Using DNA taxonomy to investigate the ecological determinants of plankton diversity: explaining the occurrence of Synchaeta spp. (Rotifera,Monogononta) in mountain lakes

1. The occurrence of unresolved complexes of cryptic species may hinder the identification of the main ecological drivers of biodiversity when different cryptic taxa have different ecological requirements. 2. We assessed factors influencing the occurrence of Synchaeta species (monogonont rotifers) in 17 waterbodies of the Trentino‐South Tyrol region in the Eastern Alps. To do so, we compared the results of using unresolved complexes of cryptic species, as is common practice in limnological studies based on morphological taxonomy, and having resolved cryptic complexes, made possible by DNA taxonomy. 3. To identify cryptic species, we used the generalised mixed Yule coalescent (GMYC) model. We investigated the relationship between the environment and the occurrence of Synchaeta spp. by multivariate ordination using two definitions of the units of diversity, namely (i) unresolved species complexes (morphospecies) and (ii) putative cryptic species (GMYC entities). Our expectation was that resolving complexes of cryptic species could provide more information than using morphospecies. 4. As expected, DNA taxonomy provided greater taxonomic resolution than morphological taxonomy. Further, environmental‐based multivariate ordination on cryptic species explained a significantly higher proportion of variance than that based on morphospecies. Occurrence of GMYC entities was related to total phosphorus (TP), whereas no relationship could be found between morphospecies and the environment. Moreover, different cryptic species within the same morphospecies showed different, and even opposite, preferences for TP. In addition, the wide geographical distribution of haplotypes and cryptic species indicated the absence of barriers to dispersal in Synchaeta.     

High levels of cryptic species diversity uncovered in Amazonian frogs

One of the greatest challenges for biodiversity conservation is the poor understanding of species diversity. Molecular methods have dramatically improved our ability to uncover cryptic species, but the magnitude of cryptic diversity remains unknown, particularly in diverse tropical regions such as the Amazon Basin. Uncovering cryptic diversity in amphibians is particularly pressing because amphibians are going extinct globally at an alarming rate. Here, we use an integrative analysis of two independent Amazonian frog clades, Engystomops toadlets and Hypsiboas treefrogs, to test whether species richness is underestimated and, if so, by how much. We sampled intensively in six countries with a focus in Ecuador (Engystomops: 252 individuals from 36 localities; Hypsiboas: 208 individuals from 65 localities) and combined mitochondrial DNA, nuclear DNA, morphological, and bioacoustic data to detect cryptic species. We found that in both clades, species richness was severely underestimated, with more undescribed species than described species. In Engystomops, the two currently recognized species are actually five to seven species (a 150-250% increase in species richness); in Hypsiboas, two recognized species represent six to nine species (a 200-350% increase). Our results suggest that Amazonian frog biodiversity is much more severely underestimated than previously thought.     

Multi-locus DNA sequence data reveal a history of deep cryptic vicariance and habitat-driven convergence in the desert night lizard Xantusia vigilis species complex (Squamata: Xantusiidae)

The lizard genus Xantusia of southwestern North America has received recent attention in relation to delimiting species. Using more than 500 lizards from 156 localities, we further test hypothesized species boundaries and clarify phylogeographical patterns, particularly in regions of potential secondary contact. We sequenced the entire mitochondrial cytochrome b gene for every lizard in the study, plus a second mitochondrial DNA (mtDNA) region and two nuclear introns for subsets of the total sample. Phylogenetic analyses of the mtDNA recover a well-resolved, novel hypothesis for species in the Xantusia vigilis complex. The nuclear DNA (nDNA) data provide independent support for the recognition of X. arizonae, X. bezyi and X. wigginsi. Differences between the respective mtDNA and nDNA topologies result from either the effects of lineage sorting or ancient introgression. Nuclear data confirm the inference that some populations of X. vigilis in northwestern Arizona converged on rock-crevice-dwelling morphology and are not X. arizonae with an introgressed X. vigilis mtDNA genome. The historical independence of ancient cryptic lineages of Xantusia in southern California is also corroborated, though limited introgression is detected. Our proposed biogeographical scenario indicates that diversification of this group was driven by vicariance beginning in the late Miocene. Additionally, Pleistocene climatical changes influenced Xantusia distribution, and the now inhospitable Colorado Desert previously supported night lizard presence. The current taxonomy of the group likely underestimates species diversity within the group, and our results collectively show that while convergence on the rock-crevice-dwelling morphology is one hallmark of Xantusia evolution, morphological stasis is paradoxically another.     

