Molecular evidence for the synonymy of two species of Apatemon Szidat, 1928, A. gracilis (Rudolphi, 1819) and A. annuligerum (von Nordmann, 1832) (Digenea: Strigeidae) parasitic as metacercariae in British fishes

The current study examined rDNA (internal transcribed spacer regions, ITS1 and ITS2) and cytochrome c oxidase subunit 1 (CO1) sequence data of Apatemon annuligerum (originating from two geographical locations) and A. gracilis metacercariae (originating from four natural piscine hosts) to determine the systematic status of these two strigeid digeneans. With the exception of short repeat motifs, the ITS1 regions sequenced demonstrated no intra- or inter-specific sequence variation. ITS2 sequences were 292 bp and CO1 sequences 366 bp in length and identical for both nominal Apatemon species. These sequence data provide strong evidence that the two species are con-specific and that A. annuligerum should be regarded as a junior synonym of A. gracilis.     

Observations on the morphology and life-cycle of Procerovum varium (Onji & Nishio, 1916) (Trematoda: Heterophyidae)

The life-cycle of Procerovum varium (Digenea: Haplorchiinae) was studied experimentally and the morphology of stages in the life-cycle has been described and illustrated. Infections with adult flukes were found in the pond heron Ardeola grayii and heavy infections with metacercariae were found, attached to the liver of the fish Oryzias melastigma (Oryziatidae) occurring in a freshwater stream situated in Visakhapatnam, India. The cercariae developing in the snail Thiara tuberculatapossessed typical haplorchiine features and were characterised by the presence of numerous cystogenous glands. Early stages of metacercarial development occurred free in the muscles of the fish intermediate host. The larvae reached the liver at 5 days post-infection, encystment commenced 2 days later and 15-day-old metacercariae were found to be infective to chicks, ducks and mice that served as suitable experimental hosts. The adult flukes obtained from natural and experimental infections showed many intraspecific variations, especially in the size and shape of the expulsor which depend on the quantity of sperm it contains. The validity of various species described in the genus and differentiated on the basis of differences in the size of the expulsor has been examined. It is concluded that only three species of the genus, namely P. varium, P. cheni and P. calderoni, are valid. "P. sisoni" of Chen (1949) is confirmed as a synonym of P. varium. P. varium is reported for the first time from India.     

The life-cycle of Plagiorchis maculosus (Rudolphi, 1802) Braun, 1902 (Trematoda: Plagiorchiidae), a parasite of swallows (Hirundinidae)

During 1984 eggs of Plagiorchis maculosus were obtained from the faeces of naturally infested nestlings of Hirundo rustica and Delichon urbica near Ulm, Federal Republic of Germany. Developmental stages and adults were reared in the laboratory using Lymnaea stagnalis and Radix auricularia for daughter-sporocysts and cercariae, larvae of Chaoborus for metacercariae, and a canary for adults. Eggs from the faeces of the canary were reared once more up to the metacercarial stage for comparison. Infection experiments were negative with Radix peregra and Galba palustris as first intermediate hosts and chickens as final hosts. The stages of the life-cycle are figured and described in detail, including the chaetotaxy of the cercaria. Characteristics distinguishing P. maculosus from related species are discussed. A medium for in vitro excystation of metacercariae is presented. It is suggested that the range of the final hosts and the geographical distribution of P. maculosus be reconsidered in the light of study of its larval stages. ac]19860715     

GEOGRAPHICAL CORRELATION OF MORPHOLOGICAL VARIATION IN SILVETIA COMPRESSA (FUCACEAE,FUCALES, PHAEOPHYCEAE)

