Ecological Differentiation of Cryptic Species within an Asexual Protist Morphospecies: A Case Study of Filamentous Green Alga Klebsormidium (Streptophyta)

Taxa of microbial eukaryotes defined on morphological basis display a large degree of genetic diversity, implying the existence of numerous cryptic species. However, it has been postulated that genetic diversity merely mirrors accumulation of neutral mutations. As a case taxon to study cryptic diversity in protists, we used a widely distributed filamentous genus, Klebsormidium, specifically the lineage E (K. flaccidum/K. nitens complex) containing a number of morphologically similar strains. Fourteen clades were recognized in the phylogenetic analysis based on a concatenated ITS rDNA + rbcL data set of more than 70 strains. The results of inferred character evolution indicated the existence of phylogenetic signal in at least two phenotypic characters (production of hydro‐repellent filaments and morphology of zoosporangia). Moreover, the lineages recovered exhibited strong ecological preferences to one of the three habitat types: natural subaerial substrata, artificial subaerial substrata, and aquatic habitats. We interpret these results as evidence of existence of a high number of cryptic species within the single morphospecies. We consider that the permanent existence of genetically and ecologically well‐defined cryptic species is enabled by the mechanism of selective sweep.     

MOLECULAR PHYLOGENY OF THE GREEN ALGAL ORDER PRASIOLALES (TREBOUXIOPHYCEAE,CHLOROPHYTA)1

The systematics of the Prasiolales was investigated by phylogenetic inference based on analyses of the rbcL and 18S rRNA genes for representatives of all four genera currently attributed to this order (Prasiococcus, Prasiola, Prasiolopsis, Rosenvingiella), including all type species. The rbcL gene had higher sequence divergence than the 18S rRNA gene and was more useful for phylogenetic inference at the ranks of genus and species. In the rbcL gene phylogeny, three main clades were observed, corresponding to Prasiola, Prasiolopsis, and Rosenvingiella. Prasiococcus was nested among species of Prasiola occurring in subaerial and supralittoral habitats. Trichophilus welckeri Weber Bosse, a subaerial alga occurring in the fur of sloths in Amazonia, was closely related to Prasiolopsis ramosa Vischer. The species of Prasiola were grouped into three well‐supported clades comprising (i) marine species, (ii) freshwater and terrestrial species with linear blades, and (iii) terrestrial species with rounded or fan‐shaped blades. Sequence divergence was unexpectedly low in the marine group, which included species with different morphologies. For the 18S rRNA gene, the phylogenetic analyses produced several clades observed for the rbcL gene sequence analysis, but, due to very little sequence variation, it showed considerably lower resolution for inference at the species and genus levels. Due to the low support of some internal branches, the results of the analyses did not allow an unambiguous clarification of the origin and the early evolution of the Prasiolales.     

PHYLOGENETIC POSITION OF CRUSTOMASTIX STIGMATICA SP. NOV. AND DOLICHOMASTIX TENUILEPIS IN RELATION TO THE MAMIELLALES (PRASINOPHYCEAE,CHLOROPHYTA)1

A new marine microalga from the Mediterranean Sea, Crustomastix stigmatica Zingone, is investigated by means of LM, SEM, TEM, and pigment and molecular analyses (nuclear‐encoded small subunit [SSU] rDNA and plastid‐encoded rbcL). Pigment and molecular information is also provided for the related species Dolichomastix tenuilepis Throndsen et Zingone. Crustomastix stigmatica has a bean‐shaped cell body 3–5 μm long and 1.5–2.8 μm wide, with two flagella four to five times the body length. The single chloroplast is pale yellow‐green, cup‐shaped, and lacks a pyrenoid. A small bright yellow stigma is located in the mid‐dorsal part of the cell under the chloroplast membrane. An additional accumulation of osmiophilic globules is at times seen in a chloroplast lobe. Cells lack flat scales, whereas three different types of hair‐like scales are present on the flagella. The main pigments of C. stigmatica are those typical of Mamiellales, though siphonein/siphonaxanthin replaces prasinoxanthin and uriolide is absent. The pigment pool of D. tenuilepis is more similar to that of Micromonas pusilla (Butcher) Manton et Parke and of other Mamiellales. The nuclear SSU rDNA phylogeny shows that the inclusion of C. stigmatica and D. tenuilepis in the Mamiellales retains monophyly for the order. The two species form a distinct clade, which is sister to a clade including all the other Mamiellales. Results of rbcL analyses failed to provide phylogenetic information at both the order and species level. No unique morphological or pigment characteristics circumscribe the mamiellalean clade as a whole nor its two daughter clades.     

