New records of the genera Leptogorgia , Pacifigorgia and Eugorgia ( Octocorallia : Gorgoniidae ) from Ecuador , with a description of a new species

1 Dpto. Biodiversidad y Biología Evolutiva. Museo Nacional de Ciencias Naturales (MNCN-CSIC), 28006 Madrid, Spain. E-mail: mar.s.hurtado@gmail.es; annie@mncn.csic.es 2 Biodiversidad y Ecología de Invertebrados Marinos, Facultad de Biología, Universidad de Sevilla, 41012, Sevilla, Spain. E-mail: pjlopez@us.es 3 Real Jardín Botánico (CSIC), Plaza de Murillo 2, 28014 Madrid, Spain. E-mail: jmunoz@rjb.csic.es 4 Centro de Biodiversidad y Cambio Climático, Universidad Tecnológica Indoamérica, Quito, Ecuador.


INTRODUCTION
The octocorals are found in marine habitats ranging from intertidal to abyssal waters and are distributed from the Arctic to the Antarctic (Bayer 1961).The gorgonian octocorals are one of the best-represented taxonomic groups in sublittoral marine ecosystems, are ecologically important and are the dominant macrofaunal group on many tropical reefs (Sánchez et al. 2003, Williams andBreedy 2004).The study of the eastern Pacific gorgonian octocorals, specifically in Ecuador, has not matched the intensity and number of publications dedicated to Caribbean species (see Bayer 1981).Despite important contributions by authors such as Breedy andGuzmán (2002, 2007), Williams and Breedy (2004) and Breedy et al. (2009) in the eastern Pacific, the knowledge on the gorgonian fauna of Ecuador is far from complete (but see Bielschowsky 1929 andSoler-Hurtado andLópez-González 2012).Four gorgoniid genera (Gorgoniidae) were previously reported in the eastern Pacific, Phycogorgia Milne-Edwards and Haime, 1850, Leptogorgia Milne-Edwards and Haime, 1857, Eugorgia Verrill, 1868 and Paci figorgia Bayer, 1951.The genus Leptogorgia, with about 60 valid species (27 species in the eastern Pacific) is one of the most frequent genera in the shallow water communities of the eastern Pacific (Breedy and Guzmán 2007), with a wide distribution from southern California to Chile.This genus is also present in the Caribbean, western and eastern South African coasts, the eastern Atlantic and the Mediterranean Sea, and one species is known from the sub-Antarctic (Williams and Lindo 1997).The genus Pacifigorgia is geographically confined to the Pacific coast of tropical America, with the exception of Pacifigorgia elegans (Milne-Edwards and Haime, 1857) from the tropical western Atlantic.Pacifigorgia includes about 36 recognized species distributed throughout the eastern tropical Pacific region (Breedy and Guzmán 2002, 2003, 2004, Williams and Breedy 2004, Guzmán and Breedy 2011).The genus Eugorgia, which includes 16 species, is considered exclusive to the eastern Pacific (from southern California to Peru) (Breedy and Guzmán 2013).Finally, the genus Phycogorgia, with only one species, Phycogorgia fucata (Valenciennes, 1846), is distributed along the west coast of Central and South America (Bayer 1953).
The aim of this report is to present new information derived from material of the genera Eugorgia, Leptogorgia and Pacifigorgia collected in Ecuador.We expand the known distribution of some species in the eastern Pacific and describe new morphological variability for several taxa.Finally, using morphological and genetic data, we describe one new species of Pacifigorgia, emphasizing the set of morphological characters considered to be of taxonomic importance for this genus (Breedy and Guzmán 2002, 2003, 2004, Williams and Breedy 2004, Guzmán and Breedy 2011).We include the sequence analysis of the barcode of mtMutS plus COI with a short, adjacent intergenic region (Igr1) proposed by McFadden et al. (2011), with its closest congeners and available sequences in GenBank.

Study area and sampling methodology
Eleven stations in Ecuadorian waters were sampled from February 2010 to June 2014 (Fig. 1).Gorgonians were collected by SCUBA diving in a depth range of between 5 and 30 m. Substrata in these habitats mainly include sand and rocky bottoms.During sampling, we took colour photographs of the living specimens in their environment; specimens were also photographed outside the water, just after collection to ensure the accuracy of our colour descriptions.Subsamples were fixed in either absolute ethanol for further molecular analyses, or in 4% buffered formalin (after previous relaxation with menthol crystals for a few hours) for the morphological study; the rest of the colonies were allowed to air dry.Buffered formalin-fixed subsamples were subsequently transferred to 70% ethanol (Soler-Hurtado and López-González 2012).

External morphology and SEM study
Fragments from different parts of the colony were prepared for study by SEM according to standard methods (Bayer andStefani 1989, Alderslade 1998).Additionally, permanent mounts were made for light microscopy observation.The colonies are described and illustrated according to standard terminology (Bayer et al. 1983, Breedy and Guzmán 2003, 2007).