Silvetia compressa ( J. Agardh) Serrão et al. is a common member of the upper intertidal fucoid community on the Pacific coast of America from Humboldt County, California, to Punta Baja, Baja California, Mexico. A relatively narrow range of morphological variability is exhibited by most mainland populations, regardless of latitude, but some mainland populations and all insular populations participate in a complex pattern that we have attempted to analyze. A few populations on the Monterey Peninsula in which the fronds are atypically delicate were described by Setchell & Gardner as f. gracilis, to which was assigned a population from Santa Catalina Island. After comparing populations from various parts of the range of the species, including all of the Channel Islands, we conclude that two subspecies may be recognized. In subsp. compressa, which includes f. gracilis as a growth form and occurs chiefly on the mainland, the frond is robust with long tapered receptacles. In the variant subspecies, which is chiefly insular but also occurs on the coast of northern Baja California, the typical frond has slender axes as in f. gracilis, but is more densely branched and has short ellipsoidal receptacles. Comparison of nucleotide sequences from the ITS regions of rDNA revealed an identical pattern for subsp. compressa from Baja California and central California, including populations assignable to f. gracilis. By contrast, the pattern for the variant subspecies differed by 2 bp (0.3%) from that of subsp. compressa.     

Variation in the morphology of adult Apatemon gracilis Rudolphi, 1819 (Digenea: Strigeidae) reared in different avian hosts

Adult Apatemon gracilis (Rudolphi, 1819) were reared experimentally in three different avian hosts: herring gulls Larus argentatus Gmelin, domestic chicks Gallus gallus (L.) and eider ducklings Somateria mollissima (L.). Comparisons of size, body proportions and fecundity were made between these and specimens obtained from a naturally infected goosander Mergus merganser L. The eider duck proved to be a suitable experimental host, rearing adults of comparable size and fecundity to those from the natural host. The growth and development of the parasite in domestic chicks and herring gulls were significantly reduced. Similar conditions are likely to exist in the natural environment and this should be taken into consideration when choosing experimental hosts for use in the laboratory or when identifying species.     

Intercontinental distributions,phylogenetic position and life cycles of species of Apharyngostrigea (Digenea,Diplostomoidea) illuminated with morphological,experimental, molecular and genomic data

When subjected to molecular study, species of digeneans believed to be cosmopolitan are usually found to consist of complexes of species with narrower distributions. We present molecular and morphological evidence of transcontinental distributions in two species of Apharyngostrigea Ciurea, 1924, based on samples from Africa and the Americas. Sequences of cytochrome c oxidase I and, in some samples, internal transcribed spacer, revealed Apharyngostrigea pipientis (Faust, 1918) in Tanzania (first known African record), Argentina, Brazil, USA and Canada. Sequences from A. pipientis also match previously published sequences identified as Apharyngostrigea cornu (Zeder, 1800) originating in Mexico. Hosts of A. pipientis surveyed include definitive hosts from the Afrotropic, Neotropic and Nearctic, as well as first and second intermediate hosts from the Americas, including the type host and type region. In addition, metacercariae of A. pipientis were obtained from experimentally infected Poecilia reticulata, the first known record of this parasite in a non-amphibian second intermediate host. Variation in cytochrome c oxidase I haplotypes in A. pipientis is consistent with a long established, wide-ranging species with moderate genetic structure among Nearctic, Neotropic and Afrotropic regions. We attribute this to natural dispersal by birds and find no evidence of anthropogenic introductions of exotic host species. Sequences of CO1 and ITS from adult Apharyngostrigea simplex (Johnston, 1904) from Egretta thula in Argentina matched published data from cercariae from Biomphalaria straminea from Brazil and metacercariae from Cnesterodon decemmaculatus in Argentina, consistent with previous morphological and life-cycle studies reporting this parasite—originally described in Australia—in South America. Analyses of the mitochondrial genome and rDNA operon from A. pipientis support prior phylogenies based on shorter markers showing the Strigeidae Railliet, 1919 to be polyphyletic.     

