Recent Speciation Events Research Articles

Turnover of bird species along the Nullarbor Plain: Insights from taxonomic, phylogenetic, and functional beta diversity

AbstractThe Nullarbor Plain constitutes one of the main biogeographic barriers of Australia, and it has been suggested to have played a key role in the disjunct distribution of numerous southern Australian species. Although previous research has shown that the origin of this barrier coincides with the timing of the speciation events in some plant lineages, it is not clear whether the uplift of this barrier promoted divergence events in vertebrates. We addressed the role of the Nullarbor barrier and its fringing semiarid habitats as drivers of beta diversity in bird assemblages. Specifically, we determined the effect of distance from the Nullarbor barrier, environmental conditions, and isolation by distance on the composition of local communities on both sides of the plain. We measured beta diversity using taxonomic, phylogenetic, and functional metrics of composition. The influence of precipitation, geographic distance, and distance to the Nullarbor barrier on these metrics was addressed using generalized dissimilarity models and a moving‐window approach. We also tested for differences in local extinction, dispersal and speciation rates, and lineage diversity between two regions, southeastern (SE) and southwestern (SW) Australia. Geological and orogenetic dynamics linked to the appearance of the Nullarbor Plain may have spurred speciation events in SE. However, evidence suggests that subsequent periods in which this region was wetter and forested favored dispersal, mainly from SE to SW. Accordingly, observed dissimilarity in species composition was lower than expected at random, suggesting the existence of considerable turnover between regions. Our results suggest that precipitation deficit (and the xeric vegetation that it promotes) was the most important predictor of beta diversity, whereas the distance to the barrier explained some variation in terms of phylogenetic composition. This study shows that the uplift of the Nullarbor barrier played a minor role in shaping present‐day bird diversity in southern Australia. Recent speciation events coupled with historical connectivity can explain the observed patterns.

Divergence between sea urchins and their microbiota following speciation

Animals have a deep evolutionary relationship with microbial symbionts, such that individual microbes or an entire microbial community can diverge alongside the host. Here, we explore these host-microbe relationships in Echinometra, a sea urchin genus that speciated with the Isthmus of Panama and throughout the Indo-West Pacific. We find that the eggs from five Echinometra species generally associate with a species-specific bacterial community and that the relatedness of these communities is largely congruent with host phylogeny. Microbiome divergence per million years was higher in more recent speciation events than in older ones. We, however, did not find any bacterial groups that displayed co-phylogeny with Echinometra. Together, these findings suggest that the evolutionary relationship between Echinometra and their microbiota operates at the community level. We find no evidence suggesting that the associated microbiota is the evolutionary driver of Echinometra speciation. Instead, divergence between Echinometra and their microbiota is likely the byproduct of ecological, geographic, and reproductive isolations.

The relationship between Otiorhynchus ukrainicus (Korotyaev, 1984), Otiorhynchus rotundus (Marseul, 1872) and Otiorhynchus smreczynskii (Cmoluch, 1968): a hybrid speciation reconstruction

Otiorhynchus rotundus (Marseul, 1872) and Otiorhynchus smreczynskii (Cmoluch, 1968: a hybrid speciation reconstruction. Folia Biologica (Kraków) 72: 16-28. Otiorhynchus smreczynskii (Cmoluch, 1968) is a ubiquitous weevil species. Despite being common, the species is relatively unknown and most research has focused on its role as a plant pest. In our work, we compared O. smreczynskii and the closely related Otiorhynchus rotundus (Marseul, 1872) and Otiorhynchus ukrainicus (Korotyaev, 1984) based on molecular data. This was the first time that the molecular data of O. ukrainicus has been obtained. We used mitochondrial CO1 and the nuclear markers CAS and ArgK. Based on this data, we created phylogenetic trees, calculated genetic distances and conducted species delimitation using the PTP method. We also analysed the allozymes, proving that all the studied specimens of O. smreczynskii are hybrid triploids. The lower interspecific divergence (COI: 0.49%) indicates a recent speciation event. These results show that with a high probability, O. smreczynskii originated from O. rotundus and O. ukrainicus.

