Neogoniolithon Setchell et L.R.Mason 1943

Neogoniolithon Setchell et L.R.Mason 1943 View in CoL

Neogoniolithon cf. mamillare (clade 1)

Specimens: Herbarium vouchers UNA 00075209, UNA 00075219, UNA 00075220 ( Tables 4 and S 1; Figures 5A – D View Figure 5 and 6A – D View Figure 6 ).

The three specimens from the Florida Keys and two specimens from Roatán (RTN19-53, RTN19-54) formed a well-supported clade on all phylogenetic trees ( Figures 2 – 4 View Figure 2 View Figure 3 View Figure 4 , S 1 View Figure 1 and S 2 View Figure 2 ). No sequences available in GenBank matched those obtained from these specimens .

Neogoniolithon cf. mamillare (clade 2)

Specimen: UNA 00075210 ( Tables 4 and S 1; Figures 5A – D View Figure 5 and 6A – D View Figure 6 ).

Although morphologically and anatomically similar to N. cf. mamillare clade 1, this specimen was considered to be a separate species based on the species delimitation methods

( Figure 3A and B View Figure 3 ).

Comments on N. cf. mamillare clades: The four specimens belonging to these two clades were encrusting to warty plants with mammillated protuberances and an adherent white margin. The thallus thickness ranged from 150 to 900 μm; uniporate conceptacles had an outer diameter (od) of 500 – 1,800 μm and a chamber diameter of 300 – 900 μm. Thallus construction was monomerous and plumose; trichocytes were single, large and scattered ( Table 4; Supplementary Material for full description). The inclusion of a specimen called N. mamillare from Mexico (herbarium voucher NCU 624389; Mateo-Cid et al. 2014) in our molecular analyses that appeared to be a different species to both clades that we called N. cf. mamillare demonstrates that morphologically and anatomically identical but phylogenetically distinct species have been given this name. Type material of N. mamillare needs to be sequenced from the syntype localities (Bahia, Brazil; Tierra del Fuego; Cabo Verde; Algoa Bay, Cape Province, South Africa) to correctly apply this name.

Neogoniolithon sp. (clade 1)

Specimen: UNA 00075215 ( Tables 4 and S 1; Figures 5E – H View Figure 5 and 6E – H View Figure 6 ).

The psbA sequence from this specimen was identical to a sequence of N. mamillare from Mateo-Cid et al. (2014; KJ637664) as indicated by both species delimitation methods. Although some features of our specimen conform to the description of N. mamillare , such as the encrusting to warty thallus with mammillated protuberances, the adherent margin with a white edge and the single and scattered trichocytes ( Table 4; Supplementary Material), the strongly coaxial hypothallus described herein and in Mateo-Cid et al. (2014) do not. For this reason, we considered this specimen Neogoniolithon sp. ( Figure 2 View Figure 2 , clade 1).

Neogoniolithon sp. (clade 2)

Specimen: UNA 00075212 ( Tables 4 and S 1; Figures 5I – L View Figure 5 and 6I – L View Figure 6 ).

The specimen belonging to this clade had an encrusting to warty thallus and a thickness that ranged from 980 to 2,680 μm. Conceptacles were uniporate with an outer diameter of 520 – 1,400 µm and a chamber diameter of 500 – 950 µm. Thallus construction was monomerous and plumose while the trichocytes were single and scattered ( Table 4; Supplementary Material). The psbA sequence from this specimen was identical to that of N. acropetum ( Mateo-Cid et al. 2014; KJ637663). However, both the original description from Foslie and Howe (1906) as well as that of Mateo-Cid et al. (2014) indicated the ability of this species to form dense and branching masses 6 – 10 cm high, a characteristic that was not observed in our specimen. Therefore, given that no type specimen of N. acropetum (type locality: Culebra Island, Puerto Rico) has been sequenced, we chose to designate this specimen as Neogoniolithon sp. ( Figure 2 View Figure 2 , clade 2).

Neogoniolithon sp. (clade 5)

Specimen: UNA 00075225 ( Tables 4 and S 1; Figures 5M – P View Figure 5 and 6M – P View Figure 6 ).

