A phylogenetic assessment of the colonisation patterns in <Emphasis Type="Italic">Spauligodon atlanticus</Emphasis> Astasio-Arbiza et al., 1987 (Nematoda: Oxyurida: Pharyngodonidae), a parasite of lizards of the genus <Emphasis Type="Italic">Gallotia</Emphasis> Boulenger: no simple answers

Parasite taxonomy traditionally relies on morphometric and life-cycle characteristics which may not reflect complex phylogenetic relationships. However, genetic analyses can reveal cryptic species within morphologically described parasite taxa. We analysed the phylogenetic variation within the nematode Spauligodon atlanticus Astasio-Arbiza, Zapatero-Ramos, Ojeda-Rosas & Solera-Puertas, 1987, a parasite of the Canarian lizard genus Gallotia Boulenger, inferring the origin of their current association. We also attempted to determine its relationship with other Spauligodon spp. Three different markers, mitochondrial COI plus nuclear 18S and 28S ribosomal RNA, were used to estimate the evolutionary relationships between these nematodes. S. atlanticus was found to be paraphyletic, suggesting that Gallotia spp. were colonised by two independent lineages of Spauligodon. Additional analyses of other Spauligodon spp. are required for a more complete interpretation of the evolution of this genus from the Canarian archipelago and its closest taxa. Our results emphasise the importance of extensive sampling and phylogenetic studies at the intrageneric level, and highlight the limitations of a morphologically based taxonomy in these parasites.     

Cryptic diversity in the pen shell Atrina pectinata (Bivalvia: Pinnidae): high divergence and hybridization revealed by molecular and morphological data

Cryptic species have been increasingly revealed in the marine realm through an analytical approach incorporating multiple lines of evidence (e.g., mtDNA, nuclear genes and morphology). Illustrations of cryptic taxa improve our understanding of species diversity and evolutionary histories within marine animals. The pen shell Atrina pectinata is known to exhibit extensive morphological variations that may harbour cryptic diversity. In this study, we investigated A. pectinata populations along the coast of China and one from Japan to explore possible cryptic diversity and hybridization using a combination of mitochondrial (cytochrome c oxidase subunit I, mtCOI) and nuclear (ribosomal internal transcribed spacer, nrITS) genes as well as morphology. Phylogenetic analyses of mtCOI ‘DNA barcoding gene’ sequences resolved six divergent lineages with intralineage divergences between 0.4% and 0.8%. Interlineage sequence differences ranged from 4.3% to 22.0%, suggesting that six candidate cryptic species are present. The nrITS gene revealed five deep lineages with Kimura 2‐parameter distances of 3.7–30.3%. The five nuclear lineages generally corresponded to mtCOI lineages 1–4 and (5 + 6), suggestive of five distinct evolutionary lineages. Multiple nrITS sequences of significant variance were found within an individual, clearly implying recent hybridization events between/among the evolutionary lineages, which contributed to cytonuclear discordance. Morphologically, five morphotypes matched the five genetic lineages, although the intermediates may well blur the boundaries of different morphotypes. This study demonstrates the importance of combining multiple lines of evidence to explore species cryptic diversity and past evolutionary histories.     

Intestinal helminths of the granite spiny lizard (Sceloporus orcutti)

Examination of the intestinal tracts of 74 granite spiny lizards (Sceloporus orcutti) from Riverside County, California (USA) revealed infection with one cestode species Oochoristica scelopori (Anoplocephalidae) and one nematode species Spauligodon giganticus (Pharyngodonidae). Helminth prevalence was 24%. The presence of Oochoristica scelopori represents a new host record.     

Morphology of the Podarcis wall lizards (Squamata: Lacertidae) from the Iberian Peninsula and North Africa: patterns of variation in a putative cryptic species complex

Cryptic species complexes represent groups that have been classified as a single species, because of the difficulty in distinguishing its members morphologically. Morphological investigation following the discovery of cryptic diversity is crucial for describing and conserving biodiversity. Here we present a detailed account of morphological variation in a group of Iberian and North African Podarcis wall lizards of the family Lacertidae, trying to elucidate the morphological patterns observed between known mitochondrial lineages. Our results reveal very high morphological variation within lineages, considering both biometric and pholidotic traits, but also indicate that lineages are significantly different from each other. The main sources of variation, both globally and between lineages, arise from body size, head dimensions, and limb length, possibly pointing to underlying ecological mechanisms. A combination of body size, body shape, and continuous pholidotic traits allows a relatively good discrimination between groups, especially when comparing one group with the rest or pairs of groups. However, ranges of variation greatly overlap between groups, thereby not allowing the establishment of diagnostic traits. The high morphological variation observed indicates that external morphology is not particularly useful for species delimitation in this group of lizards, as local adaptation seems to play a major role in within‐ and between‐group differentiation. © 2011 The Linnean Society of London, Zoological Journal of the Linnean Society, 2012, 164 , 173–193.     

