Mitochondrial 16S Research Articles

A molecular assessment of species boundaries and relationships in the Australian brine shrimp Parartemia (Anostraca: Parartemiidae).

Australian salt lakes contain a diverse range of endemic invertebrates. The brine shrimp Parartemia is among the most speciose and salt-tolerant of these invertebrates. The morphotaxonomy of Parartemia is well established but there has only been limited molecular assessment of the phylogenetic relationships and boundaries of the morphospecies. We used multiple genetic markers (nuclear 28S and mitochondrial 16S and COI ) and tree-building methods (Bayesian inference and maximum likelihood) to investigate the phylogeny of Parartemia . We also used species delimitation methods to test the validity of morphological species designations. The data set included all but 2 of the 18 described Parartemia morphospecies, collected from a total of 93 sites from across southern Australia plus some sequences from GenBank. The results identified large amounts of molecular divergence (e.g. COI P- values of up to 25.23%), some groups of closely related species (which also usually shared some morphological similarities) and some distinctive species, although the relationships among divergent lineages were generally not well resolved. The most conservative set of results from the species delimitation analyses suggests that the morphotaxonomy is largely accurate, although many morphospecies comprised divergent genetic lineages separated by COI P- values of up to 17.02%. Two putative new morphospecies, three cryptic species and one synonymy were identified. Our findings improve the knowledge of Parartemia taxonomy and will facilitate the development of future studies and conservation of this taxon.

A rapid and parallel Late Pleistocene/Holocene morphological radiation in a predaceous planktonic water flea: the case of Bythotrephes (Cladocera: Cercopagididae)

Abstract The predatory cladoceran Bythotrephes is one of North America’s most successful and impactive invasive species in freshwater plankton communities. The taxonomic status of the genus Bythotrephes Leydig, 1860 (Crustacea: Cladocera: Cercopagididae) has remained unclear and a subject of intensive debate for over 150 years. We applied an integrative taxonomy approach with multi-gene analysis (mitochondrial COI and 12S genes, and nuclear 18S and 28S genes) on 80 individuals (representing at least four morphospecies) from various regions spanning the genus distribution (North America, Europe, and Asia) to resolve the taxonomic status of species within Bythotrephes. The results of our study strongly support the hypothesis of a single species—Bythotrephes longimanus Leydig, 1860, and ecological morphs should be accepted as junior synonyms. Our work also elucidates the very recent radiation of Bythotrephes, which may have begun rapidly and parallel during the Late Pleistocene, or even after the last glaciation. Finally, we provide a comprehensive biogeographic reconstruction of Bythotrephes dispersal within the Holarctic realm. Europe likely served as a dispersal centre for Bythotrephes from where they spread relatively recently (possibly, less than 10 kyr) and we distinguish five possible dispersal events in its evolutionary history.

DNA sequencing of topotypes helps delineate species distributions in the Ischnocnema verrucosa complex (Anura, Brachycephalidae)

As many new evolutionary lineages are being discovered and formally named, sequencing topotypes when holotypes are not available becomes essential for taxonomy. This study uses a DNA-taxonomy approach to sequence new populations of the Ischnocnema verrucosa species complex (Brazilian Wart Frogs) from different locations, including, for the first time, individuals from the type localities. Phylogenetic analysis of the mitochondrial 16S gene recovered a monophyletic Ischnocnema verrucosa species series composed of three main clades. The most recent common ancestor was estimated to be 33.76 million years ago, and diversification within the three main clades occurred primarily during the Miocene. We delimited eight species-level lineages with high levels of sequence divergence (7% to 16%). Our study highlights the importance of DNA taxonomy and the necessity of protecting and sequencing topotypes in taxonomic studies. Our study also contributes to the conservation and understanding of the genus Ischnocnema and the biodiversity of the Brazilian Atlantic Forest region.

