SCHIZOPATHIDAE BROOK, 1889

Brugler, Mercer R., Opresko, Dennis M. & France, Scott C., 2013, The evolutionary history of the order Antipatharia (Cnidaria: Anthozoa: Hexacorallia) as inferred from mitochondrial and nuclear DNA: implications for black coral taxonomy and systematics, Zoological Journal of the Linnean Society 169 (2), pp. 312-361 : 336-341

SCHIZOPATHIDAE BROOK, 1889

The family Schizopathidae (11 genera, 40 species) is characterized by polyps that are extremely elongated in an axial direction and usually more than 2 mm in transverse diameter (up to 9 mm). Schizopathids also display a high degree of morphological variation in the growth form of the corallum, although in all cases this is superimposed on a pinnulate branching pattern. Colonies may be either monopodial or branched, and the pinnules can be simple or subpinnulated to varying degrees. There are no unbranched forms known. The spines are smooth-surfaced, triangular to conical and without ornamentation, although rarely they may be bifurcated or multi-lobed at the apex. In taxa for which there exists adequate field data, the tentacles of expanded polyps are extremely long and narrow to a fine tip. The family is currently divided into two subfamilies, the Parantipathinae ( Roule, 1905) (four genera) and Schizopathinae ( Brook, 1889) (seven genera) based on the size of the polyps ( Opresko, 2002); however, it was later reported that polyp size is not a consistent feature within all taxa assigned to these subfamilies ( Opresko, 2005b); therefore, the validity of these subfamilies is questionable.

Based on morphological analyses, a minimum of 16 species, representing nine genera, were obtained for sequencing, of which nine could be referred with certainty to valid nominal species ( Table 1 & Supporting information Table S1); the resulting mt-contig phylogenies revealed a paraphyletic clade. The taxa sorted into two highly supported subclades, although these did not reflect the morphologically defined subfamilies. Instead, the molecular results grouped taxa based on whether the primary pinnules are alternate ( Dendrobathypathes , Dendropathes Opresko, 2005b , Lillipathes , Parantipathes , Saropathes Opresko, 2002 , Schizopathes Brook, 1889 , and Umbellapathes Opresko, 2005b ) or subopposite [ Bathypathes , Stauropathes Opresko, 2002 , and an undescribed branched schizopathid (hereafter referred to as schizopathid sp. nov. 1)]. Note that some Bathypathes described in the literature (e.g. Bathypathes alternata Brook, 1889 ) have alternating pinnules, so the pattern is not absolute. In addition, the group displaying alternate pinnules has, in general, smaller polyps (range: 1.6– 4.0 mm; outlier is Dendrobathypathes , with maximum transverse diameter of 5 mm) than the group displaying subopposite pinnules (range: 3.0–9.0 mm), although polyp size overlaps between the two groups.

Five haplotypes were obtained for Parantipathes , four of which were found within specimens from the western North Atlantic and one of which characterized all eastern North Pacific (hereafter abbreviated ENP) specimens. However, Atlantic and ENP Parantipathes were not each other’s closest relatives (see below). Tentative species names were obtained for five specimens using a key for the seven species of Parantipathes ( Molodtsova & Pasternak, 2005) . Genetically, these five specimens had one of three haplotypes; haplotype 1 was shared by P. cf. tetrasticha ( de Pourtalès, 1868) and P. cf. wolffi Pasternak, 1977 (and also Sibopathes macrospina ), haplotype 2 was shared by P. tetrasticha and P. cf. hirondelle Molodtsova, 2006 , and haplotype 3 was seen only in P. tetrasticha (short pinnules). The single Parantipathes colony with the fourth haplotype (MAN501-1) was not available for morphological examination and thus remains unidentified to species. These results suggested that: (1) morphological plasticity is high within a single haplotype group, (2) the current mor- phological characters used to delineate species within the genus Parantipathes need to be revised, or (3) the rate of speciation is outpacing the rate of molecular evolution, and thus the genetic data have yet to reflect actual speciation events, as is evident by their morphological disparity.

