Molecular Phylogenetics and Evolution 94 (2016) 814–826
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Molecular Phylogenetics and Evolution
journal homepage: www.elsevier.com/locate/ympev
The bladed Bangiales (Rhodophyta) of the South Eastern Pacific: Molecular species delimitation reveals extensive diversity q
Marie-Laure Guillemin a,b, Loretto Contreras-Porcia c,d, María Eliana Ramírez c,e, Erasmo C. Macaya f,g, ⇑ Cristian Bulboa Contador c, Helen Woods h,i, Christopher Wyatt j,k, Juliet Brodie h, a Instituto de Ciencias Ambientales y Evolutivas, Facultad de Ciencias, Universidad Austral de Chile, Casilla 567, Valdivia, Chile b UMI 3614 Evolutionary Biology and Ecology of Algae, CNRS, Sorbonne Universite´s UPMC Univ. Paris 06, Pontificia Universidad Cato´lica de Chile, Universidad Austral de Chile c Departamento de Ecología y Biodiversidad, Facultad de Ecología y Recursos Naturales, Universidad Andres Bello, República 440, Santiago, Chile d Center of Applied Ecology & Sustainability (CAPES), Pontificia Universidad Católica de Chile, Santiago, Chile e Museo Nacional de Historia Natural, Área Botánica, Casilla 787, Santiago, Chile f Departamento de Oceanografía, Facultad de Ciencias Naturales y Oceanográficas, Universidad de Concepción, Casilla 160-C, Concepción, Chile g Millennium Nucleus Ecology and Sustainable Management of Oceanic Island (ESMOI), Coquimbo, Chile h Natural History Museum, Department of Life Sciences, Cromwell Road, London SW7 5BD, UK i Centre for Ecology and Hydrology, Edinburgh, Bush Estate, Penicuik, Midlothian, Scotland EH26 0QB, UK j EMBL/CRG Research Unit in Systems Biology, Centre for Genomic Regulation (CRG), Barcelona 08003, Spain k Universitat Pompeu Fabra (UPF), Barcelona 08003, Spain article info abstract
Article history: A molecular taxonomic study of the bladed Bangiales of the South Eastern Pacific (coast of Chile) was Received 4 June 2015 undertaken based on sequence data of the mitochondrial COI and chloroplast rbcL for 193 specimens Revised 22 September 2015 collected from Arica (18°S) in the north to South Patagonia (53°S) in the south. The results revealed for Accepted 30 September 2015 the first time that four genera, Porphyra, Pyropia, Fuscifolium and Wildemania were present in the region. Available online 17 October 2015 Species delimitation was determined based on a combination of a General Mixed Yule Coalescence model (GMYC) and Automatic Barcode Gap Discovery (ABGD) coupled with detection of monophyly in tree Keywords: reconstruction. The overall incongruence between the species delimitation methods within each gene Automatic Barcode Gap Discovery (ABGD) was 29%. The GMYC method led to over-splitting groups, whereas the ABGD method had a tendency Chile Mitochondrial COI to lump groups. Taking a conservative approach to the number of putative species, at least 18 were rec- General Mixed Yule Coalescence (GMYC) ognized and, with the exception of the recently described Pyropia orbicularis, all were new to the Chilean Morphological plasticity flora. Porphyra and Pyropia were the most diverse genera with eight ‘species’ each, whereas only a ‘single’ Plastid rbcL species each was found for Fuscifolium and Wildemania. There was also evidence of recently diverging groups: Wildemania sp. was distinct but very closely related to W. amplissima from the Northern Hemisphere and raises questions in relation to such disjunct distributions. Pyropia orbicularis was very closely related to two other species, making species delimitation very difficult but provides evidence of an incipient speciation. The difference between the ‘species’ discovered and those previously reported for the region is discussed in relation to the difficulty of distinguishing species based on morphological identification. Ó 2015 Elsevier Inc. All rights reserved.
1. Introduction due to the simple and variable morphology of specimens, and all bladed species were assigned to the genus Porphyra until The bladed Bangiales are a diverse, cosmopolitan order of mor- Sutherland et al. (2011), on the basis of a two-gene phylogeny, split phologically simple red algae, and an economically important the genus into eight genera: Boreophyllum, Clymene, Fuscifolium, resource used in the production of ‘‘nori”. Identification and taxo- Lysithea, Miuraea, Porphyra, Pyropia and Wildemania. A ninth nomic placement within the order have been highly problematic bladed genus, Neothemis, has been added recently, based on a study of the bladed Bangiales in the western Mediterranean (Sánchez et al., 2014, 2015). As a consequence of the generic revi- q This paper was edited by the Associate Editor Adrian Reyes-Prieto. sion, we are now in a position to evaluate/re-evaluate local and ⇑ Corresponding author. regional Bangiales floras. Although the difficulty of distinguishing E-mail address: [email protected] (J. Brodie). http://dx.doi.org/10.1016/j.ympev.2015.09.027 1055-7903/Ó 2015 Elsevier Inc. All rights reserved. M.-L. Guillemin et al. / Molecular Phylogenetics and Evolution 94 (2016) 814–826 815 species remains, generic circumscriptions enable better placement characterized by a diffusive overlap of biota of the two major pro- at the generic level which in turn enables us to focus on species vinces (Camus, 2001). Depending on the region, the coastline is present at the regional and local level (e.g. Mols-Mortensen et al., affected by many different factors. For example, northern and 2012; Vergés et al., 2013). central Chile is subject to abnormally cold, but highly productive The number of species in the Bangiales has been reported to be waters due to up-welling subsurface waters. This highly heteroge- well over 150 (Brodie et al., 2008a). However, where in Sutherland neous coastline also varies in terms of its tidal range, temperature, et al. (2011) approximately 58 out of 126 bladed samples included salinity and circulation patterns (Fernández et al., 2000; in the analysis were undescribed or with uncertain identity, and López-Cristoffanini et al., 2013). On the other hand, the shoreline the ongoing discovery of species (e.g. Mols-Mortensen et al., south of 42°S, consists of a dense array of islands, channels and 2012; López-Vivas et al., 2014; Ramírez et al., 2014; Sánchez fjords, strongly influenced by glacial rivers and sub-Antarctic et al., 2014), the number is anticipated to be considerably higher. oceanographic and climatic conditions. Thus, those ecological As for some other algal groups, characterized by simple morpholo- particularities make the Chilean coast a particularly interesting gies and intraspecific variability of morphological features, e.g. the area to study the macroalgal biodiversity of the rocky intertidal. red algal genus Portieria (Payo et al., 2013), the green algal genus Until recently, only six species of bladed Bangiales were Boodlea (Leliaert et al., 2009) and the brown algal genus Lobophora reported from the coast of Chile (Ramírez and Santelices, 1991): (Vieira et al., 2014), species discrimination based on morphological P. capensis Kützing, P. columbina Montagne, P. miniata (C. Agardh) analysis alone in bladed Bangiales has proven to be mostly unreli- C. Agardh f. cuneiformis Setchell & Hus, P. thurettii Dawson, able (Brodie et