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.     

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.     

Taxonomy,skull diversity and evolution in a species complex of Myotis (Chiroptera: Vespertilionidae): a geometric morphometric appraisal

Phylogenetic relationships between taxa are not necessarily reflected by morphological data due to widespread homoplasy and convergence. However, combining morphological and molecular data provides insights into the evolution of biological forms and into the potential factors involved. Here we focus on a complex of three taxa of bats with unclear taxonomic affinities: Myotis myotis, Myotis blythii and Myotis punicus. Traditional morphometric methods failed to separate them, whereas recent molecular‐based studies suggested that they constitute separate biological species. In the present study, landmark‐based geometric morphometrics methods have been used to analyse the skull variability of 218 specimens belonging to this species complex. Patterns of size and shape delimitate three morphological groups that are congruent with the proposed taxonomic assignments, and therefore support species rank for all three major groups. These morphometrics results, however, suggest that M. myotis and M. punicus share shape characteristics in the rostrum and in the posterior part of the skull that differ from M. blythii. Because previous molecular phylogenetic analyses suggested that M. myotis and M. blythii are sister species, we interpret the similitude in skull morphology between M. myotis and M. punicus as a convergence probably related to their similar feeding habits. Within the taxon M. punicus, the skull of Corsican and Sardinian populations significantly differs from that of Maghrebian ones, suggesting the existence of further cryptic taxonomic diversity. © 2008 The Linnean Society of London, Biological Journal of the Linnean Society, 2008, 95 , 529–538.     

Integrative taxonomy of the stunt nematodes of the genera Bitylenchus and Tylenchorhynchus (Nematoda,Telotylenchidae) with description of two new species and a molecular phylogeny

Stunt nematodes are characterized by phenotypic plasticity, with overlapping morphology and morphometry leading to potential misidentification. Consequently, the application of integrative taxonomic approaches is useful to species delimitation based on a combination of different perspectives, e.g. morphology and DNA sequences. We conducted nematode surveys in cultivated and natural environments in Spain and the USA, from which we identified 18 known species of the family Telotylenchidae and two new taxa within the studied samples. These species were morphologically, morphometrically, and molecularly characterized. The results of light and scanning electron microscopic observations, and molecular and phylogenetic analysis also allowed two new species to be distinguished, described herein as B itylenchus hispaniensis sp. nov. and T ylenchorhynchus mediterraneus sp. nov. The phylogenetic analysis was carried out using molecular data from nuclear ribosomal DNA genes [D2–D3 expansion segments of the large ribosomal subunit (28S), internal transcribed spacer (ITS), and partial small ribosomal subunit (18S)]. We also provide here a test of alternative hypotheses that confirms the monophyly of both Tylenchorhynchus and Bitylenchus sensu Siddiqi's classification but does not support Fortuner & Luc's conceptual view of Tylenchorhynchus as a large genus. Ancestral state reconstructions of several diagnostic morphological characters using a maximum parsimony approach showed congruence in morphological and molecular evolution for stylet knob inclination and tail tip annulation. Our analysis emphasizes some of the problems related to the taxonomy and phylogeny of nematodes of Telotylenchinae. © 2014 The Linnean Society of London     

Integrative taxonomy reveals cryptic Amazonian species of Pristimantis (Anura: Strabomantidae)

Hypotheses on the taxonomic status of two Bolivian Pristimantis with taxonomic problems are assessed by an integrative taxonomic approach that integrates three independent lines of evidence: external morphology, prezygotic reproductive barriers (advertisement calls) and reciprocal monophyly (phylogenetic analyses of partial 16S mtDNA sequences). Central Andean Bolivian populations previously assigned to either P. peruvianus or P. dundeei, and lowland Amazonian populations from southern Peru and northern Bolivia previously considered P. peruvianus do not correspond to these species. Indeed, multivariate analyses of qualitative and quantitative morphological and bioacoustic characters, and phylogenetic analyses support the hypothesis that they represent different, previously unknown, cryptic lineages. They are herein described as new species. The former is a sibling species of P. fenestratus that inhabits the Amazonian and semideciduous forests of the Andean foothills in central Bolivia. The latter is sibling to the Andean species P. danae and is parapatric to it in the Amazonian lowland forests and adjacent foothills of northern Bolivia, southern Peru and adjacent Brazil. Most species of Neotropical frogs, and especially Pristimantis, have been described by using external qualitative morphological characters only. An extended integrative taxonomic approach, as exemplified herein, may lead to the discovery of many other cryptic and sibling lineages that would increase the species numbers of tropical areas. © 2009 The Linnean Society of London, Zoological Journal of the Linnean Society, 2009, 155 , 97–122.     