For either morphological and molecular comparisons, we studied types and additional materials deposited in the Museum of Comparative Zoology, Harvard University (MCZ); Muséum National d'Histoire Naturelle, Paris (MNHN); the Natural History Museum, London (NHM); the Yale Peabody Museum of Natural History, New Haven (YPM); and the National Museum of Natural History, Smithsonian, Washington (MNH).
Museum specimens were revised for morphological comparative purposes and, in some cases, for molecular analysis.These samples are listed in the text after the newly collected material of each species.Additional museum specimens of species revised but not found in Ecuador were the following: Eugorgia ampla (Verrill, 1864): NHM 69.4.15.53, Baja California (Mexico), no further data.YPM 399, Baja California, 11-15 m depth, 1865.MCZ 65167, Mexico, no depth given, date unknown.
The newly collected specimens are deposited in the Museo Ecuatoriano de Ciencias Naturales (MECN), the octocoral reference collection of the research group "Biodiversidad y Ecología de Invertebrados Marinos" at the University of Seville (BEIM), the Museo Nacional de Ciencias Naturales in Madrid (MNCN-CSIC), the Museo de Zoología de la Universidad Tecnológica Indoamérica in Quito (UTI), and the Museu de Ciénces Naturals in Barcelona (BCN).

DNA extraction, PCR amplification and sequencing
DNA was extracted from 20-30 mg of tissue using the DNeasy extraction kit (Qiagen, Inc.) according to the manufacturer's protocol.Partial COII-COI (including Igr1 region) and MutS sequences were amplified by PCR using the following primers: COII8068F (Mc-Fadden et al. 2004), COIOCTR (France and Hoover 2002), the newly developed COI-Gorg1-R3 (5′-AGA-GAAGGTGGTAATAACCAGAAA-3′) and COI-Gorg2-F2 (5′-GATTCGGAAATTGGTTTGTG-3´) for COI + Igr1; ND42599F (France and Hoover 2002) and MUT3458R (Sánchez et al. 2003) for MutS.Amplifications were carried out in a final volume of 50 µL containing 5 µL of 10x buffer (containing 10×2 mM MgCl 2 ), 1 µL dNTPs mix (10 mM), 0.8 µL of each primer (10 µM), 0.5 µL of Taq DNA polymerase (5U/ µL) (Biotools) and 2 µL of genomic DNA.Thermocycling for the COI fragment included an initial 4-min denaturation step at 94°C, followed by 40 cycles of 45 s at 94°C, 1 min at 58°C and 1 min at 72°C.The cycle ended with 10 min of sequence extension at 72°C.For MutS, we used an initial 4-min denaturation step at 94°C, followed by 35 cycles of 90 s at 94°C, 90 s at 58°C and 1 min at 72°C.The cycle ended with 5 min of sequence extension at 72°C.The amplification products were purified by ethanol precipitation.The amplicons were sequenced for both strands using Big-Dye Terminator in an ABI 3730 genetic analyser (Applied Biosystems).The sequences obtained were edited using the Sequencher v.4.6 program (Gene Code Corporation, Ann Arbor, MI, USA).The resulting alignments were inspected by eye and manually checked Table 1.-Pacifigorgia species involved in the molecular comparisons carried out in this paper.Materials in bold are species sequenced for this study.Note that all GenBank sequences are considered here with the names as they appear in GenBank and their original publications (including numbers or letters).For sequences with duplicate complete names, we have included (1), ( 2), (3), etc., for the purpose of correctly identifying the sequence in and adjusted with Se-Al v2.0a11 (Rambaut 2002).The distance matrix was obtained using PAUP*v4.0b10(Swofford 2001) with a neighbour joining clustering algorithm (Saitou and Nei 1987).A molecular data matrix was created with the morphologically closest species, together with other published sequences for Gorgoniidae in GenBank (see Table 1).
Newly collected examined material: MECN (Ant0001 Description.The colonies measure up to 350 mm in length and 105 mm wide, irregularly pinnate; branches slender, mostly in a plane (Fig. 2A).Unbranched distal twigs up to 100 mm in length and 20 mm in diameter, compressed proximally, more cylindrical and slightly tapered distally (Fig. 2A, B).The holdfast circular, up to 5 mm in diameter.Slightly marked longitudinal grooves along the thick basal branches and near the base.The polyps retract within slightly raised polypmounds, sparsely distributed all around the branches with oblong apertures (Fig. 2B).The colour of the colony is white.The coenenchymal sclerites hyaline (Fig. 2C).The dominant sclerite type spindles up to 0.14 mm in length and 0.03 mm wide, with 4-6 whorls of tubercles; straight or bent, some with a marked waist (Figs 2C , 3A).The capstans up to 0.07 mm in length and 0.03 mm wide (Fig. 3B).Crosses not found.The anthocodial sclerites hyaline rods up to 0.08 mm in length and 0.02 mm wide, with some marginal projections (Figs 2C,3C).
Although Leptogorgia alba has already been collected in Ecuador (Galápagos Islands) (Breedy and Guzmán 2007); this is the first time that it has been found on the continental coast since the paper of Bielschowsky (1929).