Ithyoclinostomum yamagutii n. sp. (Digenea: Clinostomidae) in the great blue heron Ardea herodias L. (Aves: Ardeidae) from Mississippi,USA

With only six recognised genera, the family Clinostomidae Lühe, 1901 remains a global research interest of parasitologists and ecologists. Recent efforts have focused on providing molecular data to investigate species diversity, elucidate life-cycles, and make inferences on the group’s evolutionary history. Of the clinostomid genera, the monotypic Ithyoclinostomum Witenberg, 1926 has remained more enigmatic compared to the commonly encountered Clinostomum Leidy, 1856. Recent morphological and molecular evidence from metacercariae suggests a second Ithyoclinostomum species may exist in freshwater cichlids in Central America and Mexico. In a recent survey of great blue herons Ardea herodias L. from commercial catfish production farms in Mississippi, USA, two specimens of an abnormally large (> 20 mm) clinostomid were encountered in the oesophagus of a single bird. These specimens were identified as an Ithyoclinostomum sp. morphologically distinct from the only nominal species Ithyoclinostomum dimorphum (Diesing, 1850). Using morphological and molecular data these adult specimens were confirmed as conspecific with the larval metacercariae previously described from Central America and Mexico and represent the novel species, Ithyoclinostomum yamagutii n. sp.

     

Migration, site selection, and development of Ornithodiplostomum sp. metacercariae (Digenea: Strigeoidea) in fathead minnows (Pimephales promelas)

The metacercarial stage of trematodes is typically considered an encysted, developmentally quiescent, resting stage. Yet the metacercariae of some species of strigeoid trematode undergo extravagant development within specific tissues of their second intermediate host. Our understanding of patterns of migration, site selection and development of these types of metacercariae is known for only a few species. In this study, we characterize the invasion and development of Ornithodiplostomum sp. metacercariae in their second intermediate host, the fathead minnow, Pimephales promelas. Diplostomules completed their migration into the abdominal cavity between 15 min and 48 h p.i. Most diplostomules migrated along muscular and connective tissue then penetrated the peritoneal lining of the abdominal cavity en route to the liver or pancreas. Alternatively, some diplostomules migrated within the host’s circulatory system, including the heart and arteries of the hepatic portal system. Metacercarial development in the liver and pancreas involved distinct growth, encystment and consolidation phases. Metacercarial volume increased 15-fold between 48 h and 4 weeks p.i., presumably due to absorptive and/or ingestive feeding activities within host tissues. By 2 weeks p.i., metacercariae were enveloped within a cyst wall and they were found loosely attached to the surfaces of internal tissues or unattached within the body cavity. These results emphasize the complex nature of metacercarial migration and growth and demonstrate that their growth and encystment phases occur within different habitats within their intermediate hosts.     

Genetic variation in the soil phthiracarid mite, Steganacarus (S.) magnus (Nicolet, 1855) (Acarida, Oribatida) (Notulae Oribatologicae XL)

Genetic divergence and variability at 14 enzyme loci were examined in and between Italian populations of two edaphic Oribatid (Acarida, Oribatida) species, Steganacarus (Steganacarus) magnus (8 populations) and S. (S.) hirsutus (1 population). The seven populations belonging to S. (S.) magnus can be divided into two groups according to their phenotype, form anomala (A) (MON, ARG, AST, CDO) and form magna (M) (MAL, MAM, RIF) while another can be considered as hybrid between the two preceding groups (ZOC). Genetic identity (I) values between the S. (S.) magnus populations in spite of their morphological differences ranged from 0.977 to 1.000 showing the great genetic similarity of simple local populations while those of S. (S.) hirsutus indicated two distinct morphological species. The genetic distances between all the populations examined were very low despite the ecological differences and geographical distances between the collecting sites. Genetic variability estimates in all the populations of both species were very low when compared to those reported for most arthropods. Some explanatory considerations are suggested.     

