Comparison of whole mitochondrial genome sequences of northern and southern white rhinoceroses (<Emphasis Type="Italic">Ceratotherium simum</Emphasis>): the conservation consequences of species definitions

The probable extinction of the last confirmed population of northern white rhinoceros (Ceratotherium simum cottoni) in the world has ignited debate regarding its species status compared to the southern white rhinoceros (Ceratotherium simum simum). Previous studies, based on partial mitochondrial sequences, have reported conflicting results regarding the species status of the northern white rhinoceros. We use whole mitochondrial genomes obtained using Next Generation Sequencing of four northern white rhinoceros and three southern white rhinoceros using novel primers in three overlapping fragments. Phylogenetic relationships were constructed, using Maximum Likelihood, and recovered monophyletic clades for northern white rhinoceros and southern white rhinoceros. The divergence time between the two mitochondrial DNA lineages was estimated to be between 0.46 and 0.97 million years ago using Bayesian inferences. Since there are currently only three surviving northern white rhinoceros individuals these results put into sharp focus the ongoing debate regarding the methods of species definition, and in particular the consequences of such definitions in conservation management of endangered species and subspecies. We conclude that the designation of sub-species status is more applicable to northern and southern white rhinoceros.     

Genetic Analysis of the <Emphasis Type="Italic">Indri</Emphasis> Reveals No Evidence of Distinct Subspecies

Subspecies were traditionally defined by identifying gaps between phenotypes across the geographic range of a species, and may represent important units in the development of conservation strategies focused on preserving genetic diversity. Previous taxonomic research proposed that phenotypic variation between scattered Indri indri populations warranted the naming of two distinct subspecies, I. i. indri and I. i. variegatus. We tested these subspecific designations using mitochondrial sequence data generated from the control region or D-loop (569 bp) and a large section (2362 bp) of multiple genes and tRNAs known as Pastorini’s fragment and nuclear microsatellite markers. This study used 114 samples of I. indri from 12 rainforest sites in eastern Madagascar, encompassing the entire range of the species. These genetic samples represent multiple populations from low- and high-elevation forests from both putative subspecies. Molecular analyses of the mitochondrial sequence data did not support the two proposed subspecies. Furthermore, the microsatellite analyses showed no significant differences across the range beyond population level differentiation. This study demonstrates the utility of incorporating multiple lines of evidence in addition to phenotypic traits to define species or subspecies.     

Mitochondrial and Nuclear DNA Based Genetic Assessment Indicated Distinct Variation and Low Genetic Exchange Among the Three Subspecies of Swamp Deer (<Emphasis Type="Italic">Rucervus</Emphasis> <Emphasis Type="Italic">duvaucelii</Emphasis>)

The swamp deer (Rucervus duvaucelii) occurs, primarily, in the wet grasslands of the Himalayan foothills as well as the dry grasslands of central India. Three subspecies have been identified, namely R. duvaucelii duvaucelii, R. duvaucelii branderi and R. duvaucelii ranjitsinhi. Degradation of grassland habitats led to a drastic decline in the total swamp deer population in the early 19th century. Even though the species has recently shown signs of recovery, it is still vulnerable to the small-population paradigm. Effective management plans need to be put in place to increase the population through scientific intervention. The current genetic variation within the three subspecies of R. duvaucelii is unclear, and this is hindering effective conservation planning. We examined the genetic variability, population structure and demography of the three subspecies of swamp deer using the mtDNA control region and microsatellite analysis. Despite the spatial isolation of the populations, we found a high level of variation and weak divergence among the subspecies. The genetic differentiation (F _ST ) between the subspecies and the mismatch distribution of haplotypes indicated recent colonization by these subspecies. Population bottleneck analysis indicated that the existing subspecies and their populations are at demographic equilibrium and are stable. The study highlights the need for effective conservation management intervention to maintain the population size and genetic diversity. It also indicates that all the subspecies need to be managed as separate conservation units.     

