Delimitating cryptic species in the Gracilaria domingensis complex (Gracilariaceae,Rhodophyta) using molecular and morphological data

Species in the genus Gracilaria that display conspicuously flattened vegetative morphologies are a taxonomically challenging group of marine benthic red algae. This is a result of their species richness, morphological similarity, and broad phenotypic plasticity. Within this group, the Gracilaria domingensis complex is one of the most common, conspicuous, and morphologically variable species along the tropical western Atlantic Ocean. Previous research has identified that members of this complex belong to two distantly related clades. However, despite this increased phylogentic resolution, species delimitations within each of these clades remain unclear. Our study assessed the species diversity within this difficult complex using morphological and molecular data from three genetic markers (cox1, UPA, and rbcL). We additionally applied six single‐marker species delimitation methods (SDM: ABGD, GMYCs, GMYCm, SPN, bPTP, and PTP) to rbcL, which were largely in agreement regarding species delimitation. These results, combined with our analysis of morphology, indicate that the G. domingensis complex includes seven distinct species, each of which are not all most closely related: G. cervicornis; a ressurected G. ferox; G. apiculata subsp. apiculata; a new species, Gracilaria baiana sp. nov.; G. intermedia subsp. intermedia; G. venezuelensis; and G. domingensis sensu stricto, which includes the later heterotypic synonym, G. yoneshigueana. Our study demonstrates the value of multipronged strategies, including the use of both molecular and morphological approaches, to decipher cryptic species of red algae.     

Species diversity of the genus Osmundea (Ceramiales,Rhodophyta) in the Macaronesian region

Species diversity within the genus Osmundea in the Macaronesian region was explored by conducting a comprehensive sampling in the Azores, the Canary, and the Madeira archipelagos. Toward identification, all specimens were first observed alive to verify the absence of corps en cerise, a diagnostic character for the genus and morphometric data were measured (thallus length and width, first‐order branches length and width, branchlets length and width, cortical cell length and width in surface view, cortical cell length and width in transverse section). Specimens were sequenced for COI‐5P (39 specimens) and three species delimitation methods (Generalized Mixed Yule Coalescent, Automatic Barcode Gap Discovery method, and Poisson Tree Processes) were used to assess the threshold between infra‐ and interspecific relationships. Subsequently, one or several sequences of plastid‐encoded large subunit of RuBisCO (21 specimens) per delimited species were generated to assess the phylogenetic relationships among Macaronesian Osmundea. Moreover, for each delineated species, vegetative and reproductive anatomy was thoroughly documented and, when possible, specimens were either assigned to existing taxa or described as novel species. This integrative approach has provided data for (i) the presence of O. oederi, O. pinnatifida, and O. truncata in Macaronesia; (ii) the proposal of two novel species, O. prudhommevanreinei sp. nov. and O. silvae sp. nov.; and (iii) evidence of an additional species referred as “Osmundea sp.1,” which is a sister taxon of O. hybrida.     

Morphology and Molecular Phylogeny of Two Poorly Known Species of Protocruzia (Ciliophora: Protocruziida)

The ciliate genus Protocruzia is a highly confused group, which was formerly placed in the class Heterotrichea or Karyorelictea, and is according to the most recent system tentatively assigned to the class Spirotrichea. In the present study, the morphology, ciliary pattern, and molecular phylogeny of two poorly known species, Protocruzia tuzeti Villeneuve‐Brachon, 1940, and Protocruzia granulosa Kahl, 1933, isolated from coastal waters of China, were investigated. Protocruzia tuzeti differs from its congeners mainly in possessing 6 adoral membranelles, 8–11 somatic kineties, and postoral dikinetids. Protocruzia granulosa is characterized by its extremely slender body, three postoral kineties, and 13 or 14 somatic kineties. The morphogenesis of P. granulosa is similar to that of P. tuzeti, especially in the parakinetal mode of stomatogenesis and the reorganization of the parental paroral membrane; however, more than one somatic kinety joins in the formation of the oral primordium in P. granulosa. Phylogenetic analyses based on small subunit ribosomal RNA gene revealed that six Protocruzia species form a fully supported clade that does not belong to any ciliate class; therefore, our data support the establishment of the class Protocruziea Gao et al. (Sci. Rep., 6, 2016, 24874).     

