ARTICLE IN PRESS Organisms, Diversity & Evolution 8 (2008) 399–412 www.elsevier.de/ode DNA taxonomy of Swedish Catenulida (Platyhelminthes) and a phylogenetic framework for catenulid classification Karolina Larssona, Afsaneh Ahmadzadeha, Ulf Jondeliusb,Ã aSystematic Biology, Evolutionary Biology Centre, Norbyva¨gen 18D, 752 36 Uppsala, Sweden bDepartment of Invertebrate Zoology, Swedish Museum of Natural History, PO Box 50007, 104 05 Stockholm, Sweden Received 4 April 2008; accepted 25 September 2008 Abstract Specimens of Catenulida were collected at 34 localities in Sweden. We used 18S rDNA, 28S rDNA, ITS-5.8S, and cytochrome oxidase I (COI) nucleotide sequences to infer phylogeny from parsimony jackknifing and Bayesian analysis. Our dataset contained 74 ingroup terminals and 5111 characters. The results show a basal split between a clade consisting of the marine Retronectidae+the limnic Catenulidae, and a second clade consisting of the limnic Stenostomidae. The hypothesis of the marine Retronectidae as the sister group of the limnic Catenulida is rejected. The recently introduced genus Anokkostenostomum Noren˜a, Damborenea & Brusa, 2005 results as non-monophyletic, and Suomina Marcus, 1945 as a group inside Catenula Duge`s, 1832. Therefore, we propose to render Anokkostenostomum a new junior synonym of Stenostomum Schmidt, 1848, and Suomina a new junior synonym of Catenula. Consequently, the new combinations Catenula evelinae (Marcus, 1945), Catenula sawayai (Marcus, 1945), and Catenula turgida (Zacharias, 1902) are proposed, and 14 species are returned to their original genus, Stenostomum. The molecular phylogenetic hypothesis is used to identify and discriminate catenulid species. In our material, we found 12 species of Catenulida new to Sweden, and four species new to science, all of which are distinguishable by morphological characters. r 2008 Gesellschaft fu¨r Biologische Systematik. Published by Elsevier GmbH. All rights reserved. Keywords: Catenulida; Platyhelminthes; Molecular phylogeny; DNA taxonomy Introduction abundant. Marine Catenulida, on the other hand, are rare: only 12 species are known. Catenulids have a Catenulida Meixner, 1924 is a group of small simple anatomy and lack sclerotized parts, such as the flatworms comprising about 100 species worldwide. copulatory stylets common in, e.g., rhabdocoel flat- Freshwater Catenulida, which constitute the vast worms. Many characters (e.g. shape, size, colour) show majority of the species, live in mires, ponds, streams, high intraspecific variability, which makes species and moist terrestrial habitats, where they often are very identification problematic. Catenulids, which are very fragile, can be identified only when alive, and are rarely encountered in a sexually mature stage as they normally ÃCorresponding author. reproduce by paratomy. Many currently recog- E-mail address: [email protected] (U. Jondelius). nized catenulid species are regarded as cosmopolites 1439-6092/$ - see front matter r 2008 Gesellschaft fu¨r Biologische Systematik. Published by Elsevier GmbH. All rights reserved. doi:10.1016/j.ode.2008.09.003 ARTICLE IN PRESS 400 K. Larsson et al. / Organisms, Diversity & Evolution 8 (2008) 399–412 (e.g. Stenostomum leucops Duge`s, 1828; Catenula lemnae in some barcoding studies (e.g. Hebert et al. 2003), since Duge`s, 1832). There is, however, a potential for cryptic it generates character-based hypotheses of evolutionary diversity among the cosmopolitan morphological spe- relationships also in cases where species delimitation is cies, due to the paucity of distinguishing features. ambiguous (Will and Rubinoff 2004). DNA-barcoding The Swedish catenulid fauna is virtually unknown, studies have focused on the identification of known with only two limnic species, Stenostomum karlingi species, but a greater challenge lies in the application of (Luther 1960), and S. leucops, reported by Luther DNA-based methods to poorly characterized taxa (1960). Sterrer and Rieger (1974) recorded three (Monaghan et al. 2005) such as the Catenulida. marine species: Retronectes clio (Sterrer and Rieger 1974), R. melpomene (Sterrer and Rieger 1974), and one Retronectes sp., all found in very low numbers. Material and methods However, our studies have revealed that limnic catenulids are highly abundant in Sweden. But how many species Collection and identification of specimens are there? Here we aim to provide a phylogenetic framework for the Catenulida and to sample predom- Catenulids were sampled during 2003–2005 from inantly limnic catenulids from various habitats in 34 locations in Sweden (Table 1). The specimens were Sweden in order to establish the number of Swedish collected by searching samples of moss, other vegeta- species and their identity. tion or sediment under a stereomicroscope, and then Ideally, a phylogenetic study of the Catenulida should identified live under a microscope. Live worms were encompass specimens collected worldwide. Such mate- rial was not available to us, but even though the specimens sequenced by us had been collected exclu- Table 1. Sampling data sively within Sweden, the dataset used in the analyses, Locality Province Date (year-month-day) which was complemented by the catenulid sequences available in GenBank, included material from three of 01 Bohusla¨n 03-07-26, 04-07-15 the five families, and six of the 12 catenulid genera 02 Uppland 04-05-06 currently recognized. We were not able to collect any of 03 Smaland( 04-05-15 the five species of the Chordariidae Marcus, 1945, nor 04 Ja¨mtland 04-06-30 the single species in Tyrrheniellidae Riedl, 1959. 