Sequence-based species delineation and molecular phylogenetics of the transitional Nearctic–Neotropical grasshopper genus Taeniopoda (Orthoptera,Romaleidae)

Taeniopoda is a genus of grasshoppers currently represented by 12 species distributed from southern USA to Panama, with most of them occurring along the transitional Nearctic–Neotropical region in central and southern Mexico. Despite being a small group of conspicuous, colourful species, the systematics of Taeniopoda has been largely neglected, including its phylogenetic affinity with the morphologically similar, monotypic genus Romalea. Here, we assessed the species limits in 11 of the species of Teniopoda based on two mitochondrial (mt) markers (COI, cyt b). Phylogenetic relationships were reconstructed adding two nuclear gene markers (28S, H3). A relaxed molecular clock analysis was performed based on the mt markers. We detected nuclear mt paralogues (numts) and the probable introgression of T. tamaulipensis mtDNA in specimens of T. eques from central Mexico. Between six and 14 species of Taeniopoda were delimited by the sequence-based approaches performed (COI divergence with thresholds of 1 and 2%; General Mixed Yule-Coalescent (GMYC) model). The GMYC and 1% threshold analyses with COI were more congruent with the currently recognized morphology-based taxonomy with 10 and 11 putative species, respectively. Four of these species were regarded as ‘stable’, since they were supported by at least one of the molecular analyses and by diagnostic morphological features. The species-based phylogeny recovered Taeniopoda as paraphyletic with respect to the monotypic genus Romalea. Three morphologically and geographically congruent major clades were recovered, two with species having a considerably elevated pronotal crest and one with its members having it less elevated. The origin and subsequent diversification of Taeniopoda were estimated to occur from the mid and late Miocene to Pliocene, respectively. The current species diversity in Taeniopoda was estimated to occur during the Pleistocene, which was probably influenced by the climatic oscillations that occurred during this period and the uplift of mountain ranges in Central America.     

Multilocus phylogeny of a cryptic radiation of Afrotropical long-fingered bats (Chiroptera,Miniopteridae)

The Old World bat family Miniopteridae comprises only the genus Miniopterus, which includes 20 currently recognized species from the Afrotropical realm and 15 species from Eurasia and Australasia. Since 2003, the number of recognized Miniopterus species has grown from 19 to 35, with most newly described species endemic to Madagascar and the Comoros Archipelago. We investigated genetic variation, phylogenetic relationships and clade membership in Miniopterus focusing on Afrotropical taxa. We generated mitochondrial cytochrome-b (cyt-b) and nuclear intron data (five genes) from 352 vouchered individuals collected at 78 georeferenced localities. Including 99 additional mitochondrial sequences from GenBank, we analysed a total of 25 recognized species. Mitochondrial genetic distances among cyt-b-supported clades averaged 9.3%, representing as many as five undescribed species. Multilocus coalescent delimitation strongly supported the genetic isolation of eight of nine tested unnamed clades. A large number of sampled clades in sub-Saharan Africa are distributed wholly or partly in East Africa (nine of 13 clades), suggesting that Miniopterus diversity has been grossly underestimated. Although 25 of 27 cyt-b and 23 of 25 nuclear gene tree lineages from the Afrotropics were strongly supported as monophyletic, a majority of deep nodes were poorly resolved in phylogenetic analyses. Long terminal branches subtending short backbone internodes in the phylogenetic analyses suggest a rapid radiation model of diversification. This hypothesis needs to be tested using more phylogenetically informative data.     

Mitochondrial Diversity and Distribution of African Green Monkeys (Chlorocebus Gray, 1870)

African green monkeys (Chlorocebus) represent a widely distributed and morphologically diverse primate genus in sub‐Saharan Africa. Little attention has been paid to their genetic diversity and phylogeny. Based on morphological data, six species are currently recognized, but their taxonomy remains disputed. Here, we aim to characterize the mitochondrial (mt) DNA diversity, biogeography and phylogeny of African green monkeys. We analyzed the complete mitochondrial cytochrome b gene of 126 samples using feces from wild individuals and material from zoo and museum specimens with clear geographical provenance, including several type specimens. We found evidence for nine major mtDNA clades that reflect geographic distributions rather than taxa, implying that the mtDNA diversity of African green monkeys does not conform to existing taxonomic classifications. Phylogenetic relationships among clades could not be resolved suggesting a rapid early divergence of lineages. Several discordances between mtDNA and phenotype indicate that hybridization may have occurred in contact zones among species, including the threatened Bale monkey (Chlorocebus djamdjamensis). Our results provide both valuable data on African green monkeys’ genetic diversity and evolution and a basis for further molecular studies on this genus. Am. J. Primatol. 75:350‐360, 2013. © 2013 Wiley Periodicals, Inc.     

