Evidence For Cryptic Species Research Articles

Evidence of cryptic speciation in the invasive hydroid Cordylophora caspia (Pallas, 1771) (Cnidaria, Hydrozoa) supported by new records

Evidence of cryptic speciation in the invasive hydroid Cordylophora caspia (Pallas, 1771) (Cnidaria, Hydrozoa) supported by new records

Trichophoromyia auraensis: evidence for cryptic species and first record in the state of Maranhão, Brazil.

Trichophoromyia auraensis (Mangabeira, 1942) (Diptera, Psychodidae, Phlebotominae) has a wide geographic distribution in the western region of the Amazon biome, where it is a putative Leishmania vector. Here, we reported for the first time a population of this species in the Brazilian state of Maranhão, in the eastern Amazon, from which we DNA-barcoded and compared with previously processed specimens from Acre State, in the western Amazon. For this, we analyzed the DNA barcoding fragment (658 bp) of the mitochondrial cytochrome c oxidase subunit I (COI) gene and the nuclear internal transcribed spacer 2 (ITS2) of Trichophoromyia species using phylogeneticgene trees, and species delimitation algorithms. The analyses of COI barcodes showed high values of genetic distance (mean K2P = 5.17) and well-supported clades/MOTUs for the eastern and western populations of T. auraensis, which may indicate a possible complex of cryptic species. The western population of this taxon merged with the close-related sand fly Trichophoromyia velezbernali Posada-López, Galvis and Galati, 2018 from Colombia, which may be associated with the recent speciation history and introgression between these populations. These evidences should be evaluated with a more comprehensive sampling in terms of analyzed populations and molecular markers.

The organellar genomes of Pellidae (Marchantiophyta): the evidence of cryptic speciation, conflicting phylogenies and extraordinary reduction of mitogenomes in simple thalloid liverwort lineage

Organellar genomes of liverworts are considered as one of the most stable among plants, with rare events of gene loss and structural rearrangements. However, not all lineages of liverworts are equally explored in the field of organellar genomics, and subclass Pellidae is one of the less known. Hybrid assembly, using both short- and long-read technologies enabled the assembly of repeat-rich mitogenomes of Pellia and Apopellia revealing extraordinary reduction of length in the latter which impacts only intergenic spacers. The mitogenomes of Apopellia were revealed to be the smallest among all known liverworts—109 k bp, despite retaining all introns. The study also showed the loss of one tRNA gene in Apopellia mitogenome, although it had no impact on the codon usage pattern of mitochondrial protein coding genes. Moreover, it was revealed that Apopellia and Pellia differ in codon usage by plastome CDSs, despite identical tRNA gene content. Molecular identification of species is especially important where traditional taxonomic methods fail, especially within Pellidae where cryptic speciation is well recognized. The simple morphology of these species and a tendency towards environmental plasticity make them complicated in identification. Application of super-barcodes, based on complete mitochondrial or plastid genomes sequences enable identification of all cryptic lineages within Apopellia and Pellia genera, however in some particular cases, mitogenomes were more efficient in species delimitation than plastomes.

Evidence of cryptic speciation in the Hygrobates calliger complex (Acariformes, Hydrachnidia, Hygrobatidae) with the description of two new species

Hygrobates specimens with a coarse lineated soft integument generally have been considered as representatives of H. calliger Piersig, 1896, regarded as a common and widely distributed species in the Palaearctic. In the present study using an integrative taxonomic approach that combines morphological studies and DNA barcoding we show that Hygrobates calliger-like mites belong to six distinct lineages. Two species, Hygrobates ponticus sp. nov. (East Türkiye, Georgia) and H. tyrrhenicus sp. nov. (Corsica) are described as new to science.

Re-evaluating the case for poecilogony in the gastropod Planaxis sulcatus (Cerithioidea, Planaxidae)