Remarks.Our material is in agreement with the original description (Duchassaing and Michelotti 1864:19) and its later re-description (Breedy and Guzmán 2007:12-19).Due to its white colour, it is easily recognizable underwater.Breedy and Guzmán (2007) considered that its morphological variability, especially the type of branching, could perhaps be a response to several environmental factors.In our samples we observe a more pinnate pattern, although there is a tendency towards a dichotomous branching in some specimens.In the type material, the spindles are long, up to 0.17-0.18mm in length and 0.04-0.06mm in width, with marked and complex tubercles.However, our material shows smaller spindles (up to 0.14×0.03mm, Table 2) with a less marked ornamentation.In the same way, the anthocodial rods are considered very consistent in size and shape (long and conspicuous) in this species (up to 0.15 mm in length) (Breedy and Guzmán 2007).However, the examined Ecuadorian material shows a smaller maximum length (up to 0.08 mm).
Newly collected examined material: MECN (Ant0032 Description.The colonies are up to 670 mm in length by 950 mm in width.The branching pattern is irregularly pinnate; branches are flat, lax, pinnate, slender and on a plane (Fig. 4A).The unbranched distal twigs can reach up to 22 mm in length and 17 mm in diameter (Fig. 4A, B).The holdfast is slightly flat, up to 15 mm in diameter.The polyps retract within prominent polyp-mounds, sparsely distributed all around the branches with slit-like apertures (Fig. 4B).The colour of the colony and of the coenenchymal sclerites is red (Fig. 4A-C).The spindles are the dominant sclerite type, up to 0.11 mm in length and 0.03 mm in width,  5B).Some small, scattered crosses are found (0.04×0.05 mm) (Fig. 4C).The anthocodial sclerites are orange flattened rods, up to 0.13 mm in length and 0.02 mm in width, with lobe-like marginal projections (Figs 4C, 5C).
Geographic and bathymetric distribution.Lepto gorgia diffusa has been previously reported in California, El Salvador, Costa Rica, Panama, Colombia, and Chile at 5-30 m depth (see Verrill 1868, Bielschowsky 1929, Prahl et al. 1986, Breedy and Guzmán, 2007) (Fig. 25B).This new record fills the gap between the northern and southern records of the species.Lepto gorgia diffusa probably has a wider distribution, especially in offshore areas of Mexico and Peru, but it may have been previously overlooked.Although this species is quite frequent in Ecuador, it is usually observed isolated within multispecies assemblages and does not form large gorgonian gardens.
Remarks.The morphology of this species is very constant in all examined samples.It is easily differentiable from other Leptogorgia species by the lax, pinnate style of branching, with prominent polyp-mounds.Breedy and Guzmán (2007: 34-35) noted anthocodial sclerites that were light orange, dark pink, or both; however, our materials are only orange.The coenenchymal sclerites are a bit smaller (up to 0.11×0.03mm; Table 2) than previously described (up to 0.14-0.15×0.05mm) (Verrill 1868: 398, Breedy andGuzmán 2007: 34).In addition, the Ecuadorian material has scattered small crosses not reported in this species before.(Verrill, 1868) (Figs 6, 7)
Newly collected examined material: MECN (Ant0034 Description.The colonies measure up to 200 mm in length by 40 mm in width.The branching pattern is irregularly pinnate, mostly on a plane; branches are lank and bushy with long, slender, and flexible branches, drooping slightly at the ends (Fig. 6A).The unbranched distal twigs can reach up to 50 mm in length and 18 mm in diameter (Fig. 6A, B).The holdfast is circular, up to 10 mm in diameter.The polyps retract within nearly flat polyp-mounds, closely distributed all around the branches, with oblong apertures (Fig. 6B).The colour of the colony is brown (Fig. 6A, B).The coenenchymal sclerites are red, pink and yellow; some of them are bicoloured (Fig. 6C).The capstans are the dominant sclerite type, up to 0.07 mm in length and 0.03 mm in width (Figs 6C, 7B).The spindles reach up to 0.1 mm in length and 0.03 mm in width, with 4-6 whorls of tubercles; they are straight or bent, some with a marked waist (Fig. 7A).Some crosses or four-radiates (up to 0.03×0.03mm) (Fig. 7D) and six-radiates (up to 0.03×0.02mm) are also found.The anthocodial sclerites are pink flattened rods up to 0.16 mm in length and 0.08 mm in width, with short lobe-like marginal projections (Fig. 6D).
Geographic and bathymetric distribution.This species has been previously reported in California, El Salvador and Panama at 5-30 m depth (Verrill 1868,  Breedy and Guzmán 2007) (Fig. 25C), and thus our specimens represent a sizeable expansion to the south.
