Delimitation Of Cryptic Species Research Articles

Using molecular and morphometric data as operational criteria for the analysis of a threatened rainforest species complex shows interspecific variation, with implications for cryptic-species delimitation and conservation

Resolution of systematic associations and species boundaries is vital for developing conservation priorities for threatened taxa. A complex of Fontainea (Euphorbiaceae) populations, endemic to rainforest communities in central-eastern Australia, comprises several taxonomically challenging species. Fontainea oraria is Critically Endangered, with only one natural population of 10 mature individuals, with the closely related species F. australis being listed as Vulnerable. A recently discovered Fontainea population (currently nominated as F. sp. Coffs Harbour) with taxonomic similarities to F. oraria has been provisionally listed as Critically Endangered, with fewer than 40 individuals. This study employed an integrative approach, combining genetic data and morphometrics to determine species boundaries for three threatened Fontainea population groups as a model system for an integrative approach to delimiting cryptic species. Although our results suggest the potential subdivision of the population groups into three taxa, caution is warranted because this hypothesis remains inconclusive. Taxonomic challenges demand a careful approach, acknowledging the possibility of alternative interpretations. Contrary to supporting distinct species, our morphological and genetic data may also be interpreted as indicative of a single species with geographic variation, a phenomenon observed in genetically linked populations experiencing isolation by distance. Overall, we highlight the need for further research to establish species limits to guide conservation actions.

An assessment of new character in hoverfly species delimitation using linear and geometric morphometrics – genus Merodon Meigen, 1803 (Diptera: Syrphidae) as a case study

Detection of morphologically indistinguishable cryptic species implies using an integrative taxonomic approach with a combination of molecular, contemporary morphological, ecological and other relevant analyses. Within a contemporary morphological analysis, two approaches are commonly used in hoverfly taxonomy: a geometric morphometric analysis of the wing and surstyle shape. Here, the importance of the R4+5 vein shape is tested in cryptic species delimitation within four Merodon species groups using linear and semilandmark geometric morphometric analyses. As expected, geometric morphometrics showed a stronger resolution compared to linear morphometrics. Linear morphometrics failed to detect differences related to sexual dimorphism or differences among the species M. pruni and M. obscurus. However, all cryptic species and sexes were separated with high significance based on the R4+5 vein shape. Moreover, obtained results concurred with the landmark-defined wing shape and molecular results published in previous studies. Additionally, combining two characters, the semilandmark R4+5 vein shape and the landmark-defined wing shape, provided more detailed and precise insights into the shape differences. Our results showed that the R4+5 vein shape stands out as an important character in species delimitation of hoverflies where the sinuation of this vein is present. Therefore, it can be beneficial as a single character or in combination with a landmark-based wing shape analysis.

Integrative species delimitation helps to find the hidden diversity of the leaf-litter frog Ischnocnema manezinho (Garcia, 1996) (Anura, Brachycephalidae), endemic to the southern Atlantic Forest.

The delimitation of cryptic species is a challenge for biodiversity conservation. Anurans show high cryptic diversity levels, and molecular species delimitation methods could help identify putative new species. Additionally, species delimitation approaches can provide important results for cryptic species conservation, with integrative methods adding robustness to results. Ischnocnema manezinho was described from Santa Catarina Island (SCI), southern Brazil. More recently, some inventories indicated continental populations supposedly similar in morphology to it. If these records are confirmed as I. manezinho, it would likely change its endangered status on National Red List, removing the species from conservation agendas. We investigated the threatened frog Ischnocnema manezinho, to evaluate if the continental populations belong to this species or if they form an undescribed species complex. We used coalescent, distance, and allele-sharing-based species delimitation methods and integrative analyses of morphometric and bioacoustics traits to test evolutionary independence between I. manezinho from SCI, Arvoredo Island, and continental populations. Ischnocnema manezinho is restricted to Santa Catarina Island, while the five remaining lineages should be further investigated through a taxonomic review. Our results point to a small geographic range of Ischnocnema manezinho. Additionally, the species occurs in isolated fragments of forest in SCI surrounded by expanding urban areas, confirming its status as Endangered. Thus, the protection and monitoring of I. manezinho and the taxonomic description of the continental and Arvoredo Island candidate species should be priorities.

