2urn:lsid:arphahub.com:pub:45048D35-BB1D-5CE8-9668-537E44BD4C7Eurn:lsid:zoobank.org:pub:91BD42D4-90F1-4B45-9350-EEF175B1727AZooKeysZK1313-29891313-2970Pensoft Publishers10.3897/zookeys.199.32152837Research ArticleTurbellariaBiodiversity & ConservationFaunistics & DistributionMolecular systematicsSpecies InventoriesBalearicsEuropeIberian PeninsulaFirst report of the exotic blue land planarian,
Caenoplana coerulea (Platyhelminthes, Geoplanidae), on Menorca (Balearic Islands, Spain)BreugelmansKarin1CardonaJosep Quintana2ArtoisTom3https://orcid.org/0000-0002-2491-7273JordaensKurt45BackeljauThierry15thierry.backeljau@naturalsciences.beRoyal Belgian Institute of Natural Sciences, Vautierstraat 29, B-1000 Brussels, BelgiumRoyal Belgian Institute of Natural SciencesBrusselsBelgiumInstitut Catala de Paleontologia Miquel Crusafont, Universitat Autònoma de Barcelona, edifici ICP Campus de la UAB, s/n 08193 Cerdanyola del Vallés, Barcelona, SpainUniversitat Autònoma de BarcelonaBarcelonaSpainResearch Group Zoology: Biodiversity & Toxicology, Centre for Environmental Sciences, Hasselt University, Campus Diepenbeek, Agoralaan Building D, B-3590 Diepenbeek, BelgiumHasselt UniversityDiepenbeekBelgiumJoint Experimental Molecular Unit, Royal Museum for Central Africa, Leuvensesteenweg 13, B-3080 Tervuren, BelgiumRoyal Museum for Central AfricaTervurenBelgiumEvolutionary Ecology Group, University of Antwerp, Groenenborgerlaan 171, B-2020 Antwerp, Belgium
201246201219991105FF8FFFA9-F726-FFC8-FFB9-911E2303FF8957729710420121552012Karin Breugelmans, Josep Quintana Cardona, Tom Artois, Kurt Jordaens, Thierry BackeljauThis is an open access article distributed under the terms of the Creative Commons Attribution License 3.0 (CC-BY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
In April 2009 two specimens of a terrestrial flatworm were collected from under a rock in an orchard at Ciutadella de Menorca on the easternmost Balearic island of Menorca (Spain). Their external morphology suggested that both specimens belonged to the invasive blue planarian Caenoplana coerulea, a species which is native to eastern Australia. Sequence data of a fragment of the mitochondrial cytochrome c oxidase subunit I (COI) and of the entire 18S ribosomal RNA confirm its identification. This is one of the first records of the species in Europe where it has only been found in one locality in the United Kingdom, France and NE Spain.
Several species of terrestrial planarian are known as invasive, exotic species in soils of the northern hemisphere. For instance, in North America and the British Isles about a dozen species of exotic terrestrial planarians have been introduced (Jones 1988; Jones and Boag 1996; Ogren and Kawakatsu 1998). Many of these introduced exotic species are predators of earthworms, isopods and snails (e.g. Ogren 1995; Fiore et al. 2004; Sugiura et al. 2006; Iwai et al. 2010; Sugiura 2010). As such, these flatworms may pose a threat to local biodiversity (Santoro and Jones 2001). Because of this, and in view of their rapid dispersal as well as their wide distribution, these introduced exotic terrestrial flatworms are of serious agricultural and nature conservation concern.
The impacts of introduced exotic terrestrial flatworms may be especially detrimental in islands and archipelagos that support an endemic invertebrate fauna. This is illustrated by the terrestrial flatworm Platydemus manokwari De Beauchamp, 1962, which has been introduced in many Pacific islands (e.g. Eldredge and Smith 1995) and is considered a cause of the rapid decline of endemic land snails on these islands (Chiba 2003; Okochi et al. 2004; Ohbayashi et al. 2005; Sugiura et al. 2006; Sugiura 2009; Sugiura and Yamaura 2010). Therefore the species is of serious concern in the conservation of the unique land snail fauna of archipelagos and therefore has been included in the list of the world’s 100 worst invasive alien species (see http://www.issg.org/worst100_species.html, Lowe et al. 2000). Hence, in order to develop strategies to reduce further spread and to control their impacts on local invertebrates, rapid and accurate identifications of exotic terrestrial flatworms are essential.