Remarks.Under water, the species is characterized by its decumbent or drooping branching pattern and its brown colonies.However, in small colonies the drooping habit is not as evident, and sometimes the holdfast and basal branches are an intense yellow.Crosses or four-radiate sclerites were not mentioned by Breedy and Guzmán (2007) in their revision of different type materials (e.g.deposited at MZC and YPM).However, they are evident in our collections and in the type specimens at NHM.This type of sclerite had been noted by Verrill (1868) as well, and it should be considered diagnostic for the species.Bielschowsky, 1929 (Figs 8, 9) Synonymy.See Breedy and Guzmán (2007: 57).Description.The colonies are up to 164 mm in length by 75 mm in width.The branching pattern is irregularly dichotomous; branches are bushy, closely ramified and rigid (Fig. 8A).The unbranched distal twigs can reach up to 5 mm in length and 2.3 mm in diameter, are compressed proximally, being more cylindrical and slightly tapered at the ends (Fig. 8A,  B).The holdfast is circular, up to 14 mm in diameter.The polyps retract within prominent polyp-mounds leaving oblong apertures, and are closely distributed all around the branches (Fig. 8B).The colour of the colony is purple (Fig. 8A, B).The coenenchymal sclerites are red (Fig. 8C, D).The spindles are the dominant sclerite type, up to 0.13 mm in length and 0.04 mm in width, with 4-5 whorls of tubercles; they are straight or bent, some with a marked waist (Figs 8C, 9A).The capstans reach up to 0.08 mm in length and 0.04 mm in width (Figs 8C,9B).Some crosses up to 0.06 by 0.04 mm are found (Fig. 9C).The anthocodial sclerites are orange flattened rods, up to 0.07 mm in length and 0.02 mm in width, with lobe-like marginal projections (Figs 8D, 9D).Geographic and bathymetric distribution.Lepto gorgia obscura was known previously only from the type locality (Bahía de Caráquez, Ecuador) (Bielschowsky 1929; Fig. 25D).There is an unpublished record from Baja California (Harden 1979), although Breedy and Guzmán (2007) note that this record should be verified.The only reliable previously known depth range for the species was 4-5 m (Bielschowsky 1929), which we now expand to 15 m.This species is quite abundant in the study area on rocky bottom areas and usually grows along with Lep togorgia alba.

Leptogorgia obscura
Remarks.According to Bielschowsky (1929) and Breedy and Guzmán (2007: 57), capstans are the dominant sclerites in the type material.However, spindles are the most common sclerites in our specimens.Crosses or four-radiates also occur, a type of sclerite not reported in this species before.
Newly collected examined material: MECN (Ant0035), Los Ahorcados, Manabí (Ecuador), 1°40'44"S 80°50'08"W, 15 m depth,   Description.The colonies are up to 50 mm in length by 73 mm in width, and are formed by two fans: the first fan is the largest and the second one radiates from the holdfast and extends in parallel together with the first fan, until a certain point where both may fused (Fig. 10A).The holdfast is very small, up to 4 mm in diameter.The branches are squarish, ranging from 0.6 to 0.8 mm in diameter (25 meshes cm -2 ), and the endbranchlets are short, up to 2 mm long.The network is fine and regular; it is formed mostly by square meshes (2×2.8 mm by 2.5×3 mm) (Fig. 10B).There are no midribs; nevertheless, adult specimens have large, slightly compressed principal branches, which arise from near the base, and diverge through the fan, but often for no more than a quarter of the height.The polyps retract within slightly raised polyp-mounds with asterisklike apertures, placed in multiple rows all around the branches.The colour of the colony is reddish intermingled with yellow.The coenenchymal sclerites are red, light yellow, and bicoloured (Fig. 10C).11C).
Geographic and bathymetric distribution.Pacifi gorgia cribrum has been previously reported in Mexico (Breedy and Guzmán 2002) (Fig. 25E), with additional questionable records from New Zealand (MNHN-IK 1661), Australia (NMH 58.5.15.237), and Costa Rica (e.g.USNM 49382, collected in 1927 and identified by M.F.Bayer).In this study we observed this species at 15-30 m depth.A depth range was lacking for it in the literature.
Remarks.According to Bayer and Macintyre (2001) and Breedy and Guzmán (2002), Pacifigorgia cribrum, P. adamsii, P. agassizii and P. rutila may represent a group sharing a set of morphological features, consisting of fine, regular and closely anastomosed networks.
In addition, Pacifigorgia cribrum is morphologically close to P. arenata, both species having a similar colony colour and sclerome characteristics (red, yellow, bicoloured coenenchymal sclerites, and yellow anthocodial sclerites up to 0.10-0.13mm in length; Table 3).However, the two species differ in mesh features (presence of midrib and higher meshes in P. arenata, see Breedy and Guzmán 2002).In fact, one of the principal problems in recording the possible morphological and chromatic variability of P. arenata is that the only known specimen is the holotype, which is deposited in Paris (MNHN), and there are some fragments in the Smithsonian (USNM 49567).Breedy and Guzmán, 2003 (Figs 12, 13) Newly  Description.