Examining the phylogeny of the Australasian Lymnaeidae (Heterobranchia: Pulmonata: Gastropoda) using mitochondrial, nuclear and morphological markers

We examined the species groups relationships of the freshwater snail genus Austropeplea using mitochondrial, nuclear and morphological markers in addition to traditional methods of shell shape analysis. Based primarily on the results of a combined molecular and morphological analysis, samples of the nominal species A. tomentosa form distinct lineages. The New Zealand populations of A. tomentosa are a very distinct lineage from any of the Australian populations attributed to A. tomentosa. Furthermore, within the Australian group, three lineages, south Australia, Tasmania and eastern Australia, appear to have undergone recent and/or rapid speciation events. Samples assigned to A. lessoni were resolved as two distinct lineages, representing the eastern and northern Australian populations. Kutikina hispida was resolved within the Australian A. tomentosa clade. Molecular results for A. viridis suggests that it is also composed of at least two distinct lineages that could be treated as species. Incongruence observed between the single mitochondrial, nuclear and morphological topologies highlight the importance of using a number of different datasets in the delimitation of species-group taxa.     

Morphological and genetic distinctiveness of metallicolous and non-metallicolous populations of Armeria maritima s.l. (Plumbaginaceae) in Poland

Patterns of morphological, genetic and epigenetic variation (DNA methylation pattern) were investigated in metallicolous (M) and non‐metallicolous (NM) populations of Armeria maritima. A morphological study was carried out using plants from six natural populations grown in a greenhouse. Morphological variation was assessed using seven traits. On the basis of this study, three representative populations were selected for molecular analyses using metAFLP to study sequence‐ and methylation‐based DNA variation. Only one morphological trait (length of outer involucral bracts) was common to both metallicolous populations studied; however, the level of variation was sufficient to differentiate between M and NM populations. Molecular analyses showed the existence of naturally occurring epigenetic variation in A. maritima populations, as well as structuring into distinct between and within population components. We show that patterns of population genetic structure differed depending on the information used in the study. Analysis of sequence‐based information data demonstrates the presence of three well‐defined and genetically differentiated populations. Methylation‐based data show that two major groups of individuals are present, corresponding to the division into M and NM populations. These results were confirmed using different analytical approaches, which suggest that the DNA methylation pattern is similar in both M populations. We hypothesise that epigenetic processes may be involved in microevolution leading to development of M populations in A. maritima.     

Multidisciplinary approaches for species delimitation in Sisyrinchium (Iridaceae)

A remarkable morphological similarity in natural populations of different groups of Sisyrinchium spp. (Iridaceae) makes classification of some species difficult. The present paper focused on two morphological categories of S. sellowianum (MC‐I and MC‐II) with distinct morphological traits. Morphological, cytogenetic, reproductive biology and genetic studies were performed, aiming to elucidate and better characterize such variation. The basic chromosome number established for the species was x = 9 and diploid and tetraploid populations were observed for MC‐I, whereas MC‐II showed only diploid populations. Different pollen morphologies were recognized in each morphological category. Based on pollination experiments, MC‐I is out‐crossing, whereas MC‐II is selfing. The populations were highly differentiated (F_ST = 0.46, θ^B = 0.62). In MC‐I, observed variation was greater within populations (69%) than among populations (31%), whereas in MC‐II, 61% of the variation was among populations and 39% within populations. This study evidenced that the two morphological categories correspond to distinct and reproductively isolated species and that floral traits reflect their breeding system, with allogamous plants in MC‐I and autogamous in MC‐II. Furthermore, MC‐II is unveiled as S. platycaule, a neglected taxon of Sisyrinchium in Brazil. The tools applied in this study were substantially able to check the identity of the two morphological categories as two different species and present the variation encountered in the taxa.     

