Wing shape as a potential discriminator of morphologically similar pest taxa within the Bactrocera dorsalis species complex (Diptera: Tephritidae)

Four morphologically cryptic species of the Bactrocera dorsalis fruit fly complex (B. dorsalis s.s., B. papayae, B. carambolae and B. philippinensis) are serious agricultural pests. As they are difficult to diagnose using traditional taxonomic techniques, we examined the potential for geometric morphometric analysis of wing size and shape to discriminate between them. Fifteen wing landmarks generated size and shape data for 245 specimens for subsequent comparisons among three geographically distinct samples of each species. Intraspecific wing size was significantly different within samples of B. carambolae and B. dorsalis s.s. but not within samples of B. papayae or B. philippinensis. Although B. papayae had the smallest wings (average centroid size=6.002 mm±0.061 SE) and B. dorsalis s.s. the largest (6.349 mm±0.066 SE), interspecific wing size comparisons were generally non-informative and incapable of discriminating species. Contrary to the wing size data, canonical variate analysis based on wing shape data discriminated all species with a relatively high degree of accuracy; individuals were correctly reassigned to their respective species on average 93.27% of the time. A single sample group of B. carambolae from locality 'TN Malaysia' was the only sample to be considerably different from its conspecific groups with regards to both wing size and wing shape. This sample was subsequently deemed to have been originally misidentified and likely represents an undescribed species. We demonstrate that geometric morphometric techniques analysing wing shape represent a promising approach for discriminating between morphologically cryptic taxa of the B. dorsalis species complex.     

Discrimination of Eubazus (Hymenoptera, Braconidae) sibling species using geometric morphometrics analysis of wing venation

Abstract.  Complexes of sibling and cryptic species are encountered frequently in parasitic Hymenoptera. Geometric morphometrics is a useful tool to detect minimal morphological variations, which often are undetectable by traditional morphological studies and even by classical morphometric approaches. We applied geometric morphometrics to wing venation to assess a complex case of sibling species in the genus Eubazus (Hymenoptera, Braconidae), parasitoids of conifer bark weevils of the genus Pissodes (Coleoptera, Curculionidae). The results and methods were compared with previous taxonomic studies on the same species, involving classical multivariate morphometrics, isoenzyme analyses, cross-mating experiments and biological observations. Geometric morphometrics confirmed the previous division into four distinct species. However, this approach enabled the four species to be separated simultaneously, with a reliability of 98.6% for well-classified females and 93.1% for males. A similar result in previous studies was obtained only by combining isoenzyme analyses and several canonical variate analyses, including many morphometric characters. Furthermore, measurements of wing venation were less time-consuming, more reliable and required less prior knowledge of braconid taxonomy than the measurements needed for the classical morphometrics methods. Geometric morphometrics was used also to test the effect of host species on wing shape. Several female populations of Eubazus semirugosus originating from three different Pissodes spp. were compared. Significant differences were found in wing shape between conspecific Eubazus from different host species. The results are discussed in relation to reproductive isolation and genetic flow between the four species.     

The Praon dorsale–yomenae s.str. complex (Hymenoptera,Braconidae, Aphidiinae): Species discrimination using geometric morphometrics and molecular markers with description of a new species

In this paper we apply an integrative approach combining morphometric and molecular analyses to explore parasitoids of the Praon dorsale–yomenae s.str. complex. These parasitoids occur in natural and agricultural ecosystems throughout the Palaearctic and parasitize aphid hosts belonging to the tribe Macrosiphini. The P. dorsale–yomenae species complex represents a morphologically cryptic group, consisting of several hidden taxa with specific host adaptations and distributions. For the morphometric analyses we used a large dataset comprising 230 female specimens that emerged from 30 different species of aphid hosts throughout the Palaearctic. The molecular analysis included a reduced dataset of 44 specimens that emerged from 19 aphid hosts. The mitochondrial cytochrome oxidase subunit I (COI) and the nuclear second expansion segment of the 28S rRNA gene (28SD2) were used to estimate a genetic diversity within this complex. Although all Praon species clustered closely together in the 28SD2 tree, confirming their close relatedness, the molecular characterization based on COI identified five clearly separate taxa with sequence divergences in the range of 4.7–8.9%. These taxa also exhibited significant differences in forewing shape as revealed by geometric morphometric analyses. Classical morphometric analyses revealed the length of m-cu vein as a new taxonomic character, but suggested that one commonly used trait, the color pattern of the Rs + M and m-cu veins, cannot be used for species distinction as it was highly variable in one of the taxa. Based on the combined morphometric and genetic data, we confirm the species status of Praon dorsale, P. yomenae, P. longicorne and P. volucre, and describe and illustrate a new parasitoid species of the “dorsale–yomenae” group, i.e. Praon staticobii n.sp. associated with Staticobium limonii on Limonium angustifolium.     

