly sequenced for this study, along with four outgroup specimens) and used to delineate species. Divergences, measured as uncorrected differences, between the three species were M. arrokeana-M. grossa 11.73%, M. arrokeana-M. japonica 10.62% and M. grossa-M. japonica 10.97%. The mean intraspecific divergence within the ingroup species showed a patent gap with respect to the interspecific ones: 0.18% for M. arrokeana, 0.13% for M. grossa and 0.02% for M. japonica (ranges from 0 to 0.91%).


We conclude that there is a clear correspondence between the molecular data and distinguishing morphological characters. Our results thus indicate that some morphological characters are useful for species identification and support the potential of DNA barcoding for species identification in a taxonomic group with subtle morphological external differences.


  • DNA barcoding
  • COI gene
  • Bivalvia
  • Entocommensal nemertean
  • Malacobdella


The phylum Nemertea is a group of organisms whose identification and taxonomy requires specialized methods, mainly histology. Recently, molecular methods have been a useful tool for ascertaining the actual biodiversity of these worms and increasing our knowledge of several of the problematic species (Chen et al. 2010; Fernández-Álvarez and Machordom 2013; Kvist et al. 2013). The nemertean genus Malacobdella de Blainville 1827 originally contained 13 nominal species, of which six are currently regarded as valid (Gibson 1995; Ivanov et al. 2002). The species of the genus are entocommensal in the mantle cavity of marine bivalves, mainly from the subclass Heterodonta (Jensen and Sadeghian 2005). The phylogenetic position of the genus Malacobdella is controversial within the phylum, mainly because this genus is always represented by sequences belonging only to the species M. grossa (Thollesson and Norenburg 2003; Andrade et al. 2012), the most studied and cosmopolitan species. The Malacobdella species are distributed in distant locations around the world: M. japonica, M. macomae and M. siliquae were described in the eastern (Japan) and western (west coast of the USA) Pacific Ocean; M. grossa was described in the Pacific (west coast of the USA) and Atlantic Ocean (northern Europe); and M. arrokeana was the only southern species described in the South Atlantic Ocean (Ivanov et al. 2002 and references herein). The geographic distribution of the genus requires a huge sampling effort to work with all species. Identifying Malacobdella species is difficult because important diagnostic features were not initially recognized and thus not mentioned in earlier descriptions (Ivanov et al. 2002). In addition, the similarity of the external morphology between species (terminal sucker, number of intestinal loops (undulations), anus position and immature gonad colouration) and the illustrations provided in the original descriptions of the species do not allow reliable identification. There is little knowledge about the biology of Malacobdella species. The genus Malacobdella belongs to the hoplonemertean non-pilidiophora species (Thollesson and Norenburg 2003); this group presents direct development and non-feeding planuliform larvae (Maslakova and von Döhren 2009). However, there are no studies on its larval development, dispersion and settlement.

The host specificity of the Malacobdella species is generally high; five of the six known species have been reported from only one or two bivalve species: M. arrokeana Ivanov et al. 2002 from Panopea abbreviata (Heterodonta: Hiatelloidea) (Ivanov et al. 2002; Alfaya et al. 2013); M. japonica Takakura 1897 from Spisula sachalinensis (Heterodonta: Mactroidea) (Takakura 1897); M. macomae Kozloff 1991 from Macoma nasuta and Macoma secta (Heterodonta: Tellinoidea) (Kozloff 1991); M. minuta (Coe 1945) from Yoldia cooperii (Protobranchia: Nuculanoidea) (Coe 1945); and M. siliquae Kozloff 1991 from Siliqua patula (Heterodonta: Solenoidea) (Kozloff 1991). The sixth species, M. grossa, is the type specimen of the genus and was originally obtained from Dosinia exoleta (Linnaeus 1758) (Heterodonta: Veneroidea) (Müller 1776). However, the most complete morphological description of M. grossa, reported by Riepen (1933), was based on material from Arctica islandica (Linnaeus 1767) (Heterodonta: Arcticoidea). Nemerteans are traditionally identified and classified using morphological criteria, but the relatively low number of qualitative morphological characters, the lack of adequate fixation procedures for histological studies, vague descriptions in the original papers and the paucity of species-specific characters make species delimitation problematic, especially when comparing closely related species (Chen et al. 2010; Sundberg et al. 2010; Fernández-Álvarez and Machordom 2013; Kvist et al. 2013). The difficulty of morphological recognition of a great part of the more than 1,280 species included in the phylum Nemertea has been previously discussed (Andrade et al. 2012; Sundberg and Strand 2010; Kvist et al. 2013, among others), and different authors have advocated new tools and comprehensive studies for correctly identifying species and thus providing accurate biodiversity knowledge. The combination of molecular and morphological methods has been useful in elucidating nemertean taxonomy in other genera (Sundberg et al. 2009; Junoy et al. 2010; Puerta et al. 2010; Kajihara et al. 2011; Taboada et al. 2013). DNA barcoding accelerated the discovery ratio of new species (Wiens 2007) and identified some inconsistencies between species assignment and previously sequenced specimens (Kvist et al. 2013). Nevertheless, only a small number of nemerteans have been analysed through DNA barcoding (Sundberg et al. 2009; Chen et al. 2010; Fernández-Álvarez and Machordom, 2013; Kvist et al. 2013; Strand et al. 2014). The potential use of DNA barcoding needs to be substantiated in well-established taxonomic groups before it can be fully exploited in all nemertean genera. Currently, only 6% of the phylum Nemertea has an associated barcode sequence (Bucklin et al. 2011; Fernández-Álvarez and Machordom 2013).

