The genus Callophyllis (Kallymeniaceae, Rhodophyta) from the central-south Chilean coast (33o to 41o S), with the description of two new species

The taxonomic status of the species in the genus Callophyllis Kützing (Kallymeniaceae, Rhodophyta) from centralsouth Chile (33o to 41o S) is examined on the basis of morphological and molecular evidence. Of the four species originally cited for central Chile, C. variegata, C. pinnata, C. atrosanguinea and C. laciniata, only the presence of C. variegata has been confi rmed in this study. C. pinnata reported from Chile is found to be different from C. pinnata described from California, and it is here newly described as C. concepcionensis sp. nov. C. atrosanguinea from southern Chile (including C. linguata from the Antarctic Peninsula) is distinct from the species called C. atrosanguinea from central-south Chile, the latter which is here described as C. macrostiolata sp. nov. C. variegata, C. concepcionensis and C. macrostiolata are distinguished from one another by their external habit, the nature and distribution of their cystocarps, and the female reproductive morphology. Comparative rbcL sequence analysis corroborates the distinction of these taxa from central-south Chile and their relationships to other species worldwide.


INTRODUCTION
The genus Callophyllis Kützing (1843) contains the largest number of species in the family Kallymeniaceae, with more than 50 species (Guir y & Guir y 2011) distributed preferentially in cold and temperate waters of both hemispheres.The distinction among Callophyllis species is based on comparative vegetative features such as color, size, and branching pattern, as well as reproductive characters such as number of carpogonial branches per suppor ting cell, position and size of cystocarps and number of ostioles, and whether or not tetrasporangia are organized in sori (Setchell 1923, Dawson 1954, Norris 1957, Abbott & Norris 1965).The morphological variability found in the genus has led many authors to overestimate the number of species, as in the Pacifi c Coast of North America where most of the species are concentrated (Abbott & Norris 1965).
In the southern hemisphere, nine species of Callophyllis have been reported from the temperate Pacific Coast of South America, one to several species of Callophyllis identifi ed as C. variegata or Callophyllis spp.and known collectively as "carola", are being exported as raw material for direct consumption.

Morphological analyses
The material examined belongs to the Herbarium of the Museo Nacional de Historia Natural, Santiago, Chile (SGO), Herbarium of the Marine Science Department at University of Valparaiso, Chile (UV), and Herbarium of the University of Louisiana at Lafayette (LAF).Reference material of C. atrosanguinea was obtained from the Muséum National d'Histoire Naturelle in Paris, France (PC).Algal samples were collected in the Concepción-Arauco area, in Cocholgüe,Coronel and Lota (36º35' S,72º57' W) and Isla Santa María (36º59' S, 73º32' W).Voucher specimens are also deposited in the algal collection of the Department of Oceanography at the University of Concepcion, Chile (CON) and in the LAF Herbarium.Specimens were pressed as herbarium sheets, and duplicates were wet-preserved in 10 % formalin/seawater and subsequently transferred to 5 % formalin/seawater for long-term storage.Longitudinal and cross sections were done by hand using a razor blade.Staining was performed using the modified Wittmann's aceto-iron-haematoxylin chloral hydrate (Wittmann 1965) method of Hommersand et al. (1992), with samples mounted in 50 % Hoyer's medium (Stevens 1981).Sections were also stained with 1 % aniline blue, acidifi ed with 1 % HCl and mounted in 50 % Karo TM Syrup.Images of habit were taken using a Cannon New F-1 camera.Observations were conducted using an Olympus CHT microscope, and photographs were taken with a Nikon Coolpix 4500 and Polaroid Ie digital microscope camera, respectively.

