Several colonies of Pachyseris were collected at various localities in the Red Sea and in the Indo-Pacific. Digital images of living corals in situ were taken with a Canon G9 in an Ikelite underwater housing or a Nikon Coolpix 7900 in a Nikon WP-CP4 waterproof case. From each coral specimen collected, a 2 cm² fragment was preserved in either 95% ethanol or CHAOS solution (Sargent et al. 1986, Fukami et al. 2004) for molecular analyses. After tagging, the remaining corallum was bleached in sodium hypochlorite for 48h to remove soft parts, rinsed in freshwater, and air-dried for morphological analyses. Images of the cleaned skeletons were taken with a Canon G9 digital camera. Samples were identified following Veron and Pichon (1980), Scheer and Pillai (1983), Veron (1986), Sheppard and Sheppard (1991), and Nishihira and Veron (1995). Two colonies of Leptoseris foliosa from New Caledonia already studied by Benzoni et al. (2012) were also included in the following analyses.

Macro and micro-morphological characters of Pachyseris samples were examined using both light microscopy (Zeiss Stemi DV4 stereo-microscope) and scanning electron microscopy (SEM). For SEM, Pachyseris speciosa, Pachyseris rugosa, and Leptoseris foliosa fragments were ground, mounted on stubs using silver glue, sputter-coated with conductive gold film, and examined using Vega Tescan Scanning Electron Microscopy at the SEM Laboratory, University of Milano-Bicocca. Fragments of Pachyseris inattesa sp. n. (specimens KAUST SA492 and KAUST SA1305) were sputter-coated with Au-Pd and imaged using a Quanta 200 FEG SEM at the King Abdullah University of Science and Technology.

Twenty Pachyseris and two Leptoseris foliosa specimens were included in the molecular analyses (Table 1). Total DNA was extracted using DNAeasy® Tissue kit (Qiagen Inc., Valencia, CA, USA) for samples stored in ethanol according to the manufacturer’s protocol and a phenol-chloroform based method for samples in CHAOS (Fukami et al. 2004). Each extracted sample was quantified using a NanoDrop® 1000 spectrophotometer (Thermo Scientific) and diluted to a final concentration of 3 ng/ml. The mitochondrial intergenic spacer between COI and 16S-rRNA (hereafter IGR), previously used to evaluate species boundaries within the family Agariciidae (Luck et al. 2013), was chosen as a marker and amplified using newly designed primers AGAH (5’- GCT TGA CAG GGT TTC CAA GA - 3’) and AGAL (5’- CGC ATT GAA ACA CGA GCT TA - 3’). The same region was amplified independently by Luck et al. (2013) in agariciids, but when starting our analyses, primers designed by them for this mitochondrial intergenic spacer were still not available in literature. Reactions were conducted in a 25 µl PCR mix, composed of 1X PCR buffer, 2 mM MgCl₂, 0.4 µM of each primer, 0.1 mM dNTP mix, 2 U taq polymerase (Sigma-Aldrich Co., St. Louis, MO, USA), and 4 µl of DNA solution (10-30 ng of DNA). The thermal cycle consisted of a first denaturation phase at 94°C for 4 min, followed by 30 cycles to 94 °C for 1 min, 54 °C for 1 min, 72 °C for 1 min, and finally an elongation phase of 72 °C for 5 min. All samples were purified with Illustra ExoStar (GE Healthcare, Buckinghamshire) and directly sequenced in forward and reverse directions using an ABI 3130xl Genetic Analyzer (Applied Biosystems). Sequences produced in this study have been deposited at EMBL and accession numbers are listed in Table 1.