Our objectives are to provide the most comprehensive molecular data set for the Malacobdella genus and test the usefulness of this data in the delimitation of the three most widely distributed species of the Malacobdella genus, and to provide for the first time COI sequences for M. japonica. We also estimate the phylogenetic relationship between 64 individuals from the 3 species based on partial sequences of COI.


Specimen collection and sequences used

Thirty-eight sequences of M. arrokeana were obtained from specimens collected at three northern Patagonian gulfs in Argentina (previously studied by Alfaya et al. 2013): San Matías Gulf (n = 23), San José Gulf (n = 7) and Nuevo Gulf (n = 8) (Table 1). Each of the M. arrokeana specimens was collected from a different host specimen of P. abbreviata. Specimens of M. japonica were collected from 15 different S. sachalinensis bivalves at Shinryu Beach in Akkeshi, Hokkaido, Japan. Individuals of M. grossa (n = 11) were obtained from different A. islandica clams in the waters of Tjärnö, Skagerak, Sweden. Additionally, two M. grossa COI sequences from GenBank (HQ848591 and AJ436905, from Tjärnö, Sweden, and the White Sea, Russia, respectively) were included in the analysis (Table 1). Fresh specimens were stored in absolute ethanol, and DNA was extracted from preserved tissues using a DNeasy extraction kit (Qiagen, Inc., Hilden, Germany), following the manufacturer's protocol.
Table 1

List of species used in the analysis, including the sample locality, number of specimens analysed ( N ) and GenBank accession numbers


Specimen ID




GenBank acc. number

References or voucher numbers a

Malacobdella arrokeana


San Matías Gulf, Argentina




CNP-INV 1879




CNP-INV 1880




CNP-INV 1881




CNP-INV 1882




CNP-INV 1883




CNP-INV 1884




CNP-INV 1885




CNP-INV 1886




CNP-INV 1887




CNP-INV 1888




CNP-INV 1889




CNP-INV 1890




CNP-INV 1891




CNP-INV 1892




CNP-INV 1893




CNP-INV 1894




CNP-INV 1895




CNP-INV 1896




CNP-INV 1897




CNP-INV 1898




CNP-INV 1899




CNP-INV 1900




CNP-INV 1901


Nuevo Gulf, Argentina




CNP-INV 1902




CNP-INV 1903




CNP-INV 1904




CNP-INV 1905




CNP-INV 1906




CNP-INV 1907




CNP-INV 1908


San José Gulf, Argentina




CNP-INV 1909




CNP-INV 1910




CNP-INV 1911




CNP-INV 1912




CNP-INV 1913




CNP-INV 1914




CNP-INV 1915




CNP-INV 1916

M. japonica


Hokkaido, Japan





















































M. grossa


Tjärnö, Sweden













































Mgrossa GB 1



Andrade et al. (2012)

Mgrossa GB 2

White Sea, Russia




Thollesson and Norenburg (2003)

Ramphogordius sanguineus


Maine, USA




Andrade et al. (2012)


Anglesey, UK




Thollesson and Norenburg (2003)


Nuevo Gulf, Argentina




CNP-INV 1917




CNP-INV 1918




CNP-INV 1919




CNP-INV 1920

Amphiporus lactifloreus


Anglesey, UK




Andrade et al. (2012)

Paradrepanophorus crassus


Galicia, Spain





Geonemertes pelaensis


St. Davis, Bermuda









Mateos and Giribet (2008)

aVoucher numbers are given only for Malacobdella arrokeana specimens from the previous work of Alfaya et al. (2013), and for M. grossa and M. japonica newly sequenced here. MNCN, Museo Nacional de Ciencias Naturales (Madrid, Spain); CNP-INV, Invertebrate Collection of the Centro Nacional Patagónico (Argentina).

