A new species of alien terrestrial planarian in Spain: Caenoplana decolorata

Sampling

Specimens were collected by E. Mateos from a plant nursery named “vivers casa Paraire” in Bordils municipality (Girona province, Spain, WGS84, position: 42.0348°N; 2.8986°E). All were collected by hand from beneath pots (Figs. 1E and 1F) that contained the plants on 12 January 2012 (five specimens: PT426, PT427, PT428, PT430, PT431) and 22 October 2012 (four specimens: PT655, PT657-1, 2 and 3) (Table 1).

Specimens from 12 January 2012 and specimens PT655 and PT657-3 were preserved in absolute ethanol for further molecular analyses. Specimens PT657-1 and 2 were killed with boiling water, fixed with 10% formalin and preserved in 70% ethanol. Specimens PT426 and PT657-1 were photographed alive (Fig. 1).

Molecular methods

All the sequences used in the present work were obtained in previous studies with the exception of some Cytochrome Oxidase I (herein Cox1) sequences that were obtained from individuals collected at the Real Jardín Botánico de Córdoba (Spain) by Mónica López (Table 1). In all cases, a small section of the anterior end of specimens preserved in absolute ethanol was used for DNA extraction. The new sequences were obtained following the same protocol as in Álvarez-Presas et al. (2014).

A nucleotide alignment was obtained for the Cox1 sequences based on the AA translation as a guide using BioEdit software (Hall, 1999) and the echinoderm mitochondrial genetic code (9). A Maximum Likelihood (ML) phylogeny was inferred using the IQtree software (Nguyen et al., 2015) with the MFP+MERGE implementation and 10,000 replicates for ultrafast bootstrap search (-bb option). Then two single locus molecular species delimitation methods were applied in order to check the validity of the new species presented here and the ones already described and included in the phylogeny. Automatic Barcode Gap Discovery (ABGD) (Puillandre et al., 2012) was the first method performed, implemented in the webpage: https://bioinfo.mnhn.fr/abi/public/abgd/abgdweb.html. The default parameters were used, selecting initial partitions as they are supposed to be more stable and generally give as a result a closer number of groups described by taxonomists than recursive partitions. The second method applied was the multi-rate Poisson Tree Process (mPTP) analysis (Kapli et al., 2017). The newick tree obtained in the ML phylogenetic inference was used as input in the website http://mptp.h-its.org/#/tree.

Anatomical methods

Specimens PT657-1 and 2 were sent to HDJ and are deposited in the Natural History Museum, London, accession numbers NHMUK.2014.5.13.12-13. The larger specimen had a visible gonopore, was assumed to be mature and selected for partial sectioning. It was divided into four portions: anterior portion about 2 cm long not sectioned, in alcohol; pre-pharyngeal section, TS, five slides, two at 15 µm, three at 10 µm; posterior portion including pharynx and copulatory apparatus, LS, 16 slides (pharynx separated, HLS) at 15 µm. Sectioned portions were dehydrated in ethanol and embedded in paraffin wax. Slides were stained in Harris’s haematoxylin and eosin and mounted in Canada balsam. The second specimen, about 3.4 cm long, had no visible gonopore and remains in alcohol.

Colours are expressed as RAL colours (www.ralcolor.com).

The electronic version of this article in Portable Document Format (PDF) will represent a published work according to the International Commission on Zoological Nomenclature (ICZN), and hence the new names contained in the electronic version are effectively published under that Code from the electronic edition alone. This published work and the nomenclatural acts it contains have been registered in ZooBank, the online registration system for the ICZN. The ZooBank Life Science Identifiers (LSIDs) can be resolved and the associated information viewed through any standard web browser by appending the LSID to the prefix http://zoobank.org/. The LSID for this publication is: urn:lsid:zoobank.org:pub:B2636DF8-4372-405C-8A8C-4FBEC7396276. The LSID for the new species described is: Caenoplana decolorata sp. nov. urn:lsid:zoobank.org:act:C0CEE92F-A51E-4EDD-B18B-E7F021338667. The online version of this work is archived and available from the following digital repositories: PeerJ, PubMed Central and CLOCKSS.

Molecular results

The final dataset comprises 43 Cox1 sequences (including three outgroups, Table 1), with a final length of 822 bp. The resulting ML tree (Fig. 2) shows monophyletic groups comprising seven putative Caenoplana species. Although the bootstrap values (bb) are not high enough to give support to the relationships between these clades, the monophyly of the new species described here, C. decolorata, harbor maximum support. The results of the molecular species delimitation analyses (both mPTP and ABGD) match the same clades present in the phylogeny (Fig. 2) giving rise to seven putative Caenoplana species. Among them, we find the subject of this study, Caenoplana decolorata.

Taxonomic section

Order Tricladida Lang, 1884

 Suborder Continenticola Carranza et al., 1998

  Family Geoplanidae Stimpson, 1857

   Subfamily Rhynchodeminae von Graff, 1896

    Tribe Caenoplaninae Ogren & Kawakatsu, 1991

     Genus Caenoplana Moseley, 1877

Caenoplana decolorata new species.

 Caenoplana Ca2 Álvarez-Presas et al., 2014.