The Cytochrome Oxidase I gene confirms the presence of the nemertine Carcinonemertes conanobrieni, a parasite of the Caribbean spiny lobster (Panulirus argus) in Colombia

Morphological identifications complemented with molecular analysis are important for the delimitation of cryptic species. This document contains annotations on the molecular identification of the parasitic nemertine Carcinonemertes conanobrieni in the Caribbean spiny lobsters Panulirus argus. The collected nemerteans were found parasitizing ovigerous lobsters from the Gulf of Salamanca (Puebloviejo), Colombia. DNA was extracted from a specimen and a fragment of the mitochondrial gene Cytochrome Oxidase I (COI) was amplified using Folmer’s universal primers, the sequence was edited with ProSeq 3.5 and aligned with all the C. conanobrienisequences available in GenBank. A sequence of 488 base pairs corresponding to the section of the genetic barcode of the COI gene was obtained, the matrix of genetic distances showed little variation when comparing the obtained sequence with those available in the GenBank database. These results confirm the presence of this nemertean in the Panulirus argus lobsters in Colombia providing valuable information for the correct identification and detection of this parasitic species in the country, thus facilitating its monitoring

The Cytochrome Oxidase I gene confirms the presence of the nemertine Carcinonemertes conanobrieni, a parasite of the Caribbean spiny lobster (Panulirus argus) in Colombia

Morphological identifications complemented with molecular analysis are important for the delimitation of cryptic species. This document contains annotations on the molecular identification of the parasitic nemertine Carcinonemertes conanobrieni in the Caribbean spiny lobsters Panulirus argus. The collected nemerteans were found parasitizing ovigerous lobsters from the Gulf of Salamanca (Puebloviejo), Colombia. DNA was extracted from a specimen and a fragment of the mitochondrial gene Cytochrome Oxidase I (COI) was amplified using Folmer’s universal primers, the sequence was edited with ProSeq 3.5 and aligned with all the C. conanobrienisequences available in GenBank. A sequence of 488 base pairs corresponding to the section of the genetic barcode of the COI gene was obtained, the matrix of genetic distances showed little variation when comparing the obtained sequence with those available in the GenBank database. These results confirm the presence of this nemertean in the Panulirus argus lobsters in Colombia providing valuable information for the correct identification and detection of this parasitic species in the country, thus facilitating its monitoring

Using natural history to guide supervised machine learning for cryptic species delimitation with genetic data

The diversity of biological and ecological characteristics of organisms, and the underlying genetic patterns and processes of speciation, makes the development of universally applicable genetic species delimitation methods challenging. Many approaches, like those incorporating the multispecies coalescent, sometimes delimit populations and overestimate species numbers. This issue is exacerbated in taxa with inherently high population structure due to low dispersal ability, and in cryptic species resulting from nonecological speciation. These taxa present a conundrum when delimiting species: analyses rely heavily, if not entirely, on genetic data which over split species, while other lines of evidence lump. We showcase this conundrum in the harvester Theromaster brunneus, a low dispersal taxon with a wide geographic distribution and high potential for cryptic species. Integrating morphology, mitochondrial, and sub-genomic (double-digest RADSeq and ultraconserved elements) data, we find high discordance across analyses and data types in the number of inferred species, with further evidence that multispecies coalescent approaches over split. We demonstrate the power of a supervised machine learning approach in effectively delimiting cryptic species by creating a “custom” training data set derived from a well-studied lineage with similar biological characteristics as Theromaster. This novel approach uses known taxa with particular biological characteristics to inform unknown taxa with similar characteristics, using modern computational tools ideally suited for species delimitation. The approach also considers the natural history of organisms to make more biologically informed species delimitation decisions, and in principle is broadly applicable for taxa across the tree of life.

Are members of the Anopheles fluviatilis complex conspecific?

Anopheles fluviatilis sensu lato, a primary malaria vector in India, has been identified to be comprised of four cryptic species, provisionally designated as species S, T, U and V. However, Kumar et al. (Mol Ecol Resour, 2013;13:354-61) considered all of the then known three members of this species complex (S, T and U) conspecific. The specific status of species S and T was refuted based on the lack of sufficient barcode gap in mitochondrial-CO1 and the perceived presence of heterozygotes in populations as detected through one of the two species-specific PCR assays employed for the cryptic species identification. The existence of species U was refuted claiming that earlier investigations have already refuted their existence. Here we discuss problems associated with the CO1-based barcode approach for delimitation of cryptic species, the perceived heterozygosity between species S and T based on a species-specific PCR assay, and interpretation of published reports. We demonstrated that fixed differences do exist in the ITS2-rDNA sequence of species S and T with no evidence of heterozygotes in sympatric populations and, that the observed heterozygosity by Kumar et al. in the ITS2-based species diagnostic PCR is due to the high mispriming tendency of the T-specific primer with species S. We infer that mitochondrial DNA-based ‘barcoding gap’, an arbitrary threshold recommended for species delimitation, alone, is inadequate to delimit the members of An. fluviatilis complex.