al., 2008a; Broom et al., 2010; Kucera and Saunders, P. umbilicalis (Linnaeus) Kützing [qv Brodie et al., 2008b], 2012; Nelson, 2013; Sánchez et al., 2014). Recent studies using P. woolhousiae Harvey. A further four species were recorded by molecular markers and phylogenetic reconstruction and/or genetic González and Santelices (2003): P. linearis Greville, P. pseudolinearis distance for species identification have concluded that names have Ueda, P. lanceolata [now Pyropia lanceolata (Smith & Hollenberg) been misapplied in some regions and that bladed Bangiales species S.C. Lindstrom], Porphyra torta Krishnamurthy [now Pyropia torta could be much more diverse and regionally confined than recog- (Krishnamurthy) S.C. Lindstrom]. Another species, Porphyra kunthi- nized, especially in the Southern Hemisphere (Jones et al., 2004; ana Kützing, described from Valparaíso, Chile (Kützing, 1843), was Brodie et al., 2008a; Broom et al., 2010; Nelson and Broom, 2010). subsumed into Porphyra columbina (see Ramírez and Santelices, In hyper-diverse groups, where cryptic diversity is widespread, 1991); however, the identity of this species remains to be rapid molecular methods to delimit species have been used to cir- confirmed. A new species, Pyropia orbicularis M.E. Ramírez, cumvent the difficulties of morphology-based identification. The L. Contreras-Porcia & M.L. Guillemin, has been described recently use of DNA barcodes, while presenting problems in recently (Ramírez et al., 2014). However, with the exception of this new diverging groups or in cases of hybridization or introgression Pyropia record, there is currently no evidence to date that any of (e.g. Mols-Mortensen et al., 2012), have been demonstrated to be the other species occur in this region. For example, Porphyra a very effective tool with which to identify species (Kekkonen capensis, which was reported from Magallanes and Tierra del Fuego and Hebert, 2014). Recently, two new analytical methods have in Chile, was originally described from the Cape of Good Hope, been developed for species delineation: the General Mixed Yule- South Africa (Kützing, 1843). Sequence data for 18°S (the only coalescent (GMYC; Pons et al., 2006) and the Automatic Barcode molecular data available for this species) from South African mate- Gap Discovery (ABGD; Puillandre et al., 2012a). The first method rial showed that it grouped closely with two other Porphyra species uses the shift in branching rates along a Bayesian ultrametric gene from New Zealand (Milstein and de Oliveira, 2005). One of the tree from a Yule model (i.e. species level) to a coalescent model most commonly reported species along the coast of Chile is (i.e. population level) to propose species boundaries. The second P. columbina (Ramírez and Santelices, 1991). The name, method uses breaks in the distribution of pairwise genetic distance P. columbina, has been applied to specimens with widely ranging to split the sequence alignment into possible species. Both meth- growth forms and color states found in diverse habitats from ods allow statistical measures of confidence for species boundaries subantarctic islands to warm temperate areas of New Zealand, to be calculated. The congruence among species delimitation Australia, and South America (see review in Nelson and Broom, methods and genes can be viewed as supporting the robustness 2010; Nelson, 2013). However, molecular studies performed in of any particular grouping (Carstens et al., 2013). In algae, those the Falkland Islands (Broom et al., 2010), Chile (Ramírez et al., new methods have revealed the existence of extensive cryptic 2014) and New Zealand and the Southern Ocean (Nelson and diversity and have led to the reappraisal of the global diversity of Broom, 2010; Nelson, 2013) have confirmed that many rosette- some widespread genera, the distribution and range sizes of their like blades have been misnamed P. columbina and that this species individual species, as well as the mechanisms facilitating their could be confined to cold sub-Antarctic water and not present coexistence (Tronholm et al., 2012; Pardo et al., 2014; Payo et al., along the more temperate continental shores. On the other hand, 2013; Vieira et al., 2014). P. miniata f. cuneiformis is considered to be Wildemania cuneiformis In Chile, bladed Bangiales species are harvested from the wild (Setchell & Hus) S.C. Lindstrom which has been synonymized with and sold fresh or dry as ‘‘luche” for direct human consumption Wildemania amplissima (Kjellman) Foslie (Kucera and Saunders, (Hoffmann and Santelices, 1997). During 2013, 90 wet tons were 2012). Molecular data (rbcL) for material going under the name extracted, mostly in the central area of the country (SERNAPESCA Porphyra cuneiformis from Magallanes, indicated that this is a dis- 2013). Chile has an extensive coastline that stretches approxi- tinct species, but closely related to Wildemania amplissima mately 4200 km, from ca. 18°Sto56°S, and includes both Pacific (Brodie et al., 2008a; Brodie & Mols-Mortensen, unpublished data). and a small region of Atlantic shores (Fernández et al., 2000). The Records of specimens going under the name P. umbilicalis have Pacific coast of Chile is characterized by the presence of three been widely reported along the coast of Chile, although there is no well-defined biogeographic provinces (Camus, 2001): (i) the evidence that the species is present in the region (Brodie et al., Peruvian Province, located on the northern coast of Chile, from 2008a; Brodie unpublished data) and indeed that P. umbilicalis is Peru to a southern limit around 30–33°S, which is dominated by confined to the North Atlantic (Brodie et al., 2008b). Finally, the a warm-temperate biota; (ii) the Magellanic Province extends from status of Porphyra woolhousiae in the region remains unknown and Cape Horn (56°S) up to 41–42°S (Chiloé Island), and is dominated there are currently no molecular data available for this species. by sub-Antarctic and cold-water species, and (iii) an Intermediate Recent molecular studies, which have included a limited num- Area abutted by the Peruvian and Magellanic Provinces that is ber of Chilean taxa (Brodie et al., 2008a; Sutherland et al., 2011), 816 M.