Schizopathid sp. nov. 1 (see ‘Note Added in Proof ’) (REH103-1 and others sharing that same haplotype), which displays subopposite pinnules similar to Bathypathes patula Brook, 1889 , was genetically more closely related to Stauropathes cf. puncata Roule, 1905 (which also has subopposite pinnules) than B. patula . Morphologically, the biggest difference between schizopathid sp. nov. 1 and B. patula is the presence of branching in the former. Although only a single Stauropathes haplotype was revealed by the genetic data (seven S. cf. punctata and two S. staurocrada Opresko, 2002 colonies were analysed), we encountered several different morphologies among these specimens, ranging from a small, planar Christmas tree-like colony to a large/thick baseball glove, and thus hypothesized the presence of several different species. These two species of Stauropathes are either characterized by extreme morphological plasticity or the rate of speciation is outpacing the rate of molecular evolution (as may be the case with Parantipathes ); alternatively, current molecular markers lack the necessary variation to differentiate these species.

Of extreme interest was the inability to genetically differentiate specimens identified as Lillipathes wingi Opresko, 2005b / L. sp. (except for T-426-A6, which was unique at all three mt regions, suggesting the presence of a cryptic taxon), Dendrobathypathes boutillieri (except for J 2095-2-5-1, which was unique at cox 3- cox 1), and ENP Parantipathes sp. (hereafter abbreviated ‘trigeneric complex’; all specimens identified by D.M.O.; specimen T-426-A6 was not available for reanalysis). The genus Dendrobathypathes contains four species (the type species D. grandis Opresko, 2002 and D. isocrada Opresko, 2002 from southern oceans, D. boutillieri from the Gulf of Alaska, and D. fragilis Opresko, 2005b from Japan), while the genus Lillipathes contains four species [ L. wingi from the Pacific coast of Canada and Alaska, the type species L. lilliei ( Totton, 1923) from New Zealand, L. quadribrachiata ( van Pesch, 1914) from the Banda Sea and L. ritamariae Opresko & Breedy, 2010 from the Pacific coast of Costa Rica]. The type species of Parantipathes is P. larix (Esper, 1790) , which is found in the Mediterranean Sea. Additional species within this genus are known from the West Atlantic ( P. tetrasticha ), North-East Atlantic ( P. hirondelle ), North-West Pacific [ P. euantha ( Pasternak, 1958) ] and South-West Pacific ( P. helicosticha Opresko, 1999 , P. laricides van Pesch, 1914 , and P. wolffi ); the genus has not previously been reported in the North-East Pacific. Several specimens of Lillipathes (N = 3; does not include T-426-A6), Dendrobathypathes (N = 3; does not include J 2095-2-5-1) and ENP Parantipathes sp. (N = 5) were sequenced, and all shared identical haplotypes across the three mt regions analysed, despite all three genera (and the sampled colonies in particular) being morphologically distinct from one another ( Fig. 7 ). Dendrobathypathes is classified in the subfamily Schizopathinae Brook, 1889 (diagnosis: polyps 3 mm or more in transverse diameter), while Parantipathes and Lillipathes are classified in the Parantipathinae Roule, 1905 (diagnosis: polyps usually 2–3 mm in transverse diameter). Dendrobathypathes is defined as having a monopodial- to sparsely branched and largely planar corallum with two rows of subpinnulated primary pinnules, and polyps 2–5 mm in transverse diameter. Spines are triangular to subconical, laterally compressed, smooth, acute, simple or rarely bifurcated at the apex, and are up to 0.2 mm tall ( Fig. 8 ). Three of the four species of Lillipathes have a sparsely branched corallum with simple pinnules in four rows, and polyps 2–3.6 mm in transverse diameter. One or both anterolateral pinnules are occasionally missing on parts of the corallum, resulting in only two or three rows of pinnules. Lillipathes ritamariae forms large, multi-branched, flabellate colonies and pinnules occur in only two rows on some parts of the corallum and the typical four rows elsewhere. Similar to D. boutilleri , the spines of Lillipathes are simple, smooth, triangular to conical, and compressed, and are up to 0.12 mm tall ( Fig. 8 ). Parantipathes has a largely monopodial corallum (to very sparsely branched) with simple pinnules in six or more rows, and polyps 1.6–3 mm in transverse diameter. Parantipathes wolffi has only four rows of pinnules on some parts of the corallum, although additional rows are always present in varying degrees of regularity. Like D. boutilleri and Lillipathes , the spines of Parantipathes are simple, triangular, and compressed, and are up to 0.11 mm tall (exception: P. helicosticha at 0.22 mm tall; Fig. 8 ). Lillipathes is morphologically very similar to Parantipathes ( Opresko, 2005b) , and young colonies of the latter, having only four rows of pinnules, might be difficult to distinguish from Lillipathes . The sequenced representatives of the three genera were collected from locations separated by a great-circle distance, over open ocean, of 5215 km, or 5819 km when following the coastline (central Aleutian Islands to southern California), and by a maximum depth of 1850 m.

