The Javan tiger Panthera tigris sondaica and the Bali tiger P. tigris balica were categorized as Extinct on the IUCN Red List in both 2003 and 2008, leaving only the Sumatran subspecies P. tigris sumatrae extant in Indonesia. There have, however, been occasional, more recent reports of the Javan tiger but without conclusive evidence. Here, a potential observation in 2019 of a Javan tiger in a community plantation near the village of Cipendeuy in the forest of South Sukabumi, West Java, and a single hair found on a fence nearby, are assessed. The cytochrome b mitochondrial DNA (mtDNA) gene sequence of the putative Javan tiger hair were compared with that of a Javan tiger specimen in Museum Zoologicum Bogoriense, collected in 1930; hair samples of several tiger subspecies and the Javan leopard Panthera pardus melas were used as controls. The results showed that the genetic distances (d) of the putative Javan tiger hair with the Sumatran, Bengal P. tigris tigris and Amur P. tigris altaica tigers and the Javan leopard are 0.074 ± SE 0.009, 0.071 ± SE 0.009, 0.072 ± SE 0.009 and 0.088 ± SE 0.010, respectively, whereas the genetic distance of the putative Javan tiger hair with the Javan tiger museum specimen is 0.040 ± SE 0.006. In addition, phylogenetic trees showed that the putative Javan tiger hair sample belongs to the same group as the museum specimen of the Javan tiger, but is differentiated from other tiger subspecies and the Javan leopard. Whether the Javan tiger still occurs in the wild needs to be confirmed with further genetic and field studies.

Avian malaria is a mosquito-borne disease caused by Plasmodium spp. protozoa. Although these parasites have been extensively studied in North America and Eurasia, knowledge on the diversity of Plasmodium, its vectors and avian hosts in Africa is scarce. In this study, we report on natural malarial infections in free-ranging sparrows (Passer domesticus) sampled at Giza Governorate, Egypt. Parasites were morphologically characterized as Plasmodium cathemerium based on the examination of thin blood smears from the avian host. Sequencing a fragment of the mitochondrial cytochrome b gene showed that the parasite corresponded to lineage PADOM02. Phylogenetic analysis showed that this parasite is closely related to the lineages SERAU01 and PADOM09, both of which are attributed to P. cathemerium. Experimental infection of Culex pipiens complex was successful, with ookinetes first detected at 1-day post infection (dpi), oocysts at 4 dpi and sporozoites at 6 dpi. The massive infection of the salivary glands by sporozoites corroborates that Cx. pipiens complex is a competent vector of PADOM02. Our findings confirm that Plasmodium lineage PADOM02 infects sparrows in urban areas along the Nile River, Egypt, and corroborate that Cx. pipiens complex is a highly competent vector for these parasites. Furthermore, our results demonstrate that this lineage corresponds to the morphospecies P. cathemerium and not P. relictum as previously believed.

Species of Agonoscena (Hemiptera: Aphalaridae) are key pests of pistachio in all of the most important pistachio producing countries in the Old World. The efficiency and accuracy of DNA barcoding for the identification of Agonoscena species were tested using mitochondrial cytochrome c oxidase subunit 1 (mtCO1) and cytochrome b (cytb) gene sequences. Moreover, morphometric sexual dimorphism was studied. Finally, the potential geographical distribution of Agonoscena pistaciae, the most important pistachio pest, was calculated using the MaxEnt model. Similar relationships of clustering were found in the morphometric analysis and the molecular analyses with mtCO1 and cytb genes, with A. bimaculata and A. pistaciae being closely related, and A. pegani constituting their sister group. Although the results showed that the cytb gene is a better marker for barcoding in this group, the mtCO1 gene clearly separates the three psyllid species making mtCO1 suitable for diagnostic purposes. A geometric morphometric analysis showed that the distance between landmark number 7 (bifurcation of vein M) to the fore margin of the forewing, and the distance between landmarks number 6 (apex of vein Cu1b) and 11 (wing base), are the most important geometric characters for diagnosing the studied species. Moreover, the forewing shape of males vs females is similar in A. pistaciae and A. bimaculata but differs significantly in A. pegani. In the ecological niche modeling of the distribution of A. pistaciae, the most important contribution was made by the variable ‘minimum temperature of coldest period’. The most suitable areas for A. pistaciae are restricted to Eastern, Southern and some parts of Central Iran.

