Zoologica Scripta
Integrative taxonomy of the freshwater worm Rhyacodrilus falciformis s.l. (Clitellata: Naididae), with the description of a new species SVANTE MARTINSSON,AINARA ACHURRA,MARCUS SVENSSON &CHRISTER ERSEUS
Submitted: 2 June 2013 Martinsson, S., Achurra, A., Svensson, M., Ers eus, C. (2013). Integrative taxonomy of the Accepted: 8 July 2013 freshwater worm Rhyacodrilus falciformis s.l. (Clitellata: Naididae), with the description of a doi:10.1111/zsc.12032 new species. —Zoologica Scripta, 42, 612–622. The genetic and morphological variation within Rhyacodrilus falciformis Bretscher, 1901 (Clitellata: Naididae) in Europe was explored using an integrative approach, with three unlinked genetic markers [the mitochondrial cytochrome c oxidase subunit I (COI), the nuclear histone 3 (H3) and internal transcribed spacer region (ITS)] combined with mor- phology, to investigate whether this taxon constitutes a single or several species. Using Automatic Barcode Gap Discovery on the COI data set, the specimens were divided into seven clusters, used as hypothetical species that were further tested with the other data sources. Single-gene trees were estimated for all three markers, using coalescence analysis and they were in many parts incongruent with each other. Only one of the clusters was sup- ported by all trees; it was also morphologically differentiated from the other clusters by the shape of its modified penial chaetae. This group consists of two specimens from the Crotot Cave in south-eastern France, and morphologically they fit a previously described but inva- lid variety, ‘pigueti’, which is here described as a new species, Rhyacodrilus pigueti Achurra & Martinsson sp. n. The study highlights the fact that a single data source (e.g. COI barcodes) seldom provides a sufficient basis for taxonomic decisions such as species delimitation. Corresponding author: Svante Martinsson, Department of Biological and Environmental Sciences, University of Gothenburg, Box 463, SE-405 30 G€oteborg, Sweden. E-mail: svante.martinsson@ bioenv.gu.se Ainara Achurra, Station d’Ecologie Exp erimentale du CNRS a Moulis, USR 2936, 09200, Moulis, France and Department of Zoology and Animal Cell Biology, Faculty of Science and Technology, University of the Basque Country, Box 644, 48080, Bilbao, Spain. E-mails: [email protected] Marcus Svensson, Christer Ers eus, and Svante Martinsson, Department of Biological and Environ- mental Sciences, University of Gothenburg, Box 463, SE-405 30, G€oteborg, Sweden. E-mails: [email protected], [email protected], [email protected]
Introduction et al. 2009; Envall et al. 2012; Matamoros et al. 2012; Novo Cryptic species are species that are morphologically indis- et al. 2012). tinguishable or so similar that they have been classified The mitochondrial (mt) genome is haploid and almost under the same species name (Bickford et al. 2007). This exclusively transmitted maternally, which reduces the effec- seems to be a common phenomenon among several organ- tive population size (Ne) to generally one-fourth of that of ismal groups, not the least among clitellate worms (see nuclear markers (Birky et al. 1989), increasing the genetic review by Ers eus & Gustafsson 2009). Due to the lack of drift fourfold, resulting in faster lineage sorting and shorter externally discernible characters, especially in immature time to monophyly (Neigel & Avise 1986). This together specimens, many clitellate species have proved hard to dis- with the fact that mt genes normally evolve several times tinguish without the aid of molecular markers, and the faster than nuclear genes (Brown et al. 1979, 1982; Lin & diversity has in many cases been shown to be underesti- Danforth 2004) has made such markers widely used in mated when based on morphology alone (e.g. Gustafsson studies of recent divergence and species delimitation in
612 ª 2013 The Norwegian Academy of Science and Letters, 42, 6, November 2013, pp 612–622 S. Martinsson et al. Integrative taxonomy of Rhyacodrilus falciformis s.l. several animal groups including clitellates (e.g. Heethoff DNA barcoding, large genetic variation was observed in et al. 2004; James et al. 2010; Dozsa-Farkas et al. 2012). R. falciformis. The cytochrome c oxidase subunit I (COI) gene is the most This study’s aim is to explore the genetic and morpho- often used marker in such studies, and a fragment of COI logical variation within Rhyacodrilus falciformis in order to has been proposed as the standard ‘DNA barcode’ for iden- investigate whether this taxon comprises a complex of cryp- tifying animal taxa (Hebert et al. 2003). The identification tic species or not. is often based on the assumption that intraspecific diver- gence is distinctly lower than interspecific divergence (the Material and methods so-called barcoding gap) and is performed by comparing Taxa and specimens the sequence of an unidentified individual with known Thirty-six specimens of Rhyacodrilus falciformis were col- sequences in a database (see review by Taylor & Harris lected from various locations in France, Great Britain, 2012). However, it has been shown that the use of single Spain and Sweden, between 2003 and 2012 (Table 1). Wet mt markers for species delimitation can overestimate the soil samples and sandy sediments were taken in aquatic numbers of species if used alone, that is, without nuclear habitats (e.g. groundwater springs, caves, streams, ponds markers (e.g. King et al. 2008; Dasmahapatra et al. 2010; and lakes) for sieving and sorting under a dissecting micro- Torres-Leguizamon et al. 2012; Achurra & Ers eus 2013). scope in the laboratory. After preliminary identification, Species delimitation and species conceptualisation have specimens were divided into two parts; the anterior parts long been debated among biologists, mostly regarding the were stained with paracarmine and mounted in Canada bal- species criteria. Different schools within the field have put sam as outlined by Ers eus (1994), as hologenophore vouch- forward their own different species concepts, based on, ers (sensu Pleijel et al. 2008), and for morphological for example, reproductive isolation (Mayr 1942), specific examinations, whereas the rear ends were transferred into mate recognition (Paterson 1985), different ecological 95% ethanol for subsequent DNA extraction and sequenc- niches (Van Valen 1976), monophyly (Rosen 1979) and ing. For specimen CE14049, only a fragment was available diagnosability (Nelson & Platnick 1981). De Queiroz and used for DNA extraction. (2007) proposed a unified species concept, where being a sep- arately evolving metapopulation lineage is the sole DNA sequencing and assembly requirement of a species and where all other criteria (sec- The posterior parts of all 36 individuals were selected for ondary species criteria) are used for assessing lineage sepa- DNA extraction, using Qiagen’s DNeasy Blood & Tissue ration (species delimitation). The greater the number of Kit or Epicentre QuickExtract DNA Extraction Solution secondary species criteria supporting a divergence, the 1.0, following the manufacturer’s instructions. One speci- stronger the case is for speciation. However, one piece of men was handled by Canadian Centre for DNA Barcoding evidence, if properly examined, may be enough to estab- (CCDB; Guelph, ON, Canada), with data stored at the Bar- lish lineage separation. De Queiroz’s view, which will be code of Life Data systems (BOLD). Parts of the mitochon- shared throughout this paper, is useful when dealing with drial cytochrome c oxidase subunit I (COI), the nuclear cryptic species. histone 3 (H3) and complete internal transcribed spacer Rhyacodrilus falciformis Bretscher, 1901 (Clitellata: Naidi- region (ITS; ITS1, 5.8S rDNA and ITS2) were amplified dae sensu Ers eus et al. 2008) is widespread in the Holarctic using primers and PCR programs listed in Table S1. After region. It is found in freshwater bodies, but also in wet soil amplification by PCR, the existence of the target genes was and seems to be associated with ground water (Timm et al. tested using 1% agarose gel electrophoresis. PCR products 1996; Ers eus et al. 2005; Dumnicka 2006; Achurra & were purified using exonuclease I (Fermentas, Burlington, Rodriguez 2008), and it may therefore be regarded as a Canada) and FastAP thermosensitive alkaline phosphatase stygophilic species (Giani et al. 2011). The worm is about (Fermentas). Sequencing was carried out by Macrogen Inc. 5 mm long and can be identified by the sickle-shaped pe- (Seoul, Korea) and Eurofins MWG Operon (Ebersberg, nial chaetae, which have given the species its name. These Germany). Sequences were assembled in GENEIOUS PRO v. chaetae are located in close proximity to the male pores in 5.6.3 (Biomatters Ltd., Auckland, New Zealand) and aligned sexually mature specimens. A variety, ‘pigueti’, with straight using MAFFT v6.814b (Katoh et al. 2002) as implemented in penial chaetae was described by Juget (1967) from Lake Geneious using the auto algorithm. L eman on the border between France and Switzerland. However, as the description of this variety was published Distance analyses after 1960, the name is not available and has no nomencla- Pairwise genetic distances were calculated for the COI data torial status (ICZN 1999: §15.2). In the course of the study set in MEGA 5.1 using both uncorrected p-distances and the of the diversity of European freshwater clitellates using Tamura–Nei, 1993 (TN93) model, using pairwise deletion
ª 2013 The Norwegian Academy of Science and Letters, 42, 6, November 2013, pp 612–622 613 Integrative taxonomy of Rhyacodrilus falciformis s.l. S. Martinsson et al.