Phylogeography of the Chionodraco genus (Perciformes, Channichthydae) in the Southern Ocean

Antarctic fish of the suborder Notothenioidei represent one of the most notable examples of adaptive radiation in the marine environment. The evolutionary relationships between and within the eight families of this suborder have been well established by numerous studies, whereas the microevolutionary processes of notothenioid species remain largely unexplored. In the present paper we investigated the evolutionary relationships between three closely related species of the genus Chionodraco (family Channichthyidae), namely Chionodraco hamatus, Chionodraco rastrospinosus, and Chionodraco myersi by analysing portions of the mitochondrial genome (D-loop and 16S rRNA). The taxonomic status of C. hamatus and C. rastrospinosus as separate species has been questioned because of the limited number of key morphological characters that distinguish these two taxa. Our results, based on the analysis of several specimens belonging to both morphological groups revealed a small genetic differentiation among haplotypes, however, a clear separation between the two nominal species emerged since all individuals of each of the two taxa clustered together in distinct monophyletic groups. C. myersi appeared more distantly related in the phylogenetic analysis. For one species, C. hamatus, sampling was carried out at three different geographic locations in the area of the Ross Sea and Weddell Sea. The results showed that the partition of the genetic variation within this species is not compatible with the hypothesis of panmixia as gene flow between populations was significantly reduced.     

Post‐glacial history and introgression in Abies (Pinaceae) species of the Russian Far East inferred from both nuclear and cytoplasmic markers

Aim The main aim of the present study is to infer the post‐glacial history of Abies species from north‐east Asia and to test the hypotheses that coastal Abies populations suffered less from climatic fluctuations during Pleistocene glacial periods than their more continental counterparts, and that Sakhalin was a major area of introgression. Location Natural ranges of the fir species Abies nephrolepis, Abies sachalinensis and Abies holophylla in the Russian Far East, and of Abies gracilis, which is endemic to the Kamchatka Peninsula. Methods Nineteen populations were sampled for allozyme analysis. Seventeen of these populations were also screened for variation at two paternally inherited chloroplast DNA microsatellite loci (cpSSR) and variation at one maternally inherited mitochondrial marker (nad4‐3/4). Finally a subset of 11 populations was analysed with amplified fragment length polymorphism (AFLP). Comparisons were made with already available Abies sibirica data. For all sets of markers, we estimated genetic diversity and differentiation using an analysis of molecular variance (AMOVA). Population clustering was assessed with a Bayesian approach implemented in structure v.2.3. Results Among the three major species, A. sibirica, A. nephrolepis and A. sachalinensis, A. sachalinensis demonstrated the highest cytoplasmic and nuclear diversity and the most continental species, A. sibirica, the lowest. Both nuclear and mitochondrial DNA markers revealed the presence of a transitional zone on Sakhalin Island between A. nephrolepis and A. sachalinensis of south Sakhalin. The structure analysis delivered very clear results confirming the admixed origin of A. sachalinensis, with a genetic contribution from A. nephrolepis. No variation in cytoplasmic markers was found in A. gracilis, suggesting the occurrence of a recent bottleneck. Main conclusions There is a clear reduction of genetic diversity in Abies species from the Pacific coast into the continent. The higher diversity in A. sachalinensis could have two causes: a larger effective population size in the islands due to relatively stable climatic conditions and consequently less pronounced demographic fluctuations in population size and/or hybridization with continental and Japanese populations. Sakhalin Island is a major transitional zone for conifer species. Finally, the fir from Kamchatka, A. gracilis, should be regarded as a separate species closely related to the A. nephrolepis–A. sachalinensis complex.     

