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

<p>Molecular techniques have enhanced our ability to unravel the evolutionary history and hidden diversity of species, and to explain how historical events have helped to shape the demography and dispersal of populations. Cryptic species are typically defined as two or more genetically distinct species that are morphologically indistinguishable. The discovery of cryptic diversity has become a challenge for biologists in understanding the species concepts and biodiversity patterns. Many current studies have revealed the existence of cryptic species, but few studies have focused on their ecological and biological aspects. Bostrychia, a filamentous red alga, has long been used as a model system for studies of evolutionary process and biogeographic history. In the Southern Hemisphere, there are four endemic species: B. arbuscula, B. gracilis, B. intricata and B. vaga. Bostrychia intricata is widely distributed in the Southern Hemisphere, whereas the other three species have more restricted distributions. The aim of this study was to reveal the evolutionary history, phylogeographic pattern and eco-physiological trait within B. intricata. Phylogenetic analysis based on combined data (mitochondrial COI, chloroplast rbcL and nuclear 28S) strongly supported the monophyly of the four Southern Hemisphere Bostrychia species, with B. vaga as a sister species to the other three. Multigene phylogeny and COI-based species delimitation revealed cryptic species diversity within B. intricata and B. vaga. Additionally, a COI-based phylogeographic study indicated the existence of three cryptic B. intricata species (N2, N4 and N5) in New Zealand. Population analyses demonstrated that cryptic species N2 populations recently expanded, possibly after the Last Glacial Maximum (LGM), while N4 was more diverse, showing a stable population, which possibly persisted during the LGM. The results suggested that the contrasting pattern in population structure and demographic histories between cryptic species was probably due to the difference in the evolutionary history and survival ability. Growth experiments clearly showed that cryptic species N4 had a significantly higher specific growth rate than other two species, N2 and N5, in different salinities and temperature, suggesting physiologically differentiation between these cryptic species. Additionally, the small-scale distribution of B. intricata at Moa Point, Wellington of three cryptic species showed that N4 was found at the higher tidal position than species N2 and N5. Cryptic species N2 occurred in more wave-exposed areas than other two species. These results suggest spatial niche differences between cryptic species, possibly allowing them to sympatrically co-exist. This study highlights the fact that cryptic species are distinctly different in many biological characteristics, while maintaining \identical morphologies.</p>

<p>Molecular techniques have enhanced our ability to unravel the evolutionary history and hidden diversity of species, and to explain how historical events have helped to shape the demography and dispersal of populations. Cryptic species are typically defined as two or more genetically distinct species that are morphologically indistinguishable. The discovery of cryptic diversity has become a challenge for biologists in understanding the species concepts and biodiversity patterns. Many current studies have revealed the existence of cryptic species, but few studies have focused on their ecological and biological aspects. Bostrychia, a filamentous red alga, has long been used as a model system for studies of evolutionary process and biogeographic history. In the Southern Hemisphere, there are four endemic species: B. arbuscula, B. gracilis, B. intricata and B. vaga. Bostrychia intricata is widely distributed in the Southern Hemisphere, whereas the other three species have more restricted distributions. The aim of this study was to reveal the evolutionary history, phylogeographic pattern and eco-physiological trait within B. intricata. Phylogenetic analysis based on combined data (mitochondrial COI, chloroplast rbcL and nuclear 28S) strongly supported the monophyly of the four Southern Hemisphere Bostrychia species, with B. vaga as a sister species to the other three. Multigene phylogeny and COI-based species delimitation revealed cryptic species diversity within B. intricata and B. vaga. Additionally, a COI-based phylogeographic study indicated the existence of three cryptic B. intricata species (N2, N4 and N5) in New Zealand. Population analyses demonstrated that cryptic species N2 populations recently expanded, possibly after the Last Glacial Maximum (LGM), while N4 was more diverse, showing a stable population, which possibly persisted during the LGM. The results suggested that the contrasting pattern in population structure and demographic histories between cryptic species was probably due to the difference in the evolutionary history and survival ability. Growth experiments clearly showed that cryptic species N4 had a significantly higher specific growth rate than other two species, N2 and N5, in different salinities and temperature, suggesting physiologically differentiation between these cryptic species. Additionally, the small-scale distribution of B. intricata at Moa Point, Wellington of three cryptic species showed that N4 was found at the higher tidal position than species N2 and N5. Cryptic species N2 occurred in more wave-exposed areas than other two species. These results suggest spatial niche differences between cryptic species, possibly allowing them to sympatrically co-exist. This study highlights the fact that cryptic species are distinctly different in many biological characteristics, while maintaining \identical morphologies.</p>