Hidden Diversity in Sardines: Genetic and Morphological Evidence for Cryptic Species in the Goldstripe Sardinella,Sardinella gibbosa (Bleeker, 1849)

Cryptic species continue to be uncovered in many fish taxa, posing challenges for fisheries conservation and management. In Sardinella gibbosa, previous investigations revealed subtle intra-species variations, resulting in numerous synonyms and a controversial taxonomy for this sardine. Here, we tested for cryptic diversity within S. gibbosa using genetic data from two mitochondrial and one nuclear gene regions of 248 individuals of S. gibbosa, collected from eight locations across the Philippine archipelago. Deep genetic divergence and subsequent clustering was consistent across both mitochondrial and nuclear markers. Clade distribution is geographically limited: Clade 1 is widely distributed in the central Philippines, while Clade 2 is limited to the northernmost sampling site. In addition, morphometric analyses revealed a unique head shape that characterized each genetic clade. Hence, both genetic and morphological evidence strongly suggests a hidden diversity within this common and commercially-important sardine.     

The Necessity of DNA Taxonomy to Reveal Cryptic Diversity and Spatial Distribution of Meiofauna,with a Focus on Nemertea

Meiofauna represent one of the most abundant and diverse communities in marine benthic ecosystems. However, an accurate assessment of diversity at the level of species has been and remains challenging for these microscopic organisms. Therefore, for many taxa, especially the soft body forms such as nemerteans, which often lack clear diagnostic morphological traits, DNA taxonomy is an effective means to assess species diversity. Morphological taxonomy of Nemertea is well documented as complicated by scarcity of unambiguous character states and compromised by diagnoses of a majority of species (and higher clades) being inadequate or based on ambiguous characters and character states. Therefore, recent studies have advocated for the primacy of molecular tools to solve the taxonomy of this group. DNA taxonomy uncovers possible hidden cryptic species, provides a coherent means to systematize taxa in definite clades, and also reveals possible biogeographic patterns. Here, we analyze diversity of nemertean species by considering the barcode region of the mitochondrial gene Cytochrome Oxidase subunit I (COI) and different species delineation approaches in order to infer evolutionarily significant units. In the aim to uncover actual diversity of meiofaunal nemerteans across different sites in Central America, COI sequences were obtained for specimens assigned here to the genera Cephalothrix, Ototyphlonemertes, and Tetrastemma-like worms, each commonly encountered in our sampling. Additional genetic, taxonomic, and geographic data of other specimens belonging to these genera were added from GenBank. Results are consistent across different DNA taxonomy approaches, and revealed (i) the presence of several hidden cryptic species and (ii) numerous potential misidentifications due to traditional taxonomy. (iii) We additionally test a possible biogeographic pattern of taxonomic units revealed by this study, and, except for a few cases, the putative species seem not to be widely distributed, in contrast to what traditional taxonomy would suggest for the recognized morphotypes.     

Molecular-based rapid inventories of sympatric diversity: A comparison of DNA barcode clustering methods applied to geography-based vs clade-based sampling of amphibians

Molecular markers offer a universal source of data for quantifying biodiversity. DNA barcoding uses a standardized genetic marker and a curated reference database to identify known species and to reveal cryptic diversity within well-sampled clades. Rapid biological inventories, e.g. rapid assessment programs (RAPs), unlike most barcoding campaigns, are focused on particular geographic localities rather than on clades. Because of the potentially sparse phylogenetic sampling, the addition of DNA barcoding to RAPs may present a greater challenge for the identification of named species or for revealing cryptic diversity. In this article we evaluate the use of DNA barcoding for quantifying lineage diversity within a single sampling site as compared to clade-based sampling, and present examples from amphibians. We compared algorithms for identifying DNA barcode clusters (e.g. species, cryptic species or Evolutionary Significant Units) using previously published DNA barcode data obtained from geography-based sampling at a site in Central Panama, and from clade-based sampling in Madagascar. We found that clustering algorithms based on genetic distance performed similarly on sympatric as well as clade-based barcode data, while a promising coalescent-based method performed poorly on sympatric data. The various clustering algorithms were also compared in terms of speed and software implementation. Although each method has its shortcomings in certain contexts, we recommend the use of the ABGD method, which not only performs fairly well under either sampling method, but does so in a few seconds and with a user-friendly Web interface.     

