Assessment of natural and artificial propagation of the white-clawed crayfish (Austropotamobius pallipes species complex) in the Alpine region with nuclear and mitochondrial markers

In a joint analysis of nuclear (allozyme) and mitochondrial markers (sequence and restriction fragment length polymorphism (RFLP) analysis of the 12S and 16S genes), five genetically distinct groups of the Austropotamobius pallipes Lereboullet species complex were detected in the Alpine region. The geographical distribution of these evolutionary lineages coincided largely with several taxa (A.p. pallipes, A. berndhauseri, A.(p.) italicus) formerly defined on the basis of morphological characters. A low level of genetic variability was found within these lineages. For some populations, the combination of the two markers gave additional information about the likelihood of their natural or artificial origin. A hybrid zone was detected between A.p. pallipes and A. berndhauseri in the Lake Geneva area. The mosaic distribution of the five evolutionary lineages indicated that conservation efforts should be aimed at the level of local populations.     

Population genetic structure of the endangered crayfish Austropotamobius pallipes in France based on microsatellite variation: biogeographical inferences and conservation implications

1. One important goal in conservation biology is to characterise evolutionary lineages within endangered species before management decisions are taken. Here, we assess population differentiation in the freshwater crayfish Austropotamobius pallipes, an endangered species endemic to western Europe and provide valuable information for the conservation of French populations. 2. Analysis of five microsatellite loci in 44 populations revealed very different within population levels of genetic diversity (0.000 < H₀ < 0.564). Two groups, corresponding to northern and southern French populations, showed a high degree of genetic differentiation in both allele frequencies and allele sizes. Comparison of these results with previous studies of A. pallipes strongly suggests that the divergence between northern and southern populations could have occurred during the last glaciation period of the Pleistocene from one Atlantic and one Mediterranean refuge. 3. Evidence for genetic admixture between these two lineages was revealed by correspondence analyses in southern populations, probably as the result of artificial translocations. 4. French populations appeared significantly differentiated among the different river drainages and were highly structured within rivers. The impact of population size, population bottlenecks and founder events on the population genetic differentiation are discussed. 5. Based on these results, we propose the designation of two evolutionarily significant units for A. pallipes in France. Our data also support the maintenance of separate demographic management strategies for crayfish inhabiting different river systems. However, genetic analyses will have to be combined with demographic and ecological data for sustainable conservation programmes.     

Delimiting species boundaries and the conservation genetics of the endangered maritime ringlet butterfly (Coenonympha nipisiquit McDunnough)

Species delimitation is a difficult problem that has implications across organismal biology, yet no single method has proved wholly satisfactory. We tested the utility of combining species-delimitation methods based on phylogeny and gene flow statistics using two parapatric members of the Coenonympha tullia group as an example: the endangered maritime ringlet butterfly (Coenonympha nipisiquit McDunnough) and the common inornate ringlet butterfly (Coenonympha inornata Edwards). We reconstructed the phylogeny of the nearctic C. tullia-group taxa from mitochondrial DNA (mtDNA) sequences (cytochrome oxidase I and mitochondrial control region) to explore the ancestry of the C. nipisiquit lineage within the group. We investigated the extent of gene flow between the two taxa with F-statistics using 587 nuclear amplified fragment length polymorphism markers, accounting for the effect of potential scoring 'collisions' where a marker may represent more than one DNA fragment. Combining species-delimitation methods was especially effective because it uncovered both historical and recent evolutionary patterns. Phylogenetic analysis of mtDNA revealed the early divergence of C. nipisiquit from other C. tullia-group taxa, including the morphologically similar C. inornata. F-statistics and gene-by-gene introgression profiles demonstrated clear isolation between the two taxa and revealed strong population structure within C. nipisiquit. C. nipisiquit is the first taxon in the nearctic C. tullia group showing strong evidence of genetic isolation. The methods we used are relatively inexpensive and can be widely used to delimit taxonomic boundaries near the species level, both generally and in particular for taxa that may be targets of conservation efforts.     