Description of a new species of the Asian newt genus Tylototriton (Amphibia, Urodela, Salamandridae) from Hunan Province, China

The newt genus Tylototriton is widely distributed in east, southeast, and south Asia. Previous studies have shown that basic surveys of this genus are far from comprehensive, and unknown species still exist within the group. In this study, we described a new species of this genus, Tylototriton gaowangjienensissp. nov., from Gaowangjie National Nature Reserve in Hunan Province, China, based on evidence from molecular identification and morphological comparisons. Additionally, we conducted phylogenetic analysis of the genus to elucidate the taxonomic status of the new species. Based on combined mitochondrial 16S rRNA and ND2 gene sequences, the phylogenetic tree supported the new species as a member of the T. wenxianensis species subclade in the subgenus Yaotriton. It can be distinguished from its congeners by the combination of the following characteristics: (1) medium-sized body; (2) finger tips extending beyond the snout while forelimbs stretched forward; (3) tips of forelimbs and hindlimbs reaching and overlapping when folded towards the body; (4) snout edge round; (5) absence of small papillae in the male cloacal fissure; (6) dorsal ridges narrow, with width smaller than the eye diameter; (7) head length (HDL) greater than head width (HDW) (HDL/HDW = 1.04~1.22); (8) orange markings absence on the parotoid; (9) nodule-like warts on body sides continuous; (10) vomerine teeth not connected at the tip, extending to the occipital bone at the posterior end.

A redescription and new generic placement of the New Zealand marine-associated jumping spider Marpissa marina (Goyen, 1892) (Araneae: Salticidae: Euophryini)

ABSTRACT The jumping spider Marpissa marina (Goyen, 1892) is endemic to Aotearoa New Zealand. In this paper, we redescribe the species. Marpissa marina was placed in the Northern Hemisphere genus Marpissa due to its subjective similarity to spiders in that genus. The original description lacked modern morphological diagnoses and useful illustrations. The description was based largely on the female, and therefore lacked the taxonomically important characters seen in the male pedipalp. Previous literature has suggested M. marina may be related to the Australian genus, Maratus Karsch, 1878. We applied an integrative taxonomic approach based on diagnostic morphological characters and molecular analysis for a more detailed species description and assignment into an appropriate genus. Morphological features indicate that M. marina belongs to Maratus. This is supported by phylogenetic analyses using DNA sequences from the nuclear gene Actin 5C and the mitochondrial gene 16S ribosomal RNA. We transfer Marpissa marina into the genus Maratus, as Maratus marinus (Goyen, 1892), new combination.

A new Boulenophrys species (Anura, Megophryidae) from the coastal hills of eastern Fujian Province, China.

A new species of the genus Boulenophrys is described from the coastal hills of eastern Fujian Province, China. The new taxon can be distinguished from all recognized congeners by a combination of discrete morphological character state differences and genetic divergences in the combined mitochondrial 16S + CO1 genes. We also provide a map showing the distribution pattern of Boulenophrys species in Fujian and a provincial-specific key, which will aid their conservation by helping the local authorities accurately identify species during field identifications and data collection efforts.

Communicator whistles: A Trek through the taxonomy of the Boophis marojezensis complex reveals seven new, morphologically cryptic treefrogs from Madagascar (Amphibia: Anura: Mantellidae)