-L. Guillemin et al. / Molecular Phylogenetics and Evolution 94 (2016) 814–826 have suggested that there is a rich bladed Bangiales flora in the GENBANK accession numbers are detailed in Supplementary region, including a number of unidentified species. In both studies, Material 1. the coast of Chile was noted as a region where more detailed tax- onomic studies were required. These studies have also indicated 2.3. Phylogenetic analyses links between the Bangiales flora of Chile and other regions, including areas of the North Atlantic, New Zealand, South Africa Two data sets were created for the rbcL and the COI, respec- and the Mediterranean. Despite those first molecular insights, tively. For the rbcL, 156 sequences retrieved from GENBANK were ecological studies and aquaculture development of Chilean added to the sequences obtained for the Chilean samples. Those Bangiales rely only on morphological species identification (e.g. sequences correspond to Porphyra sp. FIH (GU165884, Broom Tala and Chow, 2014). However, a reappraisal of the diversity of et al., 2010) and all samples of Bangiales used in Sutherland et al. bladed Bangiales based on molecular data is much needed since (2011), except Pseudobangia kaycoleia for which no rbcL sequence those species are within the focus of a recently ongoing was available. Minerva aenigmata (EU570053) and Dione arcuata governmental program that aim to develop small-scale aquacul- (EU570052), which correspond to the more basal clades of ture for fishermen along the coast of Chile (Subsecretaría de Pesca Bangiales (Sutherland et al., 2011), were used as outgroups. For y Acuicultura de Chile, Legislatura 361, boletín: 9151–21). the COI, 39 sequences retrieved from GENBANK were added to In this study we have used 193 specimens collected from Arica our sequences obtained for the Chilean samples (see Supplemen- (18°S) to South Patagonia (53°S) to characterize the diversity of the tary Material 2). Since no sequences of basal Bangiales clades were bladed Bangiales of Chile using two molecular markers (COI-5P, available in GENBANK, COI trees were left unrooted. mitochondria and rbcL, chloroplast). We have applied a combina- Each sequence data set was partitioned according to codon tion of methods (including ABGD, GMYC and detection of mono- position. The best-fit models were estimated independently for phyly in tree reconstruction) to delimit species using molecular each partition by the Akaike Information Criteria (AIC) imple- data. We have also conducted phylogenetic analyses of COI-5P mented in TREEFINDER (Jobb et al., 2004) and the parameters of and rbcL data and discussed misleading ideas about the ecology the different substitution models were estimated independently of the Chilean bladed Bangiales as well as evolutionary perspec- for each partition. AIC identified the GTR(5)+G model for the first tives that emerged from our study. and the second codon position of the rbcL and the first codon posi- tion of the COI, TVM(5)+G for the third codon position of the rbcL, HKY(5)+G for the second codon position of the COI and GTR(5)+G+I 2. Material and methods for the third codon position of the COI. For each marker indepen- dently, phylogenetic relationships were inferred with a mixed 2.1. Sampling model in a maximum likelihood framework by using TREEFINDER v 2008 (Jobb et al., 2004) and support for the nodes was assessed Collections of bladed Bangiales were made from the littoral with 1000 bootstrap pseudoreplicates. Bayesian inference was per- zone of the coast of Chile from Arica (18°S) to South Patagonia formed using the general type of the best fit model parameters (53°S) from January 2005 to September 2013. Specimens were defined for each dataset using MrBayes v 3.1.2 (Huelsenbeck and pressed as vouchers after removing a small portion of the blade Ronquist, 2001). Four independent analyses were run with four that was stored in silica gel for subsequent DNA analysis. Voucher chains each, for five million generations. Trees and parameters specimens are housed in the herbarium of the National Museum of were sampled every 1000 generations and the default parameters Natural History, Chile (SGO) and in the algal herbarium at the Nat- were used to fit temperature and swapping. The first 25% of sam- ural History Museum, London (BM). Herbarium abbreviations fol- pled trees were discarded as ‘‘burn-in” to ensure stabilization. low Thiers (2014). Specimen collection information and voucher The remaining trees were used to compute a consensus topology numbers are indicated in Supplementary Material 1. and posterior probability values.
2.2. DNA extraction, sequencing and alignment 2.4. Sequence-based species delimitation
DNA extraction and amplification was undertaken according to Separate sequence-based analyses were conducted for four data the methods described in Mols-Mortensen et al. (2012) and sets: all samples of Porphyra from Chile for the COI, all samples of Ramírez et al. (2014). The 50 part of the mitochondrial Cytochrome Porphyra from Chile for the rbcL, all samples of Pyropia from Chile c Oxidase I gene (COI) was amplified using the primer pair GazF1 for the COI and all samples of Pyropia from Chile for the rbcL. (50-TCA ACA AAT CAT AAA GAT ATT GG -30) and GazR1 (50-ACT Sequences retrieved from GENBANK that presented a very TCT GGA TGT CCA AAA AAY CA -30), following the amplification pro- low genetic distance with our Chilean samples (uncorrected tocols of Saunders (2005). A partial sequence of the plastid gene p-distances < 0.002 for the COI and < 0.022 for the rbcL, as calcu- rbcL, encoding the large subunit of the ribulose-1,5-bisphosphate lated in Mega v 5; Tamura et al., 2011) were added to our data sets: carboxylase/oxygenase enzyme, was obtained by a DNA amplifica- Porphyra mumfordii (JN028502), Porphyra sp. FIG (GU165885), tion either using the primers F-rbcL(50- TTG CAT AYG ATA TTG ATY Porphyra sp. SBA (GU046414, conspecific to Porphyra sp. FIH, TAT TTG AA-30) and R-rbcS(50- RAG CTG TTT KTA AAG GWC CAC AA- Broom et al., 2010), Porphyra sp. FIH (GU165884), Pyropia sp. FIA 30) and protocols published previously (Hommersand et al., 1994; (GU046428), Pyropia sp. FIA (GU165842), Pyropia sp. FIC Fredericq and Lopez-Bautista, 2002), or with RBCL5RC (F) (50-GTG (GU046422) and Pyropia sp. FID (GU046406). Whatever the GTATTCATGCTGGTCAAA-30; Klein et al., 2003) and RBCSPC (R) (50 method used, no differences in the number of species detected CACTATTCTATGCTCCTTATTKTTAT-30; Teasdale et al., 2002). For were observed between the data set containing only the Chilean both markers, sequencing was undertaken using the primers as samples and the data sets completed with those eight GENBANK described in Mols-Mortensen et al. (2012) or Ramírez et al. (2014). sequences. Only the results obtained from the data sets completed During this study, 139 sequences of COI (614 bp) and 158 with the GENBANK sequences will be presented hereafter. sequences of rbcL (875 bp) were obtained and deposited in GEN- Two empirical methods were implemented to test species BANK. Sequence data for 14 specimens of Pyropia orbicularis from boundaries. First, a distance-based barcode approach to delimit Maitencillo beach, Valparaíso, 32°390S71°260W were added to this putative species was used. The Automatic Barcode Gap Discovery new data set of Chilean bladed Bangiales (Ramírez et al., 2014). (ABGD) method allows a barcode gap separating values of M.