Genetic structure and historical diversification of catfish Brachyplatystoma platynemum (Siluriformes: Pimelodidae) in the Amazon basin with implications for its conservation

Brachyplatystoma platynemum is a catfish species widely distributed in the Amazon basin. Despite being considered of little commercial interest, the decline in other fish populations has contributed to the increase in the catches of this species. The structure, population genetic variability, and evolutionary process that have driven the diversification of this species are presently unknown. Considering that, in order to better understand the genetic structure of this species, we analyzed individuals from seven locations of the Amazon basin using eight molecular markers: control region and cytochrome b mtDNA sequences, and a set of six nuclear microsatellite loci. The results show high levels of haplotype diversity and point to the occurrence of two structured populations (Amazon River and the Madeira River) with high values for F_ST. Divergence time estimates based on mtDNA indicated that these populations diverged about 1.0 Mya (0.2–2.5 Mya 95% HPD) using cytochrome b and 1.4 Mya (0.2–2.7 Mya 95% HPD) using control region. During that time, the influence of climate changes and hydrological events such as sea level oscillations and drainage isolation as a result of geological processes in the Pleistocene may have contributed to the current structure of B. platynemum populations, as well as of differences in water chemistry in Madeira River. The strong genetic structure and the time of genetic divergence estimated for the groups may indicate the existence of strong structure populations of B. platynemum in the Amazon basin.     

A molecular assessment of species diversity in Tympanopleura and Ageneiosus catfishes (Auchenipteridae: Siluriformes)

In order to test the congruence of genetic data to the morphologically defined Neotropical catfish genera Tympanopleura and Ageneiosus and explore species diversity, we generated 17 DNA barcodes from five of six species of Tympanopleura and 12 of 13 species of Ageneiosus. To discriminate limits between species, an automatic barcode gap discovery (ABGD), a generalised mixed yule-coalescent model (GYMC) and fixed distance thresholds Kimura two-parameter (K2P; 3%) were used to discriminate putative species limits from the DNA barcodes. The ABGD, GMYC and K2P methods agreed by each generating 13 clusters: six in Tympanopleura (five nominal plus one undescribed species) and seven in Ageneiosus. These clusters corresponded broadly to the described species, except in the case of the Ageneiosus ucayalensis group (A. akamai, A. dentatus, A. intrusus, A. ucayalensis, A. uranophthalmus and A. vittatus). Haplotype sharing and low divergences may have prevented molecular methods from distinguishing these species. We hypothesise that this is the result of a recent radiation of a sympatric species group distributed throughout the Amazon Basin. One putative new species of Tympanopleura was also supported by the molecular data. These results taken together highlight the utility of molecular methods such as DNA barcoding in understanding patterns of diversification across large geographic areas and in recognising overlooked diversity.     

Cryptic diversity revealed by DNA barcoding in Colombian illegally traded bird species

Colombia is the country with the largest number of bird species worldwide, yet its avifauna is seriously threatened by habitat degradation and poaching. We built a DNA barcode library of nearly half of the bird species listed in the CITES appendices for Colombia, thereby constructing a species identification reference that will help in global efforts for controlling illegal species trade. We obtained the COI barcode sequence of 151 species based on 281 samples, representing 46% of CITES bird species registered for Colombia. The species analysed belong to nine families, where Trochilidae and Psittacidae are the most abundant ones. We sequenced for the first time the DNA barcode of 47 species, mainly hummingbirds endemic of the Northern Andes region. We found a correct match between morphological and genetic identification for 86–92% of the species analysed, depending on the cluster analysis performed (BIN, ABGD and TaxonDNA). Additionally, we identified eleven cases of high intraspecific divergence based on K2P genetic distances (up to 14.61%) that could reflect cryptic diversity. In these cases, the specimens were collected in geographically distant sites such as different mountain systems, opposite flanks of the mountain or different elevations. Likewise, we found two cases of possible hybridization and incomplete lineage sorting. This survey constitutes the first attempt to build the DNA barcode library of endangered bird species in Colombia establishing as a reference for management programs of illegal species trade, and providing major insights of phylogeographic structure that can guide future taxonomic research.     