To test for the monophyly of the genus, we sequenced Ramphogordius sanguineus specimens collected along the Argentinean coast to use as the outgroup. We also used COI sequences available from GenBank for nemerteans that are closely related to Malacobdella and have been previously used in other phylogenetic studies (Andrade et al. 2012; Mateos and Giribet 2008; Thollesson and Norenburg 2003), including two R. sanguineus outgroup sequences (Table 1).

PCR amplification and sequencing

Partial COI sequences were amplified by PCR using the following primers: LCO1490 (5′-GGTCAACAAATCATAAAGATATTGG-3′) (Folmer et al. 1994) and COI-H (5′-TCAGGGTGACCAAAAAATCA-3′) (Machordom et al. 2003). Amplification was carried out in a 50-μl final volume reaction consisting of 5 μl buffer containing 10 × 2 mM MgCl2, 1 μl dNTP mix (10 mM), 0.4 μl of Taq DNA polymerase (5 U/μl) (Biotools, Madrid, Spain), 1–3 μl of genomic DNA and 0.8 μl of each primer (10 μM). Thermal cycling conditions were an initial 4-min denaturation at 94°C, followed by 40 cycles of 45 s at 94°C, 1 min at 45°C and 1 min at 72°C, ending with a final 10-min extension at 72°C. The products were visualized under blue light in 0.8% agarose gels stained with SYBR Safe (Invitrogen, Carlsbad, CA, USA), with co-migrating 100-bp or 1-kb ladder molecular weight markers. The amplification products (around 700 bp) were purified by ethanol precipitation. Both strands were sequenced using the BigDye Terminator sequencing kit and an ABI PRISM 3730 DNA Sequencer (Applied Biosystems, Grand Island, NY, USA).

Data analysis

Special alignment was unnecessary as the COI sequences from the analysed species did not present any gaps. Phylogenetic analyses were performed with PAUP 4.0 b10 (Swofford 2000) for maximum parsimony (MP) and maximum likelihood (ML), and with MrBayes 3.2 (Huelsenbeck 2000; Huelsenbeck and Ronquist 2001) for Bayesian inference (BI). MP parameters included a heuristic search with tree bisection-reconnection (TBR) branch swapping and ten random additions. ML was also estimated through a heuristic search, with stepwise addition, applying the TIM3 + G + I (transitional) model (Posada 2003). Two runs of 5,000,000 generations were performed for BI, sampling one tree per 1,000 replicates. The model that best fits the data (TIM3 + I + G) was found with jModelTest (Posada 2008). Branch supports for MP (1,000 replicates) and ML (150 replicates) were determined by bootstrapping (Felsenstein 1985) and by posterior probabilities (after a burn-in of 20% of the obtained trees) for BI.


According to original descriptions, the three studied species of the Malacobdella genus are very similar in their external morphology but differ in internal morphological characters. Malacobdella arrokeana differs from M. japonica and M. grossa in the origin of the proboscis retractor muscle. This muscle is curved dorsally and attaches to the internal body wall in M. arrokeana, whereas in M. grossa, it originates ventrally next to the terminal sucker (Table 2). In M. japonica, the muscle also originates ventrally but ends freely in the parenchyma (Table 2). M. japonica differs from M. arrokeana and M. grossa in the position of the nerve commissure. In M. japonica, the posterior nerve commissure is situated around the terminal sucker, while in the other two species, the nerve commissure is situated dorsally in the posterior part of the intestine just before the anus (Table 2).
Table 2

Characteristic features of the analysed Malacobdella species


M. arrokeana (Ivanov et al. 2002 )

M. japonica (Takakura 1897 )

M. grossa (Müller 1776)

Excretory pores

Dorso-lateral (40% of length)

Dorso-lateral (25% of length) (Yamaoka 1940)


Proboscis length

Most of the rhynchocoel, 80 to 90

Two thirds of the rhynchocoel length (Yamaoka 1940)

