Nipponnemertes incainca sp. n. Adoption of the new taxonomic proposal for nemerteans (Nemertea, Cratenemertidae)

Abstract A new species Nipponemertes incainca is described from the intertidal zone of Santa Marta, Colombia. A new recent approach based on both morphological and molecular characters is applied for the description. The main characteristics of the species are: red color, head shield-shaped with a mid-dorsal cephalic ridge, furrows pre-cerebral inconspicuous with few faint ridges orthogonal to furrow axis, two irregular groups of eyespots situated at lateral margins in precerebral cephalic region, proboscis provided with papillae and 12 nerves, stylet smooth supported on an oval basis, and two pouches containing 3–4 accessory stylets each. The sequence of the COI gene was analyzed as an additional support for the new species.


Introduction
Nemerteans (phylum Nemertea), commonly known as ribbon worms or Rhynchocoela, comprise a cosmopolitan group of bilateral, coelomate, and unsegmented worms (Turbeville 2002). The major synapomorphy supporting the monophyly of the phylum is the presence of an eversible proboscis housed in a fluid-filled cavity, the rhynchocoel (ibid). Around 1,300 species of nemerteans are recognized, most of which are found in marine environments; nevertheless, freshwater and terrestrial species are also known (Gibson 1995, Kajihara et al. 2007). Among nemerteans the genus Nipponnemertes contains 18 species of marine benthic worms; the interwoven muscular layers in the rhynchocoel wall, and large cerebral sense organs extending behind the brain are the two main traits that distinguish them from most other monostiliferan genera (Friedrich 1968).
Of the 36 species of nemerteans documented for the Caribbean Sea (Corrêa 1961, 1963, Kirsteuer 1973, 1974, 1977, Gonzalez-Cueto et al. 2014, 13 have been reported to be present on the Colombian coast (Kirsteuer 1977, Gonzalez-Cueto et al. 2014). However, the majority of these species have been recorded from a single locality (Santa Marta Bay) despite nemerteans being an abundant component of the macrofauna communities in Colombia (Dueñas 1998, Trujillo et al. 2009). Taxonomic studies on nemerteans from Colombia are scarce. Species identifications traditionally require a detailed study of the internal anatomy, which is considered to be difficult and time consuming. This is because several morphological characters are doubtful, subjective, poorly defined, and plastic, in addition to a lack of taxon experts (Sundberg et al. 2016a). Sundberg et al. (2016a) highlight these problems for the taxonomy of nemerteans and, to advance the study of nemerteans, they suggest transitioning from a traditional, difficult, and often unreliable taxonomy to a more integrative process of describing species based on external morphological characteristics that are easily observable combined with molecular data. Together this would facilitate more accurate species identifications, even for the non-specialist.
Herein, the method proposed by Sundberg et al. (2016a) is used to describe a new species of ribbon worm from Colombia. The external appearance of the worm and photos of the histological section of the proboscis are presented, in addition to a molecular analysis using the mitochondrial gene COI. The COI sequence of one specimen is deposited in GenBank and whole specimens fixed in formalin and other tissue pieces preserved in absolute alcohol are deposited in the "Centro de Colecciones Biológicas de la Universidad del Magdalena" for future molecular and morphological studies.