The relationship between phylogeny, range size, niche breadth and niche overlap in European orchids (Orchidaceae)

AbstractAimUnderstanding the various factors that contribute to the distribution and geographical ranges of plant and animal species has been a central issue in ecology, evolution and biogeography for more than two centuries. In this study, we investigated whether (i) niche breadth is phylogenetically conserved, (ii) niche overlap is negatively correlated with orchid evolutionary distance, and (iii) more recently diverged sister species show more niche overlap compared to older sister species.LocationEurope.TaxonOrchids (Orchidaceae).MethodsEcological niche models were created for 107 European orchid species distributed across 17 genera using occurrence and environmental data. A time‐calibrated phylogeny was reconstructed and the phylogenetic signal for range size and niche breadth was estimated. Phylogenetic distances among species were calculated to test the hypothesis that niche overlap is explained by evolutionary history. Finally, we investigated whether the divergence age of sister taxa was negatively related to niche overlap.ResultsRange size and niche breadth in both geographical and environmental space varied by more than three orders of magnitude and were strongly correlated with each other. We did not find strong evidence for phylogenetic conservatism in range size, niche breadth and niche overlap. However, sister taxa pairs with older divergence age showed less overlap in their environmental niche compared to more recently diverged sister taxa.Main ConclusionsWe conclude that orchid species that have broader ecological niches tend to display larger range sizes. Our results further show that the current distribution of orchid species across the European continent is best explained by recent speciation events and relative rapid adaptation to local environmental conditions, while deep‐level phylogenetic relationships are not correlated with ecological niche breadth.

Relevance of molecular systematics in insect pest management

Relevance of molecular systematics in insect pest management

Taxonomic status of the Lesser Woolly Horseshoe bats (Chiroptera, Rhinolophidae, Rhinolophus beddomei) in peninsular India and Sri Lanka.

Insularity provides ample opportunities for species diversification. Sri Lanka is home to a large diversity of species, many of which are endemic but morphologically similar to species found in southern India, due to recent speciation events, suggesting a complex evolutionary history. However, in some taxa although morphological diversity has been noted, the genetic level variations are minimal. Among the wide-ranging horseshoe bats such a phenomenon is noted. In this study, we used bioacoustics, morphometric and molecular data to evaluate the relationships between the taxa of lesser woolly horseshoe bats in the India and Sri Lanka. Our study reveals that the two taxa-Rhinolophus beddomei Andersen, 1905 and here we have validated the existing subspecies from peninsular India and R. sobrinus Andersen, 1918 from Sri Lanka are genetically very close to R. perniger Hodgson, 1843. Currently the taxa-beddomei and sobrinus are recognized as subspecies of Rhinolophus beddomei Andersen, 1905. We provide a detailed description of the taxa beddomei and sobrinus as the original descriptions are limited in nature.

Allopatric speciation is more prevalent than parapatric ecological divergence in a recent high-Andean diversification (Linochilus: Asteraceae).

Elucidating how species accumulate in diversity hotspots is an ongoing debate in evolutionary biology. The páramo, in the Northern Andes, has remarkably high indices of plant diversity, endemicity, and diversification rates. A hypothesis for explaining such indices is that allopatric speciation is high in the páramo given its island-like distribution. An alternative hypothesis is that the altitudinal gradient of the Andean topography provides a variety of niches that drive vertical parapatric ecological speciation. A formal test for evaluating the relative roles of allopatric and parapatric ecological speciation is lacking. The main aim of our study is to test which kind of speciation is more common in an endemic páramo genus. We developed a framework incorporating phylogenetics, species' distributions, and a morpho-ecological trait (leaf area) to compare sister species and infer whether allopatric or parapatric ecological divergence caused their speciation. We applied our framework to the species-rich genus Linochilus (63 spp.) and found that the majority of recent speciation events in it (12 events, 80%) have been driven by allopatric speciation, while a smaller fraction (one event, 6.7%) is attributed to parapatric ecological speciation; two pairs of sister species produced inconclusive results (13.3%). We conclude that páramo autochthonous (in-situ) diversification has been primarily driven by allopatric speciation.

