DNA barcoding the genus Chara: molecular evidence recovers fewer taxa than the classical morphological approach

Charophytes (Charales) are benthic algae with a complex morphology. They are vulnerable to ecosystem changes, such as eutrophication, and are red‐listed in many countries. Accurate identification of Chara species is critical for understanding their diversity and for documenting changes in species distribution. Species delineation is, however, complicated, because of high phenotypic plasticity. We used barcodes of the ITS2, matK and rbcL regions to test if the distribution of barcode haplotypes among individuals is consistent with species boundaries as they are currently understood. The study included freshly collected and herbarium material of 91 specimens from 10 European countries, Canada and Argentina. Results showed that herbarium specimens are useful as a source of material for genetic analyses for aquatic plants like Chara. rbcL and matK had highest sequence recoverability, but rbcL had a somewhat lower discriminatory power than ITS2 and matK. The tree resulting from the concatenated data matrix grouped the samples into six main groups contrary to a traditional morphological approach that consisted of 14 different taxa. A large unresolved group consisted of C. intermedia, C. hispida, C. horrida, C. baltica, C. polyacantha, C. rudis, C. aculeolata, and C. corfuensis. A second unresolved group consisted of C. virgata and C. strigosa. The taxa within each of the unresolved groups shared identical barcode sequences on the 977 positions of the concatenated data matrix. The morphological differences of taxa within both unresolved groups include the number and length of spine cells, stipulodes, and bract cells. We suggest that these morphological traits have less taxonomic relevance than hitherto assumed.     

Characterization of sexual reproductive processes in Chara braunii (Charales,Charophyceae)

Chara braunii is distributed worldwide and is the most common charalean species in Japan. This species is monoecious and produces numerous sets of sex organs, each of which consists of one antheridium and one oogonium, under laboratory culture conditions. In this study, we report that light intensity strongly affected the vegetative phase and sexual reproductive phase of this species. Under high‐light conditions (70.0 μmol photons m^?2 s^?1), thalli grew but did not form reproductive organs. Under a low‐light intensity (10.0 μmol photons m^?2 s^?1), algal bodies formed many reproductive organs. In addition, antheridia without the corresponding oogonia (lone antheridia) were observed under low‐light conditions. The absence of oogonium primordia adjacent to the lone antheridium was confirmed by several microscopic approaches. The addition of liquid fertilizer increased the total number of sex organs and growth; however, the number of lone antheridia decreased with increasing fertilizer concentrations. Exogenously applied gibberellin did not affect the number of lone antheridia. These results suggest that regulatory mechanisms for the appropriate allocation of resources exist in this alga, similar to those reported in some land plants.     

Occurrence of the endangered species Nitellopsis obtusa (Charales,Charophyceae) in western Japan and the genetic differences within and among Japanese populations

Nitellopsis obtusa (Charales, Charophyceae) are widely distributed from Europe to Asia; however, this species has been recorded in only the five lakes in central Honshu in Japan. This species was thought to be extinct in Japan, but was rediscovered in limited areas of Lake Kawaguchi in central Honshu. More recently, we discovered more Japanese populations of N. obtusa in Lake Biwa in western Honshu, and it became clear that the species had a broader distribution in Japan than originally believed. In addition, although only male or female thalli have been collected at each lake, both male and female thalli were found from Lake Biwa. This is the first report of a potentially sexual population of N. obtusa in Japan. The DNA sequences of three chloroplast DNA markers, including both coding and non‐coding regions, were identical in all specimens from Lake Kawaguchi and Lake Nojiri (Central Honshu), and differed from those of Lake Biwa and German specimens. Although Japanese and German specimens were genetically similar, Japanese specimens displayed considerable genetic diversity according to locality.     