In addition to the lack of genetic divergence across 2274 bp of mtDNA for 11 specimens representing three genera (but see Fukami et al., 2004), no divergence was likewise observed in either of the ITSs of the nuclear ribosomal tandem array. We compared four colonies using ITS1 [524 bp; two Dendrobathypathes (J 2095-2-5-1 and J 2097-2-1), one ENP Parantipathes (J 2095-2-7-6), one Lillipathes (41-100-B1)] and two colonies using ITS2 [601 bp; Dendrobathypathes (J 2097-2-1) and Lillipathes (41-100-B1) only]. There are hundreds of tandem copies of the rRNA cistron at the nucleolar organizer locus with the potential for variation among repeats, assuming that concerted evolution does not homogenize the copies ( Coleman, 2003). Previous studies of ITS sequences from corals have found high levels of intraindividual (within a polyp), intra-colonial (between polyps) and intraspecific genetic variation ( Vollmer & Palumbi, 2004; Lam et al., 2006); a predictable result given that they are part of a multigene family. Thus, finding no genetic divergence within either ITS region was an extremely surprising result. 18S and 28S sequences were also obtained for a single representative from each genus ( Dendrobathypathes : J 2095-2-5-1, ENP Parantipathes : J 2106-7-1, Lillipathes : 41-100-B1). While 18S (1754 bp) was identical among the three colonies sequenced, there was a single variable position in 28S (range of sequence lengths: 2050–3498 bp). However, at that variable position, the chromatogram for specimen J 2095-2-5-1 ( Dendrobathypathes ) showed double peaks, accounting for both subsitutions in the other specimens, thus rendering the site ambiguous and uninformative. Therefore, 18S and 28S also failed to distinguish representatives of these three genera.

