Combining geometric morphometrics with pattern recognition for the investigation of species complexes

The parasitoid Hymenoptera contain a large number of species complexes that are as yet unresolved by traditional taxonomic studies. Evolutionary studies as well as biological control programmes often require further investigations which cannot rely on simple qualitative morphological characters; in many cases, particularly with dried specimens housed in museums, molecular approaches cannot be used. Recent developments in geometric morphometrics and statistical exploratory approaches open new perspectives for the objective evaluation of morphological characters in this taxonomic context. In this study, geometric morphometrics and pattern recognition approaches were applied to the wing shape and venation of two closely related braconid species considered to differ by subtle qualitative morphological head characters. Exploratory analyses such as kernel density estimates and Gaussian mixture analyses were used to explore the structure of the data in the multivariate morphometric space. Discrimination techniques (linear discriminant functions and neural networks combined with cross‐validations) were used to estimate the taxonomic value of qualitative characters. Gaussian mixtures highlighted the existence of two non‐overlapping groups. A good congruence was found between one of the two groups and the a priori defined Bassus tumidulus. The misclassification rate was higher for B. tegularis specimens, which also appeared morphometrically heterogeneous. Discrimination between the two a priori defined species was incomplete with misclassification rates higher than, or equal to, 6%. In most cases, the lack of congruence between species and morphometrically defined subgroups could be related to specimens that exhibited ambiguous qualitative character states. In summary, if two entities are present, they still need to be defined morphologically, while B. tegularis heterogeneity calls for further investigation of specimens of known origin and hosts. © 2003 The Linnean Society of London, Biological Journal of the Linnean Society, 2003, 80, 89–98.     

Defensive strategies of soft corals (Coelenterata: Octocorallia) of the Great Barrier Reef

Summary The relationship between ichthyotoxicity and predation-related defensive functional morphology was examined in alcyonacean soft corals of the central and northern regions of the Great Barrier Reef (GBR), Australia. Approximately 170 specimens were assessed encompassing a number of genera within three families: 1) the Alcyoniidae (Lobophytum, Sarcophytum, Sinularia, Cladiella, Parerythropodium, and Alcyonium); 2) Neptheidae (Lemnalia, Paralemnalia, Capnella, Lithophyton, Nephthea, Dendronephthya, Scleronephthya, and Stereonephthya), and 3) Xeniidae (Anthelia, Efflatounaria, Cespitularia, Heteroxenia, and Xenia). Ichthyotoxicity data were derived from earlier studies which used Gambusia affinis Baird and Girard (Vertebrata, Pisces) as a test organism. These data were compared to morphological data collected from specimens in the field and laboratory. Three sets of statistical analyses were performed, each considering a progressively narrower group of taxa. The first included 68 specimens and considered 16 morphological characters in each, falling into the general categories of gross colony form, colony texture, presence of mucus, colony color, polyp retractility, and sclerite morphology and distribution. These were tested for independence against ichthyotoxicity data. The second set of analyses involved a more restricted morphological data set derived from 28 species of Sinularia in combination with 28 species within the Nephtheidae, comparing them to their respective toxicity ranks. The third analysis considered the previous two taxonomic groups separately in relation to their toxicity levels.The attempt to consider many morphological characters in a taxonomically diverse collection did not reveal any general association in the Alcyonacea between defensive morphology and toxicity, and those associations which did emerge were clearly erroneous. The second analysis, considering only Sinularia spp. and nephtheids, demonstrated a negative association between ichthyotoxicity and the morphological characters of a) polypary armament, b) microarmament of the individual polyp, and c) strong mineralization of the coenenchyme. The third analysis revealed that the negative association found between toxicity and the first two characters was derived entirely from the nephtheids while the association detected between toxicity and the third character was restricted to Sinularia. It is concluded that a relationship between toxicity and morphology can be demonstrated, but it is heavily dependent upon which specific morphological characters are being considered and at what level of taxonomic resolution the analysis is being performed. An approach utilizing many characters over many taxa is unlikely to yield significant, reliable, or meaningful results.Australian Institute of Marine Science Contribution Number 383     