The specimen belonging to this clade had an encrusting to lumpy thallus with straight, terete protuberances. Crust thickness ranged from 400 to 730 µm. Conceptacles were uniporate with an outer diameter of 480 – 540 µm. Thallus construction was monomerous and coaxial in the encrusting base and radial in the protuberances. Trichocytes were single and scattered ( Table 4; Supplementary Material). The psbA sequence of this specimen was identical to a N. rhizophorae specimen from Mexico ( Mateo-Cid et al. 2014; KJ637671) and its rbc L sequence matched perfectly with a N. rhizophorae specimen from Florida ( Richards et al. 2021a; MW452895). Although our description is comparable to that of Mateo-Cid et al. (2014), no type specimen ( N. rhizophorae type locality: Exuma Harbour, Stocking Island, Bahamas) has been sequenced to validate the attribution of this name, hence we considered this specimen Neogoniolithon sp.

( Figure 2 View Figure 2 , clade 5).

Unknown genus within the subfamily Neogoniolithoideae

Unknown Neogoniolithoideae (clade 3)

Specimens: UNA 00075213, UNA 00075218 (Tables 5 and S1; Figures 5Q – T View Figure 5 and 6Q – T View Figure 6 ).

No sequences in GenBank matched sequences from these specimens.

Unknown Neogoniolithoideae (clade 4)

Specimen: UNA 00075217 (Tables 5 and S1; Figures 5Q – T View Figure 5 and 6Q – T View Figure 6 ).

No sequences in GenBank matched sequences obtained from this specimen.

Unknown Neogoniolithoideae (clade 7)

Specimens: UNA 00075221, UNA 00075224, UNA 00075226, UNA 00075227 (Tables 5 and S1; Figures 5Q – T View Figure 5 and 6Q – T View Figure 6 ).

No sequences in GenBank matched those obtained from these specimens.

Comments on the unknown Neogoniolithoideae clades: Although both species delimitation methods separated the Florida Keys specimens of the unknown Neogoniolithoideae clade into three species ( Figures 2 – 4 View Figure 2 View Figure 3 View Figure 4 ), all specimens shared the same morphological and anatomical features. Those features are in line with what was described for the now subsumed Paragoniolithon solubile ( Adey et al. 1982) , such as the encrusting to layered to foliose thallus, the multilayered perithallus with common cell fusions, the monomerous and strongly coaxial hypothallus and the presence of trichocytes either single or loosely grouped in horizontal fields of more than three trichocytes with intervening vegetative filaments between cells ( Figures 5Q – T View Figure 5 and 6Q – T View Figure 6 ; Table 5; Supplementary Material). Nevertheless, the genus Paragoniolithon is currently in synonymy with the genus Neogoniolithon so, although these specimens appear distinct from members of Neogoniolithon , an appropriate name for these specimens does not exist. Adding to the confusion, a specimen identified as N. solubile from Mexico ( Figure 2 View Figure 2 and 3 View Figure 3 : GenBank accession KJ637678, herbarium voucher NCU 624974) was included in the analyses and the description of this specimen in Mateo-Cid et al. (2014) also matches the characters observed in the type material of Goniolithon solubile (type locality: Culebra Island, Puerto Rico), including the monomerous and coaxial hypothallus, trichocytes organized in single or in vertical and horizontal rows with intervening vegetative filaments between cells, absence of secondary pit connections, and strongly projected and apiculate conceptacles. However, the sequence from NCU 624974 nests within the Neogoniolithon clade but appears to be genetically distinct from any other Neogoniolithon sequence, as well as from any specimen sequenced in this study belonging to the unknown genus. It is therefore essential that genes from the type specimen of P. solubile are sequenced to ascertain which, if any, of these numerous, phylogenetically distinct but morphologically, anatomically and reproductively similar specimens might match.

Harveylithon A.Rösler, Perfectti, V.Peña et J.C.Braga 2016

Harveylithon munitum (Foslie et M.Howe) A.Rösler, Perfectti, V.Peña et J.C.Braga 2016

Specimen: UNA 00075223 (Tables 6 and S1; Figures 7A – D View Figure 7 and 8A – D View Figure 8 ).

This specimen had an encrusting thallus and a crust thickness that ranged from 270 to 860 μm. Conceptacles were uniporate with an outer diameter of 270 – 730 μm and a chamber diameter of 150 – 200 μm. Thallus construction was monomerous and weakly coaxial ( Table 4; Supplementary Material). Sequences from this sample perfectly matched the psbA, rbc L and LSU gene sequences from the type specimens of H. munitum (type locality: Cave Cays , Exuma Chain, Bahamas) ( Figures 3 View Figure 3 , 4 View Figure 4 , and S2 ) .