Assuming the morphological differences observed among the trigeneric complex reflect separate generic status, as opposed to morphological plasticity, several scenarios could potentially explain why the sequences we compared are identical. The first is a selective sweep, which, mediated by strong positive selection, leads to the rise of mt variants with more effective nuclear interactions (i.e. interactions between gene products encoded by the two genomes) and generally results in a depletion of mtDNA diversity ( Ballard, 2000; Wu et al., 2000; Gerber et al., 2001; Hebert, Ratnasingham & deWaard, 2003b). The mt and nuclear genome of corals may already interact at an elevated level compared with other metazoans as corals are missing 20–23 required tRNAs, which must be imported from the nuclear genome ( Beagley, Okimoto & Wolstenholme, 1998). While this hypothesis is generally accepted, Haen, Pett & Lavrov (2010) could not locate any mt-tRNA genes or their corresponding aminoacyl-tRNA synthetases in the nuclear genome of Nematostella vectensis , and thus proposed that cytosolic tRNAs are imported into the mitochondria. Demonstrating the importance of interactions between the mt and nuclear genomes, Wu et al. (2000) showed that the cox 1-3 subunits of the electron transport chain in primates must associate with ten nuclear-encoded proteins to function properly (see also Rand, Haney & Fry, 2004). If this first scenario is valid, the selective sweep would have had to occur prior to the divergence of the genera and species, as all of the species are identical (or nearly so). A second scenario involves the maintenance of ancestral polymorphism (or the lack thereof in this case) due to incomplete lineage sorting (i.e. slow evolution). More explicity, the DNA has yet to reflect divergence (as anthozoan mtDNA evolves so slowly, it will take longer to evolve species/genus-specific signals). A third scenario, which can be difficult to differentiate from the second scenario, is hybridization with subsequent introgression. Additional scenarios include the possibility that a recent bottleneck event drastically reduced genetic variation in a system that was already undergoing extensive hybridization, or that these ‘genera’ are a single taxon that displays extreme morphological plasticity. This last point deserves elaboration given that genera in the family Schizopathidae are recognized on the basis of differences in the formation of the corallum and in the number and arrangement of the pinnules and subpinnules. Dendrobathypathes has subpinnulated primary pinnules in two rows, Lillipathes has simple pinnules in four rows (but sometimes two or three rows), and Parantipathes has simple pinnules in six or more rows. If the trigeneric complex is simply a single taxon, then the number and arrangement of pinnules and subpinnules are not good characters for separating these taxa. For example, if we consider all taxa in the trigeneric complex to be Dendrobathypathes boutilleri , and assume that ENP Parantipathes and Lillipathes represent juvenile and young-adult stages, respectively, then D. boutilleri would have to lose rows of pinnules as it matures and simple pinnules would become subpinnulate. We consider this hypothesis an unlikely scenario. When considering the formation of the corallum – in particular, branching – depth may be a factor. All ENP Parantipathes (monopodial to very sparsely branched) were collected from shallower depths (<1000 m), whereas Dendrobathypathes boutilleri (extensively branched) were collected from depths> 1650 m (and the two most highly branched colonies came from> 2000 m). Lillipathes wingi (moderately branched) were collected from shallower and deeper depths (814–2664 m). Varying water flow at different depths could affect branching morphology, with weaker/slower flow in deeper water requiring more branching to allow polyps to feed over a wider area. In support of this hypothesis is that the deepest occuring Parantipathes (J 2106-7-1) is branched (937 m; it was also the only Parantipathes collected on the north side of the Aleutian Islands). If depth is indeed affecting branching, then the formation of the corallum is not a good character for separating these taxa. Given that microscopic skeletal features, as opposed to gross colony morphology, were important in differentiating antipathid and aphanipathid Stichopathes , skeletal spines may be key to unravelling the trigeneric complex. Dendrobathypathes , Lillipathes , and Parantipathes all have simple, triangular, and compressed spines ( Fig. 8 ); however, in D. boutillieri the spines are sometimes subconical, and rarely bifurcate at the apex, and in Lillipathes the spines can be conical. In terms of spine size/height, Lillipathes and Parantipathes are nearly identical (0.12 and 0.11 mm tall, respectively), whereas D. boutillieri has spines up to 0.2 mm tall. It is possible that depth is responsible for the slight differences in spine characteristics between D. boutillieri and Lillipathes / Parantipathes , but given that Lillipathes sp. (USNM 1071410) was collected at the deepest depth (2664 m), this hypothesis may not be valid. To test the effect of depth on branching and spine characteristics, a transplant study that relocates monopodial colonies from shallower water to deep water is required. Although the skeletal spines are very similar among members of the trigeneric complex, the other morphological traits discussed above (number of pinnule rows and degree of branching) lead us to conclude that the trigeneric complex is not simply a single taxon that displays extreme morphological plasticity. Thus, based on the homogenization of both mt and nuclear DNA, we propose that hybridization and introgression are responsible for the current findings. Wang et al. (2010) showed molecular and cellular evidence for rapid gene conversion, via site-specific recombination, of nuclear rRNA (ITS) into the maternal genotype within hybrid scallop. Freeland & Boag (1999), in studying Darwin’s ground finches ( Geospiza Gould, 1837 ), recognized six different species based on morphology. However, this differentiation was not reflected in either mt (control region) or nuclear DNA (ITS1) sequences. In fact, variation was largely homogenized within both markers, although not to the extent seen in this study, a result the authors attributed to ongoing hybridization involving all six species (which had been observed in the field). The authors also found that hybrids had higher fitness than non-hybrids in some years; thus, there was no long-term selective pressure favouring elimination of interspecific gene flow.