Species delimitation methods reveal cryptic diversity in the Hypnea cornuta complex (Cystocloniaceae,Rhodophyta)

The simple and convergent morphologies of many red algae make these species difficult to identify using traditional morphological characters. Many cryptic species have been described in recent years based on molecular datasets, and this has led to the application of an integrative taxonomy approach in species delimitation. Here, we performed several species delimitation methods (mBGD, ABGD, SPN, PTP, GMYCs and GMYCm) based on two different loci (COI-5P and rbcL) in species of the Hypnea cornuta complex. These methods were combined with morphological and phylogenetic data, extensive sampling, analysis of topotype material, and historically relevant herbarium samples. Our findings demonstrate that the groups morphologically assigned to H. cornuta and H. stellulifera consist of five different cryptic species. H. cornuta is a polyphyletic taxon composed of three well-separated lineages, thus requiring sequencing of type or topotype specimens to determine which one is Hypnea cornuta sensu stricto. We have revealed that the distribution of H. stellulifera is limited to Asia, while the Brazilian specimens initially assigned to this species were clarified as a new endemic species: Hypnea cryptica sp. nov. Our results indicated that only an integrative approach combining several lines of evidence, including morphology, nomenclature history, molecular data, biogeography and ecology can correctly solve the taxonomic status of widely distributed cryptic species.     

Nuclear DNA recapitulates the cryptic mitochondrial lineages of Lumbricus rubellus and suggests the existence of cryptic species in an ecotoxological soil sentinel

Mitochondrial DNA analysis has revealed two distinct phylogenetic lineages within the ecotoxological sentinel earthworm model Lumbricus rubellus Hoffmeister, 1843. The existence of these lineages could complicate ecotoxicological studies that use the species as a sentinel for soil contamination testing, as they may respond differently to contamination; however, as mitochondrial haplotypes are not always expected to segregate in the same way as chromosomal DNA in natural populations, we further investigated this issue by using nuclear DNA markers (microsatellites) to measure genetic diversity, differentiation, and gene flow in sympatric populations of the two L. rubellus lineages at two sites in South Wales. Our results show that sympatric populations of the two lineages are more genetically differentiated than geographically distant populations of the same lineage, and Bayesian clustering analysis revealed no evidence of gene flow between the lineages at either site. Additionally, DNA sequencing of these microsatellite loci uncovered substantial differentiation between lineages at homologous flanking regions. Overall our findings indicate a high degree of nuclear genetic differentiation between the two lineages of L. rubellus, implying reproductive isolation at the two study sites and therefore the potential existence of cryptic species. The existence of two cryptic taxa has major implications for the application of L. rubellus as an ecotoxicological sentinel. It may therefore be necessary to consider the lineages as separate taxa during future ecotoxicological studies. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 110 , 780–795.     

Host specificity shapes population structure of pinworm parasites in Caribbean reptiles

Host specificity is one of the potential factors affecting parasite diversification because gene flow may be facilitated or constrained by the number of host species that a parasite can exploit. We test this hypothesis using a costructure approach, comparing two sympatric pinworm parasites that differ in host specificity – Parapharyngodon cubensis and Spauligodon anolis – on the Puerto Rican Bank and St. Croix in the Caribbean. Spauligodon anolis specializes on Anolis lizards, whereas P. cubensis parasitizes Anolis lizards as well as many other species of lizards and snakes. We collected lizards from across the Puerto Rican Bank and St. Croix, sampled them for S. anolis and P. cubensis and generated nuclear and mitochondrial sequence data from the parasites. We used these data to show that P. cubensis is comprised of multiple cryptic species that exhibit limited population structure relative to S. anolis, which is consistent with our prediction based on their host specificity. We also provide evidence that the distribution of P. cubensis species is maintained by competitive exclusion, and in contrast to previous theoretical work, the parasites with the greatest number of host species also reach the highest prevalence rates. Overall, our results are consistent with the hypothesis that host specificity shapes parasite diversification, and suggest that even moderate differences in host specificity may contribute to substantial differences in diversification.     

