Incongruence in molecular species delimitation schemes: What to do when adding more data is difficult

Using multiple, independent approaches to molecular species delimitation is advocated to accommodate limitations and assumptions of a single approach. Incongruence in delimitation schemes is a potential by‐product of employing multiple methods on the same data, and little attention has been paid to its reconciliation. Instead, a particular scheme is prioritized, and/or molecular delimitations are coupled with additional, independent lines of evidence that mitigate incongruence. We advocate that incongruence within a line of evidence should be accounted for before comparing across lines of evidence that can themselves be incongruent. Additionally, it is not uncommon for empiricists working in nonmodel systems to be data‐limited, generating some concern for the adequacy of available data to address the question of interest. With conservation and management decisions often hinging on the status of species, it seems prudent to understand the capabilities of approaches we use given the data we have. Here, we apply two molecular species delimitation approaches, spedeSTEM and BPP, to the Castilleja ambigua (Orobanchaceae) species complex, a relatively young plant lineage in western North America. Upon finding incongruence in our delimitation, we employed a post hoc simulation study to examine the power of these approaches to delimit species. Given the data we collected, we find that spedeSTEM lacks the power to delimit while BPP is capable, thus allowing us to address incongruence before proceeding in delimitation. We suggest post hoc simulation studies like this compliment empirical delimitation and serve as a means of exploring conflict within a line of evidence and dealing with it appropriately.     

An empirical comparison of character‐based and coalescent‐based approaches to species delimitation in a young avian complex

The process of discovering species is a fundamental responsibility of systematics. Recently, there has been a growing interest in coalescent‐based methods of species delimitation aimed at objectively identifying species early in the divergence process. However, few empirical studies have compared these new methods with character‐based approaches for discovering species. In this study, we applied both a character‐based and a coalescent‐based approaches to delimit species in a closely related avian complex, the light‐vented/Taiwan bulbul (Pycnonotus sinensis/Pycnonotus taivanus). Population aggregation analyses of plumage, mitochondrial and 13 nuclear intron character data sets produced conflicting species hypotheses with plumage data suggesting three species, mitochondrial data suggesting two species, and nuclear intron data suggesting one species. Such conflict is expected among recently diverged species, and by integrating all sources of data, we delimited three species verified with independently congruent character evidence as well as a more weakly supported fourth species identified by a single character. Attempts to validate species hypothesis using Bayesian Phylogenetics and Phylogeography (BPP), a coalescent‐based method of species delimitation, revealed several issues that can seemingly affect statistical support for species recognition. We found that θ priors had a dramatic impact on speciation probabilities, with lower values consistently favouring splitting and higher values consistently favouring lumping. More resolved guide trees also resulted in overall higher speciation probabilities. Finally, we found suggestive evidence that BPP is sensitive to the divergent effects of nonrandom mating caused by intraspecific processes such as isolation‐with‐distance, and therefore, BPP may not be a conservative method for delimiting independently evolving population lineages. Based on these concerns, we questioned the reliability of BPP results and based our conclusions about species limits exclusively on character data.     

New Metrics for Comparison of Taxonomies Reveal Striking Discrepancies among Species Delimitation Methods in Madascincus Lizards