-L. Guillemin et al. / Molecular Phylogenetics and Evolution 94 (2016) 814–826 817 intraspecific and interspecific genetic distances to be defined, even putative species boundaries in the case of recently diverging spe- when the two distributions overlap (Puillandre et al., 2012a). The cies where incomplete lineage sorting could lead to paraphyly method uses ranked pairwise genetic distance values, from small- (Hudson and Coyne, 2002). The BP&P method accommodates the est to largest, to define a barcode gap (i.e. the first statistically sig- species phylogeny as well as lineage sorting due to ancestral poly- nificant peak in the slope of these values). ABGD computes a morphism and assumes no admixture following the speciation Primary Partition that delimits the first candidate species and then event. It allows calculation of posterior probabilities of potential recursively splits the Primary Partitions into Secondary Partitions species delimitations using a user-specific-species-tree. BP&P was using the same approach until no further gaps can be detected. run using ‘algorithm 0’, e = 5, automatically adjusted fine-tuning The analysis was performed at http://wwwabi.snv.jussieu.fr/ of the parameters and with an equal prior probability assigned to public/abgd. The JC69 distance and an X, a proxy for the minimum each species delimitation model. Because assigned prior distribu- gap width, of 1.0 were selected. The remaining parameters were tions could affect the posterior probabilities of the tested models, set to default (Pmin = 0.001, Pmax = 0.100, Steps = 10, Number of analyses were performed for three combinations of gamma priors bins = 20). In order to limit the over evaluation of the real number for ancestral population size (h) and root age (s0) distributions: of species, the number of putative species present in each data set h G(1, 10) and s0 G(1, 10); h G(2, 2000) and s0 G(2, 2000); was set using the primary partition that was detected for the h G(1, 10) and s0 G(2, 2000). The other divergence time parameters widest values of Pvalues. Second, a generalized mixed Yule coales- were assigned the Dirichlet prior (Yang and Rannala, 2010: Eq. (2)). cent (GMYC) model developed by Pons et al. (2006) to delineate Each analysis was run twice for 100,000 MCMC generations to con- genetic clusters was employed. The method establishes a threshold firm consistency between runs. A species tree was estimated using between the branching patterns of a gene tree from interspecific all COI and rbcL sequences from our Chilean samples and the pro- branches (Yule model) to intraspecific branches (neutral coales- gram ⁄BEAST (Heled and Drummond, 2010). Specimens were a pri- cent) in order to define speciation events. Repeated haplotypes ori assigned, in a conservative way, to 18 species based on the were removed from each data set, since the existence of zero results of the methods described previously (i.e. ABGD, GMYC, length branches could produce an over-partition of the dataset genealogical concordance and phylogeographic network). Two by the model (Reid and Carstens, 2012). A Bayesian phylogenetic independent ⁄BEAST analyses were performed with unlinked mod- analysis was conducted in BEAST v1.5.3 (Drummond and els for the two genes: GTR+G+I substitution model, uncorrelated Rambaut, 2007) under a GTR+I+G model with an uncorrelated log- lognormal relaxed molecular clock model and Yule species tree normal relaxed molecular clock model and using a constant Yule’s model. MCMC analyses were run for 100 million generations with speciation tree prior. Markov Chain Monte Carlo (MCMC) analyses trees sampled every 1000th generation. The output was diagnosed were run for 50 million generations for the rbcL and 200 million for convergence using Tracer v.1.5 and all model parameters of the generations for the COI. For each run, trees were sampled every combined log files for each data set reached an estimated sample 1000th generation. The output was diagnosed for convergence size (ESS) of 200. using Tracer v.1.5 and all model parameters of the combined log Inter- and intraspecific uncorrected p-distances, were calcu- files for each data set reached an estimated sample size (ESS) of lated in Mega v 5 (Tamura et al., 2011) for all the 18 genetic groups 200. The GMYC analyses were performed on maximum clade cred- defined as putative species. ibility trees calculated with TreeAnnotator v. 1.4.8 (Rambaut and Drummond, 2007) and using the SPLITS package for R (http:// 3. Results r-forge.r-project.org/projects/splits/). Both the single threshold (Pons et al., 2006) and multiple threshold (Monaghan et al., Sequence data of 193 bladed Bangiales specimens were ana- 2009) models were fitted on each ultrametric tree. lyzed in this study and 84 specimens were assigned to the genus A genealogical concordance approach to species boundaries Porphyra, 105 to Pyropia,3toWildemania and 1 to Fuscifolium (Dettman et al., 2003) was attempted using a visual inspection of (Fig. 1, Supplementary Materials 1 and 3–5). Only one specimen the COI and rbcL gene trees to search for reciprocal monophyletic was assigned to the genus Fuscifolium (POB0004 from Puerto groups that were concordantly supported by the two markers. Oscuro, 31°S, Supplementary Material 1). The three specimens For all, except three putative sister species (Pyropia orbicularis, assigned to the genus Wildemania (JBCH2011.01, JBCH2011.04 Pyropia CHJ and Pyropia CHK), sample groups were considered as and JBCH2011.13, all from Buque Quemado, 52°S, Supplementary putative species when at least half the methods employed to test Material 1) show identical rbcL sequences and we considered that their boundaries were concordant. For some species it was not pos- only one Wildemania species was present, in Southern Patagonia, sible to use all the methods due to a lack sequence in one gene or within our data set. Within the genera Porphyra and Pyropia, vari- presence of singletons in the gene trees. For a group of recently ous close by genetic groups were retrieved (Fig. 1, Supplementary diverging sequences of Pyropia the results obtained for different Materials 3–5) and we have tested species boundaries using GMYC methods using the COI or rbcL data sets were incongruent and a and ABGD methods and verified the monophyly of each group in conservative way was chosen to define only three genetic species order to define the number of species present in our data set. (Pyropia orbicularis, Pyropia CHJ and Pyropia CHK) (see results for Results for species delimitation in Porphyra and Pyropia are pre- more details). Moreover, to rule out intra-specific geographic sented below. sub-structuring as the cause of the lineage splitting between those three closely related entities, a COI phylogeographic network was reconstructed using the median-joining algorithm implemented 3.1. Species delimitation in Porphyra and Pyropia in NETWORK v 4.510 (Bandelt et al., 1999) in order to study the geographic localization of the haplogroups. The summary of the results of the GMYC and ABGD analyses for We have tested for species boundaries using a Bayesian method the COI and rbcL datasets is presented in Supplementary Material based on multilocus data set, BP&P (Rannala and Yang, 2003; Yang 6. For the COI, the GMYC model was favoured over the null model and Rannala, 2010), that allow the signal of species divergence in for both the single and the multiple thresholds regardless of the multiple gene trees to be identified, even for recently diverging genus under study (Porphyra, P-value GYMCsingle = 0.0364, groups for which monophyly has not been reached. Indeed it has P-value GYMCmultiple = 0.0156; Pyropia, P-value GYMCsin- been stated that the criteria of reciprocal monophyly and strict gle = 0.0127, P-value GYMCmultiple = 0.0053). Conversely, for the congruence between gene trees could be too strict to detect rbcL, the GMYC model was never favoured over the null model 818 .L ulei ta./MlclrPyoeeisadEouin9 21)814–826 (2016) 94 Evolution and Phylogenetics Molecular / al. et Guillemin M.-L.