Species-level diversification of African dwarf crocodiles (Genus Osteolaemus): A geographic and phylogenetic perspective

The taxonomy of the African dwarf crocodile (genus Osteolaemus) has been disputed since a novel morphotype was discovered in the early 20th Century. Because this poorly-known reptile is widely hunted throughout the forests of Central and West Africa, resolving the existence and extent of taxonomic units has important management and conservation implications. Lack of molecular data from individuals of known origin and historical disagreement on diagnostic morphological characters have hindered attempts to settle one of the most important taxonomic questions in the Crocodylia. In an effort to clarify the evolutionary relationships among dwarf crocodiles, we sequenced three mitochondrial and two nuclear genes using a large sample of dwarf crocodiles from known localities across major drainage basins of forested Africa. Concordant results from Bayesian, maximum likelihood, maximum parsimony and population aggregation analytical methods support a previously recognized division of the dwarf crocodile into a Congo Basin form (O. osborni) and a West African form (Osteolaemus tetraspis), but also reveal a third diagnosable lineage from West Africa warranting recognition as an separate taxonomic unit. Corrected genetic distances between geographic regions ranged from 0.2% to 0.6% in nuclear fragments and 10.0 to 16.2% in mitochondrial COI. Population aggregation, using fixed and alternate character (nucleotide) states to cluster or divide populations, recovered 232 such molecular characters in 4286 bp of sequence data and unambiguously aggregated populations into their respective geographic clade. Several previously recognized morphological differences coincide with our molecular analysis to distinguish Congo Basin crocodiles from the Ogooué Basin and West Africa. Discrete morphological characters have not yet been documented between the latter two regions, suggesting further work is needed or molecular data may be required to recognize taxonomic divisions in cases where putative species are morphologically cryptic. This study highlights the importance of using widespread taxon sampling and a multiple evidence approach to diagnose species boundaries and reveal cryptic diversity.     

DNA barcodes successfully delimit morphospecies in a superdiverse insect genus

Polypedilum Kieffer (Diptera: Chironomidae), with 520 currently known species worldwide, can be extremely difficult to identify species level based on the morphology. We used 3,670 cytochrome c oxidase subunit I (COI) barcodes to explore the efficiency of the COI barcodes to differentiate between species in a superdiverse aquatic insect genus. The Barcode of Life Data System (BOLD) presented 286 BIN clusters in Polypedilum, representing 163 morphospecies, of which 93 were contributed from our laboratory. Molecular operational taxonomic units (OTUs) ranged from 158 to 345, based on Automatic Barcode Gap Discovery (ABGD), the Barcode Index Number (BIN), Bayesian Poisson tree processes (bPTP), generalized mixed Yule coalescent (GMYC), jMOTU, multi‐rate Poisson tree processes (mPTP), neighbor‐joining (NJ) tree and prethreshold clustering. In comparison, GMYC, bPTP, mPTP and BIN suggested more species than warranted by morphology, while ABGD, jMOTU, NJ, prethreshold clustering and ABGD yielded a conservative number of species when setting higher thresholds. Nine species complexes with deep intraspecific divergences indicated 18 potentially cryptic species, which require further taxonomic research including complete life histories and nuclear genetic data to be resolved. The discrimination of Polypedilum species by DNA barcodes proved to be successful in 94.4% of all studied morphological species.     

Integrating a Numerical Taxonomic Method and Molecular Phylogeny for Species Delimitation of Melampsora Species (Melampsoraceae,Pucciniales) on Willows in China

The species in genus Melampsora are the causal agents of leaf rust diseases on willows in natural habitats and plantations. However, the classification and recognition of species diversity are challenging because morphological characteristics are scant and morphological variation in Melampsora on willows has not been thoroughly evaluated. Thus, the taxonomy of Melampsora species on willows remains confused, especially in China where 31 species were reported based on either European or Japanese taxonomic systems. To clarify the species boundaries of Melampsora species on willows in China, we tested two approaches for species delimitation inferred from morphological and molecular variations. Morphological species boundaries were determined based on numerical taxonomic analyses of morphological characteristics in the uredinial and telial stages by cluster analysis and one-way analysis of variance. Phylogenetic species boundaries were delineated based on the generalized mixed Yule-coalescent (GMYC) model analysis of the sequences of the internal transcribed spacer (ITS1 and ITS2) regions including the 5.8S and D1/D2 regions of the large nuclear subunit of the ribosomal RNA gene. Numerical taxonomic analyses of 14 morphological characteristics recognized in the uredinial-telial stages revealed 22 morphological species, whereas the GMYC results recovered 29 phylogenetic species. In total, 17 morphological species were in concordance with the phylogenetic species and 5 morphological species were in concordance with 12 phylogenetic species. Both the morphological and molecular data supported 14 morphological characteristics, including 5 newly recognized characteristics and 9 traditionally emphasized characteristics, as effective for the differentiation of Melampsora species on willows in China. Based on the concordance and discordance of the two species delimitation approaches, we concluded that integrative taxonomy by using both morphological and molecular variations was an effective approach for delimitating Melampsora species on willows in China.     