Ultrastructure and phylogeny of the spermatozoid ofChara vulgaris (Charophyceae)

At maturity, spermatozoids of the green algaChara vulgaris are biflagellated, contain little cytoplasm, and coil for approximately 2 1/2 gyres within the mother cell wall. The anterior of the cell contains an ovoid headpiece anchoring two slightly staggered basal bodies that are positioned above and directly in front of approximately 30 linearly arranged mitochondria. An elongated stellate pattern occupies the transition zone between the BBs and axonemes. Flagella emerge from the cell just in front of the nucleus and encircle the full length of the spermatozoid. The spline comprises a maximum of 38 microtubules surrounding the anterior mitochondria and gradually decreases posteriorly to a minimum of 11. The dense nucleus is narrow, cylindrical, and occupies the central revolution of the cell. Six starch-laden plastids and associated mitochondria are linearly arranged at the cell posterior. Phylogenetic analyses of charalean taxa and archegoniates based on spermatogenesis strongly support the orderCharales, withNitella as the sister group toChara. Diagnostic features ofChara spermatozoids include absence of a lamellar strip and axonemes embedded in the cell for almost the entire length of the anterior mitochondria. Potential relationships amongCharales, Coleochaetales and archegoniates are evaluated in regards to the probable course of evolution of streamlined biflagellated gametes.     

Review: Taxonomy of Nitella (Charales, Charophyceae) based on comparative morphology of oospores and multiple DNA marker phylogeny using cultured material

The genus Nitella is the largest group in the Charales and has the highest diversity of vegetative and oospore morphologies. In his worldwide monograph on the Charales, R. D. Wood characterized the sections and species of Nitella mainly on the basis of vegetative morphology and treated oospore wall morphology as diagnostic at the infraspecific level. Therefore, many species of Nitella exhibiting distinct external morphology of the oospore wall (EMOW) were reduced to infraspecific rank and only 53 of 204 species previously described were recognized within Nitella . However, recent morphological and molecular phylogenetic studies have demonstrated the phylogenetic validity of using EMOW for diagnosing some species of Nitella . More recently, a scanning electron microscopy (SEM) study of internal morphology of the oospore wall (IMOW) and multiple DNA marker analyses using both nuclear and chloroplast gene sequences were conducted to improve our understanding of the taxonomy of Nitella at the species level, on the basis of cultured material of a large number of species. Multiple DNA marker analyses resolved detailed and robust phylogenetic relationships within the genus and demonstrated the taxonomic and phylogenetic significance of IMOW. In addition, they supported the taxonomic decisions based on differences in oospore morphology, suggesting that an integrated approach, involving both SEM studies of the EMOW and IMOW and multiple DNA marker analyses, is appropriate to address problems at lower taxonomic levels within the genus, as well as to construct a natural taxonomic system for the genus. In this paper, recent morphological and molecular phylogenetic studies are reviewed and recent improvements in taxonomy of Nitella are summarized. Moreover, the evolution of morphological features and phylogenetic relationships within Nitella are discussed, focusing especially on oospore morphology.     

Phylogeny of extant genera in the family Characeae (Charales,Charophyceae) based on rbcL,sequences and morphology

Extant genera of Characeae have been assigned to two tribes: Chareae (Chara, Lamprothamnium, Nitellopsis, and Lychnothamnus) and Nitelleae (Nitella and Tolypella), based on morphology of the thallus and reproductive structures. Character analysis of fossil and extant oogonia suggest that Tolypella is polyphyletic, the genus comprising two sections, one in each of the two tribes. Eleven morphological characters and sequence data for the Rubisco large subunit (rbcL) were used to reconstruct the phylogeny of genera, including the two sections of Tolypella. Parsimony analysis of the rbcL data, with all positions and changes weighted equally, strongly supports the monophyly of the Characeae. The two Tolypella sections form a robust monophyletic group basal to the family. Transversion weighting yielded the same tree but with a paraphyletic Tolypella. The rbcL data strongly support monophyly of tribe Chareae but tribe Nitelleae is paraphyletic. Parsimony analysis of morphological data produced one unrooted tree consistent with monophyly of the two tribes; on this tree the Tolypella sections were paraphyletic. Combining morphological with rbcL data did not change the results derived from rbcL sequences alone. The rbcL data support the monophyly of the Characeae and Coleochaete, which together form a monophyletic sister group to embryophytes.     