Phylogenetic relationships between species were inferred using our sequences and 15 sequences of Agariciidae downloaded from GenBank based on Luck et al. (2013) in order to define the position of Pachyseris inattesa sp. n. with respect to the genus Leptoseris. Moreover, in order to root the mtDNA phylogeny, we selected Siderastrea radians (Pallas, 1776) (clade IX sensu Fukami et al. 2008) as outgroup based on the phylogeny proposed by Fukami et al. (2008). Sequences were viewed, edited, and assembled using CodonCode Aligner 4.2.5 (CodonCode Corporation, Dedham, MA, USA) and manually checked using BioEdit 7.2.5 (Hall 1999). Multiple alignments were carried out using the E-INS-i option in MAFFT 7.110 (Katoh et al. 2002, Katoh and Standley 2013) under default parameters. Invariable, polymorphic, and parsimony informative sites were detected with DnaSP 5.10.01 (Librado and Rozas 2009). Intra- and inter-specific pairwise distances (uncorrected p-distances) were calculated in MEGA 4.0.2 (Tamura et al. 2007). Phylogenetic relationships were reconstructed using Bayesian Inference (BI), Maximum Likelihood analyses (ML), and Maximum Parsimony (MP). Bayesian Inference was conducted with MrBayes 3.1.2 (Huelsenbeck and Ronquist 2001, Ronquist and Huelsenbeck 2003), Maximum Likelihood analyses using PhyML 3.0 (Guindon and Gascuel 2003), and Maximum Parsimony was based on PAUP* 4.0b10 (Swofford 2003). The best-fit substitution model was determined using the Akaike Information Criterion (AIC) as implemented in MrModeltest 2.3 (Posada and Crandall 1998) in conjunction with PAUP 4.0b10 (Swofford 2003). AIC identified the General Time Reversible (GTR) model with gamma distributed rate variation among sites (+Γ) (Γ = 1.02) as the most suitable model. Bayesian Inference analyses consisted of four parallel Markov Chains Monte Carlo (MCMC) implemented for 1, 000, 000 generations, saving a tree every 100 generations and discarding the first 2, 501 trees as burn-in. Bayesian Inference analysis was stopped when the standard deviations of split frequencies were <0.01. As an additional tool, the software Tracer 1.5 (Drummond and Rambaut 2007) was used to verify the convergence of parameters and correctly estimate the burn-in. Finally, clade support was assessed based on posterior probability. The Best Maximum Likelihood tree was reconstructed with PhyML using the default parameters and 1, 000 bootstrap replicates to verify the robustness of the internal branches of the tree. Maximum Parsimony was conducted using the TBR branch swapping method with 1000 replications, random addition for 10 replicates, nchuck = 100, chuckscore = 1. Node supports were obtained with 1, 000 bootstrap replicates.

BIBELOT IRD Biodiversité Benthique dans les iles Loyauté Expedition, Loyalty Islands, New Caledonia, 2014

BMRI Borneo Marine Research Institute, Universiti Malaysia Sabah, Malaysia

IRD Institut de Recherche pour le Développement, Nouméa, New Caledonia

KAUST King Abdullah University of Science and Technology, Thuwal, Saudi Arabia

KBEA KAUST Biodiversity Expedition to the Gulf of Aqaba, 2013

KBEF KAUST Biodiversity Expedition to the Farasan Banks and Farasan Islands, 2013

NIUGINI Niugini Biodiversity Expedition, Papua New Guinea, 2012

RMNH Coel. Rijksmuseum van Natuurlijke Historie, Coelenterate collection, Naturalis Biodiversity Center, Leiden, The Netherlands

SMEE Semporna Marine Ecological Expedition, 2010

TOE Tara Oceans Expedition, 2009-2012

UNIMIB University of Milano-Bicocca, Milan, Italy

USNM United States National Museum of Natural History, Washington DC, USA

A total of 20 Pachyseris specimens belonging to three different species, namely Pachyseris rugosa, Pachyseris speciosa, and Pachyseris inattesa sp. n., and two Leptoseris foliosa sequences were successfully sequenced and used for phylogenetic reconstruction together with 15 agariciid sequences previously used by Luck et al. (2013). The final alignment consisted of 1, 153 bp and included 83 variable sites, 76 of which were parsimony informative. A total of 124 mutations (considering only synonymous and non-synonymous substitutions) were found. Topologies resulting from BI, ML, and MP analyses were largely congruent with no contrasting signals (Figure 9) and showed high resolution at species level. Only the Bayesian phylogram with branch support indicated by Bayesian posterior probability (PP_BI), ML bootstrapping support (BT_ML), and MP bootstrapping support (BT_MP) is shown in Figure 9.