Gut cell losses contribute to overall feed efficiency due to the energy requirement for cell replenishment. Intestinal epithelial cells are sloughed into the intestinal lumen as digesta passes through the gastrointestinal tract, where cells are degraded by endonucleases. This leads to fragmented DNA being present in faeces, which may be an indicator of gut cell loss. Therefore, measuring host faecal DNA content could have potential as a non-invasive marker of gut cell loss and result in a novel technique for the assessment of how different feed ingredients impact upon gut health. Faecal calprotectin (CALP) is a marker of intestinal inflammation. This was a pilot study designed to test a methodology for extracting and quantifying DNA from pig faeces, and to assess whether any differences in host faecal DNA and CALP could be detected. An additional aim was to determine whether any differences in the above measures were related to the pig performance response to dietary yeast-enriched protein concentrate (YPC). Newly weaned (∼26.5 days of age) Large White × Landrace × Pietrain piglets (8.37 kg ±1.10, n = 180) were assigned to one of four treatment groups (nine replicates of five pigs), differing in dietary YPC content: 0% (control), 2.5%, 5% and 7.5% (w/w). Pooled faecal samples were collected on days 14 and 28 of the 36-day trial. Deoxyribonucleic acid was extracted and quantitative PCR was used to assess DNA composition. Pig genomic DNA was detected using primers specific for the pig cytochrome b (CYTB) gene, and bacterial DNA was detected using universal 16S primers. A pig CALP ELISA was used to assess gut inflammation. Dietary YPC significantly reduced feed conversion ratio (FCR) from weaning to day 14 (P<0.001), but not from day 14 to day 28 (P = 0.220). Pig faecal CYTB DNA content was significantly (P = 0.008) reduced in YPC-treated pigs, with no effect of time, whereas total faecal bacterial DNA content was unaffected by diet or time (P>0.05). Faecal CALP levels were significantly higher at day 14 compared with day 28, but there was no effect of YPC inclusion and no relationship with FCR. In conclusion, YPC reduced faecal CYTB DNA content and this correlated positively with FCR, but was unrelated to gut inflammation, suggesting that it could be a non-invasive marker of gut cell loss. However, further validation experiments by an independent method are required to verify the origin of pig faecal CYTB DNA as being from sloughed intestinal epithelial cells.

The small red brocket deer Mazama bororo is endemic to the Brazilian Atlantic Forest, a biome that has been greatly fragmented and altered by human activities. This elusive species is morphologically similar to the red brocket deer Mazama americana and the Brazilian dwarf brocket deer Mazama nana, and genetic typing is necessary for reliable identification. To determine the geographical range of M. bororo more accurately, we conducted non-invasive genetic sampling using scat detection dogs trained to locate deer faeces. We surveyed 46 protected areas located within the species’ potential distribution and collected a total of 555 scat samples in 30 of the protected areas. Using a polymerase chain reaction–restriction fragment length polymorphism approach, we genotyped 497 scat samples (89%) and detected M. bororo in seven localities in three Brazilian states. The results support a range extension of the small red brocket deer to latitudes 23 and 28°S and longitudes 47 and 49°W. We show that the species’ distribution is associated with 37,517 km2 of the Ombrophilous Dense Forest in the Brazilian Atlantic Forest, and this conclusion is supported by species distribution modelling. The small red brocket deer is the largest endemic species in Brazil and may have the smallest geographical distribution of any Neotropical deer species. This species occupies fragmented landscapes and is threatened by human encroachment, poaching, and predation by dogs, and based on our findings we recommend policy intervention for conservation planning of the Ombrophilous Dense Forest.

