DNA barcoding supports identification of Malacobdella species (Nemertea: Hoplonemertea)
© Alfaya et al.; licensee Springer. 2015
Received: 16 March 2014
Accepted: 16 December 2014
Published: 10 January 2015
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.
KeywordsDNA 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
List of species used in the analysis, including the sample locality, number of specimens analysed ( N ) and GenBank accession numbers
GenBank acc. number
References or voucher numbers a
San Matías Gulf, Argentina
Nuevo Gulf, Argentina
San José Gulf, Argentina
Mgrossa GB 1
Andrade et al. (2012)
Mgrossa GB 2
White Sea, Russia
Thollesson and Norenburg (2003)
Andrade et al. (2012)
Thollesson and Norenburg (2003)
Nuevo Gulf, Argentina
Andrade et al. (2012)
St. Davis, Bermuda
Mateos and Giribet (2008)
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).
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.
Characteristic features of the analysed Malacobdella species
M. arrokeana (Ivanov et al. 2002 )
M. japonica (Takakura 1897 )
M. grossa (Müller 1776)
Dorso-lateral (40% of length)
Dorso-lateral (25% of length) (Yamaoka 1940)
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)
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
High (only in Panopea abbreviata)
High (only in Spisula sachalinensis)
Low (27 species)
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)
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.
- Alfaya JEF, Bigatti G, Machordom A (2013) Mitochondrial and nuclear markers reveal a lack of genetic structure in the entocommensal nemertean Malacobdella arrokeana in the Patagonian gulfs. Helgoland Mar Res 67(2):407–412Google Scholar
- Andrade SCS, Strand M, Schwartz M, Chen H, Kajihara H, von Döhren J, Sun S, Junoy J, Thiel M, Norenburg JL, Turbeville JM, Giribet G, Sundberg P (2012) Disentangling ribbon worm relationships: multi-locus analysis supports traditional classification of the phylum Nemertea. Cladistics 28:141–159View ArticleGoogle Scholar
- Bucklin A, Steinke D, Blanco-Bercial L (2011) DNA barcoding of marine metazoa. Annu Rev Mar Sci 3:471–508View ArticleGoogle Scholar
- Cantell CE (1975) Anatomy, taxonomy, and biology of some Scandinavian heteronemertines of the genera Lineus, Micrura, and Cerebratulus. Sarsia 58:89–122Google Scholar
- Chen H, Strand M, Norenburg JL, Sun S, Kajihara H, Chernyshev AV, Maslakova SA, Sundberg P (2010) Statistical parsimony networks and species assemblages in Cephalotrichid nemerteans (Nemertea). PLoS ONE 5(9):e12885. doi:10.1371/journal.pone.0012885PubMed CentralPubMedView ArticleGoogle Scholar
- Coe WR (1945) Malacobdella minuta, a new commensal nemertean. J Wash Acad Sci 35:65–67Google Scholar
- de Blainville HM (1827) Sangsue. In: Levrault EG (ed) Dictionnaire des Sciences Naturelles, 47. Normant, Paris, pp 270–271Google Scholar
- Felsenstein J (1985) Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39:783–791View ArticleGoogle Scholar
- Fernández-Álvarez FÁ, Machordom A (2013) DNA barcoding reveals a cryptic nemertean invasion in Atlantic and Mediterranean waters. Helgoland Mar Res 67(3):599–605View ArticleGoogle Scholar
- Folmer O, Black M, Hoeh W, Lutz R, Vrijenhoek R (1994) DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Mol Mar Biol Biotech 3:294–299Google Scholar
