Ramicrusta paradoxa E. M. S. Pestana, G. N. Santos, Cassano & J. M. C. Nunes, 2020

Pestana, Edilene Maria Dos Santos, Lyra, Goia De Mattos, Santos, Gabriel Do Nascimento, Santos, Cibele Conceição Dos, Cassano, Valéria & Nunes, José Marcos De Castro, 2020, Integrative approach reveals underestimated Peyssonneliales diversity in Brazil: registering the first occurrence of Ramicrusta and Incendia, with the description of three new species, Phytotaxa 439 (1), pp. 39-55 : 45-50

Ramicrusta paradoxa E. M. S. Pestana, G. N. Santos, Cassano & J. M. C. Nunes sp. nov. ( Fig. 3 A-D).

Diagnosis: — Thallus crustose, prostrate, completely adherent to the substratum by multicellular rhizoids. Perithallus differentiated into two regions, the lower portion consisting of 4 to 5 layers of elongated cells, occasionally branched and the upper portion composed of 10–12 layers of drop shaped cells. Hair cells absent. Secondary pit connections absent. Oval tetrasporangia born terminally among simple paraphyses. Distinguished from other species of the genus by COI-5P sequence divergence above 8.5% and by rbc L sequence divergence above 7.8%.

Type:— Brazil. Bahia, Maraú, Ponta do Mutá, 13º52’45,61”S – 38º56’50,79”W, 27 September 2015, E. M. S. Pestana, G.N. Santos & J.M.C. Nunes ( ALCB 129747). GenBank accession number for COI-5 P MN 990094, SSU MN 966417.

Etymology: — Epithet refers to characteristics of the species, which are different from those originally described for the genus.

Description: — Thallus encrusting, reddish orange, completely calcified, strongly and completely adherent to the substratum, 200–400 μm ( Fig. 3A ). Rhizoids multicellular, up to 4 cells, 200–270 μm long and 15–20 μm thick ( Fig. 3D ). Hypothallus is monostromatic, hypothallial filaments are parallel and formed by regular rectangular cells giving rise to perithallial filaments at angles of 90º from their distal surfaces. Perithallus is formed by individualized filaments without secondary pit connections and is differentiated into upper and lower parts ( Fig. 3B ). The lower perithallus consisting of 4 to 5 layers of elongated cells, 30–50 μm long and 9–20 μm thick, occasionally with ramifications ( Fig. 3C ). Upper perithallus is composed of 10–12 layers of smaller, basally constricted cells, 23–50 μm long and 12–20 μm thick ( Fig. 3B ). Hair cells are absent. Tetrasporangial nemathecia up to 100 μm thick and irregularly distributed on the thallus surface ( Fig. 3B ). Nemathecia are formed by simple paraphyses of 3–4 cells immersed in a mucilaginous matrix. Tetrasporangia are oval, 45–50 μm long and 30–40 μm diameter, decussately/cruciately divided and are born terminally. Gametophytic specimens were not observed in the present study.

Habitat: — Epilithic specimens growing over rocks in areas protected from wave exposure in the reef, emergent during low tide periods.

Examined Material:— Brazil, Bahia: Maraú, Três Coqueiros Beach , 28 September 2015, E. M. S. Pestana, G.N. Santos & J.M.C. Nunes ( ALCB 129745 ) ; Saquaíra Beach , 29 September 2015, E. M. S. Pestana, G.N. Santos & J.M.C. Nunes , ( ALCB 129746 ) .

The original concept of Ramicrusta was based on the presence of unicellular rhizoids and secondary pit connections between perithallial filaments ( Pueschel & Saunders 2009, Dixon & Saunders 2013, Ballantine et al. 2016). Ramicrusta paradoxa , however, nested in the Ramicrusta clade, is the only Ramicrusta species known to date with multicellular rhizoids and lacking secondary pit connections among perithallial filaments.

A comparison of the morphological characteristics for Ramicrusta species is shown in Table 2.

Molecular results

A total of 27 sequences (from 16 specimens) were newly generated in the present study (14 rbc L, 11 COI-5P, and two SSU sequences). These data were aligned to 78, 62, and 26 published rbc L, COI-5P, and SSU sequences, respectively ( Table S1). Alignment lengths were 862 bp for rbc L, 551 bp for COI-5P, and 2,168 bp for SSU. Numbers of variable nucleotide positions, and parsimonious informative sites were 310 and 236 for rbc L; 226 and 216 for COI-5P; 272 and104 for SSU, respectively. For rbc L, two species of Rhodymeniales were designated as outgroups, Botryocladia botryoides (Wulfen in Jacquin 1791: 146) Feldmann (1941a: 90) and Botryocladia occidentalis (Børgesen) Kylin (1931: 18) . For COI-5P, four species of Rhodymeniales served as outgroups, Botryocladia franciscana J.A.S.Santiago, P.B.M.Carneiro, A.P.Santiago, R.G.Feijó & R.Maggioni (2016: 140) , Botryocladia skottsbergii (Børgesen) Levring (1941: 645) , Ceratodictyon repens (Kützing) R.E. Norris (1987: 245) , and Ceratodictyon scoparium (Montagne & Millardet) R.E. Norris (1987: 245) . For SSU, Asparagopsis taxiformis (Delile) Trevisan (1845: 45) , Bonnemaisoniales, was used as the outgroup.

