A Good Compromise: Rapid and Robust Species Proxies for Inventorying Biodiversity Hotspots Using the Terebridae (Gastropoda: Conoidea)

Devising a reproducible approach for species delimitation of hyperdiverse groups is an ongoing challenge in evolutionary biology. Speciation processes combine modes of passive and adaptive trait divergence requiring an integrative taxonomy approach to accurately generate robust species hypotheses. However, in light of the rapid decline of diversity on Earth, complete integrative approaches may not be practical in certain species-rich environments. As an alternative, we applied a two-step strategy combining ABGD (Automated Barcode Gap Discovery) and Klee diagrams, to balance speed and accuracy in producing primary species hypotheses (PSHs). Specifically, an ABGD/Klee approach was used for species delimitation in the Terebridae, a neurotoxin-producing marine snail family included in the Conoidea. Delimitation of species boundaries is problematic in the Conoidea, as traditional taxonomic approaches are hampered by the high levels of variation, convergence and morphological plasticity of shell characters. We used ABGD to analyze gaps in the distribution of pairwise distances of 454 COI sequences attributed to 87 morphospecies and obtained 98 to 125 Primary Species Hypotheses (PSHs). The PSH partitions were subsequently visualized as a Klee diagram color map, allowing easy detection of the incongruences that were further evaluated individually with two other species delimitation models, General Mixed Yule Coalescent (GMYC) and Poisson Tree Processes (PTP). GMYC and PTP results confirmed the presence of 17 putative cryptic terebrid species in our dataset. The consensus of GMYC, PTP, and ABGD/Klee findings suggest the combination of ABGD and Klee diagrams is an effective approach for rapidly proposing primary species proxies in hyperdiverse groups and a reliable first step for macroscopic biodiversity assessment.     

Species delimitation and phylogeny in the genus Nasutitermes (Termitidae: Nasutitermitinae) in French Guiana

Species delimitation and identification can be arduous for taxa whose morphologic characters are easily confused, which can hamper global biodiversity assessments and pest species management. Exploratory methods of species delimitation that use DNA sequence as their primary information source to establish group membership and estimate putative species boundaries are useful approaches, complementary to traditional taxonomy. Termites of the genus Nasutitermes make interesting models for the application of such methods. They are dominant in Neotropical primary forests but also represent major agricultural and structural pests. Despite the prevalence, pivotal ecological role and economical impact of this group, the taxonomy of Nasutitermes species mainly depends on unreliable characters of soldier external morphology. Here, we generated robust species hypotheses for 79 Nasutitermes colonies sampled throughout French Guiana without any a priori knowledge of species affiliation. Sequence analysis of the mitochondrial cytochrome oxidase II gene was coupled with exploratory species‐delimitation tools, using the automatic barcode gap discovery method (ABGD) and a generalized mixed Yule‐coalescent model (GMYC) to propose primary species hypotheses (PSHs). PSHs were revaluated using phylogenetic analyses of two more loci (mitochondrial 16S rDNA and nuclear internal transcribed spacer 2) leading to 16 retained secondary species hypotheses (RSSH). Seven RSSHs, represented by 44/79 of the sampled colonies, were morphologically affiliated to species recognized as pests in the Neotropics, where they represent a real invasive pest potential in the context of growing ecosystem anthropization. Multigenic phylogenies based on combined alignments (1426–1784 bp) were also reconstructed to identify ancestral ecological niches and major‐pest lineages, revealing that Guyanese pest species do not form monophyletic groups.     

New insights into and limitations of the molecular phylogeny in the taxon-rich land snail genus Montenegrina (Mollusca: Gastropoda: Clausiliidae)