Abstract The Malagasy stream-breeding treefrog species Boophis marojezensis contains bioacoustically and genetically highly divergent populations. Some of these populations have been defined as candidate species and emit somewhat bizarre advertisement calls consisting of multiple whistle-notes. We here enable a long-overdue taxonomic revision of this species complex by applying a museomics approach to sequence DNA from the holotype of B. marojezensis. Based on an integrative approach that combines divergence levels in mitochondrial DNA and in three nuclear-encoded genes, morphological data, and bioacoustic comparisons, we conclude that eight different species exist in this complex, seven of which are formally described herein as new. Although morphological differences between species are small and mainly separate small-sized from larger-sized species, conclusive evidence for the new species comes from their sympatric and sometimes syntopic occurrence without haplotype sharing in three nuclear genes and under maintenance of bioacoustic differences. Uncorrected genetic divergences in the mitochondrial 16S rRNA gene are >3% in almost all cases, and in some cases up to 8%. In reference to the otherworldly sounds by which these frogs fill Malagasy rainforests, some of them reminiscent of sounds of technical equipment in the fictional “Star Trek” universe, we here name and describe the seven new species in honor of fictional captains of starships, namely B. kirkisp. nov., B. picardisp. nov., B. siskoisp. nov., B. janewayaesp. nov., B. archerisp. nov., B. pikeisp. nov., and B. burnhamaesp. nov. The majority of these species occur in northern Madagascar, where up to three species can occur in immediate geographical proximity, e.g., B. marojezensis, B. burnhamaesp. nov. and B. pikeisp. nov. at different elevations in the Marojejy Massif. South of 16°S latitude, only B. janewayaesp. nov., B. picardisp. nov., and B. kirkisp. nov. are found, with the latter extending southwards to Ranomafana National Park. Our study confirms the existence of numerous morphologically cryptic and microendemic species among Madagascar’s amphibians, some of which are known only from unprotected sites and require adequate conservation management.

Revision of the Chinese termite genus Peribulbitermes (Blattodea, Isoptera, Termitidae): A new synonym of Ahmaditermes

The genus Peribulbitermes is a small genus in Nasutitermitinae comprising three species distributed only in China. Type specimens of Peribulbitermes dinghuensis Li, 1985 and Peribulbitermes jinghongensis Li, 1985 are obtained in our study. After examining the morphological characters of the specimens, it is found that P. jinghongensis cannot be differentiated from P. dinghuensis by the diagnostic characters reported in original description. We propose that P. jinghongensis is the junior synonym of P. dinghuensis. Additionally, molecular phylogenetic relationships of Peribulbitermes, with its morphologically similar genera, Bulbitermes, Ahmaditermes and Nasutitermes, as well as other nine genera in Nasutitermes Group of Nasutitermitinae were analysed based on mitochondrial COI, COII and 16S rRNA genes. The results show that the relationship between Peribulbitermes and Ahmaditermes is the closest. Peribulbitermes should be downgraded to synonymy of Ahmaditermes. P. dinghuensis and Peribulbitermes parafulvus (Tsai and Chen, 1963) should be transferred to Ahmaditermes as Ahmaditermes dinghuensis and Ahmaditermes parafulvus.

Improvement in Microbiota Recovery Using Cas-9 Digestion of Mānuka Plastid and Mitochondrial DNA

Understanding host-microbe interactions in planta is an expanding area of research. Amplicon sequencing of the 16S rRNA gene is a powerful and common method to study bacterial communities associated with plants. However, the co-amplification of mitochondrial and plastid 16S rRNA genes by universal primers impairs the sensitivity and performance of 16S rRNA sequencing. In 2020, a new method, Cas-16S-seq, was reported in the literature to remove host contamination for profiling the microbiota in rice, a well-studied domestic plant, by engineering RNA-programmable Cas9 nuclease in 16S rRNA sequencing. For the first time, we tested the efficiency and applicability of the Cas-16S-seq method on foliage, flowers, and seed of a non-domesticated wild plant for which there is limited genomic information, Leptospermum scoparium (mānuka). Our study demonstrated the efficiency of the Cas-16S-seq method for L. scoparium in removing host contamination in V4-16S amplicons. An increase of 46% in bacterial sequences was found using six guide RNAs (gRNAs), three gRNAs targeting the mitochondrial sequence, and three gRNAs targeting the chloroplast sequence of L. scoparium in the same reaction. An increase of 72% in bacterial sequences was obtained by targeting the mitochondrial and chloroplast sequences of L. scoparium in the same sample at two different steps of the library preparation (DNA and 1st step PCR). The number of OTUs (operational taxonomic units) retrieved from soil samples was consistent when using the different methods (Cas-16S-seq and 16S-seq) indicating that the Cas-16S-seq implemented for L. scoparium did not introduce bias to microbiota profiling. Our findings provide a valuable tool for future studies investigating the bacterial microbiota of L. scoparium in addition to evaluating an important tool in the plant microbiota research on other non-domesticated wild species.

