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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, 2012). There are currently records of 11 species of the genus Lessonia that are taxonomically accepted, distributed along the coasts of South America, New Zealand, Tasmania and Sub-Antarctic islands (Cho et al., 2006). These species have major ecological roles in the structure of benthic marine communities (Villouta & Santelices, 1984; Vásquez & Santelices, 1984), and are commercially exploited for the extraction of alginic acid (Steneck et al., 2002). Lessonia species are one of the most characteristic and abundant macroalgae (12–56°S) that inhabit the rocky shores of the Chilean coast (17–56°S) (Searles, 1978; Ávila, Hoffmann & Santelices, 1985; Villouta & Santelices, 1986; Vásquez, Camus & Ojeda, 1998; Tellier et al., 2011; Martin & Zuccarello, 2012; Mansilla et al., 2014). Currently, six species have been recorded in Chile: Lessonia nigrescens Bory, L. berteroana Montagne, L. spicata Suhr, L. trabeculata Villouta & Santelices, L. searlesiana Asensi & De Reviers and L. flavicans Bory (Guiry & Guiry, 2019). A recent morphological and molecular analysis showed that the species distributed from Peru (17°) to Puerto Montt (41°), commonly known as L. nigrescens, is actually two cryptic species; the populations distributed from Peru (17°S) to central Chile (30°S) correspond to L. berteroana Montagne, and those occurring from central Chile (29°S) to Puerto Montt (41°S) correspond to L. spicata (Suhr) Santelices (González et al., 2012; Vega, 2016). However, L. nigrescens is still a valid species, because no material of the referred species has been found near its type locality, Cape Horn.

The huiro negro kelps, which include L. berteroana and L. spicata, are heavily exploited and represent almost 70% of the kelp biomass landed annually (Vega, Broitman & Vásquez, 2014). This economic activity is mainly practiced in northern Chile (18–32°S), through a complex productive chain with high social impact and low added value (Vásquez, 2008). L. berteroana and L. spicata are exported as a natural commodity to more than 20 countries mainly due to their alginate, which has high economic value (Westermeier et al., 2019). Thus populations of huiro negro have economic interest along Chilean coasts, being essential to generate a stewardship from a local and large scale.

The Katalalixar National Reserve (KNR) is a national reserve area created in 1983. KNR comprises 674,500 ha and is located in a remote zone next to the village of Tortel. This area includes a wilderness temperate rainforest with a complex ecosystem of islands and fjords (Bell, Pedersen & Newton, 2007). The offshore area (western side) of the Magellan Sub-Antarctic Channels is one of the few places of the Magellan Biogeographic Province (MBP) (43–56°S; Camus, 2001) that has not been explored systematically by scientific expeditions (Gorny & Zapata-Hernández, 2018) (Figs. 1A and 1B). KNR is located at the southern limit of the Humboldt Current System (HCS). The HCS is a key component of the general oceanic circulation in the eastern South Pacific, being one of the most productive marine ecosystems on the earth (Thiel et al., 2007). The Humboldt Current System originated in southern Chile between 42 and 48°S and is characterized by a northward flow in front of South American coasts with a strong upwelling of cool nutrient-rich waters (Silva, Rojas & Fedele, 2009). The origin of the HCS induced a large-scale redistribution of biota, and nowadays plays a key role in the biogeography of the South Pacific (Camus, 2001). Thus KNR provides an enormous opportunity to understand the taxonomic composition and biogeography of macroalgae that inhabit the southern boundary of the Humboldt Current (Camus, 2001; Thiel et al., 2007).

Collection points of Lessonia spicata.

Figure 1: Collection points of Lessonia spicata.

(A) Map showing the location of Katalalixar National Reserve (KNR) in central Patagonia, (B) Collection sites of Lessonia spicata, Torpedo Island (red circle) and Castillo Channel (blue circle) in the oceanic margin of the Campana Archipielago (KNR).

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.