Table 1 List of specimens included in the study, collection localities and dates, GenBank accession nos. and voucher nos
GenBank accession no. COI Species ID no. cluster Collection locality Collection date COI ITS H3 Voucher no.
Rhyacodrilus falciformis CE631-1 E Vit€artk€allan Spring, Gotland, SE September KF267920 KF267973 KF267939 SMNH130504 2003 Rhyacodrilus falciformis CE637 E Vit€artk€allan Spring, Gotland, SE September KF267935 KF267974 KF267940 SMNH130505 2003 Rhyacodrilus falciformis CE795-1 F Wet soil, H€allekis, G€otene, SE May 2004 KF267926 KF267990 KF267941 SMNH130506 Rhyacodrilus falciformis CE795-2 F Wet soil, H€allekis, G€otene, SE May 2004 KF267927 KF267991 KF267942 SMNH130507 Rhyacodrilus falciformis CE795-3 F Wet soil, H€allekis, G€otene, SE May 2004 KF267928 – KF267943 SMNH130508 Rhyacodrilus falciformis CE836 F S€ave an River, Lerum, SE June 2004 KF267929 KF267988 KF267944 SMNH130509 Rhyacodrilus falciformis CE1203 D Swamp, Rya skog, Hisingen, SE November KF267910 – KF267945 SMNH130510 2005 Rhyacodrilus falciformis CE3868 F Aspen Lake, Lerum, SE April 2008 KF267913 KF267975 KF267956 SMNH130511 Rhyacodrilus falciformis CE3869 F Aspen Lake, Lerum, SE April 2008 KF267914 – KF267946 SMNH130512 Rhyacodrilus falciformis CE3870 F Aspen Lake, Lerum, SE April 2008 KF267915 KF267976 KF267947 SMNH130513 Rhyacodrilus falciformis CE4387 G Stream, Omberg, Stocklycke, Odesh€ €og, SE July 2008 KF267916 KF267977 KF267948 SMNH130514 Rhyacodrilus falciformis CE4388 G Stream, Omberg, Stocklycke, Odesh€ €og, SE July 2008 KF267917 KF267978 KF267949 SMNH130515 Rhyacodrilus falciformis CE4563 F Stream, Fr€os€on, Ostersund,€ SE July 2008 KF267918 KF267989 KF267950 SMNH130516 Rhyacodrilus falciformis CE4564 G Stream, Fr€os€on, Ostersund,€ SE July 2008 KF267919 KF267980 KF267951 SMNH130517 Rhyacodrilus falciformis CE6611 F Wet soil, Uppsala Botanical Garden, SE June 2009 IN260080a ––SMNH130518 Rhyacodrilus falciformis CE6612 F Wet soil, Uppsala Botanical Garden, SE June 2009 KF267921 KF267979 KF267957 SMNH130519 Rhyacodrilus falciformis CE6615 D Wet soil, Uppsala Botanical Garden, SE June 2009 KF267922 – KF267958 SMNH130520 Rhyacodrilus falciformis CE6631 E Pr€astfj€arden Lake, Akersberga, SE June 2009 KF267923 KF267987 KF267952 SMNH130521 Rhyacodrilus falciformis CE6632 E Pr€astfj€arden Lake, Akersberga, SE June 2009 KF267924 – KF267953 SMNH130522 Rhyacodrilus falciformis CE7737 F Stream, Guldheden, G€oteborg, SE May 2010 KF267925 – KF267954 SMNH130523 Rhyacodrilus falciformis CE8714 C Borehole, Abbotshood Farm, Devon, GB January 2010 KF267930 KF267983 KF267955 SMNH130524 Rhyacodrilus falciformis CE10955 C Spring, Greatcombe Head Farm, Devon, January 2010 KF267901 – KF267959 SMNH130525 GB Rhyacodrilus falciformis CE11311 G Ogonak€ €allan Spring, Ostansj€ €o, Hallsberg, April 2011 KF267902 KF267981 KF267960 SMNH130526 SE Rhyacodrilus falciformis CE11339 G Spring, Oglunda,€ Skara, SE April 2011 KF267903 KF267982 KF267961 SMNH130527 Rhyacodrilus falciformis CE11351 F Stream, Guldheden, G€oteborg, SE April 2011 KF267904 – KF267962 SMNH130528 Rhyacodrilus falciformis CE11366 F Ditch, Guldheden, G€oteborg, SE April 2011 KF267900 – KF267963 SMNH130529 Rhyacodrilus falciformis CE11405 E Stream,Vitsippsdalen, G€oteborg, SE March 2011 KF267905 – KF267970 SMNH130530 Rhyacodrilus falciformis CE11406 E Stream,Vitsippsdalen, G€oteborg, SE March 2011 KF267906 KF267984 KF267964 SMNH130531 Rhyacodrilus falciformis CE11451 F Ditch, Guldheden, G€oteborg, SE March 2011 KF267907 KF267992 KF267965 SMNH130532 Rhyacodrilus falciformis CE11452 D Ditch, Guldheden, G€oteborg, SE March 2011 KF267908 KF267985 KF267966 SMNH130533 Rhyacodrilus falciformis CE11489 E Ground water seepage, Br€ann as, Lerum, April 2011 KF267909 KF267986 KF267967 SMNH130534 SE Rhyacodrilus pigueti sp. CE14048 A Crotot Cave, Besancßon, Dep. Doubs FR January 2011 KF267911 KF267993 KF267968 SMNH Type- n. 8475b Rhyacodrilus pigueti sp. CE14049 A Crotot Cave, Besancßon, Dep. Doubs FR January 2011 KF267912 KF267994 KF267969 No voucher n. Rhyacodrilus falciformis CE16690 B Jivero Cave, Matienzo, Cantabria, ES October 2012 KF267932 – KF267938 SMNH130535 Rhyacodrilus falciformis CE16691 B Jivero Cave, Matienzo, Cantabria, ES October 2012 KF267931 – KF267937 SMNH130536 Rhyacodrilus falciformis CE16692 B Jivero Cave, Matienzo, Cantabria, ES October 2012 KF267933 – KF267936 SMNH130537 Rhyacodrilus coccineus CE623 – Stream, Alings as, SE July 2003 GU902110c KF267996 KF267971 No voucher Rhyacodrilus CE10572 – Spring, Willesleigh Farm, Devon, GB January 2010 KF267934 KF267995 KF267972 SMNH130538 subterraneus
SE, Sweden; GB, Great Britain; FR, France, ES, Spain. aBOLD accession no: ENSWD081-11, bHolotype, cFrom Ers eus et al. (2010).