A molecular approach to identify parrotfish (Sparisoma) species during early ontogeny.

Sparisoma species (parrotfish) comprise an important functional group contributing to coral-reef resilience. The morphological diagnostic characteristics for species identification are clearly described for adult forms but not for the early stages. Consequently, many taxonomical listings of Sparisoma larvae are restricted to the genus level. The aims of this study are to determine whether the morphological and molecular identification techniques are useful to assign the species taxonomic level to Sparisoma larvae occurring in the Gulf of Mexico and whether there is a set of diagnostic features that could be used to discriminate between species in larvae of different developmental stages. Morphological assignment of Sparisoma was performed based on morphological and meristic features for 30 larvae collected in the Gulf of Mexico from late August to mid-September 2015. To corroborate and complement the morphological assignments, molecular identification was carried out using DNA sequences from regions of two mitochondrial genes, mitochondrial cytochrome oxidase I (mtDNA COI) and mitochondrial 16S rRNA (mtDNA 16S rRNA). COI and 16S gene trees for Sparisoma and related fish taxa were constructed using sequences available in the NCBI (National Center for Biotechnology Information) GenBank and BOLD (Barcode of Life Data) databases. Two morphotypes were identified based on morphology, but no diagnostic characteristics for species discrimination were found. Molecular identification, in contrast, successfully discriminated four early development stages of Sparisoma atomarium, three stages of Sparisoma radians, and two stages of Sparisoma chrysopterum and Sparisoma aurofrenatum, therefore demonstrating the successful and necessary application of molecular taxonomic approaches for species-level identifications of Sparisoma larvae.

Carcinoplaxmistio Ng & Mitra, 2019 (Crustacea, Decapoda, Goneplacidae): additional records and genetic differentiation of allied taxa.

The goneplacid crab, Carcinoplaxmistio Ng & Mitra, 2019, was originally described from West Bengal, India, in the northern Indian Ocean. Additional material of C.mistio from off Tamil Nadu in the southeast of India revealed a high degree of size-associated variation in the structures of the anterolateral tooth of the carapace, chelipeds, and male and female pleons. In addition to an in-depth morphological examination of C.mistio, this study also records the natural coloration of the species and conducts a genetic comparison (with mitochondrial COI and 16S rRNA genes) with its close relatives, C.haswelli (Miers, 1884) and C.purpurea Rathbun, 1914. Molecular comparison of C.mistio with its morphologically closest congener, C.haswelli from northern Australia and the western Pacific, corroborates their status as separate species. The genetic sequence of C.mistio, however, is similar to that of C.purpurea from the West Pacific, although these two species can easily be distinguished by distinct carapace and ambulatory leg characters. The present study provides some possible explanations for the genetic and morphological incongruence observed between C.mistio and C.purpurea and highlights the need for a detailed molecular study for Carcinoplax H. Milne Edwards, 1852, to appreciate the evolution of various morphological characters in the genus.

Diversity and distribution of reed frogs (Hyperolius spp.) on Bioko Island, Equatorial Guinea

Reed frogs (Hyperolius spp.) are the most species-rich genus in the family Hyperoliidae, a group of frogs endemic to sub-Saharan Africa and the Gulf of Guinea islands. Three species are endemic to oceanic islands in the archipelago, but the diversity and distribution of reed frogs on Bioko – a land-bridge island in the archipelago – remains unclear. Two species, H. tuberculatus and H. ocellatus ocellatus, were formally documented on Bioko in the colonial era, however, two additional reed frog species have been detected in recent surveys. In addition, observations of heterospecific amplexus suggest one of the unidentified species and H. o. ocellatus may be hybridising on the island. In this study, we examined mitochondrial DNA (mtDNA), nuclear DNA (nuDNA), morphological and acoustic data to confirm the tentative identifications of the reed frogs and determine if there is any evidence of hybridisation. Our data confirm that the two unidentified species are H. endjami and H. fusciventris, and that Bioko H. endjami have unique genetic diversity not shared with the mainland population. Additionally, our mitochondrial (16S) and nuclear DNA (CMYC) haplotype networks suggest that H. o. ocellatus and H. endjami are not hybridising on Bioko. Finally, we report colour pattern variation in H. endjami and H. o. ocellatus that is unique from populations on the continent, and describe male advertisement calls of Bioko H. endjami and H. o. ocellatus. Our study provides a first look at the diversity and distribution of reed frogs on Bioko Island and sets the stage for future studies of the ecology, evolution, and conservation of this island community. KEYWORDS: hybridisation, polymorphism, mitochondrial and nuclear DNA, morphology, land-bridge island