Species‐delimitation and phylogenetic analyses of some cosmopolitan species of Hypnea (Rhodophyta) reveal synonyms and misapplied names to H. cervicornis,including a new species from Brazil

Hypnea has an intricate nomenclatural history due to a wide pantropical distribution and considerable morphological variation. Recent molecular studies have provided further clarification on the systematics of the genus; however, species of uncertain affinities remain due to flawed taxonomic identification. Detailed analyses coupled with literature review indicated a strong relationship among H. aspera, H. cervicornis, H. flexicaulis, and H. tenuis, suggesting a need for further taxonomic studies. Here, we analyzed sequences from two molecular markers (COI‐5P and rbcL) and performed several DNA‐based delimitation methods (mBGD, ABGD, SPN, PTP and GMYC). These molecular approaches were contrasted with morphological and phylogenetic evidence from type specimens and/or topotype collections of related species under a conservative approach. Our results demonstrate that H. aspera and H. flexicaulis represent heterotypic synonyms of H. cervicornis and indicate the existence of a misidentified Hypnea species, widely distributed on the Brazilian coast, described here as a new species: H. brasiliensis. Finally, inconsistencies observed among our results based on six different species delimitation methods evidence the need for adequate sampling and marker choice for different methods.     

Phylogeography and diversification of an Amazonian understorey hummingbird: paraphyly and evidence for widespread cryptic speciation in the Plio‐Pleistocene

Straight‐billed Hermit Phaethornis bourcieri inhabits the understorey of upland terra firme forest throughout most of the Amazon basin. Currently, two allopatric taxa regarded as subspecies are recognized: P. b. bourcieri and P. b. major. However, the validity, interspecific limits and evolutionary history of these taxa are not yet fully elucidated. We use molecular characters to propose a phylogenetic hypothesis for populations and taxa grouped under Phaethornis bourcieri. Our results showed that P. bourcieri is part of the ‘Ametrornis’ clade, along with Phaethornis philippii and Phaethornis koepckeae, and that the subspecies major is more closely related to the latter two species than to populations grouped under nominate bourcieri. Our phylogenetic hypotheses recovered three main reciprocally monophyletic clades under nominate bourcieri separated by the lower Negro River and the Branco River or the Branco–Negro interfluve (clades B and C) and the upper Amazon (Solimões) or lower Marañon/Ucayali Rivers (clades C and D). Based on multi‐locus phylogeographic and population genetics approaches, we show that P. b. major is best treated as a separate species, and that P. b. bourcieri probably includes more than one evolutionary species, whose limits remain uncertain. The diversification of the ‘Ametrornis’ clade (P. bourcieri, P. philippii and P. koepckeae) is centred in the Amazon and appears to be closely linked to the formation of the modern Amazon drainage during the Plio‐Pleistocene.     

Molecular Phylogeny and Ultrastructure of Caliculium glossobalani n. gen. et sp. (Apicomplexa) from a Pacific Glossobalanus minutus (Hemichordata) Confounds the Relationships Between Marine and Terrestrial Gregarines

Gregarines are a diverse group of apicomplexan parasites with a conspicuous extracellular feeding stage, called a “trophozoite”, that infects the intestines and other body cavities of invertebrate hosts. Although the morphology of trophozoites is very diverse in gregarines as a whole, high degrees of intraspecific variation combined with relatively low degrees of interspecific variation make the delimitation of different species based on trophozoite morphology observed with light microscopy difficult. The coupling of molecular phylogenetic data with comparative morphology has shed considerable light onto the boundaries and interrelationships of different gregarine species. In this study, we isolated a novel marine gregarine from the hepatic region of a Pacific representative of the hemichordate Glossobalanus minutus, and report the first ultrastructural and molecular data from any gregarine infecting this distinctive group of hosts. Molecular phylogenetic analyses of an SSU rDNA sequence derived from two single‐cell isolates of this marine gregarine demonstrated a strong and unexpected affiliation with a clade of terrestrial gregarines (e.g. Gregarina). This molecular phylogenetic data combined with a comparison of the morphological features in previous reports of gregarines collected from Atlantic representatives of G. minutus justified the establishment of a new binomial for the new isolate, namely Caliculium glossobalani n. gen. et sp. The molecular phylogenetic analyses demonstrated a clade of terrestrial gregarines associated with a sequence acquired from a marine species, which suggest that different groups of terrestrial/freshwater gregarines evolved independently from marine ancestors.     