Species Delimitation: Discrete Names in a Continuous World with Fuzzy Boundaries

The taxonomy of Aspergillus species has recently been revolutionized with the introduction of cryptic species and section concepts. However, their species-level identification in routine laboratories remains a challenge. The aim of this study was to prospectively assess the identification accuracy of cryptic species of Aspergillus in various laboratories using the mass spectrometry identification (MSI) platform, an independent and freely accessible online mass spectrometry database. Over a 12-month period, when a select set of MSI users identified cryptic species, they were contacted and requested to send the isolates to our laboratory for sequence-based identification. Sequence and MSI identification results were then compared. During the study period, 5108 Aspergillus isolates were identified using MSI including 1477 (28.9%) cryptic species. A total of 245 isolates that corresponded to 56 cryptic species and 13 sections were randomly selected for DNA sequencing confirmation. Agreement between the two methods was 99.6% at the section level and 66.1% at the species level. However, almost all discrepancies (72/83, 86.7%) were misidentifications between closely related cryptic species belonging to the same section. Fifty-one isolates from noncryptic species were also identified, thus yielding 100% and 92.2% agreement at the section and species level, respectively. Although the MSI fungus database is a reliable tool to identify Aspergillus at the section level, the database still requires adjustment to correctly identify rare or cryptic species at the species level. Nevertheless, the application properly differentiated between cryptic and sensu stricto species in the same section, thus alerting on possible specific isolate characteristics.

Cryptic bryophyte species exhibit a decoupling in the degree of morphological and molecular divergence, as a result of different processes, from recent divergence to stasis. Here a body of cryptic species literature comprising 110 papers published between 2000 and end 2018 is reviewed. Most studies of cryptic species focused on northern hemispheric taxa, but we do not yet have sufficient studies to assess whether a geographic bias in the distribution of cryptic species exists, and we don’t know how many cryptic bryophyte species there might be globally. Fully two-thirds of all studies on cryptic bryophyte species rested their claims of morphological crypsis on previous taxonomic investigations, without revision of morphology to confirm cryptic species status. There is more than one kind of morphological crypsis, and while quantification of morphological patterns can contribute to our understanding of crypsis this is a widely neglected component. The usage of ‘cryptic species’ as an etymological tool to flag instances where traditional species concepts are deficient devalues the term, and a distinction between genuine crypsis and business as usual revision of species circumscription should be re-established and maintained. Hybridisation is possibly an under-appreciated contributor to cryptic species, but inference of hybridization has been limited by study design. Opportunities exist in the application of geometric morphometric methods and next generation sequencing technologies to overcome intrinsic limitations in traditional morphological and molecular data sources.