Limnichthyskoreanus, a new species of creediid fish (Teleostei, Acropomatiformes, Creediidae) from Korea.

Limnichthyskoreanus sp. nov. is described on the basis of the holotype and 11 paratypes from subtidal waters of Seogwipo, Jeju Island, Korea. The new species had previously been regarded as the Northern Hemisphere population of the anti-equatorial L.fasciatus, but molecular analyses of mitochondrial COI and 16S genes recovers deep genetic divergences of 9.4% and 15.0% between the new species and topotypical specimens of L.fasciatus. Limnichthyskoreanus sp. nov. is distinguished from all other species of Limnichthys based on the following combination of colouration and morphological characteristics: 38-40 vertebrae; 0-6 dorsal saddles joining mid-lateral stripe; small infraorbital sensory pores; a single median interorbital pore; and well-developed vomerine teeth. Summary characters for comparative congeneric species are provided.

Fine-scale phylogeography of Tripneustes gratilla revealed a core-periphery pattern in the South China sea

The Core-Periphery Hypothesis (CPH) is a theoretical framework in phylogeography used to examine the spatial distribution of genetic diversity. The tropical Indian-Western Pacific (IWP), serving as a prominent marine biodiversity hotspot, constantly exports species and evolutionary novelties to peripheral habitats. Many species display genetic patterns consistent with CPH predictions from the IWP to periphery regions. However, the presence of CPH’s genetic signals in intermediate distribution zones remains underexplored. Here, we collected four sea urchin populations of Tripneustes gratilla in the South China Sea and analysed their population structure and evolutionary patterns using 13 morphological parameters, one mitochondrial 16S rRNA gene and nine polymorphic microsatellites. The results revealed substantial genetic homogeneity among the populations, implying a single origin from a glacial refugium. Nonetheless, morphological and microsatellite data distinctly differentiate the northernmost Fengjiawan population from the others, which could be attributed to increased environmental selection pressures such as temperature. Analysis of genetic diversity variation along latitudinal gradients and the relationship between geographic and genetic distances align with CPH expectations for both 16S and microsatellites, albeit to varying degrees of significance. This study enhances our understanding of tropical marine invertebrate evolution and supports the applicability of the CPH model at a fine-scale level.

A systematic survey of environmental DNA in Palau's lakes and waterfalls reveals an increase in Leptospira levels after flooding

ObjectiveLeptospirosis is an important bacterial zoonosis which is widespread in tropical and subtropical islands and influences human and animal health which has secondary economic effects. Although leptospirosis is endemic in Palau, an Oceanian Pacific Island country, few systematic surveys of potential risk factors for Leptospira infection, such as weather and host animals, have been conducted in the natural environment. We used environmental DNA metabarcoding to assess the distribution, species diversity, and abundance of pathogenic Leptospira in this endemic region to investigate the potential environmental risks. MethodsForty-two paired water samples, representing fine and rainy weather conditions, were collected from four representative waterfalls and lakes on Babeldaob Island, the largest island in Palau. High-throughput sequencing analysis was conducted for polymerase chain reaction products of leptospiral 16S rRNA and vertebrate animal mitochondrial 12S rRNA genes. ResultsWe revealed greater Leptospira diversity and abundance in samples collected after continuous rain, particularly in the presence of flooding, compared with samples collected under typhoon, monsoon, or fine weather conditions. From same samples, six mammalian species including cats (Felis catus), mice (Mus musculus), Yap flying fox (Pteropus yapensis), rats (Rattus spp.), and pigs (Sus scrofa) were repeatedly detected. These may be candidates of host animals of Leptospira in Palau; however, their detection was not clearly correlated with that of Leptospira. ConclusionWe repeatedly detected several species of pathogenic Leptospira from water samples of a wide region of Babeldaob Island. We confirmed that Leptospira contamination in freshwater environments increased under rainy conditions, particularly in the presence of flooding. This information could be used to improve public health control measures in this region.