Evolution of rough sculpin (<Emphasis Type="Italic">Cottus asperrimus</Emphasis>) genetic divergence and late Quaternary displacement on the Hat Creek fault,California, USA

The rough sculpin (Cottus asperrimus) is a threatened species whose geographic range in northwestern California, USA is disrupted by Hat Creek fault. We tested whether the fluvial barriers (rapids and waterfalls) produced by this fault have generated significant phylogeographic structure among rough sculpin populations by analyzing variation in microsatellites and mitochondrial DNA. Rough sculpin isolated on either side of Hat Creek fault exhibited significant genetic divergence (microsatellite F _ST = 0.36; mitochondrial uncorrected p distance = 1 %). Independently derived estimates for the date of divergence, based upon a molecular clock and upon the age of slip on the Hat Creek fault are concordant and indicate divergence was initiated about 0.5–1 million years ago. Based upon the findings of our genetic analysis and the Pleistocene geologic history of midsections of the Pit River, we present a model of evolution of rough sculpin genetic divergence and late Quaternary displacement on Hat Creek fault. Our findings reveal that rough sculpin exhibit significant population structure and that two management units should be recognized within the species for future conservation planning.     

The Brachial Plexus of the Sumatran Rhino (<Emphasis Type="Italic">Dicerorhinus sumatrensis</Emphasis>) and Application of Brachial Plexus Anatomy Toward Mammal Phylogeny

The peripheral nervous system is a promising resource for testing phylogeny although the branching patterns of peripheral nerves are not well documented outside of Homo sapiens. Here we describe the brachial plexus of the rare Sumatran rhinoceros (Dicerorhinus sumatrensis). We compare its brachial plexus to that of another perissodactyl (Equus asinus), an artiodactyl (Odocoileus virginianus), two carnivorans (Felis catus and Neovison vison), and one primate (Homo sapiens) and examine the phylogenetic structure of the resulting data. Brachial plexuses exhibit high rates of intraspecific polymorphism, but polymorphisms cannot be recognized from one specimen. To address concerns of error due to polymorphism, we dissected 52 mink brachial plexuses and compared them to human brachial plexus variation. Both species have numerous types of brachial plexus polymorphisms. Although most individual polymorphisms occur infrequently and unilaterally, because there are numerous types of polymorphisms, most humans and mink exhibit at least one polymorphism per brachial plexus. Parsimony analysis of 15 characters compiled from the brachial plexus data produced a tree that positions Artiodactyla and Perissodactyla as sister taxa, a result consistent with other analyses. Despite a high rate of polymorphism, the peripheral nervous system seems to carry a phylogenetic signal consistent with other morphological data. With a higher rate of taxon sampling, we suggest the brachial plexus will contribute valuable data for phylogenetic testing.     

Complete mitochondrial genome of threatened mahseer <Emphasis Type="Italic">Tor tor</Emphasis> (Hamilton 1822) and its phylogenetic relationship within Cyprinidae family

The mahseers (Tor, Neolissochilus and Naziritor) are an important group of fishes endemic to Asia with the conservation status of most species evaluated as threatened. Conservation plans to revive these declining wild populations are hindered by unstable taxonomy. Molecular phylogeny studies with mitochondrial genome have been successfully used to reconstruct the phylogenetic tree and to resolve taxonomic ambiguity. In the present study, complete mitochondrial genome of Tor tor has been sequenced using ion torrent next-generation sequencing platform with coverage of more than 1000 ×. Comparative mitogenome analysis shows higher divergence value at ND1 gene than COI gene. Further, occurrence of a distinct genetic lineage of T. tor is revealed. The phylogenetic relationship among mahseer group has been defined as Neolissochilus hexagonolepis ((T. sinensis (T. putitora, T. tor), (T. khudree, T. tambroides)).     

