Fifteen species in one: deciphering the <Emphasis Type="Italic">Brachionus plicatilis</Emphasis> species complex (Rotifera,Monogononta) through DNA taxonomy

Understanding patterns and processes in biological diversity is a critical task given current and rapid environmental change. Such knowledge is even more essential when the taxa under consideration are important ecological and evolutionary models. One of these cases is the monogonont rotifer cryptic species complex Brachionus plicatilis, which is by far the most extensively studied group of rotifers, is widely used in aquaculture, and is known to host a large amount of unresolved diversity. Here we collate a dataset of previously available and newly generated sequences of COI and ITS1 for 1273 isolates of the B. plicatilis complex and apply three approaches in DNA taxonomy (i.e. ABGD, PTP, and GMYC) to identify and provide support for the existence of 15 species within the complex. We used these results to explore phylogenetic signal in morphometric and ecological traits, and to understand correlation among the traits using phylogenetic comparative models. Our results support niche conservatism for some traits (e.g. body length) and phylogenetic plasticity for others (e.g. genome size).     

Microsatellite analysis supports the existence of three cryptic species within the bumble bee <Emphasis Type="Italic">Bombus lucorum sensu lato</Emphasis>

Mitochondrial cytochrome oxidase I (COI) partial sequences are widely used in taxonomy for species identification. Increasingly, these sequence identities are combined with modelling approaches to delineate species. Yet the validity of species delineation based on such DNA ‘barcodes’ is rarely tested and may be called into question by phenomena such as ancestral polymorphisms in DNA sequences, phylogeographic divergence, mitochondrial introgression and hybridization, or distortion of mitochondrial inheritance through such factors as Wolbachia infection. The common and widespread European bumble bee Bombus lucorum s. lato contains three distinct mitochondrial DNA lineages that are assumed to represent three cryptic species, namely Bombus cryptarum, B. lucorum s. str. and B. magnus. To test whether nuclear gene pools of the three putative species were differentiated, we genotyped 304 sympatric members of the lucorum complex (54 B. cryptarum females, 168 B. lucorum s. str. females and 82 B. magnus females, as defined using mtDNA COI haplotypes) from 11 localities spread across the island of Ireland at seven nuclear microsatellite loci. Multilocus genotypes clustered into three discrete groups that largely corresponded to the three mtDNA lineages: B. cryptarum, B. lucorum s. str. and B. magnus. The good fit of mitochondrial haplotype to nuclear (microsatellite) genotypic data supports the view that these three bumble bee taxa are reproductively isolated species, as well as providing a vindication of species identity using so-called DNA barcodes.     

Evolutionary diversification of Western Atlantic <Emphasis Type="Italic">Bathygobius</Emphasis> species based on cytogenetic,morphologic and DNA barcode data

A number of fish groups, such as Gobiidae, are highly diversified and taxonomically complex. Extensive efforts are necessary to elucidate their cryptic diversity, since questions often arise about the phylogenetic aspects of new species. Clarifications about the diversity and phylogeny of the Bathygobius species from the southwestern Atlantic are particularly needed. Evidence has been accumulating on the Brazilian coast regarding the possible presence of new species while doubts remain about the taxonomic status of others. The taxonomic identification of some species of Bathygobius has been problematic, given their generally conservative external morphology, and several species are recognized as cryptic. This situation hinders understanding the real diversity in this taxon. Taken together, genetic, cytogenetic and morphometric analyses have been effective in identifying new species of this genus. Here we describe the karyotypic features and morphological patterns of three Western South Atlantic species of Bathygobius. Furthermore, its cytochrome c oxidase I (COI) gene sequences were compared with those of species from Central America, North America and the Caribbean. The broad analyses performed demonstrated an unsuspected diversity, leading to the identification of an un-described new species (Bathygobius sp.2) and the geographic redefinition of another, Bathygobius sp.1, undoubtedly a branch of B. geminatus, hitherto inaccurately identified as B. mystacium on the coast of Brazil.     

A preliminary molecular phylogeny shows Japanese and Austrian populations of the red mite <Emphasis Type="Italic">Balaustium murorum</Emphasis> (Acari: Trombidiformes: Erythraeidae) to be closely related

The red mite Balaustium murorum (Hermann) inhabits the Western Palaearctic realm and is well adapted to man-made structures. In Japan, B. murorum had been reported more frequently after the 1980s. A molecular phylogeny based on the nuclear 18S rRNA and mitochondrial COI genes, and including B. murorum individuals from Japan and Austria and representatives of related species from Japan showed four Balaustium species-level lineages in Japan (B. murorum, Balaustium sp. 1, Balaustium sp. 2, Balaustium sp. 3). The B. murorum lineage shared identical 18S sequence and COI haplotype with the Austrian population. Balaustium sp. 1 was detected from the Tokyo and Misaki area (Honshu Island) and was the sister group to B. murorum; the other two lineages inhabited coastal environments of Erimo, Hokkaido Island (Balaustium sp. 2) and Ainan, Shikoku Island (Balaustium sp. 3). The high genetic distances among these four lineages indicate that each lineage is a distinct species, with three of the lineages representing undescribed species. Our results are compatible with the conclusion that B. murorum was introduced to Japan from Europe, although our study did not resolve the polarity or timing of migration events.     