Expanded range of Nasutitermes callimorphus Mathews, 1977 (Isoptera: Termitidae: Nasutitermitinae), comparison with N. corniger (Motschulsky, 1855) and N. ephratae (Holmgren, 1910), and synonymy of N. dasyopsis Thorne, 1989 into N. nigriceps (Haldeman, 1854)

The imago of N. callimorphus is described for the first time. Nasutitermes callimorphus occurs from Mexico to Paraguay. Nasutitermes callimorphus is smaller in all measurements but generally resembles its widely distributed sympatric congeners, N. corniger (Motschulsky, 1855) and N. ephratae (Holmgren, 1910). Molecular phylogenetic analysis of a portion of the mitochondrial 16S rRNA gene and the COII mtDNA marker revealed that N. callimorphus forms a distinct clade using both maximum parsimony and maximum likelihood analysis. Unlike N. corniger and N. ephratae, N. callimorphus does not build epigeal or arboreal carton nests. We further regard N. dasyopsis Thorne, 1989 as a junior synonym of N. nigriceps (Haldeman, 1854).

From ctenophores to scyphozoans: parasitic spillover of a burrowing sea anemone

Most host-parasite associations are explained by phylogenetically conservative capabilities for host utilization, and therefore parasite switches between distantly related hosts are rare. Here we report the first evidence of a parasitic spillover of the burrowing sea anemone Edwardsiella carnea from the invasive ctenophore Mnemiopsis leidyi to two scyphozoan hosts: the native Mediterranean barrel jellyfish Rhizostoma pulmo and the invasive Indo-Pacific nomad jellyfish Rhopilema nomadica, collected from the Eastern Mediterranean Sea. Edwardsiella carnea planulae found in these jellyfish were identified using molecular analyses of the mitochondrial 16S and nuclear 18S rRNA genes. Overall, 93 planulae were found on tentacles, oral arms, and inside of the gastrovascular canals of the scyphomedusae, whereas no infection was observed in co-occurring ctenophores. DNA metabarcoding approach indicated seasonal presence of Edwardsiella sp. in the Eastern Mediterranean mesozooplankton, coinciding with jellyfish blooms in the region. Our findings suggest a non-specific parasitic relationship between Edwardsiella carnea and various gelatinous hosts based on shared functionality rather than evolutionary history, potentially driven by shifts in host availability due to jellyfish blooms. This spillover raises questions about the ecological impacts of parasitism on native and invasive scyphozoan hosts and the potential role of Edwardsiella in controlling their populations.

Lost but not forgotten: Rediscovery of the Ukrainian Lepidurus middendorffii (Braem, 1893) (Branchiopoda: Notostraca) through integrative taxonomy

Abstract The taxonomy of the notostracan LepidurusLeach, 1819 is complicated by a large number of synonymised species and extensive intraspecific variability in many relevant characters. We demonstrate, based on an integrative taxonomic approach, that the Ukrainian populations of Lepidurus are not conspecific with any currently accepted Lepidurus species and assign them to the previously synonymised species Lepidurus middendorffiicomp. nov. (Braem, 1893). This species exemplarily highlights the problems in the taxonomy of Lepidurus. The species was originally described as Apus extensus var. middendorffii (Braem, 1893), but L. extensus is a junior synonym of Lepidurus lubbocki (Brauer, 1873), which is currently treated as a subspecies of Lepidurus apus (Linnaeus, 1758). Our molecular genetic analyses based on mitochondrial COI, 12S, 16S, and nuclear 28S, which included published data, suggest the presence of further, possibly cryptic species that await formal recognition and stress the importance for a more extensive revision of Lepidurus. The original type specimens of L. middendorffii, previously stored at the Museum of Natural History (Wrocław University, Wrocław, Poland), are lost and a neotype is designated. Lepidurus middendorffii is characterised by the dorsal organ position between the ocular tubercle margins, the sharp carapace sulcus spines, the long supra-anal plate with many median spines, a telson length to carapace length ratio 0.18–0.35, and differs by > 9% uncorrected COI p-distances from all other Lepidurus species.