Materials and methods
Four specimens were hand-collected on the rocky littoral from Inca-Inca Bay, Santa Marta,Colombia (11°12'30.2"N;74°13'54.5"W). Individuals were relaxed in 7% MgCl 2 solution isotonic to seawater and photographed "in vivo" with a digital camera Nikon D7100 with a 60 mm ED Micro-Nikkon lens. Details of morphological characters were photographed with a stereomicroscope Leica M205A with an integrated Camera Leica DFC450. Detailed images of the proboscis and stylets were obtained by pressing the specimens between a slide and a coverslip (obligating them to protrude the proboscis) and photographing them with a microscope Zeiss Axiolab A1 with an integrated camera Zeiss ERc5s. Two specimens were fixed in 100% ETOH for molecular purposes and two in 10% formalin for future morphological analysis.
Two additional specimens previously collected and deposited in the "Centro de Colecciones Biológicas de la Universidad del Magdalena, CBUMAG" (Gonzalez-Cueto et al. 2014) were also examined. Cross sections of the proboscis were obtained from one these specimens (CBUMAG:NEM: 0049). For that, the proboscis was embedded in paraffin; sectioned at 7µm thickness with an AO 820 Spencer microtome, and stained with H&E. Coverslips were mounted with Permount®.
Total DNA was extracted from one entire worm fixed in 100% ETOH, using the DNeasy Blood & Tissue ® Kit following the manufacturer's protocol (Qiagen, Valencia, CA, USA). The partial COI gene was amplified with universal primers described in Folmer et al. (1994). The PCR was performed with 2 µL template in a 25 µL volume with final concentrations of 2 mM MgCl 2 , 5X buffer PCR (no MgCl 2 BIOLINE ® ), 0.4 µM of each primer, 0.4 µM of each dNTP, and 2 units Taq (BIOLASETM, BIO-LINE ® ). The PCR conditions were: 1 min at 95 °C, followed by 35 cycles of 15 s at 95 °C, 1 min at 40 °C, 1.5 min at 72 °C, and there was a final extension period of 5 min at 72 °C. The sequence was edited with ProSeq (Filatov 2009) and aligned with all the sequences from Nipponnemertes accessible in GenBank using the ClustalW algorithm available in MEGA (Tamura et al. 2011) with default parameters. Following the barcoding approach suggested by Hebert et al. (2003), a matrix of intraspecific and interspecific evolutionary genetic distances was made using the Kimura's two parameter model K2P (Kimura 1980), also available in Mega (Tamura et al. 2011). An entire additional worm, collected in Inca-Inca beach (11°12'30.2"N, 74°13'54.5"W) was used for DNA extraction. Sequence data for 615 bp of Cytochrome C Oxidase Subunit I deposited in GenBank under accession number KX879856 (see alignments with other congeners in supplemental information).

Taxonomy
Etymology. The specific epithet refers to the "Inca-Inca beach" site from which most of the specimens were collected; this name is in apposition.
Diagnosis. Nipponnemertes incainca sp. n., like other members of Nipponnemertes, has a mid-dorsal cephalic ridge, is capable of retracting the head into the body when disturbed, and is capable of swimming. However, in this new species the anterior furrows and their secondary transverse grooves are faintly visible both macro-and microscopically and they are not visible in a ventral view.
Description. Relaxed length from 11.7 mm to 22.5 mm and width 1 to 2 mm. Dorsal side uniformly bright red color (Fig. 1A). Ventral side lighter than dorsal side ( Fig. 1). Head shield-shaped, slightly demarcated from rest of body but without V-shape Frontal organ with small cirrus. Cerebral organ furrows pre-cerebral, inconspicuous, with few faint ridges orthogonal to furrow axis. Brain distinguishable as a pale brown bilobed structure through dorsal and ventral body wall. Two irregular groups of eyespots situated at lateral margins in precerebral cephalic region ( Fig. 2A), extending beyond brain parallel to lateral nerve cords. Rhynchopore subterminal. Proboscis long and stout, with papillae ( Fig. 3B), pink in color when everted (Fig. 1B). Stylet (length: 87.4 µm) smooth, supported on an oval basis (54 × 38.3 µm); two pouches containing 3-4 accessory stylets each (Fig. 2B). Twelve proboscidial nerves present ( Fig.  3A-B). This species was found among sponges and brown algae underneath rocks, and in the crust formed by sediment inside the crevices of rocks in the littoral zone. Worms capable of swimming with strong undulating movements.
The most similar species in color, arrangement of ocelli and numbers of proboscidial nerves to Nipponnemertes incainca sp. n. is N. pulchra and it might easily represent an intraspecific variation. However, in the intraspecific and interspecific genetic distance matrix (table 2), the interspecific distance between N. incainca sp. n. and N. pulchra was 21.03%, which exceeds the highest limits given by Sundberg et al. (2016b) for the Hoplonemertea. Therefore, the fact that the new species lacks the V-shape structure formed by the cephalic grooves, and the accessory stylet in the basis of the central armature, present in N. pulchra, is enough to discriminate the two species.   (Uschakov, 1927) Chernyshev 1993 Margins of body lighter in color Inhabits muddy sand (Chernyshev 1993) Nipponnemertes bimaculatus (Coe, 1901) Gibson & Crandall, 1989 (Berg 1985) 15 Nipponnemertes occidentalis (Coe, 1905) Friedrich, 1968 Blotchy dark reddish brown or pale ground color throughout whole dorsal surface, and "ventral surface without color" (Coe 1905) Highly developed intestinal caecum. Caecal appendage in esophagus and one in stomach Nipponnemertes ogumai (Yamaoka, 1947) Crandall et al., 2001 Uniformly orange (Kajihara et al. 2014) although originally described as bright vermilion (Crandall et al. 2001 Riser, 1998 Dorsum buffy white to pale yellow to orange with reddish lines (aggregation of red blood corpuscles in blood vessels), ventral side paler, brain lobes pink 12 "Not evident" (Riser 1998) Lacking