Fig. 1. Maximum Likelihood (ML) rooted tree for rbcL sequences (875 bp) of Bangiales. Minerva and Dione were used as outgroups. For each node, ML bootstrap values and Bayesian Posterior Probabilities are indicated (ML/BPP). Only high support values (>75) are shown; ‘-’ = clade not observed in the Bayesian Inference. Next to collapsed branches are abbreviated genera names (as defined in Sutherland et al., 2011). Species of Chilean foliose Bangiales are shaded in gray. Since only the COI marker was sequenced for the Porphyra specimens assigned to the CHD group, only seven Chilean Porphyra were actually retrieved in the rbcL tree. M.-L. Guillemin et al. / Molecular Phylogenetics and Evolution 94 (2016) 814–826 819
Fig. 2. Bayesian species tree inferred using ⁄BEAST with numbers above branches representing posterior probability values (only values greater than 0.70 are shown). Speciation posterior probabilities from BP&P were calculated using three different prior combinations: 1st – large ancestral Ne and deep divergences (h G(1, 10) and s0 G(1, 10); top); 2nd – small ancestral Ne and shallow divergences (h G(2, 2000) and s0 G(2, 2000); middle) and 3rd – large ancestral Ne and shallow divergences (h G(1, 10) and s0 G
(2, 2000); bottom). Supports based on five different methods employed for species delimitation are given for the 18 Chilean Bangiales: GMYCMultiple for the COI, ABDG for the
COI, GMYCMultiple for the rbcL, ABGD for the rbcL and genealogical concordance. A picture of a representative specimen is given on the right part of the figure for each species. 820 M.-L. Guillemin et al. / Molecular Phylogenetics and Evolution 94 (2016) 814–826
(P-values ranged from 0.1461 to 0.5196 depending on the genus and CHG groups (Supplementary Materials 1, 3 and 4). Only the and the threshold model). These results indicated that there is no COI marker was sequenced for the Porphyra specimens assigned significant evidence for the predicted shift in branching rates from to the CHD group (Supplementary Materials 1 and 5). Moreover, interspecific to intraspecific events for the rbcL and resulted in an within each marker data set, the grouping of the Porphyra and extremely broad range of estimated number of GMYC taxa for both Pyropia samples were not fully congruent among ABGD and Porphyra (from 1 to 30 for single and 1 to 15 for multiple thresh- GMYC analyses. GMYC analyses tended to over-split groups when olds) and Pyropia (from 1 to 27 for single and 1 to 16 multiple compared to ADGB analyses using COI sequences (e.g. Porphyra thresholds). However, since the use of the multiple threshold CHC and CHF; Pyropia CHK and orbicularis) and rbcL sequences model allowed the confidence interval of GMYC taxa to be reduced (e.g. Porphyra FIH and Pyropia CHJ) (see Fig. 2 and Supplementary by almost two for the rbcL, it was selected over the single threshold Material 1 for details). model. In the same way, within both genera, the GMYC multiple Taking a conservative approach, sample groups were consid- threshold models had lower P-values than the single threshold ered as putative species when at least half the methods employed models and we have therefore chosen to report only the GMYC to test their boundaries were concordant (but see paragraph below multiple threshold results for the COI. for Pyropia orbicularis, Pyropia CHJ and Pyropia CHK). For five puta- For GMYC (multiple threshold), Porphyra specimens were tive species where only one gene was sequenced, ABGD and GMYC assigned to eight taxa for the COI and ten for the rbcL, whereas analyses and tree topologies were in full agreement (see Fig. 2). six ABGD clusters were detected for either gene used (Supplemen- Indeed, Porphyra CHD is represented as a singleton in the COI tree tary Material 6). The number of GMYC (multiple threshold) taxa (Supplementary Material 5) while Pyropia CHG, Pyropia FID, detected within Pyropia varied from nine for the COI to ten for Porphyra FIG and Porphyra CHE were recovered as singletons or the rbcL, while the number of ABGD clusters varied from seven well-supported monophyletic groups in the ML and IB rbcL trees for the COI to eight for the rbcL. Although the number of ABGD (Supplementary Materials 3 and 4). Three putative species, Pyropia clusters and GMYC taxa detected within each genus appeared CHH, Pyropia FIA and Porphyra CHB, were supported by both GMYC roughly similar, not all specimens were sequenced for both genes and ABGD analyses for the two genes under study (Fig. 2). Those (see Supplementary Material 1 for details) and some groups were three putative species were recovered as well-supported mono- revealed by only one molecular marker. Only rbcL sequences are phyletic groups or singletons in all ML and IB trees (Fig. 2, Supple- available for the Porphyra specimens assigned to the FIG and CHE mentary Materials 3–5). Five other putative species were groups and the Pyropia samples specimens assigned to the FID supported by at least three of the five species delineation methods
COI
Pyropia orbicularis
Los Burros, Chañaral de Aceituno Abalonera, Puerto Aldea Mina Talca Puerto Oscuro Los Vilos, Los Molles Salinas de Pullay, Maitencillo, Horcón Duao Cons tución Coliumo Lota Lebu Calfuco, Niebla Estaquilla, Carelmapu 2 Ancud Cucao Pyropia Chonchi sp. CHK
Pyropia sp. CHJ Punta Arenas
Fig. 3. Phylogenetic relationships among haplotypes of Pyropia orbicularis, Pyropia CHJ and Pyropia CHK and their geographic distribution based on COI data set. Sampled populations were located on a map and their colors correspond to sampling regions (see Supplementary Material 1 for more details). In the COI haplotype network, each circle represents a haplotype and its size is proportional to the frequency in which the haplotype was encountered. Colors correspond to sampling regions. Black squares represent hypothetical un-sampled haplotypes. For haplotypes separated by more than one mutational step, the number of steps is indicated by a number of base pairs, bp. M.-L. Guillemin et al. / Molecular Phylogenetics and Evolution 94 (2016) 814–826 821 tested: Pyropia CHI, Porphyra CHC, Porphyra FIH, Porphyra CHF and other hand, there was significant overlap without gaps when vari- Porphyra ‘mumfordii’ (see Fig. 2 for more details). ation for intra-specific and inter-specific divergence between con- Pyropia orbicularis, Pyropia CHJ and Pyropia CHK seem to be a specific individuals was calculated for the rbcL(Supplementary group of recently diverging sequences for which the results Material 7). For this gene, all intraspecific divergences are lower obtained for the different methods and the different markers used than 0.74% (i.e. value calculated in Pyropia sp. CHI). However, con- were incongruent (Fig. 2). Supporting evidence was inconclusive generic interspecific rbcL distances show large range with values as using ABGD and GMYC species delimitation methods since analy- low as 0.54 ± 0.18% between Pyropia sp. CHK and Pyropia orbicularis ses tended to over-split the COI data set while groups were lumped or 0.59 ± 0.23% between Porphyra mumfordii and Porphyra sp. CHC when using the rbcL data set (see Fig. 2 and Supplementary while the highest values were of 5.40 ± 0.23% between Porphyra sp. Material 1 for more details). For those three closely diverging CHI and FID. Divergence between species belonging to different genetic entities we have then chosen to use a more phylogeo- genera in our data set was >8.0% for the rbcL and >13% for the graphic approach and have reconstructed a median joining COI (Supplementary Material 7). haplotype network for the faster evolving gene (i.e. the COI, Fig. 3). The COI haplotype network showed the presence of three 3.3. Signal of species divergence in multiple gene trees main haplogroups: 17 bp separate Pyropia orbicularis and Pyropia CHK and 10 bp separate Pyropia CHK and Pyropia CHJ (Fig. 3). Multiple runs of BP&P with different MCMC parameters and Within each haplogroup, pairs of haplotypes were separated by guide trees gave similar results (Fig. 2). Species delimitation results 1–6 bp and no clear sub-structuring was observed (Fig. 3). The were robust to the choice of prior distributions (three combina- three haplogroups were recovered as three monophyletic groups tions of gamma priors