Existence Of Cryptic Species Research Articles

Evidence for a species complex in Indialona ganapati (Chydoridae)

Indialona ganapati (Petkovski 1966), is one of the three known cladoceran endemic species from India. It is also the only one of these species that is monotypic and endemic to central India. In this study we report on habitat shifts for this species as well as presence of parthenogenetic and ephippial females throughout the year, phenomena that are uncommon in most species of the family Chydoridae. These factors prompted us to undertake a study evaluating the taxonomic status of this species in collections from India using morphological and molecular methods. This included recognition of some degree of morphometric diversity based on sexual differentiation and reproductive patterns which were not correlated with speciation events. Analysis of our data does, however, suggest that I. ganapati can be split into two recent clades. Also, phylogenetic as well as haplotype network analysis of our data suggests the presence of a sibling species complex of I. ganapati in the river Godavari. This suggests that reconsideration of taxonomic status of this species may be appropriate. In addition, this study underscores the potential utility of using COI gene based ‘barcode’ DNA sequences for recognizing the existence of cryptic species among the cladocera.

The Mediterranean as a melting pot: Phylogeography of Loxosceles rufescens (Sicariidae) in the Mediterranean Basin

The species Loxosceles rufescens is native to the Mediterranean but considered cosmopolitan because it has been dispersed worldwide. A previous study revealed 11 evolutionary lineages across the Mediterranean, grouped into two main clades, without any clear phylogeographic pattern. The high genetic diversity within this species (p-distances of up to 7.8% in some Mediterranean lineages), together with the results obtained with different species delimitation methods (GMYC, TCS) could indicate the existence of cryptic species. Here we compare the mitochondrial and microsatellite diversity to elucidate if the lineages of L. rufescens in the Mediterranean should be considered different species (cryptic species) or populations of the same species. To do so, we analyzed the cox1 diversity of 196 individuals, of which, we genotyped 148, sampled from 19 localities across the Mediterranean. STRUCTURE analyses of microsatellite data identified two genetic clusters of L. rufescens. One cluster included individuals from Western Mediterranean localities (Iberian Peninsula, Morocco, Balearic Islands) and Israel, while the second one grouped individuals from Italian and Greek localities, including Sardinia, Sicily and Tunisia. These patterns suggest that geographic proximity is the more significant factor in the clustering with microsatellite data and shows the existence of gene flow between the nearest geographic areas, even if the individuals belong to different mitochondrial lineages or clades. The lack of correspondence between both genetic markers suggests that the evolutionary lineages found within L. rufescens should not be considered different species. We conclude that these phylogenetic linages and their distribution may be the result of the maternal evolutionary history of the species and human-mediated dispersion.

New Data on the Systematics of Comb-fin Squids Chtenopteryx spp. (Cephalopoda: Chtenopterygidae) from the Canary Islands.

Alejandro Escánez, Álvaro Roura, Rodrigo Riera, Ángel Francisco González, and Ángel Guerra (2018) The systematics of the comb-fin squid species is problematic and poorly resolved. In total, 53 specimens of comb-fin squids (Chtenopteryx spp.) were caught at depths ranging from 30 to 800 m off the Canary Islands (NE Atlantic Ocean). Mantle lengths of the individuals ranged from 18 to 43 mm and the sample included immature, mature male and mature female specimens. Two species of comb-fin squids, Chtenopteryx canariensis and C. sicula, were identified by combining traditional morphological characters with a molecular analysis of a fragment of the cytochrome c oxidase subunit I (COI) gene. Intra- and interspecific genetic distances and maximum likelihood tree analyses based on COI sequences available from GenBank suggest the existence of at least four species, two from the Pacific and two from the Atlantic Ocean. Our data expand the current geographic range of C. canariensis from the NE to NW Atlantic. In the GenBank database, several sequences of comb-fin squid in different species-specific clades have been attributed only to C. sicula, indicating the possible existence of cryptic species and the need to re-analyse these data.

