Identification Of Cryptic Species Research Articles

Possibilities for identification of cryptic species of Chiroptera using host-specific ectoparasites

The possibility of identification of the sibling species of Chiroptera by the example of Myotis daubentonii Kuhl, 1817 and Myotis petax Hollister, 1912 by their host-specific ectoparasitic fauna is discussed. Their habitat limits are defined.

Averaging v. outlier removal. Decrypting variance among cryptic Lejeunea species (Lejeuneaceae: Jungermanniopsida) using geometric morphometrics

Molecular data have revealed many morphologically cryptic species. More surprising than lack of difference, however, is that morphological variation and complex patterns of overlapping features can mask cryptic species. We employ geometric morphometric methods (GMM) to explore patterns of variation within four liverwort species, three of which were previously attributed to Lejeunea tumida Mitt. Each species exhibited considerable variation within, and overlap among, species in size and shape, independent of degree of relatedness. Most variation was expressed within individuals, suggesting that the observed breadth of variation was within the developmental capacity of single genotypes. Size and shape variation within, and consequently overlap among, individuals resulted primarily from variance in growth of shoots. Inter-specific differences were swamped by intra- and inter-individual variation. We coupled GMM with multivariate methods for outlier removal, and simple averaging of individuals to explore whether intra-individual variation could be reconciled to maximise the inter-species difference, facilitating resolution of cryptic species despite extensive morphological continuity and overlap. Unfortunately, outlier removal did not achieve separation among species, because removing extremes failed to eliminate overlap resulting from within-species variation. Individual averaging was partially successful in extracting L. tumida as a discrete entity but did not segregate the remaining three species. Although the challenges for morphology-based identification of cryptic species are significant, GMM provide one of the best sets of methods for identifying and communicating any subtle morphological differences that may exist.

Molecular identification of cryptic species of Ceratomyxa Thélohan, 1892 (Myxosporea: Bivalvulida) including the description of eight novel species from apogonid fishes (Perciformes: Apogonidae) from Australian waters

Ceratomyxa parasites from the gall bladders of 23 species of cardinalfishes (family Apogonidae) from Australian waters were examined for their taxonomic identity and phylogenetic relatedness. We identified 15 of the 23 apogonid fish species infected with species of Ceratomyxa. Although the majority of apogonid species harboured only a single Ceratomyxa species, four were found with multiple species of Ceratomyxa. This study describes eight novel species using a combination of morphological, small subunit ribosomal DNA (SSU rDNA) and biological characters. Six Ceratomyxa species are reported from single apogonid species, while two are reported from multiple host species. Molecular data were critical in identifying several morphologically cryptic species. However, our results suggest that SSU rDNA was not capable of distinguishing all the species present in the current study system and alternative genetic markers should be investigated in the future.

Genetic Structure of Vaccinium parvifolium (Ericaceae) in Northern California Reveals Potential Systematic Distinctions

Vaccinium parvifolium Sm. (Ericaceae) is an important understory shrub in conifer forests in western North America. Populations putatively classified as V. parvifolium in northern California display alternate berry morphology, consistent with a possible phenotypic diversification or cryptic speciation. Identification of cryptic species or subspecies would influence management guidelines given the limited range of some morphological variants. In order to inform management guidelines, two Vaccinium species were characterized via molecular genetic analyses. Plants of typical V. parvifolium morphology from the coastal areas of northwest California, western Oregon and Washington, atypical plants from Shasta County and the central Sierra Nevada, and one population of V. deliciosum Piper, a congener, were assessed at five nuclear microsatellite loci. Analyses of differentiation, admixture, and phylogenetic relationships indicated that populations displaying atypical morphology were more similar to V. deliciosum than to the typical V. parvifolium. Although additional data are required to determine whether these differences warrant taxonomic treatment within Vaccinium, management plans should consider three distinct gene pools among these populations.