DNA barcoding,phylogenetic relationships and speciation of snappers (genus <Emphasis Type="Italic">Lutjanus</Emphasis>)

The phylogenetic relationships of 13 snapper species from the South China Sea have been established using the combined DNA sequences of three full-length mitochondrial genes (COI, COII and CYTB) and two partial nuclear genes (RAG1, RAG2). The 13 species (genus Lutjanus) were selected after DNA barcoding 72 individuals, representing 20 species. Our study suggests that although DNA barcoding aims to develop species identification systems, it may also be useful in the construction of phylogenies by aiding the selection of taxa. Combined mitochondrial and nuclear gene data has an advantage over an individual dataset because of its higher resolving power.     

Molecular phylogeny of hinge‐beak shrimps (Decapoda: Caridea: Rhynchocinetes and Cinetorhynchus) and allies: a formal test of familiar and generic monophyly using a multilocus phylogeny

The Rhynchocinetidae (‘hinge‐beak’ shrimps) is a family of marine caridean decapods with considerable variation in sexual dimorphism, male weaponry, mating tactics, and sexual systems. Thus, this group is an excellent model with which to analyse the evolution of these important characteristics, which are of interest not only in shrimps specifically but also in animal taxa in general. Yet, there exists no phylogenetic hypothesis, either molecular or morphological, for this taxon against which to test either the evolution of behavioural traits within the Rhynchocinetidae or its genealogical relationships with other caridean taxa. In this study, we tested (1) hypotheses on the phylogenetic relationships of rhynchocinetid shrimps, and (2) the efficacy of different (one‐, two‐, and three‐phase) methods to generate a reliable phylogeny. Total genomic DNA was extracted from tissue samples taken from 17 species of Rhynchocinetidae and five other species currently or previously assigned to the same superfamily (Nematocarcinoidea); six species from other superfamilies were used as outgroups. Sequences from two nuclear genes (H3 and Enolase) and one mitochondrial gene (12S) were used to construct phylogenies. One‐phase phylogenetic analyses (SATé‐II) and classical two‐ and three‐phase phylogenetic analyses were employed, using both maximum likelihood and Bayesian inference methods. Both a two‐gene data set (H3 and Enolase) and a three‐gene data set (H3, Enolase, 12S) were utilized to explore the relationships amongst the targeted species. These analyses showed that the superfamily Nematocarcinoidea, as currently accepted, is polyphyletic. Furthermore, the two major clades recognized by the SATé‐II analysis are clearly concordant with the genera Rhynchocinetes and Cinetorhynchus, which are currently recognized in the morphological‐based classification (implicit phylogeny) as composing the family Rhynchocinetidae. The SATé‐II method is considered superior to the other phylogenetic analyses employed, which failed to recognize these two major clades. Studies using more genes and a more complete species data set are needed to test yet unresolved inter‐ and intrafamilial systematic and evolutionary questions about this remarkable clade of caridean shrimps. © 2014 The Linnean Society of London     

Examination of evolutionary relationships in the Cape fossorial skink species complex (Acontinae: Acontias meleagris meleagris) reveals the presence of five cryptic lineages

