Jazmin J. Hernandez-Kantun

A Multilocus Species Delimitation Reveals a Striking Number of Species of Coralline Algae Forming Maerl in the OSPAR Maritime Area

Maerl beds are sensitive biogenic habitats built by an accumulation of loose-lying, non-geniculate coralline algae. While these habitats are considered hot-spots of marine biodiversity, the number and distribution of maerl-forming species is uncertain because homoplasy and plasticity of morphological characters are common. As a result, species discrimination based on morphological features is notoriously challenging, making these coralline algae the ideal candidates for a DNA barcoding study. Here, mitochondrial (COI-5P DNA barcode fragment) and plastidial (psbA gene) sequence data were used in a two-step approach to delimit species in 224 collections of maerl sampled from Svalbard (78°96’N) to the Canary Islands (28°64’N) that represented 10 morphospecies from four genera and two families. First, the COI-5P dataset was analyzed with two methods based on distinct criteria (ABGD and GMYC) to delineate 16 primary species hypotheses (PSHs) arranged into four major lineages. Second, chloroplast (psbA) sequence data served to consolidate these PSHs into 13 secondary species hypotheses (SSHs) that showed biologically plausible ranges. Using several lines of evidence (e.g. morphological characters, known species distributions, sequences from type and topotype material), six SSHs were assigned to available species names that included the geographically widespread Phymatolithon calcareum, Lithothamnion corallioides, and L. glaciale; possible identities of other SSHs are discussed. Concordance between SSHs and morphospecies was minimal, highlighting the convenience of DNA barcoding for an accurate identification of maerl specimens. Our survey indicated that a majority of maerl forming species have small distribution ranges and revealed a gradual replacement of species with latitude.

DNA sequencing, anatomy, and calcification patterns support a monophyletic, subarctic, carbonate reef-forming Clathromorphum (Hapalidiaceae, Corallinales, Rhodophyta)

For the first time, morpho‐anatomical characters that were congruent with DNA sequence data were used to characterize several genera in Hapalidiaceae—the major eco‐engineers of Subarctic carbonate ecosystems. DNA sequencing of three genes (SSU, rbcL, ribulose‐1, 5‐bisphosphate carboxylase/oxygenase large subunit gene and psbA, photosystem II D1 protein gene), along with patterns of cell division, cell elongation, and calcification supported a monophyletic Clathromorphum. Two characters were diagnostic for this genus: (i) cell division, elongation, and primary calcification occurred only in intercalary meristematic cells and in a narrow vertical band (1–2 μm wide) resulting in a “meristem split” and (ii) a secondary calcification of interfilament crystals was also produced. Neopolyporolithon was resurrected for N. reclinatum, the generitype, and Clathromorphum loculosum was transferred to this genus. Like Clathromorphum, cell division, elongation, and calcification occurred only in intercalary meristematic cells, but in a wider vertical band (over 10–20 μm), and a “meristem split” was absent. Callilithophytum gen. nov. was proposed to accommodate Clathromorphum parcum, the obligate epiphyte of the northeast Pacific endemic geniculate coralline, Calliarthron. Diagnostic for this genus were epithallial cells terminating all cell filaments (no dorsi‐ventrality was present), and a distinct “foot” was embedded in the host. Leptophytum, based on its generitype, L. laeve, was shown to be a distinct genus more closely related to Clathromorphum than to Phymatolithon. All names of treated species were applied unequivocally by linking partial rbcL sequences from holotype, isotype, or epitype specimens with field‐collected material. Variation in rbcL and psbA sequences suggested that multiple species may be passing under each currently recognized species of Clathromorphum and Neopolyporolithon.

Detection of gametophytes in the maerl-forming species Phymatolithon calcareum (Melobesioideae, Corallinales) assessed by DNA barcoding

Abstract Fertile gametangial plants of Phymatolithon calcareum, which are seldom reported in the Atlantic European coasts, were collected as encrusting, epilithic plants in a subtidal maerl bed in Brittany (France). Based on their morphological features, the plants were identified as P. calcareum. This identification was further confirmed by DNA barcodes using as a reference COI-5P sequences obtained from the neotype together with recent collections from the Atlantic European maerl beds. The reproductive structures were empty but they were regarded as mature female conceptacles. Compared to the two previous records of gametangial plants of P. calcareum for the Atlantic European waters, the uniporate conceptacles observed in this study are larger, and were collected at a different time of the year. To our knowledge, this is the first time that the occurrence of gametangial plants of P. calcareum is corroborated with molecular tools (DNA barcodes).