Application of molecular genetics and geometric morphometrics to taxonomy and conservation of cave beetles in central Italy

Integration of molecular genetic techniques and geometric morphometrics represent a valuable tool in the resolution of taxonomic uncertainty and the identification of significant units for conservation. We combined mitochondrial DNA cytochrome c oxidase subunit II gene sequence data and geometric morphometric analysis to examine taxonomic status and identify units for conservation in four species of the hypogean beetle Duvalius (Coleoptera, Trechinae) using mainly museum specimens collected in central Italy. Previous taxonomic studies based on morphological traits described several subspecies often inhabiting geographically distinct caves. Phylogenetic analysis identified two well supported monophyletic lineages and a number of different clades with relatively small genetic differences, suggesting a short divergence time in line with known geological history of the study area. Geometric morphometrics, on the other hand, recovered a high level of distinctiveness among specimens. Both genetic and morphometric analyses did not entirely corroborate former taxonomic nomenclature, suggesting possible rearrangements and the definition of evolutionary significant units. Beetles of the genus Duvalius are protected by regional laws and the majority of taxa considered in this study inhabit caves located outside protected areas. Our study advocates the importance of devoting protection efforts to networks of cave ecosystems rather than single locations or species.     

When the Rule Becomes the Exception. No Evidence of Gene Flow between Two Zerynthia Cryptic Butterflies Suggests the Emergence of a New Model Group

There is increasing evidence that most parapatric cryptic/sister taxa are reproductively compatible across their areas of contact. Consequently, the biological species concept, which assumes absence of interbreeding, is becoming a not so effective criterion in evolutionary ecology. Nevertheless, the few parapatric sister taxa showing complete reproductive barriers represent interesting models to study speciation processes and the evolution of reproductive isolation. In this study, we examined contact populations in northwestern Italy of two butterfly species, Zerynthia polyxena and Z. cassandra, characterized by different genitalic morphotypes. We studied levels of divergence among 21 populations distributed from Sicily to France using three genetic markers (the mitochondrial COI and ND1 genes and the nuclear wingless gene) and genitalic geometric morphometrics. Moreover, we performed species distribution modelling to estimate different climatic requirements of Z. polyxena and Z. cassandra. We projected climatic data into glacial maximum scenarios in order to verify if and to which extent glacial cycles could have contributed to speciation processes. Genetic and morphometric analyses identified two main groups. All specimens showed a concordant pattern of diversification, including those individuals sampled in the contact area. Haplotype distribution and climatic models showed that during glacial maxima both species experienced a strong range contraction and presumably remained separated into different microrefugia in southern France, in the Italian Peninsula and on the islands of Elba and Sicily. Long term separation was probably favoured by reduced dispersal ability and high phylopatry, while genitalic diversification probably favoured interbreeding avoidance. Conversely, the aposematic wing pattern remained almost identical. We compared our results with those obtained in other species and concluded that Z. polyxena and Z. cassandra represent a valuable model in the study of speciation.     