Against this background, we here report for the first time the occurrence of the invasive blue land planarian Caenoplana coerulea Moseley, 1877 in the Balearic Islands (Menorca, Spain). Its identification was confirmed by DNA sequence analysis of the entire nuclear 18S ribosomal RNA (18S rDNA) gene and of a portion of the mitochondrial cytochrome c oxidase subunit 1 (COI) gene.
Materials and methods
In April 2009 two specimens of a terrestrial flatworm were collected by hand under a rock in an orchard at Ciutadella de Menorca on the easternmost Balearic island of Menorca (Spain, 39°57'00"N, 03°51'00"E; Figures 1 and 2). Both specimens (labelled ‘1957’ and ‘1958’) were stored in 100% ethanol.
(A) Location of the Balearic Islands in the Mediterranean Sea. Menorca is in black and indicated by an arrow. (B) Detailed map of Menorca: the locality where Caenoplana coerulea was found is indicated with the letter A.
https://binary.pensoft.net/fig/18799
One of the two specimens of Caenoplana coerulea collected on Menoca.
https://binary.pensoft.net/fig/18800
Genomic DNA was extracted using the NucleoSpin® Tissue Kit (Machery-Nagel, Düren, Germany). A 424 bp fragment of the COI gene was amplified using the primer pair flatCOIL and flatCOIH (modified from Bessho et al. 1997; Table 1). PCR was performed in a total volume of 25 µl, containing 2 µl of DNA and 0.2 µM of each primer, and using the Qiagen® Multiplex PCR Kit with HotStarTaq® DNA polymerase and a final concentration of 3 mM MgCl2. The PCR profile was 15 min at 95 °C followed by 35 cycles of 45 s at 95 °C, 45 s at 50 °C and 1 min at 72 °C, and with a final extension step of 10 min at 72 °C. The entire 18S rDNA gene was amplified using the primer pair 4F18S and 16R18S (Winnepenninckx et al. 1994, Table 1). PCR was performed in a total volume of 25 µl containing 2 µl of DNA, 0.2 µM of each primer, 200 µM of each dNTP, 0.62 units of Taq DNA polymerase (Qiagen) and mQ-H2O. Triclad flatworms are known to have two types of 18S rDNA genes (Type I and II) (Carranza et al. 1996, 1999). Therefore, 18S rDNA PCR products were cloned using TOPO TA Cloning® Kit for Sequencing (Invitrogen) following the suppliers’ instructions. Fifteen colonies of each specimen were amplified as described above.
Forward (F) and reverse (R) primers used for amplification and sequencing of the mitochondrial cytochrome c oxidase subunit I (COI) and the nuclear 18S ribosomal RNA (18S rDNA) genes of the two Caenoplana specimens in this study.