‘Who's who’ in renal sphaerosporids (Bivalvulida: Myxozoa) from common carp, Prussian carp and goldfish – molecular identification of cryptic species, blood stages and new members of Sphaerospora sensu stricto

Myxozoans are a group of diverse, spore-forming metazoan microparasites bound to aquatic environments. Sphaerospora dykovae (previously S. renicola) causes renal sphaerosporosis and acute swim bladder inflammation (SBI) in juvenile Cyprinus carpio carpio, in central Europe. A morphologically similar species with comparably low pathogenicity, S. angulata has been described from C. c. carpio, Carassius auratus auratus and Carassius gibelio. To clarify uncertainties and ambiguities in taxon identification in these hosts we decided to re-investigate differences in spore morphology using a statistical approach, in combination with SSU and LSU rDNA sequence analyses. We found that developing spores of S. angulata and S. dykovae cannot be distinguished morphologically and designed a duplex PCR assay for the cryptic species that demonstrated S. dykovae is specific to C. c. carpio, whereas S. angulata infects C. a. auratus and C. gibelio. The molecular identification of myxozoan blood stages in common carp and goldfish, which had previously been ascribed to Sphaerospora spp. showed that approximately 75% of blood stages were from non-sphaerosporid coelozoic species infecting these cyprinids and more than 10% were from an alien species, Myxobilatus gasterostei, developing in sticklebacks. We hereby report non-selective myxozoan host invasion and multi-species infections, whose role in SBI still requires clarification.

DNA barcoding to characterize Brazilian species of Senna, Cassia and Casearia

Brazil is the country which has the greatest biodiversity in the world. Nowadays DNA barcoding became an important tool to aid genetic approach as well as others biological information to the plants in different fields: forensic genetics, taxonomy and identification of cryptic species. In this work, DNA barcoding has been used to better identification the species of three genus of native plants, Senna, Cassia and Casearia, helping to classify some groups, specially Senna and Cassia, which has not been properly classified by traditional taxonomy. The results indicated differences in three Senna species: S. spectabilis, S. multijuga and S. splendida (psbA+trnH primers).

Multilocus phylogenetic study of the Scheffersomyces yeast clade and characterization of the N-terminal region of xylose reductase gene.

Many of the known xylose-fermenting (X-F) yeasts are placed in the Scheffersomyces clade, a group of ascomycete yeasts that have been isolated from plant tissues and in association with lignicolous insects. We formally recognize fourteen species in this clade based on a maximum likelihood (ML) phylogenetic analysis using a multilocus dataset. This clade is divided into three subclades, each of which exhibits the biochemical ability to ferment cellobiose or xylose. New combinations are made for seven species of Candida in the clade, and three X-F taxa associated with rotted hardwood are described: Scheffersomyces illinoinensis (type strain NRRL Y-48827T = CBS 12624), Scheffersomyces quercinus (type strain NRRL Y-48825T = CBS 12625), and Scheffersomyces virginianus (type strain NRRL Y-48822T = CBS 12626). The new X-F species are distinctive based on their position in the multilocus phylogenetic analysis and biochemical and morphological characters. The molecular characterization of xylose reductase (XR) indicates that the regions surrounding the conserved domain contain mutations that may enhance the performance of the enzyme in X-F yeasts. The phylogenetic reconstruction using XYL1 or RPB1 was identical to the multilocus analysis, and these loci have potential for rapid identification of cryptic species in this clade.

Cryptic species in a Neotropical parrot: genetic variation within the Amazona farinosa species complex and its conservation implications

The application of genetic approaches has enhanced the identification of cryptic species in a wide variety of taxa, often with immediate conservation implications. Here, we employed multilocus DNA analyses to assess genetic variation and its correspondence to taxonomy within the Mealy Amazon (Amazona farinosa), a parrot species found in Central and South America. DNA sequence data from four mitochondrial regions and two nuclear introns were used to infer relationships among all five named subspecies in this species complex. Two reciprocally monophyletic groups with strong nodal support were found; one comprised of the two Central American subspecies guatemalae and virenticeps and one including all three South American subspecies farinosa,chapmani, and inornata. Molecular characters diagnosed distinct Central American and South American lineages, with an estimated divergence time of 1.75–2.70 million years ago as inferred from cytochrome-b (3.5–5.4 % corrected distance). Our data support recognizing Central American and South American Mealy Amazons as separate species worthy of independent conservation management. Furthermore, our results suggest recognition of two separate management units within the South American clade, although further study is required. These findings have important conservation implications as Central American A. farinosa are under increased pressure from habitat destruction and collection for the pet trade, yet are listed as of Least Concern due to their current classification as subspecies’ subsumed within the species complex.