12-13
Lacking Separating strongly head from rest of body

Table 1 (continued).
Remarks about morphological and behavioral traits useful to discriminate the species of the genus Nipponnemertes.
Reference after authority in species column.   Position of cephalic furrows If single pair in front of brain lobes 1

Discussion
Approximately, 2.2 million (σ 0.18) species inhabit the marine ecosystems, yet 91% of these still await description (Tittensor et al. 2010;Mora et al. 2011). The rate at which these species become extinct has reached an unprecedented degree that is much higher than the rate of new species discovered (Dirzo and Raven 2003;Scheffers et al. 2012). The new taxonomic approach of Sundberg et al. (2016a) might help facilitate the description of new species of nemerteans, which otherwise would be underestimated or overlooked. With this approach some morphological characters and molecular data of the new species will be available to scientists in order to have a more integrative assessment of biodiversity. However this approach should be interpreted cautiously because some species, such as the one described here, might require the revision of some internal features (i.e. the number of nerves in the proboscis). Nipponnemertes incainca sp. n. was recorded as Cratenemertidae sp. by Gonzalez-Cueto et al. (2014) and probably it is also the same species recorded as Cratenemertidae spp. by Collin et al. (2005) from "Bocas del Toro (Panama)". Misidentification of nemerteans is common in the environmental assessments of marine ecosystem around the world (Sundberg et al. 2016a). In fact, in Colombia, many specimens remain named as Nemertea sp. even in biological collections such as the "Museo de Historia Natural Marina de Colombia (INVEMAR)" and the "Centro de Colecciones Biológicas de la Universidad del Magdalena". The standardization of the taxonomic and behavior-based character matrix proposed by Sundberg et al. (2016a), applied in this survey (Table 3), and the use of molecular markers (e.g. COI) increase the value of taxonomic identifications in the future. Our study expands the known number of nemertean species of the Caribbean coast of Colombia from 12 to 13. In addition, it encourages a new generation of taxonomists to begin or to continue working on this neglected group of animals. burg, who kindly improved both the English and the scientific content of the paper and helped us with the preliminary identification of the species. We are particularly indebted to Dr. Jörn von Döhren, Dr. Malin Strand and an anonymous reviewer for their constructive comments on the manuscript. This study was partially supported by Departamento Administrativo de Ciencia, Tecnología e Innovación. COLCIENCIAS under the program Convocatoria para la formación de capital humano de alto nivel para el departamento del Magdalena-2014 (convocatoria 672 Cap. 3, Maestría nacional), Fundación Para la Investigación, Conservación y Desarrollo Sostenible de Socio-Ecosistemas and the Fundación Alejandro Ángel Escobar. This is a contribution No. 6 from the Centro de Colecciones Biológicas de la Universidad el Magdalena CBUMAG.