Pacifigorgia flavimaculata
The colonies reach up to 105 mm in length by 70 mm width and are formed by two parallel fans.The holdfast was not observed (Fig. 12A).The branches are cylindrical, ranging from 1.5-2.2mm in diameter (1-3 meshes cm -2 ).The branches arise directly from the base and have incomplete anastomoses that form a loose, open and irregular network.The meshes are rounded-square, oblong or triangular (19×5 mm, 9×4 mm) (Fig. 12B).There are no distinct midribs.There are long end-branchlets (up to 11 mm).The polyps retract within prominent polyp-mounds having rounded apertures, placed all around the branches in multiple rows.The colour of the colony is light brown when alive and yellow intermingled with light brown and light purple when dry.The coenenchymal sclerites are pink and light yellow, some of them bicoloured (Fig. 12C).They are spindles (0.14×0.04 mm) having acute ends and 4-5 whorls of tubercles; blunt spindles (0.11×0.04 mm) with 4 whorls of tubercles (Figs 12C, 13A, B), and capstans (0.09×0.04 mm) with tuberculate ends, warty or smooth (Figs 12C,13B).The dominant sclerite types are straight or bent spindles, some of them with a marked waist.The anthocodial sclerites are orange flattened rods, up to 0.13-0.14mm in length and 0.02 mm in width, with pointed projections, and acute and warty ends (Figs 12C, 13C).
Geographic and bathymetric distribution.Pacifi gorgia flavimaculata has only been reported from the type locality in Costa Rica at 3 m depth (Breedy and Guzmán 2003) (Fig. 25F).In the present study, it was observed at a 3-20 m depth.Breedy and Guzmán (2003) pointed out that this species was only observed in the type locality (Punta Salsipuedes, Costa Rica), despite a significant sampling effort in the neighbouring areas.Therefore, our record from Ecuador is important in defining the geographic (and bathymetric) distribution of this species.
Remarks.Colonies of this species mainly form loose and irregular networks, sometimes almost pseudoanastomosed, yellow or brown in colour but with purplish or yellowish spots around a prominent polyp-mound, making the species easily recognizable.

Pacifigorgia irene
Newly collected examined material: BEIM ( 0091 Description.The colonies reach up to 600 mm in length by 500 mm in width, and are formed by one to several fans (Fig. 14A).The holdfast was not observed.The branches are slender, ranging from 0.3 to 0.7 mm in diameter (about 35 meshes cm -2 ).The branches form a fine and regular network.The network consists of small and squarish meshes (usually up to 1.2 by 0.9 mm) (Fig. 14B).The fans have several stout, rounded midribs.There are short end-branchlets (<1 mm long).The polyps retract within slightly raised and crowded polyp-mounds, placed all around the branches.The colour of the colony is reddish intermingled with yellow both when alive and dry, with a slight discoloration at the edges of the colony.The coenenchymal sclerites are red, lemon yellow and orange, some of them bicoloured (Fig. 14C).They consist of long spindles (0.17×0.05 mm) having acute ends and 5-6 whorls of tubercles (Figs 14A, 15A), and capstans (0.08×0.04 mm) with tuberculated ends (Fig. 15 B).In our specimens the dominant sclerite type are acute straight or bent spindles, some with a marked waist.Some crosses up to 0.06 by 0.05 mm occur as well (Fig. 15C).The anthocodial sclerites are light yellow and light pink flattened rods, up to 0.11 mm in length and 0.02 mm in width, with smooth or slightly lobed borders (Fig. 14C).
Geographic and bathymetric distribution.Pacifi gorgia irene has been previously reported in Panama and Costa Rica at 12-33 m depth (Bayer 1951, Breedy and Guzmán 2002, 2003) (Fig. 25G).Our finding represents a considerable southerly extension of its known distribution.
Remarks.This species is easily recognized by its characteristic morphology, showing a wide fan or fans, marked and thick midribs and small meshes.Some coenenchymal sclerites have been considered here as elongated capstans instead of blunt spindles, as commonly described.The morphology of some sclerites are not always easy to define in this taxon, and they can also be considered transitional forms of blunt spindles, depending on the development of the two opposite distal processes on the longitudinal axis with respect to  the two central whorls with alternate tubercles (Vargas et al. 2010a).In any case, if these sclerites are considered blunt spindles, they would have equivalent forms with respect to the original description of this species.