Morphological and mitochondrial DNA variation revealed an undescribed lineage under the genus Sperata (Bagridae) in Bangladesh

Catfishes Sperata are popular, known for its taste and nutritional value, and are found naturally in wide variety of freshwaters in South Asia. The taxonomy of Sperata spp., sampled from Hakaluki Haor in Bangladesh, was re‐evaluated based on morphological variation and DNA barcoding. The collective variation in morphometric characters and mitochondrial DNA revealed an undescribed old and well‐separated lineage under the genus Sperata along with two previously known Sperata aor and Sperata seenghala in Bangladesh. Analyses of morphological traits suggested significant differentiation among Sperata species. The variation in mitochondrial DNA supported the distinct lineage and taxonomical discrimination. Sperata aor diverged earlier from the new lineage and Sperata seenghala with a divergence of 5.39 (CI: 3.91–7.19) Mya (PP > 90). Sperata seenghala and the new lineage shared a most recent common ancestry, which diverged from each other around 3.41 (CI: 2.24–4.62) Mya (PP > 90). Thus, the newly identified lineage could be a subspecies of S. seenghala or even a species under the genus Sperata. The information of the study will be useful for conservation, sustainable management and selective breeding of the putative species, including previously reported S. aor and S. seenghala in Bangladesh.     

Species of the genus Psoroptes (Acari: Psoroptidae): A taxonomic consideration

The biosystematic status of mite species belonging to the genus Psoroptes Gervais, 1841 is difficult to determine by phenotypic methods and has been subject to taxonomic revisions and ongoing debate. At present, the existence of five species, P. cuniculi (Delafond, 1859), P. ovis (Hering, 1838), P. equi (Hering, 1838), P. cervinus Ward, 1915 and P. natalensis Hirst, 1919, is generally accepted. This classification is based mainly on the host species, the localization of the mites on their hosts and morphological characters of male mites. However, a critical review of the literature indicates that the features used to discriminate between the five species are not unequivocal: (a) the localization of mite populations on host animals is not completely strict, (b) the lengths of the outer opisthosomal setae of male mites, which are the main morphological features used for species discrimination, overlap between the five postulated species, and (c) host specificity cannot be deduced from results of transfer experiments. Rather, conspecificity of the members of the genus Psoroptes has to be presumed which is supported by molecular genetic analyses. On these grounds and on rules of priority P. cervinus Ward, 1915, P. cuniculi (Delafond, 1859), P. natalensis Hirst, 1919 and P. ovis (Hering, 1838) are seen as synonyms of P. equi (Hering, 1838).     

Integration of cytochrome c oxidase I barcodes and geometric morphometrics to delimit species in the genus Gnopharmia (Lepidoptera: Geometridae,Ennominae)

We tested the hypothesis of species taxonomy in the genus Gnopharmia (Macariini, Ennominae) that was recently established in a review based on discrete morphological characters. For this objective we integrated both DNA‐based and morphometric approaches in order to infer species boundaries. A 658‐bp fragment of the mitochondrial cytochrome c oxidase subunit 1 (CO1) (DNA barcode) was analysed from populations of five species distributed throughout the Middle East to assess their consistency with traditionally defined morphospecies. Signals in the morphological variation of the aedeagus of all relevant populations were evaluated using geometric landmarks. Consistent groupings compatible with the current taxonomic classification were found with both approaches. The results strongly support the distinction of seven closely related species. © 2013 The Linnean Society of London     

The shieldbug genus Parastrachia and its position within the Pentatomoidea (Insecta: Hemiptera)

A new hemipteran subfamily, Parastrachiinae , is proposed in the Cydnidae for the single genus Parastrachia Distant 1883. There are two included species, P. japonensis (Scott 1880) and P. nagaensis Distant 1908. The morphology, relationships, and taxonomic history of Parastrachia are discussed     

Species delimitation and phylogeny in the genus Nasutitermes (Termitidae: Nasutitermitinae) in French Guiana