57 Systematics of hydropuntia montagne (gracilariaceae,gracilariales, rhodophyta) based on comparative morphology and rbcL sequence analysis

The taxonomic validity of the genus Hydropuntia Montagne (1843) (including Polycavernosa) within the Gracilariaceae (Gracilariales, Rhodophyta) is controversial. Morphological characters that define species of Hydropuntia are said to be variable and to overlap with those of Gracilaria. Here we present a global phylogenetic study of the family based on a Bayesian analysis of a large rbcL DNA sequence dataset indicating that the genus Hydropuntia forms a well supported monophyletic clade within the family, and recognize Hydropuntia as a genus distinct from Gracilaria. We also conducted smaller phylogenetic analyses in which thirty four Hydropuntia rbcL sequences resulted in two major clades within the genus, comprising a Caribbean clade and an Indo‐Pacific clade. Diagnostic reproductive stages that separate these two clades will be illustrated.     

DEFINING TAXON BOUNDARIES IN MEMBERS OF THE MORPHOLOGICALLY AND GENETICALLY PLASTIC GENES CAULERPA (CAULERPALES,CHLOROPHYTA)1

Numerous attempts to capture the morphological variability of the genus Caulerpa have resulted in an unstable classification of numerous varieties and formae. In the present study we attempted to test taxon boundaries by investigating morphological and genetic variation within and between seven taxa of Caulerpa, supposedly belonging to four species, sampled at different sites in a Philippine reef system. Using both field and culture observations, we described the relation between the variability of a set of morphological characters and ecological parameters, such as wave exposure, light intensity, and substrate type. Statistical analyses showed that the limits between two (out of three) ecads of the C. racemosa (Forsskål) J. Agardh complex were obscured by the presence of morphological plasticity. Other studied taxa of Caulerpa (i.e. C. cupressoides [Vahl] C. Agardh, C. serrulata [Forsskål] J. Agardh, and two formae of C. sertularioides [S. Gmelin] Howe) could be grouped based on morphology despite the presence of morphological plasticity. Our results indicated a strong association between light intensity and several quantitative morphological variables. Genetic diversity of these taxa was assessed by partial sequencing chloroplast rbcL and tufA genes and the ycf10‐chlB chloroplast spacer. In all phylogenetic analyses, C. serrulata, C. cupressoides, C. sertularioides, and the three ecads of C. racemosa emerged as distinct genetic units. Despite the presence of morphological plasticity and morphological convergence, a subset of morphological characters traditionally used in taxonomic delimitation still had sufficient discriminative power to recognize the terminal phylogenetic clades.     

Hidden biodiversity: total evidence phylogenetics and evolution of morphological traits in a highly diverse lineage of endogean ground beetles,Typhlocharis Dieck, 1869 (Carabidae,Trechinae, Anillini)