A new cryptic species of Echinostoma (Trematoda: Echinostomatidae) closely related to Echinostoma paraensei found in Brazil.

Echinostoma paraensei, described in Brazil at the end of the 1960s and used as a biological model for a range of studies, belongs to the ‘revolutum’ complex of Echinostoma comprising species with 37 collar spines. However, molecular data are available only for a few isolates maintained under laboratory conditions, with molecular prospecting based on specimens originating from naturally infected hosts virtually lacking. The present study describes Echinostoma maldonadoi Valadão, Alves & Pinto n. sp., a species cryptically related to E. paraensei found in Brazil. Larval stages (cercariae, metacercariae and rediae) of the new species were found in the physid snail Stenophysa marmorata in the State of Minas Gerais, Brazil, the same geographical area where E. paraensei was originally described. Adult parasites obtained experimentally in Meriones unguiculatus were used for morphological (optical microscopy) and molecular [28S, internal transcribed spacer (ITS), nad1 and cox1] characterization. The morphology of larval and adult parasites (most notable the small-sized dorsal spines in the head collar), associated with low (0–0.1%) molecular divergence for 28S gene or ITS region, and only moderate divergence for the mitochondrial cox1 gene (3.83%), might suggest that the newly collected specimens should be assigned to E. paraensei. However, higher genetic divergence (6.16–6.39%) was found in the mitochondrial nad1, revealing that it is a genetically distinct, cryptic lineage. In the most informative phylogenetic reconstruction, based on nad1, E. maldonadoi n. sp. exhibited a strongly supported sister relationship with E. paraensei, which may indicate a very recent speciation event giving rise to these 2 species.

Multilocus species delimitation analyses show junior synonyms and deep-sea unknown species of genus Gaidropsarus (Teleostei: Gadiformes) in the North Atlantic/Mediterranean Sea area

Gaidropsarus Rafinesque, 1810 is a genus of marine teleost characterized by a high ecological diversity and by species inhabiting from the intertidal zone to the deep-sea. Several taxonomic conundrums have been historically present in this taxon due to its conservative morphology and the lack of available specimens. Species delimitation analyses were carried out in multiple datasets combining both mitochondrial (COI, CytB, ND2) and nuclear (Rho, ZIC1) genetic markers. Despite some incongruence between mitochondrial and nuclear data, the analyses supported the synonymy between Gaidropsarus biscayensis and Gaidropsarus macrophthalmus and the molecular confirmation of a recently new described species Gaidropsarus gallaeciae, in the North Atlantic Ocean. Furthermore, recent speciation events can explain the close relationships among several Gaidropsarus species, including Gaidropsarus granti–Gaidropsarus vulgaris and Gaidropsarus argentatus–Gaidropsarus ensis. These results support previous findings highlighted through DNA Barcoding analyses. Incongruences between mtDNA and nDNA have arisen between Gaidropsarus guttatus and Gaidropsarus mediterraneus, therefore, further analyses will be necessary to unravel the phenomena related to them.

Transcriptomes of Giant Sea Anemones from Okinawa as a Tool for Understanding Their Phylogeny and Symbiotic Relationships with Anemonefish.