Variable morphological characters have obscured genealogical relationships in the Cape fossorial skink Acontias meleagris meleagris species complex. Currently the species complex contains four dubious operational taxonomic units (A. meleagris meleagris, A. m. orientalis, A. percivali tasmani and the morph lineicauda) with poorly defined species boundaries. In the present study we examine the evolutionary relationships within the species complex by sampling 24 skink populations from the known geographical distribution in the Western and Eastern Cape provinces of South Africa, representing a total of 119 specimens. We used partial sequence data derived from two mitochondrial DNA genes, 16S rRNA and COI, and one nuclear DNA gene, intron β‐fibrinogen (β‐fibint 7), to examine evolutionary relationships. Phylogenetic relationships were determined using both Maximum Parsimony (MP) and Bayesian inference (BI) from the combined mtDNA, nDNA and the total evidence data. Additionally we employed Maximum likelihood (ML) analyses on the total evidence data that comprised ～1.5 kb. Topologies derived from the combined mtDNA analyses were congruent with the total evidence analyses (mtDNA + nDNA) and retrieved five major clades with strong statistical support inferred from bootstrapping and posterior probabilities. The five clades were genealogically and geographically exclusive, diagnostic at both the mtDNA and nDNA level and characterized by pronounced sequence divergence, with no shared haplotypes between clades. Collectively these results suggest the presence of five putative cryptic operational taxonomic units within the A. meleagris meleagris species complex. Constraining the traditionally recognized taxa always retrieved a statistically worse topology suggesting that considerable taxonomic revision is required. Our results indicate that traditional morphological characters need to be reassessed to define the five novel lineages in the A. meleagris meleagris species complex. The phylogeographic pattern for the fossorial skinks we retrieved was novel compared to phylogeographic studies for codistributed above ground living taxa. These results suggest that the abiotic and biotic factors that impact subterranean taxa may differ from supraterranean taxa.     

Molecular phylogenetics of slit‐faced bats (Chiroptera: Nycteridae) reveal deeply divergent African lineages

The bat family Nycteridae contains only the genus Nycteris, which comprises 13 currently recognized species from Africa and the Arabian Peninsula, one species from Madagascar, and two species restricted to Malaysia and Indonesia in South‐East Asia. We investigated genetic variation, clade membership, and phylogenetic relationships in Nycteridae with broad sampling across Africa for most clades. We sequenced mitochondrial cytochrome b (cytb) and four independent nuclear introns (2,166 bp) from 253 individuals. Although our samples did not include all recognized species, we recovered at least 16 deeply divergent monophyletic lineages using independent mitochondrial and multilocus nuclear datasets in both gene tree and species tree analyses. Mean pairwise uncorrected genetic distances among species‐ranked Nycteris clades (17% for cytb and 4% for concatenated introns) suggest high levels of phylogenetic diversity in Nycteridae. We found a large number of designated clades whose members are distributed wholly or partly in East Africa (10 of 16 clades), indicating that Nycteris diversity has been historically underestimated and raising the possibility that additional unsampled and/or undescribed Nycteris species occur in more poorly sampled Central and West Africa. Well‐resolved mitochondrial, concatenated nuclear, and species trees strongly supported African ancestry for SE Asian species. Species tree analyses strongly support two deeply diverged subclades that have not previously been recognized, and these clades may warrant recognition as subgenera. Our analyses also strongly support four traditionally recognized species groups of Nycteris. Mitonuclear discordance regarding geographic population structure in Nycteris thebaica appears to result from male‐biased dispersal in this species. Our analyses, almost wholly based on museum voucher specimens, serve to identify species‐rank clades that can be tested with independent datasets, such as morphology, vocalizations, distributions, and ectoparasites. Our analyses highlight the need for a comprehensive revision of Nycteridae.     

Molecular evolution of southern North American Cyprinidae (Actinopterygii), with the description of the new genus Tampichthys from central Mexico

Most of the recognized species of the genus Dionda inhabit drainages of the Gulf of Mexico from central Mexico to central Texas, USA, and have been considered a monophyletic group based on morphological, osteological, and allozyme investigations. Phylogenetic relationships of 15 species of Dionda and 34 species from closely related genera were inferred from one mitochondrial (cytb) and three nuclear gene sequences (S7, Rhodopsin, Rag1) totaling 4487 nucleotides. Separate analyses of all four genes yield congruent phylogenies; however the 15 putative species of Dionda evaluated were never recovered as a monophyletic group when species from nine related genera were included in the analyses. Among the ingroup taxa, one well-supported and highly divergent clade is consistently recognized and consists of six recognized and three undescribed northern species currently recognized in the genus Dionda. These nine species inhabit present or past tributaries of the Rio Grande basin of northern Mexico and southern USA, and were recovered as a basal clade in all analyses. Another large, also strongly supported clade, consisting of seven genera, include five southern recognized species currently in the genus Dionda, forming the sister group to the Codoma clade. These five species comprise the "Southern Dionda clade" and inhabit headwaters of the Pánuco-Tamesí drainage and some adjacent coastal rivers in the Tampico Embayment. The consistent and repeated identification of eight different clades recovered in most of the separate gene analyses strongly supports a division of the non-natural genus Dionda. A new genus, Tampichthys, is proposed for the clade of species endemic to east-central Mexico and formerly in Dionda. Tampichthys and the putative monotypic genus Codoma are more related to Mexican species of the genera Cyprinella and Notropis than to other species referred to Dionda sensu stricto.     