Accurate species identification and assessment of species diversity are essential for studies on phylogeny and phylogeography, adaptation and ecological function. The development of molecular methods triggered the discovery of cryptic species, i.e., genetically distinct lineages in morphologically undifferentiated species. Collembola (Arthropoda, Hexapoda) are one of the most numerous soil-living animals occurring in virtually all terrestrial ecosystems and habitats. Species delimitation is particularly difficult in Collembola due to considerable morphological conservatism, and many Collembola species comprise high genetic divergence and high cryptic species diversity. DNA-based methods provide useful tools for species delimitation, phylogenetic reconstruction, and lineage divergence time estimation. By analyzing two mitochondrial and two nuclear genes from three morphospecies of the European Lepidocyrtus lanuginosus species group (Collembola: Entomobryidae) from different geographic regions of Europe, this thesis focuses on exploring cryptic species / lineages diversity, their phylogeny, and the effects of historical geographic and climatic changes on the divergence and distribution of this species group in Europe.
\nIn chapter II, I investigated phylogenetic relationships and genetic distances between populations of the morphospecies L. lanuginosus (Gmelin, 1788) from three different habitats in Central Europe, i.e. arable fields, grasslands, and forests replicated at six locations. Geographic distances between sampling locations were considerably larger than between habitat types. All four genes clearly separated the morphospecies L. lanuginosus into three major genetic lineages, with one of these lineages being close to Lepidocyrtus cyaneus Tullberg, 1871. The three lineages were genetically as distant to each other as well separated species. Selective colonization of the three habitats by these lineages indicate that they are sorted by habitats: one lineage was common and occurred in each of the three habitat types but preferentially in arable land; the second was restricted to forest; the third, although rare, preferentially occurred in grassland. The results indicate that genetic markers are a reliable and fast method to detect cryptic species, which may facilitate taxonomic research on Collembola species and the identification of possible species-specific morphological characters.
\nIn Chapter III, I delimited species boundaries of the L. lanuginosus species group sampled across Central and Southern Europe (north and south of the Alps) by utilizing three DNA-based methods, ABGD, PTP, and BPP. Species diversity delimited by morphology was compared with that delimited by genes. Three methods based on mitochondrial COI and COII congruently identified ten and nine distinct genetic lineages in the morphospecies L. cyaneus and L. lanuginosus, respectively. ABGD delimitated species barcoding gaps with K2P distances of 0.055–0.095 and 0.06–0.115 for COI and COII, respectively, within the species group. EF1-α separated 89% of these lineages, showing a higher resolution than 28S rDNA D1–2 in distinguishing closely related genetic lineages of the species group. The phylogenetic analysis based on the four genes showed that both morphospecies L. cyaneus and L. lanuginosus are polyphyletic, suggesting that body color is insufficient for delimiting morphospecies and lineages in this Collembola species group. This study challenges the current morphology-based species delimitation in the L. lanuginosus species group and suggests that molecular approaches are needed for accurate determination of Collembola species in both taxonomic and ecological studies. Overall, the results suggest that wide geographic sampling combined with molecular phylogenetic approaches is necessary to delimit species, understand the full range of cryptic diversity, and analyze phylogenetic relationships in Collembola.
\nIn Chapter IV, I studied the phylogeography of 18 lineages of the L. lanuginosus species group using a multi-locus molecular approach. The genetic diversity and population structure of all lineages were analyzed using COII, while all four above-mentioned genes were concatenated to infer the phylogeographic origin of these lineages. Results showed that the 18 lineages did not overlap in their distribution ranges in Central, Southern, and Southeastern Europe, suggesting high genetic structure and limited gene flow between these three regions. The major lineages diverged in the Late Miocene and Pliocene (17–2.59 million years ago, Mya), i.e. before Quaternary ice ages, indicating that distinct lineages survived in multiple refugia in each sampling region during Quaternary glacial periods. The genetic structure of the 18 lineages of the group supported a model of sequential allopatric diversification within each sampling region. Further, three distinct lineages which diverged during the Pleistocene and Holocene were widely distributed across Central Europe, suggesting that glacial cycles in the Quaternary affected the spread of these lineages. Identical haplotypes of both of these lineages, occurring in localities hundreds of kilometers apart, suggest recent human-mediated dispersal across Central Europe. These results indicate that distribution patterns of Collembola in Europe are more complex than previously assumed.
\nBy utilizing DNA-based analyses, my thesis highlights a novel view of the diversity, ecology, phylogeny, and phylogeography of the three morphospecies of the European L. lanuginosus species group. DNA-based analyses rejected the three-species hypothesis based on the morphological species concept and the monophyly of each species based on the phylogenetic species concept. This suggests that historical geographic and climatic changes dating to the late Miocene as well as recent human-mediated dispersal caused the lineage divergence and shaped the present-day distribution of this species group. Environmental factors other than geographic distances likely impede gene flow among lineages. Overall, the results of this thesis suggest that soil animals likely experienced different and more complex evolutionary forces than aboveground animals and plants in shaping their genetic diversification and biogeographic distribution. Future studies need to explore the physiological characters responsible for habitat sorting of cryptic species / lineages of the L. lanuginosus species group, and global scale phylogeographic studies on a number of Collembola species are needed for a deeper understanding of the dispersal, speciation, and evolution of Collembola and of soil animals in general.