Morphological and molecular evidence for cryptic species of springsnails [genus Pseudamnicola (Corrosella) (Mollusca, Caenogastropoda, Hydrobiidae)

Several Pseudamnicola (Corrosella) populations of the central and eastern Iberian Peninsula have been ascribed to Pseudamnicola (Corrosella) astieri (Dupuy, 1851), though recent evidence demonstrates the species could be endemic to the departments of Var and Alpes-Maritimes in France. Through the identification of cryptic species using a combined morphological and phylogenetic approach, this paper provides a detailed morphological description of Pseudamnicola (Corrosella) astieri, clarifying its taxonomic boundaries and confirming it as a French endemic. In parallel, by comparing Pseudamnicola (Corrosella) populations from the provinces of Castellón and Valencia in Eastern Spain, it was observed that rather than Pseudamnicola (Corrosella) astieri they represented a new species here described as Pseudamnicola (Corrosella) hauffei sp. n. Among other characters, the two species show marked differences in shell shape, male and female genital systems, radular formula and concentration of the nervous system. Pseudamnicola (Corrosella) hauffei sp. n. was also compared morphologically to another two Pseudamnicola (Corrosella) species living in nearby areas [Pseudamnicola (Corrosella) hinzi Boeters, 1986 and Pseudamnicola (Corrosella) navasiana (Fagot, 1907)], molecularly to Pseudamnicola (Corrosella) falkneri (Boeters, 1970), the type species of the subgenus, and to the rest of the Pseudamnicola (Corrosella) species described so far. Morphological differentiation between the species is supported by a genetic divergence of 7.4% inferred from a partial sequence (658 bp) of the mitochondrial gene cytochrome c oxidase subunit I (COI). On the basis of an average 8% (5.39 to 11.15%) divergence estimated for the COI gene in other Pseudamnicola (Corrosella) species reported in GenBank, the existence of two specific entities is here proposed, which will have impact on conservation policies both in France and in Spain.

Cuticular hydrocarbon profiles as a taxonomic tool: advantages, limitations and technical aspects

Cuticular hydrocarbons (CHCs) are expressed on an insect's cuticle and are one of the major factors allowing insects to identify members of their own species, colony and gender. As a result of their species‐specificity, CHCs are increasingly used to delimit species in addition to more conventional methods, such as morphology or genetic markers, and so play an important role in chemotaxonomy. Species vary in the type of CHCs that they produce, as well as in the relative quantities of shared compounds. This review summarizes not only how taxonomists may differentiate between species based on CHC profiles, but also the incentive for using CHC composition as taxonomic tool. Benefits regarding the identification of cryptic species and early signs of reproductive isolation are then discussed, giving examples from studies of taxonomy, behaviour and biosynthesis. For taxonomic characters to reliably indicate species boundaries, their limitations need to be known. Potential problems caused by environmental effects, intra‐species variation in profiles and other technical issues are highlighted, and suggestions are made regarding their avoidance. It remains a challenge to determine the variation beyond which two species can be called independent; a problem shared by most methods of delimitation. Recently, there has been a shift towards using a combination of different taxonomic tools, both molecular and non‐molecular, to test observed species differences.