Evaluation of the molecular variability and characteristics of Paramecium polycaryum and Paramecium nephridiatum, within subgenus Cypriostomum (Ciliophora, Protista)

Although some Paramecium species are suitable research objects in many areas of life sciences, the biodiversity structure of other species is almost unknown. In the current survey, we present a molecular analysis of 60 Cypriostomum strains, which for the first time allows for the study of intra- and interspecific relationships within that subgenus, as well as the assessment of the biogeography patterns of its morphospecies. Analysis of COI mtDNA variation revealed three main clades (separated from each other by approximately 130 nucleotide substitutions), each one with internal sub-clusters (differing by 30 to 70 substitutions – a similar range found between P. aurelia cryptic species and P. bursaria syngens). The first clade is represented exclusively by P. polycaryum; the second one includes only four strains identified as P. calkinsi. The third cluster seems to be paraphyletic, as it includes P. nephridiatum, P. woodruffi, and Eucandidatus P. hungarianum. Some strains, previously identified as P. calkinsi, had COI sequences identical or very similar to P. nephridiatum ones. Morphological reinvestigation of several such strains revealed common morphological features with P. nephridiatum. The paper contains new information concerning speciation within particular species, i.e. existence of cryptic species within P. polycaryum (three) and in P. nephridiatum (six).

First morphological and molecular analysis of Eucoleus boehmi like eggs in dogs from Argentina.

The canid parasites Eucoleus aerophilus (syn. Capillaria aerophila) and Eucoleus boehmi (syn. Capillaria boehmi) parasitize the lower and the upper respiratory tract, respectively. Reports and descriptions of these nematodes are scarce in Argentina, possibly due to misdiagnosis of morphologically similar trichuroids eggs, and the lack of knowledge about the species of Eucoleus in this geographical area. Scanning electron microscopy is a useful tool for identification of E. boehmi eggs based on the characteristics of the shell structure which differentiate between species. Molecular analysis complements morphological identification. Until now, there are no studies based on the analysis of E. boehmi eggs in Argentina. The aim of the present work was to study by morphological, morphometric, and molecular analysis, eggs attributable to E. boehmi isolated from dogs naturally infected in Mar del Plata city, Argentina. Eggs isolated from two dog fecal samples were analyzed by light and scanning electron microscopy. A fragment of mitochondrial DNA (mtDNA) of the cytochrome c oxidase subunit I gene (cox1) from eggs was sequenced, and phylogenetic analysis was performed in this study. According to morphological results based on the wall surface ultrastructure, the eggs studied were assigned to E. boehmi. Molecular analysis supported the morphological identification. The divergence of 9-12% with the European isolated could suggest a new geographical genetic variation of E. boehmi, but also question the possible existence of cryptic species. This is the first characterization of E. boehmi eggs in dogs from Argentina.

Incongruence between morphological and molecular diversity in Coxicerberus fukudai (Ito, 1974) (Isopoda: Microcerberidea) from East Asia

Coxicerberus fukudai (Ito, 1974) (Isopoda) is redescribed from Korean and Japanese localities. Molecular analysis based on two mitochondrial markers (mtCOI and Cytb) of the studied populations shows two diverged lineages, reflected in high (10.5% and 9.5%) pairwise differences and high haplotype diversity. One of the clades is shared by Korean and Japanese populations with little divergence rate (0.02% and 0.03%), and another is only found in Korea. We failed to find any consistent morphological differences across the examined populations, probably indicating the existence of cryptic species. We also examined the possible influence of the endosymbiotic bacterium Wolbachia on the haplotype diversity. Infection with the bacterium shows a distinct distribution pattern, with all the southern Korea populations being infected, whereas the northern populations are not.

Phylogenetic and Phylogeographic Relationships among Lineages of the Armored Catfish Ancistrus Kner, 1854 (Loricariidae: Ancistrini), from the Amazon and Paraguay Basins

Ancistrus is one of the most diverse genera in the Ancistrini tribe, with 64 nominal species. The group is characterized by high cytogenetic variability; the diploid number of chromosomes ranges from 2n = 34 to 2n = 54. Ancistrus is widely distributed in the basins of the Uruguay, Paraguay, and Amazonian rivers; the latter two regions show the greatest diversity of Ancistrus species and karyotypes. Despite these characteristics, the group includes species for which taxonomic identification is difficult, and phylogenetic relationships and phylogeographic patterns, especially in the Paraguay and Amazon basins, have not yet been revealed. In this study, we determined the phylogenetic and phylogeographic relationships among the Ancistrus lineages in these regions. In particular, 93 concatenated sequences of mitochondrial ATPase 6/8 and COI as well as nuclear Rag2 were used for a phylogenetic analysis, and ATPase 6/8 were used for a phylogeographic analysis. The topology generated by the Bayesian method included three distinct clades subdivided into 21 groups. The clades indicated a monophyletic relationship among the lineages from the Amazon and Paraguay basins. The 21 groups had a high average genetic distance (8.4%) and were structured genetically. In the haplotype network, eight large groups were observed, seven belonging to the Paraguay basin and one corresponding to the Amazon basin, and no haplotypes were shared between the two basins. These results indicate that Ancistrus lineages form a monophyletic unit in the Paraguay and Amazon basins, and these lineages have a high level of divergence and genetic isolation. These results corroborate the existence of cryptic species in the region and emphasize the need for a taxonomic revision of the genus in these basins.