Typhlocharis is the most diverse eyeless endogean ground beetle genus known to date, with 62 species all endemic to the West Mediterranean region. The lineage is characterized by a conservative and singular body plan within Carabidae that contrasts with a high morphological diversity in many traits. We provide an exhaustive phylogeny of the lineage through the study of 92 morphological characters from all 62 described species and 45 potential new species from 70 additional populations, and the combination of morphological and available molecular data, in the first total evidence phylogenetic approach for a highly diverse endogean lineage. We tracked the evolution of morphological traits over the obtained phylogenies. Results suggest eight morphologically distinct clades, which do not correspond to the species groups proposed formerly. Ancestral state reconstructions and phylogenetic signal analyses of morphological traits revealed that some of the previously key characters to the classification of Typhlocharis, such as the umbilicate series or the apical denticles of elytra, are highly homoplasic, whereas other characters show stronger phylogenetic signal, including structures in the antennae, gula, pronotum and last abdominal ventrite. This evidence supports the split of Typhlocharis into three genera: Lusotyphlus gen. nov. ; Typhlocharis Dieck, 1869 and Microcharidius Coiffait, 1969 (revalidated), forming the subtribe Typhlocharina Jeanne, 1973.     

ASSESSING PHYLOGENETIC AFFINITIES AND SPECIES DELIMITATIONS IN KLEBSORMIDIALES (STREPTOPHYTA): NUCLEAR‐ENCODED rDNA PHYLOGENIES AND ITS SECONDARY STRUCTURE MODELS IN KLEBSORMIDIUM,HORMIDIELLA, AND ENTRANSIA1

Nuclear‐encoded SSU, group I intron, and internal transcribed spacer (ITS) rDNA sequences were obtained for 16 strains of green algae representing species of Klebsormidium, Hormidiella attenuata, and Entransia fimbriata (for taxonomic authorities, see Table S1 in the supplementary material). The SSU phylogeny resolved a well‐supported clade Klebsormidiales in the Streptophyta that comprised authentic Klebsormidium isolates described recently in a monograph by G. M. Lokhorst and various strains from culture collections. The H. attenuata and En. fimbriata pair was the sister group of Klebsormidium. Certain isolates from culture collections previously identified as “Klebsormidium” emerged as Trebouxiophyceae. Strains assigned to Koliella, Gloeotila, and Stichococcus previously allied with Klebsormidium because of shared morphological and ultrastructural characteristics also belonged to Trebouxiophyceae. Group I introns inserted at Escherichia coli position 516 were found in K. nitens and SAG strain 384‐1, and at position 1506 in H. attenuata and En. fimbriata. Introns were not observed in other Klebsormidiales. Unambiguous alignment of ITS regions of Klebsormidiales was only possible after thermodynamic folding had predicted eight conserved helical domains. The ITS phylogeny provided support for five of the morphospecies recognized by Lokhorst (K. flaccidum, K. elegans, K. bilatum, K. crenulatum, K. mucosum), but the sequences of K. dissectum, K. fluitans, and K. nitens formed an unresolved clade. The species with the earliest origin in the Klebsormidium phylogeny was K. flaccidum. The incongruence between Lokhorst’s morphology‐based cladograms and the ITS phylogenies demonstrated the need for a critical reappraisal of the taxonomy and the morphological and molecular species concept in Klebsormidium on the basis of a more extensive taxonomic and geographic sampling strategy.     

Habitat-specific composition of morphotypes with low genetic diversity in the green algal genus Klebsormidium (Streptophyta) isolated from biological soil crusts in Central European grasslands and forests