Evolutionary constraints in hind wing shape in Chinese dung beetles (Coleoptera: Scarabaeinae)

This study examines the evolution hindwing shape in Chinese dung beetle species using morphometric and phylogenetic analyses. Previous studies have analyzed the evolution of wing shape within a single or very few species, or by comparing only a few wing traits. No study has analyzed wing shape evolution of a large number of species, or quantitatively compared morphological variation of wings with proposed phylogenetic relationships. This study examines the morphological variation of hindwings based on 19 landmarks, 119 morphological characters, and 81 beetle species. Only one most parsimonious tree (MPT) was found based on 119 wing and body characters. To better understand the possible role of the hindwing in the evolution of Scarabaeinae, additional phylogenetic analyses were proposed based on the only body features (106 characters, wing characters excluded). Two MPT were found based on 106 body characters, and five nodes were collapsed in a strict consensus. There was a strong correlation between the morphometric tree and all phylogenetic trees (r>0.5). Reconstructions of the ancestral wing forms suggest that Scarabaeinae hindwing morphology has not changed substantially over time, but the morphological changes that do occur are focused at the base of the wing. These results suggest that flight has been important since the origin of Scarabaeinae, and that variation in hindwing morphology has been limited by functional constraints. Comparison of metric disparity values and relative evolutionary sequences among Scarabaeinae tribes suggest that the primitive dung beetles had relatively diverse hindwing morphologies, while advanced dung beetles have relatively similar wing morphologies. The strong correlation between the morphometric tree and phylogenetic trees suggest that hindwing features reflect the evolution of whole body morphology and that wing characters are suitable for the phylogenetic analyses. By integrating morphometric and cladistic approaches, this paper sheds new light on the evolution of dung beetle hind wings.     

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.     

The geometric approach to explore the Bactrocera tau complex (Diptera: Tephritidae) in Thailand

Specimens of the genus Bactrocera were collected from several host plants in northern and western Thailand. They were morphologically recognized as Bactrocera tau and were subdivided into eleven samples according to host plant, geographic origin and time of collection. Twelve landmarks of the right wing were described in a total of 264 males and 276 females. An exploratory analysis using kernel density estimates was performed on the multivariate morphometric space. Non-parametric classification highlighted the existence of two non-overlapping clusters within both males and females. The clusters were not congruent with geography. One cluster (cluster I) contained only one plant, Momordica cochinchinensis, the other one (cluster II) contained five different plants including M. cochinchinensis. Further morphometric analyses on selected samples indicated that the influence of the plants on the shape of the wing could not explain satisfactorily the presence of two clusters. Genetic techniques identified the presence of B. tau cryptic species C in M. cochinchinensis from cluster I, and of B. tau cryptic species A in Coccinia grandis from cluster II. Our working hypothesis is that the two clusters identified by geometric morphometrics were species A and C, respectively.     

Methods for species delimitation in bumblebees (Hymenoptera,Apidae, Bombus): towards an integrative approach

Delimitation of closely related species is often hindered by the lack of discrete diagnostic morphological characters. This is exemplified in bumblebees (genus Bombus). There have been many attempts to clarify bumblebee taxonomy by using alternative features to discrete morphological characters such as wing shape, DNA, or eco‐chemical traits. Nevertheless each approach has its own limitations. Recent studies have used a multisource approach to gather different lines of speciation evidence in order to draw a strongly supported taxonomic hypothesis in bumblebees. Yet, the resulting taxonomic status is not independent of selected evidence and of consensus methodology (i.e. unanimous procedure, majority, different weighting of evidence). In this article, we compare taxonomic conclusions for a group of taxonomically doubtful species (the Bombus lapidarius‐group) obtained from the four commonly used lines of evidence for species delimitation in bumblebees (geometric morphometric of wing shape, genetic differentiation assessment, sequence‐based species delimitation methods and differentiation of cephalic labial gland secretions). We ultimately aim to assess the usefulness of these lines of evidence as components of an integrative decision framework to delimit bumblebee species. Our results show that analyses based on wing shape do not delineate any obvious cluster. In contrast, nuclear/mitochondrial, sequence‐based species delimitation methods, and analyses based on cephalic labial gland secretions are congruent with each other. This allows setting up an integrative decision framework to establish strongly supported species and subspecies status within bumblebees.     