The many faces of Peridinium cinctum (Peridiniaceae, Peridiniales): morphological and molecular variability in a common dinophyte

ABSTRACTPeridinium cinctum is a common freshwater dinophyte with a long history of research. Erich Lindemann was the first to assess intraspecific variability in this species focusing on plate pattern variation. Since then, this issue has been neglected but with the application of DNA sequence diagnostics, a combination of morphological and molecular characters may enable taxonomic delimitations. Our aim was to identify distinct morphotypes using plate pattern as the main characteristic and then compare them to the geographic occurrence of particular ribotypes (as inferred from sequences of the Internal Transcribed Spacer: ITS) in samples from Central Europe. Approximately 200 observations were carried out under the inverse light microscope for each of a total of 15 strains. We observed two main variations from the abundant plate pattern in P. cinctum, namely an unusual position of the 2a plate and the irregular shape of the 1a plate. In 88 (predominantly clonal) strains, we identified five different ribotypes (submitted as 71 new GenBank entries) which had no clear correlation to the defined morphotypes and/or spatial occurrences. In four cases, we detected two distinct ribotypes at the same locality. However, samples collected south of the Danube River presented a different predominant morphotype from the rest of the samples, thus implying a potential biogeographic signal as inferred from morphology. In general, there is morphological and molecular variability in P. cinctum, which is under-studied and which may uncover geographic or ecological correlations or even the existence of cryptic species.

Cryptic species in the Andean hemiparasite Quinchamalium chilense (Schoepfiaceae: Santalales)

The integration of different characters (e.g. morphological, ecological, and molecular) is now recognized as important in species delimitation. In particular, genetic distances between homologous genes have been suggested as one of the main tools to identify species, especially in the case of cryptic species. Quinchamalium is morphologically variable and occupies a diverse set of biomes across its distribution in the Southern Andes. Recent work based on morphology has synonymized the entire genus as a single morphospecies, Quinchamalium chilense. This widely distributed taxon presents the opportunity to find potential cryptic species. The main objective of this study was to test the existence of cryptic species, based mainly on phylogenetic gene trees, genetic distances, and geographic patterns of haplotypes from molecular markers of the nuclear (ITS) and chloroplast (trnL-F) genomes, considering climatic and morphological characteristics. The ITS phylogeny and corresponding haplotype network resulted in three lineages with strong genetic differentiation and distinct geographic patterns. These lineages were informally named Desert, Matorral, and Mountain, based on their geographic distribution in different biomes. The trnL-F chloroplast phylogeny did not distinguish Desert from Matorral, and the haplotype network showed overlap between these last two lineages. Overall, we hypothesize the existence of two cryptic species within Quinchamalium chilense (Mountain and Matorral–Desert) that correspond to genetic, climatic, and morphological differences.

New finding of green algae with potential for algal biotechnology, Chlorococcum oleofaciens and its molecular investigation