Name
Sequence 5’-3’
Source
COI:
F: flatCOIL
GCAGTTTTTGGTTTTTTGGACATCC
modified from Bessho et al. (1997)
R: flatCOIH
GAGCAACAACATAATAAGTATCATG
modified from Bessho et al. (1997)
18S rDNA:
F: 4F18s
CTGGTTGATYCTGCCAGT
Winnepenninckx et al. (1994)
R: 10R18S
TTGGYRAATGCTTTCGC
Winnepenninckx et al. (1994)
F: 9F18S
CGCGGTAATTCCAGCTCCA
Winnepenninckx et al. (1994)
R: 3R18S
GACGGGCGGTGTGTRC
Winnepenninckx et al. (1994)
F: 14F18S
ATAACAGGTCTGTGATGCCC
Winnepenninckx et al. (1994)
R: 16R18S
CYGCAGGTTCACCTACRG
Winnepenninckx et al. (1994)
All PCR products were purified using NucleoFast 96 PCR plates (Macherey-Nagel, Düren, Germany) and bidirectionally sequenced using the BigDye Terminator v1.1 chemistry on an ABI 3130xl automated capillary DNA sequencer (Life Technologies). For the sequencing of 18S rDNA several internal primers were used (Table 1). Sequences were visually inspected and aligned in SeqScape v2.5 (Life Technologies). COI and 18S rDNA sequences from other flatworm species of the Continenticola (see e.g. Álvarez-Presas et al. 2008, Sluys et al. 2009) were imported from GenBank (See Appendix). Sequence data sets were aligned in MAFFT v6.861 (Katoh and Toh 2008) and trimmed at 296 bp for the COI and at 1765 bp for the 18S rDNA fragment. From the Menorca specimens only 18S rDNA clones that yielded sequences without ambiguous positions were retained for further analyses.
Two tree reconstruction methods were implemented: Neighbor-Joining (NJ) (Saitou and Nei 1987) and Maximum Likelihood (ML). The most appropriate nucleotide substitution models for ML were selected using JMODELTEST v0.1.1 (Posada 2008). These were the GTR+G model for the COI fragment and the GTR+I+G model for the 18S rDNA fragment. NJ trees were made in MEGA v5.0 (Tamura et al. 2007) using K2P distances and with complete deletion of indels. ML trees were made in PAUP* v4.0b10 (Swofford 2002) using a heuristic search with the tree-bisection-reconnection branch-swapping algorithm and random addition of taxa. Trees were rooted with Bdelloura candida (Girard, 1850) (Maricola, family Bdellouridae). Branch support was assessed via nonparametric bootstrapping using 1000 bootstrap replicates for NJ or 200 bootstrap replicates for ML (Felsenstein 1985). Only nodes with bootstrap values of ≥ 70% were retained and considered meaningful (Hillis and Bull 1993). P-distances were calculated with MEGA v5.0.
Both specimens have been deposited in the collections of the Royal Belgian Institute of Natural Sciences, Brussels, under catalogue number IG.32062. DNA sequences have been deposited in GenBank under accession numbers JQ639215-JQ639227 (for 18S rDNA) and JQ514564 (for COI).
Results and discussion
The dorsal dark blue ground-colour with a thin median dorsal stripe, the intense blue colour of the ventral side, and eyes that are arranged in a single row around the anterior tip and which do not extend dorsally, suggest that the two specimens belong to the species of blue land planarian, Caenoplana coerulea Moseley, 1877 (Geoplanidae). This is corroborated by our phylogenetic analysis of the COI and 18S rDNA genes. Both individuals had the same COI haplotype; as in other triclads, there were two different intra-individual types of 18S rDNA (Carranza et al. 1996, 1999). We found five type I and eight type II 18S rDNA variants. Figures 3–4 show the phylogenetic trees inferred from the COI and 18S rDNA data, respectively. The COI haplotype of the Menorcan specimens clustered with strong support with a haplotype of Caenoplana coerulea from the UK (GenBank accession number DQ666030), from which it differed by only one, ambiguous position (i.e. a G for DQ666030, while ‘N’ for the Menorcan haplotype). The mean P-distance between the COI haplotype from Menorca and the other Caenoplana coerulea haplotypes was 0.10 ± 0.02, whereas the P-distance with other Geoplanid species was higher (0.16 ± 0.03) and comparable to what we found among Geoplanidae taxa (0.17 ± 0.03). The 18S rDNA type I sequences from the Menorcan specimens formed a strongly supported clade with Caenoplana coeruleaAF033040 (from the UK) (mean P-distance: 0.008 ± 0.002), whereas those of 18S rDNA type II formed a strongly supported clade with Caenoplana sp.1 AF048765 (unknown origin) and Caenoplana sp. ‘Armidale’ AJ270156 (from Australia) (mean P-distance: 0.003 ± 0.001). The mean P-distance between the Menorcan type I and type II sequences and sequences from the other geoplanid species was substantially higher, viz. 0.019 ± 0.003 and 0.058 ± 0.005, respectively.
