The pseudodichotomous Dasya sylviae sp. nov. (Delesseriaceae, Ceramiales) from 60–90 m mesophotic reefs off Bermuda

Abstract. The red alga Dasya sylviae C.W.Schneid., M.M.Cassidy & G.W.Saunders sp. nov. is described from mesophotic depths of 60–90 m off Bermuda. Genetic sequences (COI-5P, rbcL) and morphological characteristics show that this species is distinct from other known pseudodichotomous species of Dasya. Of ten current species in the genus reported from Bermuda, only three, D. collinsiana M.Howe, D. cryptica C.W.Schneid., Quach & C.E.Lane and D. punicea (Zanardini) Menegh., share the overall pattern of pseudodichotomous branching in their axes; however, key morphological features easily distinguish them from D. sylviae sp. nov. The species most similar in habit to D. sylviae sp. nov. is D. crouaniana J.Agardh (type locality West Indies), but it bears shorter pseudolateral branches, and broader and longer tetrasporangial stichidia than the new species. Unique among the species of Dasya, D. sylviae sp. nov. lacks post-sporangial cover cells in tetrasporangial stichidia.


Introduction
Since joining the 2016 Nekton XL Catlin cruise of the R / V Baseline Explorer, the mesophotic collections of macroalgae taken off the coast of Bermuda have yielded a growing list of species new to science (Richards et al. 2018;Schneider et al. 2018Schneider et al. , 2019aSchneider et al. , 2019bSchneider et al. , 2020. In the present paper, we report another new species, this a member of the genus Dasya C.Agardh that was abundantly collected at several off shore reef sites from depths of 60-90 m (Stefanoudis et al. 2018: fi g. 53, as Dasya sp.).
The genus Dasya presently hosts 90 species from throughout the world's temperate and tropical seas (Guiry & Guiry 2020). Its axial growth is sympodial with a main leading axis being continually displaced to one side by a new axis forming from a lateral bud near the apex. The resulting lateral axis takes over as the new main axis, a pattern that is repeated over and over in axial development (Parsons 1975).
Based on appearance and not development, species of Dasya mostly look to have alternate indeterminate branching patterns. However, some species of Dasya have sympodial branching patterns that appear to be dichotomous or pseudodichotomous (subdichotomous), and they are described as having this type of branching (e.g., Schneider et al. 2017;Howe 1918;Huisman 2018). In these species, the former main axis is not as suppressed by the newly produced lateral axis as in the case of most species of Dasya and often grows nearly as long as the new leading axis, in some species to a greater degree than in others. Of the ten species historically reported from Bermuda (Schneider 2003;Schneider et al. 2017), only three, D. collinsiana M.Howe, D. cryptica C.W.Schneid., Quach & C.E.Lane and D. punicea (Zanardini) Menegh., have an at least partial pseudodichotomous branching pattern appearance of their indeterminate axes. A morphological comparison was conducted to compare all of the known large species of the genus demonstrating mostly or partially pseudodichotomous branching with collections discovered in deep water off Bermuda demonstrating pseudodichotomous branching. Using both morphological comparisons and molecular data from the off shore samples, we were able to determine that the mesophotic specimens represent a species of Dasya new to science.