Tan, D. S. H., Ang, Y., Lim, G. S., Ismail, M. R. B. & Meier, R. (2010). From ‘cryptic species’ to integrative taxonomy: an iterative process involving DNA sequences, morphology, and behaviour leads to the resurrection of Sepsis pyrrhosoma (Sepsidae: Diptera). —Zoologica Scripta, 39, 51–61.The increased availability of DNA sequences has led to a surge of ‘cryptic species’ in the literature. These units are usually proposed based on finding genetically distinct lineages within species that were initially defined based on morphological characters. However, few authors attempt to confirm whether these ‘cryptic’ units are species and even fewer authors are explicit about which species concept is applied. Here, we use an example from Sepsidae (Diptera) to demonstrate how cryptic species can be validated by an iterative process involving several data sources and an evaluation of the data under different species concepts. A phylogeographic analysis based on 50 specimens for five species of the flavimana group revealed deep mitochondrial splits within Sepsis flavimana which was suggestive of a cryptic species. We resolve the initial conflict between DNA sequences and morphology by adding new morphological data as well as behavioural evidence and tests for reproductive isolation. One cryptic species is confirmed and Sepsis pyrrhosoma, a former synonym of S. flavimana, is here shown to be a valid species under most species concepts. We can thus document that the same data can lead to similar conclusions under conflicting concepts once different kinds of data are integrated.

© 2016 International Phycological Society. Sympatric coexistence of cryptic species, indistinguishable morphological taxa, has increasingly been detected on the basis of molecular data. This discovery raises the interesting question of how cryptic species can coexist, as hypothetically they would need identical ecological resources. The red alga Bostrychia intricata is commonly found along New Zealand shores. Previous studies indicated several cryptic species within this morphospecies, and that some populations have multiple species. This study aimed to determine how coexisting cryptic B. intricata distribute at a small scale. Along the shore of Moa Point, Wellington, we conducted intensive sampling of B. intricata in different habitats with respect to tidal position, wave and sun exposure levels. Our genetic data clearly documented the coexistence of three cryptic species of B. intricata: N2, N4 and N5. Multiple samples from individual algal patches indicated that each patch was made of the same ramet. Our analyses revealed a habitat-related pattern in small-scale distribution of different cryptic B. intricata, suggesting that the distribution of these cryptic species was not random. Cryptic species N4 was found at a higher tidal position than species N2 and N5, whereas cryptic species N2 occurred in more wave-exposed areas than the other species. Discriminant analysis indicated that tidal height strongly influenced the distribution pattern among these cryptic species. Our observations demonstrated that the co-occurrence of three cryptic B. intricata can partly be explained by their occupation of different intertidal habitats, highlighting the nonrandom distribution of coexisting cryptic algal species.

