Geometric morphometrics as a tool for identifying emperor fish (Lethrinidae) larvae and juveniles

The aim of this study was to evaluate the efficiency of geometric morphometrics for describing the body shape of fish larvae and juveniles, and identifying them to species, in comparison with traditional linear measurements. Species of emperor fishes (Perciformes: Lethrinidae, genus Lethrinus) were chosen as the model group, as the late larval and early juvenile stages in this genus are particularly difficult to identify. Forty‐five individuals of different species of Lethrinus were collected from the south‐western lagoon of New Caledonia between May 2005 and March 2006. The individuals were first identified to species by their partial cytochrome‐b gene sequence. They were then morphologically characterized using eight linear measurements and 23 landmarks recorded on digital photographs. Except for a small proportion of individuals, geometric morphometrics gave better results to distinguish the different species than linear measurements. A ‘leave one out’ approach confirmed the nearly total discrimination of recently settled Lethrinus genivittatus and Lethrinus nebulosus, whereas traditional identification keys failed to distinguish them. Therefore, geometric morphometrics is a promising tool for identifying fish larvae and juveniles to species. An effective approach would require building image databases of voucher specimens associated with their DNA barcodes. These images could be downloaded by the operator and processed with the specimens to be identified.     

SYSTEMATIC EVALUATION OF THE GENUS HILDENBRANDIA (RHODOPHYTA): A SYNTHESIS OF TECHNIQUES

The evolutionary relationships among members of the red algal genus Hildenbrandia have not been well understood for several reasons. For example, the genus contains both marine and freshwater representatives, all of which are non-calcified and crustose, and few have definitive morphological characters for classification. Hildenbrandia is also assumed to be completely asexual (reproduction by tetrasporangia in marine forms and by gemmae in freshwater populations), and characters of the female gametangial system and post-fertilization structures are not available for comparative studies. Currently there are 14 marine and five freshwater species and infraspecific taxa recognized within the genus. We used phylogenetic analyses (parsimony, distance and maximum likelihood) of DNA sequences of commonly employed genes ( rbc L and 18S rRNA) to examine the evolutionary relationships among representatives of many of these taxa. In addition, we employed morphometrics (principal co-ordinates and cluster analyses) of several measured characters of these same representatives, as well as all available type specimens, to determine the number of morphologically-delimited entities within the genus. Thus far our results indicate that some characters traditionally used to distinguish species of Hildenbrandia , such as tetrasporangial division pattern, may not be useful in some cases, and a revision of the taxonomy of the genus will be necessary. Although the marine and freshwater species of Hildenbrandia appear to be well separated in our molecular analyses of European specimens, this trend was not observed for North American specimens. High sequence divergence values were calculated for both the rbc L and 18S rRNA genes of Hildenbrandia , compared to other red algal genera.     

Morphological and molecular differentiation of Clinostomum complanatum and Clinostomum marginatum (Digenea: Clinostomidae) metacercariae and adults

The separation of Clinostomum complanatum Rudolphi, 1814 and Clinostomum marginatum Rudolphi, 1819 has long been unclear. Recent data confirm the validity of the junior species, C. marginatum , by ～ 1% differences in its 18S rDNA sequences. We collected adults and metacercariae of C. complanatum and C. marginatum and found reliable morphological differences in the genital complex at both developmental stages. In addition, we identified basic morphometrics (distance between suckers, body width) in metacercariae that may be useful for discriminating the species. The morphological differences were supported by the comparison of sequences of internal transcribed spacers of ribosomal DNA and of the mitochondrial gene cytochrome c oxidase I (COI) from 39 specimens. In 36 specimens, the average divergence between the species was 7.3% in ITS and 19.4% in COI sequences. Two specimens from North America and 1 from Europe had sequences that did not allow them to be clearly allied with either species.     

