Cryptic or pseudocryptic: can morphological methods inform copepod taxonomy? An analysis of publications and a case study of the Eurytemora affinis species complex

Interest in cryptic species has increased significantly with current progress in genetic methods. The large number of cryptic species suggests that the resolution of traditional morphological techniques may be insufficient for taxonomical research. However, some species now considered to be cryptic may, in fact, be designated pseudocryptic after close morphological examination. Thus the “cryptic or pseudocryptic” dilemma speaks to the resolution of morphological analysis and its utility for identifying species. We address this dilemma first by systematically reviewing data published from 1980 to 2013 on cryptic species of Copepoda and then by performing an in‐depth morphological study of the former Eurytemora affinis complex of cryptic species. Analyzing the published data showed that, in 5 of 24 revisions eligible for systematic review, cryptic species assignment was based solely on the genetic variation of forms without detailed morphological analysis to confirm the assignment. Therefore, some newly described cryptic species might be designated pseudocryptic under more detailed morphological analysis as happened with Eurytemora affinis complex. Recent genetic analyses of the complex found high levels of heterogeneity without morphological differences; it is argued to be cryptic. However, next detailed morphological analyses allowed to describe a number of valid species. Our study, using deep statistical analyses usually not applied for new species describing, of this species complex confirmed considerable differences between former cryptic species. In particular, fluctuating asymmetry (FA), the random variation of left and right structures, was significantly different between forms and provided independent information about their status. Our work showed that multivariate statistical approaches, such as principal component analysis, can be powerful techniques for the morphological discrimination of cryptic taxons. Despite increasing cryptic species designations, morphological techniques have great potential in determining copepod taxonomy.     

THE ORIGIN AND RELATIONSHIPS OF LASTHENIA BURKEI (COMPOSITAE)

Lasthenia burkei (Compositae) is a narrowly restricted California endemic closely related to L. conjugens and L. fremontii. These three species differ from each other by pappus and phyllary characters and in geographical distribution. All are freely intercrossable, but L. fremontii forms rather sterile artificial hybrids with its two relatives which, in turn, form fairly fertile artificial hybrids with each other. Lasthenia burkei and L. conjugens have homologous chromosomes, four of which are homologous with four of those of L. fremontii. The remaining two chromosomes probably have reciprocal translocations which lead to multivalent formation during meiosis in interspecific hybrids. Pollen viability is restored in most F₂ generations, suggesting a close genetic relationship among the three species. The evolutionary relationship among these species may be a linear one with L. burkei occupying an intermediate position between L. fremontii and L. conjugens, although the direction of this linear phylogeny is not certain, or it may be one in which L. burkei has been derived from hybridization between its two relatives. Support for the latter hypothesis comes from the appearance of some individuals in F₁ progenies of L. conjugens × L. fremontii that are morphologically indistinguishable from L. burkei (although fairly sterile). The apparently rather simple genetic basis for the morphological characteristics of each of the species in this trio suggests that the morphologically heterogeneous genus Lasthenia may be considerably more homogeneous genetically than might be suspected. Because of the diverse kinds of relationships among these three Lasthenias, possible alternative taxonomies for the group are dependent upon those relationships that a taxonomist wishes to communicate. Nevertheless, the patterns of diversification in this group have led to reaffirmation of an earlier decision that three species should be recognized.     

Morphological Variability of Flowers of <Emphasis Type="Italic">Antennaria dioica</Emphasis> (L.) Gaertn.: Circumstantial Evidence for the Origin of Taxon Dioecy from Hermaphroditism through Gynodiecy

Based on the literature, we assumed the presence of several morphological forms of flowers of the dioecy species Antennaria dioica (L.) Gaertn. We investigated 6000 flowers from three sites in Europe. As a result of this study, we allocated three morphological types of flowers: female, male, and hermaphroditic (2.5% sample). Variability of the degree of reduction of different organs, especially in the case of male flowers, was detected.     