Systematics of spiny‐backed treefrogs (Hylidae: Osteocephalus): an Amazonian puzzle

Spiny‐backed tree frogs of the genus Osteocephalus are conspicuous components of the tropical wet forests of the Amazon and the Guiana Shield. Here, we revise the phylogenetic relationships of Osteocephalus and its sister group Tepuihyla, using up to 6134 bp of DNA sequences of nine mitochondrial and one nuclear gene for 338 specimens from eight countries and 218 localities, representing 89% of the 28 currently recognized nominal species. Our phylogenetic analyses reveal (i) the paraphyly of Osteocephalus with respect to Tepuihyla, (ii) the placement of ‘Hyla’ warreni as sister to Tepuihyla, (iii) the non‐monophyly of several currently recognized species within Osteocephalus and (iv) the presence of low (<1%) and overlapping genetic distances among phenotypically well‐characterized nominal species (e.g. O. taurinus and O. oophagus) for the 16S gene fragment used in amphibian DNA barcoding. We propose a new taxonomy, securing the monophyly of Osteocephalus and Tepuihyla by rearranging and redefining the content of both genera and also erect a new genus for the sister group of Osteocephalus. The colouration of newly metamorphosed individuals is proposed as a morphological synapomorphy for Osteocephalus. We recognize and define five monophyletic species groups within Osteocephalus, synonymize three species of Osteocephalus (O. germani, O. phasmatus and O. vilmae) and three species of Tepuihyla (T. celsae, T. galani and T. talbergae) and reallocate three species (Hyla helenae to Osteocephalus, O. exophthalmus to Tepuihyla and O. pearsoni to Dryaderces gen. n.). Furthermore, we flag nine putative new species (an increase to 138% of the current diversity). We conclude that species numbers are largely underestimated, with most hidden diversity centred on widespread and polymorphic nominal species. The evolutionary origin of breeding strategies within Osteocephalus is discussed in the light of this new phylogenetic hypothesis, and a novel type of amplexus (gular amplexus) is described.     

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     

Molecular systematics of the Middle American genus Hypopachus (Anura: Microhylidae)

We present the first phylogenetic study on the widespread Middle American microhylid frog genus Hypopachus. Partial sequences of mitochondrial (12S and 16S ribosomal RNA) and nuclear (rhodopsin) genes (1275 bp total) were analyzed from 43 samples of Hypopachus, three currently recognized species of Gastrophryne, and seven arthroleptid, brevicipitid and microhylid outgroup taxa. Maximum parsimony (PAUP), maximum likelihood (RAxML) and Bayesian inference (MrBayes) optimality criteria were used for phylogenetic analyses, and BEAST was used to estimate divergence dates of major clades. Population-level analyses were conducted with the programs NETWORK and Arlequin. Results confirm the placement of Hypopachus and Gastrophryne as sister taxa, but the latter genus was strongly supported as paraphyletic. The African phrynomerine genus Phrynomantis was recovered as the sister taxon to a monophyletic Chiasmocleis, rendering our well-supported clade of gastrophrynines paraphyletic. Hypopachus barberi was supported as a disjunctly distributed highland species, and we recovered a basal split in lowland populations of Hypopachus variolosus from the Pacific versant of Mexico and elsewhere in the Mesoamerican lowlands. Dating analyses from BEAST estimate speciation within the genus Hypopachus occurred in the late Miocene/early Pliocene for most clades. Previous studies have not found bioacoustic or morphological differences among these lowland clades, and our molecular data support the continued recognition of two species in the genus Hypopachus.     