The phylogenetic reconstruction resolved two main groups congruent with clade IV sensu Fukami et al. (2008) comprising Pachyseris inattesa sp. n. and the other two Pachyseris species, and clade VII sensu Fukami et al. (2008) composed by Leptoseris foliosa and the other representatives of the family Agariciidae (Figure 9). Within clade IV, Pachyseris specimens, including Pachyseris inattesa sp. n., were assigned to three well-supported main clades, namely A, B, and C. All the examined species belong to distinct lineages and their monophyly is highly supported (PP_BI = 1, BT_ML = 94, BT_MP = 100 for Pachyseris inattesa sp. n. in clade A, PP_BI = 1, BT_ML=91, BT_MP = 100 for Pachyseris rugosa in clade B, PP_BI = 0.86, BT_ML = 95, BT_MP = 100 for Pachyseris speciosa in clade C) (Figure 9). The average intraspecific genetic distances for the examined Pachyseris species are very low, in particular 0.1 ± 0.1 % for Pachyseris speciosa, 0.7 ± 0.4 % for Pachyseris rugosa, and 0.1 ± 0.1 % for Pachyseris inattesa sp. n. The genetic distances between the three clades are higher, 10 ± 2.2 % between Pachyseris speciosa and Pachyseris rugosa, 16.5 ± 2.4 % between Pachyseris speciosa and Pachyseris inattesa sp. n., and 18.2 ± 2.8 % between Pachyseris rugosa and Pachyseris inattesa sp. n. The two sequences of Leptoseris foliosa were found in clade VII together with the other agariciid sequences from GenBank. In particular, Leptoseris foliosa is closely related to Leptoseris papyracea (Figure 9) and highly divergent from Pachyseris inattesa sp. n., a result supported by the genetic distance between the two species, 42.3 ± 3.1 %. Likewise, the genetic distance between Pachyseris inattesa sp. n. and Leptoseris mycetoseroides is also high, 42.3 ± 3.1 %.

In a diagnosis of Pachyseris, Milne-Edwards and Haime (1851) remarked that the genus they described is characterized by “Polypier semblable aux agaricies, si ce n'est que les polypiérites d'une même série sont complètement confondus entre eux” [corallites similar to the agariciids, apart from the fact that the calices of the same series are completely confused between them]. Pachyseris inattesa sp. n. is, hence, quite untypical compared to the other species in the genus by having clearly recognizable calices, even when arranged in series. This is the most likely reason that the new species was previously identified as a Leptoseris. However, Pachyseris inattesa sp. n. is similar to the other two species of Pachyseris analyzed in this study on the basis of the arrangement of the carinae and the micro-morphology of the radial elements and of the columella. In particular, among its congeners, the new species is closer to Pachyseris speciosa. The two species have a similar growth form, although coralla of Pachyseris inattesa sp. n. do not form tiers. Furthermore, they have a similar structure of the columella, formed by minutely granulated processes extending from the inner ends of the radial elements into the fossa. However, the new species is devoid of the horizontal dissepiments uniting the radial elements with the columella. The most striking microstructural features shared by Pachyseris inattesa sp. n. and Pachyseris speciosa are the zigzag patterns of the upper margins of the radial elements, and their lateral faces’ ornamentation featuring clumps of granules, although the menianae and the vertically fused granules observed in Pachyseris speciosa do not form in Pachyseris inattesa sp. n.

The new species was previously collected in the Red Sea and identified as Leptoseris tenuis by Scheer and Pillai (1983: figs 7–11) and as Leptoseris foliosa by Sheppard and Sheppard (1991: fig. 85, fig 58). Moreover, in his description of Leptoseris foliosa, Veron (2000: p. 219, fig. 8) published an image of Pachyseris inattesa sp. n. from the Sinai Peninsula, Egypt. The author states that “Red Sea colonies are distinctive as they form thick figs which are usually flat”. Indeed, as mentioned above, colonies of Pachyseris inattesa sp. n. growing in well-lit conditions tend to be thick and to grow by following the underlying substrate (Figures 4c, 11b).