Crossroad between the sylvatic and domestic cycles of Mesocestoides litteratus: mesocestodiasis in dogs adopted from shelters in Türkiye.

This study aimed to report the presence of Mesocestoides litteratus in dogs adopted from shelters in Türkiye. Gravid segments were examined microscopically in the faeces of dogs from different shelters located in Ankara and Kirikkale provinces in the central region of Türkiye. Then, genomic DNA obtained from these segments, a 446-bp fragment of the mitochondrial cytochrome C oxidase subunit 1 gene, and a 350-bp fragment of mitochondrial 12S rRNA were amplified and sequenced. BLASTn search was performed. During light microscopic examination, an egg-filled paruterine organ was observed in the middle part of the segment. Thin-shelled, oval, 35-µm-diameter parasite eggs containing an oncosphere with three pairs of hooklets were observed. The gravid segments were determined as Mesocestoides spp. based on the appearance of the typical paruterine organ. PCR results supported our diagnosis; moreover, according to the BLAST results, it was detected that the species infecting two dogs was 98.01-100% similar to M. litteratus. Praziquantel-containing medication was administered to the infected dogs at a dosage of 5mg/kg. Foxes act as the final host of M. litteratus and the parasite is prevalent in wildlife; however, these animals may disperse the parasite in urban life. Veterinarians need to be made more aware of this parasite, especially if the dogs are owned from shelters.

Generalist species exhibit more genetic structure in comparison to a habitat specialist: Evidence from a phylogeographic study of two freshwater crabs (Decapoda: Potamonautidae: Potamonautes), implications for habitat conservation.

During the present study, the phylogeography of the wetland specialist, freshwater crab, Potamonautes flavusjo, was investigated and compared to that of the ubiquitous, generalist, P. sidneyi, using DNA sequence data from the mitochondrial cytochrome oxidase subunit one (COI). We inferred the evolutionary history of each species and compared their population-level genetic structure by constructing haplotype networks and using an analysis of molecular variation. Additionally, we explored the evolutionary relationship between southern African lentic and lotic freshwater crab species by examining the usefulness of carapace attributes in relation to genetic indices and a species' assumed dispersal capacity. In the lentic species, P. flavusjo, a single interconnected haplocluster characterized by shared haplotypes was observed, suggesting marked maternal dispersal, a result corroborated by the low F ST values. In contrast, for the lotic species, P. sidneyi two distinct haploclusters and marked genetic differentiation was observed indicating the absence of maternal dispersal, a result corroborated by the high F ST values. The phylogenetic relationship in P. sidneyi was further investigated using a maximum likelihood and Bayesian inference analyses with the addition of sequence data from the mitochondrial 16S rRNA locus to estimate divergence times. Three species delimitation methods (ASAP, PTP, and bGMYC) were used to explore the presence of distinct lineages in P. sidneyi. The phylogenetic results indicated that within P. sidneyi two clades were present, while divergence time estimates suggest cladogenesis during the middle to late Pleistocene. The species delimitation methods used showed moderate congruence, however all oversplit the number of putative lineages. Our results indicated that P. sidneyi is a species complex comprised of two cryptic lineages, occurring in sympatry contemporarily possibly alluding to secondary contact. Carapace height was found to have no discernible influence on the genetic indices and presumed dispersal capabilities of mainland southern African freshwater crab species. The importance of our results are discussed in terms of conservation of freshwater habitats.