Applications of ecological niche modeling for species delimitation: a review and empirical evaluation using day geckos (Phelsuma) from Madagascar

Although the systematic utility of ecological niche modeling is generally well known (e.g., concerning the recognition and discovery of areas of endemism for biogeographic analyses), there has been little discussion of applications concerning species delimitation, and to date, no empirical evaluation has been conducted. However, ecological niche modeling can provide compelling evidence for allopatry between populations, and can also detect divergent ecological niches between candidate species. Here we present results for two taxonomically problematic groups of Phelsuma day geckos from Madagascar, where we integrate ecological niche modeling with mitochondrial DNA and morphological data to evaluate species limits. Despite relatively modest levels of genetic and morphological divergence, for both species groups we find divergent ecological niches between closely related species and parapatric ecological niche models. Niche models based on the new species limits provide a better fit to the known distribution than models based upon the combined (lumped) species limits. Based on these results, we elevate three subspecies of Phelsuma madagascariensis to species rank and describe a new species of Phelsuma from the P. dubia species group. Our phylogeny continues to support a major endemic radiation of Phelsuma in Madagascar, with dispersals to Pemba Island and the Mascarene Islands. We conclude that ecological niche modeling offers great potential for species delimitation, especially for taxonomic groups exhibiting low vagility and localized endemism and for groups with more poorly known distributions. In particular, niche modeling should be especially sensitive for detecting recent parapatric speciation driven by ecological divergence, when the environmental gradients driving speciation are represented within the ecological niche models.     

Population structure and species boundary delimitation of cryptic Dioryctria moths: an integrative approach

Accurate delimitation of species boundaries is especially important in cryptic taxa where one or more character sources are uninformative or are in conflict. Rather than relying on a single marker to delimit species, integrative taxonomy uses multiple lines of evidence such as molecular, morphological, behavioural and geographic characters to test species limits. We examine the effectiveness of this approach by testing the delimitation of two cryptic Nearctic species of Dioryctria (Lepidoptera: Pyralidae) using three independent molecular markers [cytochrome c oxidase I (COI), second internal transcribed spacer unit (ITS2), and elongation factor 1alpha (EF1alpha)], forewing variation and larval host plant association. Although mitochondrial DNA (mtDNA) haplotypes do not form reciprocally monophyletic clades, restricted gene flow between COI haplotype groups, and concordance with ITS2 genotypes, forewing variation and host plant associations support delimitation of two Nearctic species: eastern Dioryctria reniculelloides and western Dioryctria pseudotsugella. Conversely, EF1alpha genotype variation was incongruent with the two previous markers. A case of discordance between COI and ITS2 was detected, suggesting either introgression due to hybridization or retained ancestral polymorphism due to incomplete coalescence. This study is consistent with other similar literature where molecular loci in closely related species progress from shared to fixed haplotypes/alleles, and from polyphyletic to reciprocally monophyletic relationships, although loci may vary in these characteristics despite maintenance of genomic integrity between distinct species. In particular, mtDNA in other studies generally showed a lower rate of fixation of differences than did X-linked or autosomal loci, reinforcing the need to use an integrative approach for delimiting species.     

Integrative taxonomy and preliminary assessment of species limits in the Liolaemus walkeri complex (Squamata,Liolaemidae) with descriptions of?three?new species from Peru

Species delimitation studies based on integrative taxonomic approaches have received considerable attention in the last few years, and have provided the strongest hypotheses of species boundaries. We used three lines of evidence (molecular, morphological, and niche envelopes) to test for species boundaries in Peruvian populations of the Liolaemus walkeri complex. Our results show that different lines of evidence and analyses are congruent in different combinations, for unambiguous delimitation of three lineages that were “hidden” within known species, and now deserve species status. Our phylogenetic analysis shows that L. walkeri, L. tacnae and the three new species are strongly separated from other species assigned to the alticolor-bibronii group. Few conventional morphological characters distinguish the new species from closely related taxa and this highlights the need to integrate other sources of data to erect strong hypothesis of species limits. A taxonomic key for known Peruvian species of the subgenus Lioalemus is provided.     

