A new deep-sea Cirripedia of the genus Heteralepas from the northeastern Atlantic

A new species of the sessile deep-sea barnacle, Heteralepas (Crustacea, Cirripedia), Heteralepas gettysburgensis sp. nov., is described. The specimens were collected at a depth of 225 m at the Gettysburg Seamount on the Gorringe Bank, located in the Portuguese Exclusive Economic Zone, approximately 200 km off the southwestern coast of mainland Portugal. Extensive morphological and molecular (COI, 12S and 16S) analyses were carried out to separate the species from its nearest congeners with similar geographic distribution, i.e., Atlantic waters.


Introduction
A hydrophone, deployed on 23 August 2015, was collected from a depth of 255 m at the Gettysburg seamount (36º34′42.46″N, 11º35′49.02″W), located in the Gorringe Bank in the Madeira-Tore geological complex (North-East Atlantic), on 28 August 2016.It was fully colonized by polyps of the hydrozoan Oceania armata Kölliker, 1853 and specimens of the new species of Heteralepas Pilsbry, 1907 proposed in this study.
Currently, twenty-three species of the genus Heteralepas are accepted at the World Register of Marine Species (Chan & Southward 2010).All species are distributed in the Atlantic, Pacifi c and Indo-Pacifi c waters.However, only six species are registered for the Atlantic, of which three are for the eastern Atlantic.
We carried out comparisons of our specimens to species with similar diagnostic characters and to species with close geographic distribution, i.e., Atlantic waters.None of the previously described species matches, neither morphologically nor genetically, the new species Heteralepas gettysburgensis sp.nov.presented here.

Material and methods
Specimens were collected from a depth of 255 m at the Gettysburg Seamount (36º34′42.46″N, 11º35′49.02″W) in the Gorringe Bank, located in the Madeira-Tore geologic complex (Fig. 1).Fifteen specimens were morphologically analyzed, of which ten were dissected for the analysis of internal characters and fi ve for molecular analyses.
For the morphological analysis, an optical microscope (Leica MZ 12) was used.Drawings were made using a camera lucida attached to the optical microscope.Pictures were obtained with a digital camera (SONY DFW-SX910) connected to an optical microscope (OLYMPUS SZX9), using an image analyser (Visilog,TNPC Software,v.4).
Sequence trace fi les were individually edited and the primer sequences were removed.The obtained sequences were aligned using Clustal W in MEGA v. 7.0 (Kumar et al. 2016).COI-5P sequences were checked for possible stop codons, indels or unusual amino acid sequences (see Song et al. 2008).BOLD Identifi cation System tool (BOLD-IDS) (Ratnasingham & Hebert 2007) and GenBank BLASTn searchs (Altschul et al. 1990) were used to confi rm if the sequences obtained for the new species match with existing taxa in the public databases.Available DNA barcodes belonging to the same genus were included in our alignment.The neighbour joining method was carried out to calculate intra-and interspecifi c distances using the Kimura-2-parameter (K2P) model (Kimura 1980).A data set was created in BOLD (Data set ID-HETSPP https://doi.org/10.5883/DS-HETSPP)comprising the COI sequences generated in this study plus the available sequences of Heteralepas specimens.All original sequences generated in this study were submitted to GenBank and correspond to accessions numbers MF695097 -MF695101, MF695104 -MF695108 and MF695113 -MF695121, including the sequences of the hydrozoan Oceania armata.

Diagnosis
Demarcation between capitulum and peduncle clear.Capitulum wider than capitulo-peduncular junction.Capitulum slightly longer than peduncle, both with folds.Carinal margin thickened.Aperture more than ⅓ and less than ½ height of the capitulum, with crenulated lips.Lips surrounding aperture clearly set off from the surrounding capitulum by a demarcation border.Mandible with four teeth covered by fi ne setae, as well as its posterior side.Lower margin of the teeth with numerous pectinations: the fi rst tooth with ten, the second, third and fourth ones with fi ve, fi ve and six pectinations, respectively.First maxilla with two strong acuminate teeth of unequal length on the upper angle.

Etymology
The specifi c epithet alludes to the location where the specimens were collected: the Gettysburg Seamount, Gorringe Bank, in the Madeira-Tore geologic complex.