BackgroundPlanaxis sulcatus has been touted as a textbook example of poecilogony, with members of this wide-ranging Indo-Pacific marine gastropod said to produce free-swimming veligers as well as brooded juveniles. A recent paper by Wiggering et al. (BMC Evol Biol 20:76, 2020) assessed a mitochondrial gene phylogeny based on partial COI and 16S rRNA sequences for 31 individuals supplemented by observations from the brood pouch of 64 mostly unsequenced individuals. ABGD and bGYMC supported three reciprocally monophyletic clades, with two distributed in the Indo-Pacific, and one restricted to the northern Indian Ocean and Red Sea. Given an apparent lack of correlation between clade membership and morphological differentiation or mode of development, the reported 3.08% maximum K2P model-corrected genetic divergence in COI among all specimens was concluded to represent population structuring. Hence, the hypothesis that phylogenetic structure is evidence of cryptic species was rejected and P. sulcatus was concluded to represent a case of geographic poecilogony.ResultsOur goal was to reassess the case for poecilogony in Planaxis sulcatus with a larger molecular dataset and expanded geographic coverage. We sequenced an additional 55 individuals and included published and unpublished sequence data from other sources, including from Wiggering et al. Our dataset comprised 108 individuals (88 COI, 81 16S rRNA) and included nine countries unrepresented in the previous study. The expanded molecular dataset yielded a maximum K2P model-corrected genetic divergence among all sequenced specimens of 12.09%. The value of 3.08% erroneously reported by Wiggering et al. is the prior maximal distance value that yields a single-species partition in ABGD, and not the maximum K2P intraspecific divergence that can be calculated for the dataset. The bGMYC analysis recognized between two and six subdivisions, while the best-scoring ASAP partitions recognized two, four, or five subdivisions, not all of which were robustly supported in Bayesian and maximum likelihood phylogenetic analyses of the concatenated and single gene datasets. These hypotheses yielded maximum intra-clade genetic distances in COI of 2.56–6.19%, which are more consistent with hypothesized species-level thresholds for marine caenogastropods.ConclusionsBased on our analyses of a more comprehensive dataset, we conclude that the evidence marshalled by Wiggering et al. in support of Planaxis sulcatus comprising a single widespread, highly variable species with geographic poecilogony is unconvincing and requires further investigation in an integrative taxonomic framework.

Ojo Guareña: A Hotspot of Subterranean Biodiversity in Spain

Ojo Guareña Natural Monument in Burgos (Spain) is an important and large karstic system. It consists of more than 110 km of surveyed galleries, and it has rich sources of organic material from the surface and permanent water circulation. It is the fourth largest cave system in the Iberian Peninsula, and one of the 10 largest in Europe. Ojo Guareña also ranks 23rd among the world’s largest caves. To date, only volcanic caves in the Canary Islands, in which between 28 and 38 subterranean species occur, are considered subterranean diversity hotspots in Spain. Here, we provide the first list of subterranean taxa present in Ojo Guareñ, which is comprised of 54 taxa that includes 46 stygobiotic and eight troglobiotic species (some still unidentified at the species level), revealing Ojo Guareña as the largest known subterranean biodiversity hotspot in Spain and Portugal. In addition, we provide a list of an additional 48 taxa, 34 stygophiles and 14 troglophiles, found in the system, whose ecological status could change with detailed biological studies, which may change the number of strictly subterranean species present in the system. Indeed, at present, these numbers are provisional as they correspond to a small part of this sizeable cave system. The biodiversity of large areas of the system remains unknown as these areas have yet to be explored from the biological point of view. In addition, a large number of samples of both terrestrial and aquatic fauna are still under study by specialists. Furthermore, evidence of cryptic species within Bathynellacea (Crustacea) indicates an underestimation of biodiversity in the karstic system. Despite these limitations, the data available reveal the typical uneven distribution of subterranean aquatic fauna, and suggest that the great heterogeneity of the microhabitats in this wide and highly connected karstic extension led to the great richness of aquatic subterranean species.

Ant crickets (Orthoptera: Myrmecophilidae) associated with the invasive yellow crazy ant Anoplolepis gracilipes (Hymenoptera: Formicidae): evidence for cryptic species and potential co-introduction with hosts