for ancestral population size (h) and root in the COI tree (Supplementary Material 5). However, for the rbcL, age (s0) distributions were tested) with the exception of one case. while Pyropia CHJ and Pyropia CHK are recovered as monophyletic Regardless of the prior distributions used, all internal nodes had groups, Pyropia orbicularis formed a basal polytomy from which the high speciation posterior probabilities (SPP = 1.0), and all but two Pyropia CHK clade emerged (Supplementary Material 4). The color- terminal lineages had high speciation posterior probabilities coding map presented in Fig. 3 show that Pyropia orbicularis and (Fig. 2; SPP > 0.84). Pyropia sp. CHG and CHH had a rather high Pyropia CHJ have been sampled in the same locality (in Consti- speciation posterior probability when priors that represented large tución, Niebla, Ancud and Cucao; Supplementary Material 1). ancestral Ne and deep or shallow divergences were used Similarly, Pyropia orbicularis and Pyropia CHK distributions overlap (SPP = 0.76 and 0.85, respectively); however, the probability was slightly between the Salinas de Pullay and Maitencillo, two much lower using the small ancestral Ne and shallow divergences localities located less than 30 km apart (Supplementary Material prior choice (SPP = 0.22, Fig. 2). Regardless of the prior distribu- 1, Fig. 3). Those three COI haplogroups present in sympatry or close tions used, BP&P results shows that all nodes separating the three parapatry in central Chile were then interpreted as three different groups of recently diverging sequences of Pyropia orbicularis, sister species (i.e. Pyropia orbicularis, Pyropia sp. CHJ and Pyropia sp. Pyropia CHJ and Pyropia CHK are supported by very high speciation CHK; Supplementary Material 1). posterior probabilities (SPP = 1.00, Fig. 2). 3.2. Intra- and interspecific variation 3.4. Species distribution and region endemicity Based on our species delimitation methods, the data set split into 18 species: one species of Fuscifolium (Fuscifolium sp. CHA), No intensive sampling was realized in the 59 localities visited one species of Wildemania (Wildemania sp. FII), eight species of Pyr- along the Chilean coast and less than 10 specimens were taken opia (Pyropia sp. CHG, CHH, CHI, CHJ, CHK, FIA, FID and orbicularis) from all the localities except Maitencillo (32°S, N = 22) and Fuerte and eight species of Porphyra (Porphyra sp. CHB, CHC, CHD, CHE, Bulnes (53°S, N = 11). Because of the low number of specimens col- CHF, FIG, FIH and ‘mumfordii’) (Figs. 1–3, Supplementary Materials lected in most of the localities and the presence of large gaps in our 1 and 3–5). For all species with a ‘‘CH” code, no identical or nearly sampling, the species distribution presented in Fig. 4 have to be identical sequences were encountered in GENBANK. Wildemania taken with caution. sp. FII, Pyropia sp. FID, Pyropia sp. FIA and Porphyra sp. FIG species In most localities only one bladed Bangiales species was included sequences already published for specimens from the Falk- observed: 17 localities for which only one specimen was land Islands (Broom et al., 2010)(Supplementary Materials 1 and sequenced and 19 localities for which two to eight specimens were 3–5). The species Porphyra sp. FIH includes one sequence published sequenced (28% and 32% of the total sampled localities, respec- from the Falkland Islands and one sequence published from the tively). Up to four species were sampled in Ancud and five in Fuerte Auckland Island (New Zealand) (Broom et al., 2010)(Supplemen- Bulnes (Fig. 4). Twelve of the 18 species encountered (i.e. 66%) tary Materials 4 and 5). The species Porphyra mumfordii included were represented by less than five individuals while the other five one sequence published from a specimen from British Columbia, species (Porphyra sp. CHF, Porphyra sp. FIH, Pyropia sp. CHK, Pyropia Canada (Kucera and Saunders, 2012)(Supplementary Material 5). orbicularis and Pyropia sp. CHJ) represented 81% of all the speci- Our results also validate the inclusion of Pyropia sp. FIC, observed mens sequenced (157 of the 193 specimens). Some species, like in the Falkland Islands (Broom et al., 2010), within Pyropia orbicu- Wildemania sp. FII, Porphyra sp. FIG, Porphyra sp. CHD, Pyropia sp. laris as proposed in Ramírez et al. (2014) (Supplementary Materials FIA, Pyropia sp. FID and Pyropia sp. CHG, seem to be restricted to 4 and 5). the southern tip of the Patagonian region along the Chilean coast Congeneric interspecific divergences for the COI range from (Fig. 4). For the five better represented species in our sampling a 3.02% to 8.92%, with uncorrected p-distance of 7.33 ± 1.61% within clearer distribution range could be drawn with two species the genus Pyropia and of 4.88 ± 1.19% within the genus Porphyra. restricted to the north of the Chilean coast (Porphyra sp. CHF, These congeneric interspecific divergences are much deeper than 23°S–36°S and Pyropia sp. CHK, 26°S–32°S) and three species pre- the intraspecific divergences which range from 0.29% in Porphyra sent only in the central and southern part of Chile (Porphyra sp. FIH up to 1.02% in Pyropia sp. CHI. These results are clearly sp. FIH, 39°S–53°S; Pyropia orbicularis,32°S–53°S and Pyropia sp. reflected by the non-overlapping distribution of intra- and inter- CHJ, 35°S–42°S) (Fig. 4). Finally, Wildemania sp. FII, Porphyra sp. specific divergences and the presence of a barcode gap located in FIG, Pyropia sp. FIA, Pyropia sp. FID, Porphyra sp. FIH, and Pyropia the range 1.5–2.5% distance (Supplementary Material 7). On the orbicularis (as Pyropia sp. FIC) were also encountered in other sub 822 M.-L. Guillemin et al. / Molecular Phylogenetics and Evolution 94 (2016) 814–826
Fig. 4. Sampling sites and bladed Bangiales species distributions along the South Eastern Pacific.
Antarctic regions (Falkland Islands and Auckland Island, Broom present in the region. Of the nine genera of bladed Bangiales et al., 2010). already reported (Sutherland et al., 2011; Sánchez et al., 2014, 2015), Porphyra, Pyropia and Wildemania are present in Chile. The 4. Discussion presence of Fuscifolium (specimen POB0004 from Puerto Oscuro) is the first report of this genus in the South Eastern Pacific. Given This study has revealed considerably more species diversity the relative distance between the rbcL sequence data with those within the bladed Bangiales for the South East Pacific than previ- for F. tasa and F. papenfussii, it could be hypothesized that the spec- ously reported and demonstrated that four different genera are imen found in Chile is in a genus closely allied to Fuscifolium and M.-L. Guillemin et al. / Molecular Phylogenetics and Evolution 94 (2016) 814–826 823 present beyond the cold temperate waters of the North Pacific recognized as monophyletic groups in the ML and the IB COI tree where the two species are recorded (Sutherland et al., 2011). reconstructions, (2) recognized as clearly separated haplogroups Indeed, for the rbcL, the divergence between POB0004 and F. tasa in the COI network reconstruction, (3) supported by very high spe- and F. papenfussii is 7.3% while the divergence between the genera ciation posterior probabilities in the multigenic BP&P analysis, and described in the work of Sutherland et al. (2011) range between (4) met the ‘‘sympatry criterion” that was shown to represent a 6.9% and 12.0%. However, we consider that it would be premature powerful tool to resolve conflictive cases in hyper-diverse groups to erect a new genus on the basis of the evidence that we have (i.e. of mollusks or insects (Puillandre et al., 2012b; Kekkonen and only one individual and only one genetic marker with comparable Hebert, 2014). When conflicting results arose using molecular spe- data set, the rbcL), and have named the specimen Fuscifolium cies delimitation methods, most authors recommend employing sp. CHA while further collections and sequence data are obtained. criteria derived from different species concepts to aid at the final Congruence across results obtained with different methods decision on the status of any ‘controversial’ species (see (monophyly in tree reconstruction, ABGD, GMYC, and BP&P analy- Puillandre et al., 2012b; Kekkonen and Hebert, 2014 and reference ses) and different