Description
The fresh specimens were translucent yellowish, allowing observation of the cirri movement inside the capitulum.Demarcation between capitulum and peduncle clear.Capitulum wider than capitulo- peduncular junction (Fig. 2A-B).Capitulum 1.2 to 1.6 times longer than peduncle, cylindrical.Both peduncle and capitulum with folds.Aperture more than ⅓ and less than ½ of the capitulum height.Lips surrounding the aperture crenulated and clearly set off from the surrounding capitulum by a demarcation border (Fig. 2C-D).Carinal margin thickened.Mandible (Fig. 3A-B) with four teeth covered by fi ne setae as well as its posterior side.Lower margin of the teeth with pectinations: fi rst tooth with ten, second, third and fourth ones with fi ve, fi ve and six pectinations, respectively (Fig. 3C-F).Distance between fi rst and second tooth 1.5 times that between second and third tooth.Latter distance similar to the distance between third and fourth tooth.First maxilla deeply notched, with two strong acuminate teeth of unequal length on upper angle, numerous setae and short to long spines on the cutting margin.Posterior margin with a dense clump of fi ne setae.(Fig. 4A-C).Second maxilla with two lobes covered with numerous long serrulate setae, those on posterior lobe longer than those on anterior lobe (Fig. 4D-E).Cirral setation lasiopod.Cirrus I separated from posterior pairs, with unequal rami.Anterior ramus slightly shorter and wider at base than posterior ramus (Fig. 5A-B).Both with numerous setae on upper and lower margins.Base of cirrus I with one fi lamentous appendage.Cirrus II with anterior ramus slightly shorter than posterior ramus (Fig. 6A).Anterior and posterior rami of cirri III and IV very similar (Fig. 6B-C).Cirri V and VI similar (Fig. 6D and 4C-D, respectively), with inner rami atrophied.Inner ramus of cirrus VI shorter than inner ramus of cirrus V. Caudal appendage slightly longer than pedicel of cirrus VI (Fig. 5E-F).Penis relatively long, annulated, with numerous long and short fi ne setae distally scattered throughout (Fig. 5G-H).Table 1 displays the measurements, ratios, mean, standard deviation and coeffi cient of variation concerning the morphological characters mentioned.

Discussion
All specimens were collected from the same site, at a depth of 255 m in the northeastern Atlantic.Seven species occur in the Atlantic: H. belli (Gruvel, 1902), H. cantelli Buhl-Mortensen & Newman, 2004, H. cornuta (Darwin, 1851), H. lankesteri (Gruvel, 1900), H. luridas Zevina, 1975, H. microstoma (Gruvel, 1901) and H. segonzaci Young, 2001, of which only three species occur in the east Atlantic (H.cornuta, H. microstoma and H. segonzaci).The new species differs distinctly from H. cornuta due to the absence of conspicuous triangular projections in the capitulum (Fig. 2A-B).The species H. microstoma has a peduncle much longer than the capitulum, being the opposite of our specimens, where the capitulum is slightly longer than the peduncle.The original description by Gruvel (1902) gives an account of the number of articles of cirrus I (inner and outer rami), the inner rami of cirri V and VI and the caudal appendage.All cirri have a greater number of articles than our specimens except the caudal appendage (Table 2).The peduncle of H. segonzaci, from a depth of 2235 m, is three times shorter than the capitulum and the aperture is more than half the length of the capitulum (Young, 2001) while our specimens have an aperture length less than half that of the capitulum, and the rami of cirri III and IV have fewer articles than H. segonzaci.Comparing with the other species recorded from the western Atlantic, H. belli has the capitulum shorter than the peduncle and the cuticle is almost smooth, with some irregular folds (Gruvel, 1902), while our specimens have the capitulum longer than the peduncle and the cuticle with numerous folds.Moreover, in H. bellii the aperture is half as high as the capitulum height (Gruvel, 1902) while in our species it is less than half its height.In H. lankesteri the capitulum is approximately as long as the peduncle (ratio length of capitulum to length of peduncle (C/P) varies from 0.6 to 1.3) (Gruvel, 1900), while in our specimens the ratio varies between 1.2 and 1.6.The cirri rami with a different number of articles and the non-pectinated mandible of H. lankesteri (Gruvel, 1900) also distinguish it from our species (Table 2).Finally, the small size of H. luridas (maximum registered size 9.5 mm), from the Caribbean, and the smaller number of articles of all cirri rami (Zevina, 1975) strongly distinguish it from our specimens (Table 2).
Regarding the species outside of the Atlantic, H. japonica Aurivillius, 1892 and H. canci Chan, Tsang & Shih, 2012 are the most similar species externally, probably due to the large morphological variability of H. japonica, unlike the species analyzed in this study (Table 1).However, they have crests on the carinal region of the capitulum, the rami of the cirri have fewer articles than our specimens and the ratio 'height of aperture/height of capitulum' is higher (Chan et al., 2009).The teeth of the mandible of H. japonica appear to be without pectinations or might have low pectinations on the lower margins of teeth 1-3, especially 2 and 3 (Buhl-Mortensen & Newman 2004), while the new species exhibits numerous pectinations on the lower margin of the four teeth.Also, the maximum length of the peduncle and capitulum of H. japonica reported by Buhl-Mortensen & Newman ( 2004) is 11.6 and 3.6 cm respectively, quite distinct from the new species described in this study (1.4 and 1.6 cm, respectively).On the other hand, the sequences of the COI obtained for our specimens had divergences of 16 to 21 % with H. japonica and 16 to 17% with H. canci (Chan et al. 2009).The intraspecifi c distances obtained for our specimens were 0.3 to 0.7%.Many studies have been carried out using the mitochondrial DNA sequences of the COI-5P, which have confi rmed that DNA barcodes (Hebert et al. 2003) are a reliable tool to discriminate species of crustaceans (Lobo et al. 2013(Lobo et al. , 2016)), including cirripedes (Chan et al. 2009).Ratnasingham & Hebert (2013) suggested that 2.2% of average intraspecifi c distance is a reference threshold for within-species boundaries, which is quite far from the divergences found in this study.In addition, the marker 12S presented divergences approximately of 6.5% and 7.5 %, with the species H. canci and H. japonica, respectively, being within the expected values for barnacle species within the same genus (Chan et al. 2007(Chan et al. , 2009)).The marker 16S also showed 7 % of genetic divergence with the only specimen of Heteralepas (unidentifi ed) available in public databases (Schiffer & Herbig 2016, GenBank accession KT947465), which is in accordance with the results obtained for other crustaceans (Brasher et al. 1992;Machado et al. 1993).H. adiposa Zevina, 1982 andH. cygnus Pilsbry, 1907, are similar to H. microstoma, but they differ from it in having numerous small calcareous knobs, four mandible teeth, and a great number of caudal segments (Zevina & Kolbasov 2000).Therefore, their diagnostic characters are also distinct from the specimens analyzed in this study.H. mystacophora Newman, 1964 has a smooth capitulum and the superior margins of the second and third teeth of the mandible support several widely spaced spinules (Zullo & Newman, 1964), while the species described here presents a wrinkled capitulum and has spinules in the inferior margin in the four teeth of the mandible.The capitulum of H. nicobarica Annandale, 1909 is indistinctly separated from the peduncle, the aperture height is ¼ of the capitulum height and the peduncle is similar to or longer than the capitulum (Annandale, 1909).Contrarily, the new species presents a clear demarcation between capitulum and peduncle, the aperture height is > ⅓ and < ½ of the capitulum height and the peduncle is shorter than the capitulum (see Table 1).In H. rex (Pilsbry, 1907), the capitulum is almost as long as the peduncle, and lips are slightly crenulated or irregularly warty and do not protrude in adults (Pilsbry, 1907).In our specimens the capitulum is slightly longer than the peduncle and the lips are clearly crenulated and protruding.In H. utinomii Newman, 1960, the length of the peduncle is one third of that of the capitulum (Newman, 1960), which distinguishes it from our specimens whose peduncle is only slightly shorter than the capitulum.(Gruvel, 1902), H. canci Chan, Tsang & Shih, 2012, H. cantelli Buhl-Mortensen & Newman, 2004, H. japonica Aurivillius, 1892, H. lankesteri (Gruvel, 1900), H. microstoma (Gruvel, 1901)