for missing data. Models were chosen after model testing The TN93 distances were analysed with the online version conducted in MEGA 5.1 (Tamura et al. 2011) using the of ABGD (Automatic Barcode Gap Discovery; Puillandre Bayesian information criterion (BIC). Histograms of dis- et al. 2012; available at http://wwwabi.snv.jussieu.fr/public/ tances were drawn in Microsoft Excel. abgd/abgdweb.html) with default settings, to delimit
614 ª 2013 The Norwegian Academy of Science and Letters, 42, 6, November 2013, pp 612–622 S. Martinsson et al. Integrative taxonomy of Rhyacodrilus falciformis s.l. mitochondrial clusters. The ABGD method detects the first Morphological study significant gap (the barcoding gap) between the two modes of The microscopically studied material included 24 mature pairwise distance distributions, where the lower distance val- specimens and seven immatures or fragments. For the ues presumably reflect the intraspecific differences and the detailed morphological analysis of the reproductive organs, higher the interspecific differences. The delimited COI clus- only completely mature specimens (i.e. with eggs or with ters were used as hypothetical species that were further tested. sperm in the spermathecal ampulla) were selected, while for external characters, we included all available specimens. Coalescent analyses This selection guarantees that differences in measurements Genealogies were estimated for the COI, ITS and H3 are not due to incompletely developed organs. Only ante- data sets under the null hypothesis that all the included rior parts (approximately segments I–XV) were examined specimens of R. falciformis constitute a single species. because posterior parts were used for the molecular study. Estimations were performed using Bayesian coalescence External characters are related to the number and size of analysis as implemented in the BEAST package (Drum- somatic chaetae, and internal characters are related to the mond & Rambaut 2007; Drummond et al. 2012). All reproductive organs: spermathecae, male ducts and penial .xml input files were created using BEAUTI v1.7.4 (Drum- chaetae. The width of segment XI was included to repre- mond & Rambaut 2007), with the TN93 + I model for sent the size of each specimen. COI, Jukes-Cantor, 1969 (JC69) + Γ model for ITS and Hasegawa–Kishino–Yano (HKY) + Γ model for H3; the Concatenated phylogenetic analysis model testing was conducted in MEGA 5.1 using the To test whether the two species found (see Results) were Bayesian information criterion (BIC). For all analyses, reciprocally monophyletic, a phylogenetic analysis was per- the following settings were used: base frequencies ‘esti- formed on a concatenated data set. mated’; clock model ‘lognormal relaxed clock (uncorrelat- The three single-gene matrices were concatenated into ed)’; tree prior ‘coalescent/constant size’; constant.popSize one data matrix using GENEIOUS PRO v. 5.6.3, with one spec- ‘lognormal: Log(Mean) = 0, Log(Stdev) = 1, offset = 0’. imen each of Rhyacodrilus coccineus and R. subterraneus added For other priors, default settings were used. The analyses as out-groups (see Table 1). A phylogenetic tree was esti- were run in BEASTMC3 v. 1.7.4 (Drummond & Rambaut mated using maximum likelihood; the analysis was per- 2007; Drummond et al. 2012), using three chains, a delta formed with PHYML 3.0 (Guindon & Gascuel 2003; value of 1.00 and swapping chains every 100 generations. Guindon et al. 2010) as implemented at the ATGC Mont- The COI and H3 analyses were run for 10 million gen- pellier bioinformatics platform (http://www.atgc-montpel- erations and the ITS analysis for 20 million generations, lier.fr/). The TN93 model with Γ parameter estimated sampling every 1000th generation. The number of gener- from the data was used after model testing in MEGA 5.2, ations was chosen to obtain sufficient effective sample and SPR+NNI was used for tree improvement. Branch size (ESS). TRACER v1.5 (Rambaut & Drummond 2007) support was calculated with the chi-square-based approxi- was used for examining ESS for parameters and deter- mative likelihood ratio test (aLRT; Anisimova & Gascuel mining the burn-in. Trees were summarised using TRE- 2006) in PHYML. EANNOTATOR v1.7.4. (Drummond & Rambaut 2007), discarding the first 10% as burn-in. Data deposition All new sequences generated in this study are deposited in Molecular species delimitation GenBank, and submission numbers are listed in Table 1; The gene trees were imported into Geneious, and in the all xml files used in the BEAST analyses, as well as log files cases where the previously delimited clusters were found from the analyses, are deposited in the Dryad Data Reposi- monophyletic, the species delimitation plug-in (Masters tory (http://www.datadryad.org/) at DOI:10.5061/dryad. et al. 2011) was used to calculate (i) P (Randomly Distinct), 1fb3d; trees and matrices are deposited in TREEBASE (http:// that is, the probability for clades to have the observed treebase.org/), submission TB2:S14391. Vouchers are degree of distinctiveness due to random coalescent pro- deposited in the Swedish Museum of Natural History, ’ cesses (Rodrigo et al. 2008) and (ii) Rosenberg sPAB that Stockholm; accession numbers are given in Table 1. expresses the probability of reciprocal monophyly of the clade of interest and its nearest defined group, under ran- Results dom branching (Rosenberg 2007). Both statistics were used DNA sequencing to test whether or not the null hypothesis of random coa- DNA from 36 samples of Rhyacodrilus falciformis s.l. was lescence can be rejected as the explanation for phylogenetic successfully amplified and sequenced with regard to COI, structure. whereas in the cases of ITS, 22 specimens, and H3, 35
ª 2013 The Norwegian Academy of Science and Letters, 42, 6, November 2013, pp 612–622 615 Integrative taxonomy of Rhyacodrilus falciformis s.l. S. Martinsson et al. specimens were successfully sequenced (Tables 1 and S2). variation between 1.3% and 6.0%. One cluster contains the More details on the alignments are given in Table S2. specimens from France (cluster A), one the Spanish speci- mens (cluster B), one the English specimens (cluster C) Distance analysis and four groups includes specimens from Sweden (clusters The maximum pairwise distance values within R. falciformis D–G). s.l. for COI were 12.5% using uncorrected p-distances and 14.0% using TN93 distances. A barcoding gap was Coalescent analyses observed between 2.9 and 4.6% pairwise differences using In the COI tree (Fig. 1B), each of the seven groups recog- uncorrected p-distances and between 3.0 and 4.9% using nised by the ABGD analysis was found monophyletic with TN93 distances (Fig. 1A). The ABGD analysis of the COI maximum support. The French specimens (cluster A) are data set yielded seven mitochondrial clusters, further used the sister group to all other R. falciformis [posterior proba- as hypothetical species, assuming maximal intraspecific bility (pp) 0.94], whereas the clade with the British
AB
CD
Fig. 1 Distribution of the genetic variation within Rhyacodrilus falciformis s.l. —A Histogram of pairwise COI distances given in both uncorrected p-distances and corrected TN93 distances. —B, C Gene trees estimated with Bayesian coalescent analysis in BEAST. Letters right of specimen codes represent COI cluster, if clusters are found monophyletic is a line followed by the letter shown right of the specimens. Letter A represents Rhyacodrilus pigueti sp.n. and B–G R. falciformis s.s. Numbers above branches are posterior probability only values above 0.50 are given. Scale shows expected numbers of substation per site. —B COI gene tree. —C ITS gene tree. —D H3 gene tree.