Morphological similarity and molecular divergence of Trochulus striolatus and T. montanus,and their relationship to sympatric congeners (Gastropoda: Pulmonata: Hygromiidae)

Shell morphology has played an important role in the differentiation of mollusc species. However, extensive morphological variation and the lack of readily available diagnostic characters often lead to confusion and controversy in taxonomy of closely related species, such as the genus Trochulus. Two nominal species Trochulus striolatus and T. montanus show only subtle differences in their reproductive systems and are indistinguishable on the basis of sole conchological traits. Therefore, we performed molecular and morphological analyses to establish robust species limits among the taxa. The canonical discriminant analysis (CDA) of shell characters confirmed strong similarity between the species. It also revealed high intraspecific variability of the shell morphology, which allowed the distinguishing of some populations as two extremely distinct forms, while simultaneously making it difficult to discriminate between the two species. Analysis of the genital morphology indicated that differences were continuous and practically negligible among the species. In contrast, phylogenetic analyses based on newly obtained 114 Trochulus sequences of the mitochondrial cytochrome c oxidase subunit I (COI) gene and 55 homologues retrieved from the GenBank database showed clear genetic divergence between T. striolatus and T. montanus. Moreover, they revealed a close relationship between some T. montanus, some T. caelatus and T. clandestinus samples, which formed a monophyletic group. Some of their haplotypes were even identical. It suggested that their recent common origin or recurrent gene flow occurred between these populations. However, T. sericeus sequences were split into independent diverged lineages, which were separated by many unidentified Trochulus species. It suggested that this taxon may represent a paraphyletic species complex.     

A description of the parthenogenetic metacercaria and cercaria of <Emphasis Type="Italic">Cercaria falsicingulae I</Emphasis> larva nov. (Digenea: Gymnophallidae) from the snails <Emphasis Type="Italic">Falsicingula</Emphasis> spp. (Gastropoda), with speculation on an unusual life-cycle

Previously unknown gymnophallid parthenogenetic metacercariae (PM), referred to as Cercaria falsicingulae I larva nov., were found in the extrapallial cavities of the snails Falsicingula mundana (Yokoyama) and F. athera (Bartsch) on coasts of Sakhalin and the Kuril Islands. Unlike all other known PM, rather than producing metacercariae infective to the definitive host, their furcocercariae emerge into the environment. The developing cercariae and metacercariae of C. falsicingulae I are described and compared with other gymnophallid larvae from littoral molluscs in the region. Experimental evidence and analysis of metacercarial group composition in naturally infected molluscs indicate that some cercariae leave their molluscan hosts and penetrate other specimens of Falsicingula in which they develop into new cercariae-producing PM. Metacercariae with developing hermaphroditic reproductive organs were never observed in naturally infected molluscs. A probable life-cycle for C. falsicingulae I is presented in which cercarial/metacercarial production is switched seasonally in order to enable the infection of the definitive hosts by PM. Importantly, the 'cercaria - PM - cercaria' component of the life-cycle appears to be somewhat autonomous.     

The life-cycle of Helicometra gibsoni n. sp. (Digenea: Opecoelidae)

The life-cycle of Helicometra gibsoni n. sp., an opecoelid trematode of marine fishes, is elucidated and the life-history stages are described. Natural infections with the cercariae, which are typically opecoelid in many respects but characterised by having very long, extensile tails, were found in the intertidal snail Anachis terpsichore collected from the Lawson's Bay Coast, Bay of Bengal. Metacercariae were found in the thoracic muscles of the shooting shrimp Alphaeus malabaricus as prominent, reddish-brown, oval cysts. Laboratory experiments demonstrated that 14 days are required for the development of cercaria into the infective metacercarial stage in shrimps. Natural infections with the adult fluke were found in Scorpaenopsis cirrhosus. Therapon jarbua served as an experimental host. Fourteen-day-old metacercariae fed to T. jarbua developed into egg-producing adults in 40–43 days. Intra-specific variations noted in the flukes obtained from natural and experimental infections are presented. H. fasciata of Madhavi (1975) is regarded as a synonym of H. gibsoni.     