- Gibson R (1967) Occurrence of the entocommensal rhynchocoelan, Malacobdella grossa, in the oval piddock, Zirfaea crispata, on the Yorkshire coast. J Mar Biol Assoc UK 49:301–317View ArticleGoogle Scholar
- Gibson R (1968) Studies on the biology of the entocommensal rhynchocoelan Malacobdella grossa. J Mar Biol Assoc UK 48:637–656View ArticleGoogle Scholar
- Gibson R (1994) Nemerteans. Synopses of the British fauna (new series, no. 24), 2nd edn. The Dorset Press, Dorchester, UKGoogle Scholar
- Gibson R (1995) Nemertean genera and species of the world: an annotated checklist of original names and description citations, synonyms, current taxonomic status, habitats and recorded zoogeographic distribution. J Nat Hist 29:1–562View ArticleGoogle Scholar
- Gibson R, Jennings JB (1969) Observations on the diet, feeding mechanisms, digestion and food reserves of the entocommensal rhynchocoelan Malacobdella grossa. J Mar Biol Assoc UK 49:17–32View ArticleGoogle Scholar
- Huelsenbeck JP (2000) MrBayes: Bayesian inference of phylogeny. University of Rochester, Rochester, NY, Department of BiologyGoogle Scholar
- Huelsenbeck JP, Ronquist F (2001) MrBayes: Bayesian inference of phylogeny. Bioinformatics 17:754–755PubMedView ArticleGoogle Scholar
- Ivanov VA, Bigatti G, Penchaszadeh PE, Norenburg JL (2002) Malacobdella arrokeana (Nemertea: Bdellonemertea), a new species of nemertean from the southwestern Atlantic Ocean entocommensal in Panopea abbreviata (Bivalvia, Heterodonta, Hiatellidae) in Argentina. P Biol Soc Wash 115:359–367Google Scholar
- Jensen K, Sadeghian PS (2005) Nemertea. In: Rohde K (ed) Marine parasitology. CSIRO press, Australia, pp 205–210Google Scholar
- Junoy J, Andrade SCS, Giribet G (2010) Phylogenetic placement of a new hoplonemertean species commensal on ascidians. Invertebr Syst 24:616–629View ArticleGoogle Scholar
- Kajihara H, Olympia M, Kobayashi N, Katoh T, Chen HX, Strand M, Sundberg P (2011) Systematics and phylogeny of the hoplonemertean genus Diplomma (Nemertea) based on molecular and morphological evidence. Zool J Linn Soc 161:695–722View ArticleGoogle Scholar
- Kozloff EN (1991) Malacobdella siliquae sp. nov. and Malacobdella macomae sp. nov., commensal nemerteans from bivalve mollusks on the Pacific coast of North America. Can J Zool 66:1612–1618View ArticleGoogle Scholar
- Kvist S, Laumer C, Junoy J, Giribet G (2013) New insights into the phylogeny, systematics and DNA barcoding of Nemertea. Invertebr Syst 28:287–308View ArticleGoogle Scholar
- Linnaeus C (1758) Systema naturae per regna tria naturae: secundum classes, ordines, genera, species, cum characteribus, differentiis, synonymis, locis, vol I, 10th edn. Laurentius Salvius, Stockholm, pp 824
- Linnaeus C (1767) Systema naturae per regna tria naturae, secundum classes, ordines, genera, species, cum characteribus & differentiis. Vol. II. Salvius, Holmiae, p 735
- Machordom A, Araujo R, Erpenbeck D, Ramos MA (2003) Phylogeography and conservation genetics of endangered European Margaritiferidae (Bivalvia: Unionoidea). Biol J Linn Soc 78:235–252View ArticleGoogle Scholar
- Maslakova SA, von Döhren J (2009) Larval development with transitory epidermis in Paranemertes peregrina and other hoplonemerteans. Biol Bull 216:273–292PubMedGoogle Scholar