A comparison of DNA‐based methods for delimiting species in a Cretan land snail radiation reveals shortcomings of exclusively molecular taxonomy

We compared the results of different approaches for delimiting species based on single‐locus DNA sequences with those of methods using binary multilocus data. As case study, we examined the radiation of the land snail genus Xerocrassa on Crete. Many of the methods based on mitochondrial sequences resulted in heavy under‐ or overestimations of the species number. The methods using AFLP data produced classifications with an on average higher concordance with the morphological classification than the methods based on mitochondrial sequences. However, the percentage of correct species classifications is low even with binary multilocus data. Gaussian clustering produced the classifications with the highest concordance with the morphological classification of all approaches applied in this study, both with single‐locus sequences and with binary multilocus data. There are two general problems that hamper species delimitation, namely rarity and the hierarchical structure of biodiversity. Methods for species delimitation using genetic data search for clusters of individuals, but do not implement criteria that are sufficient to distinguish clusters representing species from other clusters. The success of morphological species delimitation results from the potential to focus on characters that are directly involved in the speciation process, whereas molecular studies usually rely on markers that are not directly involved in speciation. © The Willi Hennig Society 2011.     

Nuclear markers confirm taxonomic status and relationships among highly endangered and closely related right whale species

Right whales (genus: Eubalaena) are among the most endangered mammals, yet their taxonomy and phylogeny have been questioned. A phylogenetic hypothesis based on mitochondrial DNA (mtDNA) variation recently prompted a taxonomic revision, increasing the number of right whale species to three. We critically evaluated this hypothesis using sequence data from 13 nuclear DNA (nuDNA) loci as well as the mtDNA control region. Fixed diagnostic characters among the nuclear markers strongly support the hypothesis of three genetically distinct species, despite lack of any diagnostic morphological characters. A phylogenetics analysis of all data produced a strict consensus cladogram with strong support at nodes that define each right whale species as well as relationships among species. Results showed very little conflict among the individual partitions as well as congruence between the mtDNA and nuDNA datasets. These data clearly demonstrate the strength of using numerous independent genetic markers during a phylogenetics analysis of closely related species. In evaluating phylogenetic support contributed by individual loci, 11 of the 14 loci provided support for at least one of the nodes of interest to this study. Only a single marker (mtDNA control region) provided support at all four nodes. A study using any single nuclear marker would have failed to support the proposed phylogeny, and a strong phylogenetic hypothesis was only revealed by the simultaneous analysis of many nuclear loci. In addition, nu DNA and mtDNA data provided complementary levels of support at nodes of different evolutionary depth indicating that the combined use of mtDNA and nuDNA data is both practical and desirable.     

Integrative taxonomy of the primitively segmented spider genus Ganthela (Araneae: Mesothelae: Liphistiidae): DNA barcoding gap agrees with morphology

Species delimitation is difficult for taxa in which the morphological characters are poorly known because of the rarity of adult morphs or sexes, and in cryptic species. In primitively segmented spiders, family Liphistiidae, males are often unknown, and female genital morphology – usually species‐specific in spiders – exhibits considerable intraspecific variation. Here, we report on an integrative taxonomic study of the liphistiid genus Ganthela Xu & Kuntner, 2015, endemic to south‐east China, where males are only available for two of the seven morphological species (two known and five undescribed). We obtained DNA barcodes (cytochrome c oxidase subunit I gene, COI) for 51 newly collected specimens of six morphological species and analysed them using five species‐delimitation methods: DNA barcoding gap, species delimitation plugin [P ID(Liberal)], automatic barcode gap discovery (ABGD), generalized mixed Yule‐coalescent model (GMYC), and statistical parsimony (SP). Whereas the first three agreed with the morphology, GMYC and SP indicate several additional species. We used the consensus results to delimit and diagnose six Ganthela species, which in addition to the type species Ganthela yundingensis Xu, 2015, completes the revision of the genus. Although multi‐locus phylogenetic approaches may be needed for complex taxonomic delimitations, our results indicate that even single‐locus analyses based on the COI barcodes, if integrated with morphological and geographical data, may provide sufficiently reliable species delimitation. © 2015 The Linnean Society of London     