molecular markers (COI and rbcL) are generally herein). The ‘‘sympatry criterion” is one of them and its use in used to support putative species delimitation (Carstens et al., resolving controversial cases is well explained in the integrative 2013; Modica et al., 2014). In our study, not all specimens were taxonomy flowcharts described by those authors. When the two sequenced for the two markers (i.e. for Porphyra sp. FIG, Porphyra ‘controversial’ species show disjunct distributions, a simple geo- sp. CHD, Porphyra sp. CHE, Pyropia sp. FID and Pyropia sp. CHG graphic structuration of within-species diversity could explain this sequences were available for only one of the two markers), which pattern and the decision should be taken to lump the two genetic limited our capacity to look for congruence between them. Within groups. However, range overlap between sister taxa could provide each marker, some differences between the ABGD and GMYC convincing evidence of a lack of gene flow and associated repro- (multiple threshold) delimitation methods have been observed in ductive isolation (i.e. the genetic groups will represent biological our data set (see Fig. 2 for more details). In six cases, the GMYC species) and in the case of sympatric ‘controversial’ species the (multiple threshold) method led to over-splitting groups while, decision should be taken to split the two genetic groups. For the in two cases, the ABGD method have lumped two groups. Over rbcL, given the paraphyly of Pyropia orbicularis and the lumping the 27 tests (i.e. 12 tests for the marker COI and 15 tests for the of Pyropia orbicularis and Pyropia CHK in one unique group for both rbcL, Fig. 2) of congruence between the ABGD and GMYC methods ABGD and GMYC species delimitation methods, it is clear that the within marker that we have realized, 29% show incongruence. This lineage sorting between those lineages have not been reached. As percentage is similar to those detected in previous studies for other bladed Bangiales (Porphyra, Niwa and Kobiyama, 2014), (Puillandre et al., 2012b; Kekkonen and Hebert, 2014, 30% and the complementary study of nuclear genes could be of help when 20% of incongruence, respectively). It is well know that GMYC delimitation between recently diverging species is intended. The could lead to an overestimation of group partitioning while ABGD integration of population genetics and phylogeographic studies is is considered as a more conservative method to delimit species clearly needed to disentangle the complexities that arise in (Puillandre et al., 2012a,b; Kekkonen and Hebert, 2014 and refer- recently diverging entities and new studies need to be undertaken ence therein). Even if some incongruence was detected between before taking a final decision about the species status of Pyropia the methods, regardless of the gene under study, all our putative orbicularis, Pyropia sp. CHJ and Pyropia sp. CHK. genetic groups (except for Porphyra mumfordii and Pyropia orbicu- Taken together, our molecular results show that at least laris in the rbcL tree, Supplementary Material 4) correspond to eighteen species of bladed Bangiales occur along the Chilean coast. highly divergent singletons or monophyletic groups, therefore rep- Porphyra and Pyropia are the most diverse genera, with 8 species resenting at least a phylogenetic species. Paraphyly could be encountered for each. A single species was found for each of the caused by incomplete lineage sorting, introgression or hybridiza- genera Fuscifolium and Wildemania. These findings adds to a very tion and could be a major problem in species delimitation in sea- large group of species, many of which are undescribed, from South weeds (e.g. Destombe et al., 2010; Neiva et al., 2010; Montecinos Africa, Australia, New Zealand, the Antarctic Peninsula and the et al., 2012). Within the genus Porphyra, the phenomena of subantarctic islands (Sutherland et al., 2011). In the review of hybridization and introgression that could even lead to speciation Sutherland et al. (2011), 71 of the 157 species of bladed Bangiales via allopolyploidization has been observed between closely related incorporated in the study (i.e. 45%, including only one specimen species from the North West Pacific (Niwa et al., 2009, 2010; Niwa from Chile ‘‘Porphyra sp. JBCH26A”) were from the cold temperate and Sakamoto, 2010; Niwa and Kobiyama, 2014). The problem of waters of the Southern Hemisphere. In this region, new species incomplete lineage sorting is more pronounced for genes charac- have recently been discovered or described (this study, Nelson, terized by a low mutation rate (e.g. the rbcL) for which the coales- 2013; Nelson and D’Archino, 2014; Ramírez et al., 2014), adding cence process is slower and reciprocal monophyly is not a to the hypothesis proposed by Broom et al. (2004) that the area necessary property of species, especially for recently diverged spe- could be considered both as a center of origin and diversification cies (Knowles and Carstens, 2007). The faster evolving rate of the for various bladed Bangiales taxa (Sutherland et al., 2011). In the COI is reflected in the presence of a clear bar-code gap in our data speciose genus Pyropia, the existence of a recent radiation crown set for this marker, while an overlap between intra- and interspeci- could clearly be linked to the profusion of species present on the fic genetic distances was observed for the rbcL. This result shows coasts of Chile, South Africa, Australia, New Zealand, the Antarctic that, even if the COI is difficult to amplify in some red algal lineages Peninsula and the subantarctic islands (this study-Supplementary (Saunders, 2008; Milstein et al., 2012; Pardo et al., 2014), it is a Material 4, Sutherland et al., 2011; Ramírez et al., 2014). The find- useful marker with which to delimit species in Rhodophyta ing of a species that belongs to Wildemania confirms the presence (Sherwood et al., 2008, 2010). However, comparison of specimens of the genus as documented before in Chile, albeit as Porphyra of Bangiales species between different floras remains problematic miniata (C. Agardh) C. Agardh f. cuneiformis Setchell & Hus, and Por- due to the limited availability of COI sequences in GENBANK. For phyra cuneiformis (Setchell & Hus) V. Krishnamurthy (see Ramírez three very recently diverging groups (i.e. Pyropia orbicularis, and Santelices, 1991; Brodie et al., 2008a). The genus is typically Pyropia sp. CHJ and Pyropia sp. CHK), delimiting putative species found in spring/summer in cold temperate waters of the Arctic/ was very difficult, even when using the COI data set. We chose to Antarctic regions and is known to be present in the South Eastern separate these 144 specimens into three putative species since Pacific (Sutherland et al., 2011). Wildemania cuneiformis has since Pyropia orbicularis, Pyropia sp. CHJ and Pyropia sp. CHK were (1) been synonymised with W. amplissima (Kucera and Saunders, 824 M.-L. Guillemin et al. / Molecular Phylogenetics and Evolution 94 (2016) 814–826