Phylogenetic Signal in the Wing Shape in the Subfamily Dolichopodinae (Diptera,Dolichopodidae)

Interspecific and sex-related variations in the wing shape of 22 species of the fly subfamily Dolichopodinae, family Dolichopodidae were analyzed using geometric morphometrics. Mapping morphometric traits onto phylogeny revealed a clear phylogenetic signal in the interspecific variation and sexual dimorphism of wing shape. In some cases, not too closely related species occupied the same portion of the morphometric space, indicating some degree of homoplasy. Interspecific variation was associated with an increased wing area due to both elongation and widening or only elongation of the wing. An increase in wing area was accompanied by extension of the posterior crossvein to the apical part of the wing. The variation in wing shape related to sexual dimorphism involved the same structures (the posterior crossvein and the apical part of CuA₁), but variation associated with sexual dimorphism was distributed in fewer dimensions than interspecific variation. The allometric component of sexual dimorphism varied between species, and in most cases it was not the leading factor in wing shape variability.     

Dealing with allometry in linear and geometric morphometrics: a taxonomic case study in the Leporinus cylindriformis group (Characiformes: Anostomidae) with description of a new species from Suriname

To achieve maximum efficacy, taxonomic studies that seek to distinguish amongst species must first account for allometric shape variation within species. Two recently developed software packages (SMATR and MorphoJ) offer regression‐based allometric approaches that are notable for their statistical power and ease of use and that may prove highly useful to taxonomists working with linear or geometric morphometric data. We investigate species delimitation of the slender‐bodied fishes in the Leporinus cylindriformis group using these programs and demonstrate the utility of the allometric corrections that they provide. Without allometric correction, many pairs of species are difficult to distinguish on the basis of morphometrics, but once regressions are used to account for marked allometric variation within species, most of the recognized species in this group can be readily distinguished with linear or geometric morphometrics, particularly using variation in the depth of the body. Both approaches returned congruent patterns of separation amongst putative species, but the geometric approach in MorphoJ distinguished amongst four more pairs of species than did the linear approach in SMATR and appears to provide slightly more statistical power. Based on distinctive morphometrics, meristics, and coloration, a highly elongate species of Leporinus from the Suriname, Corantijn, and Coppename rivers of Suriname is described herein as a new species, Leporinus apollo sp. nov. The unique L. cylindriformis holotype from Porto de Moz, Brazil differs in morphology, meristics, and pigmentation from specimens commonly referred to that species from the main basin of the Amazon; the latter specimens may represent an additional undescribed species. The L. cylindriformis holotype itself may represent a rare species or a specimen collected at the edge of its native range. Measurements of the holotype and paratype of Leporinus niceforoi, which were collected in the Amazonian slope of Colombia, differ substantially from similarly pigmented and putatively conspecific specimens from Amazonian portions of Ecuador and Peru. Recently collected specimens from Colombia are needed to determine whether the observed morphometric variation encompassed by the current concept of L. niceforoi indicates a morphocline within a single species, suggests the presence of multiple cryptic species, or results from shrinkage of the types. In all these cases, linear or geometric morphometric data can reliably differentiate amongst species, but only after one accounts for allometric shape variation. The new SMATR and MorphoJ software packages both offer easy and effective approaches to such allometrically informed taxonomy, and may prove useful to any systematist working on taxa that change shape as they grow. © 2011 The Linnean Society of London, Zoological Journal of the Linnean Society, 2011, 162 , 103–130.     

Sexual dimorphism and intraspecific variation in wing size and shape of Tongeia fischeri (Lepidoptera: Lycaenidae)

Wing morphological variations are described here for the lycaenid butterfly Tongeia fischeri. A landmark‐based geometric morphometric approach based on wing venation of 197 male and 187 female butterflies collected in Japan was used to quantify wing size and shape variations between sexes and among populations. Sexual dimorphism in wing size and shape was detected. Females had significantly larger wings than males, while males showed a relatively elongated forewing with a longer apex and narrower wing tornus in comparison to females. Intraspecific variations in wing morphology among populations were revealed for the wing shape, but not wing size. Distinct wing shape differences were found in the vein intersections area around the distal part of the discal cell where median veins originated in the forewing and around the origin of the CU1 vein in the hindwing. In addition, phenotypic relationships inferred from wing shape variations grouped T. fischeri populations into three groups, reflecting the subspecies classification of the species. The spatial variability and phenotypic relationships between conspecific populations of T. fischeri detected here are generally in agreement with the previous molecular study based on mitochondrial and nuclear sequences, suggesting the presence of a phylogenetic signal in the wing shape of T. fischeri, and thus having taxonomic implications.     