Species‐diagnostic microsatellite loci for the fig wasp genus Pegoscapus

To obtain tools for the estimation of inbreeding and assignment of offspring to matrilines, we developed 13 microsatellite loci from the fig wasps that pollinate Ficus obtusifolia. Based on morphological studies, it was thought that a single species (Pegoscapus hoffmeyeri) pollinated this fig. However, our data revealed the presence of two coexisting cryptic species. Several diagnostic microsatellite markers may be used to distinguish these two cryptic species. The new microsatellites can be used across a wide range of fig‐pollinating wasp species for both evolutionary and population genetic studies.     

Incipient genetic divergence or cryptic speciation? Procamallanus (Nematoda) in freshwater fishes (Astyanax)

Hosts provide the main environmental traits parasites have to deal with, resulting in covariation between both associates at both micro- and macro-evolutionary scales; phylogenetic analyses of highly host-specific parasites have shown that parasite and host phylogeny might be highly congruent, and adaptation of a host species to new environments may lead to concordant changes of their parasites. Procamallanus (Spirocamallanus) neocaballeroi is a highly host-specific parasitic nematode of the Neotropical freshwater fish genus Astyanax in Mexico. One of the host species of the nematode is the emblematic Mexican tetra, A. mexicanus, which exhibits two contrasting phenotypes, a cave-dwelling morph (with troglomorphic features), and the surface-dwelling morph; other congeneric species inhabit rivers and lakes, and some of them occur in sympatry, displaying trophic specializations. Here, we explored the hypothesis that contrasting environments (surface rivers vs cave rivers), and host morphological divergence (sympatric ecomorphs in a lacustrine environment) might result in the divergence of their parasites, even though the hosts maintain a cohesive genetic structure as the same species. To test the hypothesis, several populations of Astyanax spp. were sampled to search for P. (S.) neocaballeroi. The nematode was found in 10 of the 52 sampled sites; two localities corresponded to cave populations. The phylogenetic analysis based on COI sequences yielded three major lineages for P. (S.) neocaballeroi. We found no concordance between the three lineages and the habitat where they occur in Astyanax mexicanus, even considering those living in drastic environmental conditions (caves), or between these lineages and lacustrine ecomorphs of Astyanax aeneus and A. caballeroi occurring in sympatry. Instead, genetic lineages of the nematode exhibit a clearer pattern of host species association and geographical distribution; our results showed that P. (S.) neocaballeroi is experiencing an incipient divergence although the morphological study of lineages shows no conspicuous differences.     

A new species of Spauligodon (Nematoda: Oxyurida: Pharyngodonidae) in geckos from São Nicolau Island (Cape Verde) and its phylogenetic assessment

A new nematode species, Spauligodon nicolauensis n. sp., is described from geckos Tarentola bocagei and Tarentola nicolauensis on the island of S?o Nicolau, Cape Verde. The new nematode was found in the pellets obtained directly from the geckos in a non-invasive fashion, and its identity was assessed both at morphologic and genetic levels. The new species has morphological similarities with Spauligodon tarentolae Spaul, 1926, also parasitizing geckos from the Canary Islands. However, the male cloacal region in the new species is distinct, presenting a different shape of the caudal papillae. The overall resemblance probably resulted from colonization via descent from an ancestor of S. tarentolae carried by the ancestor of Cape Verde Tarentola. The analysis of nuclear DNA sequences confirms that the new species is phylogenetically distinct from all other Spauligodon species already analyzed, forming a group clearly separated from species parasitizing lacertid lizards. The COI genetic distance suggests that the S. nicolauensis n. sp. found in the 2 species of geckos in S?o Nicolau Island may have resulted from a host-switching event, when they came into contact after the unification of the island.     