The relationship between anemonefish and sea anemones is one of the most emblematic examples of mutualistic symbiosis in coral reefs. Although this is a textbook example, the major aspects of this symbiosis are still not fully understood in mechanistic terms. Moreover, since studies of this relationship have usually been focused on anemonefish, much less is known about giant sea anemones, their similarities, their phylogenetic relationships, and their differences at the molecular level. Since both partners of the symbiotic relationship are important, we decided to explore this well-known phenomenon from the perspective of giant sea anemones. Here, we report reference transcriptomes for all seven species of giant sea anemones that inhabit fringing reefs of Okinawa (Japan) and serve as hosts for six species of local anemonefish. Transcriptomes were used to investigate their phylogenetic relations, genetic differences and repertoires of nematocyte-specific proteins. Our data support the presence of three distinct groups corresponding to three genera: Entacmaea, Heteractis, and Stichodactyla. The basal position among the three groups belongs to Entacmaea, which was the first to diverge from a common ancestor. While the magnitude of genetic difference between the representatives of Entacmaea and Stichodactyla is large, intra-specific variation within Stichodactyla is much smaller and seems to result from recent speciation events. Our data reconfirms that Heteractis magnifica belongs to the genus Stichodactyla, despite an overall morphological similarity with representatives of the genus Heteractis. The availability of reference transcriptomes will facilitate further research into the fascinating relationship between sea anemones and anemonefish.

Biogeographic deconstruction of phylogenetic and functional diversity provides insights into the formation of regional assemblages

Evolutionary history and environmental filtering shape the phylogenetic and functional structure of regional assemblages. However, detecting the footprint of such eco‐evolutionary drivers is challenging because these may often counter each other's signature. Here, we examined whether a biogeographic deconstruction approach of phylogenetic (PD) and functional diversity (FD) patterns may help in identifying eco‐evolutionary signals in extant regional assemblages. As model system, we used forest understorey angiosperms found in three regions of Italy (Alpine, Mediterranean, Continental). We quantified PD and FD of all species inhabiting the three regions (regional assemblages). Then, we computed PD and FD for the subsets of species restricted to each region (biogeographic elements), also examining diversity patterns of species found across the three regions (widespread element). We used aboveground and belowground traits capturing major plant functions to calculate FD. Additionally, we assessed FD patterns decoupled from phylogeny. We found that species restricted to climatically harsh regions (Alpine and Mediterranean elements) were phylogenetically and functionally clustered, whereas widespread species were characterised by overdispersion. Species confined to the climatically intermediate (Continental) region were randomly sorted. By including all species occurring within a region, the patterns found for the region‐restricted species blurred. Phylogenetically decoupled FD patterns were qualitatively similar to non‐decoupled ones with the exception of the Alpine element, where we detected a clear signature of functional differentiation between closely related species. This suggests that recent speciation events contributed to shaping the Alpine flora. Compared to the belowground compartment, aboveground traits showed a more coherent pattern with that of all‐trait FD – likely because most biomass is allocated aboveground in forest understoreys. This biogeographic deconstruction study illustrates which type of eco‐evolutionary insights can be gained by implementing multifaceted and integrated approaches at the macroecological scale.

Intraspecific structure of the Coregonus lavaretus complex in water bodies of Siberia: a case of postglacial allopatric origin of Yukagirian whitefish

The results of morphological and genetic analyses of forms/species of the Coregonus lavaretus pidschian (Gmelin, 1789) complex from the Indigirka and Kolyma river basins are presented in the context of there being recent postglacial speciation events. It has been found that the studied whitefishes belong to the sparsely rakered and low lateral-line forms and have previously been described as Coregonus lavaretus pidschian n. jucagiricus Drjagin (Berg), 1932. Based on these characters, this whitefish does not differ from most Arctic whitefish populations (in particular from Coregonus lavaretus glacialis Kirillov, 1972). Analysis of variability of the ND1 gene (mtDNA) showed that whitefishes from the Indigirka and Kolyma basins belong to a distant phylogenetic lineage, which is significantly different from all previously studied whitefish lineages from the Ob, Yenisei, Lena, Anadyr, and Amur river basins. Analysis of variability of the ITS1 fragment (nDNA) showed that all studied forms/species (from Ob River to Amur River basins), including C. l. pidschian n. jucagiricus, have a tandem arrangement of two identical nucleotide fragments and very similar nucleotide composition of the ITS1 region. Based on contemporary data, this phylogenetic lineage of the C. pidschian complex could be considered a young postglacial allopatric species.