05 Ja¨mtland 04-06-30 In this first attempt to use molecular data to 06 Ja¨mtland 04-07-03 reconstruct the phylogeny of the Catenulida and to 07 Ja¨mtland 04-07-03, 05-06-23 provide a framework for a phylogenetic classification, 08 Ja¨mtland 04-07-06 09 Bohusla¨n 04-07-22, 05-07-27 we used the mitochondrial cytochrome oxidase I (COI) 10 Bohusla¨n 04-07-28 gene and three ribosomal markers: the 18S rDNA gene, 11 Bohusla¨n 04-07-27 the 28S rDNA gene, and the ITS1-5.8S-ITS2 rDNA 12 Bohusla¨n 04-08-02 region (ITS-5.8S). Our aim was also to identify cryptic 13 Bohusla¨n 04-08-03 diversity at the species level among these microscopic 14 Bohusla¨n 04-08-04 worms. The use of four common molecular markers 15 O¨ land 04-09-09 provided an opportunity to evaluate their performance 16 Smaland( 04-09-14 in identifying species-level taxa. We then tried to identify 17 Smaland( 04-09-14 morphological characters for the species groups, in 18 Gotland 04-09-18 order to be able to distinguish the latter based on 19 Gotland 04-09-20 morphology at the time of collection. Formal taxonomic 20 Gotland 04-09-20 21 Gotland 04-09-20 descriptions of the new species we have identified will be 22 Skane( 04-10-05 the subject of a separate study (Larsson and Willems, 23 Ja¨mtland 05-06-23 unpublished), which will also include detailed anatom- 24 Ja¨mtland 05-06-24 ical data. In the present paper, those new species are 25 Ja¨mtland 05-06-24 referred to with provisional names based on their 26 Ja¨mtland 05-06-25 morphological characteristics. 27 Ja¨mtland 05-06-25 The phylogenetic hypothesis based on our combined 28 Ja¨mtland 05-06-30 data permitted us to examine some evolutionary 29 Ja¨mtland 05-06-30 problems within the Catenulida, such as the proposed 30 Lappland 05-07-01 sister-group relationship between marine and limnic 31 Lappland 05-07-08 catenulids (Ehlers 1994). Our choice of a parsimony- 32 Bohusla¨n 05-08-01 33 Bohusla¨n 05-08-06 based method to identify cryptic diversity has an 34 Ja¨mtland 05-06-23 important advantage over distance-based methods used ARTICLE IN PRESS K. Larsson et al. / Organisms, Diversity & Evolution 8 (2008) 399–412 401 photographed and drawings were made before speci- additional sequences downloaded from GenBank in mens were preserved in 95% ethanol. Material for Table 4. The four genes were aligned separately using histological study was preserved in Bouin’s fluid. the software MUSCLE (Edgar 2004). Hypervariable regions were manually deleted from the resulting alignment. The final lengths of the datasets were DNA extraction, PCR amplification and sequencing 1803 bp (18S), 1553 bp (28S), 599 bp (COI), and 1156 bp (ITS-5.8S), respectively. DNA was extracted from 74 ethanol-preserved speci- mens using the DNeasy Tissue Kit (Qiagen), following the manufacturer’s protocol. Amplifications were per- Phylogenetic reconstruction formed with 2 ml DNA extract and 1 ml of each primer using Ready-To-Go PCR beads (Amersham Bio- Parsimony jackknifing (Farris et al. 1996) was sciences), each containing 2.5 U of PuReTaq DNA performed on the combined 18S rDNA+28S rDNA+ Polymerase, 10 mM Tris–HCl, 50 mM KCl, and 1.5 mM COI+ITS-5.8S dataset, using the software TNT MgCl2, 200 mM each of dNTP and stabilizers including (Goloboff et al. 2003). We performed 1000 jackknife bovine serum albumin. The final volume was 25 ml. 18S replicates each, with 50 random additions, TBR branch rDNA was amplified in two overlapping fragments swapping and a deletion frequency of 36%. The results using the primer combination 4fb+1806R (1200 base from parsimony jackknifing were summarized in a pairs) and 5fk+S30 (900 base pairs). 28S rDNA was majority-rule tree (Fig. 1) with the cut-off value at amplified with the primers LSU5+L1642R (1450 base 70%. We also performed parsimony-jackknife anal- pairs), COI with the primers COI3B+COI5B (600 base yses on the separate gene datasets, using TNT with the pairs), and ITS-5.8S was amplified in two fragments same parameter settings as above (Figs. 2 and 3). with the primers ITS4+ITS5 (950 base pairs) (for Bremer support (BS) values (Bremer 1988) were calcu- primer sequences and references, see Table 2). Products lated on the strict consensus tree from the parsimony were purified with the QIAquick PCR Purification Kit analysis with the combined dataset, using PAUP* 4b.10 (Qiagen) according to the manufacturer’s protocol.