In situ specimens seen during this study show a more intense reddish and yellow colour than Costa Rican specimens.Bayer (1951) described the colour of the colony as dark purple with greenish borders, but this is not the case in the Ecuadorian material, where colour only fades slightly at the edges of the fans.Description of the holotype.The colony is formed by a single fan 250 mm in length by 290 mm in width (Fig. 16A).The holdfast is circular, up to 15 mm in diameter.The branches are cylindrical, ranging from 1.5-2 mm in diameter (7-9 meshes cm -2 ) and the end-branchlets reach up to 9 mm in length, and have blunt tips.The network is regular and complete, and is formed of mostly square and rectangular meshes (4×5 mm, 10×3 mm); in some cases, meshes are oblong (Fig. 16B).There are five or six prominent, long and strong midribs, which divide into others that progressively fuse among the anastomosed structure of the mesh (Fig. 16A).The polyps retract within slightly raised or flat polyp-mounds with slit-like apertures, placed in multiple rows all around the branches.The colour of the colony is intense red-brown when dry and bluishgrey when alive.The coenenchymal sclerites are pink, light yellow and orange, some of them bicoloured (Fig. 16C).They are spindles (up to 0.20×0.04mm) having acute ends and 5-6 whorls of tubercles (Figs 16A, 17A); blunt spindles are absent, and there are capstans (up to 0.7×0.03mm) with tuberculate ends (Figs 16C,  17B).The dominant sclerites are spindles, straight or bent, some of them with a distinct waist.There are irregular crosses with different branch lengths (up to 0.11×0.05mm) (Fig. 17C).The anthocodial sclerites are pink, light orange and light yellow in colour, most of them bicoloured; flattened rods (0.11×0.02 mm) with short pointed or lobe-like marginal projections and acute or rounded ends also occur.There are also platelets (0.06×0.02 mm) (Figs 16C,17D).

Pacifigorgia machalilla
Variability.This new species is fairly constant with regard to colonial and sclerome characters.However, in some specimens there are small secondary fans grow-ing parallel to the primary fan.In addition, in some small colonies (about 100 mm in length) the midrib is not very obvious or even absent, or is only formed by two short basal branches.
Geographic and bathymetric distribution.Pacifi gorgia machalilla is only known from the type locality in Cope, Los Ahorcados, and Isla de la Plata (continental coast of Ecuador), living on rocky bottoms in shallow waters at a depth of 10-23 m.
Etymology.The new species is dedicated to the National Park of Machalilla (Ecuador) and its staff for their constant support and help during field work.Name considered as a noun in apposition.
The anthocodial sclerites are yellow flattened rods up to 0.12 mm in length and 0.02 mm in width, with short lobe-like marginal projections, and acute and warty or smooth ends (Figs 19C,20C).
Geographic and bathymetric distribution.Pacifi gorgia rubicunda was originally described by Breedy and Guzmán (2003) from Costa Rica (Fig. 25I).The geographic range of the species is here extended to Ecuador (Manabí).Its bathymetric distribution ranges from 5 to 30 m depth (Breedy and Guzmán 2003).
Remarks.There are some morphological differences between the specimens of P. rubicunda found in Ecuador and the original description of this species by Breedy and Guzmán (2003).The colour of sclerites is very similar (orange and yellow); however, in Ecuadorian material most of the sclerites are pink (bicoloured or multicoloured) and colourless sclerites are lacking.If compared with the type material (6-9 meshes cm -2 , 5-1.2×2.5-0.9 mm) (Breedy and Guzmán 2003), in our colonies the network is less compact, usually more elongate and larger, and there are no crosses.
Newly collected material of P. rubicunda from Ecuador permits the description of variability of some characters.The species is reported herein for the first time since its original description in Costa Rica.(Valenciennes, 1846) (Figs 21, 22) Synonymy.See Breedy and Guzmán (2002: 833).

Pacifigorgia stenobrochis
Newly collected examined material: BEIM (0090  Description.The colonies are up to 280 mm in length by 270 mm in width and are formed by a single fan that may divide into smaller fans.The holdfast was not observed (Fig. 21A).The branches are rounded, ranging from 1.1-2.3mm in diameter (2 meshes cm -2 ).The branches arise directly from the base and have an open and irregular network.The meshes are rectangu-lar or oblong (55×6 mm, 10×5 mm) (Fig. 21B).There are no distinct midribs.There are long end-branchlets (up to 26 mm).The polyps retract within flat polypmounds, placed throughout the branches, on both sides.The colour of the colony is light brown or dark yellow when alive and yellow intermingled with brown.The  coenenchymal sclerites are light yellow (Fig. 21C).
There are long acute spindles (0.16×0.04 mm), straight or bent, some with a marked waist, having 8 whorls of tubercles, and they are the dominant sclerite types.
Geographic and bathymetric distribution.Pacifi gorgia stenobrochis has been reported in Mexico, El Salvador, Nicaragua, Costa Rica, Panama, Peru (Verrill 1868, Breedy and Guzmán 2003) and Ecuador (current study) at 3-30 m depth.In the original description, New Zealand is indicated as the type locality, which appears to be an error (see Valenciennes 1846, Breedy and Guzmán 2002, 2003) (Fig. 25J).
Generally, colonies of this species occur both solitary and mixed with other species in the same environment.The Ecuador records nicely connect the previously known localities from Mexico and Peru.
Remarks.The chromatic variability of the colonies and sclerites of this species has been known for a long time (Hickson 1928, Breedy and Guzmán 2002, 2003).In the Ecuadorian material studied here, all the coenenchymal sclerites are yellow.Pink and grey sclerites noted by other authors are lacking.The colour of Ecuadorian colonies varies between yellow and orange hues, while it is said to be reddish purple and brown in previous descriptions.
