Background

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.

Methods

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
Full size table

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.

Results

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
Full size table

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.

Full size image

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.

Full size image

Discussion

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.

Conclusions

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.