Molecular phylogeny and DNA barcoding confirm cryptic species in the African freshwater oyster Etheria elliptica Lamarck, 1807 (Bivalvia: Etheriidae)

Recent molecular approaches to taxonomy have led to a steady increase in the identification of cryptic species. Within the Etheriidae, the species Etheria elliptica (freshwater oyster) is widespread and common and exists in most of the major African drainages. Within the African freshwater ecosystems, there are major threats to biodiversity and cryptic species complicate conservation strategies; unknown species exist and no conservation status has been assigned. Our objective here was to determine if E. elliptica from several locations in the Congo drainage are correctly classified as representing a single species. We analysed the genetic diversity at two mitochondrial loci (COI and 16S) and two nuclear loci (H3 and 28S), and estimated evolutionary relationships using phylogenetic and DNA barcoding techniques. Bayesian inference yielded three cryptic species of Etheria, and mismatch analysis revealed discrete differences between the cryptic species. We identified three cryptic species within these collections, and evidence indicates that the third species may resolve further with more sampling. In conclusion, the taxonomic history of E. elliptica makes finding cryptic species unsurprising. However, molecular studies such as this may finally help to resolve the number of species within this genus.     

Mitonuclear discordance as a confounding factor in the DNA taxonomy of monogonont rotifers

Discordance between mitochondrial and nuclear phylogenies is being increasingly recognized in animals and may confound DNA‐based taxonomy. This is especially relevant for taxa whose microscopic size often challenges any effort to distinguish between cryptic species without the assistance of molecular data. Regarding mitonuclear discordance, two strikingly contrasting scenarios have been recently demonstrated in the monogonont rotifers of the genus Brachionus. While strict mitonuclear concordance was observed in the marine B. plicatilis species complex, widespread hybridization‐driven mitonuclear discordance was revealed in the freshwater B. calyciflorus species complex. Here, we investigated the frequency of occurrence and the potential drivers of mitonuclear discordance in three additional freshwater monogonont rotifer taxa, and assessed its potential impact on the reliability of DNA taxonomy results based on commonly used single markers. We studied the cryptic species complexes of Keratella cochlearis, Polyarthra dolichoptera and Synchaeta pectinata. Phylogenetic reconstructions were based on the mitochondrial barcoding marker cytochrome c oxidase subunit I gene and the nuclear internal transcribed spacer 1 locus, which currently represent the two most typical genetic markers used in rotifer DNA taxonomy. Species were delimited according to each marker separately using a combination of tree‐based coalescent, distance‐based and allele‐sharing‐based approaches. Mitonuclear discordance was observed in all species complexes with incomplete lineage sorting and unresolved phylogenetic reconstructions recognized as the likely drivers. Evidence from additional sources, such as morphology and ecology, is thus advisable for deciding between often contrasting mitochondrial and nuclear species scenarios in these organisms.     

Patterns and processes in the genetic differentiation of the Brachionus calyciflorus complex, a passively dispersing freshwater zooplankton

Elucidating the evolutionary patterns and processes of extant species is an important objective of any research program that seeks to understand population divergence and, ultimately, speciation. The island-like nature and temporal fluctuation of limnetic habitats create opportunities for genetic differentiation in rotifers through space and time. To gain further understanding of spatio-temporal patterns of genetic differentiation in rotifers other than the well-studied Brachionus plicatilis complex in brackish water, a total of 318 nrDNA ITS sequences from the B. calyciflorus complex in freshwater were analysed using phylogenetic and phylogeographic methods. DNA taxonomy conducted by both the sequence divergence and the GMYC model suggested the occurrence of six potential cryptic species, supported also by reproductive isolation among the tested lineages. The significant genetic differentiation and non-significant correlation between geographic and genetic distances existed in the most abundant cryptic species, BcI-W and Bc-SW. The large proportion of genetic variability for cryptic species Bc-SW was due to differences between sampling localities within seasons, rather than between different seasons. Nested Clade Analysis suggested allopatric or past fragmentation, contiguous range expansion and long-distance colonization possibly coupled with subsequent fragmentation as the probable main forces shaping the present-day phylogeographic structure of the B. calyciflorus species complex.     