Diagnoses and key to the genera of the Gracilariaceae (Gracilariales,Rhodophyta)

A key to the genera of the Gracilariaceae is provided along with a short diagnosis for each genus. Features of the mature cystocarp and spermatangial configurations that separate genera are illustrated.     

Phylogeny and biogeography of Crinum L. (Amaryllidaceae) inferred from nuclear and limited plastid non-coding DNA sequences

The genus Crinum L. is the only pantropical genus of the Amaryllidaceae. Phylogenetic and biogeographical analyses of nrDNA ITS and plastid trnL-F sequences for all continental groups of the genus Crinum and related African genera are presented, with the genus Amaryllis used as outgroup. ITS indicates that C. baumii is more closely related to Ammocharis and Cybistetes than to Crinum sensu stricto . Three clades are resolved in Crinum s.s. One unites a monophyletic American group with tropical and North African species. The second includes all southern African species and the Australian endemic C. flaccidum . The third includes monophyletic Madagascar, Australasian and Sino-Himalayan clades, with southern African species. The trnL-F phylogeny resolves an American and an Asian/Madagscar clade, and confirms the relationship of C. flaccidum with species endemic to southern Africa. The salverform, actinomorphic perianths of subg. Crinum appear to have evolved several times in the genus from ancestors with zygomorphic perianths (subg. Codonocrinum ), thus neither subgenus is monophyletic. Biogeographical analyses place the origin of Crinum in southern Africa, as the region is optimized at all ancestral nodes in the tree topology, and in basal interior nodes of all but one of the major clades. The genus underwent three major waves of radiation corresponding to the three main clades resolved in our trees. Two entries into Australia for the genus are indicated, as are separate Sino-Himalayan and Australasian dispersal events.  © 2003 The Linnean Society of London, Botanical Journal of the Linnean Society , 2003, 141 , 349–363.     

Phylogeny and classification of tribe Aedini (Diptera: Culicidae)