Hidden diversity—Delimitation of cryptic species and phylogeography of the cyprinidGarraspecies complex in Northern Oman

Hidden diversity—Delimitation of cryptic species and phylogeography of the cyprinid<i>Garra</i>species complex in Northern Oman

Another stripe on the tiger makes no difference? Unexpected diversity in the widespread tiger tarantula Davus pentaloris (Araneae: Theraphosidae: Theraphosinae)

Abstract Integrative taxonomy is relevant for the discovery and delimitation of cryptic species by incorporating different sources of evidence to construct rigorous species hypotheses. The genus Davus was recently revised and it was found that Davus pentaloris presents high morphological variation across its widespread distribution. However, tarantulas usually present low dispersal capabilities that occasionally result in local endemism. In order to evaluate species boundaries within this taxon, we examine the morphological variation and, additionally, employ four strategies based on mtDNA data (COI): two distance-based [automatic barcode gap discovery (ABGD) and Neighbor-Joining (NJ)] and two tree-based methods [general mixed Yule coalescent (GMYC) and Bayesian Poisson tree process (bPTP)]. Available morphological evidence recovers 13 putative morphospecies, but the different methods based on molecular data recover a variable number of candidate species (16–18). Based on the congruence across all analyses and the available morphological data, we recognize 13 clearly diagnosable species, 12 of them new to science. We conclude that the underestimation of the diversity in D. pentaloris was mainly caused by deficient practices in taxonomy, rather than by the presence of cryptic diversity. Although COI is a functional barcoding marker and it gives reliable results in our study, we recommend combining multiple sources of evidence and strategies to construct better species delimitation hypotheses.

Ecology and morphology of mouse lemurs (Microcebus spp.) in a hotspot of microendemism in northeastern Madagascar, with the description of a new species.

Delimitation of cryptic species is increasingly based on genetic analyses but the integration of distributional, morphological, behavioral, and ecological data offers unique complementary insights into species diversification. We surveyed communities of nocturnal mouse lemurs (Microcebus spp.) in five different sites of northeastern Madagascar, measuring a variety of morphological parameters and assessing reproductive states for 123 individuals belonging to five different lineages. We documented two different non-sister lineages occurring in sympatry in two areas. In both cases, sympatric species pairs consisted of a locally restricted (M. macarthurii or M. sp. #3) and a more widespread lineage (M. mittermeieri or M. lehilahytsara). Estimated Extents of Occurrence (EOO) of these lineages differed remarkably with 560 and 1,500 km2 versus 9,250 and 50,700 km2 , respectively. Morphometric analyses distinguished unambiguously between sympatric species and detected more subtle but significant differences among sister lineages. Tail length and body size were most informative in this regard. Reproductive schedules were highly variable among lineages, most likely impacted by phylogenetic relatedness and environmental variables. While sympatric species pairs differed in their reproductive timing (M. sp. #3/M. lehilahytsara and M. macarthurii/M. mittermeieri), warmer lowland rainforests were associated with a less seasonal reproductive schedule for M. mittermeieri and M. lehilahytsara compared with populations occurring in montane forests. Distributional, morphological, and ecological data gathered in this study support the results of genomic species delimitation analyses conducted in a companion study, which identified one lineage, M. sp. #3, as meriting formal description as a new species. Consequently, a formal species description is included. Worryingly, our data also show that geographically restricted populations of M. sp. #3 and its sister species (M. macarthurii) are at high risk of local and perhaps permanent extinction from both deforestation and habitat fragmentation.

Promising Perspectives for Detection, Identification, and Quantification of Plant Pathogenic Fungi and Oomycetes through Targeting Mitochondrial DNA.