Applications of DNA barcoding to fish landings: authentication and diversity assessment

DNA barcoding methodologies are being increasingly applied not only for scientific purposes but also for diverse real-life uses. Fisheries assessment is a potential niche for DNA barcoding, which serves for species authentication and may also be used for estimating within-population genetic diversity of exploited fish. Analysis of single-sequence barcodes has been proposed as a shortcut for measuring diversity in addition to the original purpose of species identification. Here we explore the relative utility of different mitochondrial sequences (12S rDNA, COI, cyt b, and D-Loop) for application as barcodes in fisheries sciences, using as case studies two marine and two freshwater catches of contrasting diversity levels. Ambiguous catch identification from COI and cyt b was observed. In some cases this could be attributed to duplicated names in databases, but in others it could be due to mitochondrial introgression between closely related species that may obscure species assignation from mtDNA. This last problem could be solved using a combination of mitochondrial and nuclear genes. We suggest to simultaneously analyze one conserved and one more polymorphic gene to identify species and assess diversity in fish catches.     

DNA barcoding reveals taxonomic uncertainty in Salminus (Characiformes)

Salminus is a genus composed of four species of migratory fishes and top predators. Although this group has great economic and ecological importance, the species level diversity of Salminus is not yet completely clarified. Our goal was to detect if this taxonomic problem is the consequence of lineage divergence within species, and, if so, whether these divergences are sufficient to flag potentially undescribed taxa. We employed the standard DNA barcoding analyses and a generalized mixed Yule-coalescent model (GMYC) using one mitochondrial (COI) marker and Bayesian Inference (BI) reconstruction for one nuclear (RAG2) marker for all currently recognized species of Salminus, sampled across different hydrographic basins. Eight MOTUs (Molecular Operational Taxonomic Units) were determined by distance and model-based analyses, and recovered with BI analyses for COI. Only Salminus affinis and Salminus franciscanus formed monophyletic haplogroups. Salminus brasiliensis and Salminus hilarii had two and four distinct mitochondrial lineages, respectively, and higher intraspecific K2P distances than the adopted optimum threshold. The RAG2 gene tree supported two lineages of S. hilarii (S. hilarii Amazon and S. hilarii Araguaia), while the other mitochondrial lineages of S. hilarii and S. brasiliensis were not supported. All lineages of both species, corresponded to morphological variation described in previous studies. We suggest, based on the DNA barcoding analysis, a new taxonomic scenario and conservation polices for Salminus in the Brazilian territory.     

Cave-dwelling populations of Rhamdia (Siluriformes: Heptapteridae) from the Sierra de Zongolica,Veracruz, Mexico: an evidence-based checklist with comments on their evolutionary history and taxonomy

In this study, the authors present a comprehensive checklist of cave-dwelling populations of Rhamdia from the Sierra de Zongolica (SdZ) in Veracruz, Mexico, including two new records, based on permanent and verifiable evidence in the form of voucher specimens. The authors use phylogenetic and phylogeographic analyses of voucher-derived comparative cytochrome b data to shed light on the evolutionary history of these populations. The results of this study, while preliminary, suggest that hypogean forms of Rhamdia from the SdZ are cave-adapted populations of the more widespread and epigean species R. laticauda.     

Cryptic diversity in Indo-Australian rainbowfishes revealed by DNA barcoding: implications for conservation in a biodiversity hotspot candidate

The rainbowfishes of the family Melanotaeniidae represent one of the largest radiations of freshwater fishes from the Indo-Australian archipelago. A total of 75 nominal species have been described, among which several have become very popular among tropical fish hobbyists because of their tendency to form large schools of colourful individuals. Facing habitat loss and competition or predation by introduced species, this group has become a priority in the conservation of ornamental fishes in Indonesia. In this context, several expeditions have been conducted between 2007 and 2010 in Indonesian Papua with the aim to initiate a large-scale survey of the genetic resources in this group. We assessed the diversity of the Papua rainbowfishes with DNA barcoding. We sequenced the mitochondrial COI gene for 350 specimens belonging to 53 nominal species throughout the Indo-Australian archipelago. Unexpected levels of cryptic diversity and endemism were detected since additional cryptic lineages were detected in several watersheds from the Vogelkop and the Lengguru massif. DNA barcoding supports the presence of nearly 30 evolutionary lineages among the 15 nominal species sampled in the Vogelkop and all these lineages are endemic to a single lake or watershed. This result highlights that the diversity of the family has been largely underestimated and urges for the identification of conservation priorities in Papua.     