The yellow crazy ant, Anoplolepis gracilipes (Smith, 1857), is a widespread invasive ant in tropical and subtropical regions. In our study, we surveyed ant cricket species (Myrmecophilinae) associated with A. gracilipes in the Indo-Pacific region and provided a taxonomic revision using an integrative approach by combining morphological and molecular data. At least eight ant cricket species were found in A. gracilipes nests, which represents the greatest number of ant cricket species recorded for a single ant species. Some of these ant crickets were widespread across the Indo-Pacific and have an overlapping distribution with A. gracilipes. Haplotype networks showed incongruence between haplotype groupings and geographic distribution of ant cricket species, indicating co-introductions of ant cricket species with their ant hosts have occurred. A new taxonomic status of related Myrmecophilus species was given: 1) three new species were described (Myrmecophilus antilucanus sp.n., Myrmecophilus caliginosus sp.n., and Myrmecophilus ikaros sp.n.), and 2) three new synonyms and one resurrection was made (M. quadrispina Perkins, 1899 = M. formosanus Shiraki, 1930 syn.n., M. hebardi Mann, 1920 = M. leei Kistner & Chong, 2007 syn.n., M. dubius Saussure, 1877 = M. flavocinctus Wasmann, 1894 stat.n. & syn.n.; M. mayaealberti Hugel & Matyot, 2006 stat.rev.). In addition, traits that potentially promote A. gracilipes as a favorable host for ant crickets are discussed, along with potential ecological impacts associated with co-introduced ant crickets in their non-native range.Open access, licensed under CC BY 4.0. © 2020 The Author(s).DOI: 10.25849/myrmecol.news_030:103Pages: 103-129Volume: 30Year: 2020Journal: Myrmecol. News

An Examination of Shallow-Water Hydroids (Cnidaria, Hydrozoa, Hydroidolina) in Cape Breton, Nova Scotia, Using Morphology and DNA Barcoding

Hydroids are common constituents of marine environments, both as benthic polyps and planktonic hydromedusae. Despite the volume of hydroid research focused on the northwest Atlantic, the only comprehensive examinations of hydroids from Cape Breton Island were published by Charles McLean Fraser in 1913 and 1927. In this study, we reexamined the shallow-water hydroid community of Cape Breton. We collected hydroids from 19 sites between July 2016 and June 2017 spanning intertidal and shallow subtidal habitats. We sorted specimens into individual taxa based on morphology and then preserved them for further morphological and molecular analyses. We amplified the 16SrRNA barcoding gene for select specimens and used BLAST searches to determine the strongest matches in GenBank. We performed molecular phylogenetic analyses to refine identifications where necessary. We recorded 19 hydroid taxa spanning 15 genera, 10 families, and 2 orders. Species diversity was low relative to other NW Atlantic regions, likely due to sporadic sampling and our focus on nearshore, shallow-water localities. However, 3 species, Pachycordyle michaeli, Podocorynoides minima, and Lizzia blondina, were new occurrences for Atlantic Canada. Additionally, our results provided further evidence of cryptic speciation in Obelia dichotoma. This updated species inventory represents an important benchmark of hydroid diversity for future comparisons.

Evidence of cryptic speciation in South American lungfish

Evidence of cryptic speciation in South American lungfish

Molecular systematics and phylogeography of a widespread Neotropical avian lineage: evidence for cryptic speciation with protracted gene flow throughout the Late Quaternary

AbstractHere we use an integrative approach, including coalescent-based methods, isolation–migration and species distribution models, to infer population structure, divergence times and diversification in the two species of the genus Cymbilaimus (Aves, Thamnophilidae). Our results support a recent and rapid diversification with both incomplete lineage sorting and gene flow shaping the evolutionary history of Cymbilaimus. The spatio-temporal pattern of cladogenesis suggests that Cymbilaimus originated in the north/western portion of cis-Andean South America and then diversified into the Brazilian Shield and Central America after consolidation of the modern Amazonian drainage and the Andean range. This evolutionary scenario is explained by cycles of range expansion and dispersal, followed by isolation, and recurrent gene flow, during the last 1.2 Myr. Our results agree with those recently reported for other closely related suboscine lineages, whereby the window of introgression between closely related taxa remains open for up to a few million years after their original split. In Cymbilaimus, introgression was recurrent between C. lineatus and C. sanctaemariae, even after they acquired vocal and ecological differentiation, supporting the claim that at least in Neotropical suboscines, full reproductive compatibility may take millions of years to evolve and cannot be interpreted as synonymous with a lack of speciation.

DNA barcoding of commercially important reef fishes in Weh Island, Aceh, Indonesia.

Knowledge on the precise identification of fish resources is critical for sustainable fisheries management. This study employs the DNA barcoding approach to generate a molecular taxonomic catalogue of commercially important reef fishes in the waters of Weh Island (Aceh Province), the most northerly inhabited island in the biodiverse Indonesian Archipelago. The waters not only support artisanal fisheries but also a feeder for the industry in the greater island of Aceh. In total, 230 specimens from 72 species belonging to 32 genera and 17 families were DNA barcoded, representing a major segment of the captured reef fish taxa and a quarter of fish species diversity that had previously been recorded. The sequence read lengths were 639 bp revealing 359 conserved sites, 280 variable sites, 269 parsimony informative and 11 singletons. Our molecular findings paralleled the morphological identification with no evidence of cryptic species or new species discovery. This study is a significant contribution to the fisheries statistics of this area, which would facilitate assessment of species catch composition and hence for strategizing management plans. It is an important input to the DNA barcode library of Indonesian marine fishes and to the global DNA barcode entries in general.