Neighbor-Joining and ML tree of the 296 bp dataset of the mitochondrial cytochrome c oxidase subunit I gene (COI). The haplotype of the Menorcan specimens is indicated with an asterisk. Bootstrap values ≥ 70% for the NJ and ML trees are given as NJ/ML and are shown at the nodes. – indicates that the node was not supported by the analysis.
https://binary.pensoft.net/fig/18801
Neighbor-Joining and ML tree of the 1765 bp dataset of the nuclear 18S rDNA gene. The clones (cl) of the Menorcan specimens ‘1957’ and ‘1958’ are indicated with an asterisk. Bootstrap values ≥ 70% for the NJ and ML trees are given as NJ/ML and are shown at the nodes. – indicates that the node was not supported by the analysis. Note that the clades of the type I and type II 18S rRNA variants are not supported.
https://binary.pensoft.net/fig/18802
Caenoplana coerulea is native to eastern Australia but, as a result of human activities, it has been introduced to New Zealand, the United States, the United Kingdom, Norfolk Island (Australia), and France (Ogren 1989; Winsor 1998; Jones 1998, 2005), and more recently in Argentina (Luis-Negrete et al. 2011) and NE Spain (Mateos et al. 2012). After introduction, the species may expand its range rapidly. For example, since its accidental introduction into the USA prior to 1943, it has spread rapidly over a large part of the country (California: 1943, Florida: 1961, Georgia: 1972, Texas: 1978, Iowa: 1999, North Carolina: 2001) (Ogren 2001). Whether this fast expansion is due to its high intrinsic dispersal capacity or due to repeated, independent introductions, is unknown.
In the Iberian Peninsula and Balearic Islands, at present ten autochthonous species of the family Geoplanidae have been reported (Mateos et al. 1998, 2009; Vila-Farré et al. 2008, 2011). In addition, two introduced species, Bipalium kewense Moseley, 1878 (Bipaliidae; recorded from Barcelona) (Filella-Subirá 1983) and Platydemus sp. (Geoplanidae; recorded from Benamargosa, Málaga) (Vila-Farré et al. 2011), have been reported from the Iberian Peninsula but not from the Balearic Islands where only Microplana terrestris (O.F. Müller, 1774) (Geoplanidae) has been found (Minelli 1977). Hence, the present record of two specimens of Caenoplana coerulea implies the first introduced species of Geoplanidae in the Balearic Islands. Very recently, the species was also found on the Iberian Peninsula (La Garrotxa, Girona province) (Mateos et al. 2012). Also, pictures of the species that were taken in Spain (Boadilla del Monte, October 2010 and Girona, 22 December 2011) can be found at http://www.flickr.com/photos/51708886@N03/6351086047/ and http://www.biodiversidadvirtual.org/insectarium/Caenoplana-coerulea-img293381.html, respectively. In Europe, the species is further only known from a hothouse in Liverpool (Jones 1998, 2005) and one locality in France (Ogren 1989; Winsor 1998; Winsor et al. 2004).
We do not know when exactly this exotic species arrived in the Balearic Islands. The first specimens of Caenoplana coerulea were found in an orchard in April 2009. In 2011 the species had spread to a nearby garden, where it was found at shaded places. As is the case in other land planarians, its spread and distribution in newly colonized areas is probably mainly determined by moisture (Fraser and Boag 1998). Even in its native region (Australia), the distribution of Caenoplana coerulea is restricted to areas with a high humidity (Luis-Negrete et al. 2011). Even though the impact of Caenoplana coerulea on earthworm and terrestrial gastropod populations is not known, the species is at least reported to feed on isopods, diplopods, earwigs, and snails (Olewine 1972; Barnwell 1978; Terrace and Baker 1994; Jones 2005). Its broad food spectrum might facilitate the establishment and possible spread of the species in Spain and, eventually, elsewhere in Europe.