Cryptic species and morphological plasticity in long-lived bivalves (Unionoida: Hyriidae) from inland Australia

Molecular (mitochondrial DNA, isozyme) and morphological diversity of freshwater mussels (Family Hyriidae) was examined at 21 sites encompassing four large river systems, across southwest Queensland, Australia. Evidence was found for two major morphological groups. One group, which occurred in every river system, closely matched a recognized species (Velesunio ambiguus) both morphologically and in a well-supported lineage within a mitochondrial phylogeny generated from partial cytochrome c oxidase subunit I (COI) sequences. The second group most closely matched Velesunio wilsonii in shell morphology but formed three deeply divergent mitochondrial DNA lineages. All four lineages occurred sympatrically in some areas and displayed corresponding fixed differences at nuclear allozyme loci, which suggests an absence of recent hybridization and the presence of separate species.     

Multiple Pleistocene refugia and post‐glacial colonization in the European chub (Squalius cephalus) revealed by combined use of nuclear and mitochondrial markers

Aim To analyse patterns of nuclear and mitochondrial genetic variation in the European chub, Squalius cephalus (Linnaeus, 1758), in order to understand the evolutionary history of this species and to test biogeographical hypotheses for the existence of co‐distributed European freshwater fish species. Location Rivers in Europe (Finland, Poland, Czech Republic, France, Bulgaria, Spain, Italy). Methods We genotyped 12 polymorphic microsatellite markers derived from 310 individuals collected from across the distribution of S. cephalus in Europe (including a total of 15 populations) and sequenced mitochondrial DNA (mtDNA) from a subset of 75 individuals. Sequences of mtDNA cytochrome b were analysed using both phylogenetic (median‐joining networks) and population genetic methods (tests for demographic history, mismatch distributions, Bayesian coalescent analysis). Geographical structure in microsatellite loci was examined using a distance method (F_ST), factorial correspondence analysis (FCA) and a Bayesian clustering method (structure ). Results The mtDNA network showed a clear split into four different haplogroup lineages: Western (separated into Atlantic and Danubian sublineages), Eastern, Aegean (occurring in two distinct sublineages in the Balkans and in Spain) and Adriatic. Our results indicate recent population expansion in the Eastern and Western Atlantic lineages and the admixture of two previously separate sublineages (Atlantic and Danubian) in the Western lineage. Bayesian structure analysis as well as FCA results roughly corresponded to the mtDNA‐based structure, separating the sampled individuals into almost non‐overlapping groups. Main conclusions Our results support hypotheses suggesting origins of extant lineages of freshwater fishes in multiple refugia and the subsequent post‐glacial colonization of Europe via different routes. We confirmed the previously proposed two‐step expansion scenario from the Danube refuge, the existence of a secondary (Atlantic) refuge during the last glaciation (probably in the Rhone River) and population expansion of this lineage. Conspicuous divergences among Mediterranean populations reflect their different origin, as well as their low contribution to the recent genetic pool of chub in central Europe.     

Description of a new genus and three new species of Otothyrinae (Siluriformes,Loricariidae)

The genus Hisonotus was resurrected as a member of the tribe Otothyrini (actually subfamily Otothyrinae). However, phylogenetic studies based on morphological and molecular data showed that Hisonotus is not monophyletic and independent lineages can be identified, such as the group composed of the species Hisonotus insperatus, Hisonotus luteofrenatus, Hisonotus oliveirai, Hisonotus paresi and Hisonotus piracanjuba, a lineage unrelated to that containing the type species of the genus Hisonotus (Hisonotus notatus). Herein, based in molecular and morphological data, a new genus is described to accommodate the lineage mentioned above, into which are also added three new species. This new genus can be distinguished from other genera of Otothyrinae by the following combination of characters: (1) a pair of rostral plates at the tip of the snout; (2) two large pre-nasal plates just posterior to the rostral plates; (3) a supra-opercular plate that receives the laterosensory canal from the compound pterotic before the preopercle; (4) a well developed membrane at anal opening in females; and (5) a V-shaped spinelet. A key to species of Curculionichthys is provided.     