The practice of soil algology shows that algae from the order Chlamydomonadales are among the most poorly studied and difficult to identify due to the high heterogeneity of their morphology and ultrastructure. Only the involvement of molecular genetic methods usually makes it possible to determine their taxonomic status with high accuracy. At the same time, in the algae flora of Ukraine there are more than 250 species from the order Chlamydomonadales, the status of which in most cases is established exclusively on the basis of light microscopy. This work is devoted to the study of the biotechnologically promising green alga Chlorococcum oleofaciens, taking into account the modern understanding of its taxonomic status. Two new strains of this species, separated from samples of forest litter and oak forest soil (the Samara Forest, Dnipropetrovsk region), are described. The strains were studied at the morphological level by using light microscopy methods, as well as using molecular genetic methods based on the studies of the nucleotide genes sequences of the 18S ribosomal DNA (rDNA) and chloroplast rbcL genes, the topology of secondary structures of internal transcribed spacer 2 (ITS2). The obtained results helped to confirm the presence of C. oleofaciens in the algae flora of Ukraine. Also, the authors of the article discuss the differences in the secondary structure of ITS2 in different strains of C. oleofaciens associated with the presence of compensatory base change (CBC), hemi-CBC in helices I and II, as well as deletions in helix IV and providing a basis for the hypothesis of the existence of cryptic species within C. oleofaciens. The obtained data can be used at the stage of preliminary selection of biochemical research objects.

DNA barcoding of metacestodes found in the Guerlinguetus ingrami (Rodentia: Sciuridae) reveals the occurrence of Hydatigera taeniaeformis sensu stricto (Cyclophyllidea: Taeniidae) in the Americas

The existence of cryptic species in the genus Hydatigera, cyclophillid cestodes, mainly of felids, was recently described based on molecular studies of parasites from Asia, Europe and Africa. However, the occurrence of H. taeniaeformis sensu stricto (s.s.), the species more widely distributed and with a presumed specificity for murid rodents as intermediate hosts, has not been formally described in Americas. In the present study, during necropsy of an Ingram's squirrel specimen, Guerlinguetus ingrami, found dead in the municipality of Belo Horizonte, Minas Gerais state, Brazil, strobilocerci were found in the liver. The metacestodes were subjected to morphological and molecular studies. Sequences of the COI barcode region were obtained and used for phylogenetic analyses. The morphology and measures of the rostellar hooks were compatible with the ones described for H. taeniaeformis s.s. This identification was confirmed by a molecular phylogenetic approach (96.2–99.7% similarity with isolates of the parasite from Europe and Asia). This is the first molecular confirmation of the existence of H. taeniaeformis s.s. on the American continent. Moreover, the involvement of sciurid rodents in the transmission of H. taeniaeformis s.s. is discussed here as a probable case of parasite spillover.

Population growth rate of dry bulb mite, Aceria tulipae (Acariformes: Eriophyidae), on agriculturally important plants and implications for its taxonomic status

Dry bulb mite (DBM), Aceria tulipae, is an economically important mite with a worldwide distribution and a broad host range. As a generalist, it is the most important eriophyoid mite attacking bulbous plants such as garlic, onion and tulip. To date, DBM has been recorded on host plants belonging to the families Liliaceae, Amaryllidaceae, Melanthiaceae and Asparagaceae. However, a precise understanding of DBM host range is lacking as it is largely based on casual records of mites on plants, some of which may include accidental hosts. Moreover, the possible existence of cryptic species has not been considered. In this study the hypothesis that DBM may be a complex of distinct genetic lineages or cryptic species was tested by comparing the common barcode sequence marker mtDNA COI of specimens from several populations originating from the Netherlands and Poland. The population growth rate of DBM on seven agriculturally important plant species and on various parts of the garlic plant was also experimentally assessed in the laboratory. The results did not support the first hypothesis, and indicated that DBM populations originating from Poland and the Netherlands shared essentially the same genome. In addition, they indicated that DBM reached the highest population growth rate on leek and also displayed high growth rates on garlic, chive and red onion, whereas white onion and wheat were not colonized by the mites. Answering the question of whether DBM is a single polyphagous species rather than a complex of cryptic lineages is of particular importance since the misidentification of pests may lead to ineffective control strategies. Moreover, improved knowledge of DBM host range is essential for assessing risk to crops.

Early events in speciation: Cryptic species ofDrosophila aldrichi.

Understanding the earliest events in speciation remains a major challenge in evolutionary biology. Thus identifying species whose populations are beginning to diverge can provide useful systems to study the process of speciation. Drosophila aldrichi, a cactophilic fruit fly species with a broad distribution in North America, has long been assumed to be a single species owing to its morphological uniformity. While previous reports either of genetic divergence or reproductive isolation among different D. aldrichi strains have hinted at the existence of cryptic species, the evolutionary relationships of this species across its range have not been thoroughly investigated. Here we show that D. aldrichi actually is paraphyletic with respect to its closest relative, Drosophila wheeleri, and that divergent D. aldrichi lineages show complete hybrid male sterility when crossed. Our data support the interpretation that there are at least two species of D. aldrichi, making these flies particularly attractive for studies of speciation in an ecological and geographical context.