New evidence of cryptic speciation in the family Longidoridae (Nematoda: Dorylaimida)

This research was supported by grant 201740E042 “Analisis de diversidad molecular, barcoding, y relaciones filogeneticas de nematodos fitoparasitos en cultivos mediterraneos” from the Spanish National Research Council (C.S.I.C.) and by the Humboldt Research Fellowship for Postdoctoral Researchers awarded for the second author. The first author acknowledges the China Scholarship Council (CSC) for financial support. The second author is a recipient of Humboldt Research Fellowship for Postdoctoral Researchers at Helmholtz Centre for Environmental Research‐UFZ, Leipzig, Germany. The fourth author acknowledges Spanish Ministry of Economy and Competitiveness for the “Ramon y Cajal” Fellowship RYC‐2017‐22228.

Morphological Variation in the Skull of Nephelomys meridensis (Rodentia, Cricetidae): Evidence for Cryptic Species in Andean Populations from Northern South America

The genus Nephelomys comprises at least 13 taxa with altitudinal distribution ranging from 900 to 4,000 meters above sea level. Three species are currently known to occur in Venezuela, but some authors suggest that two additional taxa are present in Venezuelan Andes populations and consider N. meridensis as a species complex. We conducted geometric morphometric analyses of skulls and jaws from populations assignable to the monotypic species N. meridensis from the Venezuelan Andes, in order to examine and compare morphological variations in this species. We compared four localities (including the type locality of N. meridensis) situated in northern, central, and western portions of Cordillera de Mérida and the Tamá páramo. We created morphological landmarks on dorsal, ventral, lateral, and jaw views of each skull using the tpsDIG software. Significant differences were found in skull shape and isometric size in most of the populations examined. The discrimination and morphological differentiation of some of these groups with respect to the population from the type locality (Mérida) were aligned with the results from multivariate and cytogenetic analyses carried out previously by other authors. Our data support recognizing N. meridensis as a species complex, comprising at least three still undescribed taxa from the Yacambú National Park (cordillera de Mérida), La Trampita (cordillera de Mérida) and the Tamá páramo (Tamá massif). A more detailed taxonomic study of Nephelomys from Venezuela, molecular and linear morphometric analyses, and revision of morphological characters, are necessary to describe potential new taxa and determine the geographical distribution of the species in this genus.

Complex evolutionary history of the American Rubyspot damselfly, Hetaerina americana (Odonata): Evidence of cryptic speciation

Analyzing the magnitude and distribution of genetic variation within and among populations allows for hypothesis testing about historical demographic size changes, secondary contacts, refugia, and speciation patterns. Species distribution and genetic structure are greatly influenced by the complex life cycle and behavior of odonates. Hetaerina americana has been widely used as a model system in behavioral studies, but its population genetic structure has not been analyzed, except for a single study that included only three populations but identified the presence of markedly differentiated genetic groups, suggesting the existence of cryptic species. Here, we tested this hypothesis by assessing throughout the distribution range of H. americana the patterns of genetic and morphological variation in the male caudal appendages, due to the great importance of these structures in mate recognition. As molecular markers we used sequences of the mitochondrial cytochrome oxidase I (COI) gene and the nuclear internal transcribed spacer (ITS) region, as well as six nuclear microsatellites. We found very high population genetic differentiation (ΦST > 0.51) in the three sets of markers but with strong mitonuclear discordance. A neutrality test suggested that the mitochondrial genome might be under purifying selection in association to climatic variables (temperature seasonality). The assignment of individuals to nuclear genetic groups showed little admixture and complete congruence with morphological differentiation in the male caudal appendages. Hence, the results suggest that H. americana represents at least two different cryptic species which are isolated reproductively.

Evidence of cryptic species in the blenniid Cirripectesalboapicalis species complex, with zoogeographic implications for the South Pacific.