Species identification in the taxonomically neglected, highly diverse, neotropical parasitoid wasp genus Notiospathius (Braconidae: Doryctinae) based on an integrative molecular and morphological approach

Various DNA sequence-based methods for species delineation have recently been developed to assess the species-richness of highly diverse, neglected invertebrate taxa. These methods, however, need to be tested under a variety of conditions, including the use of different markers and parameters. Here, we explored the species diversity of a species-rich group of braconid parasitoid wasps, the Neotropical genus Notiospathius, including 233 specimens from 10 different countries. We examined sequences of two mitochondrial (mt) (COI, cyt b) and one nuclear (wg) gene fragments. We analysed them separately as well as concatenating the mt data with the general mixed Yule-coalescent (GMYC) model for species delineation using different tree-building methods and parameters for reconstructing ultrametric trees. We evaluated the performance of GMYC analyses by comparing their species delineations with our morphospecies identifications. Reconstructing ultrametric trees with a relaxed lognormal clock rate using the program BEAST gave the most congruent results with morphology for the two mt markers. A tree obtained with wg using the programs MrBayes+Pathd8 had the fewest cases of incongruence with morphology, though the performance of this nuclear marker was considerably lower than that of COI and cyt b. Species delimitation using the coalescent prior to obtain ultrametric trees was morphologically more congruent with COI, whereas the Yule prior was more congruent with cyt b. The analyses concatenating the mt datasets failed to recover some species supported both by morphology and the separate analyses of the mt markers. The highest morphological congruence was obtained with the GMYC analysis on an ultrametric tree reconstructed with cyt b using the relaxed lognormal clock rate and the Yule prior, thus supporting the importance of using alternative markers when the information of the barcoding locus (COI) is not concordant with morphological evidence. Seventy-one species were delimited based on the congruence found among COI, cyt b and morphology. Both mt markers also revealed the existence of seven potential cryptic species. This high species richness from a scattered geographical sampling indicates that there is a remarkable number of Notiospathius species that remains undiscovered.     

Unexpectedly High Levels of Cryptic Diversity Uncovered by a Complete DNA Barcoding of Reptiles of the Socotra Archipelago

Few DNA barcoding studies of squamate reptiles have been conducted. Due to the significance of the Socotra Archipelago (a UNESCO Natural World Heritage site and a biodiversity hotspot) and the conservation interest of its reptile fauna (94% endemics), we performed the most comprehensive DNA barcoding study on an island group to date to test its applicability to specimen identification and species discovery. Reptiles constitute Socotra’s most important vertebrate fauna, yet their taxonomy remains under-studied. We successfully DNA-barcoded 380 individuals of all 31 presently recognized species. The specimen identification success rate is moderate to high, and almost all species presented local barcoding gaps. The unexpected high levels of intra-specific variability found within some species suggest cryptic diversity. Species richness may be under-estimated by 13.8–54.4%. This has implications in the species’ ranges and conservation status that should be considered for conservation planning. Other phylogenetic studies using mitochondrial and nuclear markers are congruent with our results. We conclude that, despite its reduced length (663 base pairs), cytochrome c oxidase 1, COI, is very useful for specimen identification and for detecting intra-specific diversity, and has a good phylogenetic signal. We recommend DNA barcoding to be applied to other biodiversity hotspots for quickly and cost-efficiently flagging species discovery, preferentially incorporated into an integrative taxonomic framework.     

