A new record of kelp Lessonia spicata (Suhr) Santelices in the Sub-Antarctic Channels: implications for the conservation of the “huiro negro” in the Chilean coast

The Katalalixar National Reserve (KNR) lies in an isolated marine protected area of Magellan Sub-Antarctic channels, which represent an important area for marine biodiversity and macroalgal conservation. The present study is the first report of the species Lessonia spicata, “huiro negro”, in the Magellan Sub-Antarctic channels. This finding has implications for macroalgal biogeography and conservation concerns in the Chilean coast. In the ecological assessments of the KNR in 2018 we found populations of L. spicata, specifically on rocky shores of Torpedo Island and Castillo Channel. The morphological identification and molecular phylogeny based on nuclear (ITS1) sequences revealed that these populations of Lessonia are within the lineage of L. spicata of central Chile. This report increases the species richness of kelps for the Magellan Sub-Antarctic Channels from two to three confirmed species (L. flavicans, L. searlesiana and L. spicata), and it also extends the southern distribution range of L. spicata. This species has high harvest demand and is moving towards southern Chile; thus, these populations should be considered as essential for macroalgal conservation in high latitudes of South America.


INTRODUCTION
Lessonia Bory (Laminariales, Phaeophyceae) is one of the most conspicuous brown macroalgal genera that inhabit the littoral to sublittoral zone of rocky coasts (~20 m depth) in temperate-cool waters of the South Pacific Ocean (Cho et al., 2006;Martin & Zuccarello, understand the taxonomic composition and biogeography of macroalgae that inhabit the southern boundary of the Humboldt Current (Camus, 2001;Thiel et al., 2007).
The present study contributes the first report of the species L. spicata in the Magellan Sub-Antarctic Channels. The distribution of this was thought to be limited to 41 S, but appears to be extended south of the Golfo de Penas (46 59′-47 40′S). Continuing survey studies will be necessary to understand the occurrence patterns of populations of L. spicata in the MBP.

MATERIALS AND METHODS
Three individuals of Lessonia spicata were collected in the intertidal zone of Torpedo Island and Castillo Channel (Figs. 1A and 1B) in July, 2018. The specimens were air-dried and then pressed on herbarium sheets for morphological observation and molecular analysis. The Chilean Hydrographic and Oceanographic Service of the Navy (N 13270/24/337) approved field sampling.
External and internal morphological observations were made. The anatomical observations were performed by sectioning with a razor and staining with 1% aqueous aniline blue acidified with 1% diluted HCl, and mounted in 70% glycerin. Photomicrographs were taken with a Canon Powershot S5 IS camera attached to a BX 51 Olympus microscope (Canon USA, Melville, NY, USA; Olympus Corp., Tokyo, Japan, respectively). A total of 15 replicates from the three individuals were selected for measurement of cortical cell diameter following González et al. (2012); means and standard deviations were calculated. Samples of other species occurring in the Sub-Antarctic region (L. flavicans and L. searlesiana) were also analyzed for comparative purposes. Voucher specimens were deposited in the herbarium of University of Magallanes, Punta Arenas, Chile.
The phylogenic analysis was constructed using maximum likelihood (ML) and Bayesian inference (BI) analyses. The program PartitionFinder (Lanfear et al., 2012) were used to choose the best-fitting nucleotide substitution model under the Bayesian Information Criterion. The general time-reversible nucleotide substitution model with a gamma distribution and a proportion of invariable sites (GTR + Γ + I) was selected as the best substitution model. Maximum likelihood analysis was performed with the RAxML HPC-AVX program (Stamatakis, 2014) implemented in the raxmlGUI 1.3.1 interface (Silvestro & Michalak, 2012) with the statistical support obtained by 1,000 bootstrap replications. Bayesian inference was performed with the MrBayes v. 3.2.5 software (Ronquist et al., 2012) using Metropolis-coupled Markov Chain Monte Carlo (MC 3 ). The inference of Bayesian posterior probability (BPP) was inferred following Calderon & Boo (2017).
The neighbor-joining analysis was performed in MEGA5 v.6.06 with the default settings software, using 1,000 bootstrap replicates.

RESULTS
This is the first confirmed report of L. spicata in the Sub-Antarctic region, extending its distribution to the south by seven degrees of latitude ( Fig. 2A). The sporophytes collected in the two localities have cylindrical stipes, flattened toward the beginning of the blades, with a regular, almost dichotomous long lanceolate blade with a spike (Figs. 2B-2E).