TWO MORPHOLOGICAL FORMS OF CYLINDROSPERMOPSIS RACIBORSKII (CYANOBACTERIA) ISOLATED FROM SOLOMON DAM, PALM ISLAND, QUEENSLAND

Cylindrospermopsis raciborskii Woloszynska is a prominent constituent of a number of water bodies in northern Australia. In Solomon Dam, this species occurs as two distinct morphological forms, one with straight trichomes and one with coiled trichomes. Isolates of the two forms have been grown in pure culture and have been shown to maintain their respective characteristic form over successive generations. Both forms were similar with respect to cylindrospermopsin content expressed as a percentage of freeze-dried culture material, ranging from 0.14% to 0.20%, depending on the N source provided in the medium. A morphological comparison between natural populations of the two forms showed significant differences in vegetative, heterocyst, and akinete cell dimensions. These characteristics are the primary taxonomic criteria at the species level in this genus. In culture, the coiled form grew slightly faster than the straight form over the range of conditions investigated in this study. The coiled form was better suited to growth under low-light conditions. These clear and consistent morphological and physiological differences contrast with the 99.8% similarity between the two forms in their 16S rRNA gene nucleotide sequences. It is concluded that although taxonomic separation of the two forms at the species level might not be warranted, the two strains investigated are clearly distinct morphotypes, and it is recommended that they be so recorded in monitoring programs.     

Ultrastructure of the extensively developed nuchal organs of Laonice bahusiensis (Annelida: Canalipalpata: Spionidae)

The nuchal organs of annelid Laonice bahusiensis (Spionidae) from northern Europe have been studied using scanning and transmission electron microscopy. L. bahusiensis is the first spionid species in which extensively developed, continuous nuchal organs are described. The nuchal organs of this genus are the longest known among polychaete annelids. They consist of paired double bands extending from the prostomium on a mid‐dorsal caruncle for about 24–30 setigers. Their microanatomy corresponds to the general structural plan of nuchal organs: there are ciliated supporting cells and bipolar sensory cells with sensory cilia traversing an olfactory chamber. The organs are overlaid by a secondary paving‐stone‐like cover and innervated by one pair of longitudinally elongated nuchal nerves. These findings clearly favor the hypothesis that the paired, extensively developed ciliated structures found in some Spionidae are homologous with the prostomial nuchal organs characteristic of polychaete annelids. J. Morphol. 2010. © 2009 Wiley‐Liss, Inc.     

网衣属3个中国新记录种

该研究以采集于中国新疆地区的网衣科网衣属地衣标本为试验材料,采用形态解剖学、地衣化学物质检测方法和系统发育学方法,观察分析网衣属地衣的形态解剖特征、次生代谢产物以及核糖体DNA内转录间隔区(internal transcribed spacer,ITS)系统发育树,依据形态解剖数据、次生代谢产物种类以及系统发育树分类地位更为综合的鉴定种。结果显示：(1)共鉴定出网衣科网衣属3个中国新记录种——暗色网衣(Lecidea fuscoatrina Hertel & Leuckert)、珠光网衣(Lecidea perlatolica Hertel & Leuckert)和多器网衣(Lecidea polypycnidophora U. Rupr. & Türk),并提供了3个新记录的形态解剖图。(2)ITS系统发育树结果表明,暗色网衣与近缘种Lecidea atrobrunnea原种及亚种、Lecidea fuscoatra、珠光网衣与近缘种Lecidea promiscens以及Lecidea polypycnidophora与近缘种Lecidea andersonii各形成一个分支,依据分支组成可将形态差异不明显的近缘种鉴别开。     

Generic interrelationships within the Spionidae (Annelida: Polychaeta)

The phylogenetic relationships of spionid genera are estimated from parsimony analyses of morphological characters, with Trochochaetidae, Poecilochaetidae and Uncispionidae as outgroups. A first analysis of currendy recognised genera proved inconclusive and even exclusion of six of the most polymorphic genera resulted in 13 305 equally parsimonious trees and a fully collapsed consensus tree. A second analysis using only the type species of each genus, yielded four equally parsimonious trees; reduced to two after successive weighting. The topologies of these two trees indicated division of the family into four main groups: (1) Aonidella and Xandaros; (2) Prionospio (sensu fato)-complex, Laonice, Spiophanes and Aonides; (3) a large assemblage of genera, including Polydora-{senm late), Scolelepis, Malacoceros and Spio; (4) Atherospio, Pseudatherospio and Pygospiopsis. Earlier literature classifications of the group are evaluated and compared with die new results.     