The diversity and prevalence of malaria parasites of the genera Plasmodium and Haemoproteus were determined in the globally-threatened Aquatic Warbler Acrocephalus paludicola. Birds were sampled during migration in Portugal and at the wintering quarters in Senegal and parasites were detected using molecular methods. Only three generalist parasite lineages (Plasmodium) were found. There were no significant differences in the prevalence of parasites between sexes in Europe, but adults had higher prevalence than first-year birds, and birds in Europe had higher prevalence than those captured in Africa. When comparing with other Acrocephalus species and taking sample size into account, Aquatic Warblers had the lowest prevalence and, together with another threatened species, the Seychelles Warbler Acrocephalus sechellensis, the lowest diversity of malaria parasites. We hypothesize that the low diversity of parasites and absence of specialist lineages of Aquatic Warblers are caused by its small population size and fragmented distribution. Furthermore, Aquatic Warblers’ extreme habitat specialization may decrease their exposure to malaria parasites, but other explanations such as high mortality (which would constraint the sampling of infected birds) or, in contrast, very efficient immunological system in clearing the infections cannot be ruled out. This study contributes to explain variation in prevalence and diversity of malaria parasites among hosts.

Carcasses of whales provide much valuable information on their natural history. However, some specimens cannot be identified in the field due to the advanced state of decomposition. In this study, the DNA was extracted and the mitochondrial cytochrome b gene was amplified by polymerase chain reaction and sequenced for four carcasses of possible mysticeti (GEMM: 075, 088, 135 and GEMARS: 1302). A blast search using the nucleotide–nucleotide basic local alignment (blastn) search tool was conducted using the generated sequences. Samples GEMM 075 and GEMARS 1302 showed 98% identity to one sequence of Balaenoptera acutorostrata. Samples GEMM 088 and GEMM 135 showed 99% identity to sequences from Balaenoptera edeni and Megaptera novaeangliae, respectively. A neighbour-joining tree was generated using sequences from GenBank from all species of balaenopterid that occur on the coast of Brazil. The results showed that all carcasses analysed were correspondent to species from the family Balaenopteridae already recorded in Brazil.

To date, a molecular phylogenetic approach has not been used to investigate the evolutionary structure of Trogoderma and closely related genera. Using two mitochondrial genes, Cytochrome Oxidase I and Cytochrome B, and the nuclear gene, 18S, the reported polyphyletic positioning of Trogoderma was examined. Paraphyly in Trogoderma was observed, with one Australian Trogoderma species reconciled as sister to all Dermestidae and the Anthrenocerus genus deeply nested within the Australian Trogoderma clade. In addition, time to most recent common ancestor for a number of Dermestidae was calculated. Based on these estimations, the Dermestidae origin exceeded 175 million years, placing the origins of this family in Pangaea.

We characterized 15 Trypanosoma cruzi isolates from bats captured in the Amazon, Central and Southeast Brazilian regions. Phylogenetic relationships among T. cruzi lineages using SSU rDNA, cytochrome b, and Histone H2B genes positioned all Amazonian isolates into T. cruzi I (TCI). However, bat isolates from the other regions, which had been genotyped as T. cruzi II (TC II) by the traditional genotyping method based on mini-exon gene employed in this study, were not nested within any of the previously defined TCII sublineages, constituting a new genotype designated as TCbat. Phylogenetic analyses demonstrated that TCbat indeed belongs to T. cruzi and not to other closely related bat trypanosomes of the subgenus Schizotrypanum, and that although separated by large genetic distances TCbat is closest to lineage TCI. A genotyping method targeting ITS1 rDNA distinguished TCbat from established T. cruzi lineages, and from other Schizotrypanum species. In experimentally infected mice, TCbat lacked virulence and yielded low parasitaemias. Isolates of TCbat presented distinctive morphological features and behaviour in triatomines. To date, TCbat genotype was found only in bats from anthropic environments of Central and Southeast Brazil. Our findings indicate that the complexity of T. cruzi is larger than currently known, and confirmed bats as important reservoirs and potential source of T. cruzi infections to humans.