Overcoming challenges to morphological and molecular identification of Empidonax flycatchers: a case study with a Dusky Flycatcher

Flycatchers in the genus Empidonax are among the most difficult avian taxonomic groups to identify to species. Observers often rely on calls or songs in the field or detailed morphometrics in the hand to identify species. In January and February 2013, we twice captured an Empidonax flycatcher at the Virginia Zoo in Norfolk, Virginia. After being unable to identify the flycatcher to species level using morphometrics and photographs, we extracted DNA from two tail feathers collected during the second encounter to identify the individual genetically. Comparison of cytochrome c oxidase I (COI) with reference sequences in the Barcode of Life Database (BOLD) suggested that the specimen had a >99.8% probability of placement as a Dusky Flycatcher (Empidonax oberholseri). Additional comparisons of NADH dehydrogenase subunit 2 (ND2) to reference sequences in GenBank, however, suggested that the specimen was a Pine Flycatcher (Empidonax affinis), a species not represented in BOLD and confined geographically to a small area in Mexico and Guatemala. After analyzing both COI and ND2 from additional vouchered specimens, the bird caught in Virginia was determined to be a Dusky Flycatcher. We also suspect that some of the sequences in GenBank might derive from incorrectly identified specimens or otherwise could represent overlooked pseudogenes. Because the putative identification, based on GenBank sequences, would have represented the first record of Pine Flycatcher from the United States, our results reinforce the need for carefully vetted and taxonomically comprehensive molecular databases to allow definitive conclusions about sample identity. Further molecular phylogeographic review of this genus is warranted to resolve haplotype ambiguities.     

Taxonomic revision of the olingos (Bassaricyon), with description of a new species,the Olinguito

We present the first comprehensive taxonomic revision and review the biology of the olingos, the endemic Neotropical procyonid genus Bassaricyon, based on most specimens available in museums, and with data derived from anatomy, morphometrics, mitochondrial and nuclear DNA, field observations, and geographic range modeling. Species of Bassaricyon are primarily forest-living, arboreal, nocturnal, frugivorous, and solitary, and have one young at a time. We demonstrate that four olingo species can be recognized, including a Central American species (Bassaricyon gabbii), lowland species with eastern, cis-Andean (Bassaricyon alleni) and western, trans-Andean (Bassaricyon medius) distributions, and a species endemic to cloud forests in the Andes. The oldest evolutionary divergence in the genus is between this last species, endemic to the Andes of Colombia and Ecuador, and all other species, which occur in lower elevation habitats. Surprisingly, this Andean endemic species, which we call the Olinguito, has never been previously described; it represents a new species in the order Carnivora and is the smallest living member of the family Procyonidae. We report on the biology of this new species based on information from museum specimens, niche modeling, and fieldwork in western Ecuador, and describe four Olinguito subspecies based on morphological distinctions across different regions of the Northern Andes.     

Shell geometric morphometrics in Biomphalaria glabrata (Mollusca: Planorbidae) uninfected and infected with Schistosoma mansoni

Freshwater planorbid mollusks belonging to the genus Biomphalaria act as intermediate hosts for Schistosoma mansoni,the etiological agent of human intestinal schistosomiasis,in the Neotropical Region.Identification ofBiomphalaria spp.are carried out based on morphological characters,and the Schistosoma infection are determined by the presence of cercariae (verified through microscope preparation and mounting).Recently,the geometric morphometrics has proven to be a useful tool for determining shape differences in disease vectors arthropods.Due to this,we used geometric morphometrics to determine Biomphalaria glabrata shell differences (shape and size)between uninfected and infected specimens.We digitalized 12 anatomical points over the shell left side (from umbilicus to the last whorl) by combining type Ⅰ and Ⅱ landmarks and sliding semilandmarks;the coordinates were aligned by generalized Procrustes analysis.Principal component analyses were implemented for examining main variation axes,and discriminant analysis for testing group membership significance.We found significant separation between infected and uninfected shell conformation.All specimens were 100％correctly classified.The main differences occur in the peristome.The Kruskal-Wallis test finds significant differences in shell isometric size among infected and uninfected specimens.These findings correspond to other studies of traditional morphometrics,that infected snails showed the reduction in shell size in contrast to those uninfected specimens.     