The presence of thick and robust branches, shape of the mesh and absence of the midrib are diagnostic for P. stenobrochis, and readily separate it from similar species, such as P. firma.

Eugorgia daniana
Newly collected examined material: MECN (Ant0033 Description.The colonies are up to 110 mm in length by 190 mm in width and arise from a circular holdfast, 0.5 mm in diameter.They are profusely and pinnately branched in multiple planes.Main stems are 3-5 mm in diameter, compressed, and very short (up to 10 mm long) or absent (Fig. 23A).The branches arise directly from the base, and are cylindrical, ranging from 0.8 to 1.2 mm in diameter.Some of the branches are pseudoanastomosed (anastomosis to the coenenchyme level, but not including the axes).The unbranched distal twigs can reach up to 15 mm in length and are blunt at the ends (Fig. 23A, B).In some branches, there are numerous and evident yellow, longitudinal grooves.The polyps retract within prominent polyp-mounds, leaving a small bilabiate aperture; they occur in multiple rows on all sides of the branches, sometimes having yellow spots.Colonies are dark orange or bright red streaked with bright yellow on the branches or on the polyp-mounds (Fig. 23B).The coenenchymal sclerites are red, yellow or bicoloured (Fig. 23C).They are double discs reaching up to 0.07 mm in length, 0.06 mm in width (Figs 23C, 24B).The capstans reach up to 0.08 mm in length, 0.05 mm in width (Fig. 23C).The dominant sclerite types are spindles (up to 0.15 mm in length, 0.05 mm in width); discspindles (up to 0.14 mm in length, 0.05 mm in width) are also abundant, with 4-6 whorls of warty tubercles; they are straight or bent, some with a marked waist (Fig. 24A, D).The crosses are up to 0.14 mm in length, 0.06 mm in width (Figs 23C,24C).No anthocodial sclerites were obtained.
Geographic and bathymetric distribution.This species has a wide distribution along the eastern Pacific.It has been recorded in USA, Mexico, El Salvador, Nicaragua, Costa Rica, Panama, Colombia, Ecuador (Galapagos and the Gulf of Guayaquil) and Peru (Fig. 25K), at 3-30 m depth (Verrill 1868, Breedy et al. 2009, present record).
The only specimen of Eugorgia daniana previously found in continental Ecuadorian waters was collected in the Gulf of Guayaquil (identified by Bayer in 1966, and published by Breedy et al. 2009).Our record from Manabí and Santa Elena is the second finding of this species in continental Ecuador, providing information about the possible existence of widespread populations of this species in this area.Currently, Eugorgia dani ana and E. ahorcadensis Soler-Hurtado and López-González, 2012 are the only species of this genus found in Ecuadorian waters.
Remarks.Differentiation between E. daniana and E. aurantica is difficult at first sight due to a similar branching pattern and colour of their colonies.
These species, together with Leptogorgia maria rosae Soler-Hurtado and López-González, 2012, Eugorgia ahorcadensis and L. aequatorialis (Bielschowsky, 1929), constitute the current list of gorgoniid species from the continental coast of Ecuador.Despite the recent contribution of Breedy et al. (2013) suggesting the synonymization of E. ahorcadensis with E. nobilis based on the consideration of an enlargement of morphology and chromatic variability of the latter, recent molecular analyses based on the study of both type materials demonstrated that they should be considered separate taxa (Soler-Hurtado et al. unpublished).In addition, to complete the species list for Ecuadorian waters, the species described above for the Galapagos Islands, Pacifigorgia darwinii (Hickson, 1928), P. dampieri Williams and Breedy, 2004, P. symbiotica Williams and Breedy, 2004and P. rubripunctata Williams and Breedy, 2004, should be added.
According to Bayer (1961), gorgonian corals systematics relies on a combination of characters such as axial skeleton (if any), colonial form, polyp arrangement and sclerite morphology.However, the often unwieldy older literature with no or neglected illustrations and lost or badly preserved type material have played a major role in the taxonomic confusion around octocoral systematics (Sánchez 2004, Soler-Hurtado andLópez-González 2012).Currently, variation in sclerite size, sculpture, and coloration and the relative proportion of the different sclerite types are the main criteria for species delimitation in Gorgoniidae (Williams and Lindo 1997, Breedy 2001, Sánchez et al. 2007, Vargas et al. 2010a).These characters vary widely and the ranges of variation have not been established for all gorgoniid taxa, hindering a robust taxonomy for the group.
The description of sclerite type can sometimes be difficult, because of continuous variation in size and ornamentation within and between species (Williams and Lindo 1997, Sánchez et al. 2003, Vargas et al. 2010a).This continuum represents a major obstacle to the assignment of size intervals or ranges that could be used to define and separate possible species.Vargas et al. (2010b) proposed for Pacifigorgia the combined use of continuous and discrete morphological characters, in order to define the relationships between species at different resolution level, in an integrative approach with molecular data.