Sequencing type material resolves the identity and distribution of the generitype Lithophyllum incrustans, and related European species L. hibernicum and L. bathyporum (Corallinales, Rhodophyta)

DNA sequences from type material in the nongeniculate coralline genus Lithophyllum were used to unambiguously link some European species names to field‐collected specimens, thus providing a great advance over morpho‐anatomical identifi‐cation. In particular, sequence comparisons of rbcL, COI and psbA genes from field‐collected specimens allowed the following conclusion: the generitype species, L. incrustans, occurs mostly as subtidal rhodoliths and crusts on both Atlantic and Mediterranean coasts, and not as the common, NE Atlantic, epilithic, intertidal crust reported in the literature. The heterotypic type material of L. hibernicum was narrowed to one rhodolith belonging in Lithophyllum. As well as occurring as a subtidal rhodolith, L. hibernicum is a common, epilithic and epizoic crust in the intertidal zone from Ireland south to Mediterranean France. A set of four features distinguished L. incrustans from L. hibernicum, including epithallial cell diameter, pore canal shape of sporangial conceptacles and sporangium height and diameter. An rbcL sequence of the lectotype of Lithophyllum bathyporum, which was recently proposed to accommodate Atlantic intertidal collections of L. incrustans, corresponded to a distinct taxon hitherto known only from Brittany as the subtidal, bisporangial, lectotype, but also occurs intertidally in Atlantic Spain. Specimens from Ireland and France morpho‐anatomically identified as L. fasciculatum and a specimen from Cornwall likewise identified as L. duckerae were resolved as L. incrustans and L. hibernicum, respectively.

Phymatolithon lusitanicum sp. nov. (Hapalidiales, Rhodophyta): The Third Most Abundant Maerl-Forming Species in the Atlantic Iberian Peninsula

Abstract Phymatolithon lusitanicum is a new maerl species described based on an integrative systematic approach including molecular (COI-5P, psbA) and morphological data obtained from recent collections, as well as comparison of type material from the morphologically and ecologically alike NE Atlantic species P. lamii and P. laevigatum. Molecular analyses including type material of P. lamii and P. laevigatum were congruent in delimiting P. lusitanicum as an independent lineage from these crustose species. The three species shared a common external morphology of multiporate asexual conceptacles, but P. lusitanicum has been detected only unattached as maerl while P. lamii and P. laevigatum are crustose. Phymatolithon lusitanicum is particularly abundant in subtidal maerl beds of the Atlantic Iberian Peninsula (Galicia and the Algarve); however it has also been detected northwards in Ireland intertidally and in Western Mediterranean Sea (Alborán Sea, Balearic Islands) down to 64 m. Phymatolithon lusitanicum differs from other Phymatolithon species reported for the European coasts mainly by the external shape of the multiporate asexual conceptacles (pore plate flush with surface or slightly sunken without a conspicuous thick raised rim) and its unattached habit as maerl/rhodolith. In addition, the lectotype of Lithothamnion hamelii turned out to be conspecific to Phymatolithon calcareum, therefore this taxon is proposed as a heterotypic synonym of P. calcareum. Finally, our molecular analyses detected cryptic diversity within the European collections of Phymatolithon, while collections identified as P. lenormandii from Canada or P. repandum from New Zealand were resolved as unrelated to the remaining Phymatolithon. In the light of these results, it is clear that further work is necessary to resolve species diversity within the genus Phymatolithon and its relationship with related genera.

Phylogenetic analysis of rhodolith formation in the Corallinales (Rhodophyta)

Although the ecological importance of rhodolith (maerl, free-living coralline algae) beds is well-known, rhodolith-forming species have been neglected in molecular phylogenetic studies. This is the first molecular systematic study aimed at understanding whether the rhodolith habit is a fixed feature in lineages and determining the relationship (phylogenetic vs. environmental) between rhodolith and crustose habits. Phylogenetic relationships of rhodolith-forming species and encrusting coralline algae at generic and species levels were analysed using SSU rDNA and psbA sequences. Extensive sampling in the European North Atlantic, Pacific and Caribbean Mexico of Phymatolithon, Lithothamnion, Lithophyllum and Neogoniolithon taxa forming rhodoliths and crusts was accompanied by examination of type or topotype material. Phylogenetic reconstruction showed that Neogoniolithon contained a monophyletic group of rhodolith-forming species whereas other rhodolith-formers were closely related to encrusting forms in the genera Phymatolithon, Lithothamnion, Mesophyllum, Hydrolithon, Spongites and Sporolithon. DNA analysis showed that the crust-forming Lithophyllum cf. incrustans/dentatum also forms rhodoliths with a stone nucleus that occur on rocky shores. In contrast, species that form beds of non-nucleate rhodoliths (e.g. Neogoniolithon spectabile, N. strictum, Lithophyllum cf. incrustans/dentatum or sp. 1 and Phymatolithon calcareum) rarely form crusts. The rhodolith habit cannot be used to delimit species for taxonomic or identification purposes. Extensive taxonomic revision will be required to deal with problems such as the position of specimens identified as Lithophyllum margaritae in two unrelated lineages.