Molecular analyses
Total DNA was extracted from silica gel-dried specimen parts of the same samples that were formalin-fixed.Voucher specimens are deposited at the University of Louisiana at Lafayette (LAF).DNA extraction was performed with a DNeasy Plant Mini Kit (Qiagen, Valencia, CA, USA).Specific gene regions were amplified by PCR and prepared for sequencing following the protocols described in Gavio & Fredericq (2002).The rbcL primers used for amplification and sequencing reactions were as follows: FrbcLstart, F57, R753, R1150, RrbcSstart (Freshwater & Rueness 1994); F577, F753, F993 (Hommersand et al. 1994) andF64 (Lin et al. 2001).Sequencing reactions were set up using the Big Dye kit and protocol (Applied Biosystems, Foster City, CA, USA), and run on ABI 3100 Genetic Analyzers (DNA Analysis Core Facility, CMS; Biology Deparment, UL Lafayette).Sequence contigs were assembled using Sequencher (Gene Codes Corp., Ann Arbor, MI, USA) and sequence data sets were compiled and aligned using MacClade (v.4, Maddison & Maddison 2000).Phylogenetic analyses were conducted with the Maximum Parsimony (MP) algorithm as implemented in PAUP 4.0b 10 (Swofford 2002), the Maximum Likelihood (ML) algorithm using RAxML (Stamatakis et al. 2008), and the Bayesian (MB) inference as implemented in MrBayes v. 3.1.2 (Ronquist & Huelsenbeck 2003).Maximum likelihood tree was done under the General Time Reversible (GTR) + GAMMA evolutionary model of nucleotide substitution.A parametric bootstrap test was applied to the ML analysis using 100 boostrap replicates.For Bayesian trees four Metropolis-coupled Markov chain Monte Carlo (MCMC) chains (one cold and three incrementally heated) were run using the GTR + I + Γ model of sequence evolution for 5000000 generations, trees were sampled every 1000 generations, with a burn-in value of 20000 generations.Maximum parsimony trees were inferred from a heuristic search, excluding uninformative characters consisting of 1000 random sequence additions holding 10 trees at each step.MULPARS and tree-bisection-reconnection (TBR) algorithms with the MULTREES (saving multiple trees) and STEEPEST DESCENT option.Support for nodes of the MP analysis groups was determined by calculating bootstrap proportion based on 1000 replicates.Species examined, collection localities, and GenBank accession numbers for generated sequences are listed in Table 1.

Observations
Callophyllis specimens were collected in centralsouth Chile (see Appendix) and examined for their habit shape, and for their vegetative and reproductive morphology.Three distinct entities were recognizable as follows: Species 1 Habit and vegetative morphology: Pink to intense red in color, with a membranous consistency.The holdfast is small and extends in a short stipe (Figs.1A-1C) bearing fronds up to 30 cm long.The thallus is fanshaped, consisting of flat, dichotomous to subdichotomous branches (6-20 or more) in a same plane; additional blades are irregularly divided and are numerous near the basal region of the thallus (Figs.1A-1C).Cystocarps are located on the upper margins of the fronds, forming rows in some cases (Fig. 1H).Prominent cystocarps bear one (Fig. 1G) to three (Fig. 1I) ostioles.The tetrasporophytic t h a l l i a r e s u p e r f i c i a l l y s i m i l a r t o t h e gametophytes, with reproductive structures irregularly distributed throughout the fronds (Fig. 1B).Male specimens are typically thinner than the female gametophytes (Fig. 1C).
The gonimoblasts cut off chains of three to four carposporangia fi lling part of the medulla when mature and they are distributed in groups separated by abundant vegetative filaments (Fig. 1G).The cystocarps are spherical, reaching 1 mm in diameter (Figs.1H, 1I).Mature carpospores, about 15 µm in diameter, are surrounded by medullary fi laments, about 100 µm thick, that do not contribute to a pericarp.Cortical cell layers increase in number and project towards the outside forming exit pores (Fig. 1G).Cystocarps are located near the margins of the thalli, especially in young parts (Fig. 1H).Each cystocarp is topped by one to three ostioles (Fig. 1I) located on one or both sides of the fronds, with each ostiole, up to 250 µm tall, extending inwardly in a narrow channel.
C r u c i a t e l y d i v i d e d t e t r a s p o r a n g i a , measuring approximately 27 µm long x 17 µm width, are located on both sides of the tetrasporophyte fronds; they are irregularly grouped, with most concentrated near the apical region.Tetrasporangial parent cells originate from inner cortical cells.Cortical cells surrounding the tetrasporangia do not become modifi ed and do not increase the number of cell layers (Fig. 1J).Male gametophytes cut off spermatangial parent cells (Fig. 1K), 7 µm long x 5 µm wide, that are surface cells and produce spermatangia, approximately 4 µm in diameter.The spermatia are released through a disintegrating zone at the thallus surface (Fig. 1K).

Species 2
Habit and vegetative morphology: Membranous to cartilaginous consistency and pink to dark purple in color.The stipe is generally thin and short, although in some specimens the fi rst division is 2 or 3 cm removed from the holdfast, extending in a palmate or fl abellate upright frond that reaches a height of up to 35 cm, with wide main segments usually not exceeding 3 cm (Figs.2A-2C).Thalli are four to eight times dichotomously branched in a regular pattern.Although most specimens have narrow branch angles at the branching points and smooth margins, some have laciniate branches that only appear on the upper par t of the frond.The apices are generally rounded.Fronds sometimes bear few to many pinnate proliferations 1 to 4 cm long x 1 cm wide (Fig. 2A).Female gametophytes bear barely projecting cystocarps scattered over the frond.Male gametophytes are similar to female gametophytes and tetrasporophytes (Fig. 2B) in size and shape, but are thinner and sometimes more branched (Fig. 2C).Thalli are approximately 150-500 µm thick.Longitudinal sections through the frond at mid level reveal a two-layered cor tex consisting of regular, elongate surface cells 7 µm long x 3-5 µm wide.The subcortical cells are 5-10 µm long x 5-17 µm wide.The medulla consists of four layers of cells 60-80 µm long and 100-300 µm wide, surrounded by rizoidals cells, 3-15 µm wide x 12-25 µm long (Fig. 2H).
Reproductive morphology: The female gametophyte has procarps located on both sur faces of the frond, isolated from other procarps (Fig. 2D).A supporting cell originates from a subcortical cell, 7-10 µm in diameter, and cuts of f three filaments, two of them comprising two-celled subsidiar y cells (Fig. 2E).The other fi lament is a carpogonial branch of three cells with a carpogonium bearing a curved trichogyne.The procarp consists of a single carpogonial branch per supporting cell.Subsequently, cells of the procarp increase in size, most of them become lobulate with the exception of the hypogynous cell and the carpogonium.Mature procarps measure about 87 µm (Fig. 2F) in diameter.After presumed fer tilization, a fusion cell, formed from the fusion of the procarp cells, cuts off gonimoblast initials towards the medulla (Fig. 2G), that continue cutting of chains of uninucleate gonimoblast cells, each 7-17 µm long x 4-5 µm wide.
Small rhizoidal cells in the medulla tend to be globose when close to gonimoblast fi laments.Short three-celled carposporangial chains within cystocarps distributed over the frond sur face measure 1 to 2 mm in diameter and completely fi ll the medulla with carpospores 12 to 17 µm in diameter.The cystocarps are sur rounded by vegetative filaments, 65-125 µm thick, whose cells becoming dif ferentiated from the medullar y cells (Fig. 2H).Each cystocarp is topped by one to three ostioles (Fig. 2I), whose exit channels are surrounded by a few layers of cells resembling a simple breakage in the cortex.These channels extend to a depth of 125-250 µm and open to a single side of the frond.In female specimens, extensive zones bearing young or immature cystocarps are color-less.Most cystocarps are located next to the margins, but in some specimens, are scattered over the entire thallus.
T e t r a s p o r o p h y t e s p e c i m e n s b e a r tetrasporangia that are distributed irregularly throughout the thallus, abundantly at midlevel.Cruciately divided tetrasporangia, 27-37 µm long x 15-20 µm wide, originate from tetrasporangial initials in the inner cor tex on both sides of the thallus (Fig. 2J).Male gametophytes bear spermatangia, about 4 µm in diameter, cut of f from isodiametric spermatangial parent cells, 5 µm in diameter, in the outer cortex on both sides of the thallus.Colorless spermatia are released at the thallus surface, surrounded by a gelatinized cuticle (Fig. 2K).Species 3 H a b i t a n d v e g e t a t i v e m o r p h o l o g y : Cartilagenous thallus, red to brown in color, subdichotomous branching.Stipe 1.5 cm long, widening gradually to 5-7 cm until the fi rst branching point, thallus reaching 12 to 35 cm in length, branching points rounded, branch apices lightly rounded and weakly dissected (Figs.3A-3C).Female gametophytes with abundant distal segments, narrow at the base, dividing four to 13 times, some with lateral proliferations (Fig. 3A).Cystocarps prominent over the entire frond, each with a large, centrally invaginated ostiole (Fig. 3H).Tetrasporophyte thalli (Fig. 3B) and male gametophytes (Fig. 3C) less ramified than the female gametophytes.Thalli 50-150 µm in thickness, with a cor tex of four to fi ve cell layers, surface cells isodiametric to rectangular, 5-7 µm long x 4-5 µm wide, inner cells isodiametric, 7-15 µm in diameter.Cells gradually increasing in size towards the medulla.Medulla of fi ve to eight layers of ovoidal cells, 65-140 µm long x 30-70 µm wide.Abundant medullar y fi laments of small rhizoidal cells present, 7-20 µm long x 4-12 µm wide.
R e p r o d u c t i v e m o r p h o l o g y : P r o c a r p s originate on both sides of the thallus over the entire surface in groups and can be seen in different stages of development (Fig. 3D).
Suppor ting cells, formed from an internal cor tical cell 5-7 µm in diameter, each cut of f a single three-celled carpogonial branch (Fig. 3E) and sometimes a one-or two-celled subsidiar y fi lament.The carpogonium emits a thin trichogyne towards the surface.When the procarp matures, the supporting cell and cells of the subsidiar y filament increase in size, become rounded, bulbose, elongated and oriented toward the interior of the medulla.Mature procarps measure 75 µm x 37 µm (Fig. 3F).Following presumed fer tilization, the supporting cell fuses with cells of carpogonial branch for ming a fusion cell that emits extensions from which initial gonimoblasts are generated.Cells of the gonimoblast fi laments measure 7-12 µm in length x 2-5 µm in width and form chains of carposporangia in groups of three, carpospores measure 10 to 17 µm in diameter.Cystocarps 2 mm in diameter (Fig. 3H), with a large ostiole on only one side of the thallus to the thallus (Fig. 3G), central ostiole channel 140-400 µm long.Vegetative medullary fi laments surround compact cystocarps 150-350 µm in thickness, and consisting of isodiametric cells 10-40 µm in diameter.
Tetrasporangia originate in the subcortex, and are distributed over the entire sur face of the frond, on both sides of the thallus.Tetrasporangial initials developing in cruciate tetrasporangia 25-27 µm x 15-17 µm, and remaining embedded among cor tical cells, surrounded by several cells, less numerous towards the apical portions of the frond (Fig. 3I).Spermatangia occur on both sides of the frond.The spermatangial parent cells, 4 µm x 5 µm, are formed in the outer cortex, and produce spermatangia 4 µm in diameter.The liberation of the spermatia occurs together with the gelatinization of the thallus' surface (Fig. 3J).
Fig. 4: Maximum-likelihood (ML) tree from the analysis of rbcL sequence data under the (GTR) + GAMMA model, for 28 taxa including Callophyllis from central-south Chile (33º to 44º S) (in bold letters) and Kallymenia reniformis as the outgroup.Bootstrap proportion values are indicated for MP (1000 replicates) and ML (100 replicates) (above branches), and Bayesian (below branches) methods when > 50.
resulting from the ML analysis.The Maximum Parsimony (MP) analysis also recovered twelve most parsimonious trees with similar topologies.In the dataset, 257 characters (18 % of the total) were parsimony-informative, 148 characters were parsimony-uninformative, and 1014 characters were constant.The support values for the MP, ML and Bayesian analyses (PP) are shown in Fig. C. atrosanguinea from Punta Dungeness a n d P u n t a A r e n a s , s o u t h e r n C h i l e , i s conspecific with C. linguata Kylin from the Antarctic Peninsula and forms a stronglysuppor ted monophyletic clade (MP = 91, ML = 91, PP = 99) with C. macrostiolata sp.nov.from Concepción, Chile.The latter is currently reported as C. atrosanguinea but the rbcL sequence divergence between vouchers from central and southern Chile ranges from 2-2.5 %.This new species is clearly resolved as a sister taxon to the Antarctic and Chilean southernmost species of Callophyllis.This Antarctic Peninsula and Chilean clade is resolved with variously support as sister to the remainder of the Callophyllis taxa (MP = 61, ML = 70, PP = 100).A nuclear marker, rDNA LSU, explored by Harper & Saunders (2002) provided less resolution within species of Callophyllis than obtained with rbcL sequence analysis.In red algae, a rbcL sequence divergence between taxa of ≥ 2 % is the standard to recognize a separate species (see for example Gurgel & Fredericq 2004).The sequence divergence between C. concepcionensis and its closest sister taxon, C. obtusifolia is 4 %, and between C. concepcionensis and C. pinnata from type locality in Califor nia is 4.8-5 %.Hence, it is clear that C. concepcionensis, cur rently reported as C. pinnata in Chile, is a distinct species from true C. pinnata.
With a % rbcL sequence divergence of 2-2.5, C. atrosanguinea from Punta Dungeness in southern Chile (including C. linguata from the Antarctic Peninsula), is distinct from the taxon going under this name from Concepción in central Chile.We have provided a new name for the central Chilean coast, C. macrostiolata sp.nov.
In a taxonomic study of Callophyllis, Euthora and Pugetia (Kallymeniaceae) on the basis of rDNA LSU DNA sequences, Harper & Saunders (2002) (Hariot 1889, Levring 1960).The rhizoidal and basal cells in C. atrosanguinea from Punta Dungeness, Chile, are of the same size and agree with Hariot's (1889) description, a characteristic not obser ved in material from central Chile, which suggests that this species does not occur in the region.It is not possible to determine with the data at hand if C. atrosanguinea is present in other parts of the Chilean coast, but in all likelihood this species is restricted to southern Chile.Fur ther phylogenetic and biogeographic studies are needed on the Antarctic species of Callophyllis that are also reported for the southern Chilean coast, such as C. multifida, C. fastigiata, and C. tenera.The habits of these three species are well characterized (Kützing 1867, Pl. 90;Reinsch 1888, Taf II Figs. 1-5;Levring 1960, Fig. 7), and do not correspond to any of the features characterizing C. macrostiolata sp.nov., in which the frond consists of numerous thin, distal bladelets with truncated, non-laciniate apices, and in which the first branching point is located a distance away from the holdfast.The cystocarpic specimens of C. macrostiolata sp.nov.from Isla Santa María, Concepción (36º S) consist of larger fronds and are more branched than the male gametophytes and tetrasporophytes.These dif ferences between individuals were not noticeable in specimens from the coast near Osorno, Valdivia, or Chiloé (40º to 41º S).
The three taxa from central-south Chile, C. variegata, C. concepcionensis sp.nov., and C. macrostiolata sp.nov., differ in their comparative vegetative and reproductive structures (Table 2).C. macrostiolata sp.nov. is characterized by surface and internal cortical cells that are regularly isodiametric and more numerous than those found in C. variegata and C. concepcionensis sp.nov.which contain fewer cell layers, and in which the subcortical cells are periclinally wider than high.In agreement with Etcheverr y (1986), Ricker (1987), and Ramírez & Rojas (1988), cell arrangement may be a useful characteristic in the delimitation of the species.
The number of carpogonial branches per suppor ting cell is an impor tant taxonomic character to separate Callophyllis species; however, it is not applicable to differentiate the species from central south Chile since they are all monocarpogonial.Although Harper & Saunders (2002)   Whereas the distribution of cystocarps on the fronds of Callophyllis species was an impor tant characteristic for Setchell (1923) andMillar (1993) to delineate species, Dawson (1954) emphasized the size and degree of cystocarp protr usion and the number of ostioles.Besides these characteristics, the present study adds information on ostiole shape and projection, and size of the ostiole channel.Thus, the cystocarps of C. macrostiolata sp.nov.and C. variegata are protuberant, while those of C. concepcionensis sp.nov.from Chile are extended and less protuberant.Although Millar (1993) suggested that this character depends on the position of the cystocarp on the thallus and the stage of maturity, it is here confi rmed that ostiole features show up early and are maintained throughout the development of the cystocarp.
The distribution of protuberant cystocarps over the entire frond, as in C. macrostiolata sp.nov., has also been observed in specimens of C. megalocarpa Setchell & Sweezy from Mexico (Dawson 1954).However, this Mexican species bears cystocarps with one to many rostrate ostioles, a different situation from that in C. macrostiolata sp.nov., a species characterized by a single, notably invaginated ostiole.
The male reproductive str uctures of C. macrostiolata sp.nov.and C. concepcionensis sp.nov.from central-south Chile generally fit the general patter n described for the genus (Dawson 1954, Norris 1957, Womersley & Nor ris 1971).The disposition of the tetrasporangia in the cortex of C. macrostiolata sp.nov.from central Chile does not show remarkable differences to that in other species cited from this area (Etcheverry 1986, Ramírez & Rojas 1988).In C. macrostiolata sp.nov. the tetrasporangia are immersed in the cor tex whereas in C. variegata and C. concepcionensis sp.nov. the cortical cells do not completely cover the tetrasporangia due to the smaller number of cor tical cell layers.Using the presence or absence of tetrasporangial sori advocated by Dawson (1954) to characterize species of Callophyllis was not useful in this study, as all specimens analyzed had sori lacking a characteristic pattern.
Two tetrasporic specimens and a female individual from a Typological Series of C. atrosanguinea collected by J. D. The current geographical distribution of C. macrostiolata sp.nov.and C. concepcionensis sp.nov. is restricted to central-south Chile, with as northern limit the Concepción region (36º S).The range for C. variegata is confi rmed for Peru (14º S) to Magallanes, Punta Arenas (52º S).The Identifi cation key to the species of Callophyllis of the central-south (33º to 41º S) coast of Chile is presented in Table 3.
Even though C a l l o p h y l l i s has been the subject of many studies, its extreme morphological variability (especially branching pattern) and the suggested inclusion of species with undivided blades (C.firma) makes it dif ficult to clarify the generic status at this point, and fur ther phylogenetic studies are needed in this group.The intergeneric relationships among Callophyllis and other genera of Kallymeniaceae is beyond the scope of this study.Only after we have gained a better understanding of the genera and species relationships within the Kallymeniaceae, will the attempted reconstr uction of the biogeographic histor y of the family be meaningful.
4. C. concepcionensis sp.nov.(a taxon previously known as C. pinnata) from Chile and C. pinnata from California are resolved into two separate clades.C. concepcionensis sp.nov.from Chile is sister to C. obtusifolia J. Agardh from Baja California (MP = 94, ML = 96, PP = 100), and C. pinnata from California does not group with any of the other species from California and Washington.The % rbcL sequence divergence between C. pinnata from California and C. concepcionensis from Chile is 4.8-5.C. edentata Kylin from California and C. edentata from Washington form a clade with C. violacea (J.Agardh) Kylin from California (MP = 100, ML = 100, PP = 100).C. variegata, the type species of the genus, from Chile and Peru is resolved as a fully supported monophyletic group (MP = 100, ML = 100, PP = 100).The Japanese species C. crispata Okamura and C. japonica Okamura group together (MP = 58, ML = 57, PP = 97).A Bayesian phylogenetic reconstruction indicates that the Callophyllis species from Japan, USA, and Chile form a well-supported clade (PP = 75).This clade, in the Bayesian-generated tree, is weakly resolved as a sister taxon to the Australian species C. lambertii (Turner) J. Agardh (PP = 62), but variously suppor ted as a sister group to C. variegata from Peru and Chile (ML = 56, MP = 74, PP = 100).Taxa from New Zealand are spread across four dif ferent but closely related clades: C. decumbens J. Agardh (GQ376536) clusters with taxa from Japan, USA, Chile, Peru and Australia (MP = 56, ML = 67, PP = 96).The position of Callophyllis sp. from New Zealand (GQ376537) lacks support as a sister taxon in any phylogenetic analysis.The clade of the New Zealand taxa C. hombroniana (Montagne) Kützing and Pugetia delicatissima R. Norris (MP = 60, ML = 83, PP = 99) is sister to the previously mentioned group only in the Bayesian phylogenetic reconstruction (PP = 76).Another New Zealand species, C. depressa (J.Agardh) Schmitz ex Laing (GQ376535), occupies a basal position and is poorly resolved (PP = 58).