Rapa Nui, commonly known as Easter Island (Chile), is one of the most isolated tropical islands of the Pacific Ocean. The island location of Rapa Nui makes it the easternmost point of the geographic ranges for many western Pacific fish species that are restricted to the subtropical islands south of 20°S latitude. The blenniid fish species Cirripectesalboapicalis has been thought to have one of the most extensive geographic distribution ranges among these southern subtropical fish species, extending from the southern Great Barrier Reef to Rapa Nui. A phylogenetic analysis was conducted to determine the taxonomic status of the species. The results provide genetic evidence that suggests that this formerly South Pacific-wide species comprises at least three cryptic species with allopatric geographic distributions. The analyses reveal the geographic distributions of these clades and their genetic relationships with each other, and with other species within the genus Cirripectes. The processes that culminated in the current geographic distribution of this species complex and the zoogeographic implications of this finding for the South Pacific region are discussed.

Evidence of cryptic species in the genus Tinaminyssus (Acari: Rhinonyssidae) based on morphometrical and molecular data.

The study of cryptic species allows to describe and to understand biodiversity, and the evolutionary processes shaping it. Mites of the family Rhinonyssidae are permanent parasites of the nasal cavities of birds, currently including about 500 described species and 12 genera. Here, we tested the hypothesis that mites from five populations of the genus Tinaminyssus-three isolated from European turtle doves (Streptopelia turtur), and two from Eurasian collared doves (Streptopelia decaocto; Aves: Columbiformes)-are, in fact, two cryptic species inhabiting different hosts. First, we performed a morphometrical study on 16 traits. Then, we used the ITS1-5.8S rDNA-ITS2 nuclear region (ITS region), and a fragment of the mitochondrial cytochrome c-oxidase 1 (COI) to carry out phylogenetic and species delimitation analyses on Tinaminyssus species. Morphological analyses revealed a lack of biometric differentiation among Tinaminyssus populations from the two host species. However, molecular analyses indicated a high degree of genetic differentiation between populations of Tinaminyssus sp. from S. turtur and S. decaocto. Overall, results show that they can be considered as different cryptic species, suggesting a case of evolutionary stasis, likely because of the anatomical similarity between closely-related bird host species.

Widespread plant specialization in the polyphagous planthopper Hyalesthes obsoletus (Cixiidae), a major vector of stolbur phytoplasma: Evidence of cryptic speciation.

The stolbur phytoplasma vector Hyalesthes obsoletus is generally considered as a polyphagous species associated with numerous wild and cultivated plants. However, recent research in southeastern Europe, the distribution centre of H. obsoletus and the area of most stolbur-inflicted crop diseases, points toward specific host-plant associations of the vector, indicating specific vector-based transmission routes. Here, we study the specificity of populations associated with four host-plants using mitochondrial and nuclear genetic markers, and we evaluate the evolution of host-shifts in H. obsoletus. Host-plant use was confirmed for Convolvulus arvensis, Urtica dioica, Vitex agnus-castus and Crepis foetida. Mitochondrial genetic analysis showed sympatric occurrence of three phylogenetic lineages that were ecologically delineated by host-plant preference, but were morphologically inseparable. Nuclear data supported the existence of three genetic groups (Evanno’s ΔK(3) = 803.72) with average genetic membership probabilities > 90%. While populations associated with C. arvensis and U. dioica form a homogenous group, populations affiliated with V. agnus-castus and C. foetida constitute two independent plant-associated lineages. The geographical signal permeating the surveyed populations indicated complex diversification processes associated with host-plant selection and likely derived from post-glacial refugia in the eastern Mediterranean. This study provides evidence for cryptic species diversification within H. obsoletus sensu lato: i) consistent mitochondrial differentiation (1.1–1.5%) among host-associated populations in syntopy and in geographically distant areas, ii) nuclear genetic variance supporting mitochondrial data, and iii) average mitochondrial genetic distances among host-associated meta-populations are comparable to the most closely related, morphologically distinguishable species, i.e., Hyalesthes thracicus (2.1–3.3%).