Using complementary techniques to distinguish cryptic species: A new Erysimum (Brassicaceae) species from North Africa

Cryptic species are superficially morphologically indistinguishable and therefore erroneously classified under one single name. The identification and delimitation of these species is usually a difficult task. The main aim of this study is to provide an inclusive methodology that combines standard and new tools to allow accurate identification of cryptic species. We used Erysimum nervosum s.l. as a model system. Four populations belonging to E. nervosum s.l. were sampled at their two distribution ranges in Morocco (the Atlas Mountains and the Rif Mountains). Fifteen individuals per population were collected to assess standard taxonomic traits. Additionally, corolla color and shape were quantified in 30 individuals per population using spectrophotometry and geometric morphometrics, respectively. Finally, we collected tissue samples from each population per species to study the phylogenetic relationships among them. Using the standard taxonomic traits, we could not distinguish the four populations. Nonetheless, there were differences in corolla color and shape between plants from the two mountain ranges. The population differentiation based on quantitative morphological differences were confirmed and supported by the phylogenetic relationships obtained for these populations and the rest of the Moroccan Erysimum species. The joint use of the results obtained from standard taxonomic traits, quantitative analyses of plant phenotype, and molecular data suggests the occurrence of two species within E. nervosum s.l. in Morocco, one located in the Atlas Mountains (E. nervosum s.s.) and the other in the Rif Mountains (E. riphaeanum sp. nov.). Consequently, we suggest that combining quantitative and molecular approaches with standard taxonomy greatly benefits the identification of cryptic species.

Multiple morphological characters needed for field identification of cryptic long‐eared bat species around the Swiss Alps

The identification of cryptic species may significantly change our view about their distribution, abundance, ecology and therefore conservation status. In the European Alps, molecular studies have revealed the existence of three sibling species of plecotine bats Plecotus auritus, Plecotus austriacus and, very recently, Plecotus macrobullaris. Knowledge of the ecological niche partitioning of cryptic species is a requisite to develop sound conservation policies. Yet, this requests the development of unambiguous identification methods easily applicable in the field. This study investigates the reliability of several morphological methods used for species recognition and proposes a new identification key for field workers. We captured 214 Plecotus bats from 29 sites in four bioregions within Switzerland, collected biopsy punches for genetic analysis, described and measured external morphological characters. All three species occurred as mono-specific colonies, except at one site where P. auritus and P. macrobullaris shared the same church attic. Qualitative traits alone did not allow a reliable separation of the three species. A series of multivariate analyses conducted on external linear measurements resulted in a discriminant function enabling correct species classification with a 97.5% probability. Compared with genetic analysis, our multivariate morphological method represents a valuable, rapid and cost-effective alternative.

DNA barcoding: a new module in New Zealand’s plant biosecurity diagnostic toolbox

Molecular methods for identification of high risk pests and pathogens have been employed for more than a decade to supplement standard diagnostic protocols. However, as the volume of traded goods continues to increase so does the breadth of taxa that diagnosticians need to deal with. Keeping pace by introducing more molecular tests that are typically species‐group specific is not an efficient way to progress. Since 2005 classical DNA barcoding using cytochrome oxidase I sequence has been employed routinely in New Zealand for the highest risk insect species (fruit flies and lymantriid moths). Subsequently a broader range of pests have been considered. Case studies are presented here for three important lepidopteran pests, Lymantria mathura (pink gypsy moth), Conogethes punctiferalis (yellow peach moth) and Hyphantria cunea (fall web worm), as well as a trial to identify miscellaneous border interceptions. While the data support the effectiveness of DNA barcoding for border diagnostics, they also raise issues around cryptic species identification and potential species discovery that could impact on operational biosecurity systems.

Morphological Characteristics of Male Genitalia in Some Species of the Genus Eurytoma (Eurytomidae, Hymenoptera)

Morphological Characteristics of Male Genitalia in Some Species of the GenusEurytoma(Eurytomidae, Hymenoptera)Based on the analysis of morphological characteristics of related species of the genusEurytoma, several groups of cryptic species poorly distinguishing by external morphology, but clearly confined to certain hosts are shown to have morphological differences of male genitalia that are highly species-specific and can be useful for identification of cryptic species of the genusEurytoma(E. rosaeNees,E. strigifronsThomson, andE. jaceaeMayr species complexes).