616 ª 2013 The Norwegian Academy of Science and Letters, 42, 6, November 2013, pp 612–622 S. Martinsson et al. Integrative taxonomy of Rhyacodrilus falciformis s.l.
’ ≤ specimens (cluster C) is found as sister group to one of the Rosenberg sPAB 0.05, whereas P(Randomly Distinct) was clades from Sweden (cluster D; pp 1), and the Spanish between 0.47 and 0.73. cluster B is sister group to them with low support (pp 0.82). Morphological study In the ITS tree (Fig. 1C), there are five well-supported All examined mature specimens fit with previous descrip- clades (pp > 0.99). One of them is identical to the French tions of Rhyacodrilus falciformis Bretscher, 1901 (Piguet cluster (A), while the other clades either consist of speci- 1906; Hrab e 1935; Juget 1967; Kasprzak 1979; Chekanovs- mens from several clusters or contain only some of the kaya 1981; Timm et al. 1996) except for individual specimens from one cluster. Unfortunately, none of the CE14048, which shows different genital chaetae (Fig. 2). specimens from Spain and only one specimen from Eng- We compared the ranges of the values of morphological land was successfully sequenced; the latter specimen characters across the COI clusters to search for congruence (CE8714) having the same ITS haplotype as one Swedish with molecular results (Table S3). There is no evidence for specimen (CE11406). The French cluster A is nested morphological differentiation in reproductive characters among the other clades, but the relationships between most among the clusters, except the different genital chaetae in of the clades are unsupported. specimen CE14048 (cluster A). Slight differences in num- In the H3 tree (Fig. 1D), clusters A, B and C are mono- ber and size of somatic chaetae (external characters) are phyletic with maximum support. None of the other mt frequent among populations and are thus not considered clusters are recovered in the analysis, and the relationships species specific. At specimen level, such differences may be between groups are generally without support. due to the use and replacement of chaetae. The number of genital chaetae as well as their shape and size is considered Molecular species delimitation to be a diagnostic character in species of the genus Rhyaco- In the Geneious species delimitation analyses on the COI drilus Bretscher, 1901 (Timm 2009; Rodriguez & Fend genealogy, the null hypothesis of observing reciprocal 2013). monophyly by chance could be rejected for all groups ’ ≤ [Rosenberg sPAB 0.05], and the null hypothesis that the Concatenated phylogenetic analysis observed degree of distinctiveness is due to random coales- The maximum likelihood tree based on combined COI, cence processes could be rejected [P (Randomly Distinct) H3 and ITS (Fig. S1) shows a coherent, maximally sup- <0.05] for all groups except cluster G [P(Randomly Dis- ported group of all specimens of Rhyacodrilus falciformis tinct) = 0.10]. In the ITS genealogy, all five well-supported s.l. All clusters except cluster E are recovered with maxi- ’ ≤ clades (including cluster A) had a Rosenberg sPAB 0.05, mal support, and cluster E is paraphyletic with respect to whereas cluster A had a P (Randomly Distinct) of 0.12 and P a clade consisting of clusters B–D. Cluster A, that is, (Randomly Distinct) varied between 0.11 and 0.45 for the Rhyacodrilus pigueti sp.n., is found as sister group to all other clades. In the H3 genealogy, only clades A–C were other clusters, Rhyacodrilus falciformis s.s. with a support monophyletic and could be tested. For these groups, of 0.99.
A BC
Fig. 2 Differences in the penial chaetae between Rhyacodrilus pigueti sp.n. and R. falciformis. —A. Rhyacodrilus falciformis. —B. Rhyacodrilus pigueti, Holotype. —C Drawings of the penial chaetae in R. pigueti (straight chaeta) and R. falciformis (sickle-shaped chaeta). Scale bars = 50 lm.
ª 2013 The Norwegian Academy of Science and Letters, 42, 6, November 2013, pp 612–622 617 Integrative taxonomy of Rhyacodrilus falciformis s.l. S. Martinsson et al.
Other material, genetically examined. CE14049, only molecular data, no voucher available. COI barcode sequence, GenBank acc. no. KF267912; ITS sequence, GenBank acc. no. KF267994; H3 sequence, GenBank acc. no. KF267969.