Cryptomyrus: a new genus of Mormyridae (Teleostei,Osteoglossomorpha) with two new species from Gabon,West-Central Africa

We use mitochondrial and nuclear sequence data to show that three weakly electric mormyrid fish specimens collected at three widely separated localities in Gabon, Africa over a 13-year period represent an unrecognized lineage within the subfamily Mormyrinae and determine its phylogenetic position with respect to other taxa. We describe these three specimens as a new genus containing two new species. Cryptomyrus, new genus, is readily distinguished from all other mormyrid genera by a combination of features of squamation, morphometrics, and dental attributes. Cryptomyrus ogoouensis, new species, is differentiated from its single congener, Cryptomyrus ona, new species, by the possession of an anal-fin origin located well in advance of the dorsal fin, a narrow caudal peduncle and caudal-fin lobes nearly as long as the peduncle. In Cryptomyrus ona, the anal-fin origin is located only slightly in advance of the dorsal fin, the caudal peduncle is deep and the caudal-fin lobes considerably shorter than the peduncle. Continued discovery of new taxa within the “Lower Guinea Clade” of Mormyridae highlights the incompleteness of our knowledge of fish diversity in West-Central Africa. We present a revised key to the mormyrid genera of Lower Guinea.     

Discrimination of Eubazus (Hymenoptera, Braconidae) sibling species using geometric morphometrics analysis of wing venation

Abstract.  Complexes of sibling and cryptic species are encountered frequently in parasitic Hymenoptera. Geometric morphometrics is a useful tool to detect minimal morphological variations, which often are undetectable by traditional morphological studies and even by classical morphometric approaches. We applied geometric morphometrics to wing venation to assess a complex case of sibling species in the genus Eubazus (Hymenoptera, Braconidae), parasitoids of conifer bark weevils of the genus Pissodes (Coleoptera, Curculionidae). The results and methods were compared with previous taxonomic studies on the same species, involving classical multivariate morphometrics, isoenzyme analyses, cross-mating experiments and biological observations. Geometric morphometrics confirmed the previous division into four distinct species. However, this approach enabled the four species to be separated simultaneously, with a reliability of 98.6% for well-classified females and 93.1% for males. A similar result in previous studies was obtained only by combining isoenzyme analyses and several canonical variate analyses, including many morphometric characters. Furthermore, measurements of wing venation were less time-consuming, more reliable and required less prior knowledge of braconid taxonomy than the measurements needed for the classical morphometrics methods. Geometric morphometrics was used also to test the effect of host species on wing shape. Several female populations of Eubazus semirugosus originating from three different Pissodes spp. were compared. Significant differences were found in wing shape between conspecific Eubazus from different host species. The results are discussed in relation to reproductive isolation and genetic flow between the four species.     

Electrophysiological and molecular genetic evidence for sympatrically occuring cryptic species in African weakly electric fishes (Teleostei: Mormyridae: Campylomormyrus)

For two sympatric species of African weakly electric fish, Campylomormyrus tamandua and Campylomormyrus numenius, we monitored ontogenetic differentiation in electric organ discharge (EOD) and established a molecular phylogeny, based on 2222bp from cytochrome b, the S7 ribosomal protein gene, and four flanking regions of unlinked microsatellite loci. In C. tamandua, there is one common EOD type, regardless of age and sex, whereas in C. numenius we were able to identify three different male adult EOD waveform types, which emerged from a single common EOD observed in juveniles. Two of these EOD types formed well supported clades in our phylogenetic analysis. In an independent line of evidence, we were able to affirm the classification into three groups by microsatellite data. The correct assignment and the high pairwise F(ST) values support our hypothesis that these groups are reproductively isolated. We propose that in C. numenius there are cryptic species, hidden behind similar and, at least as juveniles, identical morphs.     