Our phylogenies were consistent with each other, without conflicting supported clades (over 75%), and confirmed Peyssonneliales as monophyletic, with high support values for COI-5P and rbc L ( Figs 4-6 ). Incendia , Metapeyssonnelia Boudouresque, Coppejans & Marcot (1976) , Polystrata Heydrich (1905) , Ramicrusta , Riquetophycus Denizot (1968) and Sonderophycus were also monophyletic. Peyssonnelia was polyphyletic, and its lectotype, P. squamaria (S.G.Gmelin) Decaisne ex J. Agardh (1842: 93) was placed in a clade together with P. coriacea Feldmann (1941b: 284) , P. heteromorpha (Zanardini) Athanasiadis (2016: 669) , P. replicata Kützing (1847: 775) , and P. rubra (Greville) J. Agardh (1851: 502) in the rbc L phylogeny.

Part of the Brazilian specimens were grouped in the monophyletic Ramicrusta clade, forming a subclade with full support in the rbc L phylogeny ( Fig. 4 ), with moderate support for PP (0.94) in the SSU phylogeny ( Fig. 6 ). In the COI-5P ( Fig. 5 ), Ramicrusta species formed two clades with high support, one that included six specimens from this study (87/1.00) and another that included five specimens (88/0.99). This represents the first occurrence of Ramicrusta in the Brazilian coast.

Our phylogenies support the recognition of two new species, here proposed as R. fujiiana and R. paradoxa , which was further tested with ABGD for the three genetic markers. ABGD for rbc L confirmed specimens of both species in two mutually exclusive groups. While ABGD for COI-5P recovered the group of specimens of R. fujiiana , but split R. paradoxa into two subclades. For rbc L, R. fujiiana formed a fully-supported clade, sister to another fully-supported clade that included R. aranea ( Vanuatu, type locality) and R. textilis ( Jamaica, type locality), diverging from 7.8 to 8.35%, and 7.2–7.85%, respectively. For the COI-5P phylogeny, R. fujiiana was sister to R. appressa ( Vanuatu, type locality), with divergencies of 6.0–6.5% between them. The divergence between R. paradoxa and R. australica ( Australia, type locality) was 7.8 - 8.6% for rbc L and 8.5–8.7% for COI- 5P. Intraspecific variation for rbc L ranged from 0–1.2% in R. fujiiana and from 0.55–0.9% in R. paradoxa . For COI-5P, intraspecific variation in R. fujiiana ranged from 0–0.14%, and R. paradoxa ranged from 0–0.18%.

One Brazilian specimen nested in the monophyletic Incendia clade with full support in the rbc L phylogeny. Therefore, we propose the new species Incendia yoneshigueana , hypothesis that was also tested by ABGD for COI-5P and rbc L gene matrices, which confirmed our specimen in one exclusively separated group. Divergence of Incendia yoneshigueana from its sister species, Incencia glabra from Vanuatu (type locality), was 7.9%. Only two rbc L sequences of Incendia species were available in GenBank, nevertheless our taxonomic decision of describing this new species, as well as the two Ramicrusta species, is also robustly based on morphological analyses.

Intra and interspecific variation are shown in Suplementary Tables 2, 3 and 4.

Figures

FIGURE 3. Ramicrusta paradoxa. (A) Habit. (B) Radial vertical section of thallus, evidencing perithallus, tetrasporangial nemathecium (arrow) and tetrasporangia (arrowhead). (C) Lower perithallus cells branched, giving rise to two upper perithallus cells (arrows). (D) Multicellular rhizoids. Scale bar: A, 1 cm; B, 100 μm; C and D, 30 μm.

FIGURE 4. Maximum likelihood (ML) tree of rbcL sequences. Bootstrap values (BP) and posterior probabilities (PP) are shown at the nodes ML/PP. Samples generated in this study are in bold; – indicates lack of bootstrap support or values under 75; * indicates full support. Inset displays ABGD reconstructions.

FIGURE 5. Maximum likelihood (ML) tree of COI-5P. Bootstrap values (BP) and posterior probabilities (PP) are shown at the nodes BP/PP. Samples generated in this study are in bold; – indicates lack of bootstrap support or values under 75; * indicates full support. Inset displays ABGD reconstructions.

FIGURE 6. Maximum likelihood (ML) tree of SSU. Bootstrap values (BP) and posterior probabilities (PP) are shown at the nodes BP/PP. Samples generated in this study are in bold; – indicates lack of bootstrap support or values under 75; * indicates full support.