The influence of spatial scale on the congruence of classifications circumscribing morphological units of biodiversity

Aim The ‘taxonomic impediment’ has led to a growing trend in ecology and conservation biology to use operational surrogates for species within the context of a particular research project. Because such ‘parataxonomic’ classifications are typically spatially limited in scope, we examined the influence of increasing spatial scale on the congruence of two such approaches with a more traditional taxonomic classification. Location Sturt National Park, north‐western New South Wales, Australia. Methods Specimens of two ant genera, Camponotus and Rhytidoponera, were classified by three independent methods. The ‘parataxonomic’ classification assigned specimens to morphospecies without specialist taxonomic expertise; the ‘taxonomic’ classification assigned specimens to either described species or, where this was not possible, to operational taxonomic units (OTUs) using specialist taxonomic expertise; the ‘phenetic’ classification assigned specimens to putative species using a K‐means partitioning algorithm on basic morphometric data. Specimens of each genus were pooled into ‘assemblages’, which were defined at multiple spatial scales using a nested sampling design. Congruence in the interspecimen relationships of the different classifications was tested for each assemblage using pair‐wise Mantel correlations. Results Classification congruence tended to decrease with increasing spatial scale. There were, however, clear differences between the genera. Parataxonomic–taxonomic congruence was consistently greater for Camponotus, while phenetic–taxonomic congruence showed the opposite pattern. Conclusions Observed patterns in classification congruence are attributed to two principal causes: (i) within‐species morphological variation, including ecotypic variation in Rhytidoponera and caste polymorphism in Camponotus; and (ii) a limit to the morphological similarity of potentially competing species at small spatial scales. Regardless of cause, the decline in agreement as the spatial scale of observation is increased has important implications for the measurement of biodiversity, particularly when comparing samples over regional, continental, and global scales.     

Integrative insect taxonomy based on morphology,mitochondrial DNA,and hyperspectral reflectance profiling

Integrative taxonomy is considered a reliable taxonomic approach of closely related and cryptic species by integrating different sources of taxonomic data (genetic, ecological, and morphological characters). In order to infer the boundaries of seven species of the evacanthine leafhopper genus Bundera Distant, 1908 (Hemiptera: Cicadellidae), an integrated analysis based on morphology, mitochondrial DNA, and hyperspectral reflectance profiling (37 spectral bands from 411–870 nm) was conducted. Despite their morphological similarities, the genetic distances of the cytochrome c oxidase subunit I (COI) gene among the tested species are relatively large (5.8–17.3%). The species‐specific divergence of five morphologically similar species (Bundera pellucida and Bundera spp. 1–4) was revealed in mitochondrial DNA data and reflectance profiling. A key to identifying males is provided, and their morphological characters are described. Average reflectance profiles from the dorsal side of specimens were classified based on linear discriminant analysis. Cross‐validation of reflectance‐based classification revealed that the seven species could be distinguished with 91.3% classification accuracy. This study verified the feasibility of using hyperspectral imaging data in insect classification, and our work provides a good example of using integrative taxonomy in studies of closely related and cryptic species. © 2015 The Linnean Society of London     

Patterns of cryptic host specificity in duck lice based on molecular data

Documenting patterns of host specificity in parasites relies on the adequate definition of parasite species. In many cases, parasites have simplified morphology, making species delimitation based on traditional morphological characters difficult. Molecular data can help in assessing whether widespread parasites harbour cryptic species and, alternatively, in guiding further taxonomic revision in cases in which there is morphological variation. The duck louse genus Anaticola (Phthiraptera: Philopteridae), based on current taxonomy, contains both host‐specific and widespread species. Mitochondrial and nuclear DNA sequences of samples from this genus were used to document patterns of host specificity. The comparison of these patterns with morphological variations in Anaticola revealed a general correspondence between the groups identified by DNA sequences and morphology, respectively. These results suggest that a more thorough taxonomic review of this genus is needed. In general, the groups identified on the basis of molecular data were associated with particular groups of waterfowl (e.g. dabbling ducks, sea ducks, geese) or specific biogeographic regions (e.g. North America, South America, Australia, Eurasia).     