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     

Patterns of cryptic hybridization revealed using an integrative approach: a case study on genets (Carnivora, Viverridae, Genetta spp.) from the southern African subregion

Recent years have seen the development of molecular‐based methodologies to investigate hybridization and its impact on the evolutionary process. However, morphological characterization of hybrid zones has only scantily been considered, especially in zootaxa. Thus, the level of congruence between molecular and morphological characters when attempting to detect hybrids remains a poorly tackled area. The genets (genus Genetta) provide an ideal case study for further investigation of the respective contribution of morphology and DNA in hybrid zone characterization because (1) their morphology has recently been exhaustively explored and (2) the existence of hybrid zones in southern Africa was proposed in the literature. We assessed levels of hybridization among the southern African genets, and questioned the role of ecological factors on the hybridization patterns detected. We used an integrative approach involving nine discrete morphological characters and a diagnostic discriminant function, geometric morphometrics and sequences of cytochrome b including collection specimens. The combination of independent materials allowed us to accurately reassess the level of hybridization in southern African genets, and revealed cryptic, interspecific gene flows. Morphology unambiguously detected a low number of G. maculata × G. tigrina hybrids and rejected the hypothesis of a large intergradation zone in KwaZulu‐Natal, thus supporting the species status of the two genets. Cytochrome b analyses revealed: (1) cryptic, massive hybridization between G. tigrina and the sympatric G. felina, and (2) a trace of reticulation (one sequence) between G. tigrina and the allopatric G. genetta. The type specimen of G. mossambica Matschie, 1902 is considered to be a morphological hybrid between G. maculata and G. angolensis. Remarkably, the morphological approaches (discrete characters and morphometrics) proved complementary to conclusions derived from cytochrome b sequences. Whilst morphometrics was generally unable to accurately identify all putative hybrids, this approach revealed diagnostic cranial shape differences between recognized species as well as the cryptic G. ‘letabae’ (included in the super‐species G. maculata). Morphometrics also confirmed the diagnostic value and age dependency of discrete characters. Our integrative approach appeared necessary to the detection of cryptic hybridizations and to the comprehensive characterization of hybrid zones. The recurrent detection of hybrids exhibiting tigrina‐like coat patterns may suggest (1) asymmetric hybridization of G. tigrina males to females of other species and (2) positive selection for tigrina‐like phenotype in South African habitats, but these hypotheses will have to be further tested using other sources of evidence. Despite the precise mosaic of hybrid zones identified in southern African genets, the environmental factors that shape patterns of distribution of hybrids remain unclear. Nevertheless, in the light of our range reassessment, it appears that seasonality of precipitation and periods of annual frost may play stringent roles in the distribution of genets. The complementarity of our results based on morphology and molecules is regarded as encouraging for the further development of integrative approaches in order to better understand the complex phenomena that underlie hybridization processes. © 2005 The Linnean Society of London, Biological Journal of the Linnean Society, 2005, 86 , 11–33.     

Taxonomic status of southern South American Conepatus (Carnivora: Mephitidae)

Despite recent taxonomic evaluations of Mephitidae and North American hog‐nosed skunks, southern South American species of Conepatus have not been thoroughly examined in a systematic context. Conepatus chinga and Conepatus humboldtii were described more than 150 years ago, based on external characters such as hair coloration and size. Although historically recognized as valid species, to date no detailed systematic analysis has been performed for either of these taxa. Herein, we evaluated the taxonomic status of C. chinga and C. humboldtii within the southern part of South America using geometric morphometrics of the skull and mandible, mitochondrial DNA analysis using the cytochrome b and cytochrome oxidase c subunit I genes, and also control region and pelage pattern variation. We failed to find morphological (skull shape and pelage coloration patterns) or molecular differences between these two species; thus, we considered that the specimens assigned to C. chinga and C. humboldtii belong to the same species. Our results indicate that environmental variation seems to be responsible for shape and size variation in Conepatus skulls from southern South America. © 2013 The Linnean Society of London     