Highly diversified population structure of the spider <Emphasis Type="Italic">Lycosa ishikariana</Emphasis> inhabiting sandy beach habitats

Sandy beach ecosystems are decreasing worldwide and organisms living there are becoming threatened. The burrowing wolf spider Lycosa ishikariana is one such example. To establish effective conservation strategies under habitat fragmentation, we examined population genetic structure of L. ishikariana from mitochondrial cytochrome oxidase I gene and 6 microsatellite loci. Mitochondrial sequence data revealed 6 population subgroups with very high fixation indices, indicating that L. ishikariana has a clear phylogeographic structure and that the level of differentiation among regions is considerable. In particular, one subgroup in the western Honshu mainland (clade G) has a highly distinct genetic structure, despite having no clear geographic barriers from its parapatric population. Moreover, the distribution ranges of the other two subgroups (clades D and E) were highly restricted, suggesting their vulnerability to local human impacts and highlighting their high conservation priorities. Microsatellite data revealed 10 subgroups that were compatible with the clades identified from the mitochondrial data. Fixation indices among these groups were very high, indicating a limited gene flow induced by male spiders. Based on these results, we proposed six conservation units of L. ishikariana and effective conservation/restoration strategies in the face of ongoing coastal armoring.     

Population expansion,current and past gene flow in Gould’s petrel: implications for conservation

Seabird life-history traits such as long generation time, low annual fecundity and delayed sexual maturation make them more prone to population loss and consequently to extinction; petrels are indeed amongst the most threatened birds. Based on coalescence and multiloci inference this study examines the extent of genetic differentiation of a vulnerable New Caledonia (caledonica) and Australia (leucoptera) subspecies of Pterodroma leucoptera (Gould’s Petrel), and whether the genetic relationship between them results from the influence of past events like variation in sea level, or is dominated by contemporary gene flow. Sequences of two mitochondrial genes and five nuclear introns were obtained from 86 individuals from both populations. Haplotype networks were used to infer relationships between the haplotypes of both populations. The demographic history of the P. leucoptera complex was studied using neutrality tests and Extended Bayesian Skyline Plots. A weak population differentiation was revealed. The Extended Bayesian Skyline plot suggested a population expansion approximately 80,000 years before present (bp) for caledonica and 30,000 years bp for leucoptera. The split was dated to 30,000 years bp by means of multilocus inference through *BEAST. Despite genetic similarity of the two taxa, we advocate to consider them as independent units for conservation management, given their strong ecological distinctiveness (foraging distribution, winter distribution, breeding phenology and breeding distribution).     

Genetic distinctiveness of brown pelicans (<Emphasis Type="Italic">Pelecanus occidentalis</Emphasis>) from the Galápagos Islands compared to continental North America

We examined population differentiation across a substantial portion of the range of the brown pelican (Pelecanus occidentalis) to assess (1) the genetic distinctness of the Galápagos subspecies (P. o. urinator) and (2) genetic differentiation between subspecies that inhabit the coasts of North and Central America (P. o. californicus and P. o. carolinensis). Birds were sampled from coastal California, coastal Florida, and the Galápagos Islands. Using a 957 bp (bp) fragment of the NADH dehydrogenase subunit 2 (ND2) gene, 661 bp of the mitochondrial control region, and eleven microsatellite loci we characterize population genetic differentiation among 158 brown pelicans. The Galápagos subspecies is genetically distinct from the sampled continental subspecies, possessing a unique ND2 haplotype and unique mitochondrial control region haplotypes. Samples from the two continental subspecies all possessed the same ND2 haplotype and shared four mitochondrial control region haplotypes. Bayesian clustering in STRUCTURE placed the Galápagos subspecies in a distinct genetic group with high probability, but could not differentiate the continental subspecies from one another. Estimates of migration rates from BayesAss indicated substantial migration between continental subspecies, but no migration between the Galápagos subspecies and either continental subspecies. There are clearly two Evolutionarily Significant Units within the range of the brown pelican, which warrants conservation attention. Further investigation should determine how the un-sampled subspecies (P. o. murphyi and P. o. occidentalis) fit into the broader picture.     