- Mateos E, Giribet G (2008) Exploring the molecular diversity of terrestrial nemerteans (Hoplonemertea, Monostilifera, Acteonemertidae) in a continental landmass. Zool Scr 37:235–243View ArticleGoogle Scholar
- Müller OF (1776) Zoologiae Danicae prodromus, seu Animalium Daniae et Norvegiae indigenarum characters, nomina, et synonyma imprimis popularium. Typis Hallageriis, Havniae, Copenhagen
- Posada D (2003) Using Modeltest and PAUP* to select a model of nucleotide substitution. In: Baxevanis AD, Davison DB, Page RDM, Petsko GA, Stein LD, Stormo GD (eds) Current protocols in bioinformatics. John Wiley & Sons, New York, pp 6.5.1–6.5.14Google Scholar
- Posada D (2008) jModelTest: phylogenetic model averaging. Mol Biol Evol 25:1253–1256PubMedView ArticleGoogle Scholar
- Puerta P, Andrade SCS, Junoy J (2010) Redescription of Lineus acutifrons Southern, 1913 (Nemertea) and comments on its phylogenetic position. J Nat Hist 44:37–40View ArticleGoogle Scholar
- Riepen O (1933) Anatomie und histologic von Malacobdella grossa (Müll.). Z Wiss Zool Abt A 143:323–496Google Scholar
- 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. Biol J Linn Soc 86:201–212View ArticleGoogle Scholar
- Strand M, Hererra-Bachiller A, Nygren A, Kånneby T (2014) A new nemertean species: what are the useful characters for ribbon worm descriptions? J Mar Biol Assoc UK 94:317–330View ArticleGoogle Scholar
- Sundberg P, Strand M (2010) Nemertean taxonomy - time to change lane? J Zool Syst Evol Res 48:283–284Google Scholar
- Sundberg P, Thuróczy Vodoti E, Zhou H, Strand M (2009) Polymorphism hides cryptic species in Oerstedia dorsalis (Nemertea, Hoplonemertea). Biol J Linn Soc 98:556–567View ArticleGoogle Scholar
- Sundberg P, Vodoti E, Strand M (2010) DNA barcoding should accompany taxonomy the case of Cerebratulus spp. (Nemertea). Mol Ecol Res 10:274–281View ArticleGoogle Scholar
- Swofford DL (2000) PAUP*. Phylogenetic analysis using parsimony (*and other methods), Version 4. Sinauer Associates, Sunderland, Massachusetts, USA
- Taboada S, Junoy J, Andrade SCS, Giribet G, Cristobo J, Avila C (2013) On the identity of two Antarctic brooding nemerteans: redescription of Antarctonemertes valida (Burger, 1893) and description of a new species in the genus Antarctonemertes Friedrich, 1955 (Nemertea, Hoplonemertea). Polar Biol 36:1415–1430View ArticleGoogle Scholar
- Takakura U (1897) On a new species of Malacobdella (M. japonica). Annot Zool Japon 1:105–112Google Scholar
- Teso SV, Bigatti G, Bazterrica MC, Ciocco NF, Penchaszadeh PE (2006) The reproductive cycle of the entocommensal nemertean Malacobdella arrokeana and its symbiosis with the geoduck Panopea abbreviata. Invertebr Biol 125(4):314–324View ArticleGoogle Scholar
- Thollesson M, Norenburg JL (2003) Ribbon worm relationships: a phylogeny of the phylum Nemertea. P Roy Soc Lond B Biol 270:407–415View ArticleGoogle Scholar
- Thornhill DJ, Mahon AR, Norenburg JL, Halanych KM (2008) Open-ocean barriers to dispersal: a test case with the Antarctic Polar Front and the ribbon worm Parborlasia corrugatus (Nemertea: Lineidae). Mol Ecol 17:5104–5117PubMedView ArticleGoogle Scholar
- Wiens JJ (2007) Species delimitation: new approaches for discovering diversity. Syst Biol 56:875–878PubMedView ArticleGoogle Scholar
- Yamaoka T (1940) The fauna of Akkeshi Bay. IX. Nemertini. J Fac Sci, U Hokkaido 6(7):205–261Google Scholar
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited.