Revista chilena de historia natural
versión ISSN 0716-078X
Rev. chil. hist. nat. vol.84 no.4 Santiago dic. 2011
Revista Chilena de Historia Natural 84: 481-499, 2011
© Sociedad de Biología de Chile
The genus Callophyllis (Kallymeniaceae, Rhodophyta) from the central-south Chilean coast (33° to 41° S), with the description of two new species
El género Callophyllis (Kallymeniaceae, Rhodophyta) de la costa central y sur de Chile (33° a 41° S), con la descripción de dos nuevas especies
NATALIA ARAKAKI1,3· *, KRISLER ALVEAL1, MARÍA ELIANA RAMÍREZ2 & SUZANNE FREDERICQ3
1 Facultad de Ciencias Naturales y Oceanográficas, Universidad de Concepción, Casilla 160-C, Concepción, Chile.
2 Museo Nacional de Historia Natural, Casilla 787, Santiago, Chile.
3 University of Louisiana at Lafayette, Lafayette LA 70504-2451, USA.
*Corresponding author: email@example.com
The taxonomic status of the species in the genus Callophyllis Kützing (Kallymeniaceae, Rhodophyta) from central-south Chile (33° to 41° 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 confirmed 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.
Key words: Callophyllis, Chile, Kallymeniaceae, rbcL, Rhodophyta, systematics.
Se examina el estatus taxonómico de las especies del género Callophyllis Kützing (Kallymeniaceae, Rhodophyta) de la costa centro-sur de Chile (33° a 41° S) en base a caracteres morfológicos y moleculares. De las cuatro especies citadas para Chile central, C. variegata, C. pinnata, C. atrosanguinea y C. laciniata, solo la presencia de C. variegata ha sido confirmada en este estudio. C. pinnata de Chile muestra diferencias con C. pinnata descrita para California y con el resto de las especies de Callophyllis hasta ahora conocidas, constituyendo así una nueva especie, C. concepcionensis sp. nov. C. atrosanguinea del sur de Chile (incluyendo C. linguata de la Península Antártica) muestra diferencias con la especie llamada C. atrosanguinea de la costa centro-sur de Chile, esta última especie es descrita como C. macrostiolata sp. nov. C. variegata, C. concepcionensis y C. macrostiolata se distinguen una de otra por la morfología externa del talo, la distribución y morfología de los cistocarpos, incluyendo la morfología reproductiva femenina. El análisis comparativo de las secuencias de la subunidad mayor del gen Rubisco (rbcL), corrobora la distinción de los taxa procedentes de la zona central y sur de Chile y su relación con otras especies del género provenientes de otras partes del mundo.
Palabras clave: Callophyllis, Chile, Kallymeniaceae, rbcL, Rhodophyta, sistemática.
The genus Callophyllis Kützing (1843) contains the largest number of species in the family Kallymeniaceae, with more than 50 species (Guiry & Guiry 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 supporting 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 Pacific 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, namely the generitype C. variegata (Bory) Kützing, C. atrosanguinea (J.D. Hooker & Harvey) Hariot, C. fastigiata (J. Agardh) J. Agardh, C. laciniata (Hudson) Kützing, C. multifida (Reinsch) Kylin, C. pinnata Setchell & Swezy, C. tenera J. Agardh, C. violacea J. Agardh, and Callophyllis sp. (Ramírez & Santelices 1991). Most of these species are reportedly distributed in the southern part of the continent (Hooker & Harvey 1847, Hariot 1889, Skottsberg 1923, Pujals 1963, Lee 1964, Ramírez 1982, Wiencke & Clayton 2002). For the central-south Chilean coast only four species have been reported: C. variegata, C. pinnata, C. laciniata and C. atrosanguinea (Ramírez & Santelices 1991). The distribution of C. variegata also includes the Falkland Islands, Fuegia, Iles Crozet, Iles Kerguelen, Macquarie Islands, New Zealand, South Africa, South Georgia, South Orkney Islands, and Tristan da Cunha Islands (Ricker 1987). According to Ricker (1987), large numbers of often poorly defined species of Callophyllis occur in New Zealand and subantarctic regions. Morphological studies on C. variegata by Montagne (1852) and Etcheverry (1986) emphasized a polymorphic thallus and the position of the cystocarps on the blade margins as remarkable features for this species. Hooker & Harvey (1847) described six varieties of C. variegata for the subantarctic region of South America, with one of them (var. atrosanguinea) later elevated to species level by Hariot (1889).
C. atrosanguinea is reported from both south and central Chile (Ramírez & Santelices 1991). This species shares with C. variegata a circumpolar distribution (Adams 1994), being cited for the Falkland Islands, Kerguelen Island, and New Zealand. According to Levring (1960) and Etcheverry (1986), C. atrosanguinea and C. variegata are conspecific based on thallus shape; Adams (1994), however, suggested that the two taxa are distinct species that could be distinguished in New Zealand by the texture and color of the thallus while downgrading the importance of whether cystocarps are scattered over the blade, as in C. atrosanguinea, or restricted to the margins, as in C. variegata.
C. pinnata was described by Setchell & Swezy in Setchell (1923) from Duxbury reef, Marin County, California, for a North Pacific species occurring from Whidbey Island, Washington, to Baja California (Abbott & Norris 1965); the species was later reported for the central-south coast of Chile (Ramírez & Rojas 1988). The description by Ramírez & Rojas (1988) of the morphology, anatomy and some reproductive attributes of C. pinnata from Chile agrees with the one given by Abbott & Norris (1965) and Abbott & Hollenberg (1976) for C. pinnata from the Pacific coast of North America. However, a molecular analysis conducted by Harper & Saunders (2002) noted that two isolated specimens identified as "C. pinnata" from Chile and California do not group together in a phylogenetic tree on the basis of LSU rDNA sequence analysis, suggesting that perhaps the South American taxon could represent either a previously unrecognized species or perhaps a robust form of C. violacea J. Agardh.
C. laciniata from the central coast of Chile (Valparaíso) and Peru (Callao) has not been reported since Montagne (1852). Ramírez & Santelices (1991) point out that Howe (1914) had already expressed doubts about the Peruvian record. The geographical distribution of C. laciniata is reported to be restricted to Western Europe (Bert 1967) and the Islas Orcadas del Sur (Orkney Islands, in the southern Atlantic Ocean) (Pujals 1963).
An important goal of this study is to describe and illustrate the reproductive system of Callophyllis species from central-south Chile as these diagnostic characters are missing in reports of the taxa from this region. Since the Callophyllis species from Chile have not been the subject of an up-to-date systematic revision, the present study is a critical examination of the species reported from the central-south coast of Chile (33° to 41° S), C. variegata, C. pinnata, C. laciniata and C. atrosanguinea, on the basis of a comparative study of vegetative and reproductive structures, and of rbcL sequence data.
It is especially important that the species of Callophyllis are well characterized as in Chile, one to several species of Callophyllis identified as C. variegata or Callophyllis spp. and known collectively as "carola", are being exported as raw material for direct consumption.
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 (36°35' S, 72°57' W) and Isla Santa María (36°59' S, 73°32' 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, acidified with 1 % HCl and mounted in 50 % KaroTM 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.
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) and F64 (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 Mr Bayes 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.
Callophyllis specimens were collected in central-south Chile (see Appendix) and examined for their habit shape, and for their vegetative and reproductive morphology. Three distinct entities were recognizable as follows:
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 fan-shaped, 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 thalli are superficially similar to the gametophytes, with reproductive structures irregularly distributed throughout the fronds (Fig. 1B). Male specimens are typically thinner than the female gametophytes (Fig. 1C).
Fronds are 50-140 μπι thick, with a cortex of three layers of unordered cortical cells (Fig. 1E), the outer cells isodiametric (approximately 5 μη1 in diameter) to rectangular (7 μη1 long x 5 μη1 wide). The subcortex is comprised of isodiametric cells, 7 μη1 long x 7-15 μη1 wide. The medulla consists of three to four layers of ovoid cells (Fig. 1G), 65-90 μη1 x 35-50 μη1, surrounded by rhizoidal cells (Fig. 1G, arrow), 17-25 μη1 long x 5-15 μη1 wide, that are more abundant in mature parts of the thallus.
Reproductive morphology: Female gametophytes bear procarps on both sides of the thallus, near the frond margin (Fig. 1D). A supporting cell, originated from a subcortical cell, forms two to four filaments comprising one to three two-celled unfertile subsidiary cells (Fig. 1E). The other filament corresponds to a three-celled carpogonial branch with a carpogonium bearing a trichogyne directed towards the surface. A single carpogonial branch per supporting cell is produced (Fig. 1F) per procarp. Mature procarps measure aproximately 87 μη1 in diameter. The supporting cell, the subsidiary cells and the basal cell of the carpogonial branch become lobulated, the hypogynous cell remains isodiametric and smaller than the rest of the procarp cells, and a triangular-shaped carpogonium contains a distinct nucleus (Fig. 1F). A fusion cell forms after presumed fertilization, established by the fusion of the subsidiary cells, the basal cell of the carpogonial branch and the supporting cell. The fusion cell cuts off protrusions towards the medulla that continue to grow into gonimoblast filaments of uninucleate cells, 5-12 μη1 long x 3-5 μη1 wide. Gonimoblast filaments develop between medullary cell spaces.
The gonimoblasts cut off chains of three to four carposporangia filling 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 μη1 in diameter, are surrounded by medullary filaments, about 100 μη1 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 μη1 tall, extending inwardly in a narrow channel.
Cruciately divided tetrasporangia, measuring approximately 27 μη1 long x 17 μη1 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 modified and do not increase the number of cell layers (Fig. 1J). Male gametophytes cut off spermatangial parent cells (Fig. 1K), 7 μη1 long x 5 μη1 wide, that are surface cells and produce spermatangia, approximately 4 μη1 in diameter. The spermatia are released through a disintegrating zone at the thallus surface (Fig. 1K).
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 first division is 2 or 3 cm removed from the holdfast, extending in a palmate or flabellate 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 part 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 μη1 thick. Longitudinal sections through the frond at mid level reveal a two-layered cortex consisting of regular, elongate surface cells 7 μη1 long x 3-5 μη1 wide. The subcortical cells are 5-10 μη1 long x 5-17 μη1 wide. The medulla consists of four layers of cells 60-80 μη1 long and 100-300 μη1 wide, surrounded by rizoidals cells, 3-15 μη1 wide x 12-25 μΓη long (Fig. 2H).
Reproductive morphology: The female gametophyte has procarps located on both surfaces of the frond, isolated from other procarps (Fig. 2D). A supporting cell originates from a subcortical cell, 7-10 μη1 in diameter, and cuts off three filaments, two of them comprising two-celled subsidiary cells (Fig. 2E). The other filament 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 μη1 (Fig. 2F) in diameter. After presumed fertilization, 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 μη1 long x 4-5 μη1 wide.
Small rhizoidal cells in the medulla tend to be globose when close to gonimoblast filaments. Short three-celled carposporangial chains within cystocarps distributed over the frond surface measure 1 to 2 mm in diameter and completely fill the medulla with carpospores 12 to 17 μη1 in diameter. The cystocarps are surrounded by vegetative filaments, 65-125 μη1 thick, whose cells becoming differentiated from the medullary 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 μη1and 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.
Tetrasporophyte specimens bear tetrasporangia that are distributed irregularly throughout the thallus, abundantly at mid-level. Cruciately divided tetrasporangia, 2737 μη1 long x 15-20 μη1 wide, originate from tetrasporangial initials in the inner cortex on both sides of the thallus (Fig. 2J). Male gametophytes bear spermatangia, about 4 μη1 in diameter, cut off from isodiametric spermatangial parent cells, 5 μη1 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).
Habit and vegetative morphology: Cartilagenous thallus, red to brown in color, subdichotomous branching. Stipe 1.5 cm long, widening gradually to 5-7 cm until the first 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 50150 μη1 in thickness, with a cortex of four to five cell layers, surface cells isodiametric to rectangular, 5-7 μη1 long x 4-5 μη1 wide, inner cells isodiametric, 7-15 μη1 in diameter. Cells gradually increasing in size towards the medulla. Medulla of five to eight layers of ovoidal cells, 65-140 μη1 long x 30-70 μη1 wide. Abundant medullary filaments of small rhizoidal cells present, 7-20 μη1 long x 4-12 μη1 wide.
Reproductive morphology: Procarps originate on both sides of the thallus over the entire surface in groups and can be seen in different stages of development (Fig. 3D).
Supporting cells, formed from an internal cortical cell 5-7 μη1 in diameter, each cut off a single three-celled carpogonial branch (Fig. 3E) and sometimes a one- or two-celled subsidiary filament. The carpogonium emits a thin trichogyne towards the surface. When the procarp matures, the supporting cell and cells of the subsidiary filament increase in size, become rounded, bulbose, elongated and oriented toward the interior of the medulla. Mature procarps measure 75 μη1 x 37 μη1 (Fig. 3F). Following presumed fertilization, the supporting cell fuses with cells of carpogonial branch forming a fusion cell that emits extensions from which initial gonimoblasts are generated. Cells of the gonimoblast filaments measure 7-12 μη1 in length x 2-5 μη1 in width and form chains of carposporangia in groups of three, carpospores measure 10 to 17 μη1 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 μη1 long. Vegetative medullary filaments surround compact cystocarps 150-350 μη1 in thickness, and consisting of isodiametric cells 10-40 μη1 in diameter.
Tetrasporangia originate in the subcortex, and are distributed over the entire surface of the frond, on both sides of the thallus. Tetrasporangial initials developing in cruciate tetrasporangia 25-27 μη1 x 15-17 μη1, and remaining embedded among cortical 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 μη1 x 5 μη1, are formed in the outer cortex, and produce spermatangia 4 μη1 in diameter. The liberation of the spermatia occurs together with the gelatinization of the thallus' surface (Fig. 3J).
The rbcL dataset included in the analyses consists of 1419 characters for 29 taxa; Kallymenia reniformis, the type of the family, was selected as the outgroup. The Maximum Likelihood (ML) analysis resulted in a tree indicating that most species relationships are strongly supported (Fig. 4). The Bayesian analysis produced a majority rule consensus tree that corroborates the overall tree topologyresulting 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. 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 supported 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 different 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).
C. atrosanguinea from Punta Dungeness and Punta Arenas, southern Chile, is conspecific with C. linguata Kylin from the Antarctic Peninsula and forms a strongly-supported 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).
This study recognizes three distinct species of Callophyllis from central-south Chile (33o to 41o 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, Etcheverry 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 & Harvey) 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).
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 California is 4.8-5 %. Hence, it is clear that C. concepcionensis, currently 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) also found that C. pinnata from Isla Chiloé (Chile) and C. pinnata from Piedras Blancas, California (USA) comprise different 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. 4,10); 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. pinnata from Moss Beach, California, near to the type locality (Duxbury Reef, Marin Co.), as this species is characterized by being loosely and irregularly branched with long branches 2-3 cm wide that terminate in straight to somewhat pointed apices (Abbott & Hollenberg 1976). Since C. concepcionensis sp. nov. from Chile occupies a position in the rbcL tree removed from C. pinnata from California, the recognition of two species is fully supported.
A close relationship between C. macrostiolata sp. nov., C. atrosanguinea (including C. linguata) from southern Chile and the Antarctic region, and an undescribed species from the Antarctic Peninsula is found in all the phylogenetic analyses conducted. C. macrostiolata sp. nov. was previously identified from central Chile as C. atrosanguinea by Etcheverry (1986), a species reported from New Zealand (Antipodes Islands, Auckland Islands, Campbell Island), the Falkland Islands (type locality), Kerguelen Islands, and Tierra del Fuego (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 observed 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. Further 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 (36° S) consist of larger fronds and are more branched than the male gametophytes and tetrasporophytes. These differences between individuals were not noticeable in specimens from the coast near Osorno, Valdivia, or Chiloé (40° to 41° 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 Etcheverry (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 supporting cell is an important 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) suggested a close relationship between the polycarpogonial species, the rbcL-based analyses indicate that monocarpogonial and polycarpogonial species can group together, such as C. edentata (polycarpogonial species) from California with C. violacea (monocarpogonial) from California. Norris (1957) considered C. variegata, the type species, as monocarpogonial although in one instance he observed a procarp that had two complete carpogonial branches attached to one supporting cell; this prompted him to view the presence of only one carpogonial branch per supporting cell as not being a reliable diagnostic character for Callophyllis. Later, he reconsidered his earlier opinion because Abbott & Norris (1965) reported the existence of both mono- and polycarpogonial species. The current study shows the presence of only monocarpogonial species occurring in central-south Chile (C. macrostiolata sp. nov., C. variegata, C. concepcionensis sp. nov.), even though Ramírez & Rojas (1988) had suggested that C. 'pinnata' (= C. concepcionensis sp. nov.) from Chile was polycarpogonial. C. pinnata from Pacific North America is polycarpogonial (Abbott & Norris 1965) and occupies a different position from C. concepcionensis sp. nov. in the rbcL tree.
Womersley and Norris (1971) proposed that the procarps are relatively uniform in Callophyllis species. As shown in this study, C. variegata and C. concepcionensis sp. nov. from central-south Chile have procarps with lobulate cells and one to three subsidiary filaments, in contrast to the procarp of C. macrostiolata sp. nov. that is comprised of rounded, bulbose cells that are non-lobulate and have almost not subsidiary filaments. This attribute was previously used by Millar (1993) for differentiating species of Callophyllis and by Ganesan (1976) for differentiating species of Kallymenia.
Whereas the distribution of cystocarps on the fronds of Callophyllis species was an important characteristic for Setchell (1923) and Millar (1993) to delineate species, Dawson (1954) emphasized the size and degree of cystocarp protrusion 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 confirmed 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 structures of C. macrostiolata sp. nov. and C. concepcionensis sp. nov. from central-south Chile generally fit the general pattern described for the genus (Dawson 1954, Norris 1957, Womersley & Norris 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 cortex whereas in C. variegata and C. concepcionensis sp. nov. the cortical cells do not completely cover the tetrasporangia due to the smaller number of cortical 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. 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 Etcheverry (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 well-defined and invaginated ostiole, and markedly different from C. atrosanguinea, a species with extended cystocarps and diffuse ostioles. A taxon identified 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.
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 (36° S). The range for C. variegata is confirmed for Peru (14° S) to Magallanes, Punta Arenas (52° S). The Identification key to the species of Callophyllis of the central-south (33° to 41° S) coast of Chile is presented in Table 3.
Even though Callophyllis 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 difficult to clarify the generic status at this point, and further 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 reconstruction of the biogeographic history of the family be meaningful.
ACKNOWLEDGEMENTS: We thank Gloria Collantes (University of Valparaiso, Chile), Bruno de Reviers (National Museum of Natural History, Paris), Patrick Martone (Stanford University), and all the collectors listed in Table I for providing specimens used in this study. This research was funded by a University of Concepcion grant (PI 96-112-036-1.0), and NSF DEB-0743024, DEB-0919508 and DEB-0936855 to SF.
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Associate Editor: Sylvain Faugeron
Received May 7, 2010; accepted August 30, 2011.
Description, typification and list of representative specimens and species of Callophyllis examined from the central-south Chilean coast (33° to 41° S).
Species 1 = Callophyllis variegata (Bory) Kützing.
Basionym: Halymenia variegata Bory, 18261828: 179.
Type: Herbarium Montagne, PC. Isotype (UC 435656, Ç) collected by Binder.
Type locality: Valparaíso, Chile.
Representative specimens examined: MAGALLANES, PUNTA ARENAS: M. Codoceo, 9.ii.1954 (SGO 095804, Ç); Seno Otway, Punta Prat, U.M.A.G., 20.xi.1985 (SGO 105382); Punta Dungeness, D. Díaz, 24.iv.1986 (SGO 105352, Θ). CHILOÉ: Abtao, I. Meza, ii.1984 (SGO 105133); Ancud, Mar Brava, M.E. Ramírez & G. Rojas, 4.xii.1990 (SGO 122249). VALDIVIA: Playa Blanca, D. Contreras, 4.i.1979 (SGO 096039, Θ); Los Molinos, B. Parra, 20.ii.1985 (SGO 103978, Ç); Punta Misión, G. Rojas, 30.i.1987 (SGO 106151), M.E. Ramírez, 22.x.1988 (SGO 109824). CONCEPCIÓN: Bahía Coliumo, D.H. Kim, 14.v.1969 (SGO 088128, Ç); Los Morros, H. Romo, 3.iv.1976 (CON 1105, Ç), J. Cid, 3.iv.1976 (CON 1107, 1110, Ç); Bahía San Vicente, zona sur, H. Etcheverry, ix.1959 (UV, θ); Las Escaleras, D.H. Kim, 2.vi.1976 (CON 1767, Ç; 1109, 1115, θ); Golfo de Arauco, J. Vásquez, i.1981 (SGO 095732, θ); Punta Lavapie, K. Alveal, 30.i.1991 (CON, θ); Raqui, W. Wilkomirsky, iii.1974 (CON 194, θ), Llico, Isla Verde, K. Alveal, 30.i.1991 (CON, Ç); Yana, K. Alveal, iv.1978 (CON 557, θ); Trauco, J. Cid, xi.1975 (CON 551, 559, θ); Lebu, A.
Alveal & K. Alveal, ii.1985 (CON 526, Ç); Bahía Concepción, Tumbes, K. Alveal, xi.1972 (CON 548, 593, 1101, θ); Cocholgüe, K. Alveal, v.77 (CON 545, 547, 591, θ), L. Chuecas, 22.vi.1982 (SGO, θ), C. Werlinger, iv.1987 (CON 603, Ç), J. Valenzuela & M. Figueroa, 20.x.1989 (CON 1157, 1342, 2448, θ), K. Alveal, 19.i.1990 (CON 1102, 1155, Ç); Caleta Leandro, J. Cid, 9.vi.1976 (CON 1113, θ), H. Romo, 9.vi.1976 (CON 1114, θ); Isla Santa María, W. Wilkomirsky, iii.1974 (CON 549, θ); Cocholgüe, Puertos de Coronel and Lota e Isla Santa María, N. Arakaki, iii.1998, 9.x.1998, 3.xi.1998, 7.iv.2000, 25.vii.2000, 28.vii.2000, 4.x.2000, 11.xi.2000, 16.xi.2000 (CON, Ç, θ, 22 specimens). VALPARAÍSO: Navidad, La Boca, Punta Perros, M.E. Ramírez, 24.x.1980 (SGO 095640); Cartagena, M. Espinoza, i.1917 (SGO 081074, θ), Ventana, M. Figueroa, H. Romo & K. Alveal, iii.1971 (CON 534, θ) Maitencillo, H. Etcheverry, ii.1954 (UV, Ç). Los Vilos, Bahía Conchalí, A. Llaña, v.1945 (UV 1247, θ). COQUIMBO: Bahía Tongoy, H. Etcheverry, ii.1954 (UV, Ç ); Puerto Aldea, J. Vásquez & P. Ojeda, 22.i.1981 (SGO 081153, θ); Bahía Herradura, E. Fonck, 23.ix.1976 (SGO 097104, Ç ), H. Black & M.E. Ramírez, 13.xii.1982 (SGO 102514); Bahía Herradura, A. Llaña, 20.vi.1945 (UV 1208, θ). LA SERENA: Peñuelas, H. Etcheverry & K. Alveal, x.1959 (UV, Ç ). ANTOFAGASTA: Bahía Mejillones del Sur, M.E. Ramírez, 13.i.1987 (SGO 106175). IQUIQUE: Cavancha, R. Pinto, xii.1987 (SGO 107308, Ç ). Additional material: 108 specimens from different localities of central-south Chile (SGO and CON, Ç, θ).
Species 2 = Callophyllis concepcionensis Arakaki, Alveal et Ramírez sp. nov.
Thallus membranaceus usque ad cartilagineus, 4-8 dichotome ramosus ordinate ad 35 cm alt. Stipes brevis extensus in thallum palmatum aut flabellatum usque ad 35 cm longum, segmentis principalibus largis plerumque non plus quam 3 cm., angusti ramorum anguli, margines laeves, aut rami laciniati solum primi visi supra thalli, apices rotundati interdum prolificationibus pinnatis paucibus vel multibus. Cortex 2-5 stratus, cellulis isodiametricus. Cellula sustinens monocarpogonialis, procarpium 2 ramulis subsidiariis bicellularibus 3-cellulari ramo carpogoniali, cellulae procarpii lobatae. Cystocarpia plana protuberantia, 1-2 mm diam., super lamina dispersa, cum 1-3 simplicibus ostiolis. Thalli feminei zonati cystocarpiis immaturis albidis. Tetrasporangia cruciata, 27-37 μm x 15-20 μm, orienda in subcortice. Spermatangia, 4 μm, irregulariter dispersa in cortice externo.
Membranous to cartilaginous thallus, four to eight times dichotomously branched in a regular pattern. Short stipe,extending in an upright palmate or flabellate frond up to 35 cm in height, with wide main segments usually not more than 3 cm. Branch angles narrow with smooth margins, branch apices rounded, or branches laciniate at the upper part of the frond, sometimes with few to many pinnate proliferations. Cortex of two-five layers of isodiametric cells. Procarp monocarpogonial, consisting of three-celled carpogonial branch and two two-celled subsidiary filaments, procarp cells lobulated. Cystocarp flat, protuberant, 1-2 mm in diam., scattered over the frond, each topped by one to three simple exit channel ostioles. Female specimens with extensive zones bearing young or immature off-white cystocarps. Tetrasporangia cruciately divided, 27-37 μm x 15-20 μm, originating in the subcortex. Spermatangia originating in the outer cortex, irregularly distributed, 4 μm diam.
Etymology: C. concepcionensis is named for Concepción, Central Chile, an area in which the new species is common.
Holotype: Herbarium of Museo de Historia Natural, Santiago (SGO 100665), Ç , Concepción, Golfo de Arauco, J. Vásquez, i.1981 (Fig. 2A).
Type locality: Concepción, Chile (36°59' S; 73°32' W).
Paratypes: CHILOÉ: Abtao, I. Meza, 7.ii.1984 (SGO 105299, Ç); Ancud, Mar Brava M.E.
Ramírez & G. Rojas, 4.xii.1990 (SGO 136438, Ç ); Estuario Pudeto, A. Llaña, xii.1943 (UV 631, 687, θ). VALDIVIA: Los Molinos, B. Parra, 1.xi.1984 (SGO 103999, θ), 20.i.1985 (SGO 104010, Ç ), B. Parra, i.1985 (CON 1773, Ç ); San Carlos, Corral, B. Parra, 7.v.1985 (SGO 104008, 104009, Ç ), B. Parra, iv.1983 (CON 2676, Ç ).
D.H. Kim, 30.iv.1976 (CON, Ç ); Dichato, K. Alveal, iii.1986 (CON 589, θ); Golfo de Arauco, J. Vásquez, i.1981 (SGO 100665, 100666, 100349, Ç ); Trauco, K. Alveal, 30.i.1991 (CON, Ç ); Punta Lavapié, K. Alveal, 30.i.1991 (CON, Ç ); Lebu, A. Alveal, ii.1985 (CON 594, 1700, θ); Las Escaleras, D.H. Kim, 2.vi.1976 (CON 1765, Ç ); Pichiraqui, W. Wilkomirsky, 5.v.1974 (CON 553, θ; CON, Ç ); Bahía Concepción, Punta Parra, E. Rebolledo, v.1986 (SGO, Ç ); Cocholgüe, K. Alveal, iii.1977 (CON 192, θ), H. Romo & K. Alveal, iii.1977 (CON 552, 555, θ), X. Silva, 14.xi.1981 (SGO 105310, θ), L. Ortiz, 22.vi.1982 (SGO 103986, Ç ), J. Vargas, 23.vi.1982 (SGO, Ç ), O. Mendoza, 2.vii.1982 (SGO 103989, Ç ), K. Alveal, xii.1991 (CON 2494, θ), K. Alveal, 1.iii.2000 (CON, Ç); Cocholgüe, Puertos de Coronel y Lota e Isla Santa María, N. Arakaki, 7.iv.2000, 31.v.2000, 28.vii.2000, 11.xi.2000, 16.xi.2000, 18.xi.2000, 24.ii.2001, 5.iii.2001, 24.iii.2001 (CON, Ç, θ, 154 specimens). Additional material: 77 specimens from different localities of central-south Chile (SGO and CON, Ç, θ).
Species 3 = Callophyllis macrostiolata Arakaki, Alveal et Ramírez sp. nov.
Thallus cartilagineus, subdichotome ramosus, ad 35 cm alt. Stipites elongati ad 7 cm longi, segmentis ramulosis attenuate base, axillae rotundatae, apices rotundati vel truncati, raro dentati. Cortex 4-5 stratus, cellulis isodiametricus. Cellula sustinens monocarpogonialis, procarpium 0 vel 1 ramulis subsidiariis bicellularibus 3-cellulari ramo carpogoniali, cellulae procarpii rotundae, bulbosae non lobatae. Cystocarpia magna (ad 2 mm lat.) protuberantia, super lamina dispersa, cum ostiolo singulari profundo cum margine valde inflexo. Area circa cystocarpium 150-350 μm crassum, compactum, cellulis ovoideis. Tetrasporangia cruciata, 25-27 x 15-17 μm, in cellulis periphericis inclusa et ad cellulas corticales subsuperficias affixa. Spermatangia, 4 μm, in pagina thalli irregulariter dispersa efferentia.
Cartilaginous thallus weakly dentate, with subdichotomous branching, up to 35 cm in height. Stipes elongated up to 7 cm in length, distally branched segments narrow at the base, with rounded branching points and apices. Cortex of four-five layers of isodiametric cells. Procarp monocarpogonial, supporting cell bearing a three-celled carpogonial branch and zero-one two-celled subsidiary filaments; procarp cells rounded, bulbose, non-lobulate. Cystocarps large, protuberant, up to 2 mm diam., distributed over the entire frond, each bearing a single, deep ostiole with strongly curved margins. Area surrounding cystocarp 150-350 μm thick, compact, of ovoid cells. Tetrasporangia cruciately divided, 25-27 μm x 15-17 μm, originating in the subcortex. Spermatangia originating in the outer cortex, irregularly distributed, 4 μm diam.
Etymology: C. macrostiolata is named for its large, invaginated cystocarp ostiole.
Holotype: Herbarium of Museo de Historia Natural, Santiago (SGO 159068), Ç, Isla Santa María, Concepción, Chile, coll. N. Arakaki, 17.iii.2001 (Fig. 3A).
Type locality: Isla Santa María, Concepción, Chile (36°59' S; 73°32' W).
Paratypes: CHILOÉ: Ancud, M. Robles, xi.1997 (CON, Ç ); Maullín, M. Robles, xi.1997 (CON, Ç), ix.1997 (CON, θ). OSORNO: Playa Pucatrihue, G. Arriagada, 22.i.1981 (SGO 105135, 105137, Ç; 105136, θ). VALDIVIA: San Carlos, Corral, C. Moreno, 8.iii.1980 (SGO, Ç), 10.iv.1980 (SGO, Ç). CONCEPCIÓN: Isla Santa María, N. Arakaki, 7.iv.2000, 16.xi.2000, 5.iii.2001 and 17.iii.2001 (CON, Ç, θ, 26 specimens).