Neoplecostomus doceensis: a new loricariid species (Teleostei,Siluriformes) from the rio Doce basin and comments about its putative origin

A new species of Neoplecostomus is described from the rio Doce basin representing the first species of this genus in the basin. The new species is distinguished from its congeners by having enlarged, fleshy folds between dentaries, two or three series of developed papillae anterior to premaxillary teeth and a adipose-fin membrane present, and by lacking enlarged odontodes along snout lateral margins in mature males, a well-developed dorsal-fin spinelet wider than dorsal-fin spine base, lower number of lateral-line figs and developed membrane on the dorsal portion of the first, second and third pelvic-fin branched rays. Additionally, we present a brief discussion of biogeographic scenarios that may explain the distribution of the new species in the rio Doce basin. We suggested that the ancestral lineage of the new species reached the rio Doce from the upper portions of rio Paraná drainages about 3.5 Mya (95% HPD: 1.6–5.5) indicating a colonization route of the N. doceensis ancestral lineage from the south end of Serra do Espinhaço, probably as a result of headwater capture processes between the upper rio Paraná and rio Doce basins.     

DNA barcoding uncovers extensive cryptic diversity in the African long-fin tetra Bryconalestes longipinnis (Alestidae: Characiformes)

To investigate the presence of cryptic diversity in the African longfin-tetra Bryconalestes longipinnis, we employed DNA barcoding in a phylogeographic context, as well as geometric morphometrics, documenting for the first time genetic and body shape variation in the species. Analysis of cytochrome oxidase I gene (coI) sequence variation exposed extremely high levels of genetic differentiation among samples from across the geographic range of the species (up to 18%), certainly much greater than the traditionally employed c. 3% sequence divergence heuristic threshold for conspecifics. Phylogeographic analyses of coI data revealed eight clusters/clades that diverge by >4% and up to 18% (p-distance), potentially representing cryptic members of a species complex. A clear biogeographic pattern was also uncovered, in which the two main coI lineages corresponded geographically with the upper Guinea (UG) and lower Guinea (LG) ichthyofaunal provinces of continental Africa, respectively. Within each of these main lineages, however, no apparent phylogeographic structuring was found. Despite strong genetic differentiation, there is considerable overlap in body shape variation between UG and LG populations. For the most part, morphological variation does not match the strength of the molecular phylogeographic signal. Therefore, the ability to reliably utilise external body shape for regional delimitation remains elusive. Further anatomical investigation appears necessary to establish whether compelling diagnostic morphological features do exist between the divergent lineages of the B. longipinnis complex uncovered in this study.     

Cryptic biodiversity and phylogenetic relationships revealed by DNA barcoding of Oriental black flies in the subgenus Gomphostilbia (Diptera: Simuliidae)

Understanding the medical, economic, and ecological importance of black flies relies on correct identification of species. However, traditional taxonomy of black flies is impeded by a high degree of morphological uniformity, especially the presence of cryptic biodiversity, historically recognized by details of chromosomal banding patterns. We assess the utility of DNA barcoding, based on cytochrome c oxidase subunit 1 (COI) sequences, for identifying 13 species of Oriental black flies in the subgenus Gomphostilbia. Samples of larvae fixed in Carnoy's solution were used to gather molecular and chromosomal data from the same individual. We found that larvae refrigerated in Carnoy's fixative for as long as 11 years can be used for DNA study. Levels of intraspecific genetic divergence, based on the Kimura-2 parameter, range from 0% to 9.28%, with a mean of 2.75%, whereas interspecific genetic divergence ranges from 0.34% to 16.05%. Values of intraspecific and interspecific genetic divergence overlap in seven species owing to incomplete lineage sorting and imperfect taxonomy, implying that DNA barcoding to identify these species will be ambiguous. Despite a low level of success, we found that DNA barcoding is useful in revealing cryptic biodiversity, potentially facilitating traditional taxonomy. Phylogenetic analyses indicate that species groups currently recognized on morphological criteria are not monophyletic, suggesting a need to reevaluate the classification of the subgenus Gomphostilbia.     