Symbiotic associations in the phenotypically-diverse brown alga Saccharina japonica

The brown alga Saccharina japonica (Areschoug) Lane, Mayes, Druehl et Saunders is a highly polymorphic representative of the family Laminariaceae, inhabiting the northwest Pacific region. We have obtained 16S rRNA sequence data in symbiont microorganisms of the typical form (TYP) of S. japonica and its common morphological varieties, known as "longipes" (LON) and "shallow-water" (SHA), which show contrasting bathymetric distribution and sharp morphological, life history traits, and ecological differences. Phylogenetic analysis of the 16S rRNA sequences shows that the microbial communities are significantly different in the three forms studied and consist of mosaic sets of common and form-specific bacterial lineages. The divergence in bacterial composition is substantial between the TYP and LON forms in spite of their high genetic similarity. The symbiont distribution in the S. japonica forms and in three other laminarialean species is not related to the depth or locality of the algae settlements. Combined with our previous results on symbiont associations in sea urchins and taking into account the highly specific character of bacteria-algae associations, we propose that the TYP and LON forms may represent incipient species passing through initial steps of reproductive isolation. We suggest that phenotype differences between genetically similar forms may be caused by host-symbiont interactions that may be a general feature of evolution in algae and other eukaryote organisms. Bacterial symbionts could serve as sensitive markers to distinguish genetically similar algae forms and also as possible growth-promoting inductors to increase algae productivity.     

Extensive cryptic diversity in the cosmopolitan sludge worm <Emphasis Type="Italic">Limnodrilus hoffmeisteri</Emphasis> (Clitellata,Naididae)

Limnodrilus hoffmeisteri Claparède, 1862 is a common freshwater worm, often regarded as an indicator of organic pollution. The taxonomic status of this species is controversial due to great variation in morphological features. Numerous morphological forms of L. hoffmeisteri are recorded in the literature, especially from Europe and North America. Today, DNA-based species delimitation assumes that species boundaries can be more objectively and effectively estimated using genetic data rather than with morphological data alone. To investigate if L. hoffmeisteri is a single species, 295 worms identified as either L. hoffmeisteri or other similar (congeneric) morphospecies, using currently accepted morphological criteria, were collected from 82 locations in the northern hemisphere. The number of primary species hypotheses (PSHs) was first explored with cytochrome oxidase subunit I (COI), the proposed DNA barcode for animal species, and with data for all specimens. Both automatic barcoding gap discovery (ABGD) and the Bayesian general mixed Yule coalescent (bGMYC) model revealed the existence of ≥25 distinct PSHs (COI lineages) in our dataset. Then, smaller samples of individuals representing major COI lineages were used for exploration of a nuclear locus, the internal transcribed spacer (ITS) region. In the ITS gene tree (81 sequences), generated by BEAST, 16 well-supported terminal groups were found, but not all of these groups were congruent with the PSHs found in the COI tree. As results across these different analyses were inconsistent, we resorted to analyzing reciprocal monophyly between gene trees and used a minimum consensus of all evidence, suggesting that there are 13 separately evolving lineages (=13 species) within our sample. The smallest uncorrected COI p-distance between these species is 12.1%, and the largest intraspecific p-distance is 16.4%, illustrating the problem of species delimitation with a DNA-barcoding gap as the sole criterion. Ten of these species are morphologically identified as “L. hoffmeisteri,” the remaining three can be attributed to morphologically distinct congeneric species. An individual from the type locality in Switzerland was designated as a neotype of L. hoffmeisteri sensu stricto. This worm belongs to one of the ten species, and this lineage is widely distributed in Europe, Asia, and North America. The remaining nine species show a mixed distribution pattern; some appear to be endemic to a restricted area, others are Holarctic. Our results provide clues to the future revalidation of some of the nominal species today placed in synonymy with L. hoffmeisteri. A BEAST analysis, based on previously published and newly generated 16S data, suggested that this complex contains also other species than those studied by us. By integrating additional genetic data, it will be possible to identify these and additional specimens in future studies of Limnodrilus, and the neotype provides a baseline for further revisions of the taxonomy of the L. hoffmeisteri complex.     

A molecular phylogenetic and phylogeographic study of two forms of Calliptamus barbarus (Costa 1836) (Orthoptera: Acrididae,Calliptaminae) from two regions of Algeria

Calliptamus barbarus (Orthoptera: Acrididae) is the most polymorphic species within the genus Calliptamus. It shows a morphological polymorphism (three hind femoral spots, or only one hind femoral spot). Several studies have been made in order to distinguish the two forms: morphometry, number of ovarioles, sound production, protein and enzyme system. The aim of our work is to assess whether the two forms can be considered as different taxa and to perform a molecular phylogenetic study of two populations of C. barbarus collected from two different Algerian localities. No clear genetic differentiation was found between the samples with different morphologies. Additionally, the samples from Algeria do not form a monophyletic sister clade compared to the one formed by the sequences from GenBank from other geographical regions. Despite the morphological differences shown between the two populations, our molecular study indicates that there are no differences at a molecular level using the two mitochondrial genes COI and 16S.     