Leaf morphology and anatomy of Camellia section Camellia (Theaceae)

The delimitations of species in Camellia section Camellia have been disputed for many years, resulting from uncertain relationships among species. Leaf morphological and anatomical characters for 54 species and three varieties in this section were investigated to reveal the relationships. Principal component analysis and cluster analysis were conducted using the transformed data for quantitative and qualitative characters from leaf morphology and anatomy. Combining the results of statistical analysis with comparative leaf characters of morphology and anatomy, we discussed the taxonomic treatment of section Camellia by Chang compared with that of Ming and we conclude that section Camellia consists of c. 50 species. © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 159 , 456–476.     

How useful are the recommended counts and indices in the systematics of the Octopodidae (Mollusca: Cephalopoda)

A morphological dataset based on 14 standard counts and indices was constructed for 68 specimens comprising 12 species of octopuses. This was used to construct distance matrices based on morphological characters. These matrices were compared with genetic distance matrices compiled during molecular phylogenetic analyses of the same 12 species using four mitochondrial and two nuclear genes. Mantel tests showed that there was significant congruence between the phenetic and genetic matrices, suggesting that the genetic signal is reflected in the morphological data set. Matrices of geographical distance were constructed for the 12 species based on the latitude, longitude, and depth of capture of 1726 individuals. These matrices never showed significant congruence with genetic data or with morphological data. Multivariate analysis of the morphological dataset suggests that these counts and indices, traditionally used for discriminating between species in cephalopods, do not show great discrimination at species level, but provide excellent discrimination at the generic level, and, as such, might be useful for resolving the generic placement of some problematic taxa. © 2008 The Linnean Society of London, Biological Journal of the Linnean Society, 2008, 95 , 205–218.     

Molecular phylogenetics and morphological variation reveal recent speciation in freshwater mussels of the genera Arcidens and Arkansia (Bivalvia: Unionidae)

Intra‐ and interspecific morphological variation due to both phenotypic plasticity and evolutionary convergence hinder the work of taxonomists and lead to over‐ and underestimates of species richness. Nevertheless, most species on Earth are recognized solely based on morphological characters. We used molecular phylogenetic and morphometric techniques to examine two freshwater mussel species. One is common and widespread, while the other is imperiled and endemic to the Interior Highlands of the USA. Phylogenetic and molecular clock analyses revealed that divergence of Arcidens confragosus and Arkansia wheeleri is small and relatively recent. Divergence in these and other taxa is probably due to isolation of streams in the Interior Highlands. Morphometric analyses showed distinct shell shapes using traditional morphometrics, but not through geometric morphometrics. Outlined shell shapes are indistinguishable; geometric morphometrics could not capture a three‐dimensional component. Our analyses support the validity of these two species as congeners, with the nomen Arcidens (Simpson 1900) having priority. Because shell morphologies are both heritable and environmentally determined, our study emphasizes the importance of considering both molecular and morphometric analyses for identification of freshwater molluscs of conservation concern. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 112 , 535–545.     

Phylogenetic interrelationships,taxonomy, and reductive evolution in the Neotropical electric fish genus Hypopygus (Teleostei,Ostariophysi, Gymnotiformes)

A phylogenetic reconstruction of the Neotropical electric fish genus Hypopygus based on 47 parsimony‐informative morphological characters is presented. A series of synapomorphies support the hypothesis of monophyly of Hypopygus, and partially resolve species‐level relationships within the genus. Hypopygus species are recognized here as miniaturized fishes based on two criteria; first, a derived condition of diminutive body size, and; second, the presence of a suite of reductive morphological characters, including partial or total losses, simplifications, and reductions of the anal‐fin rays, scales, cranial bones, and laterosensory canal system. Reductive characters associated with miniaturization comprise 45% of the total number of characters in the phylogenetic reconstruction of the genus. Miniaturization and reductive morphological evolution in Hypopygus are discussed here in the phylogenetic context. A taxonomic revision of Hypopygus is presented, in which five new species are described, two species previously assigned to the genus are redescribed, and a single known species of Stegostenopos is redescribed and included in Hypopygus as a junior synonym. Distribution maps and a key for all eight valid species of Hypopygus are provided, based on the examination of 5014 catalogued museum specimens. © 2011 The Linnean Society of London, Zoological Journal of the Linnean Society, 2011, 163 , 1096–1156.     