Ultrastructure of the body wall of three species of Grania (Annelida: Clitellata: Enchytraeidae)

De Wit P., Erséus C. and Gustavsson L.M. 2011. Ultrastructure of the body wall of three species of Grania (Annelida: Clitellata: Enchytraeidae). —Acta Zoologica (Stockholm) 92 : 1–11. The body wall of three species of Grania, including the cuticle, epidermis and the musculature, are studied using TEM. The cuticle is similar to previously studied enchytraeids, with an orthogonal grid pattern of collagen fibers. This pattern is also seen in Crassiclitellata, which has been suggested as the sister taxon of Enchytraeidae. Variation of epicuticular and fiber zone patterns seen in Naididae (formerly Tubificidae and Naididae) seem to be lacking in Enchytraeidae. The fiber thickness, however, varies between Grania species and may be a phylogenetically informative character. The epidermis consists of supporting cells, secretory cells and sensory cells. Basal cells, typical for Crassiclitellata, were not observed. The clitellum of Grania seems to consist of two types of gland cells, which develop from regular epidermal tissue. It is possible that more cell types exist in different regions of the clitellum, however. The body wall musculature is arranged somewhat differently from that of closely related taxa; this refers to the reduction of circular and outer, triangular longitudinal muscle fibers, while the inner, ribbon‐shaped longitudinal muscle fibers are well‐developed. A search was conducted for the cause of the peculiar green coloration of Grania galbina De Wit and Erséus 2007, and it was concluded that neither cyanobacteria nor epidermal pigment granules were present in the fixed material.     

Phylogenetic relationships and biogeographic history of the unique Saxifraga sect. Irregulares (Saxifragaceae) from eastern Asia

Saxifraga L. is the largest genus in Saxifragaceae and a characteristic component of the herbaceous flora of the temperate and alpine mountains in the Northern Hemisphere. Section Irregulares is a small group of 15–20 species, representing one of the early‐diverged lineages in the genus characterized with unique zygomorphic flowers. We used both nuclear internal transcribed spacer and chloroplast DNA regions (psbA‐trnH, trnL‐F, and matK) to reconstruct its species relationships, estimate divergence times, and infer its historical biogeography. Our phylogenetic results corroborate the monophyly of sect. Irregulares and its sister relationship to sect. Heterisia from North America. The section was well resolved into two lineages corresponding to their morphological features and biogeographic distributions. One represents ser. Stoloniferae including S. stolonifera Curtis and S. nipponica Makino with long‐creeping stolons/rhizomes and small petals with spots and the other comprises the remaining taxa (ser. Rufescentes) which lack long‐creeping rhizomes. Spots on leaves (abaxially spotted vs. abaxially without spots) and spots on petals (without spots vs. with spots) are inferred to be phylogenetically informative within ser. Rufescentes. Divergence time estimates and ancestral area analysis suggested a western North American origin of sect. Irregulares with migration into East Asia by way of the Bering land bridge in the Middle Oligocene. The development of drying and desertification belt in the late Miocene could have played an important role in the subsequent restriction and separation of the north and south lineages within eastern Asia.     

Conservation genetics and demographic history of the endangered Cape Fear shiner (Notropis mekistocholas)

We examined allelic variation at 22 nuclear-encoded markers (21 microsatellites and one anonymous locus) and mitochondrial (mt)DNA in two geographical samples of the endangered cyprinid fish Notropis mekistocholas (Cape Fear shiner). Genetic diversity was relatively high in comparison to other endangered vertebrates, and there was no evidence of small population effects despite the low abundance reported for the species. Significant heterogeneity (following Bonferroni correction) in allele distribution at three microsatellites and in haplotype distribution in mtDNA was detected between the two localities. This heterogeneity may be due to reduced gene flow caused by a dam built in the early 1900 s. Bayesian coalescent analysis of microsatellite variation indicated that effective population size of Cape Fear shiners has declined in recent times (11-25 435 years ago, with highest posterior probabilities between 126 and 2007 years ago) by one-two orders of magnitude, consistent with the observed decline in abundance of the species. A decline in effective size was not indicated by analysis of mtDNA, where sequence polymorphism appeared to carry the signature of an older expansion phase that dated to the Pleistocene ( approximately 12 700 > 1 million years ago). Cape Fear shiners thus appear to have undergone an expansion phase following a glacial cycle but to have declined significantly in more recent times. These results suggest that rapidly evolving markers such as microsatellites may constitute a suitable tool when inferring recent demographic dynamics of populations.     