A multivariate morphometric analysis and systematic review of Pseudonaja (Serpentes,Elapidae, Hydrophiinae)

Pseudonaja is a clade of seven nominal species of elapid snakes distributed throughout Australia and in southern New Guinea. The species‐level systematics of this group is generally considered to be problematic. A recent phylogenetic analysis of mitochondrial DNA sequences for a geographically extensive series of Pseudonaja specimens revealed nine major clades, of which six largely coincide with nominal species (P. affinis, P. guttata, P. inframacula, P. ingrami, P. modesta and P. textilis). The three remaining clades are composed of specimens currently referred to P. nuchalis. This paper presents a multivariate analysis of 30 morphometric variables recorded for 220 specimens, representing the P. affinis, P. inframacula, P. textilis and three P. nuchalis clades (P. guttata, P. ingrami and P. modesta are well‐demarcated species and, accordingly, were not considered). The morphometric data readily separate these putative lineages, affording compelling evidence that they constitute evolutionary species. The names aspidorhyncha and mengdeni are resurrected for two of the three species presently recognized as P. nuchalis. These species, P. affinis, P. inframacula, P. nuchalis and P. textilis are redescribed. © 2009 The Linnean Society of London, Zoological Journal of the Linnean Society, 2009, 155 , 171–197.     

Systematics of North American Froelichia (Amaranthaceae subfam. Gomphrenoideae) II: Phylogeny and biogeographic speciation patterns inferred from nrITS sequence data

Molecular phylogenetic analyses of the nuclear ribosomal DNA internal transcribed spacer (ITS 1 and ITS 2) and the 5.8S gene were used to infer a phylogeny among the ten recognized taxa of Froelichia in North America. Analyses using both maximum parsimony (MP) and maximum-likelihood (ML) depicted a low level of sequence divergence though it was sufficient in most cases to differentiate taxa. Froelichia xantusii, a species restricted to southern Baja California was shown to be the basalmost member of the group subtending three clades. Two of the clades received good bootstrap support in the MP analysis and corresponded to a genetically homogeneous F. interrupta, and a clade comprising the two species F. latifolia and F. texana. A third clade receiving low bootstrap support contained F. floridana, F. gracilis, F. arizonica, and F. drummondii. Species diversity within the genus was centered within the Tamaulipan Brushland region of north-east Mexico and the southern portion of the US state of Texas where taxa from two of the three principal clades occurred, indicating a region of high speciation and diversification within the genus.     

Molecular data reveal cryptic lineages within the northeastern Atlantic and Mediterranean small mussel drills of the Ocinebrina edwardsii complex (Mollusca: Gastropoda: Muricidae)

We used a molecular phylogenetic approach to investigate species delimitations and diversification in the mussel drills of the Ocinebrina edwardsii complex by means of a combination of nuclear (internal transcribed spacer 2, ITS2) and mitochondrial [cytochrome oxidase subunit I (COI) and 16S] sequences. Our sample included 243 specimens ascribed to seven currently accepted species from 51 sites. Five of the samples were from either the type locality of a nominal species or a close nearby locality (O. edwardsii from Corsica, O. carmelae and O. piantonii from the Kerkennah Islands, O. hispidula from the Gulf of Gabès and O. leukos from the Canary Islands), one from the inferred original locality (O. ingloria from Venice Lagoon), and specimens assigned in the recent literature to O. nicolai. We used a combination of distance‐ and tree‐based species delimitation methods to identify Molecular Operational Taxonomic Units (MOTUs) to compare with the a priori species identifications. The consensus tree obtained by BEAST on the COI alignment allows the recognition of several distinct clades supported by the three species delimitation methods employed. The eight‐MOTUs scenario, shared by the Automatic Barcode Gap Discovery (ABGD) and Generalized Mixed Yule‐Coalescent (GMYC) methods, comprises the following major clades: clade A contains the south Tunisian species Ocinebrina piantonii Cecalupo, Buzzurro & Mariani from which the sympatric taxon O. carmelae Cecalupo, Buzzurro & Mariani (new synonym) cannot be separated; clades B and C bring together all populations from the Aegean Sea and some from the Ionian Sea, respectively; clade D groups, on the one hand, the south Tunisian samples morphologically assigned to O. hispidula Pallary and, on the other, Atlantic and Alboran Sea samples (including the Canarian taxon O. leukos Houart); clade E includes a sample from the type locality of O. edwardsii and several samples from the Tyrrhenian Sea; clades F and G correspond to a few samples from the Venice Lagoon and the Tyrrhenian Sea, respectively; clade H groups the bulk of samples from the Adriatic Sea, including samples from the Venice Lagoon morphologically identified as Ocinebrina ingloria (Crosse), and some from the Ionian Sea. No final conclusions could be reached to reconcile the currently recognized morphological taxa with the clades suggested by the COI data. The geographical structure proposed by the mitochondrial markers is similar to that found in other marine invertebrates and partially corresponds to the species defined by shell characters. We propose here a framework for the revision of the Ocinebrina edwardsii species complex, suggesting a geographical pattern for the diversification of this group in the studied area. © 2013 The Linnean Society of London     