Rapid adaptive radiation in a hillstream cyprinid fish in the East African White Nile River basin.

Adaptive radiation of freshwater fishes was long thought to be possible only in lacustrine environments. Recently, several studies have shown that riverine and stream environments also provide the ecological opportunity for adaptive radiation. In this study, we report on a riverine adaptive radiation of six ecomorphs of cyprinid hillstream fishes of the genus Garra in a river located in the Ethiopian Highlands in East Africa. Garra are predominantly highly specialized algae-scrapers with a wide distribution ranging from Southeast Asia to West Africa. However, adaptive phenotypic diversification in mouth type, sucking disc morphology, gut length and body shape have probably been found among these ecomorphs in a single Ethiopian river. Moreover, we found two novel phenotypes of Garra ("thick-lipped" and "predatory") that had not been discovered before in this species-rich genus (>160species). Mitochondrial and genome-wide data suggest monophyletic, intrabasin evolution of Garra phenotypic diversity with signatures of gene flow from other local populations. Although sympatric ecomorphs are genetically distinct and can be considered to being young species as suggested by genome-wide single nucleotide polymorphism data, mitochondrial DNA was unable to identify any genetic structure suggesting recent and rapid speciation events. Some data suggest a hybrid origin of the novel "thick-lipped" ecomorph. Here we highlight how, driven by ecological opportunity, an ancestral trophically highly specialized lineage is likely to have rapidly radiated in a riverine environment promoted by the evolution of novel feeding strategies.

Using 3D geometric morphometrics to aid taxonomic and ecological understanding of a recent speciation event within a small Australian marsupial (Antechinus: Dasyuridae)

Abstract Taxonomic distinction of species forms the foundation of biodiversity assessments and conservation priorities. However, traditional morphological and/or genetics-based taxonomic assessments frequently miss the opportunity of elaborating on the ecological and functional context of species diversification. Here, we used 3D geometric morphometrics of the cranium to improve taxonomic differentiation and add ecomorphological characterization of a young cryptic divergence within the carnivorous marsupial genus Antechinus. Specifically, we used 168 museum specimens to characterize the recently proposed clades A. stuartii ‘south’, A. stuartii ‘north’ and A. subtropicus. Beyond slight differences attributable to overall size (and, therefore, not necessarily diagnostic), we also found clear allometry-independent shape variation. This allowed us to define new, easily measured diagnostic traits in the palate, which differentiate the three clades. Contrary to previous suggestions, we found no support for a latitudinal gradient as causing the differentiation between the clades. However, skull shape co-varied with temperature and precipitation seasonality, suggesting that the clades may be adapted to environmental variables that are likely to be impacted by climate change. Our study demonstrates the use of 3D geometric morphometrics to improve taxonomic diagnosis of cryptic mammalian species, while providing perspectives on the adaptive origins and potential future threats of mammalian diversity.

Integrative species delimitation based on COI, ITS, and morphological evidence illustrates a unique evolutionary history of the genus Paracercion (Odonata: Coenagrionidae).