CHEMOTAXONOMY IN MARINE ALGAE: CHEMICAL SEPARATION OF SOME LAURENCIA SPECIES (RHODOPHYTA) FROM THE GULF OF CALIFORNIA1

Species separation in the genus Laurencia (Rhodomelaceae, Rhodophyta) is complicated by the high degree of morphological variation within the species. Chemical investigations on a worldwide basis of over 15 species indicate that 1 or more of the halogenated natural products synthesized by Laurencia are unique to each species. Our chemical investigations of Laurencia pacifica, as presently understood from the Gulf of California, indicate that more than 1 species had been included under this name. Thin layer chromatographic (TLC) comparisons of the halogenated components of 3 recognizable forms of “L. pacifica” were completed. The results revealed 3 distinct forms, with halogenated products unique to each form. In each form the observed chemical characters had been previously isolated and identified and could now be positively assigned to their algal source. Comparisons were also conducted with L. pacifica Kylin (1941) from the type locality of the species, La Jolla, California, and revealed that it contained another halogenated product different from those isolated from the Gulf species. We conclude that 3 species of Laurencia have been elucidated in the Gulf of California and these are separate from L. pacifica Kylin. Each species can be distinguished by its characteristic array of halogenated compounds. Comparative thin layer chromatography of the lipid components of morphologically similar Laurencia species should prove to be a useful new taxonomic aid.     

Erysiphe azerbaijanica and E. linderae: Two new powdery mildew species (Erysiphales) belonging to the Microsphaera lineage of Erysiphe

Two new species, Erysiphe azerbaijanica on Castanea sativa and E. linderae on Lindera praecox, both belonging to the Microsphaera lineage of the genus Erysiphe are described based on morphological and molecular data. Erysiphe azerbaijanica is distinguished from other Erysiphe species occurring on Castanea spp. by its cylindrical conidia with a length/width ratio of 2–3.6, longer conidiophore, and foot-cells. Molecular analyses indicated that this species forms a clade of its own, supporting the morphological observations. Molecular phylogenetic analyses showed that E. blasti s. lat. is divided into two genetically differentiated groups associated with different host species. Based on the sequence differences in the 28S rRNA gene and ITS region, connected with differences in the number and length of appendages, the fungus on L. praecox is described as a new species, E. linderae.     

The Use of Chemical and Infra-red Spectral Analyses to Verify the Identification of Two Species of Duckweed, Lemna minor L. and Lemna gibba L. Grown Under Defined Conditions

Flat forms of the fronds of Lemna gibba L., used widely in laboratoryexperiments, can be easily confused with Lemna minor Chemicalanalyses of the two species, grown under defined laboratoryconditions, showed clear distinctions between the two speciesin relation to total protein content and the content of individualmonosaccharides. Infra-red spectral analysis confirmed the chemicaldata for protein content and also revealed differences in thetypes of proteins present in the two species. Chemical and infra-redspectral analyses, particularly the amide I: carbohydrate absorbanceratio, provide a rapid and reliable means of verifying betweenthe two species, which are often difficult to identify withcertainty from morphological differences. Key words: Gibbosity, infra-red spectroscopy, Lemna gibba, Lemna minor, monosaccharides, proteins     

中国苔类孢子形态的研究

通过电镜扫描方法 ,研究了 3个属 9种的中国叶状体苔类植物 :稀枝钱苔 (RicciahuebenerianaLindb .)、宽翅钱苔 (R .hantamensisPerold .)、曲脊钱苔 (R .cavernosaHoffm .)、R .warnstorfiiLimpr.exWarnst.、小孢钱苔 (R .frostiiAust.)、中华钱苔 (R .chinensisHerz .)、东亚花萼苔 (Asterellayoshinagana (Horik .)Horik .)、矮网花萼苔 (A .sanguiniaL .etL .)、背托苔 (Preissiaquadrata) (Scop .)Nees.)。对上述种类的孢子形态特征进行了描述。研究发现 ,上述 3属的孢子形态特征具有明显差异。而且同一属的孢子形态特征也存在差异 ,甚至可以作为种类划分的依据之一。同时采自不同地点的同一种类其孢子形态有时具微小的差别。这可能是由于生态环境差异所造成的     