Disentangling the impact of demographic factors on population differentiation of an endangered freshwater crayfish (Austropotamobius pallipes) using population density and microsatellite data

1. Habitat fragmentation of stream ecosystems often results in decreased connectivity between populations and lower population sizes. Hence, understanding how habitat fragmentation affects genetic erosion is important for the preservation of freshwater biodiversity, in particular, as small populations suffer from loss of genetic diversity through genetic drift and loss of fitness because of inbreeding, increasing the risk of extinction. 2. Here, we assess the impact of demographic factors on population differentiation in the endangered freshwater crayfish Austropotamobius pallipes by analysing population genetic structure, estimating effective population sizes and comparing levels of polymorphism at five microsatellite loci with density estimates of 10 populations within a small French catchment that has become progressively confined to headwaters over the last six decades. 3. Levels of expected heterozygosity and allelic richness per population were relatively low (0.214–0.396 and 1.6–2.6, respectively). We found strong genetic differentiation between these geographically close populations (F_ST = 0.283), with weak statistical evidence for a pattern of isolation by distance. Estimates of effective population size were low (<150) in most populations, but potentially reached several thousands in three populations. 4. Population density and allelic richness were strongly positively correlated. A robust relationship between population density and heterozygosity values was also noted, but only after discarding two populations for which significant genetic signatures of a recent bottleneck were found; these two populations displayed high expected heterozygosity compared with a very low density. Populations with the highest densities of individuals had the highest effective population size estimates and vice versa. 5. Our results clearly show the importance of demographic factors and genetic drift on A. pallipes populations. Furthermore, analysis of genetic and population density data is a pragmatic and efficient approach to corroborate inferences from genetic data and can be particularly useful in the identification of populations experiencing a bottleneck and therefore in conservation genetics studies aiming at identifying priority populations for conservation.     

Population history of the Hispaniolan hutia Plagiodontia aedium (Rodentia: Capromyidae): testing the model of ancient differentiation on a geotectonically complex Caribbean island

Hispaniola is a geotectonically complex island consisting of two palaeo-islands that docked c. 10 Ma, with a further geological boundary subdividing the southern palaeo-island into eastern and western regions. All three regions have been isolated by marine barriers during the late Cenozoic and possess biogeographically distinct terrestrial biotas. However, there is currently little evidence to indicate whether Hispaniolan mammals show distributional patterns reflecting this geotectonic history, as the island's endemic land mammal fauna is now almost entirely extinct. We obtained samples of Hispaniolan hutia (Plagiodontia aedium), one of the two surviving Hispaniolan land mammal species, through fieldwork and historical museum collections from seven localities distributed across all three of the island's biogeographic regions. Phylogenetic analysis using mitochondrial DNA (cytochrome b) reveals a pattern of historical allopatric lineage divergence in this species, with the spatial distribution of three distinct hutia lineages biogeographically consistent with the island's geotectonic history. Coalescent modelling, approximate Bayesian computation and approximate Bayes factor analyses support our phylogenetic inferences, indicating near-complete genetic isolation of these biogeographically separate populations and differing estimates of their effective population sizes. Spatial congruence of hutia lineage divergence is not however matched by temporal congruence with divergences in other Hispaniolan taxa or major events in Hispaniola's geotectonic history; divergence between northern and southern hutia lineages dates to c. 0.6 Ma, significantly later than the unification of the palaeo-islands. The three allopatric Plagiodontia populations should all be treated as distinct management units for conservation, with particular attention required for the northern population (low haplotype diversity) and the south-western population (high haplotype diversity but highly threatened).     

Isolation and characterization of nine polymorphic microsatellite loci in the endangered shrub Disanthus cercidifolius var. longipes (Hamamelidaceae)

Nine polymorphic microsatellite loci were isolated and characterized from an AC-enriched genomic library of Disanthus cercidifolius var. longipes. Microsatellite polymorphism was investigated using 24 individuals from one natural population. The observed number of alleles per locus ranged from two to four. Observed and expected heterozygosities ranged from 0.17 to 0.92 and from 0.16 to 0.72, respectively. These polymorphic microsatellite loci provide useful tools for the ongoing population genetic studies of D. cercidifolius var. longipes.     