Delimiting and describing species is fundamental to numerous biological disciplines such as evolution, macroecology, and conservation. Delimiting species as independent evolutionary lineages may and often does yield different outcomes depending on the species criteria applied, but methods should be chosen that minimize the inference of objectively erroneous species limits. Several protocols exploit single-gene or multi-gene coalescence statistics, assignment tests or other rationales related to nuclear DNA (nDNA) allele sharing to automatically delimit species. We apply seven different species delimitation protocols to a taxonomically confusing group of Malagasy lizards (Madascincus), and compare the resulting taxonomies with two newly developed metrics: the Taxonomic index of congruence C_tax which quantifies the congruence between two taxonomies, and the Relative taxonomic resolving power index R_tax which quantifies the potential of an approach to capture a high number of species boundaries. The protocols differed in the total number of species proposed, between 9 and 34, and were also highly incongruent in placing species boundaries. The Generalized Mixed Yule-Coalescent approach captured the highest number of potential species boundaries but many of these were clearly contradicted by extensive nDNA admixture between sympatric mitochondrial DNA (mtDNA) haplotype lineages. Delimiting species as phenotypically diagnosable mtDNA clades failed to detect two cryptic species that are unambiguous due to a lack of nDNA gene flow despite sympatry. We also consider the high number of species boundaries and their placement by multi-gene Bayesian species delimitation as poorly reliable whereas the Bayesian assignment test approach provided a species delimitation highly congruent with integrative taxonomic practice. The present study illustrates the trade-off in taxonomy between reliability (favored by conservative approaches) and resolving power (favored by inflationist approaches). Quantifying excessive splitting is more difficult than quantifying excessive lumping, suggesting a priority for conservative taxonomies in which errors are more liable to be detected and corrected by subsequent studies.     

Phylogenetic Molecular Species Delimitations Unravel Potential New Species in the Pest Genus Spodoptera Guenée, 1852 (Lepidoptera,Noctuidae)

Nowadays molecular species delimitation methods promote the identification of species boundaries within complex taxonomic groups by adopting innovative species concepts and theories (e.g. branching patterns, coalescence). As some of them can efficiently deal with large single-locus datasets, they could speed up the process of species discovery compared to more time consuming molecular methods, and benefit from the existence of large public datasets; these methods can also particularly favour scientific research and actions dealing with threatened or economically important taxa. In this study we aim to investigate and clarify the status of economically important moths species belonging to the genus Spodoptera (Lepidoptera, Noctuidae), a complex group in which previous phylogenetic analyses and integrative approaches already suggested the possible occurrence of cryptic species and taxonomic ambiguities. In this work, the effectiveness of innovative (and faster) species delimitation approaches to infer putative species boundaries has been successfully tested in Spodoptera, by processing the most comprehensive dataset (in terms of number of species and specimens) ever achieved; results are congruent and reliable, irrespective of the set of parameters and phylogenetic models applied. Our analyses confirm the existence of three potential new species clusters (for S. exigua (Hübner, 1808), S. frugiperda (J.E. Smith, 1797) and S. mauritia (Boisduval, 1833)) and support the synonymy of S. marima (Schaus, 1904) with S. ornithogalli (Guenée, 1852). They also highlight the ambiguity of the status of S. cosmiodes (Walker, 1858) and S. descoinsi Lalanne-Cassou & Silvain, 1994. This case study highlights the interest of molecular species delimitation methods as valuable tools for species discovery and to emphasize taxonomic ambiguities.     

Species delimitation through DNA barcoding of freshwater leeches of the Glossiphonia genus (Hirudinea: Glossiphoniidae) from Eastern Siberia,Russia

The study of biodiversity is a priority task of biological science. The structural unit of biodiversity is a species that has a clear identification in a taxonomic system. Morphological features are traditionally the main criteria for species discrimination in zoological studies. However, the presence of inter- and intraspecific polymorphism and phenotypic plasticity makes it difficult to identify species in many groups of invertebrates. To solve this problem, in this research, we analyzed morphological and genetic data in combination to delimit species among the Eastern Siberia Glossiphonia leeches using different approaches. Morphology analysis revealed phenetically distinct groups, suggesting the existence of at least two species in the region, G. verrucata, a rare Palaearctic species, and a potentially new species Glossiphonia sp. Moreover, sequence-based species delimitation methods congruently supported eight distinct species groups (including two Siberian species) within the available molecular dataset of the Glossiphonia world fauna, using phylogenetic (ML and BI), coalescent (ABGD and GMYC) methods, and pairwise analysis of sequences. The detected p-distances (modal value of 0.11) between these 8 groups and the level of genetic polymorphism (max. 0.0041) within groups indicate that the groups are 8 independent species according to the DNA barcoding. Our results once again proved the usefulness of molecular systematics. At the same time, we detected several inaccuracies in the leech species identification, as well as many ambiguous sites in sequences uploaded on GenBank, which affects the analysis and impedes progress of DNA barcoding technology.     