Paracercion are common ‘blue and black’ colored damselflies. We explore the species boundaries of Paracercion (Odonata: Coenagrionidae) using ABGD, bPTP, GMYC and Distance-based clustering. We finally got the molecular data of all nine species of Paracercion. P. hieroglyphicum and P. melanotum were combined into one putative species based on cytochrome c oxidase I (COI). However, they were separated into two putative species based on the nuclear segment including ITS1-5.8S-ITS2 (ITS). This suggests the introgression of mtDNA in Paracercion. Paracercion barbatum and Paracercion melanotum can be separated into two species based on COI, whereas they were combined into one putative species based on ITS, which suggests a hybridization event between them. The lower interspecific divergence (COI: 0.49%) between P. barbatum and Paracercion v-nigrum indicates a recent speciation event in Paracercion. Paracercion sieboldii and P. v-nigrum can be separated into two putative species based on COI, while they were frequently merged into the same putative species based on ITS. This can be explained by incomplete lineage sorting in nDNA. Besides, P. pendulum and P. malayanum were synonymized as junior synonyms of P. melanotum. P. luzonicum was confirmed not to belong to Paracercion. The possibility of introgression, hybridization, recent speciation and incomplete lineage sorting makes species delimitation, based on molecular data, difficult and complicates understanding of the evolutionary history of Paracercion. The discordance in COI and ITS also indicates the value of using markers from different sources in species delimitation studies.

Taxogenomic and Comparative Genomic Analysis of the Genus Saccharomonospora Focused on the Identification of Biosynthetic Clusters PKS and NRPS.

Actinobacteria are prokaryotes with a large biotechnological interest due to their ability to produce secondary metabolites, produced by two main biosynthetic gene clusters (BGCs): polyketide synthase (PKS) and non-ribosomal peptide synthetase (NRPS). Most studies on bioactive products have been carried out on actinobacteria isolated from soil, freshwater or marine habitats, while very few have been focused on halophilic actinobacteria isolated from extreme environments. In this study we have carried out a comparative genomic analysis of the actinobacterial genus Saccharomonospora, which includes species isolated from soils, lake sediments, marine or hypersaline habitats. A total of 19 genome sequences of members of Saccharomonospora were retrieved and analyzed. We compared the 16S rRNA gene-based phylogeny of this genus with evolutionary relationships inferred using a phylogenomic approach obtaining almost identical topologies between both strategies. This method allowed us to unequivocally assign strains into species and to identify some taxonomic relationships that need to be revised. Our study supports a recent speciation event occurring between Saccharomonospora halophila and Saccharomonospora iraqiensis. Concerning the identification of BGCs, a total of 18 different types of BGCs were detected in the analyzed genomes of Saccharomonospora, including PKS, NRPS and hybrid clusters which might be able to synthetize 40 different putative products. In comparison to other genera of the Actinobacteria, members of the genus Saccharomonospora showed a high degree of novelty and diversity of BGCs.

Incipient hybrid inferiority between recently introduced, diverging dung beetle populations

AbstractUnderstanding why and how certain clades emerge as speciation hotspots is a fundamental objective of evolutionary biology. Here we investigate divergences between exotic Onthophagus taurus, a dung beetle introduced into the USA and Australia in the 1970s, as a potential model for the widespread recent speciation events characterizing the genus Onthophagus. To do so, we hybridized O. taurus derived from Eastern US (EUS) and Western Australian (WA) populations, and assessed fitness-relevant trait expression in first- and second-generation hybrids. We found that dams invest more in offspring provisioning when paired with a sire from the same population, and that WA dams crossed with EUS sires produce smaller and lighter F1 hybrids, with an unexpectedly male-biased sex ratio. Furthermore, fewer F2 hybrids with vertically inherited WA cytoplasm and microbiome emerged compared with WA backcrosses with WA cytoplasm/microbiome, suggesting that combinations of nuclear genome, cytoplasm and/or microbiome may contribute to hybrid viability. Lastly, we document a dominance of WA genotypes over body size at the point of inflection between minor and major male morphs, a trait of significance in mate competition, which has diverged remarkably between these populations. We discuss our results in light of the evolutionary ecology of onthophagine beetles and the role of developmental evolution in clade diversification.

