Dictyota falklandica sp. nov. (Dictyotales, Phaeophyceae) from the Falkland Islands and southernmost South America

ABSTRACT Surveys of the seaweed flora of the Falkland Islands and of Tierra del Fuego revealed the presence of a new species of brown alga. Dictyota falklandica sp. nov. inhabits the shallow rocky infralittoral in sheltered localities and the lower intertidal in more exposed sites. Dictyota falklandica has a regular to irregular habit of dichotomously branched blades, forming erect thalli composed of a single-layered cortex and medulla, with margins in the apical parts dotted with dormant apical cells. Sporangia occur in irregular groups or longitudinal lines on the thallus surface. Molecular phylogenies based on chloroplast psbA and rbcL and mitochondrial cox1 sequences showed that the species from the Falkland Islands is sister to a clade formed by D. korowai, recently described from New Zealand and D. kunthii known from both the Pacific coast of South America and New Zealand. Temperature tolerance experiments, showing mortality at 25 °C but survival at 20 °C, confirm the cold-temperate affinity of this taxon. Its relationship to other cold-temperate Southern Hemisphere species is discussed, with its closest relatives living in regions with sea surface temperatures of at least 7–10 °C higher.


INTRODUCTION
Dictyota species are common members of the benthic communities in warm-temperate and tropical seas, and the genus is widely considered to have warm-water affinities (Tronholm et al. 2010). Among the 97 Dictyota species currently recognized, 87 are described from warm-temperate and tropical seas (Guiry & Guiry 2019). Their effective chemical defense against grazing based on diterpenes (Hay et al. 1987;Wiesemeier et al. 2007) and high constitutive hydrogen peroxide release, likely for the control of bacterial biofilms (Küpper et al. 2002), as well as their ability to propagate through fragmentation (Herren et al. 2006), enables them to form sizeable populations even in localities under high grazing pressure such as Ascension Island (Tsiamis et al. 2017). Significantly, defense against amphipods is inducible in Chilean Dictyota kunthii (C.Agardh) Greville (Macaya & Thiel 2008), which may be a more widespread feature in this genus.
In contrast to the ubiquity of Dictyota in warm seas, few reports exist of Dictyota species in cold-temperate seas of the southern Hemisphere (Fig. S1). Asensi & Küpper (2012) reported a Dictyota sp. from Puerto Deseado (Patagonia, Argentina), where it was present year-round, growing on calcareous algae. There is a high chance that this report corresponds to the entity from Argentina identified as D. dichotoma (Hudson) J. V.Lamouroux by Lopes-Filho et al. (2017) using DNA sequence information. Dictyota dichotoma is primarily known from Europe, but is also present in South Africa and the temperate Atlantic coast of Brazil and Argentina (Tronholm et al. 2010;O. De Clerck, pers. obs.). Dictyota kunthii occurs in cold to warm-temperate regions along the South American Pacific coast from Peru to Chile, the Juan Fernandez Islands (Ramírez & Osorio 2000;Rodríguez-Ruiz et al. 2017), and in New Zealand (Hoffmann & Santelices 1997;Nelson 2013;Ramírez & Santelices 1991). The most southern Dictyota described so far is D. decumbens (Ricker) Hörnig, Schnetter et Prud'homme van Reine from Macquarie Island (54°S, Ricker 1987). The species was originally described as Dilophus decumbens Ricker, because the medulla comprised 2-3, irregularly organized layers of cells near the margins. Dilophus decumbens was subsequently transferred to Dictyota (Hörnig et al. 1992b(Hörnig et al. , 1992a. Recent work (Mystikou et al. 2016) reported a hitherto undescribed Dictyota sp. from the eastern tip of Tierra del Fuego (Argentina) and from two localities in the Falkland Islands. In a psbA phylogeny, this unknown taxon was sister to D. binghamiae J.Agardh from Mexico and California (Mystikou et al. 2016). However, more research was needed before it could be formally described as a separate species, especially its relationships with Southern Hemisphere taxa (Chile, Juan Fernandez) such as D. phlyctaenodes Montagne and D. kunthii, which was the rationale for the present study. In this paper, we report on our recent observations and collections of Dictyota from the Falkland Islands, including a formal description as a new species, underpinned by temperature tolerance experiments with cultured isolates.