Morphological analysis ofAlouatta seniculus species group (Primates,Cebidae). A comparison with biochemical and karyological data

Fifteen cranial measurements were taken from wild caught specimens ofAlouatta seniculus seniculus, A. s. stramineus andA. s. macconnelli. A morphological analysis showed sex dimorphism in these three groups. A multivariate analysis discriminated among these taxa; males being more clearly discriminated than females. Our data showed that these taxa can be separated on the basis of quantitative cranial traits, biogeographic distribution, karyological differences, and biochemical characters. We therefore propose a new taxonomic arrangement, changing their taxonomic status to the species level (Alouatta seniculus, A. macconnelli, andA. stramineus).     

淫羊藿属分类学研究:进展、问题与展望

淫羊藿是我国特有且传统的重要药用植物,逐渐步入大宗品种行列。物种的准确鉴定是药效保障和用药安全的前提,为促进其资源的合理开发和利用,该文对淫羊藿属分类学研究进行系统梳理,并对其中存在的问题及存疑类群进行阐述。淫羊藿属共发表68种,中国58种(85.3%),其中57种为特有分布,具有显著的资源优势。淫羊藿属中国类群的分类学研究较为特殊,共26种集中发表于1990s,共31种(53.4%)为国外研究者命名,且绝大多数依据少量栽培个体命名。由于缺乏广泛的形态调查和性状变异分析,导致大量类群的形态描述不准确或不全面,后续20个类群被归并或降级。依据栽培个体命名的类群是补充描述和分类修订的重点。花色、根茎类型、花茎叶的数量及着生方式等性状在中国类群中存在广泛变异。经分类修订后,该属目前包括46种、1亚种和2变种。淫羊藿属中国组类群仍处于活跃进化中,其形态变异复杂,种间关系无法得到解决,为该属分类的最大挑战。但种质资源的研究和利用需要建立在清晰的分类学基础上。未来研究应基于居群调查,完善各物种的形态描述;在此基础上,整合形态变异特征、地理分布格局和基因序列特征,检测自然种间杂交事件,从而揭示物种的...     

The taxonomic status of Astragalus mokiacensis (Fabaceae), a species endemic to the southwestern United States

Astragalus mokiacensis has been a problematic species since it was first described in 1877. Every major revision delimited this taxon differently. A principal components analysis of morphological data from herbarium specimens was used to determine the affinities between type specimens and extant populations ofA. mokiacensis. The taxon recently recognized asA. lentiginosus var.trumbullensis is morphologically similar to the lectotype ofAstragalus mokiacensis. Astragalus lentiginosus var.trumbullensis is herein recognized as a lowelevation minor variant and considered a synonym ofA. mokiacensis. A taxonomic key and complete synonymy are included.     

Molecular and morphological patterns across Acanthocyclops vernalis‐robustus species complex (Copepoda,Cyclopoida)

Bláha, M., Hulák, M., Slouková, J. & Tě?itel, J. (2010). Molecular and morphological patterns across Acanthocyclops vernalis‐robustus species complex (Copepoda, Cyclopoida).—Zoologica Scripta, 39, 259–268. Morphological traits within Acanthocyclops (Kiefer, 1927) are highly variable, and morphology is too constrained to give complete information of phylogenetic relationships. This study combined morphological and molecular techniques to investigate the taxonomic and phylogenetic relationships of three species of Acanthocyclops (Acanthocyclops trajani, Acanthocyclops einslei and Acanthocyclops vernalis) inhabiting continental Europe. Morphological indices subjected to principal component analysis (PCA) separated sample populations into three distinct clusters corresponding with the taxonomic status of the species analysed. In addition, the taxonomy status of A. trajani and A. einslei was in agreement with molecular data; however, the intraspecific variation in sequences of 12S rRNA was lower in contrast to specimens morphologically determined as A. vernalis, which were divided into two deeply divergent clades, based on mtDNA sequence divergences. Moreover, high sequence divergence (26%) between these clades indicated the existence of another species that may not be a sister taxon of A. vernalis s.s. Results point to the need for further taxonomic work on Acanthocyclops.     