2012). However, the taxon in Chile represents a closely related spe- physiology of both the macroscopic thallus stage and the micro- cies to W. amplissima but undescribed (this study, Mols-Mortensen, scopic conchocelis stage, the study of the development of repro- pers. comm.). ductive structure in the laboratory and population genetic In our study, only two species, Porphyra mumfordii and Pyropia analyses need to be undertaken in order to understand where orbicularis, are named and none of the six taxonomic species that and how these species coexist. Conclusions relating to the species had been cataloged for Chile and for which COI or rbcL sequences range limits along the South Eastern Pacific and the possible link were available in GENBANK were encountered (i.e. Porphyra miniata, between the Chilean flora and those present in other part of the P. columbina, P. umbilicalis, P. thurettii). The discovery that none of world are limited by the sparse sampling and the lack of extensive the species recorded matched those that had been reported for this studies of the Bangiales flora especially in the Southern Hemi- region (except for the recently described Pyropia orbicularis, sphere. Within Chile, our results indicate that probably not all spe- Ramírez et al., 2014) is further evidence of the problem of distin- cies are present everywhere. However, the possible restriction of guishing species based on morphology and the misuse of names their distribution to only one or two of the recognized biogeo- that have been applied to specimens in different parts of the world. graphic regions (i.e. the Peruvian province: 5°S to 30–33°S, the A similar situation was recently reported by Sánchez et al. (2014), Intermediate area: 30–33°Sto42°S and the Magellanic Province: who undertook a re-evaluation of the bladed Bangiales from the 42–56°S) is not clear. It is of note that the distribution of some spe- western Mediterranean. They found that most of the Porphyra cies seems to stop at 30–33°S(Pyropia sp. CHK, Pyropia orbicularis species that had been reported from the region were not present and Pyropia sp. CHI) or 41–42°S(Porphyra mumfordii, Pyropia sp. and that there was greater diversity than previously described CHH, Pyropia orbicularis and Pyropia sp. CHJ) which corresponds (even one new genus, Neothemis was discovered). In the Falkland well to documented biogeographical limits (Meneses and Islands, apart for Pyropia columbina, all eight species encountered Santelices, 2000; Camus, 2001; Thiel et al., 2007). Even if the during the study of Broom et al. (2010) remain unnamed. majority of the species of bladed Bangiales encountered along Because our sampling is very limited in some regions and few the South Eastern Pacific have not been reported outside this study, thalli are available for most of the species delimited (less than five there is a pattern of connection that arises from our new data set. specimens are available for 12 of the 18 species), we have chosen Six species that seems to be circumscribed to cold-cool water in not to describe new species on the basis of these collections at pre- Chile (Wildemania sp. FII, Porphyra sp. FIG, Porphyra sp. FIH, Pyropia sent. Moreover, all statistical methods used for delimiting species sp. FIA, Pyropia sp. FID and Pyropia orbicularis –asPyropia sp. FIC) are highly sensitive to the lack of information about within- were also encountered in other subantarctic regions (Falkland species genetic variation (Puillandre et al., 2012b; Kekkonen and Islands and Auckland Island, Broom et al., 2010). Those results fully Hebert, 2014). In the case of low taxon coverage, as in our study, support the multiple line of evidence of the importance of recur- better sampling is critical to strengthen the confidence in the pro- rent events of dispersal by rafting in shaping the flora and fauna posed species delimitation. Even if a more complete morphological of the Southern Hemisphere since the opening of the Drake Passage study were undertaken, the striking morphological plasticity and and the initiation of the West Wind Drift effects in the region convergence of these undescribed species would make them diffi- (Hommersand and Fredericq, 2001; Fraser et al., 2009). Similarly cult to distinguish using morphology alone (see representative for the genus Pyropia, most species of Porphyra encountered in specimens of each species in Supplementary Material 8). The exis- Chile are positioned within clades consisting of several species tence of such morphological plasticity is particularly compelling in from the Southern Pacific (e.g. Porphyra sp. CHB, Porphyra sp. Porphyra sp. CHF, Porphyra sp. FIH, Pyropia sp. CHK, Pyropia orbicu- CHE, Porphyra sp. CHF, Supplementary Material 3). However, the laris and Pyropia sp. CHJ, the five species for which a high number Porphyra mumfordii samples have identical COI and rbcL sequences of specimens are available and were sampled in different regions from the North East Pacific (see for example JN028947: (Supplementary Material 8). As stated by Verbruggen (2014) British Columbia, Canada; EU223113: Oregon, USA; EU223112: ‘‘morphological features [...] should not be trusted to be informa- Washington, USA for the rbcL or JN028502: British Columbia, tive about species boundaries [...] and one should not assume that Canada for the COI, Kucera and Saunders, 2012). Porphyra because two individuals look alike, they are going to belong to the mumfordii was originally described from British Columbia and until same species”. our work, was only known from the North-East Pacific. This species Many more specimens and localities exist in our collections for is very closely related to P. umbilicalis and P. linearis in the which we do not have molecular data. The presence of one or only North Atlantic, which are considered to be sibling species a few species in a collection may not be a true reflection of the local (Mols-Mortensen et al., 2014; Mols-Mortensen, personal commu- diversity. In this study, collecting has tended to be haphazard and nication). This lack of genetic divergence from the sequences opportunistic and whole regions south of Chiloe to the southern retrieved along the coast of Chile and the ones available from the part of the land mass remain unexplored. Molecular studies of North East Pacific and close relationship with species from the unexplored vouchers collection and new sampling campaigns are North Atlantic suggest a recent dispersal and could support a likely to expand the distribution range of most species and to scenario of recent introduction in Chile. change our estimation of local and regional diversity and level of Given the high number of rare taxa along the coast of Chile, it is sympatry between bladed Bangiales. In our study up to five species highly unlikely that we have sampled all the bladed Bangiales spe- were encountered at the same sampling point but, a fine habitat cies of the South East Pacific. Areas of the coastline in particular sampling over different seasons in Brighton Beach (New Zealand), that need to be sampled include the northern coast (from Pan de have observed that a huge diversity of morphologically similar Azúcar to the border of Peru), between Chiloé and the Straits of bladed Bangiales occurs in sympatry (i.e. nine species in an area Magellan and the tip of Chile, although these areas are extensive of 3800 m2, Schweikert et al., 2012). In Japan, two cryptic species and remain logistically challenging. Furthermore, there is a real of Pyropia have even been reported to grow in sympatry on the need for collections to be made throughout the year, as some spe- same rock (Niwa et al., 2014). The mechanisms facilitating cies are only present for a relatively short time on the shore. These coexistence could include restriction to microhabitat, differences results continue to point to the need to undertake a global evalua- in life cycle and reproductive strategies and difference in seasonal- tion of the bladed Bangiales both from where they have been ity for growth or reproduction. In the case of the bladed Bangiales described in the past and also from unexplored regions. Until this along the coast of Chile, population sampling along transects has been undertaken, endemics and species boundaries will coupled with habitat description, investigation into the cellular remain uncertain. M.-L. Guillemin et al. / Molecular Phylogenetics and Evolution 94 (2016) 814–826 825
Acknowledgments Hudson, R.R., Coyne, J.A., 2002. Mathematical consequences of the genealogical species concept. Evolution 56, 1557–1565. Huelsenbeck, J.P., Ronquist, F., 2001. MrBayes: Bayesian inference of phylogenetic Fieldwork in Chile for JB was funded by the Natural History trees. Bioinformatics 17, 754–755. Museum Enhancement Fund. Sampling from Salinas de Pullay to Jobb, G., von Haeseler, A., Strimmer, K., 2004. TREEFINDER: a powerful graphical Los Burros was made with the help of the project FONDECYT NO analysis environment for molecular phylogenetics. BioMed Central Evol. Biol. 4, 18. 1130797 (CONICYT, Comisión Nacional de Investigación Científica Jones, W.A., Griffin, N.J., Jones, D.T., Nelson, W.A., Farr, T.J., Broom, J.E., 2004. y Tecnológica, Gobierno de Chile) to MLG. This research also was Phylogenetic diversity in South African Porphyra (Bangiales, Rhodophyta) supported by the Instituto Antártico Chileno (INACH) projects determined by nuclear SSU sequence analysis. 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