Fungi and oomycetes encompass many pathogens affecting crops worldwide. Their effective control requires screening pathogens across the local and international trade networks along with the monitoring of pathogen inocula in the field. Fundamentals to all of these concerns are their efficient detection, identification, and quantification. The use of molecular markers showed the best promise in the field of plant pathogen diagnostics. However, despite the unquestionable benefits of DNA-based methods, two significant limitations are associated with their use. The first limitation concerns the insufficient level of sensitivity due to the very low and uneven distribution of pathogens in plant material. The second limitation pertains to the inability of widely used diagnostic assays to detect cryptic species. Targeting mtDNA appears to provide a solution to these challenges. Its high copy number in microbial cells makes mtDNA an attractive target for developing highly sensitive assays. In addition, previous studies on different pathogen taxa indicated that mitogenome sequence variation could improve cryptic species delimitation accuracy. This review sheds light on the potential application of mtDNA for pathogen diagnostics. This paper covers a brief description of qPCR and DNA barcoding as two major strategies enabling the diagnostics of plant pathogenic fungi and oomycetes. Both strategies are discussed along with the potential use of mtDNA, including their strengths and weaknesses.

Delimitation of cryptic species drastically reduces the geographical ranges of marine interstitial ghost-worms (Stygocapitella; Annelida, Sedentaria)

The recognition of cryptic species concealed in traditionally established species may reveal new biogeographical patterns and alter the understanding of how biodiversity is geographically distributed. This is particularly relevant for marine ecosystems where the incidence of cryptic species is high and where species distribution data are often challenging to collect and interpret. Here, we studied specimens of the ‘cosmopolitan’ interstitial meiofaunal annelid Stygocapitella subterranea Knöllner, 1934 (Parergodrilidae, Orbiniida), obtaining data from four coastlines in the Northern hemisphere. Using phylogenetic tools and several species-delimitation methods (haplotype networks, GMYC, bPTP, maximum likelihood, posterior probability and morphology) we describe eight new Stygocapitella species. With one exception, all species are present along a single coastline, ultimately challenging the idea that Stygocapitella subterranea has a cosmopolitan distribution. We found evidence for several oceanic transitions having occurred in the past as well as a recent translocation, potentially due to human activity. No diagnostic characters were found, and qualitative and quantitative morphological data do not allow an unequivocal differentiation of the identified cryptic species. This suggests that (i) neither traditional diagnostic features nor quantitative morphology suffice to recognise species boundaries in cryptic species complexes, such as the Stygocapitella species complex; and that (ii) the recognition and description of cryptic species is of seminal importance for biodiversity assessments, biogeography and evolutionary biology.

An Integrative Approach Using Phylogenomics and High-Resolution X-Ray Computed Tomography for Species Delimitation in Cryptic Taxa.

Morphologically cryptic taxa have proved to be a long-standing challenge for taxonomists. Lineages that show strong genomic structuring across the landscape but are phenotypically similar pose a conundrum, with traditional morphological analyses of these cryptic lineages struggling to keep up with species delimitation advances. Micro X-ray computed tomography (CT) combined with geometric morphometric analyses provides a promising avenue for identification of morphologically cryptic taxa, given its ability to detect subtle differences in anatomical structures. However, this approach has yet to be used in combination with genomic data in a comparative analytical framework to distinguish cryptic taxa. We present an integrative approach incorporating genomic and geometric morphometric evidence to assess the species delimitation of grassland earless dragons (Tympanocryptis spp.) in north-eastern Australia. Using mitochondrial and nuclear genes (ND2 and RAG1, respectively), along with $>$8500 SNPs (nuclear single nucleotide polymorphisms), we assess the evolutionary independence of target lineages and several closely related species. We then integrate phylogenomic data with osteological cranial variation between lineages using landmark-based analyses of three-dimensional CT models. High levels of genomic differentiation between the three target lineages were uncovered, also supported by significant osteological differences. By incorporating multiple lines of evidence, we provide strong support for three undescribed cryptic lineages of Tympanocryptis in north-eastern Australia that warrant taxonomic review. Our approach demonstrates the successful application of CT with integrative taxonomic approaches for cryptic species delimitation, which is broadly applicable across vertebrates containing morphologically similar yet genetically distinct lineages. Additionally, we provide a review of recent integrative taxonomic approaches for cryptic species delimitation and an assessment of how our approach can value-add to taxonomic research.

Techniques and tools for species identification in ciliates: a review.