Empirical evidence for fast temperature-dependent body size evolution in rotifers

Organisms tend to decrease in size with increasing temperature by phenotypic plasticity (the temperature-size rule; ectotherms) and/or genetically (Bergmann’s rule; all organisms). In this study, the evolutionary response of body size to temperature was examined in the cyclically parthenogenetic rotifer Brachionus plicatilis. Our aim was to investigate whether this species, already known to decrease in size with increasing temperature by phenotypic plasticity, presents a similar pattern at the genetic level. We exposed a multiclonal mixture of B. plicatilis to experimental evolution at low and high temperature and monitored body size weekly. Within a month, we observed a smaller size at higher temperature, as compared to body size at lower temperature. The pattern was consistent for the size of both mature females and eggs; rotifers kept at high temperature evolved to be on average 14% (after 2 weeks) and 3% (after 3 weeks) smaller than the ones kept at low temperature (10 and 5% in the case of eggs, respectively). We therefore found that B. plicatilis is genetically programmed to adjust its body size-to-environmental temperature.     

Establishing species and species boundaries in <Emphasis Type="BoldItalic">Sabellastarte</Emphasis> Krøyer, 1856 (Annelida: Sabellidae): an integrative approach

Sabellastarte Krøyer, 1856 (Sabellidae), a morphologically homogeneous group distributed in warm and temperate coasts of the Indo-Pacific and Caribbean Sea, is characterized by the presence of a unique combination of features. To date, the genus comprises eight species, but morphological characters traditionally used in diagnostics have shown intra-specific variability, making species boundaries and distributions unclear. The present study constitutes the first attempt to test the monophyly of Sabellastarte and its relationships to other sabellid genera by combining molecular (COI and 16S) and morphological data. Results include placement of a clade containing Stylomma, Sabella, Branchiomma and Bispira as the sister group to Sabellastarte. Phylogenetic analyses and genetic divergence among specimens from several localities around the world indicate the presence of at least six lineages within Sabellastarte. In the context of a discussion of species boundaries and diagnostic features, the distribution of some of those lineages can be explained by the presence of cryptic species and potential introductions.     

Authentication of five <Emphasis Type="Italic">Barilius</Emphasis> species from Indian waters using DNA barcoding

Authentic identification of fish species is essential for conserving them as a valuable genetic resource in our environment. DNA barcoding of living beings has become an important and ultimate tool for establishing their molecular identity. Among cyprinids, Barilius is an important genus having nearly 23 species in Indian region whose morphological identification is often difficult due to minute differences in their features. Five species collected from Indian waters and primarily identified as Opsarius bakeri (syn. Barilius bakeri), B. gatensis, B. vagra, B. bendelisis and B. ngawa were authenticated by their DNA barcoding based on mitochondrial COI gene sequences. Five individuals of each species were taken for barcode preparation by COI gene sequencing which yielded one barcode for B. ngawa, two barcodes each for O. bakeri, B. gatensis, B. bendelisis and three barcodes for B. vagra. The order of inter and intra-specific variation was estimated to know a preliminary status of variation prevailing in these cold stream fish species significant for evolution and conservation of these valued species of our ichthyofauna. Average variation within genera was found to be 13.6% with intra-specific variation ranging from 0.0% (B. ngawa) to 0.6% (B. gatensis). These distance data are in the same order found by various researchers globally using COI barcode sequences in different fish species. Phylogenetic relatedness among Barilius species and some other cyprinids validate their status of individual species as established by conventional taxonomy.     