We investigate the degree of between-population genetic differentiation in the Mediterranean field cricket Gryllus bimaculatus, as well as the possible causes of such differentiation. Using cytochrome b mtDNA sequences, we estimate genetic variation in G. bimaculatus from seven South African and two Mediterranean populations. Within-population genetic variation in Europe (two haplotypes, one unique to a single individual) suggest low effective population size and strong bottlenecks with associated founder effects, probably due to cold winter environments in Europe that limit reproduction to a short part of the summer. The likely cause for this is the daily maxima in winter temperatures that fall below the critical level of 16°C (enabling normal calling and courtship behaviour) in Mediterranean Europe, whereas the equivalent temperatures in southern Africa are above this limit and enable reproduction over a large part of the year. European genetic variants were either shared with Africa or closely related to African haplotypes. For survival, European populations are probably dependent on immigration from other areas, including Africa. South African populations have low but measurable gene flow with Europe and show significant between-population genetic differentiation (30 haplotypes). Isolation-by-distance is not sufficient to explain the degree of between-population genetic differences observed, and a large degree of dispersal is also required in order to account for the observed patterns. Differences in morphology and calling behaviour among these populations are underlied by these genetic differences.

The Cyprinidae is the largest freshwater fish family in Vietnam, with over 220 recognised species, many of which play an important role in aquaculture or are harvested from the wild. Despite numerous studies on the taxonomy of this family based on traditional morphological data, the relationships between major cyprinid groups is poorly understood and the taxonomic validity of a number of these groups is under debate. While an increasing number of molecular studies on cyprinid relationships have been conducted many have used restricted sampling and none have incorporated Vietnamese species. In this study, mitochondrial 16S rRNA, D-loop and cytochrome b gene sequences from 25 species of cyprinids collected from Vietnam were obtained and combined with sequences of cyprinids available in GenBank, in order to investigate the taxonomic validity of subfamilies within Cyprinidae and their phylogenetic relationships. The molecular data supported traditional division of the Cyprinidae into two major lineages: Cyprinines and Leuciscines. The placement of the Danioninae as the sister lineage to this grouping was not supported. Many of the subfamily boundaries were questioned and doubt was raised on some of the generic level classifications. The validity of species designation in Cyprinus, Tor and Cyclocheilichthys was also questioned. This study will need to be extended with greater taxon and gene sampling to further consolidate our understanding of cyprinid relationships and classification.

Malaria parasites in the genus Plasmodium are now placed within 11 subgenera based on morphology under the light microscope, life-history traits, and host taxon. The phylogenetic significance of these characters, however, is problematic because the observed variation could be homoplasious. Using Plasmodium infections found in 2632 birds of many avian families collected in the USA, and several samples from other locations, we compared identifications to subgenus based on morphology in blood smears with a 2-gene molecular phylogeny (the first for avian Plasmodium) to determine if the 5 avian Plasmodium subgenera represent monophyletic groups. Phylogenetic trees recovered by parsimony, likelihood, and Bayesian methods presented nearly identical topologies. The analysis allowed testing the hypothesis of monophyly for the subgenera. Monophyly of the subgenera Haemamoeba, Huffia, and Bennettinia was supported by the analysis. The distinctive morphology of Haemamoeba species appears to have evolved once. Most samples identified to Novyella also fell within a monophyletic clade with the exception of 2 samples that fell basal to all other avian Plasmodium. Samples of the subgenus Giovannolaia did not form a monophyletic group. Thus, the characters used by parasitologists for over a century to define subgenera of Plasmodium vary in their phylogenetic significance.