Genetic distinctiveness of the damselfly <Emphasis Type="Italic">Coenagrion puella</Emphasis> in North Africa: an overlooked and endangered taxon

North African odonates are facing conservation challenges, not only by increased degradation and loss of habitat, but also by having poorly understood taxonomy. Coenagrion puella is a widely distributed damselfly but there is debate about the taxonomic status of North African populations, where the species is very rare. We evaluate the genetic distinctiveness of North African C. puella using mitochondrial and nuclear genetic markers. We found a clear genetic differentiation between North African and European populations (3.4 % mtDNA) and a lack of shared haplotypes between individuals from the two continents. These results suggest that the damselfly C. puella comprises two genetically distinct phylogenetic lineages: one in Europe and one in North Africa, and re-invigorate the debate on the validity of the North African endemic C. puella kocheri. We propose that these two lineages of C. puella should be managed as distinct molecular operational taxonomic units. More generally, this study reinforces the important role of North Africa as centre of speciation and differentiation for odonates, and highlights the relevance of incorporating genetic data to understand the evolutionary history and taxonomy for effective biodiversity conservation.     

The Role of Geographical and Ecological Factors on Population Divergence of the Neotropical otter <Emphasis Type="Italic">Lontra longicaudis</Emphasis> (Carnivora,Mustelidae)

The geographic distribution of the populations of a species are influenced by the spatial structure of the ecosystems, the environmental factors and the presence of geographic barriers. The Neotropical otter, Lontra longicaudis, is widely distributed throughout the Americas, where a wide range of environmental conditions and geographical features could promote genetic and morphological variation on the three currently recognized subspecies. In this study, we combined phylogeographic, morphometric and environmental niche modelling analyses to examine whether: (1) genetic variation is associated with the presence of barriers to gene flow and/or hydrography; (2) genetic and morphologic variation are associated with environmental variation; and (3) the observed variation in L. longicaudis populations corresponds to the previously defined subspecies. We found strong phylogeographic structure between the northern (L. l. annectens) and the two-southern subspecies (L. l. longicaudis and L. l. enudris), and although shallower, we also detected genetic differentiation between the two South American subspecies. Such genetic differentiation corresponds to the hydrography and to the geographical barriers characteristic of the distributional area of the species. We found a correlation between the shape of the skull and mandible with the environmental variation through the distribution of the species, and we rejected the hypothesis of niche equivalency and similarity between the three identified genetic lineages, suggesting adaptations to different environmental conditions. Our results support that the variation in environmental conditions, in concert with geographical barriers to gene flow and hydrography, have led to population divergence of L. longicaudis along the Neotropics. These results have important taxonomic implications for the species and its conservation.     

Genetic structure and environmental niche modeling confirm two evolutionary and conservation units within the western spadefoot (<Emphasis Type="Italic">Spea hammondii</Emphasis>)

The western spadefoot (Spea hammondii) is a Species of Special Concern in California and is now under review by the U.S. Fish and Wildlife Service for listing under the Endangered Species Act. We delineated potential conservation units within S. hammondii by analyzing spatial genetic structure across the species’ range using five nuclear and one mitochondrial loci. For both nuclear and mitochondrial markers we found that S. hammondii consists of two genetically distinct, allopatric clusters divided by the Transverse Ranges. To corroborate the northern and southern genetic clusters as conservation units from an ecological perspective, we applied a niche identity test to environmental niche models of the two groups. We found that the niche models of the northern and southern clusters were significantly different, suggesting they may be ecologically non-exchangeable. Given our demonstration of significant genetic and ecological differentiation between allopatric clusters of S. hammondii, we recommend that ongoing conservation efforts consider each as a separate unit with potentially unique management needs.     

The contrasting genetic patterns of two sympatric flying fox species from the Comoros and the implications for conservation