The phylogeny and classification of tribe Aedini are delineated based on a cladistic analysis of 336 characters from eggs, fourth‐instar larvae, pupae, adult females and males, and immature stage habitat coded for 270 exemplar species, including an outgroup of four species from different non‐aedine genera. Analyses of the data set with all multistate characters treated as unordered under implied weights, implemented by TNT version 1.1, with values of the concavity constant K ranging from 7 to 12 each produced a single most parsimonious cladogram (MPC). The MPCs obtained with K values of 7–9 were identical, and that for K = 10 differed only in small changes in the relationships within one subclade. Because values of K < 7 and > 10 produced large changes in the relationships among the taxa, the stability of relationships exemplified by the MPC obtained from the K = 9 analysis is used to interpret the phylogeny and classification of Aedini. Clade support was assessed using parsimony jackknife and symmetric resampling. Overall, the results reinforce the patterns of relationships obtained previously despite differences in the taxa and characters included in the analyses. With two exceptions, all of the groups represented by two or more species were once again recovered as monophyletic taxa. Thus, the monophyly of the following genera and subgenera is corroborated: Aedes, Albuginosus, Armigeres (and its two subgenera), Ayurakitia, Bothaella, Bruceharrisonius, Christophersiomyia, Collessius (and its two subgenera), Dahliana, Danielsia, Dobrotworskyius, Downsiomyia, Edwardsaedes, Finlaya, Georgecraigius (and its two subgenera), Eretmapodites, Geoskusea, Gilesius, Haemagogus (and its two subgenera), Heizmannia (and subgenus Heizmannia), Hopkinsius (and its two subgenera), Howardina, Hulecoeteomyia, Jarnellius, Kenknightia, Lorrainea, Macleaya, Mucidus (and its two subgenera), Neomelaniconion, Ochlerotatus (subgenera Chrysoconops, Culicelsa, Gilesia, Pholeomyia, Protoculex, Rusticoidus and Pseudoskusea), Opifex, Paraedes, Patmarksia, Phagomyia, Pseudarmigeres, Rhinoskusea, Psorophora (and its three subgenera), Rampamyia, Scutomyia, Stegomyia, Tanakaius, Udaya, Vansomerenis, Verrallina (and subgenera Harbachius and Neomacleaya), Zavortinkius and Zeugnomyia. In addition, the monophyly of Tewarius, newly added to the data set, is confirmed. Heizmannia (Mattinglyia) and Verrallina (Verrallina) were found to be paraphyletic with respect to Heizmannia (Heizmannia) and Verrallina (Neomacleaya), respectively. The analyses were repeated with the 14 characters derived from length measurements treated as ordered. Although somewhat different patterns of relationships among the genera and subgenera were found, all were recovered as monophyletic taxa with the sole exception of Dendroskusea stat. nov. Fifteen additional genera, three of which are new, and 12 additional subgenera, 11 of which are new, are proposed for monophyletic clades, and a few lineages represented by a single species, based on tree topology, the principle of equivalent rank, branch support and the number and nature of the characters that support the branches. Acartomyia stat. nov. , Aedimorphus stat. nov. , Cancraedes stat. nov. , Cornetius stat. nov. , Geoskusea stat. nov. , Levua stat. nov. , Lewnielsenius stat. nov. , Rhinoskusea stat. nov. and Sallumia stat. nov., which were previously recognized as subgenera of various genera, are elevated to generic status. Catageiomyia stat. nov. and Polyleptiomyia stat. nov. are resurrected from synonymy with Aedimorphus, and Catatassomyia stat. nov. and Dendroskusea stat. nov. are resurrected from synonymy with Diceromyia. Bifidistylus gen. nov. (type species: Aedes lamborni Edwards) and Elpeytonius gen. nov. (type species: Ochlerotatus apicoannulatus Edwards) are described as new for species previously included in Aedes (Aedimorphus), and Petermattinglyius gen. nov. (type species: Aedes iyengari Edwards) and Pe. (Aglaonotus) subgen. nov. (type species: Aedes whartoni Mattingly) are described as new for species previously included in Aedes (Diceromyia). Four additional subgenera are recognized for species of Ochlerotatus, including Oc. (Culicada) stat. nov. (type species: Culex canadensis Theobald), Oc. (Juppius) subgen. nov. (type species: Grabhamia caballa Theobald), Oc. (Lepidokeneon) subgen. nov. (type species: Aedes spilotus Marks) and Oc. (Woodius) subgen. nov. (type species: Aedes intrudens Dyar), and seven are proposed for species of Stegomyia: St. (Actinothrix) subgen. nov. (type species: Stegomyia edwardsi Barraud), St. (Bohartius) subgen. nov. (type species: Aedes pandani Stone), St. (Heteraspidion) subgen. nov. (type species: Stegomyia annandalei Theobald), St. (Huangmyia) subgen. nov. (type species: Stegomyia mediopunctata Theobald), St. (Mukwaya) subgen. nov. (type species: Stegomyia simpsoni Theobald), St. (Xyele) subgen. nov. (type species: Stegomyia desmotes Giles) and St. (Zoromorphus) subgen. nov. (type species: Aedes futunae Belkin). Due to the unavailability of specimens for study, many species of Stegomyia are without subgeneric placement. As is usual with generic‐level groups of Aedini, the newly recognized genera and subgenera are polythetic taxa that are diagnosed by unique combinations of characters. The analysis corroborates the previous observation that ‘Oc. (Protomacleaya)’ is a polyphyletic assemblage of species.     