Genetic Diversity of Urban Camponotus Mayr (Hymenoptera: Formicidae) Ants Revealed by Capture of Alates and DNA Sequencing.

The Camponotus Mayr genus of carpenter ants is one of the largest in species number and widely represented in the Neotropical Region. Most species are generalists and capable of exploiting diverse habitats including urban environments. Urban green areas can act as a repository of regional biodiversity, thus we investigated whether this is valid for the largest city in South America. We compared the richness of Camponotus spp. in two green areas in regions with distinct urbanization profiles and also with previous surveys made in smaller cities and in natural areas of the original Atlantic Forest. Besides the usual capture of worker specimens, we included capture of alates to improve the species richness sampling. Morphological identification of Camponotus spp. is challenging, even more when alates are included. To assist in specimen identification, we performed DNA sequencing of mitochondrial and nuclear markers. The richness observed in the less stressed urban area was higher than in the more stressed one. Camponotus spp. reported in natural areas are largely represented in the urban area. DNA sequencing for specimen identification is hampered by the lack of corresponding sequences in the GenBank, but it helped to associate workers and alates of the same species and indicated the existence of cryptic species in the genus. Capture of alates allowed detection of several species for which workers were not sampled; therefore, it is a valuable tool for surveying diversity of Camponotus or other ant taxa with arboreal or hypogeic habits.

Islands in the desert: Species delimitation and evolutionary history of Pseudotetracha tiger beetles (Coleoptera: Cicindelidae: Megacephalini) from Australian salt lakes

The Australian salt lakes are a natural archipelago-like laboratory for investigating evolutionary and population processes. Their environmental conditions have not undergone relevant changes since the aridification of Australia 10–5 million years ago. The genus Pseudotetracha, a group of nocturnal tiger beetles found on these remote salt lakes, includes 20 described species. Recent studies based on molecular markers and cytogenetics hinted at the existence of cryptic species within this group. Here we use various species delimitation algorithms to detect a high number of cryptic and undescribed taxa, and challenge the validity of the taxonomic characters traditionally used for discerning species in this group. Our analyses show that the divergence dates of the clades, between 10 and 5 million years ago, correspond to the period in which Australia was undergoing an aridification process that probably isolated the ancestral Pseudotetracha populations to individual lakes or palaeodrainage basins. This implies an important role of the isolation, produced by the aridification of Australia, in the speciation and divergence of Pseudotetracha, which underwent a remarkable radiation as the populations became geographically restricted.

Porcelain crabs of the genera Pachycheles Stimpson and Neopisosoma Haig (Decapoda : Anomura : Porcellanidae): new premises based on molecular data and comments on phylogenetic relationships in the family

Porcellanidae Haworth, 1825 is a family of marine anomuran crabs distributed throughout tropical and temperate regions of all the oceans, typically littoral and sublittoral waters, with a considerable diversity of lifestyles, habitats and colouration. Pachycheles Stimpson, 1858 and Neopisosoma Haig, 1960 share, among other morphological characteristics, the fragmentation of the lateral carapace walls. The morphology of this body region was key to supporting the establishment of Neopisosoma, which increased uncertainty about the taxonomic status of these genera due to the high intraspecific variation of this character. Our study reconstructs the phylogenetic relationship between Pachycheles and Neopisosoma based on mitochondrial and nuclear DNA sequences to evaluate whether these are valid taxa. While Pachycheles seems to be monophyletic, the position of Neopisosoma mexicanum (Streets, 1871) indicates that the group is polyphyletic, and deserves further investigation. Pachycheles is revealed to be older than Neopisosoma, and likely originated in the Indo-Pacific, later spreading to the American continent during the early Tertiary. Neopisosoma may have arisen much later in the Caribbean Province before the closure of the Isthmus of Panama, explaining its distribution, which is restricted mainly to Central America. The inclusion of a considerable number of species from both genera represents a significant advance in the study of this controversial group. The phylogenetic reconstruction of Pachycheles unveiled clades corroborated by morphology, but also revealed unclear relationships, which may indicate the potential existence of cryptic species.