Internal anatomy
Our specimens showed several layers of cortical tissue with cells of smaller diameter compared to L. searlesiana (Figs. 3B, 3E, and 3H) and L. flavicans (Figs. 3C, 3F, and 3I), moreover no lacunas were observed in our samples, unlike L. flavicans (Figs. 3C and 3H). The medulla was composed of elongated medullary cells with filamentous elements (Fig. 3G). The internal anatomy was composed of a narrow cortex (Fig. 3A), with cortical cell diameter of 25.91 ± 2.90 for the individual 1, 28.22 ± 2.10 for individual 2 and 27.02 ± 2.27 for the individual 3 (Table 2).

Phylogenetic analysis
The ITS phylogeny placed our specimens within the lineage of L. spicata of central Chile (Fig. 4A). The phylogenetic trees constructed by ML and BI had the same topology except for the phylogenetic position of L. corrugata and L. variegata from northeastern South Is. The three specimens analyzed consistently formed a strongly supported clade with     positions (16.8%) were parsimoniously informative. Intraspecific divergence of L. spicata from three different populations ranged between 0.0% and 0.2% (0-3 bp). L. spicata differed by 0.8-1.0% from L. berteroana and by 1.1-1.3% from L. trabeculata. L. variegata is a non-monophyletic species complex of four different species.

DISCUSSION
We confirm here the presence of L. spicata both morphologically and genetically, whose individuals correspond to the central Chile lineage described by González et al. (2012).
Morphologically these features correspond to those described for L. spicata by von Suhr (1839) and González et al. (2012). These values also agree with those mentioned by González et al. (2012) for L. spicata. Genetically our phylogeny is consistent with those of previous studies that show Lessonia as a monophyletic lineage (Lane et al., 2006, Martin & Zuccarello, 2012. Lessonia species are a characteristic component of benthic ecosystems in this region (Searles, 1978;Martin & Zuccarello, 2012). We highlight two aspects about the importance of this report of L. spicata for this area: (a) we increase the knowledge of the species richness of kelps for the Sub-Antarctic Channels, and (b) this species has a strong extraction activity which we hypothesize that will move southward in the near future, therefore these populations should be properly preserved in order to prevent high risk of human impact.
The name L. spicata was proposed because it was the oldest name available to assign the lineage of central Chile, populations between 29 and 43 S. However, L. spicata would be a provisional name mainly because no representative specimens of L. nigrescens have been found near the type locality Cape Horn. Therefore, if the true L. nigrescens belongs to one of the lineages already described or to a new one, this name would have priority (González et al., 2012). In the MBP L. nigrescens has been recorded not only for Cape Horn; Searles (1978) reported a population in the Trinidad Channel (Puerto Alert 49 53.6 ′S), and two others in the Aysén region (Searles, 1978). Puerto Alert is 126 km south of Castillo Channel where we found the population of L. spicata. Therefore, it is likely that Searles' records (1978) correspond to populations of L. spicata. Finally, it is important to mention that, like González et al. (2012), in recent expeditions to the Diego Ramirez and Cape Horn archipelago-which are related to the characterization of the Diego Ramírez-Drake Passage Marine Park (Rozzi et al., 2017)-we have not found populations of L. nigrescens, only individuals of L. flavicans (Rozzi et al., 2017). Therefore, in the absence of biological material from the type locality the status of L. nigrescens is still in doubt, and the lineage of central Chile that now extends south of 43 S should continue to be named as L. spicata.
Several bio-geographical breaks have been described along the coast of Chile (Santelices & Meneses, 2000;Tellier et al., 2009;Fraser et al., 2010); one of the most relevant for many taxa is at 42 S (Brattström & Johanssen, 1983;Lancellotti & Vásquez, 1999;Valdovinos, Navarrete & Marquet, 2003). For macroalgae and particularly for kelp species such as Durvillaea antarctica, a marked divergence is present south of 43 S, where populations between 49 and 55 S are genetically different from the rest of the populations occurring in the Chilean coast (32 and 43 S) (Fraser et al., 2010). These authors suggested that although D. antarctica has a high dispersion capacity due to its buoyancy (rafting), it could only colonize free coasts, since it would have limited potential to increase gene flow between established populations. Therefore, it is interesting that although L. spicata has a low-dispersal capacity in comparison to D. antarctica (Oppliger et al., 2012), since it does not have the buoyancy capacity, there is a single genetic unit in the individuals collected in this study and individuals from the central zone of Chile. L. spicata must have some physiological adaptations which allowed it to colonize and inhabit areas of high latitudes. In this sense, this species has been described as a perennial seaweed and has not been found in the "bank of microscopic forms" in the Chilean central coast (boulders and water from tidal pools) (Santelices et al., 1995;Santelices, Aedo & Hoffmann, 2002). However, it has been observed that microscopic form of L. spicata can survive up to 90 days in total darkness and propagules can germinate in total absence of light (Santelices, Aedo & Hoffmann, 2002). This high capacity for tolerance to darkness could be a key strategy to colonize new areas with a significant seasonal changes in daylight hours and luminosity (Photosynthetically Active Radiation) during the winter period (Ojeda et al., 2019). Nevertheless, future studies and a greater number of samples along the Chilean coast (mainly the area between 41 and 48 S) will help to elucidate its biogeographic history and how much structure and connectivity the populations of L. spicata present throughout their distribution (29-48 S).
The harvesting pressure on the genus Lessonia has increased alarmingly along the Chilean coast, so we should take a precautionary approach to potential harvesting of L. spicata in its austral distribution range. L. berteroana (sister species of L. spicata) is currently the most exploited seaweed in South America; the main landings are in northern Chile (Westermeier et al., 2019). Lessonia is socially important in this region because many artisanal fishers depend directly or indirectly on its harvest (Vega, Broitman & Vásquez, 2014). However, high demand, lack of oversight and harvest methods have created a concerning scenario for kelp forests (Vega, Broitman & Vásquez, 2014;Westermeier et al., 2019). The extraction of L. spicata in southern Chile began in 2012, and its extractive pressure has been moving southward, mainly between 33 and 41 S (SERNAPESCA, 2019). In the Chilean Los Lagos Region (41 S), between 2014 and 2017 landing increased from 494 to 747 dry tons of L. spicata (SERNAPESCA, 2019). This gradual increase should draw attention to kelp forest conservation, since there is evidence on sustainability problems that Lessonia populations have experimented and their biodiversity in northern Chile (Vega, Asorey & Piaget, 2016). This concern acquires significant relevance if we consider that the Magellan Sub-Antarctic Channels are the austral distribution range of L. spicata, where kelp forest populations are important for sustainability of small-scale fisheries (e.g., king crab; Cárdenas, Cañete & Mansilla, 2007), indigenous traditions (Ojeda et al., 2018) and terrestrial and marine biodiversity (Darwin, 1839;Rosenfeld et al., 2014).