Phylogenetic analysis of nuclear and mitochondrial DNA reveals a complex of cryptic species in Crassicutis cichlasomae (Digenea: Apocreadiidae), a parasite of Middle-American cichlids

We obtained nuclear ITS-1 and mitochondrial cox1 sequences from 225 Crassicutis cichlasomae adults collected in 12 species of cichlids from 32 localities to prospect for the presence of cryptic species. This trematode is commonly found in species of cichlids over a wide geographic range in Middle-America. Population-level phylogenetic analyses of ITS-1 and cox1, assessments of genetic and haplotype diversity, and morphological observations revealed that C. cichlasomae represents a complex of seven cryptic species for which no morphological diagnostic characters have been discovered thus far. Bayesian and Maximum Likelihood analyses of concatenated datasets (906 bp) recovered eight lineages of C. cichlasomae, all with high posterior probabilities and bootstrap branch support. Values of genetic divergence between clades ranged from 1.0% to 5.2% for ITS-1, and from 7.2% to 30.0% for cox1. Morphological study of more than 300 individuals did not reveal structural diagnostic traits for the species defined using molecular evidence. These observations indicate that some traditional morphological characters (e.g., testes position) have substantial intra-specific variation, and should be used with caution when classifying C. cichlasomae and their sister taxa. Additionally, phylogenetic analyses did not reveal a strict correlation between these cryptic species and their host species or geographic distribution, however it appears that genetic distinctiveness of these cryptic species was influenced by the diversification and biogeographical history of Middle-American cichlids.     

DNA barcoding for community ecology--how to tackle a hyperdiverse, mostly undescribed Melanesian fauna

Background

Trigonopterus weevils are widely distributed throughout Melanesia and hyperdiverse in New Guinea. They are a dominant feature in natural forests, with narrow altitudinal zonation. Their use in community ecology has been precluded by the “taxonomic impediment”.

Methodology/Principal Findings

We sampled >6,500 specimens from seven areas across New Guinea; 1,002 specimens assigned to 270 morphospecies were DNA sequenced. Objective clustering of a refined dataset (excluding nine cryptic species) at 3% threshold revealed 324 genetic clusters (DNA group count relative to number of morphospecies = 20.0% overestimation of species diversity, or 120.0% agreement) and 85.6% taxonomic accuracy (the proportion of DNA groups that “perfectly” agree with morphology-based species hypotheses). Agreement and accuracy were best at an 8% threshold. GMYC analysis revealed 328 entities (21.5% overestimation) with 227 perfect GMYC entities (84.1% taxonomic accuracy). Both methods outperform the parataxonomist (19% underestimation; 31.6% taxonomic accuracy). The number of species found in more than one sampling area was highest in the Eastern Highlands and Huon (Sørensen similarity index 0.07, 4 shared species); ⅓ of all areas had no species overlap. Success rates of DNA barcoding methods were lowest when species showed a pronounced geographical structure. In general, Trigonopterus show high α and β-diversity across New Guinea.

Conclusions/Significance

DNA barcoding is an excellent tool for biodiversity surveys but success rates might drop when closer localities are included. Hyperdiverse Trigonopterus are a useful taxon for evaluating forest remnants in Melanesia, allowing finer-grained analyses than would be possible with vertebrate taxa commonly used to date. Our protocol should help establish other groups of hyperdiverse fauna as target taxa for community ecology. Sequencing delivers objective data on taxa of incredible diversity but mostly without a solid taxonomic foundation and should help pave the road for the eventual formal naming of new species.     

Review: Taxonomy of Nitella (Charales, Charophyceae) based on comparative morphology of oospores and multiple DNA marker phylogeny using cultured material

The genus Nitella is the largest group in the Charales and has the highest diversity of vegetative and oospore morphologies. In his worldwide monograph on the Charales, R. D. Wood characterized the sections and species of Nitella mainly on the basis of vegetative morphology and treated oospore wall morphology as diagnostic at the infraspecific level. Therefore, many species of Nitella exhibiting distinct external morphology of the oospore wall (EMOW) were reduced to infraspecific rank and only 53 of 204 species previously described were recognized within Nitella . However, recent morphological and molecular phylogenetic studies have demonstrated the phylogenetic validity of using EMOW for diagnosing some species of Nitella . More recently, a scanning electron microscopy (SEM) study of internal morphology of the oospore wall (IMOW) and multiple DNA marker analyses using both nuclear and chloroplast gene sequences were conducted to improve our understanding of the taxonomy of Nitella at the species level, on the basis of cultured material of a large number of species. Multiple DNA marker analyses resolved detailed and robust phylogenetic relationships within the genus and demonstrated the taxonomic and phylogenetic significance of IMOW. In addition, they supported the taxonomic decisions based on differences in oospore morphology, suggesting that an integrated approach, involving both SEM studies of the EMOW and IMOW and multiple DNA marker analyses, is appropriate to address problems at lower taxonomic levels within the genus, as well as to construct a natural taxonomic system for the genus. In this paper, recent morphological and molecular phylogenetic studies are reviewed and recent improvements in taxonomy of Nitella are summarized. Moreover, the evolution of morphological features and phylogenetic relationships within Nitella are discussed, focusing especially on oospore morphology.     