Reassessment of branched Lithophyllum spp. (Corallinales, Rhodophyta) in the Caribbean Sea with global implications

Abstract: Plastid-encoded rbcL and psbA sequences from branched, Caribbean Sea Lithophyllum specimens indicate that four species are present, not one. Short (263 base pairs) rbcL sequences from an isolectotype of L. kaiseri (Gulf of Suez) and the holotypes of L. congestum, L. daedaleum and L. platyphyllum (Caribbean Sea) show that L. congestum and L. daedaleum are conspecific with L. kaiseri, the last having nomenclatural priority. Lithophyllum platyphyllum, currently considered a synonym of L. congestum, is recognised as a valid species. Lithophyllum stictaeforme, originally described from the Mediterranean Sea, is not conspecific with L. kaiseri (as L. congestum) as previously suggested. Lithophyllum neocongestum sp. nov. and L. pseudoplatyphyllum sp. nov. are proposed. Together with L. platyphyllum, these three branched species are so far endemic to the Caribbean Sea. This is the first report, documented by DNA sequence data, of a coralline species (L. kaiseri) widespread through the tropical Indo-West Pacific Oceans, Red Sea and Caribbean Sea.

Branched Halymenia species (Halymeniaceae, Rhodophyta) in the Indo-Pacific region, including descriptions of Halymenia hawaiiana sp. nov. and H. tondoana sp. nov.

Several species in the red algal genus Halymenia from the Indo-Pacific have been described with branched thalli, toothed margins, spinose proliferations on the blade, and a firm gelatinous texture. Previous works have synonymized many of these morphologically similar species with H. durvillei. Our increased taxon sampling and molecular data indicate that the taxonomy of the Indo-Pacific Halymenia species is in need of revision, and that several aspects of taxonomies proposed by previous authors now seem unlikely. Thus, the aim of the present work was to analyse species delimitation in branched Halymenia species. Molecular and morphological data for specimens from the coral triangle and peripheral Indo-Pacific localities (East African coast, Hawaii) were used to understand species delimitation for selected branched Halymenia spp. Phylogenetic analyses based on 29 rbcL gene sequences grouped the specimens in four well-supported clusters at the species level with high p-distances (2.7–5.3%). After the morphological analysis, five features were retained as diagnostic to identify the four species studied. Our analyses led to the recognition and description of two new species, H. hawaiiana from the Hawaiian Islands (previously erroneously called H. formosa) and H. tondoana from the Philippines. In addition, H. harveyana (currently treated as a subspecies of H. floresii in Australia) is reassessed and recognized at the species level. Specimens with seven orders of branching and a thick cortex (70–150 µm) formed a monophyletic group, including sequences from previous work, with mostly well-supported branches and with high p-distances at the species level. We propose to call this group the H. durvillei complex until further reassessment is completed. None of the sequences studied here grouped with H. floresii from the Mediterranean, suggesting that previous Indo-Pacific reports of the species were erroneous.

North Atlantic Rhodolith Beds

Beds of coralline algal sediment form ecologically and economically important habitats in the North Atlantic. These habitats can occur from the intertidal down to 60 m depth, and they are locally abundant in several countries. Thirteen species of coralline algae form rhodoliths or maerl in this region; Lithothamnion corallioides, L. glaciale, L. tophiforme and Phymatolithon calcareum are the most widely recorded. The structure and biodiversity of these habitats is destroyed by dredging and can be degraded by towed demersal fishing gear and by mussel and salmon farming. Legislation has been passed in the European Union (EU) to protect P. calcareum and L. corallioides which should be extended to include the other maerl species from the region. Outside the EU there is a lack of baseline information concerning the importance of these habitats: a fuller understanding of their role may lead to protection in Scandinavia, Iceland and the Atlantic coasts of Canada and the United States. The design of such protected areas would need to consider the ongoing effects of invasive species, ocean warming and acidification.

Genetic and morphological variation in an ecosystem engineer, Lithophyllum byssoides (Corallinales, Rhodophyta)

Lithophyllum byssoides is a common coralline alga in the intertidal zone of Mediterranean coasts, where it produces biogenic concretions housing a high algal and invertebrate biodiversity. This species is an ecosystem engineer and is considered a target for conservation efforts, but designing effective conservation strategies currently is impossible due to lack of information about its population structure. The morphological and molecular variation of L. byssoides was investigated using morphoanatomy and DNA sequences (psbA and cox2,3) obtained from populations at 15 localities on the Italian and Croatian coasts. Lithophyllum byssoides exhibited a high number of haplotypes (31 psbA haplotypes and 24 cox2,3 haplotypes) in the central Mediterranean. The psbA and cox2,3 phylogenies were congruent and showed seven lineages. For most of these clades, the distribution was limited to one or a few localities, but one of them (clade 7) was widespread across the central Mediterranean, spanning the main biogeographic boundaries recognized in this area. The central Mediterranean populations formed a lineage separate from Atlantic samples; psbA pair‐wise divergences suggested that recognition of Atlantic and Mediterranean L. byssoides as different species may be appropriate. The central Mediterranean haplotype patterns of L. byssoides were interpreted as resulting from past climatic events in the hydrogeological history of the Mediterranean Sea. The high haplotype diversity and the restricted spatial distribution of the seven lineages suggest that individual populations should be managed as independent units.