Wing Morphometry Helps Diagnose Cryptic Species and ResurrectMindarus pinicolus(Hemiptera: Aphididae)

AbstractWing venation, two-dimensional and with easily recognized reference points at vein junctions, presents an opportunity for the development of automated insect identification. Using a suite of continuous characters, I investigated the use of wing morphometry for computerized insect identification of cryptic species of the aphid genus Mindarus. A priori groups were determined using cytochrome oxidase 1 DNA barcodes. Discriminant function analysis of 24 wing measurements consistently grouped individuals of unknown taxonomic affinity with the correct a priori groups. The results suggest that diagnostic signal is present in wing morphometry, but the signal is considerably stronger with the addition of morphometry from other aphid appendages, namely, 10 leg and antennal segments. Almost all Mindarus collected in eastern North America have been determined as the balsam twig aphid, Mindarus abietinus Koch (Hemiptera: Aphididae), but molecular diagnostics reveals that the Palearctic species is not present in the Western Hemisphere. Schizoneura pinicola Thomas has been considered a North American synonym of M. abietinus. Morphometric discriminant function analysis suggests that the Abies-feeding eastern North American population is M. pinicolus. The species is here reinstated with a new combination and redescribed.

Identification of cryptic species of Culicoides (Diptera: Ceratopogonidae) in the subgenus Culicoides and development of species-specific PCR assays based on barcode regions

Culicoides biting midges (Diptera: Ceratopogonidae) are vectors of important diseases affecting wild and domestic animals. During the last decade they have played a major role in the epidemiology of the largest bluetongue epizootic ever recorded in Europe, the disease is transmitted between hosts almost exclusively by bites of Culicoides midges and affects both domestic and wild ruminants however severe disease usually occurs in certain breeds of sheep and some species of deer. An accurate vector identification is of major importance in arthropod borne diseases surveillance, as great differences in vectorial capacity are found even between close species. Unfortunately, specialized taxonomic knowledge of Culicoides identification is rarely available in routine surveillance, mainly based on wing morphology. Recently, some European species of Culicoides belonging to the subgenus Avaritia Fox, 1955 and Culicoides Latreille, 1809 have been described as new bluetongue virus vectors. In the present study, by using a fragment of the barcode region (COI gene) we report the presence of up to 11 species within the subgenus Culicoides in Catalonia (NE Spain), a region recently affected by a bluetongue epizootic. The molecular analysis revealed new non-described cryptic species which were grouped in three complexes of morphologically similar species, two in the Pulicaris complex resembling Culicoides pulicaris, two in the Fagineus complex resembling Culicoides fagineus and three in the Newsteadi complex resembling Culicoides newsteadi. The phylogenetic relationships among them showed that cryptic species detected in both Pulicaris and Fagineus complexes were closely related, whereas those in the Newsteadi complex were more distant. Accurate analysis of all species using morphological and molecular approaches resulted in the detection of diagnostic metric traits for cryptic species and the design of several new species-specific single and multiplex PCR assays to identify unambiguously all the species, most of them still lacking a specific molecular diagnosis.

Systematics and evolutionary relationships of the mountain lizard Liolaemus monticola (Liolaemini): how morphological and molecular evidence contributes to reveal hidden species diversity