The taxonomic status of some Atlanto‐Mediterranean species in the subgenus Holothuria (Echinodermata: Holothuroidea: Holothuriidae) based on molecular evidence

Molecular and morphological data were used to evaluate the taxonomic status of the species Holothuria tubulosa Gmelin, 1790 , Holothuria stellati Delle Chiaje, 1823 , Holothuria mammata Grube, 1840 , and Holothuria dakarensis Panning, 1939, belonging to the nominate subgenus Holothuria (Holothuria) (family Holothuriidae) from the Mediterranean Sea and Atlantic Ocean. A 16S rRNA marker distinguished three well‐supported clades with clear genetic differentiation amongst them. The morphometric characters, although they reflected the clades, showed great variability, and some specimens from different clades overlapped. The morphological data and the literature suggest that the clades correspond to H. dakarensis (from Cape Verde Islands), H. mammata (from the Atlanto‐Mediterranean area) and H. tubulosa (from the Mediterranean Sea). Holothuria stellati is considered here to be a junior subjective synonym of H. tubulosa. Great morphological intraspecific variation within H. tubulosa and H. mammata explains the confusion in the literature. Holothuria tubulosa includes specimens with distinctive ossicles, but others are similar to H. mammata. In these cases, the presence or absence of Cuvierian tubules proved a reliable indicator to the identity of these species; unfortunately this character is difficult to assess in preserved material. According to the results of discriminant analysis we propose a set of ossicle morphometric variables that permit the optimum assignation of individuals to the clades. Our results present a new perspective on the taxonomic status of species in Holothuria (Holothuria), and show how a molecular approach, combined with a morphological approach, can solve taxonomic problems.     

Reuse of voucher specimens provides insights into the genomic associations and taxonomic value of wing colour and genitalic differences in a pest group (Lepidoptera: Tortricidae: Choristoneura)

Subtle morphological differences can be essential to diagnosing closely related species, and an understanding of the genetic basis of these characters can contribute to understanding their divergences. We used voucher specimens from previous genetic analyses of population structure to subsequently analyse genome-wide associations linking morphology to genetic variation in spruce budworms, a group of economically important and morphologically similar forest pests. In particular, we assessed the taxonomic value and genetic architecture of two morphological traits (wing pattern and genitalic spicule abundance) that have been reported to differ among spruce budworm species. Our results suggest that phallic spicule number has greater taxonomic utility than wing pattern for distinguishing Choristoneura fumiferana (Clemens) from Choristoneura occidentalis occidentalis Freeman and Choristoneura occidentalis biennis Freeman. However, there was considerable overlap among taxa for all phenotypic characters analysed. In a genome-wide association study, wing pattern variation was significantly associated with four single nucleotide polymorphism (SNP) loci, including two adjacent SNPs. One SNP was flanked by sequence resembling RNA-directed DNA polymerase from mobile element jockey-like. This locus is a promising candidate for the study of wing pattern development in spruce budworms, as jockey-like transposable elements and polymerases have documented roles in wing spot production in other Lepidoptera. Our study links classical taxonomic characters and genomic data to provide insights into the potential genetic architecture of species differences. It also demonstrates previously untapped morphological and taxonomic value in voucher specimens from earlier molecular genetic analyses.     