Terrestrial filamentous green algae of the widely distributed, cosmopolitan genus Klebsormidium (Klebsormidiophyceae, Streptophyta) are typical components of biological soil crusts (BSCs). These communities occur in all climatic zones and on all continents, where soil moisture is limited or where there has been disturbance. BSCs form water-stable aggregates that have important ecological roles in primary production, nitrogen fixation, nutrient cycling, water retention and stabilization of soils. Although available data on Klebsormidium are limited, its functional importance in BSCs is regarded as high. Therefore, in the present study Klebsormidium strains were isolated from BSCs sampled from various grassland and forest plots of different land use intensities in Central Europe, as provided by the Biodiversity Exploratories, and its intraspecific genetic diversity was evaluated. Previous phylogenetic analyses revealed a relationship between sequence similarity and habitat preference with a higher genetic diversity than expected from a morphological classification. We isolated and sequenced 75 Klebsormidium strains. The molecular phylogeny based on the ITS regions showed that all strains belong to either the previously described clade B/C or clade E. This classification was supported by morphological characteristics: strains assigned to clade B/C were identified as Klebsormidium cf. flaccidum or Klebsormidium cf. dissectum, and strains from clade E as K. nitens or Klebsormidium cf. subtile. Within one clade the strains showed low sequence divergences. These minor differences were independent of the sampling region and land use intensity. Interestingly, most of the strains assigned to clade E were isolated from forest sites, whereas strains from clade B/C occurred equally in grassland and forest sites. Therefore, it is reasonable to assume that habitat with its microenvironmental conditions, and not biogeography, controls genetic diversity in Klebsormidium.     

GLOBAL SYSTEMATIC AND PHYLOGENETIC ANALYSIS OF SARGASSUM IN THE GULF OF MEXICO,CARIBBEAN AND PACIFIC BASIN

Sargassum is one of the most species‐rich genera in the brown algae with over 400 described species worldwide. The bulk of these species occurs in Pacific‐Indian ocean waters with only a small portion found on the Atlantic side of the Isthmus of Panama. Sargassum also has one of the most subdivided and complex taxonomic systems used within the algae. Systematic distinctions within the genus are further complicated by high rates of phenotypic variability in several key morphological characters. Molecular analyses in such systems should allow testing of systematic concepts while providing insights into speciation and evolutionary patterns. Global molecular phylogenetic analyses using both conserved and variable regions of the Rubisco operon (rbcL and rbcL‐IGS‐rbcS) were performed with species from the Gulf of Mexico, Caribbean, and Pacific basin. Results confirm earlier analyses based on rbcL‐IGS‐rbcS from Pacific species at the subgeneric and sectional level while providing additional insights into the systematics and phylogenetics on a global scale. For example, species east of the Isthmus of Panama form a distinct well‐resolved clade within the tropical subgenus. This result in sharp contrast to traditional systematic treatments but provides a window into the evolutionary history of this genus in the Pacific and Atlantic Ocean basins and a possible means to time speciation events.     

Phylogeny in Echinocereus (Cactaceae) based on combined morphological and molecular evidence: taxonomic implications

Echinocereus is a morphologically diverse genus that includes 64 species grouped into eight taxonomic sections based on morphological traits. In previous molecular phylogenetic analyses, the relationships amongst Echinocereus species were not entirely revealed and useful characters to recognize clades were not provided. The inclusion of several sources of evidence in a phylogenetic analysis is likely to produce more supported hypotheses. Therefore, we performed a combined phylogenetic analysis with a set of 44 morphological characters and six chloroplast DNA sequences. Topologies from parsimony and Bayesian analyses were mostly congruent. However, the relationships of E. poselgeri were not consistent between analyses. A second Bayesian analysis using a long-branch extraction test resulted in a topology with the morphological position of E. poselgeri congruent with that in parsimony analysis. Parsimony and Bayesian analyses corroborated the monophyly of Echinocereus, which included eight monophyletic groups. The combined phylogeny integrated into different clades those taxa that were not determined in previous analyses and changed the relationships of some recognized clades. The clades did not recover the recent infrageneric classification. In the present study, a new sectional classification for Echinocereus is proposed based on the eight recovered clades, which is supported by a combination of morphological and molecular characters. An identification key for sections in the genus is included.     