8 The delimitation of species is a major issue in systematic biology and has been a re-emerging discipline in the last decade. A number of studies have shown that the use of multiple data sets is critical for the identification of cryptic species, particularly in groups with complex evolutionary histories. Liolaemus monticola is a montane lizard species distributed in central Chile (32°-42°S), with four described subspecies in a latitudinal gradient from north to south: L. m. monticola, L. m. chillanensis, L. monticola ssp. and L. m. villaricensis. In order to test the systematic status and phylogenetic relationships of the taxa included in the L. monticola group, we analysed morphological (morphometric and meristic) and molecular (allozyme and mitochondrial DNA) data sets. The results of the morphological analyses showed that meristic variables correctly assigned individuals with higher accuracy than did morphometric characters. The results of the analyses of allozyme data revealed eight diagnostic loci that are evidence for significant differences among the four L. monticola subspecies. Phylogenetic analyses with mitochondrial DNA data, including additional species, showed that the L. monticola group is polyphyletic. We postulate that the four current subspecies represent independent evolutionary lineages and must be raised to the specific level as L. monticola, L. chillanensis and L. villaricensis. The taxonomic status of the unnamed L. monticola ssp. remains unresolved, although we provide a preliminary proposal. © 2009 The Linnean Society of London, Biological Journal of the Linnean Society, 2009, 96, 635-650.

Lemurs

Lemurs

Ecological niche modelling of two cryptic bat species calls for a reassessment of their conservation status

1. Conservation priorities are set on the basis of existing knowledge of the species concerned. Identification of cryptic species within former single taxa may significantly change the perspective on distribution and abundance which, in turn, might impact the species? conservation status. 2. In the 1990?s it was discovered that one of the most abundant bat species in Europe actually consists of two cryptic species, P. pipistrellus and P. pygmaeus. We systematically recorded echolocation calls from both species along road transects driven across Switzerland. Subsequently, we have used presence data obtained to analyse the species? habitat selection and respective ecological niches by GIS-modelling and by discriminant analysis. 3. Habitats mostly associated with P. pygmaeus presence are wide rivers and lakes, human settlements and open woodland habitats. P. pygmaeus is patchily distributed and only occurs in its optimum habitats, i.e. in areas characterized by the simultaneous presence of all habitats mentioned above. 4. P. pipistrellus has similar habitat preferences, but unlike P. pygmaeus, it is very tolerant with regard to deviations from its optimal habitat. The overall niche of P. pipistrellus encompasses nearly the entire global habitat availability. In contrast, the niche of P. pygmaeus is narrow and mostly included in one end of the niche of P. pipistrellus. 5. Due to its wide distribution and abundance P. pipistrellus sensu lato was ranked «not endangered» in Switzerland. In this study we found P. pipistrellus to occur in a 30-fold higher abundance than P. pygmaeus. However the rarity of P. pygmaeus and its restricted distribution significantly change the conservation status of this cryptic species. Extrapolated habitat suitability maps help to focus conservation efforts to protect important hunting areas (i.e. riparian woodlands) in regions with highest probability of occurrence. 6. Synthesis and applications. The present example points out that conservation status attributed to taxa encompassing sibling species may be misleading and needs to be changed constantly according to scientific advances. Progress in systematics will spawn more cryptic species in the near future. Ecological studies are required to assess their environmental requirements and provide the necessary baseline information for appropriate conservation.

Utility of external morphometrics for cryptic species identification: A case study of two species in Poecilus 'samurai' (Coleoptera: Carabidae)

Even in species which are distinguishable by mainly internal structures or molecular data (e.g., cryptic species), external morphology could be useful for species identification. This paper describes the morphology and distribution of two species in Poecilus ‘samurai’ (Coleoptera: Carabidae) and tested the utility of external morphometrics for identification of cryptic species. The examination of the membranous parts of genitalia revealed that P. ‘samurai’ is composed of two externally similar species: P. sp. 1 (Hokkaido and Honshu) and P. sp. 2 (Hokkaido, Kyushu, Russia and Korea). With a focus on the identification of individuals from Hokkaido where the two species sympatrically occur, discriminant analyses based on measurements of ten external characteristics were performed between P. sp. 1 (Hokkaido) and P. sp. 2 (Hokkaido), and between P. sp. 1 (Hokkaido and Honshu) and P. sp. 2 (Hokkaido). In the former, all individuals were correctly classified into their own species, whereas in the latter, some individuals were incorrectly classified, suggesting that discriminant analyses using focal populations are more effective. A morphological phylogeny of five Poecilus species (including sp. 1 and 2) was provided, and the distribution pattern in the Japanese Archipelago was discussed.