Worldwide sampling reveals low genetic variability in populations of the freshwater ciliate <Emphasis Type="Italic">Paramecium biaurelia</Emphasis> (<Emphasis Type="Italic">P. aurelia</Emphasis> species complex,Ciliophora, Protozoa)

Species (or cryptic species) identification in microbial eukaryotes often requires a combined morphological and molecular approach, and if possible, mating reaction tests that confirm, for example, that distant populations are in fact one species. We used P. biaurelia (one of the 15 cryptic species of the P. aurelia complex) collected worldwide from 92 sampling points over 62 years and analyzed with the three above mentioned approaches as a model for testing protistan biogeography hypotheses. Our results indicated that despite the large distance between them, most of the studied populations of P. biaurelia do not differ from each other (rDNA fragment), or differ only slightly (COI mtDNA fragment). These results could suggest that in the past, the predecessors of the present P. biaurelia population experienced a bottleneck event, and that its current distribution is the result of recent dispersal by natural or anthropogenic factors. Another possible explanation for the low level of genetic diversity despite the huge distances between the collecting sites could be a slow rate of mutation of the studied DNA fragments, as has been found in some other species of the P. aurelia complex. COI haplotypes determined from samples obtained during field research conducted in 2015–2016 in 28 locations/374 sampling points in southern Poland were shared with other, often distant P. biaurelia populations. In the Kraków area, we found 5 of the 11 currently known COI P. biaurelia haplotypes. In 5 of 7 reservoirs from which P. biaurelia was obtained, two different COI haplotypes were identified.     

Susceptibility of germinating cruciferous seeds to <Emphasis Type="Italic">Bagrada hilaris</Emphasis> (Hemiptera: Pentatomidae) feeding injury

Bagrada hilaris (Burmeister) (Hemiptera: Pentatomidae) is a serious pest that attacks both germinating and seedling stages of a variety of cruciferous crops grown in the Central Coast of California. B. hilaris feeding on germinating seeds can cause severe stunting and plant mortality, and little is known about the feeding preference of B. hilaris for germinating seeds of major cruciferous hosts and varieties of hosts. No-choice and choice experiments were conducted in which germinating seeds in soilless and soil settings were exposed to B. hilaris adults for 7 days. Susceptibility scores were developed using B. hilaris feeding injury sites, distorted leaves, and deformed and dead plants to determine the overall B. hilaris preference for germinating host seeds. Based on the scores, the order of preference was arugula (Eruca sativa L.)?>?turnip (B. rapa L. var. rapa)?>?mizuna (B. rapa L. nipposinica)?>?kale (B. oleracea L. acephala)?>?choi (Brassica rapa L. var. chinensis)?>?broccoli (B. oleracea var. italica Plenck)?>?cauliflower (B. oleracea L. var. botrytis)?>?lettuce (Lactuca sativa L.)?>?sweet alyssum (Lobularia maritima [L.] Desv.). The lowest feeding injury was recorded on germinating lettuce and sweet alyssum seeds. Furthermore, no-choice and choice experiments were conducted with four varieties each of arugula and mizuna, twelve varieties each of kale and choi, and nine varieties/types of leafy Asian greens. The arugula varieties ‘Wild Rocket’ and ‘Spirit’ were more damaged by B. hilaris than other varieties tested. Among mizuna varieties, ‘Beira F1’ was more attractive to B. hilaris than ‘Scarlet’ or ‘Starbor F1.’ The choi varieties ‘Tokyo Bekana,’ ‘Feng Qing Choi F1,’ ‘Joi Choi F1,’ and ‘Win-Win Choi F1’ were more attractive than ‘Rosie F1.’ The leafy Asian greens variety ‘Carlton F1’ was more attractive to B. hilaris than ‘Yukina Savon,’ ‘Tatsoi OG,’ ‘Komatsuna Summerfest F1,’ ‘Red Rain F1,’ and ‘Shungiku.’ Therefore, the results suggest that not all varieties were equally susceptible to B. hilaris feeding and possibly be utilized for further field evaluation as a trap crop or developing more resistant varieties to B. hilaris.     

Mite color alteration and acaricidal activity of 3,7-dimethyl-2,6-octadienal and its structural analogues against the stored food pest mite <Emphasis Type="Italic">Tyrophagus putrescentiae</Emphasis>

The study of cryptic species allows to describe and to understand biodiversity, and the evolutionary processes shaping it. Mites of the family Rhinonyssidae are permanent parasites of the nasal cavities of birds, currently including about 500 described species and 12 genera. Here, we tested the hypothesis that mites from five populations of the genus Tinaminyssus—three isolated from European turtle doves (Streptopelia turtur), and two from Eurasian collared doves (Streptopelia decaocto; Aves: Columbiformes)—are, in fact, two cryptic species inhabiting different hosts. First, we performed a morphometrical study on 16 traits. Then, we used the ITS1-5.8S rDNA-ITS2 nuclear region (ITS region), and a fragment of the mitochondrial cytochrome c-oxidase 1 (COI) to carry out phylogenetic and species delimitation analyses on Tinaminyssus species. Morphological analyses revealed a lack of biometric differentiation among Tinaminyssus populations from the two host species. However, molecular analyses indicated a high degree of genetic differentiation between populations of Tinaminyssus sp. from S. turtur and S. decaocto. Overall, results show that they can be considered as different cryptic species, suggesting a case of evolutionary stasis, likely because of the anatomical similarity between closely-related bird host species.     