Blood-feeding, susceptibility to infection with Schmallenberg virus and phylogenetics of Culicoides (Diptera: Ceratopogonidae) from the United Kingdom

BackgroundCulicoides biting midges (Diptera: Ceratopogonidae) are responsible for the biological transmission of internationally important arboviruses of livestock. In 2011, a novel Orthobunyavirus was discovered in northern Europe causing congenital malformations and abortions in ruminants. From field studies, Culicoides were implicated in the transmission of this virus which was subsequently named Schmallenberg virus (SBV), but to date no assessment of susceptibility to infection of field populations under standardised laboratory conditions has been carried out. We assessed the influence of membrane type (chick skin, collagen, Parafilm M®) when offered in conjunction with an artificial blood-feeding system (Hemotek, UK) on field-collected Culicoides blood-feeding rates. Susceptibility to infection with SBV following blood-feeding on an SBV-blood suspension provided via either (i) the Hemotek system or via (ii) a saturated cotton wool pledglet was then compared. Schmallenberg virus susceptibility was defined by RT-qPCR of RNA extractions of head homogenates and related to Culicoides species and haplotype identifications based on the DNA barcode region of the mitochondrial cytochrome c oxidase 1 (cox1) gene.ResultsCulicoides blood-feeding rates were low across all membrane types tested (7.5% chick skin, 0.0% for collagen, 4.4% Parafilm M®, with 6029 female Culicoides being offered a blood meal in total). Susceptibility to infection with SBV through membrane blood-feeding (8 of 109 individuals tested) and pledglet blood-feeding (1 of 94 individuals tested) was demonstrated for the Obsoletus complex, with both C. obsoletus (Meigen) and C. scoticus Downes & Kettle susceptible to infection with SBV through oral feeding. Potential evidence of cryptic species within UK populations was found for the Obsoletus complex in phylogenetic analyses of cox1 DNA barcodes of 74 individuals assessed from a single field-site.ConclusionsMethods described in this study provide the means to blood-feed Palaearctic Culicoides for vector competence studies and colonisation attempts. Susceptibility to SBV infection was 7.3% for membrane-fed members of the subgenus Avaritia and 1.1% for pledglet-fed. Both C. obsoletus and C. scoticus were confirmed as being susceptible to infection with SBV, with potential evidence of cryptic species within UK Obsoletus complex specimens, however the implications of cryptic diversity in the Obsoletus complex on arbovirus transmission remains unknown.

Six species in one: evidence of cryptic speciation in the Hygrobates fluviatilis complex (Acariformes, Hydrachnidia, Hygrobatidae)

Water mites of the genus Hygrobates are widely distributed in all biogeographic regions except the Antarctic. Palaearctic Hygrobates species with reticulated soft integument generally have been considered as representatives of one common and widely distributed species, Hygrobates fluviatilis Strøm, 1768. Based on partial COI sequences (DNA-barcodes) and statistical analysis of morphological data, we show that these mites belong to six distinct lineages. Two of them are widely distributed in Central Europe: Hygrobates fluviatilis here redescribed based on a neotype designated from the type locality in Norway, and a species new to science, H. arenarius Smit & Pešić. The four remaining lineages represent additional species new to science that appear to have more restricted distributions: H. corsicus Pešić & Smit (Corsica, Sardinia), H. marezaensis Pešić & Dabert (Montenegro, Albania, Croatia), H. turcicus Pešić, Esen & Dabert (Turkey), and H. persicus Pešić & Asadi (Iran, E Turkey). Statistical morphometric analysis reveals that the latter two species cannot be separated on morphological characters and should be considered true cryptic species. We provide data concerning biology and geographical distributions together with a key to all species of the complex.

Globally intertwined evolutionary history of giant barrel sponges

Three species of giant barrel sponge are currently recognized in two distinct geographic regions, the tropical Atlantic and the Indo-Pacific. In this study, we used molecular techniques to study populations of giant barrel sponges across the globe and assessed whether the genetic structure of these populations agreed with current taxonomic consensus or, in contrast, whether there was evidence of cryptic species. Using molecular data, we assessed whether giant barrel sponges in each oceanic realm represented separate monophyletic lineages. Giant barrel sponges from 17 coral reef systems across the globe were sequenced for mitochondrial (partial CO1 and ATP6 genes) and nuclear (ATPsβ intron) DNA markers. In total, we obtained 395 combined sequences of the mitochondrial CO1 and ATP6 markers, which resulted in 17 different haplotypes. We compared a phylogenetic tree constructed from 285 alleles of the nuclear intron ATPsβ to the 17 mitochondrial haplotypes. Congruent patterns between mitochondrial and nuclear gene trees of giant barrel sponges provided evidence for the existence of multiple reproductively isolated species, particularly where they occurred in sympatry. The species complexes in the tropical Atlantic and the Indo-Pacific, however, do not form separate monophyletic lineages. This rules out the scenario that one species of giant barrel sponge developed into separate species complexes following geographic separation and instead suggests that multiple species of giant barrel sponges already existed prior to the physical separation of the Indo-Pacific and tropical Atlantic.