The genus Leptoseris is characterized, like many other scleractinian taxa, by a great variability in the macro-morphology of the colonies and by a notable interspecific phenotypic variability that led to the identification of several nominal species and an enduring taxonomic confusion (Veron and Pichon 1980, Dinesen 1980, Luck et al. 2013). The two nominal species with which Pachyseris inattesa sp. n. has been confused, Leptoseris tenuis (Scheer and Pillai 1983) and Leptoseris foliosa (Sheppard and Sheppard 1991, Veron 2000), are a typical case of such a problematic taxonomic history. Leptoseris foliosa was described by Dinesen (1980) to include Leptoseris tenuis mainly because the holotype described by van der Horst (1921) was different from the actual specimen deposited as the holotype. Without engaging into an evaluation of the taxonomic action itself, it is clear that the same species was initially called Leptoseris tenuis and later Leptoseris foliosa. This species was recently examined in detail by Benzoni et al. (2012) who re-established Craterastrea levis Head, 1983, previously synonymized with Leptoseris foliosa as a result of molecular, micro-morphological, and microstructural analyses showing that the two nominal species were two distinct and valid taxonomic entities characterized by a striking convergence of traditional macro-morphological features. Leptoseris foliosa was included in the present paper in order to address its morphologic and molecular affinities with Pachyseris inattesa sp. n. Although the latter never forms upwards concave colonies as often observed in Leptoseris foliosa (Benzoni et al. 2012: Figure 10), the two species can form similarly shaped colonies (cf. Benzoni et al. 2012: Figure 8 with Figure 4b herein). However, despite a superficial similarity in terms of corallum shape, corallite arrangement in series (Figures 10a, b), and the formation of carinae separating series of calices, the micro-morphology of the radial elements and of the structure of the columella in Pachyseris inattesa sp. n. is substantially different from that of Leptoseris foliosa (Figures 10c–f). In Pachyseris inattesa sp. n. the columella is composed of one or more spatula-like processes extending from the margins of the radial elements into the fossa (Figure 10c), while in Leptoseris foliosa the columella is formed by one solid boss in all the corallites with the exception of the protocorallite (Benzoni et al. 2012) (Figure 10d). Moreover, while the upper margin of the radial elements in Pachyseris inattesa sp. n. is typically arranged in a zigzag fashion and the lateral ornamentation consists of clumps of radially arranged granules (Figure 10e), in Leptoseris foliosa the upper margin of the radial elements is straight and the granulations can be separated or elongated aggregations of parallel granules (Figure 10f), which can form or merge into menianae parallel to the growing septal margin (Benzoni et al. 2012: figs 28–32). Hence, similar to the case of Leptoseris foliosa and Craterastrea levis, and despite some macroscopic similarities we could find substantial micro-morphological differences substantiated by a significant genetic distance also between Leptoseris foliosa and Pachyseris inattesa sp. n. (Figure 9).

Leptoseris foliosa was not encountered in the Saudi Arabian reefs in 2013, and since, as discussed above, previous records of this species in the Red Sea turned out to be Pachyseris inattesa sp. n., the presence of this species in the region is currently not confirmed. In the Red Sea Pachyseris inattesa sp. n. co-occurs with Pachyseris speciosa (Figures 11a–d) and with the common Leptoseris mycetoseroides. In the original description of this species, Wells (1954) noted some resemblance of this species to Pachyseris, such as the development of concentric carinae with Pachyseris-like rows of calices and the presence in some coralla of fig-like columellae (Veron and Pichon 1980). This species bears some superficial similarities with Pachyseris inattesa sp. n. in the colony morphology in situ (Figure 11c) but the typical ridges intersecting the carinae in Leptoseris mycetoseroides (Figure 11f) (Scheer and Pillai 1983) are never observed in the new species, which also has smaller calices and thinner and more numerous radial elements reaching the fossa (Figure 11). Our molecular results support these morphological gaps, revealing an important genetic distance between these two species (Figure 9).