MATERIALS & METHODS
Specimens of Dictyota were collected in the shallow subtidal zone at two localities in East Falkland ( Fig. 1 Total genomic DNA was extracted from silica gel-dried samples using a modified CTAB-extraction method (Steen et al. 2017). Sequences were generated for the plastidencoded PSII reaction centre protein D1 (psbA) and the mitochondrial-encoded cytochrome oxidase subunit 1 (cox1) gene. PCR primers and conditions are described in Tronholm et al. (2010). Sequences were aligned by eye using MEGA 7 (Kumar et al. 2016) and added to a reference species-level alignment of Dictyota and related genera (Canistrocarpus, Dictyotopsis, Dilophus, Scoresbyella and Rugulopteryx). The matrix, a multi-locus alignment of mitochondrial cox1 and cox3 genes, and chloroplast psaA, psbA and rbcL genes, consisted of 64 taxa and 5446 positions. The matrix was 81% filled at the gene level (Table S1).
A maximum-likelihood (ML) tree was generated from the concatenated alignment, partitioned by organelle and codon position. The partition scheme and substitution models were estimated using PartitionFinder 2 (Lanfear et al. 2017). ML analyses were conducted using RAxML v8.1 under a GTR + GAMMA model (Stamatakis 2014). The robustness of the resulting phylogenies was tested using 1000 replicates of a rapid bootstrap heuristic (Stamatakis 2006). In addition, a Bayesian tree was estimated using MrBayes 3.2. (Ronquist et al. 2012), applying a GTR +GAMMA model applied to every partition. Two runs, consisting of four chains each, were run for 10 million generations. Stationarity and convergence of runs was assessed visually using  For all specimens, we collected mean sea surface temperature (SST mean) based on locality information and environmental layers present in Bio-Oracle v2.0 (Assis et al. 2018). Averaged mean surface temperatures correlate equally well or better with marine species distribution ranges compared to average minimum or maximum temperatures as demonstrated by Bosch et al. (2018). Ancestral states of SST affinities were reconstructed and visualised on the phylogeny using the fastAnc and contMap functions of the R package phytools (Revell 2012).
Three unialgal isolates (coded FI 17-182 and FI 17-186 from San Carlos, and FI 17-203 from North Arm) were obtained from thallus apices. The culture medium was autoclaved, half-strength Provasoli-enriched sea water (Coelho et al. 2012), which for the first weeks contained 4 mg l −1 GeO 2 to inhibit diatom growth. During isolation, the algae received natural daylight near a north-facing window, and the temperature ranged between 12-14 ºC.
In a laboratory-based experiment, the gross temperature tolerance was determined with cultures pre-cultivated at 15°C. On day 1, a dish (10 ml) of each of the three isolates  was placed at 15°C, 20°C and 25°C under white light of 30 μmol m −1 sec −1 . On day 11, the medium was changed in all cultures, and those which had been incubated at 20 and 25°C were transferred to 15°C (where all cultures were kept). On days 24 and 38, the medium was changed in all dishes, and all cultures were continued at 15°C until final examination two months after start of the experiment.

In situ observations
At both San Carlos and North Arm, Dictyota falklandica grew on rocky substrata at 1-3 m beneath low tide level, typically under a canopy of Macrocystis pyrifera (Linnaeus) C.Agardh (Figs. 2-5). Synchronous with our subtidal collections on the Falkland Islands we examined the rocky intertidal zone close to the two sites. We did not find the species on the rocks or in shallow tide pools.

Phylogeny
The maximum-likelihood and Bayesian phylogenies (Fig. 6) were in good agreement, differing only in parts of the tree that received low support. Dictyota falklandica sp. nov. was resolved sister to a clade formed by D. korowai, a species recently described from northern New Zealand (Nelson et al. 2019) and D. kunthii from the Pacific coast of South America and New Zealand. Another closely related species is D. binghamiae from the northeastern  Taken together, this suggests that this is a species new to science, which is formally described below:  MK516759 -MK516759 (cox1) and MK516799-MK516800 (psbA), and MK516815 (rbcL). ISOTYPE CULTURE: A unialgal culture isolated on 31 January 2017 from the type material collected at San Carlos was deposited in the Culture Collection of Algae and Protozoa (no. CCAP 1335/1).

Vegetative and reproductive morphology
Dictyota falklandica grew upright, but lacked a conspicuous base. The species was attached by patches of rhizoids, present near basal parts of thalli (Fig. 8). Apices were obtuse, with protruding lens-shaped apical cells. Irregular branching in the apical parts of thalli might be related to regained meristematic activity of additional apical cells which dot the margins of the apical parts of the thallus (Figs. 7, 9). Dichotomies were evenly spaced every 13-15 mm. The branching angle was approximately 45-50°. Surface and margins were smooth, and lacked teeth or proliferations (Fig. 11).
Male and female gametophytes were not observed. Immature sporangia occurred in irregular groups or longitudinal lines on the thallus surface (Fig. 12). Sporangia were not surrounded by an involucrum and were borne on a single stalk cell. Thalli had tufts of hairs randomly scattered on both surfaces (Fig. 13).

Temperature tolerance
Two months after the start of the experiment, cultures of the three isolates exposed to 25°C for 11 days showed clear signs of stresstwo were dead, and one culture was mostly bleached with only small tissue parts remaining pigmented. In contrast, the cultures at 20 or 15°C for the same duration had healthy (browndark brown) pigmentation and growth.