Molecular analyses

Genomic DNA was extracted from ~5 mg of dried thallus ground in liquid nitrogen using a NucleoSpin Plant II Kit (Macherey-Nagel, Düren, Germany) according to the manufacturer’s protocol. The PCR primers for the ITS were ITSP1-ITSRi (Tai, Lindstrom & Saunders, 2001; Martin & Zuccarello, 2012) and KP5- KG4 (Lane et al., 2006). Polymerase chain reaction products were purified using a NucleoSpin Gel and PCR Clean-up (Macherey-Nagel, Düren, Germany) and commercially sequenced (Macrogen, Seoul, South Korea). The electropherograms were edited using the Chromas v1.45 software (McCarthy, 1998) and the new generated sequences were deposited in GenBank (www.ncbi.nlm.nih.gov/genbank/).

A total of 34 ITS sequences (731 bp) were included in the construction of the phylogeny: 31 sequences belonging to the genus Lessonia and three outgroups, Cymathaere triplicata (Postels & Ruprecht) J. Agardh, Ecklonia cava Kjellman and Macrocystis pyrifera (Linnaeus) C. Agardh (Table 1). Sequences were aligned using the MUSCLE algorithm in MEGA5 v.6.06 software using the default settings (Tamura et al., 2013).

Table 1:
List of species used in DNA analyses, information on collections and accession numbers in GenBank (sequences generated in the present study are shown in bold).
Species Collection site Voucher code ITS
LesA Torpedo island, Aysen, Chile MN061669
LesB Channel Castillo, Aysen Chile MN061670
LesC Channel Castillo, Aysen, Chile MN061671
Lessonia adamsiae South Promontory, The Snares, New Zealand A626 GU5938021
Lessonia adamsiae Tahi, The Snares, New Zealand A614 GU5937991
Lessonia berteroana (as L. nigrescens northern lineage) San Marcos, Tarapaca, Chile B858 GU5937811
Lessonia berteroana (as L. nigrescens northern lineage) San Marcos, Tarapaca, Chile B859 GU5937821
Lessonia brevifolia Smoothwater Bay, Campbell Is., New Zealand A548 GU5938031
Lessonia brevifolia Antipodes, New Zealand A973 GU5938041
Lessonia brevifolia Perseverance Harbour, Campbell Is., New Zealand B296 GU5938051
Lessonia corrugata Gov. Is. Reserve, Tasmania, Australia AY8579022
Lessonia corrugata Bicheno, Tasmania, Australia A985 GU5937941
Lessonia corrugata Skeleton Pt., Tasmania, Australia C057 GU5937951
Lessonia flavicans Rookery Bay, East Falkland, Falkland Islands A634 GU5937861
Lessonia flavicans (as Lessonia vadosa) Punta Arenas, Patagonia, Chile B985 GU5937891
Lessonia spicata (as L. nigrescens IA lineage) La Pampilla, Coquimbo, Chile A581 GU5937751
Lessonia spicata (as L. nigrescens IA lineage) Bahia Mansa, Osorno, Chile B719 GU5937801
Lessonia tholiformis Wharf reef, Owenga, Chatham Is, New Zealand A518 GU5937971
Lessonia tholiformis Wharekauri, Chatham Is, New Zealand A532 GU5937981
Lessonia trabeculata Punihuil, Chiloe Is, Chile B715 GU5937831
Lessonia trabeculata Punihuil, Chiloe Is, Chile B716 GU5937841
Lessonia variegata (as L. variegata lineage N) North Cape, Northland, New Zealand A557 GU5938081
Lessonia variegata (as L. variegata lineage N) Maitai Bay, Northland, New Zealand B129 GU5938091
Lessonia variegata (as L. variegata lineage N) The Sailors Grave, Coromandel, New Zealand B312 GU5938101
Lessonia variegata (as L. variegata lineage K) South Bay, Kaikoura, New Zealand A138 GU5938171
Lessonia variegata (as L. variegata lineage K) New Wharf, Kaikoura, New Zealand A606 GU5938181
Lessonia variegata (as L. variegata lineage S) Curio Bay, Catlins, New Zealand A434 GU5938201
Lessonia variegata (as L. variegata lineage S) Causet Cove, Doubtful Sound, New Zealand C154 GU5938211
Lessonia variegata (as L. variegata lineage W) Princess Bay, Wellington, New Zealand A001 GU5938111
Lessonia variegata (as L. variegata lineage W) Cape Palliser, Wairarapa, New Zealand A613 GU5938151
Lessonia variegata (as L. variegata lineage W) Riversdale Beach, Wairarapa, New Zealand A025 GU5938161
Cymathaere triplicata Whiffen Spit, Sooke, BC, Canada AY8578842
Macrocystis pyrifera California, USA AF3190373
Ecklonia cava GU5937731
DOI: 10.7717/peerj.7610/table-1