South American fire ant decapitating flies in the genus Pseudacteon (Diptera: Phoridae) are potential biocontrol agents of the invasive fire ants Solenopsis invicta and S. richteri in the United States and other regions of the world due to their high host specificity and the direct and indirect damage to their host ants. Despite their importance and the fact that several flies have already been released in the US, little is known about the genetic variability and phylogenetic relationships of Pseudacteon flies parasitizing South American fire ants in the Solenopsis saevissima species-group. A species delimitation analysis was conducted using a distance-based method (ABGD) and two tree-based methods (GMYC and mPTP) using COI sequences of 103 specimens belonging to 20 of the 22 Pseudacteon species known from southern South America. Additionally, phylogenetic relationships between the already described and new candidate species were inferred using mitochondrial (COI) and nuclear (wingless) sequences. The species delimitation analysis suggests that species richness in these flies has been previously underestimated, due to the existence of putative cryptic species within nominal Pseudacteon obtusus, P. pradei, P. tricuspis, P. cultellatus, and P. nudicornis. Geographic distribution and host fire ant species seem to support cryptic lineages, though additional morphological data are needed to corroborate these results. All phylogenetic analyses reveal that South American fire ant decapitating flies are grouped into two main clades, with Pseudacteon convexicauda sister and well differentiated relative to these clades. Neither host nor geographic association appeared to be related to the differentiation of these two main clades within South American fire ant decapitating flies. This work provides information that will allow testing whether the putative cryptic phorid fly species show differences in their effectiveness as biocontrol agents against the highly invasive imported fire ants.

:Sympatric coexistence of cryptic species, indistinguishable morphological taxa, has increasingly been detected on the basis of molecular data. This discovery raises the interesting question of how cryptic species can coexist, as hypothetically they would need identical ecological resources. The red alga Bostrychia intricata is commonly found along New Zealand shores. Previous studies indicated several cryptic species within this morphospecies, and that some populations have multiple species. This study aimed to determine how coexisting cryptic B. intricata distribute at a small scale. Along the shore of Moa Point, Wellington, we conducted intensive sampling of B. intricata in different habitats with respect to tidal position, wave and sun exposure levels. Our genetic data clearly documented the coexistence of three cryptic species of B. intricata: N2, N4 and N5. Multiple samples from individual algal patches indicated that each patch was made of the same ramet. Our analyses revealed a habitat-related pattern in small-scale distribution of different cryptic B. intricata, suggesting that the distribution of these cryptic species was not random. Cryptic species N4 was found at a higher tidal position than species N2 and N5, whereas cryptic species N2 occurred in more wave-exposed areas than the other species. Discriminant analysis indicated that tidal height strongly influenced the distribution pattern among these cryptic species. Our observations demonstrated that the co-occurrence of three cryptic B. intricata can partly be explained by their occupation of different intertidal habitats, highlighting the nonrandom distribution of coexisting cryptic algal species.

© 2016 International Phycological Society. Sympatric coexistence of cryptic species, indistinguishable morphological taxa, has increasingly been detected on the basis of molecular data. This discovery raises the interesting question of how cryptic species can coexist, as hypothetically they would need identical ecological resources. The red alga Bostrychia intricata is commonly found along New Zealand shores. Previous studies indicated several cryptic species within this morphospecies, and that some populations have multiple species. This study aimed to determine how coexisting cryptic B. intricata distribute at a small scale. Along the shore of Moa Point, Wellington, we conducted intensive sampling of B. intricata in different habitats with respect to tidal position, wave and sun exposure levels. Our genetic data clearly documented the coexistence of three cryptic species of B. intricata: N2, N4 and N5. Multiple samples from individual algal patches indicated that each patch was made of the same ramet. Our analyses revealed a habitat-related pattern in small-scale distribution of different cryptic B. intricata, suggesting that the distribution of these cryptic species was not random. Cryptic species N4 was found at a higher tidal position than species N2 and N5, whereas cryptic species N2 occurred in more wave-exposed areas than the other species. Discriminant analysis indicated that tidal height strongly influenced the distribution pattern among these cryptic species. Our observations demonstrated that the co-occurrence of three cryptic B. intricata can partly be explained by their occupation of different intertidal habitats, highlighting the nonrandom distribution of coexisting cryptic algal species.