Barcoding,molecular taxonomy,and exploration of the diversity of shrews (Soricomorpha: Soricidae) on Mount Nimba (Guinea)

We tested the efficiency of cytochrome oxidase I (COI)‐barcoding as a taxonomic tool to discriminate and identify sympatric shrew species on Mount Nimba (Guinea). We identified 148 specimens at the species level using morphological characters and comparison with type specimens, including several taxa from Mount Nimba. We identified ten morphospecies and tested aspects of genetic diversity and monophyly using genetic data from three mitochondrial (16S, cytochrome b, and COI) and one nuclear marker (the breast cancer gene, BRCA). Nine morphospecies were validated under the phylogenetic and genetic species concepts, including the recently diverged species Crocidura buettikoferi, Crocidura theresae, and Crocidura grandiceps. Under the same concepts, our analyses revealed the presence of two cryptic species amongst animals identified as Crocidura muricauda. We then tested the efficiency of barcoding thanks to commonly used phenetic methods, with the 148 specimens representing 11 potentially valid species based on morphological and molecular data. We show that COI‐barcoding is a powerful tool for shrew identification and can be used for taxonomic surveys. The comparison of genetic divergence values shows the presence of a barcoding gap (i.e. difference between the highest intraspecific and the lowest interspecific genetic divergence values). Given that only a few COI sequences are available for Afrotropical shrews, our work is an important step forward toward their enrichment. We also tested the efficiency of the three other sequenced markers and found that cytochrome b is as efficient as COI for barcoding shrews. © 2012 The Linnean Society of London, Zoological Journal of the Linnean Society, 2012, 166 , 672–687.     

The validation of specific status of the sakhalin sturgeon <Emphasis Type="Italic">Acipenser mikadoi</Emphasis> (Acipenseridae) in the light of recent genetic and morphological data

The comparative morphological study in different Acipenseriform species demonstrates that there are only two morphological features combining the kaluga and the great sturgeon and separating them from other sturgeons, namely the shape of a mouth and the manner attaching of gill membranes to isthmus in adult specimens; whereas many morphological characters are different in these species. The relations of morphological similarity/distinction among different sturgeon species and polyphyletic origin of genus Huso revealed by both molecular and cytogenetic studies presume the restoration of initial taxonomic states for the great sturgeon and kaluga as members of the same genus Acipenser, namely A. huso and A. dauricus.     

One species in eight: DNA barcodes from type specimens resolve a taxonomic quagmire

Each holotype specimen provides the only objective link to a particular Linnean binomen. Sequence information from them is increasingly valuable due to the growing usage of DNA barcodes in taxonomy. As type specimens are often old, it may only be possible to recover fragmentary sequence information from them. We tested the efficacy of short sequences from type specimens in the resolution of a challenging taxonomic puzzle: the Elachista dispunctella complex which includes 64 described species with minuscule morphological differences. We applied a multistep procedure to resolve the taxonomy of this species complex. First, we sequenced a large number of newly collected specimens and as many holotypes as possible. Second, we used all >400 bp examine species boundaries. We employed three unsupervised methods (BIN, ABGD, GMYC) with specified criteria on how to handle discordant results and examined diagnostic bases from each delineated putative species (operational taxonomic units, OTUs). Third, we evaluated the morphological characters of each OTU. Finally, we associated short barcodes from types with the delineated OTUs. In this step, we employed various supervised methods, including distance‐based, tree‐based and character‐based. We recovered 658 bp barcode sequences from 194 of 215 fresh specimens and recovered an average of 141 bp from 33 of 42 holotypes. We observed strong congruence among all methods and good correspondence with morphology. We demonstrate potential pitfalls with tree‐, distance‐ and character‐based approaches when associating sequences of varied length. Our results suggest that sequences as short as 56 bp can often provide valuable taxonomic information. The results support significant taxonomic oversplitting of species in the Elachista dispunctella complex.     