Material and methods
On the 2016 Nekton XL Catlin cruise of the R / V Baseline Explorer (BEX) off the coast of Bermuda, collections from living low-profi le reefs in the mesophotic zone (Stefanoudis et al. 2018) were made by a team of technical rebreather divers (Global Underwater Explorers (GUE), High Springs, Florida, USA) equipped with closed-circuit JJ-CCR CE Edition rebreathers (JJ-CCR ApS, Copenhagen, Denmark) modifi ed to GUE confi guration, and Triton 1000-2 class submersibles (Vero Beach, Florida, USA) with mechanical collecting arms. Approximately 30 specimens of a common species of Dasya were taken at fi ve collecting sites from depths of 60-90 m. Vouchers of type specimens are deposited in the herbaria noted in the Material examined section below; herbarium abbreviations follow the online Index Herbariorum (Thiers, continuously updated). Collection site locations on the BEX were recorded using a Beier Radio DP1 (dynamic positioning, Beier Integrated Systems, Mandeville, Louisiana, USA) to receive shipboard GPS. After living specimens were chosen for DNA analysis, they were photographed using a Canon Powershot s90 digital camera (Canon Inc., Tokyo, Japan), and fragments of each were then dried in silica gel for later DNA extraction. The remainder of the DNA specimens were dried on herbarium paper as permanent vouchers. Hand-cut sections were mounted in 30% corn syrup with acidifi ed 1% aniline blue in a ratio of 20 :1. Dried specimens were scanned on an Epson ET-2650 scanner (Seiko Epson Corporation, Suwa, Nagano, Japan), and photomicrographs were taken using a Zeiss Axioskop 40 microscope (Oberkochen, Germany) equipped with a Spot Idea 28.2 5MP digital camera (Diagnostic Instruments, Sterling Heights, Michigan, USA).
Specimens generated for use in our molecular analysis are listed in Table 1. When quick drying single or multiple isolates of associated fi eld collection numbers, the silica gel samples were designated with unique 'BDA' numbers. DNA extractions of BDA numbers followed Saunders & McDevit (2012) and PCR amplifi cation and sequencing of COI-5P and rbcL were as detailed in Saunders & Moore (2013). These markers initially identifi ed fi ve specimens assignable to a new genetic group. To place this new species into a wider phylogenetic context, additional COI-5P and rbcL sequences were generated for a variety of species of Dasya and the taxonomically related genera Dasysiphonia I.K.Lee & J.A.West, Heterosiphonia Mont. and Rhodoptilum J.Agardh from Canada, Australia and the US (Table 1). To expand further our analyses, COI-5P and rbcL data were also downloaded from GenBank for additional taxonomically related species, and their accession numbers are included in Fig. 1. Two single-gene alignments were generated: COI-5P with 28 sequences of 664 bp (base pairs) and rbcL with 36 sequences and 1272 bp. These alignments were analysed separately in Geneious ver. 2021.0.1 (https://www.geneious.com; Kearse et al. 2012) with maximum likelihood (GTR+I+G) using RAxML (Stamatakis 2014) with partitioning by codon and 500 bootstrap replicates. Since no confl icts were detected, a concatenated COI-5P and  (2021) rbcL alignment was constructed (38 sequences, 1936 bp) with analyses as described for the single gene alignments, but with partitioning by gene and codon and with 1000 bootstrap replicates (Fig. 1). In all cases, the trees were rooted on the lineage defi ned as the Heterosiphonia group (Choi et al. 2002).

Phylogenetic analysis
In the speciose genus Dasya worldwide, there is a total of eight known corticated species taller than 3 cm at maturity that bear indeterminate axes appearing mostly or partially pseudodichotomously branched. A comparative review of these species is summarized in Table 2. None of these bear a suite of characteristics that is comparable with the mesophotic specimens collected off Bermuda.
We successfully generated COI-5P (664 bp) for fi ve individuals of this novel species and all had identical sequences except for one substitution in BDA2011, or 0 -0.15% divergence within this species. The nearest neighbor identifi ed through a BLAST search in GenBank was Dasya adela Heggøy, Rueness & Sjøtun that demonstrated a 6% divergence. Similarly, we generated an rbcL sequence for two specimens, which were identical over 1358 bp and 2.9% divergent from D. adela. Phylogenetic analyses placed the new genetic group solidly in a clade with D. adela and an undescribed species from the euphotic zone off Bermuda (Fig. 1, Dasya sp. 1Bda). Of further note, none of the genera included in the current tree were monophyletic except for the monospecifi c Rhodoptilum, which nonetheless fell solidly in a group with the generitype of Dasya, D. baillouviana (S.G.Gmel.) Mont. (Fig. 1). Clearly considerable taxonomic work remains to be done on the genera included in our phylogenetic analyses, but this does not detract from our clear discovery of a novel species best included in the genus Dasya. Therefore, based upon our molecular comparisons and phylogenetic analysis, we here describe the following unique mesophotic species of Dasya for Bermuda and the western Atlantic.

Diagnosis
Diff ering from most species of Dasya by its pronounced pseudodichotomous branching pattern ( Fig. 2A-C), and from its most similar congener in habit, D. crouaniana J.Agardh, by its longer pseudolaterals, narrower and shorter tetrasporangial stichidia and axes fully covered with pseudolaterals to barely denuded proximal axes. The new taxon diff ers from all species of Dasya by its lack of post-sporangial cover cells.

Etymology
The species is named after Dr Sylvia A. Earle, pioneering phycologist, scientist and open-water diver, 50 years after she led the fi rst all-female team of aquanauts in Tektite II on the fl oor of the Caribbean Sea (Earle 1972a(Earle , 1972b.