Evaluating multilocus Bayesian species delimitation for discovery of cryptic mycorrhizal diversity

The increasing availability of DNA sequence data enables exciting new opportunities for fungal ecology. However, it amplifies the challenge of how to objectively classify the diversity of fungal sequences into meaningful units, often in the absence of morphological characters. Here, we test the utility of modern multilocus Bayesian coalescent-based methods for delimiting cryptic fungal diversity in the orchid mycorrhiza morphospecies Serendipita vermifera. We obtained 147 fungal isolates from Caladenia, a speciose clade of Australian orchids known to associate with Serendipita fungi. DNA sequence data for 7 nuclear and mtDNA loci were used to erect competing species hypotheses by clustering isolates based on: (a) ITS sequence divergence, (b) Bayesian admixture analysis, and (c) mtDNA variation. We implemented two coalescent-based Bayesian methods to determine which species hypothesis best fitted our data. Both methods found strong support for eight species of Serendipita among our isolates, supporting species boundaries reflected in ITS divergence. Patterns of host plant association showed evidence for both generalist and specialist associations within the host genus Caladenia. Our findings demonstrate the utility of Bayesian species delimitation methods and suggest that wider application of these techniques will readily uncover new species in other cryptic fungal lineages.     

Ancient drainages divide cryptic species in Australia's arid zone: morphological and multi-gene evidence for four new species of Beaked Geckos (Rhynchoedura)

Deserts and other arid zones remain among the least studied biomes on Earth. Emerging genetic patterns of arid-distributed biota suggest a strong link between diversification history and both the onset of aridification and more recent cycles of severe aridification. A previous study based on 1 kb of mtDNA of the monotypic gecko genus Rhynchoedura identified five allopatric clades across the vast Australian arid zone. We supplemented this data with 2.2kb from three nuclear loci and additional mtDNA sequences. Phylogenetic relationships estimated from the mtDNA data with ML and Bayesian methods were largely concordant with relationships estimated with the nDNA data only, and mtDNA and nDNA data combined. These analyses, and coalescent-based species-tree inference methods implemented with (?)BEAST, largely resolve the relationships among them. We also carried out an examination of 19 morphological characters for 268 museum specimens from across Australia, including all 197 animals for which we sequenced mtDNA. The mtDNA clades differ subtly in a number of morphological features, and we describe three of them as new species, raise a fourth from synonymy, and redescribe it and the type species, Rhynchoedura ornata. We also describe a morphologically distinctive new species from Queensland based on very few specimens. The distribution of arid zone clades across what is now relatively homogeneous sand deserts seems to be related to a topographic divide between the western uplands and eastern lowlands, with species' distributions correlated with dryland rivers and major drainage divides. The existence of five cryptic species within the formerly monotypic Rhynchoedura points to ancient divergences within the arid zone that likely were driven by wet phases as well as dry ones.     

Testing species delimitation in sympatric species complexes: the case of an African tropical tree, Carapa spp. (Meliaceae)

Plant species delimitation within tropical ecosystems is often difficult because of the lack of diagnostic morphological characters that are clearly visible. The development of an integrated approach, which utilizes several different types of markers (both morphological and molecular), would be extremely useful in this context. Here we have addressed species delimitation of sympatric tropical tree species that belong to Carapa spp. (Meliaceae) in Central Africa. We adopted a population genetics approach, sampling numerous individuals from three locations where sympatric Carapa species are known to exist. Comparisons between morphological markers (the presence or absence of characters, leaf-shape traits) and molecular markers (chloroplast sequences, ribosomal internal transcribed spacer region (ITS) sequences, and nuclear microsatellites) demonstrated the following: (i) a strong correlation between morphological and nuclear markers; (ii) despite substantial polymorphism, the inability of chloroplast DNA to discriminate between species, suggesting that cytoplasmic markers represent ineffective DNA barcodes; (iii) lineage sorting effects when using ITS sequences; and (iv) a complex evolutionary history within the genus Carapa, which includes frequent inter-specific gene flow. Our results support the use of a population genetics approach, based on ultra-polymorphic markers, to address species delimitation within complex taxonomic groups.     