Harveylithon sp. clade 2

Specimen: UNA 00075228 (Tables 6 and S1; Figures 7E – H View Figure 7 and 8E – H View Figure 8 ).

The psbA gene failed to amplify for this specimen but the sequence obtained from the rbc L gene was most closely related to another sample from the Florida Keys (UNA 00075216) and phylogenetically close to other Harveylithon species ( Figure 4 View Figure 4 , clade M). Sequences from both ribosomal genes also placed it within clade M ( Figures S1 View Figure 1 and S 2 View Figure 2 ), and related to RNT19-46 and UNA 00075223 ( H. munitum ). Although no trichocytes were present in this specimen, other morphological features conform to the description of Porolithon pachydermum (now P. antillarum ; Gabrielson et al. 2018), such as the thick thallus, the small conceptacle size, the single layered epithallus and the monomerous and plumose thallus construction, but its gene sequences were distinct from any Porolithon sequences.

Hydrolithon (Foslie) Foslie 1909

Hydrolithon cf. boergesenii (clade 3)

Specimen: UNA 00075211 (Tables 6 and S1; Figures 7I – L View Figure 7 and 8I – L View Figure 8 ).

This specimen had an encrusting thallus with a crust thickness ranging from 200 to 700 μm. The uniporate conceptacles had an outer diameter of 160 – 400 μm and a chamber diameter of 150 – 370 μm. Thallus construction was dimerous while trichocytes were present and loosely packed ( Table 4; Supplementary Material). The psbA sequence from this specimen was identical to that of H. boergesenii from Brazil (KY485316; Jesionek et al. 2016), although the type specimen (type locality: St. Croix, US Virgin Islands) of H. boergesenii has yet to be sequenced. However, the Hydrolithon clade (clade P) formed by samples from both the Florida Keys and Roatán was separated by species delimitation methods into three (rbc L) or four (psbA) species. Only a single study (see Richards et al. 2021b) previously suggested that more than one species of NGCA in the western tropical Atlantic with the same morphological and anatomical features as H. boergesenii , might be included under this name. DNA sequences from additional samples are needed to confirm how many Hydrolithon species are present in the western Atlantic.

Titanoderma Nägeli 1858

Titanoderma cf. prototypum

Specimen: UNA 00075214 (Tables 6 and S1; Figures 7M – P View Figure 7 and 8M – P View Figure 8 ).

The psbA gene sequence for this specimen was identical to that of T. prototypum from Brazil (KY485319; Jesionek et al. 2016) but was phylogenetically distinct from two Roatán samples in the psbA tree (RTN19-32, RTN19-51; Figure 3 View Figure 3 ) and from four samples in the SSU tree (RTN19-31, RTN19-32, RTN19-35, RTN19-51; Figure S1 View Figure 1 ). Although the rbc L and LSU genes from this specimen did not amplify, the morphological and molecular data support the designation of T. cf. prototypum including the encrusting to layered thallus, a crust thickness ranging from 25 to 50 µm, small uniporate conceptacles (od = 120 – 250 μm), the dimerous thallus construction composed of palisade cells, and the retained epithallial cells readily visible in SEM cross section images. Nevertheless, the type specimen (type locality: St. Croix, US Virgin Islands) of T. prototypum has yet to be sequenced to unambiguously apply this name.

Lithophyllum Philippi 1837

Lithophyllum sp. (clade 1)

Specimen: UNA 00075222 (Tables 6 and S1; Figures 7Q – T View Figure 7 and 8Q – T View Figure 8 ).

Sequences obtained from the psbA and LSU genes placed this specimen, along with a specimen from Roatán (RTN19- 50), close to members of the genus Lithophyllum ( Figures 3 View Figure 3 and S 2 View Figure 2 ). In the SSU tree, however, a temperate specimen called Titanoderma ‘pustulatum’ (AF093409) from the UK also fell within clade H and confirmed the uncertainties related to these two taxa that were discussed in previous studies ( Bittner et al. 2011; Richards et al. 2014). Because of the presence of predominantly non-palisade hypothallial cells ( Figure 7Q – T View Figure 7 ), we consider this to be a Lithophyllum sp.