Although the mitochondrial genome of Scleractinia species is usually characterized by a slow evolution rate resulting in low levels of intraspecific variation (Shearer et al. 2002, Hellberg 2006, Huang et al. 2008), recent molecular studies have demonstrated that two mitochondrial intergenic non-coding regions, i.e., the putative control region located between ATP8 and COI and an open reading frame of yet unknown function located between ATP6 and NAD4 genes, exhibit high levels of sequence variation and can provide high resolution within the pocilloporid genera Pocillopora Lamarck, 1816, Seriatopora Lamarck, 1816, and Stylophora Schweigger, 1820 (Flot and Tillier 2007, Flot et al. 2008, 2011, Schmidt-Roach et al. 2012, Pinzón et al. 2013). Moreover, Luck et al. (2013) demonstrated that the mitochondrial spacer between COI and 16S-rRNA is powerful in resolving species boundaries in the agariciid genera Pavona and Leptoseris. In our study we present the first phylogenetic analysis of the genus Pachyseris based on the mitochondrial intergenic spacer between COI and 16S-rRNA. The region shows high variability and resolves species-level relationships within the genus.

The molecular phylogenetic reconstruction corroborates our micro-morphological results, confirming Pachyseris inattesa sp. n. as a monophyletic lineage belonging to Pachyseris, and not related to the family Agariciidae including the genus Leptoseris. This study confirms the utility of a combined morpho-molecular approach in resolving phylogenetic relationships among scleractinian species (Benzoni et al. 2007, 2011, 2014; Kitahara et al. 2012a, b; Huang et al. 2014; Arrigoni et al. 2014a, b).

As researchers increasingly combine traditional morphological with modern molecular taxonomic approaches on reference and museum collections (Rainbow 2009), previously overlooked taxa will continue to be discovered, or re-discovered (Arrigoni et al. 2014b) clarifying phylogenetic relationships and biogeography. This is particularly true in understudied regions such as the Red Sea, where corals and other organisms are currently being investigated for the first time through a morpho-molecular approach. For example, DiBattista and Randall (2013) have identified that a Chromis species (family Pomacentridae) once thought to be widespread through the Red Sea and Indian Ocean is actually two distinct species, one endemic to the Red Sea and one widespread throughout the Indian Ocean. Studies considering the evolutionary history of several fishes suggest that endemism in this region may be higher than currently thought (DiBattista et al. 2013).

The present study indicates that the same pattern may be true for a wider range of taxa including scleractinian corals, i.g., Acropora hemprichii (Ehrenberg, 1834), Acropora variolosa (Kluzinger, 1879), and Cantharellus doederleini (von Marenzeller, 1907) and provides some insight to more general biogeographic trends in the Red Sea (Hoeksema 1989, Wallace 1999). Specifically, Pachyseris inattesa sp. n. was not recorded in the Farasan Islands in the southern Red Sea (six sites), although the species was found in central and northern Red Sea sites (17), including the northern end of the Farasan Banks (nine sites) (Figure 8). Moreover, during the 2013 expeditions that led to the discovery of Pachyseris inattesa sp. n., remarkable differences in coral assemblages composition and discontinuities in species distribution were noted between the Farasan Banks and the Farasan Islands (Benzoni pers. comm). There is emerging evidence that the environmental shift between the Farasan Banks and the Farasan Islands (separated by ~110km) may represent a more general biogeographic barrier, with both a fish and a sponge species showing marked genetic structure correlated with environmental gradients (such as temperature, salinity, or productivity) between these habitats (Nanninga et al. 2014, Giles 2014).

Moreover, further investigations into the taxonomic status of marine species throughout the wider Red Sea, particularly with reference collections to facilitate morphological and molecular examinations (see Rocha et al. 2014), will help to delineate specific marine biogeographic province boundaries (as defined by Briggs 1974) as well as elucidate the evolutionary or ecological mechanisms involved. The role of the Red Sea in broader Indo-Pacific biogeographic patterns for scleractinian corals and other reef dwelling organisms remains intriguing (Bowen et al. 2013), potentially functioning as an exporter of biodiversity, i.e., a region in which some species have originated and then subsequently expanded their range beyond the Red Sea (e.g. DiBattista et al. 2013).