Genetic diversity of Philaenus spumarius and P. tesselatus (Hemiptera,Aphrophoridae): implications for evolution and taxonomy

The meadow spittlebugs Philaenus spumarius and P. tesselatus are closely related taxa with uncertain taxonomic position in the light of previous morphological, ecological, cytological and molecular research. Despite morphological homogeneity of P. spumarius (with the exception of high colour and pattern polymorphism) across its wide Holarctic range, it is possible that additional taxa (species or subspecies) exist. Philaenus spumarius is a potentially important pest in parts of its range where it was introduced. We used DNA markers to describe the genetic diversity of P. spumarius and P. tesselatus and to verify the taxonomic status of P. tesselatus and remote populations of the former species. The mitochondrial (cytochrome B) data showed that there are two main groups encompassing a northeastern (Asia and north‐central Europe) and a southwestern (Mediterranean area and western Europe, including North American specimens) clade. According to the elongation factor‐1α gene, there are three main clades: northeastern (Eurasiatic clade, E1), southeastern (east Mediterranean – Caucasus clade, E2) and southwestern (Iberian clade, E3). These two or three mitochondrial and nuclear clades could be considered as separate taxonomic units. On the other hand, all studied individuals of both species possessed the same internal transcribed spacer 2 haplotype. American specimens most probably originated from some western European populations. All studied specimens of P. tesselatus belong to the southwestern clade and western Mediterranean cluster. Therefore, together with cytological data, its species status is doubtful. To definitively solve the taxonomic status of P. tesselatus and populations of P. spumarius, further research using more samples and more genetic markers are needed.     

Phylogenomic analysis of pseudocryptic diversity reveals the new genus Deltalsia (Rhodomelaceae,Rhodophyta)

Molecular analyses, in combination with morphological studies, provide invaluable tools for delineating red algal taxa. However, molecular datasets are incomplete and taxonomic revisions are often required once additional species or populations are sequenced. The small red alga Conferva parasitica was described from the British Isles in 1762 and then reported from other parts of Europe. Conferva parasitica was traditionally included in the genus Pterosiphonia (type species P. cloiophylla in Schmitz and Falkenberg 1897), based on its morphological characters, and later transferred to Symphyocladia and finally to Symphyocladiella using molecular data from an Iberian specimen. However, although morphological differences have been observed between specimens of Symphyocladiella parasitica from northern and southern Europe they have yet to be investigated in a phylogenetic context. In this study, we collected specimens from both regions, studied their morphology and analyzed rbcL and cox1 DNA sequences. We determined the phylogenetic position of a British specimen using a phylogenomic approach based on mitochondrial and plastid genomes. Northern and southern European populations attributed to S. parasitica represent different species. Symphyocladiella arecina sp. nov. is proposed for specimens from southern Europe, but British specimens were resolved as a distant sister lineage to the morphologically distinctive Amplisiphonia, so we propose the new genus Deltalsia for this species. Our study highlights the relevance of using materials collected close to the type localities for taxonomic reassessments, and showcases the utility of genome-based phylogenies for resolving classification issues in the red algae.     

Gracilaria phuquocensis sp. nov., a new flattened Gracilaria species (Gracilariales,Rhodophyta), previously recognized as G. mammillaris,from the southern coast of Vietnam

Flattened Gracilaria species are widely distributed along the coasts of the South China Sea with more than 20 species recorded. Within the South China Sea, Gracilaria mammillaris has only been reported from Vietnam, but this species is likely restricted to the western Atlantic. This study aimed to reevaluate the taxonomic status of Vietnamese specimens of ‘G. mammillaris’ using combined morphological and molecular data. Our data clearly indicated that Vietnamese specimens were morphologically and genetically distinct from authentic G. mammillaris from the western Atlantic, and also other described flat Gracilaria species. We, therefore, propose that specimens from Vietnam originally identified as G. mammillaris be designated as a new species, Gracilaria phuquocensis sp. nov. Morphologically, G. phuquocensis can be distinguished from other flat Gracilaria species by its small thallus size, narrower blades, many medullary layers, abundant basal nutritive filaments within mature cystocarps, and tetrasporangial nemathecium. Our rbcL sequence analyses showed that the new species was sister to Gracilaria rhodymenioides from Thailand, and these two species formed a clade with cylindrical Gracilaria species. Our study contributes to clarification of the taxonomic status of misidentified specimens attributed to the flattened Gracilaria species in the South China Sea.