Phylogeny of Impatiens (Balsaminaceae): integrating molecular and morphological evidence into a new classification

Impatiens L. is one of the largest angiosperm genera, containing over 1000 species, and is notorious for its taxonomic difficulty. Here, we present, to our knowledge, the most comprehensive phylogenetic analysis of the genus to date based on a total evidence approach. Forty‐six morphological characters, mainly obtained from our own investigations, are combined with sequence data from three genetic regions, including nuclear ribosomal ITS and plastid atpB‐rbcL and trnL‐F. We include 150 Impatiens species representing all clades recovered by previous phylogenetic analyses as well as three outgroups. Maximum‐parsimony and Bayesian inference methods were used to infer phylogenetic relationships. Our analyses concur with previous studies, but in most cases provide stronger support. Impatiens splits into two major clades. For the first time, we report that species with three‐colpate pollen and four carpels form a monophyletic group (clade I). Within clade II, seven well‐supported subclades are recognized. Within this phylogenetic framework, character evolution is reconstructed, and diagnostic morphological characters for different clades and subclades are identified and discussed. Based on both morphological and molecular evidence, a new classification outline is presented, in which Impatiens is divided into two subgenera, subgen. Clavicarpa and subgen. Impatiens; the latter is further subdivided into seven sections.     

Developments in the taxonomy of silica‐scaled chrysophytes – from morphological and ultrastructural to molecular approaches

Taxonomy in silica‐scaled chrysophytes has gone through three morphological phases. From primary studies of the cell morphology in the 18th century, the focus was in the 20th century replaced by studies of the silica structures of the cell envelope. Now, in the latest decades the importance of DNA sequencing has been recognized, not only to support the taxonomic framework but also to obtain new understanding of taxonomic relations among particular taxa. In the first part of this review, we provide a historical overview of the developments in the taxonomy of scale‐bearing chrysophytes. In the second part, we present a phylogenetic reconstruction of chrysophyte algae, updated by newly obtained SSU rDNA and rbcL sequences of several isolated Synura, Mallomonas and Chrysosphaerella species. We detected significant incongruence between the phylogenies obtained from the different datasets, with the SSU rDNA phylogram being the most congruent with the morphological data. Significant saturation of the first rbcL codon position could indicate the presence of positive selection in the rbcL dataset. Within the Synurales, the relationships revealed by the phylogenetic analyses highlight the artificial infragenetic classification of Mallomonas and Synura, and the occurrence of cryptic diversity within a number of traditionally defined species. Finally, three new combinations are proposed based on the phylogenetic analyses: Tessellaria lapponica, Synura asmundiae and S. bjoerkii.     

MOLECULAR AND MORPHOMETRIC DATA PINPOINT SPECIES BOUNDARIES IN HALIMEDA SECTION RHIPSALIS (BRYOPSIDALES,CHLOROPHYTA)1

Molecular systematic studies have changed the face of algal taxonomy. Particularly at the species level, molecular phylogenetic research has revealed the inaccuracy of morphology‐based taxonomy: Cryptic and pseudo‐cryptic species were shown to exist within many morphologically conceived species. This study focused on section Rhipsalis of the green algal genus Halimeda. This section was known to contain cryptic diversity and to comprise species with overlapping morphological boundaries. In the present study, species diversity within the section and identity of individual specimens were assessed using ITS1–5.8S–ITS2 (nrDNA) and rps3 (cpDNA) sequence data. The sequences grouped in a number of clear‐cut genotypic clusters that were considered species. The same specimens were subjected to morphometric analysis of external morphological and anatomical structures. Morphological differences between the genotypic cluster species were assessed using discriminant analysis. It was shown that significant morphological differences exist between genetically delineated species and that allocation of specimens to species on the basis of morphometric variables is nearly perfect. Anatomical characters yielded better results than external morphological characters. Two approaches were offered to allow future morphological identifications: a probabilistic approach based on classification functions of discriminant analyses and the classical approach of an identification key.     