Two thirds of the rhynchocoel length

Proboscis retractor muscle

Curved dorsally and attached to the body muscular wall

Originates ventrally and ends freely in the parenchyma (Yamaoka 1940)

Originates ventrally next to the terminal sucker (Riepen 1933)

Ovary colour (mature)

White or purple (Teso et al. 2006)

Rosy (Yamaoka 1940)

Olive green or yellowish green (Gibson 1968)

Testicle colour (mature)

Pale rose (Teso et al. 2006)

White (Yamaoka 1940)

Rosy or pinkish hue (Gibson 1968)

Nerve commissure

Dorsal in the posterior part of the intestine just before the anus

Far behind the anus along the posterior margin of the sucker (Yamaoka 1940) (posterior around the terminal sucker)

Dorsal in the posterior part of intestine, above the anus

Host specificity

High (only in Panopea abbreviata)

High (only in Spisula sachalinensis)

Low (27 species)

Geographic distribution

South Atlantic Ocean (from Uruguay to north Patagonian gulfs)

North Pacific Ocean (northern Japan)

North Atlantic Ocean (Europe and North America) North Pacific Ocean (North America)

M. grossa also differs from M. arrokeana and M. japonica in the position of the excretory pores, being ventro-lateral in M. grossa and dorso-lateral in M. arrokeana and M. japonica. The rhynchocoel occupies most of the body length in the three species, up to 80% to 90% with the proboscis occupying two thirds of the rhynchocoel. Gonad colouration varies according to species and stage of gonad development. Generally, the gonads are dark olive green in colour in mature specimens of M. grossa, rosy or purple in mature M. arrokeana and rosy in mature M. japonica (Figure 1, Table 2). However, gonad colouration is always white in immature specimens (Figure 1A).
Figure 1
Figure 1

Specimens of the different Malacobdella species here analysed. Malacobdella arrokeana: (A) unrelaxed mature female, (B) relaxed male, (C) original illustration (Ivanov et al. 2002) and (D) immature specimen (scale bars: A, B: 10 mm; C: 3.5 mm, D: 1 mm). Malacobdella japonica: (E) relaxed mature female and (F) original illustration (Yamaoka 1940) (scale bars: E: 10 mm; F: 1 mm). Malacobdella grossa: (G) unrelaxed mature female, (H) immature specimen and (I) original illustration (Riepen 1933) (scale bars: A: 10 mm, B: 4 mm; C: 1 mm). Abbreviations: ts, terminal sucker; u, gut undulations; o, ovaries; t, testes.

The COI fragments sequenced for M. arrokeana, M. grossa and M. japonica were 658 bp in length. These sequences were deposited in GenBank under accession numbers KF597241 to KF597264. The different phylogenetic analyses resulted in the same topology, with high support within the ingroup (Figure 2). The divergences, measured as uncorrected distances (percentage of nucleotide substitutions), between the three species were as follows: M. arrokeana-M. grossa 11.73%, M. arrokeana-M. japonica 10.62% and M. grossa-M. japonica 10.97%. There was a clear gap between these interspecific divergences and the intraspecific ones: the mean intraspecific divergence within the ingroup species was 0.18% for M. arrokeana (range 0% to 0.92%), 0.13% for M. grossa (range 0% to 0.61%) and 0.02% for M. japonica (range 0% to 0.15%). It is noteworthy that within the R. sanguineus sequences selected as the outgroup (Table 1), the divergence between one (GenBank accession number HQ848580) of the sequences and the rest of the sequences of this species (GenBank AJ436938 and those collected by the authors) was more than 15% (15.75%), while the maximum divergence between the others was 100-fold lower (around 0.15%), suggesting that the COI sequence HQ848580 from GenBank is not actually from R. sanguineus. The five substitutions found among the M. grossa haplotypes were synonymous, as was the unique change among specimens of M. japonica, while of the 15 substitutions found among the haplotypes of M. arrokeana, three lead to amino acid changes.
Figure 2
Figure 2

Haplotype phylogeny estimated by Bayesian analysis of the Malacobdella species studied. The numbers above the branches represent the posterior probabilities; the numbers below the branches represent the bootstrap percentages of the MP and ML analysis, respectively. When a certain node was not recovered by one of the methods, a hyphen was added. Ma, GSJ, M. arrokeana; Mja, M. japonica; Mgrossa, M. grossa; Li and R.s, Ramphogordius sanguineus, from Argentina and GenBank respectively; A.l, Amphiporus lactifloreus; P.c, Paradrepanophorus crassus; G.p, Geonemertes pelaensis.