The bumble-bee bat Craseonycteris thonglongyai of the monospecific family Craseonycteridae is categorized as Endangered on the IUCN Red List. First discovered in 1973, it was until recently only known from a small population of approximately 2,300 individuals restricted to the catchment area of the River Kwai, Thailand. However, in 2001 a single craseonycterid was discovered in Mon State, Myanmar, extending its geographical range by approximately 250 km. In October and November 2002 a survey was undertaken to examine the status of C. thonglongyai in Myanmar and assess its geographical distribution and population size. C. thonglongyai calls were recorded from bats emerging from nine of 19 caves surveyed; the population size was estimated to be 1,500. The phylogenetic relationships between the Thai and Myanmar populations were investigated using molecular, morphological and acoustic data. Morphologically, the two populations are indistinguishable. However, there is an 8–10 kHz echolocation call divergence between the populations. Cytochrome b data suggest that the two populations are similar and that the Myanmar population may be monophyletic. Annual surveys of the known bat roosts and in situ education programmes for local people are recommended together with the establishment of an integrated, transboundary Myanmar/Thai conservation strategy.

More than 200 species of avian Haemosporidia (genera Plasmodium, Haemoproteus, and Leucocytozoon) have been described based primarily on morphological characters seen in blood smears. Recent molecular studies, however, suggest that such methods may mask a substantial cryptic diversity of avian haemosporidians. We surveyed the haemosporidians of birds sampled at 1 site in Israel. Parasites were identified to species based on morphology, and a segment of the parasite's cytochrome b gene was sequenced. We compared 3 species concepts: morphological, genetic, and phylogenetic. Fifteen morphological species were present. Morphological species that occurred once within our dataset were associated with a unique gene sequence, displayed large genetic divergence from other morphological species, and were not contained within clades of morphological species that occurred more than once. With only 1 exception, morphological species that were identified from multiple bird hosts presented identical sequences for all infections, or differed by few synonymous substitutions, and were monophyletic for all phylogenetic analyses. Only the morphological species Haemoproteus belopolskyi did not follow this trend, falling instead into at least 2 genetically distant clades. Thus, except for H. belopolskyi, parasites identified to species by morphology were supported by both the genetic and phylogenetic species concepts.

Assessment of pinniped predation most often relies on analysis of the hard, undigested prey remains evident in faecal (scat) samples. For many prey species this method can yield valuable information on predator–prey interactions. For some genera, however, species diagnostic characteristics are lost during the process of prey digestion, thereby preventing morphological identification of fish prey species. Here, the feasibility of using faecal DNA to detect the presence of salmonids in pinniped scat samples and to distinguish reliably between sea trout Salmo trutta and Atlantic salmon S. salar was assessed. Novel salmonid mitochondrial DNA (mtDNA) primers were designed to amplify 162 bp of the 16S rDNA and a 327 bp section of the cytochrome b gene. Species-specific banding patterns were obtained by digestion of the cytochrome b PCR product with the restriction endonuclease AluI, and confirmed by the species-specific amplification of the 16S rDNA fragment from Atlantic salmon. Scats collected from captive grey seals Halichoerus grypus fed on known monospecific diets used to validate the PCR-RFLP assay indicated a probability of at least 95.8% (23 of 24 faecal extracts) of detecting salmonids using DNA extracted from the scat matrix. Implemented alongside conventional prey remains analyses, this technique presents a promising new method for examining prey composition and assessing pinniped predation on salmonids.

Birds harbouring several malarial parasites are common in the wild, and resolving such multiple infections is important for our understanding of host–parasite relationships. We propose a simple and reasonably accurate method for detecting and resolving multiple infections, based on the analysis of parasite cytochrome b DNA sequences: genetically mixed infections are first identified by double nucleotide peaks on sequence electropherograms, and later retrieved by TA-cloning. We applied this method to wild birds, and to experimentally created mixes with varying proportion of two parasites (Plasmodium spp. and Haemoproteus spp.). In general, the method was very efficient in detecting and resolving multiple infections, but some problems were encountered. Several multiple infections were erroneously scored as simple, either because one of the parasite lineages was a better target for the primers used, or because it was much more abundant in the mix. On the other hand, single nucleotide substitutions and template switching during PCR produced artificial sequences in some clones. We discuss the utility of the method, and propose a framework for its use when screening for genetically diverse avian malarial parasites.