Integrative taxonomy uncovers hidden species diversity in the rheophilic genus Potamometra (Hemiptera: Gerridae)

The genus Potamometra Bianchi, 1896 represents big rheophilic semi-aquatic bugs that typically inhabit middle-altitude mountainous streams. Here, we integrated molecular and morphological data for delimiting species boundaries and understanding the evolutionary history of the genus Potamometra. Twenty-seven complete mitochondrial genomes of Potamometra were sequenced, with samples representing most of the known geographically distributed locations around the Sichuan Basin. The results of different species delimitation methods (ABGD, bPTP, GMYC and BPP) based on the monolocus or multilocus data strongly supported the existence of two cryptic new species (Potamometra anderseni Zheng, Ye & Bu, sp. nov. and Potamometra zhengi Zheng, Ye & Bu, sp. nov.) although more entities were found in the tree-based delimitation methods. The two new species were successfully validated using morphological characters within a detailed taxonomic framework. Phylogenetic analyses supported the reciprocal monophyly of the seven highly node-supported clades, which were matched with the five known species and two new taxa. A novel gene arrangement pattern that two trnF (trnF1 and trnF2) genes separated by an intergenic spacer (IGS) were found in all the species except the sister group of Potamometra berezowskii Bianchi, 1896 and Potamometra linnavuorii Chen, Nieser & Bu, 2016. This gene rearrangement event could be explained by the tandem duplication and random loss (TDRL) model. Our study emphasized that the combination of molecular sequence data, morphological characters and mitochondrial structural information could improve the accuracy of species delimitation.     

PHYLOGENETIC ANALYSIS OF THE GENUS EUGLENA (EUGLENOPHYCEAE) WITH PARTICULAR REFERENCE TO THE TYPE SPECIES EUGLENA VIRIDIS1

Euglena viridis (subgenus Euglena) serves as the type species for the genus Euglena. In this study, molecular phylogenetic analyses using a small subunit (SSU) and a combined SSU–partial large subunit rDNA data set for members of the genus Euglena showed that strains identified as E. viridis on the basis of morphology are distributed between two separate nonsister clades. Although all the E. viridis strains examined were morphologically indistinguishable and possessed spherical mucocysts and stellate chloroplasts with one paramylon center, there was a high degree of sequence divergence between the E. viridis strains in different clades, making this a cryptic species. Like E. viridis, all taxa from the subgenus Euglena are characterized by having one or more stellate chloroplasts with paramylon grains clustered around the center of the chloroplast. These additional taxa were divided into four clades in all the molecular analyses. Strains of Euglena stellata formed two nonsister clades whose members had a single aggregate chloroplast with paramylon center and spindle‐shaped mucocysts. A geniculata clade included species with one or two stellate chloroplasts with paramylon centers and spherical mucocysts, and the cantabrica clade had members with one stellate chloroplast with paramylon center and spherical mucocysts often arranged in spiral rows. Interspersed among these were three additional clades bearing taxa from the subgenus Calliglena that contains members with discoid plastids and pyrenoids that may or may not be capped with paramylon. These taxa formed a laciniata clade, mutabilis clade, and gracilis clade. This study demonstrates that E. viridis and E. stellata are cryptic species that can only be distinguished at the molecular level. Because E. viridis is the designated type species for the genus Euglena, we designated an epitype for E. viridis.