Etymology: “decolorata” indicating that live specimens resemble C. coerulea but are comparatively pale and discolored.

NHMUK.2014.5.13.12-13

E. Mateos collection code PT657-1 and PT657-2. Locality: Bordils (Girona, Spain), position N42.0348049 E2.8986153, date 22 October 2012.

Preserved dimensions: holotype (PT657-1): length 46 mm; width 2 mm; height 1 mm; anterior to mouth 28 mm (61% of body length); anterior to gonopore 39 mm (85% of body length); paratype (PT657-2): length 34 mm; width 2.1 mm; anterior to mouth 17 mm (50%); apparently immature.

All other specimens (with a small section of the anterior end removed) are deposited in the collection of M. Riutort at the University of Barcelona.

External characters

Live specimens are ‘mahogany brown’ (RAL 8106) with a narrow ‘cream’ (RAL 9001) mid-line dorsally, merging to ‘beige brown’ (RAL 8024) laterally. The anterior end is ‘copper brown’ (RAL 8004). The ventral mid-line is ‘pastel turquoise’ (RAL 6034) merging into the lateral ‘beige brown.’

Eyes in a sparse uniserial row round the anterior end, biserial for a short distance behind the anterior end and sparse staggered uniserial to the posterior end. Sole nearly the whole of the ventral surface.

Anatomy

Transverse sections (Fig. 3A) are about 1.3 mm high and 2 mm wide. The ciliated creeping sole is about 80% of the width. The cilia are about 5 µm long. The ventral epidermis is a monolayer about 30 µm thick and has few rhabdites. Ventral sub-epidermal muscle consists of a layer of circular muscle fibres about 10 µm thick and longitudinal muscle in bundles about 30 µm thick. Dorsal to the longitudinal muscle bundles is a ventral nerve plexus. There is a distinct, compact layer of parenchymal longitudinal muscle ventrally, 40–50 µm thick, 150 µm in from ventral surface. Ventral nerve cords are about 750 µm centre to centre, about 120 µm in diameter, with transverse commissures. Laterally and dorsally the parenchymal longitudinal muscle is less compact and 10–20 µm thick. Dorsal epidermis is 45 µm thick, non-ciliated and has numerous rhabdites. Dorsal and lateral sub-epidermal circular muscle is about 10 µm thick, and longitudinal muscle in bundles about 35 µm thick. Rhabdites are numerous dorsally and laterally ental to the sub-epidermal muscle, but in the mid-dorsal region, the rhabdites layer is slightly deeper (Figs. 3A and 3B), presumed to be coincident with the pale midline visible in the living animal.

The retracted cylindrical pharynx occupies the whole length of the pharyngeal pouch and is about 2.5 mm long, 0.9 mm in diameter. The pouch is 5.4% of body length. The pharyngeal aperture is about half way along the pharyngeal pouch. Pharyngeal musculature consists of an outer layer of circular muscle about 10 µm thick, a layer of mixed longitudinal and radial muscle about 360 µm thick and an inner layer of circular muscle about 30 µm thick.

The anterior portion containing the ovaries has not been sectioned. Ovovitelline ducts are about 500 µm apart on the inner dorsal surface of the ventral nerve cords (Figs. 3A and 3D). Vitellaria are not distinguishable with certainty. Their outer and inner diameters are about 25 µm and 7 µm respectively. They run to about 800 µm behind the gonopore, turn dorsally and open into the common female duct about 800 µm long which extends forwards with little differentiation to open into the common antrum above the gonopore (Figs. 4A, 4C and 4E). There is little or no shell gland tissue surrounding the common female duct.

Testes are numerous, ventral, ovate, about 200 µm wide and 300 µm high (Figs. 4A, 4D and 4E) and run almost to the copulatory apparatus. The sperm ducts cannot be distinguished with certainty in transverse sections. They enter the anterior end of the muscular bulb of the eversible penis, widen slightly and contain small amounts of stored sperm (Fig. 4B). They separately enter the anterior end of the ejaculatory duct which is complex, long and sinuous, about 1.5 mm from its anterior end to the gonopore (Figs. 4A, 4B, 4D and 4F). It has several regions, for ease of reference they are here arbitrarily numbered 1–7 from anterior to posterior as follows. (1) A small chamber (seminal vesicle) which extends transversely through 10 × 15 µm sections, thus about 150 µm wide, the two sperm ducts entering on either lateral extremity. (2) A narrow duct extending posteriorly and turning ventrally and opening into, (3) a sinus-like duct wide laterally, 23 × 15 µm thus 345 µm wide, but only 35 µm in the antero-posterior direction. This duct initially turns ventrally then narrows and curves posteriorly to be almost U-shaped (second arm shorter). The ejaculatory duct continues into, (4) a narrow sinus-like lumen surrounded by strongly eosinophilic cells forming a structure roughly spherical in outline about 400 µm in diameter. The cells of this region appear to be elongate with nuclei mostly adjacent to the lumen (Fig. 4G). This in turn opens into, (5) a portion about 400 µm long with sinuous margins, which in turn opens via, (6) a small papilla-like opening into, (7) a longer and wider duct about 600 µm long with sinuous walls which can be considered to be the male antrum. This in turn opens to the common antrum above the gonopore.