Molecular characterization of eukaryotic algal communities in the tropical phyllosphere based on real-time sequencing of the 18S rDNA gene

Backgroud

Foliicolous algae are a common occurrence in tropical forests. They are referable to a few simple morphotypes (unicellular, sarcinoid-like or filamentous), which makes their morphology of limited usefulness for taxonomic studies and species diversity assessments. The relationship between algal community and their host phyllosphere was not clear. In order to obtain a more accurate assessment, we used single molecule real-time sequencing of the 18S rDNA gene to characterize the eukaryotic algal community in an area of South-western China.

Result

We annotated 2922 OTUs belonging to five classes, Ulvophyceae, Trebouxiophyceae, Chlorophyceae, Dinophyceae and Eustigmatophyceae. Novel clades formed by large numbers sequences of green algae were detected in the order Trentepohliales (Ulvophyceae) and the Watanabea clade (Trebouxiophyceae), suggesting that these foliicolous communities may be substantially more diverse than so far appreciated and require further research. Species in Trentepohliales, Watanabea clade and Apatococcus clade were detected as the core members in the phyllosphere community studied. Communities from different host trees and sampling sites were not significantly different in terms of OTUs composition. However, the communities of Musa and Ravenala differed from other host plants significantly at the genus level, since they were dominated by Trebouxiophycean epiphytes.

Conclusion

The cryptic diversity of eukaryotic algae especially Chlorophytes in tropical phyllosphere is very high. The community structure at species-level has no significant relationship either with host phyllosphere or locations. The core algal community in tropical phyllopshere is consisted of members from Trentepohliales, Watanabea clade and Apatococcus clade. Our study provided a large amount of novel 18S rDNA sequences that will be useful to unravel the cryptic diversity of phyllosphere eukaryotic algae and for comparisons with similar future studies on this type of communities.

     

Phylogenetic relationships in southern African Bryde’s whales inferred from mitochondrial DNA: further support for subspecies delineation between the two allopatric populations

Bryde’s whales (Balaenoptera edeni) are medium-sized balaenopterids with tropical and subtropical distribution. There is confusion about the number of species, subspecies and populations of Bryde’s whale found globally. Two eco-types occur off South Africa, the inshore and offshore forms, but with unknown relationship between them. Using the mtDNA control region we investigated the phylogenetic relationship of these populations to each other and other Bryde’s whale populations. Skin, baleen and bone samples were collected from biopsy-sampled individuals, strandings and museum collections. 97 sequences of 674 bp (bp) length were compared with published sequences of Bryde’s whales (n?=?6) and two similar species, Omura’s (B. omurai) and sei (B. borealis) whales (n?=?3). We found eight haplotypes from the study samples: H1–H4 formed a distinct, sister clade to pelagic populations of Bryde’s whales (B. brydei) from the South Pacific, North Pacific and Eastern Indian Ocean. H5–H8 were included in the pelagic clade. H1–H4 represented samples from within the distributional range of the inshore form. Pairwise comparisons of the percentage of nucleotide differences between sequences revealed that inshore haplotypes differed from published sequences of B. edeni by 4.7–5.5% and from B. brydei by 1.8–2.1%. Ten fixed differences between inshore and offshore sequences supported 100% diagnosability as subspecies. Phylogenetic analyses grouped the South African populations within the Bryde’s-sei whale clade and excluded B. edeni. Our data, combined with morphological and ecological evidence from previous studies, support subspecific classification of both South African forms under B. brydei and complete separation from B. edeni.     

Assessing the distribution and conservation status of a long-horned beetle with species distribution models