The systematics of Lobophora (Dictyotales,Phaeophyceae) in the western Atlantic and eastern Pacific oceans: eight new species

Lobophora is a common tropical to temperate genus of brown algae found in a plethora of habitats including shallow and deep‐water coral reefs, rocky shores, mangroves, seagrass beds, and rhodoliths beds. Recent molecular studies have revealed that Lobophora species diversity has been severely underestimated. Current estimates of the species numbers range from 100 to 140 species with a suggested center of diversity in the Central Indo‐Pacific. This study used three molecular markers (cox3, rbcL, psbA), different single‐marker species delimitation methods (GMYC, ABGD, PTP), and morphological evidence to evaluate Lobophora species diversity in the Western Atlantic and the Eastern Pacific oceans. Cox3 provided the greatest number of primary species hypotheses(PSH), followed by rbcL and then psbA. GMYC species delimitation analysis was the most conservative across all three markers, followed by PTP, and then ABGD. The most informative diagnostic morphological characters were thallus thickness and number of cell layers in both the medulla and the dorsal/ventral cortices. Following a consensus approach, 14 distinct Lobophora species were identified in the Western Atlantic and five in the Eastern Pacific. Eight new species from these two oceans were herein described: L. adpressa sp. nov., L. cocoensis sp. nov., L. colombiana sp. nov., L. crispata sp. nov., L. delicata sp. nov., L. dispersa sp. nov., L. panamensis sp. nov., and L. tortugensis sp. nov. This study showed that the best approach to confidently identify Lobophora species is to analyze DNA sequences (preferably cox3 and rbcL) followed by comparative morphological and geographical assessment.     

Toward Modern Classification of Eustigmatophytes,Including the Description of Neomonodaceae Fam. Nov. and Three New Genera1

The class Eustigmatophyceae includes mostly coccoid, freshwater algae, although some genera are common in terrestrial habitats and two are primarily marine. The formal classification of the class, developed decades ago, does not fit the diversity and phylogeny of the group as presently known and is in urgent need of revision. This study concerns a clade informally known as the Pseudellipsoidion group of the order Eustigmatales, which was initially known to comprise seven strains with oval to ellipsoidal cells, some bearing a stipe. We examined those strains as well as 10 new ones and obtained 18S rDNA and rbcL gene sequences. The results from phylogenetic analyses of the sequence data were integrated with morphological data of vegetative and motile cells. Monophyly of the Pseudellipsoidion group is supported in both 18S rDNA and rbcL trees. The group is formalized as the new family Neomonodaceae comprising, in addition to Pseudellipsoidion, three newly erected genera. By establishing Neomonodus gen. nov. (with type species Neomonodus ovalis comb. nov.), we finally resolve the intricate taxonomic history of a species originally described as Monodus ovalis and later moved to the genera Characiopsis and Pseudocharaciopsis. Characiopsiella gen. nov. (with the type species Characiopsiella minima comb. nov.) and Munda gen. nov. (with the type species Munda aquilonaris) are established to accommodate additional representatives of the polyphyletic genus Characiopsis. A morphological feature common to all examined Neomonodaceae is the absence of a pyrenoid in the chloroplasts, which discriminates them from other morphologically similar yet unrelated eustigmatophytes (including other Characiopsis-like species).     

Phylogeny,biogeography, and morphological ancestral character reconstruction in the Mediterranean genus Fumana (Cistaceae)

Fumana is a diverse genus of the Cistaceae family, consisting of 21 currently accepted species. In this study, nuclear (ITS) and plastid (matK, trnT‐L) molecular markers were used to reconstruct the phylogeny and to estimate divergence times, including 19 species of Fumana. Phylogenetic analyses (Bayesian Inference, Maximum Parsimony and Maximum Likelihood) confirmed the monophyly of Fumana and did not support the infrageneric divisions previously established. The results support four main clades that group species that differ in vegetative and reproductive characters. Given the impossibility to define morphological characters common to all species within the clades, our proposal is to reject infrageneric divisions. Molecular dating and ancestral area analyses provide evidence for a Miocene diversification of the genus in the north‐western Mediterranean. Ancestral state reconstructions revealed ancestral character states for some traits related to xeric and arid habitats, suggesting a preadaptation to the Mediterranean climate.