Final Remarks
Specimens of Heteralepas are not encountered often since they live in deep-sea environments.The combination of the morphological and molecular data, reveals that the new species Heteralepas gettysburgensis sp.nov. is distinct from all the species described so far for this genus.It is recorded from the type locality at a depth of 255 m in the Gettysburg Seamount of the Gorringe Bank only, located in the Portuguese Exclusive Economic Zone, approximately 200 km off the southwestern coast of mainland Portugal.Due to its outstanding biodiversity, including several priority species and habitats, the Portuguese Institute for Nature Conservation and Forests, proposed in 2015 to declare the Gorringe Bank as a Site of Community Importance (SCI) under the Natura 2000 network.The present work not only contributes to improve the knowledge of the benthic biodiversity in this deep-sea environment, but also provides both a morphological description and genetic data (three mitochondrial markers: COI-5P, 12S and 16S), which will be a relevant contribution to help clarifying potential issues regarding the taxonomy of the genus Heteralepas.

Fig. 1 .
Fig. 1.Map showing the location of the sampling site (red dot).

Fig. 2 .
Fig. 2. External morphology of Heteralepas gettysburgensis sp.nov.(holotype) A-B.Side views showing the capitulum and peduncle.C. Side view of the aperture.D. Front view of the aperture.

Table 1 .
External and internal morphological variation for specimens analyzed in this study.(-lack of  information.)

Table 2 .
Comparison of diagnostic characters between the new species H. gettysburgensis sp.nov.and H. belli Young, 2001nzaciYoung, 2001.The table was based onBuhl-Mortensen & Newman (2004).C/P = Ratio of length of capitulum (C) to length of peduncle (P); A/C = Ratio of height of aperture (A) to height of capitulum (C).Characters coincident with the new species are underlined.