Etymology. Named after Emile Piguet, a specialist on aquatic oligochaetes of the early 20th century. The new material fits the description of R. falciformis var. pigueti Ju- get, 1967, but as varieties published after 1961 are invalid, Juget’s name pigueti is not available (§15.2 and §45.5 ICZN 1999). Therefore, the name pigueti is here proposed as new for this taxon, now regarded as a species. According to Ju- get 1967, Emile Piguet had once found specimens with the characters of Juget’s variety, but Piguet only identified Fig. 3 Summary of the results, indicating the amount of support them as Rhyacodrilus sp. and never formally described them for each COI cluster. For COI, ITS and H3 clusters that were monophyletic are indicated with a grey bar, pale grey indicates a taxonomically. ’ ≤ ’ ≤ Rosenberg sPAB 0.05 and dark grey both a Rosenberg sPAB 0.05 and a P(Randomly Distinct) ≤0.05. Cluster A represents Rhyacodrilus Diagnosis. This species can be distinguished from other pigueti sp.n. and clusters B–G R. falciformis s.s. species in Rhyacodrilus by the shape of the penial chaetae (Fig. 2) and by genetic data (Fig. 3). See also Remarks and Discussion below. Therefore, in the light of molecular and morphological data (results summarised in Fig. 3) and further discussed in Description. Incomplete specimen, with 16 anteriormost Discussion, we consider cluster A as a separate species from segments 1.93 mm long, body width 265 lm in segment R. falciformis s.s. The shape of the penial chaetae of V, 380 lm in segment XI. Prostomium 120 lm long, CE14048 is in accordance with the description of R. falci- 132 lm wide. Coelomocytes abundant within coelomic cav- formis var. pigueti Juget, 1967. However, a new name pub- ity of all segments, round, 27 lm diameter, granulated, lished after 1960 expressly as a ‘variety’ is considered to be when nuclei stained with paracarmine. Clitellum indistin- infrasubspecific and as such does not formally exist (ICZN guishable (poorly developed?). Spermathecal pores midlat- 1999: §15.2). Therefore, a new species is proposed below eral, located anteriorly in segment X. One pair of male for this form, including the two specimens in cluster A. pores open ventrally in segment XI. Dorsal and ventral chaetae of identical shape, bifid, with Taxonomy distal tooth somewhat thinner and longer than proximal. Genus Rhyacodrilus Bretscher, 1901 Anteriorly 3–5 chaetae per bundle, 1–2 in segments XI–XV Rhyacodrilus pigueti Achurra & Martinsson, and 2–3 in segment XVI; maximum length 92 lm, 2–3 lm sp. n. (Figure 2A, C). thick. Modified penial chaetae in segment XI, a single Rhyacodrilus falciformis, var. pigueti Juget, 1967: Fig. 2B, chaeta per ‘bundle’, straight, somewhat spoon-shaped Table 1. (Fig. 2A, C); chaeta 150 lm long, 15 lm thick in proximal and middle parts, narrowing to 10 lm before widening to Holotype. CE14048, whole-mounted worm, anterior part 15 lm in the distal portion. Proximal end of penial chaeta (segments I–XVI only), stained in paracarmine and associated with conspicuous chaetal gland (diameter 75 lm) mounted in Canada balsam. Collected by Michel C. des and strong muscular strands. Distal end located within Chatelliers, 26 Jan 2011. Deposited in Swedish Museum of male pore. Penial chaetae orientated with their distal ends Natural History (SMNH), Stockholm, accession no. towards segment X. SMNH Type-8475. COI barcode sequence, GenBank acc. Atria paired in segment XI, close to septum XI/XII, no. KF267911; ITS sequence, GenBank acc. no. elongate, pear-shaped, ampullae 150 lm long, maximum KF267993; H3 sequence, GenBank acc. no. KF267968. diameter 50 lm, with about 10 lm-thick muscular layer. Proximal end of atrium barely observed due to the pres- Type locality. Crotot Cave, north-east of Besancßon, ence of the assembly of penial chaeta + chaetal D epartment du Doubs, France (47°26′44.52′′N,6°24′31.64′′ gland + muscular strands. Abundant prostatic cells covering E, DMS datum WGS84; elevation: 375 m). proximal and middle parts of atrium. Spermathecae paired,
618 ª 2013 The Norwegian Academy of Science and Letters, 42, 6, November 2013, pp 612–622 S. Martinsson et al. Integrative taxonomy of Rhyacodrilus falciformis s.l. ampullae 96 lm diameter, 60 lm long, with a single-layer and mixed in different clades. In the case of ITS, five well- – l fi ’ ≤ epithelium 5 10 m thick; lumen lled with random mass supported clades were found, with a Rosenberg sPAB 0.05, of sperm; duct 80 lm long, 60 lm diameter. whereof one constitutes cluster A, that is, Rhyacodrilus pigueti. Distribution and habitat. Crotot Cave is a large cavity The molecular operational taxonomic units (MOTUs; (5800 m long), in Jurassic limestone, on a plateau near the Floyd et al. 2002) that will be defined thus differ consider- valley of the Doubs River (Departement du Doubs), east- ably between the genes, both in numbers and constitution. ern France. Following Juget (1967), the new species has This highlights the problems with using a single genetic also been found in the deep zone of Lake L eman (Lake marker for delimiting taxonomic units. Instead using Geneva), in Switzerland and France, approximately 100 km an integrative approach (see review by Schlick-Steiner away from the Crotot Cave. et al. 2010), combining several genetic markers as well as other data sources can result in more robust taxonomic Remarks. Rhyacodrilus pigueti sp. n. is well distinguished hypotheses. from R. falciformis s.s. by the shape of the penial chaetae. The support for the hypothesis that the delimited COI- The chaetae are sickle-shaped in R. falciformis, while they based groups represent separately evolving lineages varies. are straight and somewhat spoon-shaped in the new species Some of the groups are supported by COI only, others by (Fig. 2). In R. falciformis s.s., the ratio of the distance from two loci, but only cluster A, here regarded as Rhyacodrilus the focal point of the sickle to the dorsal axis of the shaft pigueti sp. n., is supported by all genes as well as morphol- (indicated in Fig. 2C) and the chaetal length (mean ratio is ogy (Fig. 3). The differences in topology between the gene 0.06 in R. falciformis, 0.02–0.10, n = 7; ratio is 0.03 in trees suggest that gene flow is present between some of the R. piguet, n = 1). However, this measurement should be different COI-based groups and that speciation – if at all in interpreted with caution as it seems to vary with the orien- progress – is not yet complete between them. However, tation of the chaeta. The distal end of the penial chaeta is recent speciation events, where lineage sorting of nuclear more pointed and slender in R. falciformis than in the new genes still is incomplete and morphological differences not species, in which the distal part is widening proximal to the yet have accumulated to any discernible degree, cannot be apex. In addition, the penial chaetae are 150 lm long in the ruled out, especially not as some of the COI clusters show new species, while they are between 100 and 140 lm long a clear geographical pattern. in our specimens of R. falciformis. Other measurements in The shape of the penial chaetae is the only morphologi- the literature also show slightly shorter penial chaetae for cal diagnostic character that we found to separate the new R. falciformis: 100–138 lm (Piguet 1906; Hrab e 1935; Juget species from R. falciformis s.s., but together with the molec- 1967; Kasprzak 1979; Chekanovskaya 1981; Timm et al. ular data, it gives us strong support for R. pigueti being a 1996), but no measurements of the penial chaetae are given separately evolving lineage. The integration of independent in the original description (Bretscher 1901). data sources is here crucial for a proper determination of the species boundaries in R. falciformis s.l.; neither individ- Discussion ual gene trees nor the morphological data alone provide Based on the results from the ABGD and species delimita- enough support to clearly delimit the two species. New tion tests on the COI data set, it