Ciliates are highly divergent unicellular eukaryotic organisms with nuclear dualism and a highly specialized ciliary pattern. They inhabit all biotopes and play crucial roles in regulating microbial food webs as they prey on bacteria, protists and even on microscopic animals. Nevertheless, subtle morphological differences and tiny sizes hinder proper species identification for many ciliates. In the present review, an attempt has been made to elaborate the various approaches used by modern day ciliate taxonomists for species identification. The different approaches involved in taxonomic characterization of ciliates such as classical (using live-cell observations, staining techniques, etc.), molecular (involving various marker genes) and statistical (delimitation of cryptic species) methods have been reviewed. Ecological and behavioural aspects in species identification have also been discussed. In present-day taxonomy, it is important to use a 'total evidence' approach in identifying ciliates, relying on both classical and molecular information whenever possible. This integrative approach will help in the mergence of classical methods with modern-day tools for comprehensive species description in future.

AmphiNom: an amphibian systematics tool

Large-scale comparative and systematic studies rely on the seamless merging of multiple datasets. However, taxonomic nomenclature is constantly being revised making it problematic to combine data from different resources or different years of publication, which use different synonyms. This is certainly true for amphibians, which have experienced a spike in taxonomic revisions in part as the result of the widespread use of DNA barcoding to resolve cryptic species delimitation issues and large-scale collaborative efforts to revise the entire amphibian tree. The ‘Amphibian Species of the World Online Reference’ (ASW) is one of the most widely used and most regularly updated databases for amphibian taxonomy, but existing R tools for querying synonyms such as ‘taxize’ do not include this resource. ‘AmphiNom’ is a tool suite written in the R programming language designed to facilitate batch-querying amphibian species names against the ASW database. This facilitates the merging of datasets that use different nomenclature and its functionality is easily integrated into customizable R workflows. Moreover, it allows direct querying of the ASW website using R and straightforward reporting of summary information on current amphibian systematics.

Cryptic species as a window into the paradigm shift of the species concept.

The species concept is the cornerstone of biodiversity science, and any paradigm shift in the delimitation of species affects many research fields. Many biologists now are embracing a new "species" paradigm as separately evolving populations using different delimitation criteria. Individual criteria can emerge during different periods of speciation; some may never evolve. As such, a paradigm shift in the species concept relates to this inherent heterogeneity in the speciation process and species category-which is fundamentally overlooked in biodiversity research. Cryptic species fall within this paradigm shift: they are continuously being reported from diverse animal phyla but are poorly considered in current tests of ecological and evolutionary theory. The aim of this review is to integrate cryptic species in biodiversity science. In the first section, we address that the absence of morphological diversification is an evolutionary phenomenon, a "process" counterpart to the long-studied mechanisms of morphological diversification. In the next section regarding taxonomy, we show that molecular delimitation of cryptic species is heavily biased towards distance-based methods. We also stress the importance of formally naming of cryptic species for better integration into research fields that use species as units of analysis. Finally, we show that incorporating cryptic species leads to novel insights regarding biodiversity patterns and processes, including large-scale biodiversity assessments, geographic variation in species distribution and species coexistence. It is time for incorporating multicriteria species approaches aiming to understand speciation across space and taxa, thus allowing integration into biodiversity conservation while accommodating for species uncertainty.

Integrative analyses of Nervilia (Orchidaceae) section Linervia reveal further undescribed cryptic diversity in Thailand

The delimitation of cryptic species is necessary to accurately classify and appropriately conserve biodiversity. Integrative analyses can be incisive in detecting and circumscribing cryptic diversity, especially in species complexes whose members are delineated by minor or overlapping morphological variation. We adopt an integrative approach to assess species relationships and resolve species boundaries in the taxonomically difficult Nervilia adolphi/punctata species alliance of N. sect. Linervia, an Old World complex of reduced, one-flowered terrestrial orchids that is both species-rich and poorly known in tropical and warm temperate Asia. We sampled 12 of the 27 known species of the alliance in Asia, including all four species reported from Thailand and a further 20 plants collected in that country that could not be satisfactorily identified using morphology alone. Phylogenetic analyses using one nuclear (ITS) and two plastid (matK and trnL-F) markers confirmed both N. sect. Linervia and the alliance itself as monophyletic, and corroborated 11 of the 12 sampled species; N. punctata proved polyphyletic, with the Thai samples referred to this Indonesian species falling sister to the Himalayan N. mackinnonii. The 20 unidentified Thai samples formed three distinct, strongly supported clades. STACEY, a Bayesian coalescence approach to species delimitation, resolved the same three clusters, but provided evidence suggesting that one comprised two distinct sub-clades. Building on this genetic evidence, we identify subtle morphological differences and invoke a diagnosable species concept to circumscribe three previously unrecognized cryptic species from Thailand. This objective approach to species delimitation validates ostensibly minor morphological differences as a basis for differentiating species within the alliance, paving the way for a global analysis of species boundaries throughout the genus as a whole.