Species delimitation and identification can be arduous for taxa whose morphologic characters are easily confused, which can hamper global biodiversity assessments and pest species management. Exploratory methods of species delimitation that use DNA sequence as their primary information source to establish group membership and estimate putative species boundaries are useful approaches, complementary to traditional taxonomy. Termites of the genus Nasutitermes make interesting models for the application of such methods. They are dominant in Neotropical primary forests but also represent major agricultural and structural pests. Despite the prevalence, pivotal ecological role and economical impact of this group, the taxonomy of Nasutitermes species mainly depends on unreliable characters of soldier external morphology. Here, we generated robust species hypotheses for 79 Nasutitermes colonies sampled throughout French Guiana without any a priori knowledge of species affiliation. Sequence analysis of the mitochondrial cytochrome oxidase II gene was coupled with exploratory species‐delimitation tools, using the automatic barcode gap discovery method (ABGD) and a generalized mixed Yule‐coalescent model (GMYC) to propose primary species hypotheses (PSHs). PSHs were revaluated using phylogenetic analyses of two more loci (mitochondrial 16S rDNA and nuclear internal transcribed spacer 2) leading to 16 retained secondary species hypotheses (RSSH). Seven RSSHs, represented by 44/79 of the sampled colonies, were morphologically affiliated to species recognized as pests in the Neotropics, where they represent a real invasive pest potential in the context of growing ecosystem anthropization. Multigenic phylogenies based on combined alignments (1426–1784 bp) were also reconstructed to identify ancestral ecological niches and major‐pest lineages, revealing that Guyanese pest species do not form monophyletic groups.     

Ecotypic differentiation and phenotypic plasticity in reproductive traits of Armadillidium vulgare (Isopoda: Oniscidea)

Armadillidium vulgare differed in growth and survivorship on two field sites. Growth rates were higher at a site with consistently higher quality food than at the other site where less high-quality food was produced and which was less predictably accessible. Survivorship was higher at the second site where temperature fluctuations were consistently smaller. Individuals from the two populations were kept for 6 months under the same food and temperature conditions and patterns of resource allocation to reproductive traits analysed. Members of the population from the site with good growth conditions had significantly higher reproductive allocation, by 13.5%, and larger broods, by 9.1%, than those from the site with poor growth conditions. Contrary to theoretical predictions, they also had significantly larger offspring, by 7.5% dry mass. Larger offspring survived better than small ones. This differential survivorship, by 20% for a 3.4% difference in live mass, was much more pronounced under conditions of moisture stress and under fluctuating temperature regimes. Larger offspring would therefore be at a selective advantage on the site with more severe temperature fluctuations. Phenotypic plasticity in reproductive traits in response to experimental changes in food quality, temperature and crowding were monitored. Reproductive allocation was increased by 20.8% under conditions of higher food quality, by 14.7% at higher temperatures, and by 12.5% under less crowded conditions. Brood size, but not offspring dry mass, increased when food quality increased. When crowding increased by 25.0%, the size of broods remained the same but the dry mass of individual offspring decreased by 11.2%. Members of the population from the site with more variable access to high-quality food showed more plasticity in reproductive traits in response to changes in food supply than members of the population from the site with the more predictable food supply. Members of the population from the site with more stable temperatures showed less plasticity to temperature changes than members of the population from the site with greater temperature fluctuations. It is concluded that the observed microevolutionary processes and phenotypic plasticity have adaptative value as responses to spatial and temporal heterogeneity in environmental conditions.     

Exploring phenotypic plasticity and biogeography in emerald moths: A phylogeny of the genus Nemoria (Lepidoptera: Geometridae)