Rock-dwelling gastropods are usually patchily distributed in limestone habitats, presumably have low active and passive dispersal ability and often represent narrow-ranged endemic taxa. Their current taxonomy is predominantly shell morphology based, and it remains unknown whether the morphologically differentiated and geographically separated populations represent phylogenetic clades. In this study, we analysed the hyperdiverse, terrestrial door snail genus Montenegrina. Based on the current taxonomy defined by shell morphology, it contains 29 species and 106 subspecies distributed in the Balkan region. The constructed phylogenetic tree using three mitochondrial markers was used to test whether it agrees with the current taxonomy. In this comprehensive tree, about half of the species and subspecies are monophyletic. Some of the paraphylies could be reasonably resolved by taxonomic changes; that is, some subspecies should be reassigned or raised to species level. Other incongruencies probably arose due to introgression even between distant clades. The histone genes turned out to be unsuitable for elucidating the phylogeny of Montenegrina. In the species-delimitation tests, considerably more molecular operational taxonomic units were delimited than the number of presently described species. The present data indicate that (a) shell morphology-based taxonomy and taxon recognition can be problematic in such a large and morphologically highly variable genus; (b) the potential error due to incomplete sampling presents a problem in a genus as variable as Montenegrina; (c) multi-locus analyses should be conducted to arrive at a better basis for species delimitation; and (d) integrative approaches including genetic as well as morphological/anatomical data from a comprehensive geographic sample are necessary.     

Species delimitation in the Andean grasshopper genus Orotettix Ronderos & Carbonell (Orthoptera: Melanoplinae): an integrative approach combining morphological,molecular and biogeographical data

The reciprocal illumination nature of integrative taxonomy through hypothesis testing, corroboration and revision is a powerful tool for species delimitation as more than one source has to support the hypothesis of a new species. In this study, we applied an integrative taxonomy approach combining molecular and morphological data sets with distributional patterns to examine the level of differentiation between and within the grasshopper Orotettix species. Orotettix was described based on five valid species distributed in the Andes of Peru. In our study, initially a molecular‐based hypothesis was postulated and tested against morphological data and geographical patterns of distribution. Results from molecular and morphological analyses showed agreement among the species delimitation in Orotettix, and were also consistent with the geographical distribution. The analyses allowed us to delimit five new species for the genus ( O. lunatus sp. nov. , O . astreptos sp. nov. , O. colcaensis sp. nov. , O . paucartambensis sp. nov. and O . dichrous sp. nov. ) from the Eastern and Western Cordilleras of Peru. We also provide critical knowledge on the phylogenetic relationships and distribution of the genus and conduct a revision of Orotettix. © 2015 The Linnean Society of London     

An integrative approach to species delimitation in Benthomangelia (Mollusca: Conoidea)

DNA sequences are currently used to propose primary hypotheses of species delimitation, especially when morphological variability is difficult to assess. In an integrative taxonomy framework, these hypotheses are then compared with other characters, such as morphology or geography, to produce robust species delimitations. For this purpose, the cytochrome oxidase subunit I (COI) gene has been sequenced for almost 50 specimens of the genus Benthomangelia , a deep-sea marine gastropod genus, collected in the South-West Pacific. Five genetic groups, displaying low and high genetic distances respectively within and between groups, were defined. COI hypotheses were compared with both the results obtained with the independent nuclear 28S gene and with an elliptic Fourier analysis of the shape of the last whorl of the shell. 28S gene analysis confirmed the same well-supported groups as COI, and elliptic Fourier analysis identified several morphological characters that vary similarly to genetic variability.  © 2009 The Linnean Society of London, Biological Journal of the Linnean Society , 2009, 96 , 696–708.     

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.     

Taking the discovery approach in integrative taxonomy: decrypting a complex of narrow‐endemic Alpine harvestmen (Opiliones: Phalangiidae: Megabunus)

Species delimitation is fundamental for biological studies, yet precise delimitation is not an easy task, and every involved approach has an inherent failure rate. Integrative taxonomy, a method that merges multiple lines of evidence, can profoundly contribute to reliable alpha‐taxonomy and shed light on the processes behind speciation. In this study, we explored and validated species limits in a group of closely related Megabunus harvestmen (Eupnoi, Phalangiidae) endemic to the European Alps. Without a priori species hypotheses, we used multiple sources of inference, including mitochondrial and multilocus nuclear DNA, morphometrics and chemistry. The results of these discovery approaches revealed morphological crypsis and multiple new species within two of the five hitherto known species. Based on our analyses, we discussed the most plausible evolutionary scenarios, invoked the most reasonable species hypotheses and validated the new species limits. Building upon the achieved rigour, three new species, Megabunus cryptobergomas Muster and Wachter sp. nov., Megabunus coelodonta Muster and Steiner sp. nov., and Megabunus lentipes Muster and Komposch sp. nov., are formally described. In addition, we provide a dichotomous morphological key to the Megabunus species of the Alps. Our work demonstrates the suitability of integrative, discovery‐based approaches in combination with validation approaches to precisely characterize species and enabled us to implement nomenclatural consequences for this genus.     