Allotetraploid cryptic species in Asplenium normale in the Japanese Archipelago, detected by chemotaxonomic and multi-locus genotype approaches.

Delimitation of cryptic species provides an understanding of biodiversity and opportunities to elucidate speciation processes. Extensive flavonoid variation has been reported in the tetraploid cytotype of the fern, Asplenium normale, although related species have no intraspecific variations in flavonoid composition. We hypothesized that Japanese A. normale still harbors multiple cryptic species with different flavonoid compositions, and tested this hypothesis using chemotaxonomic and multilocus genotyping approaches. We determined the multilocus genotypes (MLGs) of 230 samples from 37 populations for one chloroplast DNA region and three nuclear genes. MLGs were used to delimit reproductively isolated lineages by population-genetic approaches. We also tested the correspondence between genetically recognized groups and flavonoid compositions. To identify the origins of putative cryptic species, we conducted phylogenetic analysis of the DNA markers used in genotyping. The genetic clusters and flavonoid compositions showed clear correspondence. We recognized three putative cryptic species in tetraploid Asplenium normale in Japan. Phylogenetic analyses revealed that cryptic species I and III originated from allopolyploidization between a diploid A. normale and an unknown diploid of A. boreale, and cryptic species II originated from allopolyploidization between a diploid A. normale and A. oligophlebium. Our study demonstrated that intraspecific variation of secondary metabolites can be a good indicator of cryptic species in ferns. The presence of the two cryptic species having the same progenitor diploid pair suggests that speciation between allopolyploid lineages of independent origin may be more common than previously considered.

Cryptic characters for cryptic taxa: On the taxonomic utility of the genital chaetae in earthworms (Oligochaeta, Hormogastridae)

Hard anatomical characters have successfully been used in invertebrate systematics. Besides their chaetae, this kind of characters is virtually absent from earthworms (a taxonomically challenging group). This work explores the taxonomic utility of genital chaetae, which have been demonstrated to play an important role in earthworm reproduction. Genital chaetae were collected from different clades in the family Hormogastridae, some of them comprising cryptic complexes. The morphology of these structures was studied in detail with scanning electron microscopy and differential interference contrast microscopy with the aim of finding relevant phylogenetic information at different levels: putative diagnostic characters for clades, phylogenetic relationships between clades, and support for cryptic species delimitation. Our results revealed wide morphological variability in the distal tip of the genital chaetae between the different clades (summarized in five qualitative characters) and cryptic lineages. Canonical Variate Analysis based on dorsal landmarks was able to separate chaetae from the cryptic lineages in non-overlapping clusters and Discriminant Function Analysis allowed to assess its reliability. Some complex structures in the tip of genital chaetae, such as denticles and pores, highlighted the necessity of further research on their role in the reproduction of the different earthworm families. Our results support the potential of genital chaetae as a systematic character to resolve conflicting phylogenetic relationships of earthworms, being also potentially suitable for disentangling cryptic species complexes.

A Combination of Divergence and Conservatism in the Niche Evolution of the Moorish Gecko, Tarentola mauritanica (Gekkota: Phyllodactylidae).

The quantification of realized niche overlap and the integration of species distribution models (SDMs) with calibrated phylogenies to study niche evolution are becoming not only powerful tools to understand speciation events, but can also be used as proxies regarding the delimitation of cryptic species. We applied these techniques in order to unravel how the fundamental niche evolved during cladogenesis within the Tarentola mauritanica species-complex. Our results suggest that diversification within this complex, during the Miocene and Pleistocene, is associated with both niche divergence and niche conservatism, with a pattern that varies depending on whether the variables involved are related to the mean or seasonality of temperature and humidity. Moreover, climatic variables related to humidity and temperature seasonality were involved in the niche shift and genetic diversification of the European/North African clade during the Pleistocene and in its maintenance in a fundamental niche distinct from that of the remaining members of the group. This study further highlights the need for a taxonomic revision of the T. mauritanica species-complex.