TOWARD A NATURAL SYSTEM OF SPECIES IN CHLOROGONIUM (VOLVOCALES, CHLOROPHYTA): A COMBINED ANALYSIS OF MORPHOLOGICAL AND rbcL GENE SEQUENCE DATA

The taxonomy of species of Chlorogonium (Volvocales, Chlorophyta) was studied based on comparative light and electron microscopy and DNA sequence data of 23 strains from five major algal culture collections. All of the 23 strains showed pyrenoids under photoautotrophic conditions, but 17 of the 23 exhibited marked reduction in size of pyrenoids, or pyrenoids were absent under photoheterotrophic conditions. The strains could clearly be delineated into six species, C. euchlorum, C. elongatum, C. fusiforme, C. capillatum, C. neglectum, and C. kasakii on the basis of differences in cell shape, number of contractile vacuoles, number and stability of pyrenoids, and ultrastructure of pyrenoids and stigmata. This distinction of species based on morphology was also supported by analyses of rbc L gene sequences. The later strongly showed that each species, C. euchlorum (seven strains), C. elongatum (three strains), and C. capillatum (10 strains), forms a robust clade. Although some morphological differences were noted within different strains of C. euchlorum and C. capillatum, these features were regarded as strain-specific because they were not reflected in the rbc L gene phylogenies. In addition, the rbc L gene trees strongly suggested that C. neglectum and C. kasakii are closely related to each other, consistent with the similarity of the ultrastructure of pyrenoids and stigmata between the two species. However, C. kasakii can be distinguished clearly from C. neglectum by its multiple pyrenoids in the chloroplast and acute anterior and posterior ends in the vegetative cell.     

贵州高原6种掌突蟾形态特征差异分析

本文通过形态指标测量,比较了分布于贵州高原的6种掌突蟾属(Leptobrachella)物种的形态特征差异.结果 显示,头体长比较,6种掌突蟾的头体长从大到小依次为赤水掌突蟾(L.chishuiensis)、绥阳掌突蟾(L.suiyangensis)、金沙掌突蟾(L.jinshaensis)、毕节掌突蟾(L.bijie...     

Investigation on Pollen Morphology of Aconitum L.

Pollen morphology of 62 species in Aconitum L. was investigated with light and scanning electron microscopies. The pollen grains in this genus are 3-colpate and exhibit spinulate surface pattern in all the species. According to the shape of pollen grains in equatorial view Aconiturn L. may be divided into three types. Type Ⅰ pollen grains are wide elliptic; Type Ⅱ are narrow-elliptic and type Ⅲ are rectangular or close to square in shape. The pollen characteristics among species are very similar. The morphological information of the pollen grains shows that species of Aconiturn L. are a natural taxon.     

Validity Analysis of the Morphological Identification of Three Highly Morphologically Similar Species Belonging to the Genus Niviventer (Rodentia: Muridae)

Cryptic species are prevalent among mammals, and identifying morphological methods or measurements that can effectively distinguish cryptic species has high significance in taxonomy. Three of the white-bellied rats, Niviventer confucianus, N. fulvescens and N. huang (Rodentia: Muridae), have such similar morphological characteristics that taxonomists have not been able to effectively identify them according to their morphologies. Recent studies have determined that the N. fulvescens species complex contains multiple species, indicating that previous morphological comparisons were potentially based on inaccurate species identifications, leading to false conclusions. To clarify whether the morphological differences among these three species can be used for identification, a variety of morphological methods and measurements were combined with molecular species identification to ensure accurate identifications prior to comparing morphological characteristics. The results showed that: (1) only N. confucianus has a white tail tip, distinguishing this species from the other two species but not N. fulvescens from N. huang; and (2) the tail length of N. fulvescens and ear length of N. confucianus are greater than those of the other species. Traditional morphological methods cannot differentiate these species well, with the discrimination rate reaching more than 90%; therefore, they can only be used as auxiliary methods for morphological identification. Additionally: (3) the geometric morphological results of the four skull views can be combined to distinguish the three species, among which the discrimination rate of lateral view of mandible reaches 98%, making it effective for differentiating these species; and (4) the phallic morphologies of the three species differed significantly in the presence of a dorsal groove, thickness of the distal baculum, and the positional relationship between the distal and proximal baccula; thus, this approach can completely distinguish these species. This study examines the applicability of various morphological measurements for the identification of highly morphologically similar species, provides a reference for distinguishing cryptic species produced by convergent evolution or incomplete differentiation by morphology, and suggests that phallic morphology should be the primary characteristic for differentiating such species in the future.