Data shortfalls on species distribution affect species differently, but it is frequent among insects. Species distribution models (SDMs) are important tools to fill biogeographic deficits and provide support for practical conservation actions, particularly for cryptic or hard to survey species. We employed SDMs to evaluate one such species, the long-horned beetle (Macrodontia cervicornis), listed as ‘vulnerable’ in the IUCN’s Red List of Threatened Species. Given new distributional data for this species, we applied three different SDMs to: (1) provide the first assessment of this species’ distribution and potential dispersal routes; (2) evaluate the effectiveness of the current South American protected areas system for its conservation; and (3) discuss its potential distribution, as well as historical, biogeographical, and taxonomic issues related to it. Our models reached fair True Skilled Statistics values (TSS > 0.5), with the core area for M. cervicornis located in the Amazon forest, although suitable areas were also predicted along the Atlantic forest. Areas in the dry diagonal South American corridor (dry biomes of Cerrado, Caatinga, and Pampas) in South America were not predicted as suitable. The preference of M. cervicornis for humid areas with high temperatures may guarantee a better physiological control for dehydration, considering that large insects are more affected by water loss. In general, approximately 15 % of the distribution of M. cervicornis is in humid protected areas. The disconnected distribution of the long-horned beetle may be an indication of the existence of cryptic species under the same classification. We suggest that similar studies with other insect groups (e.g. butterflies, bees) should be conducted to properly assess their distributions, conservation status, and responses to hot-humid gradients throughout South America.     

Assessment of the genetic distances between some species of the family Bradybaenidae (Mollusca,Pulmonata)

On the basis of inter-simple sequence repeat (ISSR) loci and the nucleotide sequences of nuclear (18S and ITS-1) and mitochondrial genes (COI and 16S), a phylogenetic analysis of the three species of terrestrial mollusks of the family Bradybaenidae (Mollusca, Pulmonata), Bradybaena fruticum Müll., Bradybaena schrencki Midd., and Bradybaena transbaicalia Shileyko, was conducted to clarify their taxonomic status. The analysis showed that Br. fruticum was far apart from the other two species (Br. schrencki and Br. transbaicalia). The genetic distance between the latter puts in doubt their status as distinct species. It is suggested that the species Br. transbaicalia can be treated as a form of Br. schrencki var. transbaicalia.     

Identification of Japanese <Emphasis Type="Italic">Lymantria</Emphasis> species (Lepidoptera: Lymantriidae) based on DNA sequences

We examined the utility of the cytochrome c oxidase subunit I (COI) and II (COII) genes of mitochondrial DNA (mtDNA) as a tool to identify nine Japanese Lymantria species, including four Asian gypsy moth species [Lymantria dispar japonica (Motschulsky), Lymantria umbrosa (Butler), Lymantria albescens Hori and Umeno, and Lymantria postalba Inoue]. In phylogenetic trees constructed for the COI and COII genes using the maximum likelihood methods, we could identify seven out of the nine Lymantria species (L. albescens and L. postalba could not be identified). These results suggest that the DNA sequences of the COI and COII genes are useful for identifying Japanese Lymantria species.     

Revised classification and phylogeny of an Afrotropical species group based on molecular and morphological data,with the description of a new genus (Coleoptera: Scarabaeidae: Onthophagini)

The worldwide distributed Onthophagus genus comprises at present more than 2000 species, that often show a complicated and uncertain systematic history. In particular, the many Afrotropical species included in this genus have never been entirely reviewed after the division into 32 species groups proposed by d’Orbigny in 1913, although subsequent researches focusing on some of these species suggested that Onthophagus constituted a nonmonophyletic taxon. In order to highlight their phylogenetic relationships, the various Afrotropical species groups of d’Orbigny must thus be examined, and it would be advisable to study them separately to avoid misunderstanding. In this framework, the taxonomic position of the three species currently included in the 21st d’Orbigny group was examined. Both morphological and biomolecular analyses contributed in confirming that these species (i.e., Onthophagus caffrarius d’Orbigny, 1902; Onthophagus quadraticeps Harold, 1867; and Onthophagus signatus Fåhraeus, 1857) constituted a well-defined monophyletic group that cannot be maintained within the genus Onthophagus. Therefore, the Kurtops gen.n. is here described to accommodate these Afrotropical species, that are nevertheless always included within the Onthophagini tribe. On the basis of the phylogenetic relationships here elucidated, it was also emphasized that the new genus is strictly related to Digitonthophagus and Phalops; thus, it was proposed to include the three genera into a single clade of suprageneric rank naming it as Phalops complex.     

DNA barcoding of fishes in Irtysh River China

DNA barcoding was a molecular diagnostic method that provided rapid and accurate species identification. The 650 bp-length cytochrome c oxidase subunit I (COI) gene of 33 species in Irtysh River China was sequenced and analyzed in this study. The average intra-species, -genus, -family, and -order of Kimura two parameter (K2P) distances were 0.003, 0.060, 0.163 and 0.240, respectively. The genetic distance between genus Barbatula and Cobitis was the largest whereas that between genus Hypophthalmichthys and Aristichthys was the smallest. The neighbour-joining tree constructed by all 44 haplotypes was divided into two major clusters: Cypriniformes fishes and other fishes. A cryptic species of Barbatula barbatula was detected according to 2% genetic threshold.