Phylogeography of the endangered rosewood Dalbergia nigra (Fabaceae): insights into the evolutionary history and conservation of the Brazilian Atlantic Forest

The Brazilian rosewood (Dalbergia nigra) is an endangered tree endemic to the central Brazilian Atlantic Forest, one of the world''s most threatened biomes. The population diversity, phylogeographic structure and demographic history of this species were investigated using the variation in the chloroplast DNA (cpDNA) sequences of 185 individuals from 19 populations along the geographical range of the species. Fifteen haplotypes were detected in the analysis of 1297 bp from two non-coding sequences, trnV-trnM and trnL. We identified a strong genetic structure (F_ST=0.62, P<0.0001), with a latitudinal separation into three phylogeographic groups. The two northernmost groups showed evidence of having maintained historically larger populations than the southernmost group. Estimates of divergence times between these groups pointed to vicariance events in the Middle Pleistocene (ca. 350 000–780 000 years ago). The recurrence of past climatic changes in the central part of the Atlantic forest, with cycles of forest expansion and contraction, may have led to repeated vicariance events, resulting in the genetic differentiation of these groups. Based on comparisons among the populations of large reserves and small, disturbed fragments of the same phylogeographic group, we also found evidence of recent anthropogenic effects on genetic diversity. The results were also analysed with the aim of contributing to the conservation of D. nigra. We suggest that the three phylogeographic groups could be considered as three distinct management units. Based on the genetic diversity and uniqueness of the populations, we also indicate priority areas for conservation.     

Island history affects faunal composition: the treeshrews (Mammalia: Scandentia: Tupaiidae) from the Mentawai and Batu Islands,Indonesia

The Mentawai and Batu Island groups off the west coast of Sumatra have a complicated geological and biogeographical history. The Batu Islands have shared a connection with the Sumatran ‘mainland’ during periods of lowered sea level, whereas the Mentawai Islands, despite being a similar distance from Sumatra, have remained isolated from Sumatra, and probably from the Batu Islands as well. These contrasting historical relationships to Sumatra have influenced the compositions of the respective mammalian faunas of these island groups. Treeshrews (Scandentia, Tupaiidae) from these islands have, at various times in their history, been recognized as geographically circumscribed populations of a broadly distributed Tupaia glis, subspecies, or distinct species. We used multivariate analyses of measurements from the skull and hands to compare the island populations from Siberut (Mentawai Islands) and Tanahbala (Batu Islands) with the geographically adjacent species from the southern Mentawai Islands (T. chrysogaster) and Sumatra (T. ferruginea). Results from both the skull and manus of the Siberut population show that it is most similar to T. chrysogaster, whereas the Tanahbala population is more similar to T. ferruginea, confirming predictions based on island history. These results are further corroborated by mammae counts. Based on these lines of evidence, we include the Siberut population in T. chrysogaster and the Tanahbala population in T. ferruginea. Our conclusions expand the known distributions of both the Mentawai and Sumatran species. The larger geographical range of the endangered T. chrysogaster has conservation implications for this Mentawai endemic, so populations and habitat should be re‐evaluated on each of the islands it inhabits. However, until such a re‐evaluation is conducted, we recommend that the IUCN Red List status of this species be changed from ‘Endangered’ to ‘Data Deficient’. Published 2013. This article is a U.S. Government work and is in the public domain in the USA, Biological Journal of the Linnean Society, 2014, 111 , 290–304.     

PERMANENT GENETIC RESOURCES: Development and characterization of 14 polymorphic microsatellite loci in the endangered tree Euptelea pleiospermum (Eupteleaceae)

Fourteen polymorphic microsatellite loci were developed and characterized for the endangered and tertiary relict tree, Euptelea pleiospermum. A genomic DNA enrichment protocol was used to isolate microsatellite loci and polymorphism was explored using 32 individuals from one natural population. The observed number of alleles ranged from two to nine. The ranges of observed and expected heterozygosities were 0.25-1.00 and 0.22-0.85, respectively. These microsatellite markers provide powerful tools for the ongoing conservation genetic studies of E. pleiospermum.     