Species delimitation: inferring gaps in morphology across geography

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

Species delimitation and digit number in a North African skink

Delimitation of species is an important and controversial area within evolutionary biology. Many species boundaries have been defined using morphological data. New genetic approaches now offer more objective evaluation and assessment of the reliability of morphological variation as an indicator that speciation has occurred. We examined geographic variation in morphology of the continuously distributed skink Chalcides mionecton from Morocco and used Bayesian analyses of nuclear and mitochondrial DNA (mtDNA) loci to examine: (i) their concordance with morphological patterns, (ii) support for species delimitation, (iii) timing of speciation, and (iv) levels of gene flow between species. Four digit individuals were found at sites between Cap Rhir (in the south) and the northern extreme of the range, whereas five‐digit individuals were found in two disjunct areas: (i) south of Cap Rhir and (ii) the north of the range where they were often syntopic with four‐digit individuals. The pattern of variation in generalized body dimensions was largely concordant with that in digit number, suggesting two general morphotypes. Bayesian analyses of population structure showed that individuals from sites south of Cap Rhir formed one genetic cluster, but that northern four‐ and five‐digit individuals clustered together. Statistical support for delimitation of these genetic clusters into two species was provided by a recent Bayesian method. Phylogenetic–coalescent dating with external time calibrations indicates that speciation was relatively recent, with a 95% posterior interval of 0.46–2.66 mya. This postdates equivalent phylogenetic dating estimates of sequence divergence by approximately 1 Ma. Statistical analyses of a small number of independent loci provide important insights into the history of the speciation process in C. mionecton and support delimitation of populations into two species with distributions that are spatially discordant with patterns of morphological variation.     

Bayesian species delimitation in West African forest geckos (Hemidactylus fasciatus)

Genealogical data are an important source of evidence for delimiting species, yet few statistical methods are available for calculating the probabilities associated with different species delimitations. Bayesian species delimitation uses reversible-jump Markov chain Monte Carlo (rjMCMC) in conjunction with a user-specified guide tree to estimate the posterior distribution for species delimitation models containing different numbers of species. We apply Bayesian species delimitation to investigate the speciation history of forest geckos (Hemidactylus fasciatus) from tropical West Africa using five nuclear loci (and mtDNA) for 51 specimens representing 10 populations. We find that species diversity in H. fasciatus is currently underestimated, and describe three new species to reflect the most conservative estimate for the number of species in this complex. We examine the impact of the guide tree, and the prior distributions on ancestral population sizes (θ) and root age (τ₀), on the posterior probabilities for species delimitation. Mis-specification of the guide tree or the prior distribution for θ can result in strong support for models containing more species. We describe a new statistic for summarizing the posterior distribution of species delimitation models, called speciation probabilities, which summarize the posterior support for each speciation event on the starting guide tree.     

Phylogeny and species reassessment of Hyalopterus (Aphididae,Aphidinae)

The broadly distributed genus Hyalopterus currently comprises three formally recognized species that are highly similar morphologically and hence difficult to be identified with certainty. This group has undergone multiple revisions in the past century, but none of these has assessed species from Asia, which has hampered our understanding of the species diversity within this genus. Based on a comprehensive data set from morphological data and host-associated data, and by coalescent-based delimitation approaches, the Hyalopterus species boundaries, distribution and diversity were clarified here to further reveal the composition of the species. Two single-locus (ML-GMYC and mPTP) and two multilocus (BPP and STACEY) delimitation methods were conducted based on extensive sampling. Then, the phylogenetic relationships and morphological divergence were assessed. Our data strongly supported that the number of recognized species in Hyalopterus had likely been underestimated. The phylogenetic analyses recovered four major clades, which corresponded to distinct host-plant preferences. Also, the morphological analyses showed significant differentiation for only one of the newly recognized candidate species uncovered by the delimitation approaches, suggesting the existence of at least two independent evolutionary lineages within Hyalopterus arundiniformis, which showed different patterns of host association. Moreover, based on our data, the taxonomic misidentification of H. arundiniformis in China was corrected here. This study lays the groundwork for the thorough taxonomic revision of Hyalopterus and for future evolutionary studies and underlines the importance of an integrated framework for species determination.     