CONCLUSION
Despite the geographical distance and the presence of important biogeographic breaks (41 and 46 S), our results confirm that the individuals collected in the coastal zone of the Katalalixar Reserve are the species L. spicata. The strong morphological and genetic evidence are indicating that the individuals analyzed are associated with the lineage of central Chile, and the populations of L. spicata would inhabit the area exposed to the Pacific.
With diverse industrial uses, including providing phycocolloids in the form of alginate L. spicata is a potentially important economic resource in the Chilean coast. However, with extractive pressure moving to the south, caution is needed given that this kelp serves not only as a habitat for many animals but also as a spawning ground for some benthic (e.g., gastropods) species.
Fabio Mendez conceived and designed the experiments, contributed reagents/materials/ analysis tools, approved the final draft, help in the expedition. Martha S. Calderon conceived and designed the experiments, performed the experiments, analyzed the data, contributed reagents/materials/analysis tools, authored or reviewed drafts of the paper, approved the final draft. Francisco Bahamonde conceived and designed the experiments, performed the experiments, contributed reagents/materials/analysis tools, prepared figures and/or tables, approved the final draft. Juan Pablo Rodríguez conceived and designed the experiments, contributed reagents/ materials/analysis tools, prepared figures and/or tables, approved the final draft. Jaime Ojeda conceived and designed the experiments, contributed reagents/materials/ analysis tools, prepared figures and/or tables, authored or reviewed drafts of the paper, approved the final draft. Johanna Marambio conceived and designed the experiments, contributed reagents/ materials/analysis tools, prepared figures and/or tables, authored or reviewed drafts of the paper, approved the final draft. Matthias Gorny conceived and designed the experiments, contributed reagents/ materials/analysis tools, approved the final draft, help in the expedition. Andrés Mansilla conceived and designed the experiments, contributed reagents/ materials/analysis tools, approved the final draft, he contributed with the funding.

Field Study Permissions
The following information was supplied relating to field study approvals (i.e., approving body and any reference numbers): The field samplings were approved by the Chilean Hydrographic and Oceanographic Service of the Navy (SHOA). This species is not protected by the Chilean Fishery Subsecretary and has not been included in the Chilean fishery statistics in the Aysen Region. The permission to undertake field studies and to collect specimens was issued by the Chilean Hydrographic and Oceanographic Service of the Navy Sub-Director (Felipe Barrios Burnett), under the technical memorandum N 13270/24/337.

DNA Deposition
The following information was supplied regarding the deposition of DNA sequences: The Lessonia spicata ITS sequences are accessible via GenBank: LesA MN061669, LesB MN061670, LesC MN061671.

Data Availability
The following information was supplied regarding data availability: The detailed information on the new records of Lessonia spicata are available in File S1.

Supplemental Information
Supplemental information for this article can be found online at http://dx.doi.org/10.7717/ peerj.7610#supplemental-information.