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 (MC3). 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. 2B2E).

Distribution of Lessonia spicata (interspersed bars), showing its previously known distribution (solid bars).

Figure 2: Distribution of Lessonia spicata (interspersed bars), showing its previously known distribution (solid bars).

(A) Habitat of specimen collected in both sites (Torpedo Island and Castillo Channel). We included the Chilean biogeographical classification of Camus (2001). (B) Habitat of specimen collected in Torpedo Island (LMS000001). (C) Discoid holdfasts of specimen collected in Torpedo Island (LMS000001), (D) Blades of specimen collected in Torpedo Island (LMS000001), (E) Habitat of specimen collected in Castillo Channel (LMS000002, LMS000003).

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).

Cross section of the medial part of mature fronds of Lessonia species who inhabit the Sub-Antarctic channels.

Figure 3: Cross section of the medial part of mature fronds of Lessonia species who inhabit the Sub-Antarctic channels.

Cross section of the medial part of mature fronds of Lessonia spicata collected in the Katalalixar Reserve (A, D and G), of L. searlesiana from Fuerte Bulnes (B, E and H) and L. flavicans from Horn Island (C, F and I); mer = meristoderm, co = cortex and me = medulla, l = lacuna.
Table 2:
Morphological measurements (mean ± SE) of individuals collected in Torpedo Island and Castillo Channel.
External measurements Internal measurements
TL DD NS DC
Individual Site
1 Torpedo Island 68 10 9 25.91 ± 2.90 This study
2 Castillo Channel 166 21 6 28.22 ± 2.10 This study
3 Castillo Channel 55 5 13 27.02 ± 2.27 This study
Average 96.33 ± 60.68 12 ± 8.19 9.33 ± 3.51 27.05 ± 1.15 This study
L. spicata
Maintencillo 150 ± 13.3 25.7 ± 1.4 (González et al., 2012)
Matanzas 160 ± 5.0 27 ± 1.6 (González et al., 2012)
Calfuco 120 ± 7.2 30 ± 1.8 (González et al., 2012)
DOI: 10.7717/peerj.7610/table-2

Note:

External morphological data: TL, thallus length (cm); DD, disc diameter; NS, number of stipes. Internal morphological data: DC, diameter of cortical cells.

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 sequences of L. spicata (97% for ML and 0.96 for BPP) collected in Chile; having to L. berteroana and L. trabeculata as sister taxa. The cladogram was consistent with the phylogenetic tree (Fig. 4B). Variable sites occurred at 201 positions (27.5%), and 123 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.

Phylogenetic tree of ITS sequences obtained by maximum likelihood (ML) inference.

Figure 4: Phylogenetic tree of ITS sequences obtained by maximum likelihood (ML) inference.

(A) Phylogenetic tree of ITS sequences obtained by maximum likelihood (ML) inference. ML bootstrap values (≥50%) and Bayesian posterior probabilities (≥0.90) are indicated next to branches. (B) Cladograms of ITS sequences obtained by the neighbor joining (NJ) method. Bootstrap values (≥50%) are indicated next to branches. The sequence for taxa in bold was generated in this study.

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.

Supplemental Information

Biodiversity dataset and related metadata from this study.

DOI: 10.7717/peerj.7610/supp-1

ITS sequences of the individual of Lessonia spicata used in the phylogenetic analysis.

DOI: 10.7717/peerj.7610/supp-2