Taxonomic revision of the South American fish genus Chalceus Cuvier (Teleostei: Ostariophysi: Characiformes) with the description of three new species

The Neotropical characid fishes of the genus Chalceus Cuvier, 1817, are reviewed. In total, five species are recognized (including three new species). Chalceus epakros sp. nov. is the most widespread geographically, occurring in many rivers of the Amazon basin, the Río Orinoco and in the Essequibo River drainage in Guyana. Chalceus guaporensis sp. nov. is restricted to the upper Rio Madeira, Rio Guaporé and Río Madre de Dios, of Brazil, Bolivia and Peru, respectively. Chalceus spilogyros sp. nov. occurs only in the Rio Trombetas, lower Rio Tapajós and lower Rio Madeira drainages. Chalceus macrolepidotus Cuvier occurs in the Rio Negro drainage, middle and lower Río Orinoco basin and in the rivers of the Atlantic slopes of the Guianas, and Chalceus erythrurus Cope in the Rio Amazonas and Rio Solimões to Río Ucayali drainage in Peru. Chalceus ararapeera Cuvier & Valenciennes and Creagrutus pellegrini Puyo are considered synonyms of C. macrolepidotus , and Pellegrinina heterolepis Fowler is placed into the synonymy of C. erythrurus . Characters pertaining to the monophyly of Chalceus are discussed and a key to species is presented.  © 2004 The Linnean Society of London, Zoological Journal of the Linnean Society , 2004, 140 , 103−135.     

Population structure and biogeography of migratory freshwater fishes (Prochilodus: Characiformes) in major South American rivers

Mitochondrial DNA (mtDNA) sequences were used to infer the phylogenetic relationships of Prochilodus species in the Paraná, Amazonas, Orinoco, and Magdalena basins. Sequences of ATPase subunits 6 and 8 (total 840 bp) were obtained for 21 Prochilodus specimens from the four river systems. Using Semaprochilodus as an outgroup, phylogenetic analyses showed that: (i) each river basin contains a monophyletic group of mtDNA lineages; and (ii) the branching order places Magdalena in a basal position with subsequent branching of Orinoco, Amazon and Paraná. The mitochondrial control region was sequenced for 26 P. lineatus (from the Paraná basin) and six other Prochilodus specimens from the Magdalena, Orinoco and Amazon. All 26 control region haplotypes were unique with sequence divergence ranging from 0.3 to 3.6%. The control region phylogeny is well resolved but phylogenetic structure is not associated with geography. For example, mtDNA haplotypes from the upper Paraná (Mogui Guassú) and the upper Bermejo, separated by at least 2600 km, have close genealogical ties. Phylogeographic analyses, including nested clade analysis, suggest high levels of gene flow within this basin.     

New Ecuadorian records of the eyeless banjo catfish Micromyzon akamai (Siluriformes: Aspredinidae) expand the species range and reveal intraspecific morphological variation

Two specimens of Micromyzon akamai, an eyeless and miniaturized species previously known only from the deep channels of the eastern Amazon basin in Brazil, are reported from the Curaray River, a tributary of the Napo River in Ecuador. The new specimens are the first records of Micromyzon in the headwaters of the Amazon River and the first records of M. akamai outside Brazil. External morphological characters and a phylogenetic analysis of cytochrome c oxidase I (coI) gene support the identification of the new specimens as M. akamai. Nevertheless, the new specimens also indicate that some features previously hypothesized to be apomorphic for M. akamai are intraspecifically variable.     

Cryptic diversity in flathead fishes (Scorpaeniformes: Platycephalidae) across the Indo‐West Pacific uncovered by DNA barcoding

Identification of taxonomical units underpins most biological endeavours ranging from accurate biodiversity estimates to the effective management of sustainably harvested, protected or endangered species. Successful species identification is now frequently based on a combination of approaches including morphometrics and DNA markers. Sequencing of the mitochondrial COI gene is an established methodology with an international campaign directed at barcoding all fishes. We employed COI sequencing alongside traditional taxonomic identification methods and uncovered instances of deep intraspecific genetic divergences among flathead species. Sixty‐five operational taxonomic units (OTUs) were observed across the Indo‐West Pacific from just 48 currently recognized species. The most comprehensively sampled taxon, Platycephalus indicus, exhibited the highest levels of genetic diversity with eight lineages separated by up to 16.37% genetic distance. Our results clearly indicate a thorough reappraisal of the current taxonomy of P. indicus (and its three junior synonyms) is warranted in conjunction with detailed taxonomic work on the other additional Platycephalidae OTUs detected by DNA barcoding.     