Systematics and taxonomy are the backbone of all components of biology and ecology, yet cryptic species present a major challenge for accurate species identification. This is especially problematic as they represent a substantial portion of undiscovered biodiversity, and have implications for not only species conservation, but even assaying potential risk of zoonotic spillover. Here, we use integrative approaches to delineate potential cryptic species in horseshoe bats (Rhinolophidae), evaluate the phenotypic disparities between cryptic species, and identify key traits for their identification. We tested the use of multispecies coalescent models (MSC) using Bayesian Phylogenetic and Phylogeography (BPP) and found that BPP was useful in delineating potential cryptic species, and consistent with acoustic traits. Our results show that around 40% of Asian rhinolophid species are potentially cryptic and have not been formally described. In order to avoid potential misidentification and allow species to be accurately identified, we identified quantitative noseleaf sella and acoustic characters as the most informative traits in delineating between potential cryptic species in Rhinolophidae. This highlights the physical differences between cryptic species that are apparent in noseleaf traits which often only qualitatively described but rarely measured. Each part of the noseleaf including the sella, lateral lappets, and lancet furrows, play roles in focusing acoustic beams and thus, provide useful characteristics to identify cryptic Rhinolophus species. Finally, species delimitation for cryptic species cannot rely on genetic data alone, but such data should be complemented by other evidence, including phenotypic, acoustic data, and geographic distributions to ensure accurate species identification and delineation.

Life-history traits, abiotic environment and coexistence: The case of two cryptic rotifer species

Molecular approaches and the concept of species and species complexes in lichenized fungi

BackgroundThe subfamily Phlebotominae comprises 1028 species of sand fly, of which only 90 are recognized as vectors of pathogenic agents such as Trypanosoma, Leishmania, and Bartonella. In Thailand, leishmaniasis—a sand fly-borne disease—is currently endemic, with 36 documented sand fly species. However, many cryptic species likely remain unidentified. To improve our understanding of the distribution, habitat preferences, and role in disease transmission of these sand flies, further research is necessary.MethodsSand flies were collected using CDC light traps from 13 locations across four provinces in Thailand between October 2022 and October 2023. Initially, species identification was based on morphological characteristics, employing identification keys, and subsequently confirmed through mitochondrial cytochrome oxidase c subunit I (COI) and cytochrome b (Cytb) sequencing. Species identities were verified using BLASTN and BOLD searches. Species delimitation was conducted using Automatic Barcode Gap Discovery (ABGD) and Assemble Species by Automatic Partitioning (ASAP) with three substitution models. Additionally, intraspecific and interspecific genetic variation, neutrality tests (including Tajima’s and Fu and Li’s D* tests), phylogenetic analyses, and TCS haplotype network analysis were performed using the obtained sequences.ResultsA total of 3693 phlebotomine sand flies were collected, with 2261 (61.22%) identified as female. Integrative analyses combining morphological data, BLASTN searches, phylogenetic assessments, and species delimitation confirmed the identification of four genera: Sergentomyia, Grassomyia, Phlebotomus, and Idiophlebotomus, encompassing 12 species: Sergentomyia anodontis, Se. sylvatica, Se. perturbans, Se. barraudi, Se. hivernus, Se. khawi, Se. siamensis, Grassomyia indica, Phlebotomus barguesae, Ph. stantoni, Idiophlebotomus asperulus, and Id. longiforceps. Furthermore, molecular analysis revealed cryptic and complex species, including two putatively novel species, Se. sp. 1 and Se. sp. 2, as well as a unique haplotype.ConclusionsThis study, which integrated genetic and morphological identification techniques, identified 12 sand fly species and unveiled cryptic and complex species, including two putatively novel species (Se. sp. 1 and Se. sp. 2) and a unique haplotype. The findings underscore the utility of mitochondrial genes, combined with species delimitation methodologies, as reliable approaches for identifying diverse sand fly species.Graphical