Despite the rebirth of invertebrate systematics due to the ever-increasing availability of DNA sequence characters (Mallet and Willmott 2003, Sánchez 2004, Due-ñas and Sánchez 2009), evaluation of the effectiveness of molecular barcodes in octocorals is largely hindered by lack of knowledge regarding species boundaries in these organisms.One reason for this is that the rate of octocoral mitochondrial gene evolution is very slow.It is estimated to be 10-100× slower than in other metazoans (Chen et al. 2009, Brockman andMcFadden 2012), resulting in insufficient resolution to discriminate species within many genera (Sánchez et al. 2003, Lepard 2003in Cairns and Bayer 2005).Specimens identified as different morphospecies can share the same barcode, which should motivate additional taxonomic work to test species boundaries and quantify intraspecific morphological and molecular variation (McFadden et al. 2010).For instance, Sánchez et al. (2003), exploring mitochondrial genes ND2, ND6 and MutS, observed that in Eunicea spp.and Plexaura spp. the number of substitutions supporting nodes was low (less than 0.005 substitutions/site), and the clade formed by these two genera could be considered unresolved from a molecular point of view.In addition, McFadden et al. (2011) showed that congeners of the genera Isidella, Keratoisis and Lepidisis were identical at COI and Igr1 sequences, and only varied 0%-2% for MutS.
In this study, we found that pairs of morphologically different species, Pacifigorgia exilis and P. media (Verrill, 1864), and P. bayeri Breedy, 2001 and P. cat edralensis Breedy and Guzmán, 2004, showed no interspecific variation (0%, in COII+Igr+COI+MutS, cf.Table 4); while others, P. firma and P. catedralensis, P. rubicunda and P. exilis, and P. rubicunda and P. me dia, showed a very low genetic divergence (0.006%).In fact, the maximum genetic divergence observed in this study is 1.3%, between P. media and P. firma, and between P. exilis and P. firma, confirming the reduced variability in the genetic regions examined.
The analysis combining MutS and Igr1-COI for DNA barcoding indicates that these markers are not always suitable and conclusive for species-level identification of eastern Pacific octocorals (Vargas et al. 2014).However, we can say that these regions can at least define different species groups.This may serve as a supporting tool for morphological findings among species within and between groups.According to our results, based on strong morphological data and molecular tests with sufficient resolution for this set of octocoral species, we propose Paci figorgia machalilla as a new species.
In conclusion, we recommend the use of molecular tools as a necessary complement to morphological identification for future descriptions of new species.Although mitochondrial markers are known to evolve at much lower rates than in other zoological groups, they still provide information that should not be neglected.These mitochondrial markers can be complemented with nuclear regions (e.g.28S, ITS and SRP54, among others) that have been demonstrated to be especially useful for some octocoral families and genera (Sánchez et al. 2007, McFadden et al. 2014, Wirshing and Baker 2015).
Although much more work is needed to fully understand the morphological diversification of octocorals, a combination of molecular and morphological data is a very promising approach to disentangling phylogenetic relationships among species (Breedy et al. 2013, Mc-Fadden and Van Ofwegen 2013, López-González et al. 2015) and intraspecific population ecology (Calderón et al. 2006, Prada et al. 2008, Prada and Hellber 2013).Molecular and morphological analytic tools will be essential to quantify the variety of evolutionary pathways within these groups (Sánchez et al. 2003, McFadden et al. 2006, Concepción et al. 2010).
However, even when combined, the two approaches do not seem to fully solve the taxonomic problems in this group, and more informative characters in both disciplines must be identified to distinguish closely related morphological species (e.g.McFadden and Van Ofwegen 2013 on stoloniferous octocorals), as well as to obtain more natural classifications, reducing the number of taxonomic synonyms (e.g.Grajales and Rodríguez 2016, on sea anemones).This will help to clarify the evolution of a zoological group that is very important in the structural functioning of benthic marine communities (Conell 1978, Fabricius and Alderslade 2001, Fabricius 2005).
Finally, the presence and abundance of these and other species of octocorals in shallow waters could be used as an environmental indicator, suggesting potential areas for protection.Intensive sampling on the coast of Ecuador in biotopes such as submarine ridges and coralligenous bottoms, rich in gorgonian species, could reveal unrecorded gorgonian species in this area.Therefore, much work remains to elucidate Gorgoniidae taxonomy on the coast of Ecuador.The octocorals are an important structural component of the rocky reef fauna of these waters.The present survey contributes towards resolving issues concerning the distribution and taxonomy of this fauna in this region.

Fig. 4 .
Fig. 4. -Leptogorgia diffusa (MNCN 2.04/1172).A, colony; B, detail of a branch; C, light micrograph of sclerites.Table 2. -Comparative general features of the species of the genera Leptogorgia and Eugorgia collected in this study, on the coast of Ecuador.

Fig. 25 .
Fig. 25.-Geographical distribution of the Leptogorgia, Pacifigorgia and Eugorgia species mentioned in the paper.The solid circle shows the relative position of the species in the countries where they have been recorded.The empty circles indicate the first record for the species in Ecuador.