Use of RAD sequencing for delimiting species

RAD-tag sequencing is a promising method for conducting genome-wide evolutionary studies. However, to date, only a handful of studies empirically tested its applicability above the species level. In this communication, we use RAD tags to contribute to the delimitation of species within a diverse genus of deep-sea octocorals, Chrysogorgia, for which few classical genetic markers have proved informative. Previous studies have hypothesized that single mitochondrial haplotypes can be used to delimit Chrysogorgia species. On the basis of two lanes of Illumina sequencing, we inferred phylogenetic relationships among 12 putative species that were delimited using mitochondrial data, comparing two RAD analysis pipelines (Stacks and PyRAD). The number of homologous RAD loci decreased dramatically with increasing divergence, as >70% of loci are lost when comparing specimens separated by two mutations on the 700-nt long mitochondrial phylogeny. Species delimitation hypotheses based on the mitochondrial mtMutS gene are largely supported, as six out of nine putative species represented by more than one colony were recovered as discrete, well-supported clades. Significant genetic structure (correlating with geography) was detected within one putative species, suggesting that individuals characterized by the same mtMutS haplotype may belong to distinct species. Conversely, three mtMutS haplotypes formed one well-supported clade within which no population structure was detected, also suggesting that intraspecific variation exists at mtMutS in Chrysogorgia. Despite an impressive decrease in the number of homologous loci across clades, RAD data helped us to fine-tune our interpretations of classical mitochondrial markers used in octocoral species delimitation, and discover previously undetected diversity.     

Coalescence-based species delimitation using genome-wide data reveals hidden diversity in a cosmopolitan group of lichens

Although there is an abundance of species delimitation methods on the market, most approaches depend on predefined assignment of specimens to species or populations. Assignment-free methods, which can simultaneously infer boundaries and relationships among species, are of high importance in cases, when correct pre-assignment is difficult or not at all possible. In this study, we use assignment-free multispecies coalescent-based species delimitation (STACEY, tr2-delimitation, and BP&P), phylogenetic methods, and clustering algorithms to investigate the inter- and infraspecific relationships within a common and widespread group of lichens with contentious species boundaries. The Cetraria aculeata group presents a good example of extreme morphological variability and unclear species delimitation in lichens. Based on DNA-sequence data from 26 fungal loci and 10 microsatellite loci, as well as morphological and chemical data, our results provide evidence for the occurrence of five different taxa within the group and highlight the difficulties of morphologically distinguishing these species. We discovered a separate lineage (clade C) within C. aculeata s. str., which does not fully coincide with any of the a priori identified species C. aculeata, C. crespoae, or C. steppae and conclude that this clade constitutes a semi-cryptic, genetically isolated lineage within C. aculeata. We recognize this lineage at subspecific rank as C. aculeata subsp. steppae and synonymize Cetraria crespoae with C. aculeata subsp. aculeata. Epitypes are designated for all involved names to stabilize their usage. The PKS8 gene locus is recommended as a barcode for the separation of C. aculeata subsp. aculeata and subsp. steppae. We demonstrate the potential use of microsatellite data for species delimitation in lichens that might offer an alternative insight or be used to test species delimitation hypotheses, when dealing with closely related or potentially cryptic species. Our results also confirm the presence of an undescribed sister lineage to C. odontella previously misidentified as C. muricata and extend the known range of this lineage to Central Asia (Altay Mts.) and the Central European Alps (France, Switzerland), which calls for a critical reappraisal of records of C. aculeata and C. muricata from these mountain ranges.