DISCUSSION
The order Dictyotales is frequent in temperate to tropical seas, and particularly diverse in Australia and New Zealand (Guiry & Guiry 2019;Nelson et al. 2019;Phillips 2001). However, the nearby subantarctic region is largely devoid of this order, and it is missing in Antarctica (Papenfuss 1964;Wiencke & Clayton 2002;Wiencke et al. 2014). This study highlights that the overall diverse seaweed flora of the Falkland Islands still has significant potential for new discoveries, warranting further surveys and taxonomic studies (Küpper & Kamenos 2018). Dictyota falklandica sp. nov. from the Falkland Islands and southernmost South America is one of the few species able to grow in cold-temperate waters, and it appears to be geographically confined to this cold-temperate region. Its closest relatives, D. korowai, D. binghamiae and D. kunthii, live in regions with sea surface temperatures at least 7-10 ºC higher. In the Falkland Islands, surface water temperatures oscillate between 5 and 9°C (Arkhipkin et al. 2013), similar to temperatures present in eastern Tierra del Fuego (Rivas & Pisoni 2010). One of the collecting sites, North Arm, lies in the SE part of the Falkland Islands, which is strongly influenced by cold subantarctic waters. In high-temperature tolerance experiments, our cultures of D. falklandica survived 20°C but not 25 ºC for an 11-day duration. It resembles other subantarctic species from South America, which have upper survival temperatures between 19.9 and 24.5 but not 25°C (Peters & Breeman 1993). Such species, albeit adapted to the cold-temperate subantarctic environment, have a large 'safety margin' which may allow occurrence in milder habitats such as shallow pools or the intertidal. At Bahía Thetis (Tierra del Fuego; Mystikou et al. 2016), D. falklandica had been collected in the mid-intertidal zone of a more exposed site, as judged from the presence of Durvillaea antarctica (Chamisso) Hariot. Mystikou et al. (2016) concluded that D. falklandica sp. nov. (as Dictyota sp.) is morphologically and genetically different from other Dictyota species. In the present study, Dictyota falklandica was resolved as sister to D. kunthii from the Pacific coast of South America, the Juan Fernandez Islands, New Zealand and Australia (Guiry & Guiry 2019) and D. korowai from northern New Zealand (Nelson et al. 2019). However, the phylogenetic relationship of D. falklandica to D. decumbens endemic to subantarctic Macquarie Island is unknown, as no sequences are available of D. decumbens. The hydrographic conditions at Macquarie Island just north of the Antarctic Convergence resemble those at the Falkland Islands, with even slightly lower sea surface temperatures of 3.0-7.3°C (Ricker 1987). However, although one can hypothesize that D. falklandica sp. nov. may be Figs 7-14. Herbarium specimens and micrographs of Dictyota falklandica sp. nov. Fig. 7. Isotype (PC accession no. 0776066). Scale bar = 5 cm. Fig. 8. Surface view, with walls of larger medullar cells indicated by black lines. Scale bar = 50 µm. Fig. 9. Holotype specimen (BM accession no. BM013828094). Scale bar = 5 cm. Fig. 10. Apical region of branching thallus, with actively growing apical cells (asterisks) as well as marginal dormant apical cells (arrowheads). Scale bar = 1 mm. Fig. 11. Spores on thallus surface in irregular longitudinal groups. Scale bar = 50 µm. Fig. 12. Cross section of thallus with two hair tufts. Scale bar = 100 µm. Fig. 13. Cross section of central blade area. Scale bar = 100 µm. Fig. 14. Cross section of blade margin. Scale bar = 100 µm. related to D. decumbens, the geographic distance and morphological differences (the latter was initially described as Dilophus decumbens due to a double-layered medulla; Ricker 1987) suggests they are not conspecific. A number of recent studies on macroalgae with a circum-Antarctic distribution show that only large species or taxa that are good drifters [Macrocystis pyrifera, Durvillaea antarctica, Adenocystis utricularis (Bory) Skottsberg] and possibly their symbionts and pathogens such as Maullinia braseltonii P.Murúa, P.Goecke & S.Neuhauser (Blake et al. 2017;Murua et al. 2017), Herpodiscus durvilleae (Lindauer) G.R.South  and Laminariocolax aecidioides (Rosenvinge) Peters (Bernard et al. 2018) are genetically similar in the different land masses and islands, whereas less good drifters and smaller algae [e.g. Lessonia spp., the cryptic species included in Bostrychia intricata (Bory) Montagne] rather form different species in the distant localities Martin & Zuccarello 2012;Muangmai et al. 2014). Like D. decumbens, D. falklandica sp. nov. was found in the subtidal zone. However, D. decumbens has a prostrate habit. It forms several cm thick mats on boulders or other macroalgae (Ricker 1987), whereas the alga described here was erect and always epilithic. Available herbarium specimens of D. decumbens had been fixed in formalin before mounting (Ricker 1987) and are therefore unsuitable for the extraction of DNA. We prefer to describe the Falkland Dictyota as a separate species based on morphological differences and geographic separation.