Clonal identification by microsatellite loci in sporadic flowering of a dwarf bamboo species, <Emphasis Type="Italic">Sasa cernua</Emphasis>

Dwarf bamboo species are monocarpic. They flower simultaneously and die after several decades. The type of flowering in the genus Sasa ranges from sporadic to gregarious. In order to determine whether or not the sporadic flowering of dwarf bamboo is fixed genetically, we investigated the distribution of clones using eight microsatellite (SSR) loci in a sporadic flowering patch of Sasa cernua Makino, a major dwarf bamboo species found in central Hokkaido. In May 2006, flowering occurred on 60.5% of living culms in a 1600 m² patch. We established a 50 × 10 m study plot in this patch and noted 1267 clumps consisting of 2529 living culms. We investigated all 1267 clumps and identified six multilocus genotypes as clones using five variable SSR loci. All flowering clumps belonged to the same clone. On the other hand, non-flowering vegetative clumps were also discovered to be of the same clone. These data suggest that all flowering culms originated from a single clone of a sporadic flowering patch of S. cernua. Clonal analysis for investigation of sporadic flowering of S. cernua revealed that only a portion of a clone flowers and dies instead of the whole clone.     

Effects of conspecific and heterospecific floral density on the pollination of two related rewarding orchids

Variation in within-population floral density can affect interactions between plants and pollinators, resulting in variable pollen export for plants. We investigated the effects of conspecific and heterospecific floral densities on pollination success both of two related, self-compatible, nectar-rewarding orchid species in Ireland, Spiranthes romanzoffiana (rare and listed as endangered) and its congener, S. spiralis (more abundant and not of conservation concern). Floral densities, insect visitation rates, and orchid pollen transport were recorded in multiple quadrats in four populations of both orchid species over their flowering season. We found that conspecific and heterospecific co-flowering plant density affected pollination in both orchid species. For S. romanzoffiana, higher heterospecific density increased pollen removal. For S. spiralis, higher conspecific visitation increased pollen removal and increased heterospecific density decreased pollen deposition. In addition, increased conspecific density increased pollen deposition in both species. This study shows that plants may interact to facilitate or compete for different components of the pollination process, namely; pollinator attraction, pollen removal and deposition. Such interactions have immediate consequences for endangered plant species, as increases in both conspecific and heterospecific coflowering density may ameliorate the negative effects of rarity on pollination, hence overall reproductive success.     

Genetic variation within the endangered mangrove species Sonneratia paracaseolaris (Sonneratiaceae) in China detected by inter-simple sequence repeats analysis

Sonneratia paracaseolaris, is a critically endangered mangrove species in China. Using inter-simple sequence repeats (ISSR) markers, we compared the genetic variation of introduced populations with that of natural populations to check whether the genetic diversity has been conserved. At the species level, genetic diversity was relatively high (P = 81.37%, He = 0.2241, and SI = 0.3501). Genetic variation in introduced populations (P = 75.78%, He = 0.2291, and SI = 0.3500) was more than that in natural populations (P = 70.81%, He = 0.1903, and SI = 0.2980). Based on Nei's G_ST value, more genetic differentiation among natural populations was detected (G_ST = 0.3591). Our data show that the genetic diversity of S. paracaseolaris was conserved in introduced populations to some extent, however, owing to the small natural populations and the threats they encountered, more plants should be planted to enlarge and restore the populations.     

Genetic diversity of the endangered coastal violet <Emphasis Type="Italic">Viola grayi</Emphasis> Franchet et Savatier (Violaceae) and its genetic relationship to the species in subsection <Emphasis Type="Italic">Rostratae</Emphasis>

Viola grayi, a coastal violet, is found on sandy seashores in limited regions in Japan, but not on rocky sea cliffs or in inland areas. This stemmed violet is classified in the subsection Rostratae. Because of recent human activity on sandy seashores, this species is now recorded as endangered. The present status of this species was surveyed and its genetic diversity was analyzed using 17 newly developed simple sequence repeat markers. The results are compared with those for two commonly found inland violet species in the same subsection, V. grypoceras and V. kusanoana. The coastal violet populations showed markedly lower genetic diversity than those of the common species, V. grypoceras, whereas V. kusanoana showed intermediate genetic diversity. About a half of the total genetic variation of V. grayi is found among populations in analysis of molecular variance. In contrast, only less than 25% of the total variation is found among populations for the two common violets. These three species showed a remarkably high inbreeding coefficient, which indicates high inbreeding under natural mating conditions, although these species have both chasmogamous and cleistogamous flowers. In a population-based dendrogram, V. grayi was clustered in a single and concise group, whereas V. grypoceras and V. kusanoana were found in somewhat discrete positions. In the Bayesian analysis of genetic structure of the coastal violet, delta K was the highest at K = 7, and the seven clusters almost correspond to seven populations studied here. Implications of the conservation of this endangered coastal violet are presented.     