TABLE 1
Callophyllis species and associated taxa included in the molecular analyses.Especies de Callophyllis y otros taxa incluidos en los análisis moleculares.

TABLE 1
This study recognizes three distinct species of Callophyllis from central-south Chile (33 o to 41 o S) on the basis of comparative morphology and anatomy and sequence analysis of chloroplast-encoded rbcL: Species 1 is here confirmed as representing C. variegata (see Appendix), but there is incongruence with the current taxonomy (Abbott & Hollenberg 1976, Etchever r y 1986) of two additional species growing in central Chile.The two species that do not fit the concept of the type of C. pinnata Setchell & Swezy and of the type of C. atrosanguinea (J.D. Hooker & Har vey) Hariot are here assigned new species rank.Species 2 that goes under the name C. pinnata in central Chile is here newly described as C. concepcionensis sp.nov.(see Appendix).Species 3 that goes under the name C. atrosanguinea in central Chile is here newly described as C. macrostiolata sp.nov.(see Appendix). DISCUSSION also found that C. pinnata from Isla Chiloé (Chile) and C. pinnata from Piedras Blancas, California (USA) comprise dif ferent taxa.They did not determine the identity of the Chilean taxon although based on previous morphological studies they suggested a similarity with C. violacea from Peru or C. obtusifolia from North America.Our phylogenetic analysis includes C. obtusifolia from Baja California and C. violacea from California and supports the distinction of the Chilean "C.pinnata" as C. concepcionensis sp.nov.The habit of C. obtusifolia has been well characterized as comprising long, linear blades that are rarely palmately divided with terminal dichotomies longer than penultimate dichotomies (Dawson 1954, Pl. 35 and Pl.36; Abbott & Hollenberg 1976, Fig. 410); in contrast, C. concepcionensis sp.nov.from Concepción, Chile, has straight palmate fronds with narrow branch angles or with laciniate branches that only appear at the upper part of the dissected frond.These characters do not fit the habit pattern described for C.

TABLE 2
Comparison of relevant morphological and anatomical characters of Callophyllis from central-south Chile (33º to 41º S). de caracteres morfológicos y anatómicos relevantes de Callophyllis de centro-sur de Chile (33º a 41º S).
Hooker in the Falkland Islands (Cape Pembroke) and Tierra del Fuego (Rade de Goreé, Point Guanaco) housed in PC, France, were examined in this study.The material analyzed by Etcheverr y (1986) from Isla Santa María, Chile, and identified as C. atrosanguinea, does not fit the characteristics of C. atrosanguinea and instead corresponds to C. macrostiolata sp.nov.Although C. macrostiolata sp.nov.resembles C. atrosanguinea in that both are dark red, subdichotomously branched, with rounded apical regions that are poorly laciniate, and with cystocarps distributed over the entire frond, the frond of C. macrostiolata sp.nov.bears protuberant cystocarps, each with a welldefi ned and invaginated ostiole, and markedly dif ferent from C. atrosanguinea, a species with extended cystocarps and diffuse ostioles.A taxon identifi ed as C. atrosanguinea from Punta Dungeness, Chile, is included in this study; as this material was not fertile, cystocarp comparison was not possible.In conclusion, C. macrostiolata is morphologically, reproductively and phylogeneticaly distinct from the other species of Callophyllis present in central Chile, including C. variegata and C. concepcionensis, another new species.