Integration of molecular, ecological, morphological and endosymbiont data for species delimitation within the Pnigalio soemius complex (Hymenoptera: Eulophidae)

Integrative taxonomy is a recently developed approach that uses multiple lines of evidence such as molecular, morphological, ecological and geographical data to test species limits, and it stands as one of the most promising approaches to species delimitation in taxonomically difficult groups. The Pnigalio soemius complex (Hymenoptera: Eulophidae) represents an interesting taxonomical and ecological study case, as it is characterized by a lack of informative morphological characters, deep mitochondrial divergence, and is susceptible to infection by parthenogenesis‐inducing Rickettsia. We tested the effectiveness of an integrative taxonomy approach in delimiting species within the P. soemius complex. We analysed two molecular markers (COI and ITS2) using different methods, performed multivariate analysis on morphometric data and exploited ecological data such as host–plant system associations, geographical separation, and the prevalence, type and effects of endosymbiont infection. The challenge of resolving different levels of resolution in the data was met by setting up a formal procedure of data integration within and between conflicting independent lines of evidence. An iterative corroboration process of multiple sources of data eventually indicated the existence of several cryptic species that can be treated as stable taxonomic hypotheses. Furthermore, the integrative approach confirmed a trend towards host specificity within the presumed polyphagous P. soemius and suggested that Rickettsia could have played a major role in the reproductive isolation and genetic diversification of at least two species.     

DNA barcodes for the pipefish genus Corythoichthys (Actinopterygii: Syngnathiformes) from the Indian Ocean provide insights into cryptic diversity

The syngnathiform genus Corythoichthys comprises a group of taxonomically complex, tail-brooding (Syngnathinae) pipefishes widely distributed in the Indo-Pacific region. Due to the presence of overlapping interspecific morphological characters, reliable taxonomic information on Corythoichthys is still lacking. Using 52 CO1 sequences, including seven newly generated, a phylogenetic analysis was carried out to understand the genetic diversity, distribution and ‘species groups’ within the genus Corythoichthys. Species delimitation using Automatic Barcode Gap Discovery (ABGD) analysis confirmed the presence of 13 species which include ‘species-complexes’ previously considered as a single taxon. Our results revealed the presence of three species groups, ‘C. amplexus’, ‘C. conspicillatus’ and ‘C. haematopterus’ and four unidentified/undescribed species in the wider Indo-Pacific realm. Interestingly, 60 sequences and a mitogenome identified as Corythoichthys in GenBank are misidentified at the genus level. Based on our findings, we suggest that the taxonomy and systematics of Corythoichthys need to be re-examined and validated using integrative methods, and care should be taken while selecting specimens for genetic studies.     

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     

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     

Species complex or complex species? Integrative taxonomy of the land snail genus Rossmaessleria (Gastropoda,Helicidae) from Morocco and Gibraltar

We applied an integrative approach to re-evaluate the taxonomy of the conchologically highly diverse land snail genus Rossmaessleria from the Rif Mountains in Morocco and from Gibraltar, which has been classified into 12 nominal species so far. An analysis of cox1 and 16S rDNA sequences using the General Mixed Yule-coalescent approach with a single or multiple thresholds, its Bayesian implementation as well as the Automatic Barcode Gap Discovery method indicated that all Rossmaessleria populations can be classified into a single species, R. scherzeri (Zelebor, 1867). This result is confirmed by the lack of diagnostic differences in the genitalia as shown in a principal component analysis of the genital measurements. The variation of shell characters also does not allow an unambiguous subdivision of the complex. However, the populations of a mountain or a mountain ridge share characteristic combinations of shell characters so that they can be classified as geographic subspecies. The delimitation of the subspecies and their distribution is discussed and three subspecies are described as new to science: R. scherzeri periclitata ssp. nov. R. scherzeri ingae ssp. nov., and R. scherzeri eleanorae ssp. nov.

http://zoobank.org/urn:lsid:zoobank.org:pub:187FE235-A257-423F-8FC3-957117546400     

Diversity of Siphonaria Sowerby I, 1823 (Gastropoda,Siphonariidae) in the Seychelles Bank and beyond