Cryptic or pseudocryptic: can morphological methods inform copepod taxonomy? An analysis of publications and a case study of the Eurytemora affinis species complex

Interest in cryptic species has increased significantly with current progress in genetic methods. The large number of cryptic species suggests that the resolution of traditional morphological techniques may be insufficient for taxonomical research. However, some species now considered to be cryptic may, in fact, be designated pseudocryptic after close morphological examination. Thus the “cryptic or pseudocryptic” dilemma speaks to the resolution of morphological analysis and its utility for identifying species. We address this dilemma first by systematically reviewing data published from 1980 to 2013 on cryptic species of Copepoda and then by performing an in‐depth morphological study of the former Eurytemora affinis complex of cryptic species. Analyzing the published data showed that, in 5 of 24 revisions eligible for systematic review, cryptic species assignment was based solely on the genetic variation of forms without detailed morphological analysis to confirm the assignment. Therefore, some newly described cryptic species might be designated pseudocryptic under more detailed morphological analysis as happened with Eurytemora affinis complex. Recent genetic analyses of the complex found high levels of heterogeneity without morphological differences; it is argued to be cryptic. However, next detailed morphological analyses allowed to describe a number of valid species. Our study, using deep statistical analyses usually not applied for new species describing, of this species complex confirmed considerable differences between former cryptic species. In particular, fluctuating asymmetry (FA), the random variation of left and right structures, was significantly different between forms and provided independent information about their status. Our work showed that multivariate statistical approaches, such as principal component analysis, can be powerful techniques for the morphological discrimination of cryptic taxons. Despite increasing cryptic species designations, morphological techniques have great potential in determining copepod taxonomy.     

Cryptic species in the Andean hemiparasite Quinchamalium chilense (Schoepfiaceae: Santalales)

The integration of different characters (e.g. morphological, ecological, and molecular) is now recognized as important in species delimitation. In particular, genetic distances between homologous genes have been suggested as one of the main tools to identify species, especially in the case of cryptic species. Quinchamalium is morphologically variable and occupies a diverse set of biomes across its distribution in the Southern Andes. Recent work based on morphology has synonymized the entire genus as a single morphospecies, Quinchamalium chilense. This widely distributed taxon presents the opportunity to find potential cryptic species. The main objective of this study was to test the existence of cryptic species, based mainly on phylogenetic gene trees, genetic distances, and geographic patterns of haplotypes from molecular markers of the nuclear (ITS) and chloroplast (trnL-F) genomes, considering climatic and morphological characteristics. The ITS phylogeny and corresponding haplotype network resulted in three lineages with strong genetic differentiation and distinct geographic patterns. These lineages were informally named Desert, Matorral, and Mountain, based on their geographic distribution in different biomes. The trnL-F chloroplast phylogeny did not distinguish Desert from Matorral, and the haplotype network showed overlap between these last two lineages. Overall, we hypothesize the existence of two cryptic species within Quinchamalium chilense (Mountain and Matorral–Desert) that correspond to genetic, climatic, and morphological differences.     

Cryptic species in the Pyropia yezoensis complex (Bangiales,Rhodophyta): Sympatric occurrence of two cryptic species even on same rocks

In a previous study on wild populations of Pyropia, the occurrence of two possible new species (Pyropia sp. 2 and Pyropia sp. 3) which are closely related to the two commercially important Pyropia species, P. yezoensis and P. tenera, was confirmed as the result of molecular phylogenetic analyses. To characterize the morphological features of the two wild Pyropia species, we collected Pyropia blades in a natural population in which Pyropia sp. 3 was known to occur, and carried out molecular identification before detailed morphological observations. Through the molecular identification we found, unexpectedly, that Pyropia sp. 2 blades grew sympatrically in the same site. Therefore, after molecular identification, we examined in detail the external morphology and anatomy of the two wild Pyropia species using more than 10 blades each. As a result, it is concluded that all of the blades of the two species are morphologically identical to P. yezoensis, but distinct from P. tenera. It is therefore considered that both of the two wild Pyropia species are cryptic species within the P. yezoensis complex. Furthermore, this study revealed that the two cryptic species grew sympatrically, even on the same rocks within the natural habitat.     

Cryptic speciation in the field vole: a multilocus approach confirms three highly divergent lineages in Eurasia