Assessing a generic synapomorphy of Pseudodebis Forster, 1964 (Lepidoptera : Nymphalidae : Satyrinae) and a recent speciation with a shift in elevation between two new species in the western Andes

The field of systematics and our understanding of phylogenetic relationships have been invigorated by the use of molecular data, but analyses based on DNA sequence data are not always corroborated by diagnostic morphological characters. In particular, several taxonomic changes in butterflies (Papilionoidea) have been made solely on the basis of molecular data without identifying morphological synapomorphies that might have aided in diagnosing taxa from butterfly collections or specimens with no accessible DNA. We here focus on the butterfly genus Pseudodebis Forster, 1964 in the so-called ‘Taygetis clade’, which is one of the major clades in the diverse Neotropical nymphalid subtribe Euptychiina. We inferred the evolution of a male genitalic character using the most comprehensive molecular phylogeny for the ‘Taygetis clade’ to date. This approach allowed us to identify a synapomorphy for Pseudodebis Forster, 1964, which can be used to morphologically diagnose this genus and to distinguish it from other genera in the ‘Taygetis clade’. In addition, we describe two new species of Pseudodebis, P. nakamurai Nakahara & Willmott, sp. nov. and P. pieti Nakahara & Willmott, sp. nov., recovered as sister species based on molecular data, with an estimated time of divergence of 0.3 Ma (Bayesian confidence interval 0.03–1.61 Ma). Despite the low genetic divergence between these two Pseudodebis species, they can be readily distinguished by wing morphology. Pseudodebis nakamurai, sp. nov. and P. pieti, sp. nov. occur in partial sympatry across an elevational gradient along the western Andes, and the inferred recent speciation event might be related to a shift in elevation and possibly a change in larval hostplant preference. urn:lsid:zoobank.org:pub:38B4AF76-79E9-4D4D-BF16-FCD8F53A7277

Código de barras de ADN

Se ha impuesto la imagen del código de barras para hacer referencia a una secuencia de ADN mitocondrial que es utilizada para medir la distancia genética entre poblaciones animales. Según la interpretación esencialista de esta analogía, este método de medición serviría, por sí mismo, no solo para la rápida identificación de ejemplares, o muestras fragmentarias, de especies previamente clasificadas, sino también para el descubrimiento de nuevas especies. Los taxónomos integrativos han rechazado este último uso del método del código de barras de ADN, dado que la complejidad biológica impide aplicarlo con precisión en eventos de especiación recientes. Argumentaré, sin embargo, que el método falla por razones conceptuales y no meramente empíricas. Aunque no existiera ese impedimento empírico, el código de barras de ADN no podría ser un método de descubrimiento de especies.

Five new pseudocryptic land planarian species of Cratera (Platyhelminthes: Tricladida) unveiled through integrative taxonomy.

Background Cratera is a genus of land planarians endemic to the Brazilian Atlantic forest. The species of this genus are distinguished from each other by a series of external and internal characters, nonetheless they represent a challenging taxonomic issue due to the extreme alikeness of the species analysed in the present work. To resolve these difficulties, we have performed morphological analyses and used three nuclear markers (ribosomal 18S and 28S, Elongation Factor, a new anonymous marker named Tnuc813) and two mitochondrial fragments (Cytochrome c oxidase subunit I gene, and a fragment encompasing NADH deshydrogenase subunit 4 gene, trnF and the beginning of the Cytochrome c oxidase I gene) in an integrative taxonomic study.MethodsTo unveil cryptic species, we applied a molecular species delimitation approach based on molecular discovery methods, followed by a validation method. The putative species so delimited were then validated on the basis of diagnostic morphological features.ResultsWe discovered and described four new species, namely Cratera assu, C. tui, C. boja, and C. imbiri. A fifth new species, C. paraitinga was not highly supported by molecular evidence, but was described because its morphological attributes are unique. Our study documents for the genus Cratera the presence of a number of highly similar species, a situation that is present also in other genera of land planarians. The high number of poorly differentiated and presumably recent speciation events might be explained by the recent geological history of the area.