Distribution and habitat
At present, endemic to mesophotic reefs off Bermuda, western Atlantic Ocean.

Discussion
The genus Dasya is characterized by the development of 2-4 pre-and post-sporangial cover cells that partially cover tetrasporangia in their stichidium (Parsons 1975). Dasya sylviae sp. nov. appears to be unique among its congeners as it completely lacks these cover cells, thus tetrasporangia sit naked on their whorl branches (Table 2, Fig. 2G). Among the presently accepted 90 species of Dasya (Guiry & Guiry 2020), only eight corticated species of Dasya that reach at least 3 cm tall at maturity, including three from Bermuda (D. collinsiana, D. cryptica and D. punicea), appear to share an overall axial pattern of pseudodichotomous branching with D. sylviae sp. nov. (Table 2, Fig. 2A-C). However, key morphological characteristics easily distinguish them from the new species presented here. Among the few species with longer pseudolaterals approaching the length of those in the new species (to 5.5 mm long), D. anastomosans (Weber Bosse) M.J.Wynne and D. cryptica demonstrate a short bushy habit with more dense pseudolateral growth, larger tetrasporangia and longer stichidia ( Table 2).
The species most similar to Dasya sylviae sp. nov. in overall habit is D. crouaniana (type locality West Indies), but the latter species is characterized by its striking loss of deciduous pseudolaterals in the lower half of its main axes contrasted by densely enveloped distal portions as illustrated in Taylor (1928: pl. 35 fi g. 5;1960: pl. 71 fi g 1). These pseudolaterals are shorter in length (1-2 μm) but more densely packed than the longer ones of D. sylviae sp. nov. (1.7-5.5 μm). Furthermore, the tetrasporangial stichidia of D. crouaniana are both longer and broader (to 1 mm × 80-120 μm) than in the new species (269-305 μm × 73-80 μm). Unfortunately, we do not have genetic information for this species, but its morphological diff erences distinguish it from D. sylviae sp. nov.
While Dasya punicea (type locality Venice) appears to be somewhat similar to D. sylviae sp. nov. in habit, the Mediterranean species can be diff erentiated by its subverticillate pseudolaterals, slightly longer tetrasporangial stichidia (300-400 μm vs 269-305 μm) and slightly larger tetrasporangia (30-40 μm vs 21-29 μm). Its long pseudolaterals (to 4.4 mm) are reminiscent of the new species. Dasya punicea was reported from Bermuda by Collins & Hervey (1917) who stated that their specimens had a tendency to issue ramelli [pseudolaterals] "in more or less distinct whorls," a condition dissimilar to that in specimens from the eastern Atlantic where the pseudolaterals were spirally arranged (Maggs & Hommersand 1993). Ballantine & Aponte (2004) argued that the entity fi rst reported in the western Atlantic from Bermuda by Collins & Hervey (1917) as D. punicea was diff erent from eastern Atlantic and Mediterranean isolates. Three archival specimens left by A. Hervey as D. punicea (Collins & Hervey 1917;NY 2178604) are an admixture of two species on a single sheet, one representing a young Dasya spinuligera Collins & Herv., the remaining representative of Wrangelia C.Agardh (Wrangeliaceae J.Agardh), not Dasya.
The mesophotic specimens described here as a new taxon were collected along with two other species of Dasya at these depths, D. cf. baillouviana (S.G.Gmel.) Mont. (58-77 m) and D. spinuligera (60 m). Unlike D. sylviae sp. nov., both of these species with diff erent and distinctive morphologies are also known on shallow subtidal reefs in Bermuda. Including D. collinsiana, D. cryptica and D. spinuligera, D. sylviae sp. nov. represents the fourth species of the genus with its type locality in Bermuda (Collins & Hervey 1917;Howe 1918;Schneider et al. 2017).
Genetically, Dasya sylviae sp. nov. falls in a clade with the recently described D. adela, a species discovered in a landlocked fjord in Norway (Sjøtun et al. 2016), and the alternately to irregularly branched Dasya sp. 1Bda from the shallow subtidal of Bermuda (Schneider et al. 2017) (Fig. 1). Dasya adela is signifi cantly smaller (to 3 cm) than D. sylviae sp. nov., and develops "radially to irregularly set side [indeterminate] branches" (Sjøtun et al. 2016) and cover cells for tetrasporangia in stichidia. These two species are morphologically easy to diff erentiate even if their habitats weren't also disparate.