As previously mentioned, nemerteans of the Malacobdella genus are very difficult to identify based only on external characters. Rather, it is the internal morphology that provides the main taxonomic features used for identification, which can be differentiated only after a rigorous histological procedure. The genetic analysis performed here clearly shows that the distinguishing internal morphological differences used to separate the three species of Malacobdella analysed are concordant with differences in a fragment of the COI gene. However, previous DNA barcoding studies with nemerteans of the genera Tetrastemma and Cerebratulus revealed a lack of concordance between morphological and molecular characters (see Strand and Sundberg 2005; Sundberg et al. 2010). These authors argued that this difference could be the result of intraspecific variation or changes in the external morphology during development (Cantell 1975). They also concluded that the morphological characters used to describe Tetrastemma and Cerebratulus species are inadequate to identify evolutionary lineages. Our results showed that the divergence found between the sequences of the Malacobdella species (10% to 11% between the three species) are consistent with their status as distinct species. The values here found for intra- and interspecific divergences are in agreement with values found for other nemertean groups (e.g. Sundberg et al. 2010; Chen et al. 2010; Andrade et al. 2012; Kvist et al. 2013). The only exception was the divergence obtained between samples of R. sanguineus, 100-fold higher than other intraspecific values, suggesting that the COI sequence from GenBank (HQ848580) is not actually from R. sanguineus.

The phylogenetic analysis presented here showed that M. arrokeana and M. japonica are more closely related to each other than to M. grossa. Further studies using more molecular markers could clarify the phylogeny of the Malacobdella genus, taking into account the high host specificity of the majority of the species versus the cosmopolitan distribution and low host specificity of M. grossa. In this sense, the sequence of this last species from many hosts throughout its entire distribution would clarify whether M. grossa is indeed a single cosmopolitan species or a complex of species.

Based on the available literature by Takakura (1897), Yamaoka (1940), Riepen (1933), Gibson (1967, 1968, 1994), Gibson and Jennings (1969), Kozloff (1991), Ivanov et al. (2002) and the present work, the positions of the proboscis muscle and the nerve commissure appear to be good diagnostic characters for identifying species within the genus Malacobdella (since they are unique characteristic autapomorphies).

Several authors have concluded that nemertean species identification based only on morphological characters could be unreliable (see Strand and Sundberg 2005; Sundberg et al. 2009; Thornhill et al. 2008; Chen et al. 2010; Fernández-Álvarez and Machordom 2013; Kvist et al. 2013). More comprehensive future studies using the results presented here may strengthen species identification of other Malacobdella species by DNA barcoding.


Analysis of the COI (DNA barcoding) sequences presented in this work is clearly a powerful tool for species identification, at least of the three Malacobdella species studied. These molecular data are congruent with identifications based on internal morphological characters used in the original descriptions and re-descriptions of these three species and could be used to delineate between species in this genus with a similar external morphology.



The authors thank CONICET, Secretaría de Ciencia Tecnología e Innovación Productiva de la Provincia de Chubut, Agencia Española de Cooperación Internacional y Desarrollo (AECID: A/023484/09 and A/032441/10) and the iBOL-CONICET project, for financial support. We are grateful also to Melinda Modrell for her conscientious revision of the English.

Authors’ Affiliations

LARBIM, IBIOMAR- Centro Nacional Patagónico (CENPAT), CONICET, Bvd. Brown 2915, U9120ACV Puerto Madryn, Chubut, Argentina
Facultad de Ciencias Naturales, Universidad Nacional de la Patagonia San Juan Bosco (UNPSJB), Bvd. Brown S/N, U9120ACV Puerto Madryn, Chubut, Argentina
Faculty of Science, Hokkaido University, Kita 10, Nishi 8, Kita-ku, Sapporo 060-0810, Japan
The Swedish Species Information Centre, SLU, Bäcklösavägen 10, Box 7007, SE-750 07 Uppsala, Sweden
Department of Biological and Environmental Sciences, University of Gothenburg, Medicinaregatan 18, PO Box 463, SE-40530 Gothenburg, Sweden
Museo Nacional de Ciencias Naturales (MNCN), CSIC, José Gutiérrez Abascal, 2, 28006 Madrid, Spain


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