While ENP Parantipathes could not be genetically differentiated from Dendrobathypathes or Lillipathes , their sequences were distinct enough from western North Atlantic Parantipathes that they grouped within a different subclade. Although identified as ‘ Parantipathes sp. ’ by D.M.O., no currently described species of Parantipathes occur in the North-East Pacific. We conclude that there is a monophyletic group of taxa in the North Atlantic that is probably Parantipathes , and another group of taxa in the North-East Pacific that are morphologically similar to Parantipathes , but not their closest relative, and thus should not be included in that genus.

Finding a single unique mt haplotype of Dendrobathypathes boutilleri (J 2095-2-5-1) within an indistinguishable trigeneric complex was a surprising result (the sequence was confirmed via re-extraction, amplification, and sequencing of cox 3- cox 1). Specimen J 2095-2-5-1 displayed two nucleotide peaks at the single variable position in the 28S alignment. This result suggests that specimen J 2095-2-5-1 may have been part of reciprocally hybridizing populations that were currently undergoing, or recently underwent, speciation. Alternatively, this variability could be evidence of intraspecific variation (i.e. if the trigeneric complex is evolving as a single species). Additional nuclear markers, including microsatellites, are necessary to more thoroughly address this finding.

The nuc-contig phylogenies contained eight representatives of the Schizopathidae . Similar to the mt-contig phylogenies, the Schizopathidae was separated into two subclades: subclade 1 contained Dendrobathypathes / Parantipathes (Pacific) / Lillipathes , Parantipathes (Atlantic) , Schizopathes , and Dendropathes , and subclade 2 contained Stauropathes and Bathypathes . While subclade 2 was well supported (BS: 98.1; BPP: 99–100), subclade 1 was not, and formed a polytomy in the PhyML and PhyloBayesbased phylogenetic reconstructions. Also similar to the mt-contig phylogenies, the Schizopathidae grouped sister to the Cladopathidae (represented by a single individual) with strong support (BS: 96.1; BPP: 99–100).

Figures

Figure 7. In-situ and laboratory photographs of the indistinguishable trigeneric complex, comprising Parantipathes, Lillipathes, and Dendrobathypathes (all collected from the eastern North Pacific Ocean). A, branched Parantipathes sp. (J2106-7-1; 937 m); B, small colony of D. boutillieri with lab photo inset (J2097-2-1; 1734 m); C, unbranched Parantipathes sp. (J2012-6-3; 862 m); D, unbranched Parantipathes sp. (J2095-2-7-4; 843 m); E, D. boutillieri (J2095-2-5-1; 2162 m); F, Lillipathes sp. (specimen not included in this study; photo reproduced with permission from http://mcbi.marine -conservation.org; © NOAA/MBARI; 1520 m in the eastern Gulf of Alaska). Photos A, B, C courtesy of NOAA.

Figure 8. SEM photographs of the indistinguishable trigeneric complex, comprising Parantipathes, Lillipathes, and Dendrobathypathes (all collected from the eastern North Pacific Ocean). A, D. boutillieri (USNM 1014186; scale bars 0.1 mm); B, L. wingi (USNM 1014106; scale bars 0.1 mm); C–E, Parantipathes sp. (J2095-2-7-6). C, individual spine.