Dispersal,fragmentation, and isolation shape the phylogeography of the European lineages of Polyommatus (Agrodiaetus) ripartii (Lepidoptera: Lycaenidae)

Polyommatus ripartii is a biogeographically and taxonomically poorly understood species of butterfly with a scattered distribution in Europe. Recently, it has been shown that this species includes several European endemic and localized taxa (galloi, exuberans, agenjoi) that were previously considered species and even protected, a result that poses further questions about the processes that led to its current distribution. We analysed mitochondrial DNA and the morphology of P. ripartii specimens to study the phylogeography of European populations. Three genetically differentiated but apparently synmorphic lineages occur in Europe that could be considered evolutionarily significant units for conservation. Their strongly fragmented and counterintuitive distribution seems to be the result of multiple range expansions and contractions along Pleistocene climatic oscillations. Remarkably, based on the 79 specimens studied, these genetic lineages do not seem to extensively coexist in the distributional mosaic, a phenomenon most evident in the Iberian Peninsula. One of the important gaps in the European distribution of P. ripartii is reduced by the discovery of new Croatian populations, which also facilitate a better understanding of the biogeography of the species. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 109 , 817‐829.     

Comparative ultrastructural study of the cuticle and spermatozoa in Propappus volki Michaelsen, 1916 (Annelida: Clitellata)

The ultrastructure of the cuticle and mature spermatozoa of the oligochaete Propappus volki Michaelsen, 1916 is described with the aim of providing additional data for clarifying the systematic position of the taxon. P. volki is a fresh-water species living in streams, and is easily recognized by its proboscis on the pre-segmental prostomium and, in mature specimens, by a clitellum covering the segments XII–XIV. The cuticle is composed of a proximal fibre zone and a distal layered epicuticle covered with membrane-bound epicuticular projections. The fibre zone consists of collagenous fibres in a matrix, arranged in either densely packed parallel layers with the fibres oriented in the same direction, or with more loosely distributed fibres, although with the same main orientation. The epicuticular projections are pyramidal with the base leaning on the outer surface of the epicuticle. The cuticle covering the proboscis differs in morphology from that of the rest of the worm; the fibre zone is composed of thin and short fibrils running in all directions, and the epicuticular projections are longer and more narrow than the projections in other regions of the worm.

The spermatozoa are filiform cells formed, in sequence, by an acrosome, an elongated nucleus, a long midpiece, and a flagellum. The acrosomal tube is short and straight with a completely external acrosomal vesicle. Following the acrosome is a apically corkscrew-shaped and basally straight nucleus. The midpiece is twisted and formed by five mitochondria. The flagellum shows a prominent central sheath arrangement.

A comparison with ultrastructurally described cuticles and spermatozoa from other clitellate species reveals most similarities with enchytraeids.     

Phylogeography of a pearl oyster (Pinctada maxima) across the Indo‐Australian Archipelago: evidence of strong regional structure and population expansions but no phylogenetic breaks

This study investigates the genetic structure and phylogeography of a broadcast spawning bivalve mollusc, Pinctada maxima, throughout the Indo‐West Pacific and northern Australia. DNA sequence variation of the mitochondrial cytochrome oxidase subunit I (COI) gene was analysed in 367 individuals sampled from nine populations across the Indo‐West Pacific. Hierarchical AMOVA indicated strong genetic structuring amongst populations (Φ_ST = 0.372, P < 0.001); however, sequence divergence between the 47 haplotypes detected was low (maximum 1.8% difference) and no deep phylogenetic divergence was observed. Results suggest the presence of genetic barriers isolating populations of the South China Sea and central Indonesian regions, which, in turn, show patterns of historical separation from northern Australian regions. In P. maxima, historical vicariance during Pleistocene low sea levels is likely to have restricted planktonic larval transport, causing genetic differentiation amongst populations. However, low genetic differentiation is observed where strong ocean currents are present and is most likely due to contemporary larval transport along these pathways. Geographical association with haplotype distributions may indicate signs of early lineage sorting arising from historical population separations, yet an absence of divergent phylogenetic clades related to geography could be the consequence of periodic pulses of high genetic exchange. We compare our results with previous microsatellite DNA analysis of these P. maxima populations, and discuss implications for future conservation management of this species. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 107 , 632–646.     