seems that Rhyacodrilus techniques may reveal other putative diagnostic anatomical falciformis consists of 6–7 different species, one of which structures that are currently not observable. For instance, morphologically conforms to the ‘pigueti’ form reported by Cuadrado & Martinez-Ansemil (2001), using scanning elec- Juget (1967). However, the barcoding gap is small, 1.7– tron microscopy (SEM), found several bundles of cilia near 1.9% depending on model, and there is no barcode gap the male pores in specimens of R. falciformis that had not between Rhyacodrilus pigueti and R. falciformis s.s. Several been observed with other techniques. Such ultrastructural other studies have found a much larger barcoding gap details may deserve attention in future taxonomic work on between clitellate species (e.g. Gustafsson et al. 2009; Kvist cryptic species of aquatic clitellates. However, SEM is a et al. 2010; Richard et al. 2010; Envall et al. 2012; Matamo- destructive and rather expensive technique and may there- ros et al. 2012). The difference in barcoding gap between fore be of little help in specimen identification. our study and others may be explained by the age of the It is possible that there are physiological and/or ecologi- lineages. For instance, the split between Rhyacodrilus pigueti cal differences between the two species as has been shown and R. falciformis s.s. could be distinctly younger than the for the different lineages within Tubifex tubifex (Sturmbauer splits between the taxa in the other studies. In the nuclear et al., 1999; Beauchamp et al. 2002). gene trees, members of the hypothetical species suggested The separation of R. pigueti seems to render R. falciformis by the mtDNA analyses are in many cases found scattered paraphyletic in the ITS and H3 trees, but the support for
ª 2013 The Norwegian Academy of Science and Letters, 42, 6, November 2013, pp 612–622 619 Integrative taxonomy of Rhyacodrilus falciformis s.l. S. Martinsson et al. the topologies is weak or lacking, and in the concatenated et al. 2012; Torres-Leguizamon et al. 2012). Nevertheless, analysis, R. falciformis is found monophyletic with high sup- we certainly recognise the usefulness of DNA barcoding in port. However, even if the topologies should prove to be specimen identification, once species boundaries have been correct despite the low or lacking support, non-monophyly properly established. of species in single-gene trees is not uncommon (Crisp & Chandler 1996; Funk & Omland 2003), and the reason in Acknowledgements this case is likely to be recent separation and incomplete This collaborative effort was made possible by a Zoologica lineage sorting. The Ne of the nuclear genes is greater than Scripta Travel Fellowship, from The Norwegian Academy that of mitochondrial genes and it takes a longer time for of Science and Letters to the second author. Further, we such genes to reach complete lineage sorting and reciprocal are grateful to Alexander B€ar, Michel C. de Chatelliers, monophyly (see Introduction). Evidence of reciprocal Tim Jones, Lee Knight, Emilie Lindquist and Patrick Mar- monophyly in several gene trees is a strong indication that tin for collecting and/or otherwise making material avail- groups are separately evolving lineages, that is, different able to us; to Anna Ansebo, Alexander B€ar, Ida Envall, species. However, reciprocal monophyly is not a necessity Magnus Johansson, Emelie Lindquist and Maria Lindstrom€ for species delimitation (see e.g. Doyle 1995; Helbig et al. for assistance in the laboratory; and to the Swedish 2002) except under a strict monophyletic species concept. Research Council, The Swedish Taxonomy Initiative (Art- The divergence in COI sequences observed in the Swed- Databanken), the University of The Basque Country UPV/ ish specimens of could be due to allopatric R. falciformis EHU (Contratacion de Doctores Recientes hasta su Integ- divergence with secondary contact. This could be the result racion en Programas de Formacion Postdoctoral del Vicer- of recolonisation from different refugia after deglaciation, as rectorado de Investigacion) and The Basque Goverment has been discussed for other animal groups (e.g. Verovnik (Programa Posdoctoral de perfeccionamiento de doctores) et al. 2005; De Wit & Erseus 2010; Achurra & Erseus 2013). for financial support. The combination of higher mutation rate and a faster genetic drift in mitochondrial markers compared with nuclear ones could explain why divergent haplotype clusters are formed to References a higher extent in the COI data set compared with the Achurra, A. & Erseus, C. (2013). DNA barcoding and species delimitation: the Stylodrilus heringianus case (Annelida: Clitellata: nuclear data sets. These haplotype clusters will then be Lumbriculidae). Invertebrate Systematics, 27, 118–128. fl retained after a secondary contact, but if gene ow between Achurra, A. & Rodriguez, P. (2008). Biodiversity of groundwater them resumes, the unlinked nuclear and mitochondrial hap- oligochaetes from a karst unit in northern Iberian Peninsula: lotype clusters will be mixed in the populations. The nuclear ranking subterranean sites for conservation management. Hydro- genome will also further be affected by recombination biologia, 605, 159–171. increasing the blending between haplotype clusters. Speci- Anisimova, M. & Gascuel, O. (2006). Approximate likelihood-ratio mens from different Swedish COI clusters are found at the test for branches: a fast, accurate, and powerful alternative. Sys- tematic Biology, 55, 539–552. same locality in several cases (see Table 1). Beauchamp, K. A., Gay, M., Kelley, G. O., El-Matbouli, M., Kath- The western Alps and adjacent areas seem to have a rich man, R. D., Nehring, R. B. & Hedrick, R. P. (2002). Prevalence Rhyacodrilus fauna both in surface and in ground water (e.g. and susceptibility of infection to Myxobolus cerebralis, and genetic Juget 1984, 1987; Lafont & Juget 1993) including the here differences among populations of Tubifex tubifex. Diseases of aqua- described R. pigueti, which together with R. falciformis is also tic organisms, 51, 113–121. found in Lake L eman (Juget 1967). From Lake L eman, also Bickford, D., Lohman, D. J., Sodhi, N. S., Ng, P. K., Meier, R., a seemingly undescribed species was reported and illustrated Winker, K., Ingram, K. K. & Das, I. (2007). Cryptic species as a window on diversity and conservation. Trends in Ecology & by Piguet and Bretscher (1913: Fig. 13). It has straight penial Evolution, 22, 148–155. chaetae of approximately the same length as the somatic Birky, C. W. Jr, Fuerst, P. & Maruyama, T. (1989). Organelle chaetae and lack hair chaetae, like both R. falciformis and gene diversity under migration, mutation, and drift: equilibrium R. pigueti. Future studies of the clitellate fauna in this area expectations, approach to equilibrium, effects of heteroplasmic may reveal an even larger diversity of Rhyacodrilus. cells, and comparison to nuclear genes. Genetics, 121, 613–627. € Finding unexpected genetic divergence within a morpho- Bretscher, K. (1901). Beobachtungen uber die Oligochaeten der – species can be a starting point for further investigation Schweiz. Revue Suisse Zoologie, 9, 189 223. Brown, W. M., George, M. Jr & Wilson, A. C. (1979). Rapid evo- using an integrative approach with various data sources. lution of animal mitochondrial DNA. Proceedings of the National However, COI alone should not be used to claim that Academy of Sciences of the United States of America, 76, 1967–1971. cryptic speciation has occurred, as several studies have Brown, W. M., Prager, E. M., Wang, A. & Wilson, A. C. (1982). found deep mitochondrial divergence within populations Mitochondrial DNA sequences of primates: tempo and mode of with retained gene flow (e.g. Webb et al. 2011; Hogner evolution. Journal of Molecular Evolution, 18, 225–239.
620 ª 2013 The Norwegian Academy of Science and Letters, 42, 6, November 2013, pp 612–622 S. Martinsson et al. Integrative taxonomy of Rhyacodrilus falciformis s.l.