Geographic variation in the long-nosed snake, Rhinocheilus lecontei (Colubridae): beyond the subspecies debate

Scalation, colour pattern, linear and geometric morphometrics were used to quantify geographical differentiation in the long-nosed snake, Rhinocheilus lecontei, and to test the hypothesis that all four subspecies are morphologically distinct. Also investigated were potential associations between morphological (scalation, colour pattern, linear measurements) and environmental variables (climate, vegetation, soil). Sexual dimorphism was weakest for geometric and strongest for linear morphometric variables. Morphological variables differed widely in their ability to differentiate subspecies. Linear morphometric variables achieved the most statistically significant pairwise Mahalanobis distances between subspecies, while geometric morphometrics largely failed to differentiate them. Colour pattern showed the strongest and linear morphometrics the weakest correlation with environment. Several characters varied continuously along latitudinal or longitudinal gradients, such that, in some cases, the clines for closely related traits were discordant. No one subspecies was consistently divergent in all analyses, leading to the conclusion that the three mainland subspecies are not sufficiently distinct to warrant separate subspecies status. The island subspecies, though not always statistically distinct, is geographically separate from other populations and differs in characters related to size. Given the small number of specimens available, a decision regarding its taxonomic status (i.e. elevation to species level) is best deferred until additional specimens can be examined and data on molecular variation can be analysed.  © 2004 The Linnean Society of London, Biological Journal of the Linnean Society , 2004, 83 , 65–85.     

Integrative taxonomy of the primitively segmented spider genus Ganthela (Araneae: Mesothelae: Liphistiidae): DNA barcoding gap agrees with morphology

Species delimitation is difficult for taxa in which the morphological characters are poorly known because of the rarity of adult morphs or sexes, and in cryptic species. In primitively segmented spiders, family Liphistiidae, males are often unknown, and female genital morphology – usually species‐specific in spiders – exhibits considerable intraspecific variation. Here, we report on an integrative taxonomic study of the liphistiid genus Ganthela Xu & Kuntner, 2015, endemic to south‐east China, where males are only available for two of the seven morphological species (two known and five undescribed). We obtained DNA barcodes (cytochrome c oxidase subunit I gene, COI) for 51 newly collected specimens of six morphological species and analysed them using five species‐delimitation methods: DNA barcoding gap, species delimitation plugin [P ID(Liberal)], automatic barcode gap discovery (ABGD), generalized mixed Yule‐coalescent model (GMYC), and statistical parsimony (SP). Whereas the first three agreed with the morphology, GMYC and SP indicate several additional species. We used the consensus results to delimit and diagnose six Ganthela species, which in addition to the type species Ganthela yundingensis Xu, 2015, completes the revision of the genus. Although multi‐locus phylogenetic approaches may be needed for complex taxonomic delimitations, our results indicate that even single‐locus analyses based on the COI barcodes, if integrated with morphological and geographical data, may provide sufficiently reliable species delimitation. © 2015 The Linnean Society of London     

Geometric morphometric and phylogenetic analyses of Arizona Sky Island populations of Scaphinotus petersi Roeschke (Coleoptera: Carabidae)

Scaphinotus petersi Roeschke, 1907 (Carabidae) is a ground beetle endemic to Sky Islands in south‐eastern Arizona. Previous taxonomic studies described several subspecies with morphological differences inhabiting geographically isolated mountain ranges. We combined molecular sequence data and morphometric data, especially head and pronotum shape analyses, to examine the variation and divergence in subspecies and isolated montane populations. In this study, we employ a combination of distance morphometrics as well as geometric morphometrics to quantify the level of morphological variation, and to test the hypothesis that geographically distinct populations of S. petersi are phenotypically distinct. Results suggest that these isolated populations have diverged morphologically and genetically. Phylogenetic analyses identified two monophyletic lineages within the species that correspond generally to pronotum shape. We observed significant morphological variation among most montane populations in of S. petersi, with the pronotum shape as the clearest delimiting trait. © 2015 The Linnean Society of London     