Validation of a cheap and simple nondestructive method for obtaining AFLPs and DNA sequences (mitochondrial and nuclear) in amphibians

The use of nondestructive methods for obtaining DNA from amphibians (e.g. buccal swabs) allows genetic studies to be performed without affecting the survival of the studied individuals. In this study, we compared two methods of nondestructive DNA sampling, buccal swabs and interdigital membrane or toe‐clipping, in several amphibian species of different size: Rhinella spinulosa, R. atacamensis, six species of the genus Telmatobius and Pleurodema thaul. We evaluated the integrity of the DNA extracted by sequencing fragments of mitochondrial and nuclear genes and by generating amplified fragment length polymorphisms markers (AFLPs). In all cases, we obtained an adequate amount of DNA (mean range 55–298 ng/μL). We obtained identical DNA sequences from buccal swab and interdigital membrane/toe‐clip for all individuals. The differences in the coding of AFLP markers between the tissues were similar to those reported for replicas of the same type of sample in similar analyses in other species of amphibians. In conclusion, the use of buccal swabs is a trustworthy and inexpensive method to obtain DNA for mitochondrial and nuclear sequencing and AFLP analyses. Given the types of markers evaluated, buccal swabs may be used for phylogenetic, phylogeographic and population genetic studies, even in small amphibians (<33 mm).     

Nuclear genes (but not mitochondrial DNA barcodes) reveal real species: Evidence from the Brenthis fritillary butterflies (Lepidoptera,Nymphalidae)

Discordance between entities revealed by nuclear versus mitochondrial genes is a common phenomenon in evolutionary and taxonomic studies. However, little attention has been paid to analysis of how such discordant entities correspond to traditional species detected through investigation of their morphology, ecology, and distribution. Here, we used one mitochondrial (COI, DNA barcode fragment) and four nuclear (CAD, Ca‐ATPase, arginine kinase, wg) genes to analyze the genetic structure of the taxonomically well‐studied butterfly genus Brenthis (Lepidoptera, Nymphalidae). Analysis of COI revealed multiple diverged allopatric and sympatric mitochondrial lineages within the known Brenthis species hinting at possible presence of unrecognized cryptic species. However, these multiple‐species hypotheses were not supported by further studies of nuclear genes and phenotypic traits. The discovered mitochondrial lineages did not correspond to the clusters revealed by nuclear genes. Simultaneously, we found a complete congruence between (a) traditional species boundaries, (b) clusters recognized by nuclear genes, and (c) clusters identified via cladistic analysis of phenotypic traits (genitalia and wing pattern characters, ecological preferences, and chromosome numbers). We conclude that in case of the genus Brenthis, nuclear genes rather than mtDNA barcodes reveal real species boundaries. Additionally, we suggest to support each DNA barcode‐based taxonomic conclusion by analysis of phased alleles of nuclear genes, avoiding widely used practice of nuclear and mitochondrial genes concatenation without any examination of interaction of these different types of data.     

Morphology,Ontogeny, and Molecular Phylogeny of Two Freshwater Species of Deviata (Ciliophora,Hypotrichia) from Southern China

The morphology and partial morphogenesis of two freshwater hypotrichous ciliates, Deviata brasiliensis Siqueira‐Castro et al., 2009 and Deviata rositae Küppers et al., 2007, isolated from southern China, were investigated using live observation and protargol staining. Our populations resemble the original ones in terms of their live characters and ciliary patterns. The main determinable morphogenetic features of D. brasiliensis basically correspond with those of the type population. However, the origin of anlage V for either proter or opisthe is ambiguous: whether anlage V for the proter originates from parental frontoventral row 2 (the same as in the original population) or parental frontoventral row 3 (the same as in Deviata abbrevescens) or even de novo is not clear; the anlage V for the opisthe is possibly derived from frontoventral row 3 and further migrates to frontoventral row 2, like that in D. abbrevescens. In addition, the SSU rRNA gene was first sequenced for both species. Molecular phylogenetic analyses suggest that the genus Deviata is non‐monophyletic and has a close relationship with Perisincirra paucicirrata.     