     

Lycoperdon rupicola and L. subumbrinum: two new puffballs from Europe

Lycoperdon rupicola and L. subumbrinum are here described as new species. The former is a morphologically characteristic species forming small subglobose to pyriforme fruiting bodies with a prominent, protruding and stellately lobed stoma. In the phylogenetic analyses the six sequences of L. rupicola form a clade with 100% BS and 1.0 BPP, clustering with L. dermoxanthum. The proposed species grows among mosses on siliceous boulders and flat rocks as well as on open, sandy soil in temperate and hemiboreal regions. The records to date indicate its presence in Fennoscandia and Spain (Catalunya). L. subumbrinum morphologically resembles L. umbrinum but differs in having coarser ornamentation of its spores. In the phylogenetic analyses the five sequences of L. subumbrinum form a clade with 100% BS and 1.0 BPP, clustering with L. muscorum. The records to date, originating from southernmost Sweden and NW Slovakia, indicate that L. subumbrinum is a species of rich deciduous forests. L. muscorum is recorded for the first time from the Iberian peninsula (Spain).     

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.     

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.     

Species delimitation with abc and other coalescent-based methods: a test of accuracy with simulations and an empirical example with lizards of the liolaemus darwinii complex (squamata: liolaemidae)

Species delimitation is a major research focus in evolutionary biology because accurate species boundaries are a prerequisite for the study of speciation. New species delimitation methods (SDMs) can accommodate nonmonophyletic species and gene tree discordance as a result of incomplete lineage sorting via the coalescent model, but do not explicitly accommodate gene flow after divergence. Approximate Bayesian computation (ABC) can incorporate gene flow and estimate other relevant parameters of the speciation process while testing alternative species delimitation hypotheses. We evaluated the accuracy of BPP, SpeDeSTEM, and ABC for delimiting species using simulated data and applied these methods to empirical data from lizards of the Liolaemus darwinii complex. Overall, BPP was the most accurate, ABC showed an intermediate accuracy, and SpeDeSTEM was the least accurate under most simulated conditions. All three SDMs showed lower accuracy when speciation occurred despite gene flow, as found in previous studies, but ABC was the method with the smallest decrease in accuracy. All three SDMs consistently supported the distinctness of southern and northern lineages within L. darwinii. These SDMs based on genetic data should be complemented with novel SDMs based on morphological and ecological data to achieve truly integrative and statistically robust approaches to species discovery.     

Integrative Taxonomic Approach for Describing a New Cryptic Species of Bush Frog (Raorchestes: Anura: Rhacophoridae) from the Western Ghats,India

A new cryptic species of bush frog Raorchestes honnametti sp. nov. is described from the south-eastern part of the Western Ghats, India. This newly described species belongs to the Charius clade and is morphologically similar to other clade members—R. charius and R. griet. Therefore, an integrative taxonomic approach based on molecular and bioacoustic analysis along with morphology was used to delimit the new species. Raorchestes honnametti sp. nov., is currently known only from Biligiri Rangaswamy Temple Tiger Reserve, a part of Biligiri Rangaswamy horst mountain range (a mountain formed due movement of two faults) formed during the Late Quaternary period (1.8–2.58 Ma). Discovery of cryptic species from a highly speciose and well-studied genus Raorchestes hints at the possible existence of several more cryptic species in this genus. We discuss the possible reasons for crypsis and emphasize the need for continued systematic surveys of amphibians across the Western Ghats.     

Species delimitation and phylogeography of Aphonopelma hentzi (Araneae, Mygalomorphae, Theraphosidae): cryptic diversity in North American tarantulas

Background

The primary objective of this study is to reconstruct the phylogeny of the hentzi species group and sister species in the North American tarantula genus, Aphonopelma, using a set of mitochondrial DNA markers that include the animal “barcoding gene”. An mtDNA genealogy is used to consider questions regarding species boundary delimitation and to evaluate timing of divergence to infer historical biogeographic events that played a role in shaping the present-day diversity and distribution. We aimed to identify potential refugial locations, directionality of range expansion, and test whether A. hentzi post-glacial expansion fit a predicted time frame.