Evolutionary relationships of wing venation and wing size and shape in Aphidiinae (Hymenoptera: Braconidae)

We explored evolutionary changes in wing venation and wing size and shape in Aphidiinae, one of the well-known groups of parasitic wasps from the family Braconidae. Forewings of 53 species from 12 genera were examined, for which a molecular phylogeny was constructed on the basis of the mitochondrial barcoding gene COI. By covering all types of wing venation within the subfamily Aphidiinae and by using landmark-based geometric morphometrics and phylogenetic comparative methods, we tested whether evolutionary changes in wing shape correlate to the changes in wing venation and if both changes relate to wing size. The relationship between wing morphology and host specificity has been also investigated. We found that six types of wing venation, with different degree of vein reduction, could be recognized. Wing venation type is largely genus specific, except in the case of maximal reduction of wing venation which could be found across examined Aphidiinae taxa. The reconstruction of evolutionary changes in wing venation indicates that evolutionary changes in wing shape are related to the changes in wing size, indicating that miniaturization play a role in evolution of wing morphology while host specialization does not affect the wing shape within the subfamily Aphidiinae.     

Geometric craniometric analysis of sexual dimorphism and ontogenetic variation: A case study based on two geographically disparate species, Aethomys ineptus from southern Africa and Arvicanthis niloticus from Sudan (Rodentia: Muridae)

Non-geographic morphometric variation, particularly at the level of sexual dimorphism and ontogenetic (age-related) variation, has been documented in rodents, and useful for establishing whether to analyse sexes separately or together, and for selecting adult specimens for subsequent data recording and analysis. However, such studies have largely been based on traditional morphometric analyses of linear measurements that mainly focus on overall size, rather than shape-related morphometric variation. Unit-free, landmark/outline-based geometric morphometric analyses are considered to offer a more appropriate tool for assessing shape-related morphometric variation. In this study, we used geometric cranial morphometric analysis to assess the nature and extent of sexual dimorphism and age variation within the Tete veld rat, Aethomys ineptus (Thomas and Wroughton, 1908) from southern Africa and the African Nile rat, Arvicanthis niloticus (Desmarest, 1822) from Sudan. The results obtained were in turn compared with previously published results based on independent geometric and traditional cranial morphometric data from the same sampled populations examined in the present study. While our geometric morphometric results detected statistically significant sexual dimorphism in cranial shape within Ar. niloticus only, previously published results based on traditional morphometric data failed to detect significant sexual dimorphism within this species. However, similar to previously published traditional morphometric data, our geometric morphometric results detected statistically significant age-related variation in cranial shape and size within both Ae. ineptus and Ar. niloticus, with individuals of age classes 5 and 6 being considered to represent adult specimens. Our results highlight the importance of carefully evaluating both size- and shape-related non-geographic morphometric variation prior to the analysis of geographic variation and the delineation of species. Erroneous conclusions of non-geographic variation may have implications in the interpretation of geographic and evolutionary processes that may be responsible for morphological differences at both the inter- and intra-specific levels.     

Bat Species Comparisons Based on External Morphology: A Test of Traditional versus Geometric Morphometric Approaches

External morphology is commonly used to identify bats as well as to investigate flight and foraging behavior, typically relying on simple length and area measures or ratios. However, geometric morphometrics is increasingly used in the biological sciences to analyse variation in shape and discriminate among species and populations. Here we compare the ability of traditional versus geometric morphometric methods in discriminating between closely related bat species – in this case European horseshoe bats (Rhinolophidae, Chiroptera) – based on morphology of the wing, body and tail. In addition to comparing morphometric methods, we used geometric morphometrics to detect interspecies differences as shape changes. Geometric morphometrics yielded improved species discrimination relative to traditional methods. The predicted shape for the variation along the between group principal components revealed that the largest differences between species lay in the extent to which the wing reaches in the direction of the head. This strong trend in interspecific shape variation is associated with size, which we interpret as an evolutionary allometry pattern.     