Aphid parasitoid generalism: development, assessment, and implications for biocontrol

Host specialization in aphid parasitoids is important both from a theoretical and an applied point of view. It arises from various ecological mechanisms involving their interactions with aphids, host plants, and endosymbiotic bacteria, as well as with potential competitors and enemies. From an applied point of view, host specialization in aphid parasitoids has a great importance as it determines the biological control they provide through their capacity to switch between different hosts, to persist in the agrosystem in the absence of the pest, and to regulate pest outbreaks in a rapidly changing system. It also conditions the risk of undesirable effects on non-target species in the case of introduction or augmentation of populations of parasitoids. Biocontrol literature that looks at the benefits of generalist and specialists natural enemies is mainly focused on differences between different guilds of natural enemies and does not consider the differences in host specialization within a single guild. This review synthesizes the mechanisms related to host-use by aphid parasitoids, focusing on the differences between generalist and specialist species. Second, this work describes the difficulty to determine the host range of generalist parasitoid species. Our review points out some observational artifacts, as is the existence of cryptic species or spatiotemporal variability in host acceptance, which may lead to misinterpretations about host specialization and result in pest management failures. Regarding biological control services, moderately generalized species that could use various host species to sustain their populations may ensure the long-term control, whereas specialist species would provide higher parasitism rates. At the community level, the co-occurrence of specialist and generalist parasitoids may maximize biological control services both in terms of efficiency and in terms of stability in space and time.

How many DNA markers are needed to reveal cryptic fungal species?

In the fungal kingdom there is a high prevalence of morphologically defined species that includes closely related ‘cryptic’ biological species with similar phenotypes. Due to evolutionary processes like incomplete lineage sorting and introgression through hybridization, several independent DNA markers are essential to resolve closely related fungal species. In this study we wanted to analyze how many independent loci are necessary to reveal the cryptic species, using the genus Serpula as a model system. DNA sequences from ten different DNA loci, eight nuclear and two mitochondrial DNA markers, were obtained from various cryptic species within Serpula. The inclusion of five loci gave a highly confident separation of the cryptic species. Several other loci performed better than the standard DNA barcoding marker ITS in separating the cryptic species. The DNA loci tub, hsp, rpb2 and tef gave, on average, best support for the different cryptic species in single gene trees. We conclude that the analyses of a few but informative independent DNA loci, such as tub, hsp, rpb2 and tef in addition to the standard DNA barcode ITS, may give a good indication about the existence of cryptic species in fungi.

Morphometric Analysis of Longipalpis (Diptera: Psychodidae) Complex Populations in Mato Grosso do Sul, Brazil.

Owing to the existence of cryptic species that are difficult to distinguish morphologically, the search for new taxonomic characters and methods for identifying and classifying sand flies continues. Lutzomyia longipalpis (Lutz & Neiva, 1912) and Lutzomyia cruzi (Mangabeira, 1938) (Diptera: Psychodidae) are two such species that occur in sympatry in some regions of Mato Grosso do Sul State (MS). Twenty females and twenty males from each of the five populations of Lu. longipalpis and one population of Lu. cruzi from MS were examined. An outlying population of Lu. longipalpis from Estrela de Alagoas, State of Alagoas, was used to compare the degree of divergence among the groups in MS. Specimens were cleared, mounted on slides, identified, and measured using LAS-Leica. The principal component analysis of morphometric characters showed a high degree of variation among females, while males varied to a lower degree. The populations of Alagoas and Miranda demonstrated the greatest variation. The first region, Alagoas, is geographically distant from the others and occurs under distinctly different ecological conditions, which likely accounts for the variation. Further studies should be made to elucidate the factors that contribute to the differences found between the populations of MS.

Brittle stars from the Saint Peter and Saint Paul Archipelago: morphological and molecular data

Morphological and molecular data on brittle stars from the Saint Peter and Saint Paul Archipelago, a very small isolated group of islets situated between South America and Africa, are hereby presented. We found no endemic ophiuroids. Instead, the five species, Amphipholis squamata , Ophiactis lymani, Ophiactis savignyi, Ophiocomella ophiactoides and Ophiothrix ( Ophiothrix ) angulata , are either cosmopolitan or derived from the equatorial western Atlantic region. Results indicated a western Atlantic colonization and highlighted the existence of cryptic species in the genus Ophiactis .