Acknowledgements
We would like to thank Dr. Leigh Winsor (Condon, Australia) for providing part of the literature and for information on the distribution of Caenoplana coerulea and two anonymous referees for their valuable comments. This work was supported by FWO grant G.0208.08N and the Belgian Network for DNA Barcoding (FWO contract number W0.009.11N).
ReferencesÁlvarez-PresasMBaguñàJRiutortM (2008) Molecular phylogeny of land and freshwater planarians (Tricladida, Platyhelminthes): From freshwater to land and back.47: 555-568. doi: 10.1016/j.ympev.2008.01.032BarnwellGM (1978)Geoplana vaga: a sexually-reproducing terrestrial planarian in San Antonio.23: 151-152. doi: 10.2307/3669990BesshoYTamuraSHoriHTanakaHOhamaTOsawaS (1997) Planarian mitochondria sequence heterogeneity: relationships between the type of cytochrome c oxidase subunit I gene sequence, karyotype and genital organ.6: 129-136. doi: 10.1046/j.1365-294X.1997.00162.xCarranzaSBaguñàJRiutortM (1999) Origin and evolution of paralogous rRNA gene clusters within the flatworm family Dugesiidae (Platyhelminthes, Tricladida).49: 250-259. doi: 10.1007/PL00006547CarranzaSGiribetGRiberaCBaguñàJRiutortM (1996) Evidence that two types of 18S rDNA coexist in the genome of Dugesia (Schmidtea) mediterranea (Platyhelminthes, Turbellaria, Tricladida).13: 824-832. doi: 10.1093/oxfordjournals.molbev.a025643ChibaS (2003) Species diversity and conservation of Mandarina, an endemic land snail of the Ogasawara Islands.7: 29-37.EldredgeLGSmithBD (1995)FelsensteinJ (1985)doi: 10.2307/2408678Filella-SubiráE (1983)FioreCTullJLZehnerSDuceyPK (2004) Tracking and predation on earthworms by the invasive terrestrial planarian Bipalium adventitium (Tricladida, Platyhelminthes).67: 327-334.FraserPMBoagB (1998) The distribution of lumbricid earthworm communities in relation to flatworms: a comparison between New Zealand and Europe.42: 542-553.HillisDMBullJJ (1993) An emperical test of bootstrapping as a method for assessing confidence in phylogenetic analysis.42: 182-192.IwaiNSugiuraSChibaS (2010) Predation impacts of the invasive flatworm Platydemus manokwari on eggs and hatchlings of land snails.76: 275-278. doi: 10.1093/mollus/eyq007JonesHD (1988) The status and distribution of British terrestrial planarians.36: 511-516.JonesHD (1998) The African and European land planarian faunas, with an identification guide for field workers in Europe.42: 477-489.JonesHD (2005) British land flatworms.16: 189-194.JonesHDBoagB (1996) The distribution of New Zealand and Australian terrestrial flatworms (Platyhelminthes: Turbellaria: Tricladida: Terricola) in the British Isles – the Scottish survey and MEGALAB WORMS.30: 955-975. doi: 10.1080/00222939600770511KatohKTohH (2008) Recent developments in the MAFFT multiple sequence alignment program.9: 286-298. doi: 10.1093/bib/bbn013LoweSBrowneMBoudjelasSDe PoorterM (2000)Luis-NegreteLHBrusaFWinsorL (2011) The blue land planarian Caenoplana coerulea, an invader in Argentina.82: 287-291.MateosEGiribetGCarranzaS (1998) Terrestrial planarians (Platyhelminthes, Tricladida, Terricola) from the Iberian Peninsula: first records of the family Rhynchodemidae, with the description of a new Microplana species.67: 267-276. doi: 10.1111/j.1463-6409.2009.00398.xMateosECabreraCCarranzaSRiutortM (2009) Molecular analysis of the diversity of terrestrial planarians (Platyhelminthes, Tricladida, Continenticola) in the Iberian Peninsula.38: 