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

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

Nuclear simple sequence repeat markers are superior to DNA barcodes for identification of closely related Rhododendron species on the same mountain

Accurate species delimitation of sampled biological material is critical for a range of studies. Although the DNA barcodes developed in recent years are useful for identifying numerous well differentiated species that have not experienced frequent gene flow, they fail to delimit recently diverged species, especially those with extensive introgressions. Here we use five Rhododendron species growing together on the same mountain as a model system to compare the species delimitation effectiveness of the DNA barcodes (internal transcribed spacer, matK, psbA‐trnH, and rbcL) previously proposed versus 15 pairs of microsatellite markers. Using these markers, we genotyped 129 individuals, which were members of five species according to morphological identification. We identified five simple sequence repeat genetic clusters (independently evolving lineages) corresponding to the morphological identification. However, we found that numerous individuals contained cryptic hybrid introgressions from the other species. The four DNA barcodes could not delimit three out of four closely related species that showed clear morphological differentiation and cryptic introgressions. Even after excluding all cryptic hybrids, two closely related species could not be successfully identified. The low discrimination ability of the DNA barcodes for closely related Rhododendron species could result from two, not mutually exclusive factors: introgressive hybridization and incomplete lineage sorting. Our results highlight the importance of simple sequence repeat markers in delimiting closely related species and identifying cryptic introgressions in the absence of morphological changes.     

Character- and tree-based delimitation of species in the 'Cichlasoma' facetum group (Teleostei, Cichlidae) with the description of a new genus

The 'Cichlasoma' facetum group is part of the taxonomically complex group of Neotropical cichlid fishes of the tribe Heroini. Many species groups and unplaced species of heroines are still left without a generic name following the revision of the genus Cichlasoma . We describe here the 'Cichlasoma' facetum group as a new genus, Australoheros , and provide evidence for its monophyly based on phylogenetic analyses of morphological and mtDNA characters. Australoheros is morphologically characterized by the lowest values in meristic characters among heroines and by three apomorphic characters in coloration pattern. In addition to the three described species of Australoheros , our results of species delimitation based on a combination of tree- and character-based approaches identify seven putatively new species of Australoheros . Several coding schemes of morphological characters are used to recover the intrageneric relationships within the genus, resulting in very similar topologies. Discovery of additional species within the genus is expected once material from the whole distribution area is studied.     

Multi-locus species delimitation in closely related animals and fungi: one marker is not enough

Despite taxonomy’s 250‐year history, the past 20 years have borne witness to remarkable advances in technology and techniques, as well as debate. DNA barcoding has generated a substantial proportion of this debate, with its proposition that a single mitochondrial sequence will consistently identify and delimit species, replacing more evidence‐rich and time‐intensive methods. Although mitochondrial DNA (mtDNA) has since been the focus of voluminous discussion and case studies, little effort has been made to comprehensively evaluate its success in delimiting closely related species. We have conducted the first broadly comparative literature review addressing the efficacy of molecular markers for delimiting such species over a broad taxonomic range. By considering only closely related species, we sought to avoid confusion of success rates with those due to deeply divergent taxa. We also address whether increased population‐level or geographic sampling affects delimitation success. Based on the results from 101 studies, we found that all marker groups had approximately equal success rates (～70%) in delimiting closely related species and that the use of additional loci increased average delimitation success. We also found no relationship between increased sampling of intraspecific variability and delimitation success. Ultimately, our results support a multi‐locus integrative approach to species delimitation and taxonomy.