Detection of morphometric differentiation between isolated up- and downstream populations of Siah Mahi (<Emphasis Type="Italic">Capoeta capoeta gracilis</Emphasis>) (Pisces: Cyprinidae) in the Tajan River (Iran)

It has been postulated that the building of the Shahid-Rajaei dam on the Tajan River around 1995 has lead to the morphological divergence of Siah Mahi Capotes capoeta gracilis (Pisces) of up- and downstream populations due to the isolation. A 13-landmark morphometric truss network system was used to investigate the hypothesis. Univariate analysis of variance showed significant differences between the means of the two groups for 45 standardized morphometric measurements out of 78 characters studied. In linear discriminant function analysis (DFA), the overall assignment of individuals into their original groups was 87.6%. The proportion of individuals correctly classified into their original groups was 90.3% for upstream and 83.7% for downstream population. The principal component analysis (PCA) scatter plot of individual component scores between PC1 and PC2 showed 121 fish specimens grouped into two areas but with a relativity high degree of overlap between two populations. Clustering analysis based on Euclidean square distances among groups of centroids using an UPGMA resulted into two main clusters indicating two populations of C. c. gracilis. The present study indicated the presence of two morphologically different populations of C. c. gracilis in the Tajan River across the Shahid-Rajaei dam, probably due to their limited downstream dispersal and the elimination of upstream migration altogether, due the construction of the dam.     

Kelp flies and species concepts – the case of Coelopa frigida (Fabricius, 1805) and C. nebularum Aldrich, 1929 (Diptera: Coelopidae)

The beaches of the North Atlantic and North Pacific are home to kelp flies of the Coelopa frigida/nebularum complex, which consists of one to three different species depending on whether the two nominal species are accepted and a cryptic species proposed by Remmert is counted. The morphological differences between two populations of C. frigida ( Fabricius, 1805 ) from the North Sea and the Baltic Region and two populations of C. nebularum Aldrich, 1929 from Alaska and Japan are described and discussed for small, medium, and large specimens. Crossing experiments are used to demonstrate that, under laboratory conditions, no isolation mechanisms between either population exist. Coelopa frigida and C. nebularum are therefore regarded as a single biological species, a conclusion that is congruent with the observation that the genetic distances based on Ef 1‐α and 16S rDNA indicate lower levels of differentiation within C. frigida/nebularum than between undisputed Coelopa species. The substantial morphological, breeding and genetic information on the C. frigida/nebularum species complex is then applied to six different species concepts popular in the modern systematic literature. According to the Biological, Hennigian and Recognition Species Concepts, only a single species would be recognized. The Evolutionary Species Concept is too vague to be applicable and under two variants of the Phylogenetic Species Concept, C. frigida and C. nebularum would constitute separate species. This result confirms that Phylogenetic Species Concepts lead to a higher species number than concepts based on reproductive isolation. Practical and theoretical problems with the various species concepts are briefly discussed.     

CULTIVATION‐INDEPENDENT SPECIES IDENTIFICATION OF DINOBRYON SPECIES (CHRYSOPHYCEAE) BY MEANS OF MULTIPLEX SINGLE‐CELL PCR1

With the discovery of a high molecular diversity of protists, a discrepancy between morphological and molecular species richness estimates became apparent. Solving the current concerns requires a comparative analysis of different sequences combined with morphological analyses of single cells originating from preserved field samples. We refined a single‐cell PCR (SC‐PCR) protocol for analyzing cells from field samples preserved with Lugol’s iodine solution. We linked microscopic screening with multiplex PCR targeting the SSU rDNA, internal transcribed spacer 1 (ITS1), 5.8S rDNA, internal transcribed spacer 2 (ITS2), and the mitochondrial cytochrome oxidase 1 (CO1) in a single PCR reaction. Using this method, we investigated the intraspecific molecular variation in Dinobryon populations originating from two lakes in the Salzkammergut area of Austria. All investigated genetic markers showed two separated clusters within the investigated populations of Dinobryon divergens O. E. Imhof, indicating a reproductive isolation of the two coexisting populations. Based on these findings, we describe a lineage, which is morphologically similar to D. divergens but, based on the molecular data, is reproductively isolated.