Molecular phylogenetic studies have shown that the characters of the reduced shell of the false limpets of the genus Siphonaria Sowerby I, 1823 are highly variable and often insufficient for species delimitation. The taxonomy and distribution of Siphonaria in the Indian Ocean are poorly known. We sampled Siphonaria in the Seychelles Bank to check the occurrence of recorded species using DNA sequences and to study the paths through which Siphonaria species have colonised the Seychelles Bank by reconstructing their phylogenetic relationships. Analyses of a dataset comprising 16 S rRNA gene sequences of 33 specimens from the Seychelles Bank and 300 additional Siphonaria sequences from other regions from GenBank with various methods for species delimitation resulted in 19–102 primary species hypotheses. Assemble Species by Automatic Partitioning provided a conservative estimate of the species number (42) in which several indisputable species were lumped. The results of Automatic Barcode Gap Discovery depended strongly on the assumed prior maximum intraspecific divergence, whereas the tree-based methods Generalised Mixed Yule Coalescent and Poisson Tree Processes resulted in high overestimates. The specimens from the Seychelles Bank represent three clades, belonging to the Siphonaria ‘atra’ group, the Siphonaria ‘normalis’ group and a possibly undescribed species recorded previously only from Hainan. At least two of the three species recorded from the Seychelles Bank came from the east, i.e., from the Coral Triangle in the Indo-Australian Archipelago, the region with the highest marine biodiversity worldwide. A major transport mechanism across the Indian Ocean was probably the South Equatorial Current.     

Species delimitation: inferring gaps in morphology across geography

Species are commonly delimited on the basis of gaps in patterns of morphological variation, but there seems to be little recent work on methods to objectively assess such gaps. Here, we introduce a statistical approach that uses measurements of continuous morphological characters and geographic variation in those characters to (i) measure the strength of the evidence for the existence of a gap in morphological variation between two hypothesized species and (ii) examine if a gap in morphological variation between two hypothesized species can be explained by an alternative hypothesis of geographic variation within a species. This approach is based on recent developments in analyses of multivariate normal mixtures, estimates of multivariate tolerance regions, and principal coordinates of neighboring matrices. We demonstrate the application of the approach by examining previously proposed hypotheses of species limits in the plant genus Escallonia. We discuss the main features of the method, including potential limitations, in relation to other approaches that use gaps in morphological variation as a criterion for species delimitation. The method we propose can help strengthen the link between the theory and practice of species delimitation by increasing the transparency and consistency of taxonomic decisions based on morphology, thus contributing to integrative approaches for species delimitation that consider morphological and geographic data on an equal footing with other kinds of information.     

Cryptic diversity in Brevipalpus mites (Tenuipalpidae)

Defining the taxonomic identity of organisms is a prerequisite for their study, and in the case of economically important species, misidentification may lead to the application of inappropriate prevention and control strategies. Flat mites of the Brevipalpus genus include several crop pests and the systematics of these mites represents a challenge for acarologists. Many of the most economically important Brevipalpus species have repeatedly been inaccurately identified. Such problematic classification has been attributed to the likely occurrence of cryptic species in the genus. In this study, we used an integrative approach that combined molecular analyses, including sequence‐based species delimitation, with detailed morphological identification using traits that have recently showed to be taxonomically informative. Sequences of mitochondrial cytochrome c oxidase subunit I (COI) were obtained from individuals collected from host plants belonging to 14 genera and 13 families across 29 locations in the Americas (Brazil, Chile, USA). The phylogenetic analyses included previously published Brevipalpus sequences from GenBank, and the final data set was classified into 44 haplotypes. Six putative species were recognised by COI‐based species delimitation analysis, and morphological evidence supported each of these species. The integrative approach revealed the occurrence of cryptic species in the Brevipalpus genus and contributed to the clarification of previously noted incongruences. The results presented here allow for the evaluation of taxonomic characteristics in a phylogenetic context and indicate new characters for the differentiation of Brevipalpus species. In addition, Brevipalpus incognitus n. sp. Ferragut & Navia, a cryptic species detected in this study, is described based on morphological and molecular traits. Implications of the advances in Brevipalpus systematics presented herein with respect to pest management are briefly discussed.     