Population genetic diversity and structure of two rare vernal pool grasses in central California

Vernal pool ecosystems are declining throughout California, with only 10% of historic habitat remaining. This has endangered many specialist endemic plant species, leaving extant populations fragmented, isolated, and threatened or endangered. Recovery plans for the increasing number of endangered vernal pool species require information on their genetic and ecological status to guide conservation and restoration efforts. Federally threatened Neostapfia colusana (Colusa grass) and federally endangered Tuctoria greenei (Greene’s tuctoria) are two endemic vernal pool grasses of high conservation concern in central California. Remaining populations are highly fragmented due to range-wide habitat destruction. Using five polymorphic microsatellite markers for each species, we performed genetic surveys of 240 individuals from eight vernal pools for N. colusana, and 317 individuals from 13 vernal pools for T. greenei. We detected high within-population genetic diversity for both species, with average allelic diversities of 24 alleles/locus (mean Hobs = 0.68, mean Hexp = 0.71) for N. colusana, and 19 alleles/locus (mean Hobs = 0.77, and mean Hexp = 0.79) for T. greenei. Bayesian clustering and AMOVA indicated two genetically distinct population groups for N. colusana (Fst = 0.268, P < 0.0001), and three for T. greenei (Fst = 0.11, P < 0.0001). We found very slight temporal genetic structure at one N. colusana (Fst = 0.013, P < 0.05) and two T. greenei (Fst = 0.015, Fst = 0.018, P < 0.05) pools. These estimates of population genetic diversity and structure are critical measures for both species that will help inform recovery management actions.     

Avoidance of extinction through nonexistence: the use of museum specimens and molecular genetics to determine the taxonomic status of an endangered freshwater crayfish

We investigated the endangered status and taxonomic status of the freshwater crayfish Procambarus ferrugineus, a crayfish species considered for the candidate list of the Endangered Species Act. This species has a narrow distribution from central Arkansas, USA and is codistributed with its presumed sister species, Procambarus liberorum. We sampled extensively throughout the ranges of both primary burrowing species and collected mitochondrial DNA from a hypervariable fragment of the 16S gene from 109 individuals across 22 sites. We also collected data from a variable region of the 12S gene from a subset of the resulting 16S haplotypes. Due to our inability to sample what we considered P. ferrugineus in the field, we included museum specimens from the United States Natural History Museum of both P. ferrugineus and P. liberorum. Analyses of the resulting data suggested that these two species are indeed the same and we therefore synonymize them under the name of priority—P. liberorum. Additionally, our sampling discovered three new cryptic species from southwestern Arkansas all from the genus Procambarus. Nested clade phylogeographic analysis coupled with population genetic analyses suggested that P. liberorum has had three rounds of range expansion throughout the inferred evolutionary history. Using IUCN Red List criteria for conservation assessment, we conclude that the species P. liberorum should be considered stable, but with special concern because of habitat fragmentation and urbanization, small restricted range, and a moderate level of genetic diversity. Procambarus reimeri should be considered endangered due to its limited geographic range and the potential for a decline in suitable habitat. The three potentially newly discovered species should be considered data deficient until more information is obtained on their distributional limits and habitat requirements. Our study highlights the importance of thorough geographic and taxonomic sampling coupled with the utility of collecting data from museum specimens to reach robust taxonomic and conservation conclusions for endangered species.     

Mitochondrial haplotype diversity in the tortoise species <Emphasis Type="Italic">Testudo graeca</Emphasis> from North Africa and the Middle East

Background  

To help conservation programs of the endangered spur-thighed tortoise and to gain better insight into its systematics, genetic variation and evolution in the tortoise species Testudo graeca (Testudines: Testudinidae) was investigated by sequence analysis of a 394-nucleotide fragment of the mitochondrial 12S rRNA gene for 158 tortoise specimens belonging to the subspecies Testudo graeca graeca, Testudo graeca ibera, Testudo graeca terrestris, and a newly recognized subspecies Testudo graeca whitei. A 411-nucleotide fragment of the mitochondrial D-loop was additionally sequenced for a subset of 22 T. graeca, chosen because of their 12S gene haplotype and/or geographical origin.     