Species are generally described from morphological features, but there is growing recognition of sister forms that show substantial genetic differentiation without obvious morphological variation and may therefore be considered ‘cryptic species’. Here, we investigate the field vole (Microtus agrestis), a Eurasian mammal with little apparent morphological differentiation but which, on the basis of previous sex‐linked nuclear and mitochondrial DNA (mtDNA) analyses, is subdivided into a Northern and a Southern lineage, sufficiently divergent that they may represent two cryptic species. These earlier studies also provided limited evidence for two major mtDNA lineages within Iberia. In our present study, we extend these findings through a multilocus approach. We sampled 163 individuals from 46 localities, mainly in Iberia, and sequenced seven loci, maternally, paternally and biparentally inherited. Our results show that the mtDNA lineage identified in Portugal is indeed a distinct third lineage on the basis of other markers as well. In fact, multilocus coalescent‐based methods clearly support three separate evolutionary units that may represent cryptic species: Northern, Southern and Portuguese. Divergence among these units was inferred to have occurred during the last glacial period; the Portuguese lineage split occurred first (estimated at c. 70 000 bp ), and the Northern and Southern lineages separated at around the last glacial maximum (estimated at c. 18 500 bp ). Such recent formation of evolutionary units that might be considered species has repercussions in terms of understanding evolutionary processes and the diversity of small mammals in a European context.     

Hidden in the Arabian Mountains: Multilocus phylogeny reveals cryptic diversity in the endemic Omanosaura lizards

An increase in studies in the Hajar Mountains from the southeastern Arabian Peninsula has revealed a high richness of endemic evolutionary lineages with many cryptic taxa. Omanosaura is the only lacertid lizard genus endemic to the Hajar Mountains, with two species O. cyanura and O. jayakari distributed throughout this mountain range. The phylogenetic relationships and genetic diversity between and within these species have been poorly studied. In this study, we collected mitochondrial (12S, cytb, and nd4) and nuclear (cmos and mc1r) sequences for 25 specimens of Omanosaura, including 15 individuals of O. jayakari and 10 of O. cyanura. We performed phylogenetic analyses based on network reconstruction, maximum likelihood and Bayesian inference to estimate the relationships and intraspecific genetic diversity of these species. We estimated the time of divergence between the two species in the Miocene, around 8.5 million years ago. Omanosaura jayakari shows little genetic diversity, while O. cyanura presents two differentiated lineages. These are reciprocally monophyletic at mitochondrial and nuclear genes and present a high genetic distance between them. These two lineages are associated with the geographic features of the Hajar Mountains, with one lineage distributed in the northernmost part of the Hajar Mountains and the other in the rest of the western Hajars, the Jebel Akhdar, and the eastern Hajars. This geographic relationship has been recovered previously in other reptile taxa and is generally associated with high levels of local genetic diversity. Our results suggest the existence of cryptic diversity within O. cyanura and support a general biogeographic pattern of high diversity and endemism in the northern Hajar Mountains that certainly deserves additional research in the future.     

Unmasking cryptic species: morphometric and phylogenetic analyses of the Ibero‐North African Linaria incarnata complex

Linaria incarnata has been treated as a widely distributed Ibero‐North African species in the latest taxonomic reviews. Morphological and phylogenetic disparity between populations of this species has been previously reported. Here we present new morphological and phylogenetic evidence for the disintegration of L. incarnata into three distinct species: L. incarnata from the western Iberian Peninsula; L . mamorensis sp. nov. from north‐western Morocco; and L. onubensis from south‐western Spain. The relatively poor morphological differentiation between these taxa (which can be regarded as cryptic species) and their distinct phylogenetic positions indicate that characters of the L. incarnata morphotype have been acquired multiple times in the evolution of Linaria section Versicolores. © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 00, 000–000.     

The divergence of two independent lineages of an endemic Chinese gecko,Gekko swinhonis,launched by the Qinling orogenic belt

The genetic structure and demographic history of an endemic Chinese gecko, Gekko swinhonis, were investigated by analysing the mitochondrial cytochrome b gene and 10 microsatellite loci for samples collected from 27 localities. Mitochondrial DNA data provided a detailed distribution of two highly divergent evolutionary lineages, between which the average pairwise distance achieved was 0.14. The geographic division of the two lineages coincided with a plate boundary consisting of the Qinling and Taihang Mts, suggesting a historical vicariant pattern. The orogeny of the Qinling Mts, a dispersal and major climatic barrier of the region, may have launched the independent lineage divergence. Both lineages have experienced recent expansion, and the current sympatric localities comprised the region of contact between the lineages. Individual‐based phylogenetic analyses of nucDNA and Bayesian‐clustering approaches revealed a deep genetic structure analogous to mtDNA. Incongruence between nucDNA and mtDNA at the individual level at localities outside of the contact region can be explained by the different inheritance patterns and male‐biased dispersal in this species. High genetic divergence, long‐term isolation and ecological adaptation, as well as the morphological differences, suggest the presence of a cryptic species.     

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

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