Methods and Findings

A Bayesian phylogenetic approach was used to analyze a 2051 base pair (bp) mtDNA data matrix comprising aligned fragments of the gene regions CO1 (1165 bp) and ND1-16S (886 bp). Multiple species delimitation techniques (DNA tree-based methods, a “barcode gap” using percent of pairwise sequence divergence (uncorrected p-distances), and the GMYC method) consistently recognized a number of divergent and genealogically exclusive groups.

Conclusions

The use of numerous species delimitation methods, in concert, provide an effective approach to dissecting species boundaries in this spider group; as well they seem to provide strong evidence for a number of nominal, previously undiscovered, and cryptic species. Our data also indicate that Pleistocene habitat fragmentation and subsequent range expansion events may have shaped contemporary phylogeographic patterns of Aphonopelma diversity in the southwestern United States, particularly for the A. hentzi species group. These findings indicate that future species delimitation approaches need to be analyzed in context of a number of factors, such as the sampling distribution, loci used, biogeographic history, breadth of morphological variation, ecological factors, and behavioral data, to make truly integrative decisions about what constitutes an evolutionary lineage recognized as a “species”.     

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

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

Integration of conflict into integrative taxonomy: fitting hybridization in species delimitation of Mesocarabus (Coleoptera: Carabidae)

In species differentiation, characters may not diverge synchronously, and there are also processes that shuffle character states in lineages descendant from a common ancestor. Species are thus expected to show some degree of incongruence among characters; therefore, taxonomic delimitation can benefit from integrative approaches and objective strategies that account for character conflict. We illustrate the potential of exploiting conflict for species delimitation in a study case of ground beetles of the subgenus Carabus (Mesocarabus), where traditional taxonomy does not accurately delimit species. The molecular phylogenies of four mitochondrial and three nuclear genes, cladistic analysis of the aedeagus, ecological niche divergence and morphometry of pronotal shape in more than 500 specimens of Mesocarabus show that these characters are not fully congruent. For these data, a three‐step operational strategy is proposed for species delimitation by (i) delineating candidate species based on the integration of incongruence among conclusive lines of evidence, (ii) corroborating candidate species with inconclusive lines of evidence and (iii) refining a final species proposal based on an integrated characterization of candidate species based on the evolutionary analysis of incongruence. This procedure provided a general understanding of the reticulate process of hybridization and introgression acting on Mesocarabus and generated the hypothesis of seven Mesocarabus species, including two putative hybrid lineages. Our work emphasizes the importance of incorporating critical analyses of character and phylogenetic conflict to infer both the evolutionary history and species boundaries through an integrative taxonomic approach.     

Examination of prezygotic and postzygotic isolating barriers in tropical Ulva (Ulvophyceae,Chlorophyta): evidence for ongoing speciation

Phylogenetic clades based on DNA sequences such as the chloroplast rbcL gene and the nuclear ITS region are frequently used to delimit algal species. However, these molecular markers cannot accurately delimit boundaries among some Ulva species. Although Ulva reticulata and Ulva ohnoi occasionally bloom in tropical to warm‐temperate regions and are clearly distinguishable by their reticulate or plain blade morphology, they have few or no sequence divergences in these molecular markers and form a monophyletic clade. In this study, to clarify the speciation and species delimitation in the U. reticulata‐ohnoi complex clade, reproductive relationships among several sexual strains from the Philippines and Japan including offspring that originated from the type specimen of U. ohnoi were examined by culturing and hybridization in addition to the ITS‐based analysis. As a result, both prezygotic and postzygotic reproductive isolation were revealed to occur between genetically perforated U. reticulata and imperforate U. ohnoi. They were also separated on the basis of sequence analysis of the ITS region. That strongly supports that the two taxa are independent biological species. Although no prezygotic barrier among the Philippine and Japanese strains of U. reticulata was observed, unexpectedly zoospores produced by hybrid sporophytes in some of their combinations mostly failed to develop, indicating partial formation of a postzygotic barrier despite a 0.2% divergence in the ITS sequence. These findings suggest speciation is still ongoing in U. reticulata.