Pteropus livingstonii and Pteropus seychellensis comorensis are endemic fruit bat species that are among the most threatened animals in the Comoros archipelago. Both species are pollinators and seed dispersers of native and cultivated plants and are thus of crucial importance for the regeneration of natural forests as well as for cultivated plantations. However, these species are subject to strong anthropogenic pressures and face one of the highest rates of natural habitat loss reported worldwide. Yet little is known about the population genetic structure of these two species, making it difficult to define relevant conservation strategies. In this study, we investigated for the two flying fox species (1) the level of genetic diversity within islands, as well as across the archipelago and (2) the genetic structure between the two islands (Anjouan and Mohéli) for P. livingstonii and between the four islands of the archipelago (Anjouan, Mohéli, Grande Comore and Mayotte) for P. s. comorensis using mitochondrial and microsatellite markers. The results revealed contrasting patterns of genetic structure, with P. s. comorensis showing low genetic structure between islands, whereas P. livingstonii exhibited high levels of inter-island genetic differentiation. Overall, the genetic analyses showed low genetic diversity for both species. These contrasting genetic patterns may be the result of different dispersal patterns and the populations’ evolutionary histories. Our findings lead us to suggest that in terms of conservation strategy, the two populations of P. livingstonii (on Anjouan and Mohéli islands) should be considered as two separate management units. We recommend focusing conservation efforts on the Anjouan population, which is the largest, exhibits the highest genetic diversity, and suffers the greatest anthropogenic pressure. As for P. s. comorensis, its four populations could be considered as a single unit for conservation management purposes. For this species, we recommend protecting roosting trees to reduce population disturbance.     

Characterizing range-wide divergence in an alpine-endemic bird: a comparison of genetic and genomic approaches

The delineation of intraspecific units that are evolutionarily and demographically distinct is an important step in the development of species-specific management plans. Neutral genetic variation has served as the primary data source for delineating “evolutionarily significant units,” but with recent advances in genomic technology, we now have an unprecedented ability to utilize information about neutral and adaptive variation across the entire genome. Here, we use traditional genetic markers (microsatellites) and a newer reduced-representation genomic approach (single nucleotide polymorphisms) to delineate distinct groups of white-tailed ptarmigan (Lagopus leucura), an alpine-obligate species that is distributed in naturally fragmented habitats from Alaska to New Mexico. Five subspecies of white-tailed ptarmigan are currently recognized but their distinctiveness has not been verified with molecular data. Based on analyses of 436 samples at 12 microsatellite loci and 95 samples at 14,866 single nucleotide polymorphism loci, we provide strong support for treating two subspecies as distinct intraspecific units—L. l. altipetens, found in Colorado and neighboring states; and L. l. saxatilis, found on British Columbia’s Vancouver Island—but our findings reveal more moderate patterns of divergence within the remainder of the species’ range. Results based on genetic and genomic datasets generally agreed with one another, indicating that in many cases microsatellite loci may be sufficient for describing major patterns of genetic structure across species’ ranges. This work will inform future conservation and management decisions for the white-tailed ptarmigan, a species that may be vulnerable to future changes in climate.     

Genetic variation of Manchurian pheasant (<Emphasis Type="Italic">Phasianus colchicus pallasi</Emphasis> Rotshild, 1903) inferred from mitochondrial DNA control region sequences

Sequence variation of the mitochondrial DNA control region was studied in Manchurian pheasants (Phasianus colchicus pallasi Rotshild, 1903) representing three geographic populations from the southern part of the Russian Far East. Extremely low population genetic differentiation (F _ST = 0.0003) pointed to a very high gene exchange between the populations. Combination of such characters as high haplotype diversity (0.884 to 0.913), low nucleotide diversity (0.0016 to 0.0022), low R2 values (0.1235 to 0.1337), certain patterns of pairwise-difference distributions, and the absence of phylogenetic structure suggested that the phylogenetic history of Ph. c. pallasi included passing through a bottleneck with further expansion in the postglacial period. According to the data obtained, it was suggested that differentiation between the mitochondrial lineages started approximately 100000 years ago.     