Seven new species within western Atlantic Starksia atlantica, S. lepicoelia, and S. sluiteri (Teleostei, Labrisomidae), with comments on congruence of DNA barcodes and species

Specimens of Starksia were collected throughout the western Atlantic, and a 650-bp portion of the mitochondrial gene cytochrome oxidase-c subunit I (COl) was sequenced as part of a re-analysis of species diversity of western Central Atlantic shorefishes. A neighbor-joining tree constructed from the sequence data suggests the existence of several cryptic species. Voucher specimens from each genetically distinct lineage and color photographs of vouchers taken prior to dissection and preservation were examined for diagnostic morphological characters. The results suggest that Starksia atlantica, Starksia lepicoelia, and Starksia sluiteri are species complexes, and each comprises three or more species. Seven new species are described. DNA data usually support morphological features, but some incongruence between genetic and morphological data exists. Genetic lineages are only recognized as species if supported by morphology. Genetic lineages within western Atlantic Starksia generally correspond to geography, such that members of each species complex have a very restricted geographical distribution. Increasing geographical coverage of sampling locations will almost certainly increase the number of Starksia species and species complexes recognized in the western Atlantic. Combining molecular and morphological investigations is bringing clarity to the taxonomy of many genera of morphologically similar fishes and increasing the number of currently recognized species. Future phylogenetic studies should help resolve species relationships and shed light on patterns of speciation in western Atlantic Starksia.     

Molecular and morphological delimitation of Australian <Emphasis Type="Italic">Triops</Emphasis> species (Crustacea: Branchiopoda: Notostraca)—large diversity and little morphological differentiation

Australia has a very rich and diverse large branchiopod fauna with approximately 140 described or provisionally delimited species, but only one species of Triops, Triops australiensis (Spencer and Hall 1895), is currently recognized. Previous studies identified extensive genetic diversity within T. australiensis that suggested the presence of cryptic species. Herein, we employed an integrative approach to taxonomy to delimit putative species, integrating COI and EF1α sequence data and morphological data. Putative species were initially delimited based on COI by two computational approaches (GMYC and ABGD). The results were interpreted in the light of several species concepts, with particular emphasis on reproductive isolation. Twenty to 27 genetic lineages were delimited. Of these, up to 26 represent species following an evolutionary or phylogenetic species concept. Eighteen are biological species, though reproductive isolation could not be unambiguously established for allopatric species or species without known males. The level of co-occurrences was exceptionally high for Triops, with up to three syntopic and six sympatric species. Species delimitation was impeded by extensive overlap between intraspecific variability and interspecific variation in the genetic as well as morphological datasets. Without prior delimitation of putative species via COI, morphological delimitation would have been impossible. A potential explanation for the morphological variability is the retention of ancestral polymorphisms over long periods of time and across multiple speciation events without subsequent differentiation.     

Genetic analysis of an endemic archipelagic lizard reveals sympatric cryptic lineages and taxonomic discordance

The importance of genetic data in biodiversity conservation is well established, and knowledge of standing genetic variation within and between populations is important for designing conservation strategies. We investigated partitioning of genetic diversity in an endemic lizard (Leiocephalus psammodromus) distributed in the Turks and Caicos archipelago using mtDNA and AFLP data from 259 individuals sampled across 13 islands. Current taxonomy identifies six or more subspecies of L. psammodromus within the archipelago, several of which have undergone recent drastic reductions in range due to extirpation. However, our results indicate the presence of two independent lineages, one on each of the Turks and Caicos banks, and a third sympatric cryptic lineage on both banks. These lineages do not correspond to current taxonomy and alter our understanding of diversity and conservation of this species. Gross morphological data (mass and snout-vent length) indicate some variation in female size among lineages, indicating the possibility of cryptic morphological variation. Instead of initiating separate conservation measures for nominate subspecies, we recommend a more thorough investigation of the morphology and genetics of this group and a more inclusive conservation program. Our surprising results indicate that other endemic squamates in the Bahamas Archipelago might also exhibit sympatric cryptic diversity that does not correspond to current taxonomic understanding and could have significant impacts on our approach to conservation in this region.