637-649.MateosETudóAÁlvarez-PresasMRiutortM (2012)MinelliA (1977) A taxonomic review of the terrestrial planarians of Europe.44: 399-419. doi: 10.1080/11250007709429278OgrenRE (1989) Redescription and a new name for the blue land planarian Geoplana vaga Hyman now considered conspecific with Caenoplana coerulea Moseley from Australia (Turbellaria: Tricladida: Geoplanidae).63: 135-142.OgrenRE (1995) Predation behavior of land planarians.305: 105-111. doi: 10.1007/BF00036370OgrenRE (2001)OgrenREKawakatsuM (1998)OhbayashiTOkochiISatoHOnoT (2005) Food habit of Platydemus manokwari De Beauchamp, 1962 (Tricladida: Terricola: Rhynchodemidae), known as a predatory flatworm of land snails in the Ogasawara (Bonin) Islands, Japan.40: 609-614. doi: 10.1303/aez.2005.609OkochiISatoHOhbayashiT (2004) The cause of mollusk decline on the Ogasawara Islands.13: 1465-1475. doi: 10.1023/B:BIOC.0000021334.39072.2dOlewineDA (1972)PosadaD (2008) jModelTest: Phylogenetic model averaging.25: 1253-1256. doi: 10.1093/molbev/msn083SaitouNNeiM (1987) The Neighbor-Joining method – a new method for reconstructing phylogenetic trees.4: 406-425.SantoroGJonesHD (2001)doi: 10.1078/0031-4056-00089SluysRKawakatsuMRiutortMBaguñàJ (2009) A new higher classification of planarian flatworms (Platyhelminthes, Tricladida).43: 1763-1777. doi: 10.1080/00222930902741669SugiuraS (2009) Seasonal fluctuations of invasive flatworm predation pressure on land snails: Implications for the range expansion and impacts of invasive species.142: 3013-3019. doi: 10.1016/j.biocon.2009.07.032SugiuraS (2010) Prey preference and gregarious attacks by the invasive flatworm Platydemus manokwari.12: 1499-1507. doi: 10.1007/s10530-009-9562-9SugiuraSYamauraY (2010) Potential impacts of the invasive flatworm Platydemus manokwari on arboreal snails.11: 737-742. doi: 10.1007/s10530-008-9287-1SugiuraSOkochiITamadaH (2006) High predation pressure by an introduced flatworm on land snails on the oceanic Ogasawara Islands.38: 700-703. doi: 10.1111/j.1744-7429.2006.00196.xSwoffordDL (2002)TamuraKDudleyJNeiMKumarS (2007) MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0.24: 1596-1599. doi: 10.1093/molbev/msm092TerraceTEBakerGH (1994) The blue land planarian, Caenoplana coerulea Moseley (Tricladida: Geoplanidae), a predator of Ommatoiulus moreleti (Lucas) (Diplopoda: Julidae) in Southern Australia.33: 371-372. doi: 10.1111/j.1440-6055.1994.tb01250.xVila-FarréMMateosESluysRRomeroR (2008) Terrestrial planarians (Platyhelminthes, Tricladida, Terricola) from the Iberian Peninsula: new records and description of three new species.1739: 1-20.Vila-FarréMSluysRMateosEJonesHDRomeroR (2011) Land planarians (Platyhelminthes: Tricladida: Geoplanidae) from the Iberian Peninsula: new records and description of two new species, with a discussion on ecology.45: 869-891. doi: 10.1080/00222933.2010.536267WinnepenninckxBBackeljauTDe WachterR (1994) Small ribosomal-subunit RNA and the phylogeny of the Mollusca.108: 98-110.WinsorL (1998) The Australian terrestrial flatworm fauna (Tricladida: Terricola).42: 457-463.WinsorLJohnsPMBarkerGM (2004) Terrestrial planarians (Platyhelminthes: Tricladida: Terricola) predaceous on terrestrial gastropods. In: BarkerGM (Ed.). Natural Enemies of Terrestrial Molluscs.: 227-278. doi: 10.1079/9780851993195.0227Appendix
List of samples used in this study with GenBank accession numbers and sampling locality (if known). The classification follows Sluys et al. (2009).