Using multilocus approach to uncover cryptic diversity within Pseudotrapelus lizards from Saudi Arabia

The agamid Pseudotrapelus lizards inhabit the mountainous areas of the Arabian Peninsula and eastern North Africa. Currently six Pseudotrapelus species are recognised, though diagnostic morphological characters are still lacking, creating great difficulty in describing new species. Recently, two specimens of Pseudotrapelus were collected from the vicinity of Riyadh in central Saudi Arabia, an area that was not sampled in previous phylogenetic studies. In here we used both mitochondrial and nuclear data to investigate the phylogenetic position of the new samples, and assess their phylogenetic relationships with the other recognised species of Pseudotrapelus from across the distribution range of the genus. We used a multilocus approach of haplotype networks, concatenated datasets and species trees, performed mitochondrial and nuclear species delimitation analyses, and estimated divergence times. In general, our results support previous molecular studies and uncover the presence of cryptic diversity within Pseudotrapelus. The phylogenetic structure of the genus is of two major clades and within them seven distinct, delimited phylogenetic groups belonging to the six recognised species and the seventh to the individuals from Riyadh. The Riyadh specimens were distinct in all analyses performed. We suggest that the new specimens from the Riyadh area are a distinct lineage, forming a clade with their phylogenetic relatives, P. sinaitus and P. chlodnickii. The clade formed by these three species diverged during the Late Miocene around 6.4 Ma, with cladogenesis possibly facilitated by vicariance and isolation caused due to climatic fluctuations and the progression of sandy areas. Our results suggest further morphological research is necessary to revise the taxonomic status of this lineage and of the entire genus.     

A species delimitation approach in the Trochulus sericeus/hispidus complex reveals two cryptic species within a sharp contact zone

Background  

Mitochondrial DNA sequencing increasingly results in the recognition of genetically divergent, but morphologically cryptic lineages. Species delimitation approaches that rely on multiple lines of evidence in areas of co-occurrence are particularly powerful to infer their specific status. We investigated the species boundaries of two cryptic lineages of the land snail genus Trochulus in a contact zone, using mitochondrial and nuclear DNA marker as well as shell morphometrics.     

Eighteen microsatellite loci for endemic African weakly electric fish (Campylomormyrus,Mormyridae) and their cross species applicability among related taxa

We describe isolation and characterization of the first microsatellite loci specifically developed for African weakly electric fish (Mormyridae), for the genus Campylomormyrus. Seventeen of our 18 loci are polymorphic within the Campylomormyrus numenius species complex. The polymorphic loci showed four to 15 alleles per locus, an expected heterozygosity between 0.46 and 0.94, and an observed heterozygosity between 0.31 and 1.00. Most primers also yield reproducible results in several other mormyrid species. These loci comprise a set of molecular markers for various applications, from moderately polymorphic loci suitable for population studies to highly polymorphic loci for pedigree analysis in mormyrids.     

Application of molecular genetics and geometric morphometrics to taxonomy and conservation of cave beetles in central Italy

Integration of molecular genetic techniques and geometric morphometrics represent a valuable tool in the resolution of taxonomic uncertainty and the identification of significant units for conservation. We combined mitochondrial DNA cytochrome c oxidase subunit II gene sequence data and geometric morphometric analysis to examine taxonomic status and identify units for conservation in four species of the hypogean beetle Duvalius (Coleoptera, Trechinae) using mainly museum specimens collected in central Italy. Previous taxonomic studies based on morphological traits described several subspecies often inhabiting geographically distinct caves. Phylogenetic analysis identified two well supported monophyletic lineages and a number of different clades with relatively small genetic differences, suggesting a short divergence time in line with known geological history of the study area. Geometric morphometrics, on the other hand, recovered a high level of distinctiveness among specimens. Both genetic and morphometric analyses did not entirely corroborate former taxonomic nomenclature, suggesting possible rearrangements and the definition of evolutionary significant units. Beetles of the genus Duvalius are protected by regional laws and the majority of taxa considered in this study inhabit caves located outside protected areas. Our study advocates the importance of devoting protection efforts to networks of cave ecosystems rather than single locations or species.     

长臀鮠属鱼类多变量形态分析及物种有效性研究

采用形态度量学方法对分布于我国珠江水系、元江水系及海南岛诸水系的长臀属3种鱼类,共66尾个体的 43个测量性状进行了主成分分析。研究结果表明长臀属鱼类不能在形态上进行区别。作者认为长臀属鱼类 只存在一个有效种。     

Morphologic and genetic differentiation of night monkey (Aotus infulatus, Primates, Platyrrhinians, Cebidae) populations from the right and left banks of Rio Tocantins (Brazil)

The cranial morphology of 28 specimens of night monkeys (genus Aotus) was examined using three-dimensional geometrical morphometrics. New results of the morphological differences between two populations of Aotus infulatus from both banks of the Rio Tocantins are proposed. These morphological results totally agree with the genetic distinction of these populations proposed by Schneider -- and Sampaio --, and probably point out recent rapid evolutive changes for this species. Our morphometric results can be used for taxonomic, but also for medical research, as the susceptibility to malaria of night monkeys is variable between species.     