Maximum likelihood (ML) and Bayesian inference (BI) analyses of the complete nucleotide sequences of the cytochrome b gene (cytb) were used to examine the phylogenetic position of the rare snake Xenophidionschaeferi within the alethinophidian snakes. The cytochrome b sequence of this representative of the poorly known family Xenophiidae was compared with those of a large and comprehensive suite of alethinophidian taxa. The research presented here represents the first time all families of alethinophidian and caenophidian snakes have been included in a single molecular phylogenetic study. Results from ML and BI analyses suggest a possible sister taxon relationship between Xenophidion schaeferi and the Bolyeridae. Moreover, strong statistical support also indicates that Xenophiidae is a member of a clade that contains Pythonidae, Loxocemidae, Uropeltidae, Xenopeltidae and Bolyeriidae. Additionally, maximum parsimony and BI analyses of previously published morphological data revealed that these anatomical character states and potentially low taxonomic sampling produced little phylogenetic information valuable to understanding the relationship of Xenophiidae among the Alethinophidia.

The Cytochrome b (Cyt b) gene has proved to be useful for identification and classification of many mammals and plants. In order to evaluate the utility of this gene for discrimination of Leishmania parasites as well as for exploring their phylogenetic relationships, we determined the nucleotide sequences of the Cyt b gene from 13 human-infecting Leishmania species (14 strains) from the New and Old Worlds. The Cyt b genes, approximately 1080 base pairs, were found to be A/T rich, and their 5′ terminal-editing regions were highly conserved. The nucleotide sequence variation among them was enough to discriminate parasite species; 245 nucleotide positions were polymorphic and 190 positions were parsimony informative. The phylogenetic relationships based on this gene, showed good agreement with the classification of Lainson & Shaw (1987) except for the inclusion of L. (L.) major in the L. (L.) tropica complex and the placement of L. tarentolae in another genus. These data show that the Cyt b gene is useful for phylogenetic study of Leishmania parasites.

The African rodent genus Saccostomus is common and widespread in the savannas, scrubby areas and cultivated fields from south Ethiopia and Somalia through East Africa down to the Cape Province. Its systematics and taxonomy are however poorly known, with two species currently recognized, S. mearnsi and S. campestris, the former is typical of the Somali–Maasai domain and the latter is typical of Zambezian–South African. The karyotypic findings and the analysis of the mitochondrial gene cytochrome b for some populations from Somalia, Tanzania, Zambia and South Africa are presented showing that the diversity within the genus is much higher than previously reported and believed, and that S. mearnsi and S. campestris rather represent species complexes, still actively speciating. The times of cladogenetic events and the extent of chromosomal variability within the genus that may be coupled with speciation are discussed. Finally, the possible taxonomy is considered in the context of the wide karyotypic variation shown by each species, the extent and the boundaries of which are still largely unknown, limiting therefore their definitive taxonomic assignment.

The agrimi goat Capra aegagrus cretica is unique to Crete and its offshore islands. It has been identified as a sub-species of the wild bezoar goat Capra aegagrus aegagrus Erxleben, 1777, which it closely resembles in horn shape, body form and coloration. This classification has been disputed by some researchers who claim that the agrimi are feral goats, derived from early domestic stock brought to the island by the first Neolithic settlers. In order to clarify this issue, DNA analyses (cytochrome b and D loop sequences) were carried out on tissue of live and skeletonized agrimi and compared to sequences of wild and domestic caprines. Results conclusively show the agrimi to be a feral animal, that clades with domestic goats (Capra hircus) rather than with wild Asiatic bezoar. This study demonstrates that morphometric criteria do not necessarily reflect genetic affinities, and that the taxonomic classification of agrimi should be revised.