Molecular phylogeny and species delimitation of Stachyuraceae: Advocating a herbarium specimen‐based phylogenomic approach in resolving species boundaries

Species concept and delimitation are fundamental to taxonomic and evolutionary studies. Both inadequate informative sites in the molecular data and limited taxon sampling have often led to poor phylogenetic resolution and incorrect species delineation. Recently, the whole chloroplast genome sequences from extensive herbarium specimen samples have been shown to be effective to amend the problem. Stachyuraceae are a small family consisting of only one genus Stachyurus of six to 16 species. However, species delimitation in Stachyurus has been highly controversial because of few and generally unstable morphological characters used for classification. In this study, we sampled 69 individuals of seven species (each with at least three individuals) covering the entire taxonomic diversity, geographic range, and morphological variation of Stachyurus from herbarium specimens for genome‐wide plastid gene sequencing to address species delineation in the genus. We obtained high‐quality DNAs from specimens using a recently developed DNA reconstruction technique. We first assembled four whole chloroplast genome sequences. Based on the chloroplast genome and one nuclear ribosomal DNA sequence of Stachyurus, we designed primers for multiplex polymerase chain reaction and high throughput sequencing of 44 plastid loci for species of Stachyurus. Data of these chloroplast DNA and nuclear ribosomal DNA internal transcribed spacer sequences were used for phylogenetic analyses. The phylogenetic results showed that the Japanese species Stachyurus praecox Siebold & Zucc. was sister to the rest in mainland China, which indicated a typical Sino‐Japanese distribution pattern. Based on diagnostic morphological characters, distinct distributional range, and monophyly of each clade, we redefined seven species for Stachyurus following an integrative species concept, and revised the taxonomy of the family based on previous reports and specimens, in particular the type specimens. Furthermore, our divergence time estimation results suggested that Stachyuraceae split from its sister group Crossosomataceae from the New World at ca. 54.29 Mya, but extant species of Stachyuraceae started their diversification only recently at ca. 6.85 Mya. Diversification time of Stachyurus in mainland China was estimated to be ca. 4.45 Mya. This research has provided an example of using the herbarium specimen‐based phylogenomic approach in resolving species boundaries in a taxonomically difficult genus.     

Taxonomy of Mechanitis (F.) (Lepidoptera: Nymphalidae) from the West Colombian Andes: an Integrative Approach

Species identification in the butterfly genus Mechanitis (F.) (Lepidoptera: Nymphalidae) becomes difficult when it is based only on wing color patterns, a common practice in butterfly taxonomy. Difficulties in Mechanitis taxonomy are related to the widespread mimicry and polymorphism among species belonging to this genus. Species recognition and inventories of Mechanitis genus in geographic areas as the Andean region of Colombia are of particular interest and the use of more than one character for taxonomic identification is desirable. In this study, we included morphological, ecological, and mitochondrial DNA data to identify the occurring species in this region. Species of Mechanitis were studied from ecological, morphological, and molecular perspectives considering host plant identification, oviposition behavior, and life cycles under laboratory conditions. Immature morphology, patterns of wing color, and genital structures of adults were also studied. The genetic barcoding region of the cytochrome oxidase I mitochondrial gene was sequenced and used to verify the limits between species previously defined by the other characters and to validate its usefulness for species delimitation in this particular genus. The integrative approach combining independent datasets successfully allowed species identification as compared to the approach based on a single dataset. Three well-differentiated species were found in the studied region, Mechanitis menapis (Hewitson), Mechanitis polymnia (Linnaeus), and Mechanitis lysimnia (Fabricius). New valuable characters that could improve taxonomic identification in this genus are considered.     

A taxonomic revision and species delimitation of the genus Purpuraria Enderlein, 1929 (Orthoptera: Pamphagidae) using an integrative approach

Recent studies on the endemic Canarian genus Purpuraria have shown that the taxonomy of its only recognized species (P. erna) is probably erroneous. In this study, an integrative revision of the genus is performed, based on a large number of specimens and geographical sampling. As a result, (1) the diagnostic characters at the genus level are re‐described, (2) Purpuraria magna n. sp. based on morphological, morphometric and genetic data is described and (3) the taxonomic status of a formerly described subspecies is clarified. Intraspecific and interspecific morphometric differences have been found, indicating that the genus is undergoing a process of morphological diversification. Nevertheless, the possibility of interspecific mating between individuals of the two species is suggested, because no significant differences have been found between their respective calling songs. Genetic analyses using mitochondrial and nuclear DNA sequences suggest that P. erna and P. magna are recent species with evidences of secondary contact episodes in the past.