Morphology and Phylogeny of Three Pseudovorticella Species (Ciliophora: Peritrichia) from Brackish Waters of China

The biodiversity of peritrich ciliates from brackish biotopes is rarely investigated, especially members of the genus Pseudovorticella. Here, the morphology of three species of Pseudovorticella, i.e. P. cf. vestita (Stokes, 1883) Jankowski, 1976, P. spathulata sp. n., and P. qinghaiensis sp. n. isolated from brackish waters were studied. Pseudovorticella cf. vestita is characterized by inverted bell‐shaped cell; a J‐shaped macronucleus; a single contractile vacuole ventrally located; P3 three‐rowed; pellicle striated with highly developed pellicular vesicles; 18–22 transverse silverlines between peristome and aboral trochal band, and 9–13 between aboral trochal band and scopula. Pseudovorticella spathulata sp. n. differs from its congeners by the following combination of characters: elongate‐elliptical cell; a single contractile vacuole near ventral wall of infundibulum; a J‐shaped macronucleus; P3 three‐rowed; 24–34 silverlines between oral area and aboral trochal band and 6–10 between aboral trochal band and scopula. Pseudovorticella qinghaiensis sp. n. is characterized by: cell with an oval outline; a single contractile vacuole near ventral wall of infundibulum; a C‐shaped macronucleus; P3 three‐rowed; 30–35 and 9–11 transverse silverlines above and below the trochal band, respectively. The SSU rDNA sequences of five Pseudovorticella species, namely P. annulata, P. monilata, P. parakenti, P. spathulata sp. n., and P. cf. vestita, plus that of Zoothamnium hartwigi, are reported for the first time and their evolutionary relationships are investigated. Five undefined Pseudovorticella forms are considered might be conspecific with P. monilata. Two congeners are conspecific with P. spathulata sp. n. Phylogenetic analyses based on SSU rDNA sequences reveal that Pseudovorticella is not monophyletic and Z. hartwigi clusters with its congeners as expected.     

The complete chloroplast genome of Gracilariopsis lemaneiformis (Rhodophyta) gives new insight into the evolution of family Gracilariaceae

The complete chloroplast genome of Gracilariopsis lemaneiformis was recovered from a Next Generation Sequencing data set. Without quadripartite structure, this chloroplast genome (183,013 bp, 27.40% GC content) contains 202 protein‐coding genes, 34 tRNA genes, 3 rRNA genes, and 1 tmRNA gene. Synteny analysis showed plasmid incorporation regions in chloroplast genomes of three species of family Gracilariaceae and in Grateloupia taiwanensis of family Halymeniaceae. Combined with reported red algal plasmid sequences in nuclear and mitochondrial genomes, we postulated that red algal plasmids may have played an important role in ancient horizontal gene transfer among nuclear, chloroplast, and mitochondrial genomes. Substitution rate analysis showed that purifying selective forces maintaining stability of protein‐coding genes of nine red algal chloroplast genomes over long periods must be strong and that the forces acting on gene groups and single genes of nine red algal chloroplast genomes were similar and consistent. The divergence of Gp. lemaneiformis occurred ~447.98 million years ago (Mya), close to the divergence time of genus Pyropia and Porphyra (443.62 Mya).     

Phylogeny and classification of Muscini (Diptera, Muscidae)