Chekanovskaya, O. V. (1981). Aquatic Oligochaeta of the USSR. Funk, D. J. & Omland, K. E. (2003). Species-level paraphyly and New Delhi: Amerind Publishing Co. Pvt. Ltd. polyphyly: frequency, causes, and consequences, with insights Crisp, M. D. & Chandler, G. T. (1996). Paraphyletic species. Telo- from animal mitochondrial DNA. Annual Review of Ecology, Evo- pea, 6, 813–844. lution, and Systematics, 39,7–423. Cuadrado, S. & Martinez-Ansemil, E. (2001). External structures Giani, N., Sambugar, B., Mart ınez-Ansemil, E., Martin, P. & Sch- used during attachment and sperm transfer in tubificids (Annel- melz, R. M. (2011). The groundwater oligochaetes (Annelida, ida, Oligochaeta). Hydrobiologia, 463, 107–113. Clitellata) of Slovenia. Subterranean Biology, 9,85–102. Dasmahapatra, K. K., Elias, M., Hill, R. I., Hoffman, J. I. & Mal- Guindon, S. & Gascuel, O. (2003). A simple, fast, and accurate let, J. (2010). Mitochondrial DNA barcoding detects some spe- algorithm to estimate large phylogenies by Maximum Likeli- cies that are real, and some that are not. Molecular Ecology hood. Systematic Biology, 52, 696–704. Resources, 10, 264–273. Guindon, S., Dufayard, J. F., Lefort, V., Anisimova, M., Hordijk, De Queiroz, K. (2007). Species concepts and species delimitation. W. & Gascuel, O. (2010). New algorithms and methods to esti- Systematic Biology, 56, 879–886. mate maximum-likelihood phylogenies: assessing the perfor- De Wit, P. & Ers eus, C. (2010). Genetic variation and phylogeny mance of PhyML 3.0. Systematic biology, 59, 307–321. of Scandinavian species of Grania (Annelida: Clitellata: Enchyt- Gustafsson, D. R., Price, D. A. & Ers eus, C. (2009). Genetic varia- raeidae), with the discovery of a cryptic species. Journal of Zoo- tion in the popular lab worm Lumbriculus variegatus (Annelida: logical Systematics and Evolutionary Research, 48, 285–293. Clitellata: Lumbriculidae) reveals cryptic speciation. Molecular Doyle, J. J. (1995). The Irrelevance of Allele Tree Topologies for Phylogenetics and Evolution, 51, 182–189. Species Delimitation, and a Non-Topological Alternative. Sys- Hebert, P. D., Cywinska, A., Ball, S. L. & deWaard, J. R. (2003). tematic Botany, 20, 574–588. Biological identifications through DNA barcodes. Proceedings of Dozsa-Farkas, K., Porco, D. & Boros, G. (2012). Are Bryodrilus the Royal Society of London, Series B, 270, 313–321. parvus Nurminen, 1970 and Bryodrilus librus (Nielsen and Chris- Heethoff, M., Etzold, K. & Scheu, S. (2004). Mitochondrial COII tensen, 1959) (Annelida: Enchytraeidae) really different species? sequences indicate that the parthenogenetic earthworm Octolasion A revision based on DNA barcodes and morphological data Zoo- tyrtaeum (Savigny 1826) constitutes of two lineages differing in taxa, 3276,38–50. body size and genotype. Pedobiologia, 48,9–13. Drummond, A. J. & Rambaut, A. (2007). BEAST: Bayesian evolu- Helbig, A. J., Knox, A. G., Parkin, D. T., Sangster, G. & Collinson, tionary analysis by sampling trees. BMC Evolutionary Biology, 7, M. (2002). Guidelines for assigning species rank. Ibis, 144, 518–525. 214. Hogner, S., Laskemoen, T., Lifjeld, J. T., Porkert, J., Kleven, O., Drummond, A. J., Suchard, M. A., Xie, D. & Rambaut, A. (2012). Albayrak, T., Kabasakal, B. & Johnsen, A. (2012). Deep sympat- Bayesian phylogenetics with BEAUti and the BEAST 1.7. Molec- ric mitochondrial divergence without reproductive isolation in ular Biology and Evolution, 29, 1969–1973. the common redstart Phoenicurus phoenicurus. Ecology and Evo- Dumnicka, E. (2006). Composition and abundance of oligochaetes lution, 2, 2974–2988. € (Annelida: Oligochaeta) in springs of Krakow-Czestochowa Hrab e, S. (1935). Uber Moraviodrilus pygmaeus n. g. n. sp., Rhyaco- upland (Southern Poland): effect of spring encasing and environ- drilus falciformis Br., Ilyodrilus bavaricus Oschm. und Bothrioneu- mental factors. Polish Journal of Ecology, 54, 231–242. rum vejdovskyanum Stolc. Publications de la Facult e des Sciences de Envall, I., Gustavsson, L. M. & Erseus, C. (2012). Genetic and l’Universit e Masaryk, 209,1–19. chaetal variation in Nais worms (Annelida, Clitellata, Naididae). ICZN (1999). International Code of Zoological Nomenclature. London: Zoological Journal of the Linnean Society, 165, 495–520. The International Trust for Zoological Nomenclature. Ers eus, C. (1994). The Oligochaeta. In J. A. Blake & B. Hilbig James, S. W., Porco, D., Decaens, T., Richard, B., Rougerie, R. & (Eds.) Taxonomic Atlas of the Bentic Fauna of the Santa Maria Basin Ers eus, C. (2010). DNA barcoding reveals cryptic diversity in and Western Santa Barbara Channel Volume 4 Oligochaeta to Poly- Lumbricus terrestris L., 1758 (Clitellata): resurrection of L. hercu- chaeta: Phyllodocida (Phyllodocidae to Paralacydoniidae) (pp. 5–38). leus (Savigny, 1826). PLoS ONE, 5, e15629. Santa Barbara, CA: Santa Barbara Museum of Natural History. Juget, J. (1967). Quelques donn ees nouvelles sur les oligoch etes du Ers eus, C. & Gustafsson, D. (2009). Cryptic speciation in clitellate l eman: composition et origine du peuplement. Annales de Limnol- model organisms. In D. H. Shain (Ed.) Annelids in Modern Biol- ogie, 3, 217–229. ogy (pp. 31–46). Hoboken, New Jersey: John Wiley & Sons, Inc. Juget, J. (1984). Oligochaeta of the epigean and underground fauna Ers eus, C., Rota, E., Timm, T., Grimm, R., Healy, B. & Lund- of the alluvial plain of the French upper Rhone^ (biotypological berg, S. (2005). Riverine and riparian clitellates of three drain- trial). Hydrobiologia, 115, 175–182. ages in southern Sweden. International Journal of Limnology, 41, Juget, J. (1987). Contribution to the study of the Rhyacodrilinae 183–194. (Tubificidae, Oligochaeta), with description of two new stygobi- Ers eus, C., Wetzel, M. J. & Gustavsson, L. (2008). ICZN rules—a ont species from the alluvial plain of the French upper Rhone, farewell to Tubificidae (Annelida, Clitellata). Zootaxa, 1744,66– Rhyacodrilus amphigenus, sp. n. and Rhizodriloides phreaticola, 68. g. n., sp. n. Hydrobiologia, 155, 107–118. Ers eus, C., Rota, E., Matamoros, L. & De Wit, P. (2010). Molecu- Kasprzak, K. (1979). Oligochaetes (Oligochaeta) of the Pien-iny lar phylogeny of Enchytraeidae (Annelida, Clitellata). Molecular Mountains. II. Naididae, Tubificidae, Haplotaxidae, Lumbriculi- Phylogenetics and Evolution, 57, 849–858. dae, Branchiobdellidae. Fragmenta Faunistica, 24,57–80. Floyd, R., Abebe, E., Papert, A. & Blaxter, M. (2002). Molecular Katoh, K., Misawa, K., Kuma, K. & Miyata, T. (2002). MAFFT: a barcodes for soil nematode identification. Molecular Ecology, 11, novel method for rapid multiple sequence alignment based on 839–850. fast Fourier transform. Nucleic Acids Research, 30, 3059–3066.
ª 2013 The Norwegian Academy of Science and Letters, 42, 6, November 2013, pp 612–622 621 Integrative taxonomy of Rhyacodrilus falciformis s.l. S. Martinsson et al.