The use of molecular and morphological characters to resolve the taxonomic identity of cryptic species: the case of Miniopterus manavi (Chiroptera, Miniopteridae)

Based on recent molecular phylogenetic studies, the Old World bat family Miniopteridae, composed of species in the genus Miniopterus , has been shown to contain complex paraphyletic species, many of which are cryptic based on convergent morphological characters. Herein we resolve the phylogenetic relationships and taxonomy of the species complex M . manavi on Madagascar and in the Comoro Archipelago, where these animals occur in different bioclimatic zones. First using mitochondrial cytochrome- b sequence data to define clades and then morphology to corroborate the molecular data, including comparisons to type specimens, we demonstrate that animals identified as this taxon are a minimum of three species: M . manavi sensu stricto occurs in at least the central portion of the Central Highlands; M . griveaudi has a broad distribution in lowland northern and central western Madagascar and the Comoros (Anjouan and Grande Comore), and M . aelleni sp. n. has been found in northern and western Madagascar and the Comoros (Anjouan). In each case, these three clades were genetically divergent and monophyletic and the taxa are diagnosable based on different external and craniodental characters. One aspect that helped to define the systematics of this group was isolation of DNA from one of the paratypes of M. manavi collected in 1896 and new topotypic material. Miniopterus manavi is most closely allied to a recently described species, M. petersoni . At several localities, M . griveaudi and M . aelleni have been found in strict sympatry, and together with M. manavi sensu stricto show considerable convergence in morphological characters, but are not immediate sister taxa. In defining and resolving the systematics of cryptic species, such as miniopterid bats, the process of defining clades with molecular tools, segregating the specimens accordingly, and identifying corroborative morphological characters has been notably efficient.     

Systematics of Pipizini and taxonomy of European Pipiza Fallén: molecular and morphological evidence (Diptera,Syrphidae)

Vuji?, A., Ståhls, G., A?anski, J., Bartsch, H., Bygebjerg, R. & Stefanovi?, A. (2013). Systematics of Pipizini and taxonomy of European Pipiza Fallén: molecular and morphological evidence (Diptera, Syrphidae). —Zoologica Scripta, 42, 288–305. In the present work the monophyly and molecular phylogenetic relationships of the genera of tribe Pipizini (Syrphidae) were investigated based on mitochondrial cytochrome c oxidase subunit I (COI) and nuclear 28S rDNA sequences, and the relationships among species of genus Pipiza Fallén, 1810 based on mtDNA COI sequences. Molecular phylogenetic analyses of Pipizini supported Pipiza as monophyletic and as sister group to all other Pipizini, and resolved other Pipizini genera as monophyletic lineages except for genus Heringia Rondani, 1856. To recognize the distinctness and maintain the monophyly the genus Heringia was redefined, generic rank was assigned to Neocnemodon Goffe, 1944 stat. n., and the genus Claussenia Vuji? & Ståhls gen. n., type‐species Claussenia hispanica (Strobl, 1909), was described. A revision of the European Pipiza species, including a discussion of taxonomic characters and a morphological redefinition of all included species, is presented. One new species, Pipiza laurusi Vuji? & Ståhls sp. n. was described. The taxa Pipiza carbonaria Meigen, 1822; Pipiza fasciata, Meigen 1822; Pipiza lugubris (Fabricius, 1775), Pipiza noctiluca (Linneaues, 1758), Pipiza notata Meigen, 1822 were redefined. Lectotypes are designated for 17 taxa, and neotypes were designated for seven taxa. Fourteen new synonymies were proposed. Male genitalia were illustrated for all the species, and a key of the 12 European species for males and females was provided. Geometric morphometrics of wing landmarks and extended sampling of mtDNA COI sequences was employed to delimitate taxa of the P. noctiluca and P. lugubris complexes. Despite subtle morphological differences, wing geometric morphometrics variables of wing size and shape showed highly significant differences among species within P. noctiluca and P. lugubris complexes, which were supported by the molecular data.