Worldwide in distribution, the tribe Muscini comprises 21 accepted genera and about 350 species. In the present study, a cladistic analysis based upon adult morphological characters is carried out in order to discuss the monophyly of the tribe and its genera, the intergeneric relationships and, in some cases, also the intrageneric relationships. As a result, Muscini is supported as a monophyletic tribe sister-group of Stomoxyini. Except for Morellia Robineau-Desvoidy, Curranosia Paterson, and Eudasyphora Townsend, all the remaining genera are monophyletic. The results are dubious for Polietes Rondani, which was then provisionally kept unchanged. Morellia was broadened to include the Neotropical endemic genera Parapyrellia Townsend, Trichomorellia Stein, and Xenomorellia Malloch. Therefore, a new classification is proposed for Morellia in which it is divided into four subgenera: Morellia s.s. , Parapyrellia , Trichomorellia , and Xenomorellia . Furthermore, the previously proposed subgenus Dasysterna Zimin is given new status as a genus; however, as it is preoccupied by Dasysterna Dejean, the new replacement name Ziminellia nom. nov. is proposed herewith. Eudasyphora was found to be a paraphyletic group relative to Dasyphora Robineau-Desvoidy; both genera are hence synonymized, and Dasyphora is classified in three subgenera: Dasyphora s.s. , Eudasyphora , and Rypellia Malloch. The analysis demonstrated that the traditional classification of Musca Linnaeus into subgenera is artificial and, moreover, that the use of characters from male genitalia could be strongly informative for classifying the genus in phylogeny-supported species groups. Finally, the new classification proposal for Muscini recognizes 18 genera and, furthermore, two undescribed genus-ranked taxa are indicated.  © 2007 The Linnean Society of London, Zoological Journal of the Linnean Society , 2007, 149 , 493–532.     

Genetic and morphological differentiation of Porphyra and Pyropia species (Bangiales,Rhodophyta) coexisting in a rocky intertidal in Central Chile

A recent molecular taxonomic study along the Chilean coast (18° S–53° S) described 18 candidate species of bladed Bangiales of which only two were formally described. Few studies focused on local genetic and morphological diversity of bladed Bangiales and attempted to determine their intertidal distribution in contrasting habitats, and none were performed in Chile. To delimit intertidal distributions of genetic species, 66 samples of bladed Bangiales were collected at Maitencillo (32° S) in four zones: a rocky platform, a rocky wall, and two boulders zones surrounded by sandy and rocky bottoms, respectively. These samples were identified based on sequences of the mitochondrial COI and chloroplast rbcL markers. We also collected 87 specimens for morphological characterization of the most common species, rapidly assessing their putative species identity using newly developed species‐diagnostic (PCR‐RFLP) markers. Eight microscopic and two macroscopic morphological traits were measured. We described and named three of four species that predominate in Maitencillo (including Pyropia orbicularis): Pyropia variabilis Zapata, Meynard, Ramírez, Contreras‐Porcia, sp. nov., Porphyra luchea Meynard, Ramírez, Contreras‐Porcia sp. nov., and Porphyra longissima Meynard, Ramírez, Contreras‐Porcia, sp. nov. With the exception of Po. longissima restricted to boulders surrounded by sandy bottom, and a morphotype of Py. variabilis restricted to rocky walls, the other species/morphotypes have overlapping intertidal distributions. Except for Po. longissima, which is clearly differentiated morphologically (longest and thinnest blades), we conclude that morphology is not sufficient to differentiate bladed Bangiales. Our findings underscore the importance of refining our knowledge of intrinsic and environmental determinants on the distribution of bladed Bangiales.     

Polyphyly of Limoniastrum (Plumbaginaceae): evidence from DNA sequences of plastid rbcL, trnL intron and trnL-F intergene spacer

Phylogenetic relationships of Limoniastrum and other genera of subfamily Staticoideae (Plumb-aginaceae) were studied using parsimony analysis of the plastid gene rbc L, the intron of trn L and the intergene spacer of trnL-trn F. Our analysis showed that Limoniastrum was polyphyletic. Limoniastrum ifniense , in both rbc L and combined data analyses, is sister to Armeria and Psylliostachys , whereas in the trn L-F (intron and spacer combined) analysis it is sister to a clade composed of Acantholimon, Dictyolimon and the remaining species of Limoniastrum . In all analyses, the five remaining species of Limoniastrum (excluding Limoniatrum ifniense ) formed a clade with two groups of species: L. monopetalum+L. guyonianum and those sometimes considered as the segregate genus Bubania ( L.feei, L. weygandiorum and L. rechingeri ). Levels of sequence divergence among these three groups of Limoniatrum were greater than for other well supported genera in the family and, in combination with morphological differences and paucity of synapomorphies, led us to conclude that separate generic status for each of the three clades is warranted.