King, R. A., Tibble, A. L. & Symondson, W. O. (2008). Opening Rosen, D. E. (1979). Fishes from the uplands and intermontane a can of worms: unprecedented sympatric cryptic diversity within basins of Guatemala: revisionary studies and comparative geogra- British lumbricid earthworms. Molecular Ecology, 17, 4684–4698. phy. Bulletin of the American Museum of Natural History, 162, Kvist, S., Sarkar, I. N. & Ers eus, C. (2010). Genetic variation and 267–376. phylogeny of the cosmopolitan marine genus Tubificoides (Annel- Rosenberg, N. A. (2007). Statistical tests for taxonomic distinctive- ida: Clitellata: Naididae: Tubificinae). Molecular Phylogenetics and ness from observations of monophyly. Evolution, 61, 317–323. Evolution, 57, 687–702. Schlick-Steiner, B. C., Steiner, F. M., Seifert, B., Stauffer, C., Lafont, M. & Juget, J. (1993). Description de Rhyacodrilus ardierae n. Christian, E. & Crozier, R. H. (2010). Integrative taxonomy: a sp.(Oligochaeta, Tubificidae) r ecolt ee dans l’Ardi eres (affluent de multisource approach to exploring biodiversity. Annual Review of la Saone).^ Bulletin de la Soci et e zoologique de France, 118, 115–123. Entomology, 55, 421–438. Lin, C.-P. & Danforth, B. N. (2004). How do insect nuclear and Sturmbauer, C., Opadiya, G. B., Niederst€atter, H., Riedmann, A. mitochondrial gene substitution patterns differ? Insights from & Dallinger, R. (1999). Mitochondrial DNA reveals cryptic oli- Bayesian analyses of combined datasets Molecular phylogenetics gochaete species differing in cadmium resistance. Molecular Biol- and evolution, 30, 686–702. ogy and Evolution, 16, 967–974. Masters, B. C., Fan, V. & Ross, H. A. (2011). Species Delimita- Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M. & tion–a Geneious plugin for the exploration of species bound- Kumar, S. (2011). MEGA5: molecular evolutionary genetics aries. Molecular Ecology Resources, 11, 154–157. analysis using maximum likelihood, evolutionary distance, and Matamoros, L., Rota, E. & Ers eus, C. (2012). Cryptic diversity maximum parsimony methods. Molecular Biology and Evolution, among the achaetous Marionina (Annelida, Clitellata, Enchyt- 28, 2731–2739. raeidae). Systematics and Biodiversity, 10, 509–525. Taylor, H. R. & Harris, W. E. (2012). An emergent science on Mayr, E. (1942). Systematics and the Origin of Species from the View- the brink of irrelevance: a review of the past 8 years of DNA point of a Zoologist. New York: Columbia University Press. barcoding. Molecular Ecology Resources, 12, 377–388. Neigel, J. E. & Avise, J. C. (1986). Phylogenetic relationships of Timm, T. (2009). A guide to the freshwater Oligochaeta and Poly- mitochondrial DNA under various demographic models of spe- chaeta of Northern and Central Europe. Lauterbornia, 66,1–235. ciation. In E. Nevo & S. Karlin (Eds.) Evolutionary Processes and Timm, T., Ers eus, C. & Lundberg, S. (1996). New and Unusual Theory (pp. 515–534). New York: Academic Press. Records of Freshwater Oligochaeta from the Scandinavian Pen- Nelson, G. & Platnick, N. I. (1981). Systematics and Biogeography. insula. Nordic Journal of Freshwater Reasearch, 72,15–29. New York: Columbia University Press. Torres-Leguizamon, M., Mathieu, J., Livet, A., Decaens, T. & Novo, M., Fernandez, R., Marchan, D. F., Monica, G. & Cosin, Dupont, L. (2012). Isolation of polymorphic microsatellite mark- D. J. (2012). Compilation of morphological and molecular data, ers in Aporrectodea icterica (Savigny 1826). Soil Biology & Biochem- a necessity for taxonomy: the case of Hormogaster abbatissae sp. istry, 51,16–19. n. (Annelida, Clitellata, Hormogastridae). Zookeys, 242,1–16. Van Valen, L. (1976). Ecological species, multispecies, and oaks. Paterson, H. E. H. (1985). The recognition concept of species. In Taxon, 25, 233–239. E. S. Vrba (Ed.) Species and Speciation (pp. 21–29). Pretoria: Verovnik, R., Sket, B. & Trontelj, P. (2005). The colonization of Transvaal Museum. Europe by the freshwater crustacean Asellus aquaticus (Crusta- Piguet, E. (1906). Oligoch etes de la Suisse francßaise. Revue Suisse cea: Isopoda) proceeded from ancient refugia and was directed de Zoologie, 14, 389–403. by habitat connectivity. Molecular Ecology, 14, 4355–4369. Piguet, E. & Bretscher, K. (1913). Oligoch etes. Catalogue des Webb, W. C., Marzluff, J. M. & Omland, K. E. (2011). Random Invert ebr es de la Suisse. Gen eve: Georg & Cie. interbreeding between cryptic lineages of the Common Raven: Pleijel, F., Jondelius, U., Norlinder, E., Nygren, A., Oxelman, B., evidence for speciation in reverse. Molecular Ecology, 20, 2390– Schander, C., Sundberg, P. & Thollesson, M. (2008). Phylogenies 2402. without roots? A plea for the use of vouchers in molecular phylo- genetic studies Molecular Phylogenetics and Evolution, 48, 369–371. Puillandre, N., Lambert, A., Brouillet, S. & Achaz, G. (2012). Supporting Information ABGD, Automatic Barcode Gap Discovery for primary species Additional Supporting Information may be found in the delimitation. Molecular Ecology, 21, 1864–1877. Rambaut, A. & Drummond, A. J. (2007). Tracer v1.5. Available via online version of this article: http://beast.bio.ed.ac.uk/Tracer. Figure S1. Maximum Likelihood tree based on com- Richard, B., Decaens, T., Rougerie, R., James, S. W., Porco, D. & bined COI, H3 and ITS data from Rhyacodrilus falciformis Hebert, P. D. (2010). Re-integrating earthworm juveniles into s.l. obtained with PhyML, numbers at branches are aLRT fi soil biodiversity studies: species identi cation through DNA bar- branch support. – coding. Molecular Ecology Resources, 10, 606 614. Table S1. Primers, sequences and programs used for Rodrigo, A., Bertels, F., Heled, J., Noder, R., Shearman, H. & amplification of the mitochondrial COI and nuclear ITS Tsai, P. (2008). The perils of plenty: what are we going to do with all these genes? Philosophical Transactions of the Royal Society and H3. of London. Series B, Biological sciences, 363, 3893–3902. Table S2. Details of alignments of the mitochondrial Rodriguez, P. & Fend, S. V. (2013). New species of Rhyacodrilus COI and the nuclear H3 and ITS genes. (Annelida: Clitellata: Rhyacodrilinae) of North America, with Table S3. Ranges (lm) for morphological characters of re-description of R. sodalis (Eisen, 1879). Zootaxa, 3664,1. the COI-clusters (A–G).
622 ª 2013 The Norwegian Academy of Science and Letters, 42, 6, November 2013, pp 612–622