The moth genus Nemoria (Lepidoptera: Geometridae) includes 134 described species whose larvae and adults display a considerable range of phenotypic plasticity in coloration and morphology. We reconstructed the phylogeny of 54 species of Nemoria and seven outgroups using characters from the mitochondrial genes, Cytochrome Oxidase I and II (COI and COII), and the nuclear gene, Elongation Factor-alpha (EF-1alpha). Maximum parsimony, maximum likelihood and Bayesian inference were used to infer the phylogeny. The 54 ingroup species represented 13 of the 15 recognized species groups of Nemoria [Ferguson, D.C., 1985. Fasc. 18.1, Geometroidea: Geometridae (in part). In: Dominick, R.B. (Ed.), The Moths of America North of Mexico, Fasc. 18.1. Wedge Entomological Research Foundation, Washington; Pitkin, L.M., 1993. Neotropical emerald moths of the genera Nemoria, Lissochlora and Chavarriella, with particular reference to the species of Costa Rica (Lepidoptera: Geometridae, Geometrinae). Bull. Br. Mus. Nat. Hist. 62, 39-159], and the seven outgroups came from four tribes of Geometrinae. These data support Nemoria as a monophyletic group and largely recover the species groupings proposed in previous taxonomic analyses using morphological characters. Phenotypic plasticity of larvae is not correlated with plasticity of adults among those species of Nemoria where life histories are known, and appears to be evolutionarily labile for both life history stages: Species exhibiting larval phenotypic plasticity, such as N. arizonaria and N. outina, are placed in several distinct clades, suggesting that this trait has evolved multiple times, and species displaying adult phenotypic plasticity are likewise distributed throughout the phylogeny. A comparative analysis of the biogeographic history of Nemoria supports a South American origin for the genus with multiple introductions into North America, and an application of published substitution rates to the phylogram provides an age estimate of 7.5 million years.     

Resurrecting Helix straminea,a forgotten escargot with trans‐Adriatic distribution: first insights into the genetic variation within the genus Helix (Gastropoda: Pulmonata)

Helix is a genus of large Western Palaearctic land snails, particularly diverse in the Mediterranean region. Despite the large size and attractiveness of its members, it has an unsettled taxonomy, and no data are available on its intrageneric phylogenetic relationships. One of the problematic Helix taxa is the widely distributed, economically important, and conchologically very variable H. lucorum. Two distinct forms may be encountered under this name in the Apennine Peninsula: a typical one in the north, and a form originally described as Helix straminea in central and southern Italy. To evaluate the status of H. straminea and its relationships to Italian and Balkan Helix fauna, we combined shell morphology, geometric morphometrics, and phylogenetic analysis based on two mitochondrial genes. Distribution data were improved by drawing information from unambiguously identifiable photographs posted online. Based on our results, Helix straminea is redescribed as a separate species, and we find it to have a disjunctive trans‐Adriatic range with closest relatives in the western Balkans. We provide insight into relationships and intraspecific variability within Helix, a first step towards a comprehensive revision of the genus. On the example of Italian Helix fauna, we demonstrate how understanding of snail zoogeography may change with improving taxonomy. © 2014 The Linnean Society of London     

The tribe Bryocorini (Insecta: Heteroptera: Miridae: Bryocorinae): phylogeny,description of a new genus,and adaptive radiation on ferns

A cladistic analysis of the tribe Bryocorini based on 68 morphological characters is conducted. Bryocorini are supported as a monophyletic group with Eccritotarsini as their sister taxon. Based on the phylogenetic analysis, we redefine the tribe Bryocorini to contain the following seven genera: Bryocorella Carvalho, 1956, Bryocoris Fallén, 1829, Bryophilocapsus Yasunaga, 2000, Cobalorrhynchus Reuter, 1906 gen. dist., D iplazicoris gen. nov. , Hekista Kirkaldy, 1902, and Monalocoris Dahlbom, 1851. The genus Bryocorella is transferred to Bryocorini from the tribe Eccritotarsini. The subgenus Cobalorrhynchus is treated as a separate genus. Diplazicoris is described as monotypic to accommodate D iplazicoris lombokianus sp. nov. An updated diagnosis of the tribe, a key to genera, and a diagnosis of each recognized genus are presented. Selected photomicrographs, scanning micrographs, and illustrations of the pretarsus, metepisternal scent efferent system, metafemoral trichobothria, and morphology of head, pronotum, and male and female genitalia are provided. Mapping of the host data on the revealed tree shows that Bryocorini represent one of the very few currently known examples of the adaptive radiation of a fairly large insect group on ferns. © 2015 The Linnean Society of London