DNA barcoding revealed a wider distribution of Alburnoides holciki (Teleostei: Leuciscidae) in the inland waters of Uzbekistan

Alburnoides holciki Coad & Bogutskaya, 2012 was originally described to be found in the Hari River basin. So far, this species is known to occur in tributaries of the Hari River in Afghanistan, Iran and Turkmenistan, and also in the upper reaches of the Amu Darya in Tajikistan. Populations from the inland waters of Uzbekistan were previously considered as A. eichwaldii. Our study recognized specimens of Alburnoides from the inland waters of Uzbekistan as A. holciki based on both morphological data and the molecular data. The novelty of the study is the confirmation of A. holciki for the inland waters of Uzbekistan, which constitutes the northernmost record of this species.     

甘肃省鱼类资源现状及DNA条形码在鱼类物种鉴定中的应用

为了摸清甘肃省土著鱼类资源与分布现状, 探索DNA条形码在鱼类辅助物种鉴定中的适用性, 2012年6-9月对甘肃境内黄河水系、嘉陵江水系和河西内陆河水系进行了较全面的鱼类调查。共采集鱼类标本3,087尾, 隶属于5目10科38属64种, 以鲤科种类最多, 为30种, 占总种数的46.88%。物种多样性分析表明, 在黄河水系的夏河和庄浪河多样性指数是所有调查点中最低的, 分别为1.38和1.09。嘉陵江水系各河段的多样性指数较高(H = 2.15-3.27), 其次为河西内陆河水系(H = 2.01-2.83)。在河西内陆河水系中, 疏勒河的均匀度指数最高, 为1.10, 黑河最低(0.68)。庄浪河的优势度指数最高, 为0.34, 而嘉陵江干流两当段的优势度指数在所有调查点中最低, 为0.04。利用DNA条形码分析了49种662尾标本的COI基因部分序列, 大部分种类在neighbor-joining系统树中形成各自的单系, 种内平均遗传距离0.88%, 种间平均遗传距离为9.99%, 在种内和种间COI序列遗传距离之间形成明显的条形码间隙, 斯氏高原鳅(Triplophysa stoliczkae)与达里湖高原鳅(T. dalaica), 甘肃高原鳅(T. robusta)与似鲇高原鳅(T. siluroides), 嘉陵裸裂尻鱼(Schizopygopsis kialingensis)与黄河裸裂尻鱼(S. pylzovi)之间的遗传距离低于2%, 甘肃高原鳅与似鲇高原鳅不能通过COI基因片段区分开, 其他两对物种可以采用核苷酸诊断法来进一步区分。斯氏高原鳅和拉氏鱼岁(Phoxinus lagowskii)种内遗传分歧较大, 揭示种内可能存在隐存种。结果表明, 对某些近缘种和不同地理种群差异较大的物种, 要将分子、形态和地理分布特点结合起来才能准确鉴定。     

The legacy of a vanished sea: a high level of diversification within a European freshwater amphipod species complex driven by 15 My of Paratethys regression

The formation of continental Europe in the Neogene was due to the regression of the Tethys Ocean and of the Paratethys Sea. The dynamic geology of the area and repetitious transitions between marine and freshwater conditions presented opportunities for the colonization of newly emerging hydrological networks and diversification of aquatic biota. Implementing mitochondrial and nuclear markers in conjunction with a large‐scale sampling strategy, we investigated the impact of this spatiotemporal framework on the evolutionary history of a freshwater crustacean morphospecies. The Gammarus balcanicus species complex is widely distributed in the area previously occupied by the Paratethys Sea. Our results revealed its high diversification and polyphyly in relation to a number of other morphospecies. The distribution of the studied amphipod is generally characterized by very high local endemism and divergence. The Bayesian time‐calibrated reconstruction of phylogeny and geographical distribution of ancestral nodes indicates that this species complex started to diversify in the Early Miocene in the central Balkans, partially in the shallow epicontinental sea. It is possible that there were several episodes of inland water colonization by local brackish water lineages. Subsequent diversification within clades and spread to new areas could have been induced by Alpine orogeny in the Miocene/Pliocene and, finally, by Pleistocene glaciations. The present distribution of clades, in many cases, still reflects Miocene palaeogeography of the area. Our results point out that investigations of the historical aspect of cryptic diversity in other taxa may help in a general understanding of the origins of freshwater invertebrate fauna of Europe.