Rapid genetic differentiation between ex situ and their in situ source populations: an example of the endangered Silene otites (Caryophyllaceae)

Ex situ cultivation in botanic gardens could be one possibility to preserve plant species diversity and genetic variation. However, old ex situ populations are often sparsely documented. We were able to retrieve three different ex situ populations and their source in situ populations of the endangered plant species Silene otites after 20–36 years of isolation. Furthermore, three additional wild populations were included in the analysis. Population genetic diversity and differentiation were analysed using AFLP markers. Genetic variation in the ex situ populations was lower than the variation found in the in situ populations. Strong differentiation (F_ST = 0.21–0.36) between corresponding in situ and ex situ populations was observed. Bayesian clustering approach also showed a distinct genetic separation between in situ and ex situ populations. The high genetic differentiation and loss of genetic diversity during spatial and temporal isolation in the ex situ populations can be attributable to small population sizes and unconscious selection during cultivation. Therefore, adequate sampling prior to ex situ cultivation and large effective population sizes are important to preserve genetic diversity. Near‐natural cultivation allowing for generation overlap and interspecific competition without artificial selection is recommended as being best for the maintenance of the genetic constitution. © 2011 The Linnean Society of London, Botanical Journal of the Linnean Society, 2011, ??, ??–??.     

Comparative allozyme genetics and range history of the European river barbel (Teleostei,Cyprinidae: Barbus barbus) in the Rhine/upper Danube contact area1

Enzymes, representing 23 genetic loci, were studied in n = 320 European river barbels (Barbus barbus L. 1758) from the contact zone of the Rhine and the upper Danube. Barbel revealed the highest heterozygosity estimate (H_e = 0.137) of 10 sympatric freshwater fishes (range H_e = 0.017–0.106). The geographical genetic subdivision in barbel (G_ST = 0.1690) surpassed the respective estimates in two other cyprinid species (G_ST ≤ 0.1415) and in three anadromous fishes (range G_ST = 0.0330–0.1290) from the same study area, but it fell short of the spatial genetic fragmentation of two resident salmonid species (G_ST ≥ 0.2678) and of sculpins (G_ST = 0.8489). The theoretical gene flow in the unobstructed Danube was N_em^DAN = 4.98, and across the weirs in the southern Rhine N_em^RHE = 1.84. The Rhine/Danube watershed exceeded any other isolation effect on the gene pool despite a low genetic distance (D^RHE/DAN = 0.0321) among Rhenish and Danubian samples. The range history in central Europe might imply the colonization of the study area from a Danubian and from a Rhenish or a Rhôdanian refuge. The concluded admixture of eastern and western barbels has produced a weakly pronounced cline of increasing allozymic affinity of Rhenish to Danubian barbels in the southern Rhine system. Oberschwaben has served as an important colonization pathway in the phylogeographical exchange between the Danube and west Europe. The influence of stocking for fisheries on the allozymes is not known precisely, but it appears to be of minor importance.     

A new species of Caracolus (Pulmonata: Camaenidae) from the Oligocene of Nebraska and the biotic history of the American camaenid land snails

A fossil land snail from the Oligocene White River Group of Nebraska is described as Caracolus aquilonaris sp nov. Living members of the genus Caracolus occur in tropical forest on the islands of the Greater Antilles. It is likely that C. aquilonaris occupied a similar habitat and that the present restricted occurrence of the genus Caracolus is a fragment of a previously wider range. The American carnaenid genera Pleurodonte, Polydontes and Zachrysia form a well defined monophyletic group, but there is an unresolved trichotomy involving this group, Labyrinthus and Caracolus . Well characterized fossil camaenids assigned to Pleurodonte (Jamaica, Carriacou) and the related Pleurodontites (Florida) occur in the Miocene. Antecedents to Labyrinthus were isolated in South America and to Caracolus and Pleurodonte in North America, possibly in the late Cretaceous. The latter genera reached the Antilles where Caracolus has persisted relatively unchanged. Ancestral Pleurodonte has diversified producing the distinctive subgenera Eurycratera and Thelidomus on Jamaica and the genus Polydontes on the other Greater Antilles. Drier conditions on Cuba have resulted in the divergence of Zachysia from Polydontes .