Morphological and molecular description of new species of squat lobster (Crustacea: Decapoda: Galatheidae) from the Solomon and Fiji Islands (South‐West Pacific)

The family Galatheidae is among the most diverse families of anomuran decapod crustaceans, and the South‐West Pacific is a biodiversity hot spot for these squat lobsters. Attempts to clarify the taxonomic and evolutionary relationships of the Galatheidae on the basis of morphological and molecular data have revealed the existence of several cryptic species, differentiated only by subtle morphological characters. Despite these efforts, however, relationships among genera are poorly understood, and the family is in need of a detailed systematic review. In this study, we assess material collected in different surveys conducted in the Solomon Islands, as well as comparative material from the Fiji Islands, by examining both the morphology of the specimens and two mitochondrial markers (cytochrome oxidase subunit I, COI, and 16S rRNA). These two sources of data revealed the existence of eight new species of squat lobster, four of which were ascribed to the genus Munida, two to the genus Paramunida, one to the genus Plesionida, and the last species was ascribed to the genus Agononida. These eight species are described along with phylogenetic relationships at the genus level. Our findings support the taxonomic status of the new species, yet the phylogenetic relationships are not yet fully resolved. Further molecular analysis of a larger data set of species, and more conserved genes, will help clarify the systematics of this group. © 2009 The Linnean Society of London, Zoological Journal of the Linnean Society, 2009, 156 , 465–493.     

Multivariate analyses of morphological characters of Leptolaena Thouars s.l. subgenera Mediusella and Xerochlamys (Sarcolaenaceae)

Leptolaena Thouars is one of the first described genera within Sarcolaenaceae, but represents the last great species-level taxonomic challenge in this endemic Malagasy plant family. As currently circumscribed, this genus comprises three subgenera, two of which, Mediusella Cavaco and Xerochlamys (Baker) Cavaco, exhibit patterns of morphological variation that have been understood differently and have led to an unstable taxonomy, such that a consensus on the delimitation of species has yet to be reached. Principal component analysis of morphological characters was performed on 211 herbarium specimens in order to evaluate the morphological variation within the two subgenera and to clarify historically problematical species boundaries. The results showed that ten entities could be distinguished on the basis of vegetative, floral, and fruit characters, and that they provide a robust basis for proposing a new species-level taxonomic framework.  © 2008 The Linnean Society of London, Botanical Journal of the Linnean Society , 2008, 157 , 559–574.     

Multivariate analysis of morphological and anatomical characters of Calophyllum (Calophyllaceae) in South America

Calophyllum (Calophyllaceae), previously placed in Clusiaceae, is easily recognizable by its opposite entire leaves with close parallel venation alternating with resin canals. However, distinction between species has been difficult, because of infraspecific variation in tepal and stamen number and resemblance among species that share similar habitats. Here, I report the results of multivariate analyses of morphological and anatomical characters for Calophyllum in South America, and provide a taxonomic treatment for the genus in South America, the first since that of Vesque in 1893. Thirteen preliminary morphogroups were identified. Thirty‐two morphological characters of the leaf, flower and fruit from 401 specimens, and 17 anatomical leaf characters from 45 specimens were measured and analysed using principal component analyses (PCAs) and discriminant analyses (DAs). PCAs were used to find groups and DAs were used to validate those PCAs that were potential groups. Two main subgroups were identified in the general analysis. Subgroup M1 has terete stems and smaller leaves and flowers than subgroup M2, which, instead, has quadrangular stems. Only subgroup M2 showed distinctive clusters in regional and local analyses. Distinctive clusters and morphological and anatomical characters helped us to recognize four species in South America, including a new species, Calophyllum pubescens sp. nov. . In addition, a new species, Calophyllum mesoamericanum sp. nov. , is described from Central America. © 2013 The Linnean Society of London,