Characterizing genetic integrity of rear-edge trout populations in the southern Appalachians

Vertebrate populations at the periphery of their range can show pronounced genetic drift and isolation, and therefore offer unique challenges for conservation and management. These populations are often candidates for management actions such as translocations that are designed to improve demographic and genetic integrity. This is particularly true of coldwater species like brook trout (Salvelinus fontinalis), whose numbers have declined greatly across its historic range. At the southern margin, remnant wild populations persist in isolated headwater streams, and many have a history of receiving translocated individuals through either stocking of hatchery reared fish, relocation of wild fish, or both during restoration attempts. To determine current genetic integrity and resolve the genetic effects of past management actions for brook trout populations in SC, USA, we genetically assessed all 18 documented remaining brook trout populations along with individuals acquired from six hatcheries with recorded stocking events in SC. Our results indicated that six of the 18 streams showed signs of hatchery admixture (range 57–97%) and restored patches retained genetic signatures from multiple source populations. Populations had among the lowest genetic diversity (min average H_E?=?0.147) and effective number of breeders (mean N_b?=?31.2) estimates observed throughout the native brook trout range. Populations were highly differentiated (mean pair-wise F_ST?=?0.396), and substantial genetic divergence was evident across major river drainages (max pair-wise F_ST?=?0.773). The lowest local genetic diversity and highest genetic differentiation ever reported for this species make its conservation a challenging task, particularly when combined with other threats such as climate change and non-native species. We offer recommendations on managing peripheral populations with depleted genetic characteristics and provide a reference for determining which existing populations will best serve as sources for future translocation efforts aimed at enhancing or restoring wild brook trout genetic integrity.     

Geographic population structure and subspecific boundaries in a tidal marsh sparrow

Subspecific genetic diversity is a source for ongoing evolutionary processes, can be predictive of a population’s ability to respond to conservation challenges, and may represent the raw material for incipient speciation. As such, diagnosable subspecies are increasingly recognized as an important unit for conservation. Differentiating among subspecies can be particularly difficult in ecosystems characterized by recent phenotypic divergence, such as the tidal marshes of North America. These systems require approaches that can capture selective changes which occurred over only a few millennia as species adapted to new habitats following the Holocene glacial retreat. Here we test for genetic differentiation in morphologically distinct tidal-marsh-endemic subspecies of the swamp sparrow (Melospiza georgiana) using nuclear microsatellites. This case study serves as a test approach for the quantification of conservation units for tidal marsh ecosystems. Though prior surveys of mtDNA variation showed no detectable phylogeographic structure, we found evidence of genetic differentiation in seven microsatellite loci between two M. georgiana subspecies. The most likely model of population structure suggested two clusters in western Maryland/Pennsylvania and Delaware/Chesapeake Bays, with a zone of uncertain population assignment in New Jersey. The microsatellite intergrade zone is broader than the known area of morphological intergrades. We show that microsatellites can be used to support a subspecific status for tidal-marsh taxa such as the swamp sparrow, where changing post-glacial environments likely selected for locally adapted traits while neutral genetic structure is weak. This approach thus allows for the identification and conservation of hot spots that foster ongoing evolutionary change.     

Conservation genomics of the silktail (Aves: <Emphasis Type="Italic">Lamprolia victoriae</Emphasis>) suggests the need for increased protection of native forest on the Natewa Peninsula,Fiji

Effective conservation relies on accurate taxonomy, because we cannot protect what we do not know. Species limits among phenotypically differentiated and allopatrically distributed populations on Southwest Pacific islands are poorly understood. This likely has led to an underestimate of species richness in the Southwest Pacific, and, consequently, a biased application of conservation effort. The silktail Lamprolia victoriae is a bird species endemic to Fiji. Two subspecies are known from Vanua Levu and Taveuni Islands, but uncertainty remains whether they should be considered one or two species. If the latter, increased conservation effort is warranted to protect forest habitat where isolated populations occur only on the Natewa Peninsula. Here, we address this question by examining 8859 single nucleotide polymorphisms produced by restriction-site associated DNA sequencing. We find that the silktail is best considered two species, due to high genetic differentiation and low gene flow between the two subspecies. These differences match known phenotypic differences (size and plumage), as well as allopatric island distributions. We suggest that the silktail be used as an icon for conservation efforts of the heavily degraded forest habitats on the Natewa Peninsula. Finally, we reassess the divergence age estimates of Lamprolia and its relatives, Chaetorhynchus and Rhipidura, in light of new phylogenomic evidence from oscine passerines.