Nomimoscolex suspectus n. sp. (Eucestoda: Proteocephalidea: Zygobothriinae) with morphological and molecular phylogenetic analyses of the genus

We describe a new species of Nomimoscolex from the Amazon siluriform fishes Brachyplatystoma filamentosum, B. flavicans and B. vaillanti. It differs from N. piraeeba in a lower mean number of testes, the paramuscular position of the vitelline follicles, the ovarian width/proglottis width ratio and the cirrus-pouch length/proglottis width ratio. Protein electrophoresis assays performed for 25 enzymatic systems showed that specimens of N. suspectus n. sp. from the three host species form a homogenous population which was genetically isolated from N. piraeeba and N. dorad. Moreover, the latter two species, synonymised by Rego (1991) because of their close morphological similarity, could be separated at eight loci. We thus restore N. dorad as a valid species. We finally examined the composition of the genus Nomimoscolex using DNA sequences from the 5.8S rRNA, ITS-2 and 28S rRNA nuclear ribosomal genes and a matrix of 24 morphological characters. Phylogenetic relationships were inferred for nine species of the genus, five members of other monticelliid genera and two outgroup species. The results of the phylogenetic analyses performed on morphological and molecular characters converged with those from allozyme studies and showed that N. suspectus, N. piraeeba and N. dorad clustered in a distinct clade that excluded other members of the genus. We therefore recognised them as an aggregate of species to reflect an isolation supported by both morphological and genetic data. Because relationships among the remaining Nomimoscolex representatives and other genera were generally poorly resolved, regardless of the database analysed, no action was taken to reorganise them into alternative groupings.     

Molecular identification key for pest species of Scirtothrips (Thysanoptera: Thripidae)

Effective plant quarantine and biological control initiatives require rapid and accurate identification of exotic and potentially invasive taxa that may cause high economic losses or environmental damage. The genus Scirtothrips Shull includes several species that are serious agricultural pests, and, because of their minute size and cryptic behavior, prone to undetected transport through international trade of plant material. Although assigning specimens to the genus Scirtothrips is straightforward using traditional taxonomic methods, identification of species is much more difficult and requires expert knowledge of the genus. Furthermore, the validity of some Scirtothrips species is questionable. Therefore, an easy, accurate, and highly reliable technique is desirable for Scirtothrips identification. Here, we provide a simple molecular key based on the internal transcribed spacer regions 1 and 2 (ITS1 and ITS2) of nuclear ribosomal DNA. Individual specimens can be identified by amplification of their ITS1 and ITS2 regions with general primers and determining the size of the products by using standard agarose gel electrophoresis, followed in some instances by DNA digestion with the restriction enzymes SacII or PspOM I. The advantage of this identification system is that nonspecialists are able to quickly and cheaply identify individual specimens. Material analyzed for this work was collected in the United States (California), India, South Africa, Kenya, Mexico, Guatemala, Honduras, Nicaragua, Costa Rica, Panama, Australia, New Zealand and Raiatea (Society Islands French Polynesia). We have identified seven pest species with the molecular-based methods described here. It is hoped that this system can be extended to other members of the genus as their ITS1 and ITS2 sequences become available. We also provide molecular confirmation for two new Scirtothrips species, one species from Honduras and one species from New Zealand.     

Sexually dimorphic body proportions in the catshark genus Scyliorhinus (Chondrichthyes: Carcharhiniformes: Scyliorhinidae)

Intersexual differences in morphometrics were investigated in five species of the catshark genus Scyliorhinus. ANCOVA was used to test 59 measurements, considering capture location and total length as covariates. In all examined species, pelvic–anal distances and pelvic-fin inner margin lengths were greater in males than in females, representing a clear pattern for the genus.