Using patterns of genetic structure based on microsatellite loci to test hypotheses of current hybridization,ancient hybridization and incomplete lineage sorting in Conradina (Lamiaceae)

Hybridization and/or incomplete sorting of ancestral polymorphism are commonly implicated to explain discordant phylogenetic analyses of closely related species complexes. One genus in which these phenomena have been suggested to have played major roles based on phylogenetic data is Conradina, a genus of mints (Lamiaceae) endemic to the southeastern USA containing several endangered species. The goals of this study were to use microsatellite data to better understand patterns of genetic structure in Conradina, to test hypotheses of recent or ancient hybridization and incomplete lineage sorting, and to clarify species boundaries. Individuals from 55 populations representing all Conradina species were genotyped using 10 microsatellite loci. Analyses of the patterns of genetic structure in Conradina revealed a clear differentiation of populations following recognized species boundaries, indicating that species have diverged from one another genetically and interspecific hybridization has not occurred recently. Neither ancient hybridization nor incomplete lineage sorting is supported as the sole cause of species nonmonophyly, suggesting that both may have contributed to patterns found in phylogenetic trees; however, analyses of other types of data may be more appropriate to distinguish between these two hypotheses. Because all described species appear to be valid entities, the current listing status of most endangered species of Conradina is appropriate; however, populations of Conradina canescens are genetically differentiated into three groups, each of which may merit species status, and several recently discovered populations of Conradina in Dunn's Creek State Park in Florida are highly differentiated genetically and also appear to represent a new species.     

Comparing Morphological and Molecular Estimates of Species Diversity in the Freshwater Genus Synura (Stramenopiles): A Model for Understanding Diversity of Eukaryotic Microorganisms

We performed a comparison of molecular and morphological diversity in a freshwater colonial genus Synura (Chrysophyceae, Stramenopiles), using the island of Newfoundland (Canada) as a case study. We examined the morphological species diversity in collections from 79 localities, and compared these findings to diversity based on molecular characters for 150 strains isolated from the same sites. Of 27 species or species-level lineages identified, only one third was recorded by both molecular and morphological techniques, showing both approaches are complementary in estimating species diversity within this genus. Eight taxa, each representing young evolutionary lineages, were recovered only by sequencing of isolated colonies, whereas ten species were recovered only microscopically. Our complex investigation, involving both morphological and molecular examinations, indicates that our knowledge of Synura diversity is still poor, limited only to a few well-studied areas. We revealed considerable cryptic diversity within the core S. petersenii and S. leptorrhabda lineages. We further resolved the phylogenetic position of two previously described taxa, S. kristiansenii and S. petersenii f. praefracta, propose species-level status for S. petersenii f. praefracta, and describe three new species, S. vinlandica, S. fluviatilis, and S. cornuta. Our findings add to the growing body of literature detailing distribution patterns observed in the genus, ranging from cosmopolitan species, to highly restricted taxa, to species such as S. hibernica found along coastal regions on multiple continents. Finally, our study illustrates the usefulness of combining detailed morphological information with gene sequence data to examine species diversity within chrysophyte algae.     

Morphological characterization and phylogenetic relationships of Indochinese box turtles—The Cuora galbinifrons complex

The members of the Indochinese box turtle complex, namely Cuora galbinifrons, Cuora bourreti, and Cuora picturata, rank the most critically endangered turtle species on earth after more than three decades of over‐harvesting for food, traditional Chinese medicine, and pet markets. Despite advances in molecular biology, species boundaries and phylogenetic relationships, the status of the C. galbinifrons complex remains unresolved due to the small number of specimens observed and collected in the field. In this study, we present analyses of morphologic characters as well as mitochondrial and nuclear DNA data to reconstruct the species boundaries and systematic relationships within the C. galbinifrons complex. Based on principal component analysis (PCA) and statistical analysis, we found that phenotypic traits partially overlapped among galbinifrons, bourreti, and picturata, and that galbinifrons and bourreti might be only subspecifically distinct. Moreover, we used the mitochondrial genome, COI, and nuclear gene Rag1 under the maximum likelihood criteria and Bayesian inference criteria to elucidate whether C. galbinifrons could be divided into three separate species or subspecies. We found strong support for a sister relationship between picturata and the other two species, and consequently, we recommend maintaining picturata as a full species, and classifying bourreti and galbinifrons as subspecies of C. galbinifrons. These findings provide evidence for a better understanding of the evolutionary histories of these critically endangered turtles.     

Structure of the food web including the endangered lycaenid butterfly Shijimiaeoides divinus asonis (Lepidoptera: Lycaenidae)

The structure of the food web including the endangered lycaenid butterfly Shijimiaeoides divinus asonis (Matsumura) was analyzed to identify species contributing most to maintaining the equilibrium of the food web. Twenty‐seven species belonging to 17 families fed on Sophora flavescens Aiton, the host‐plant of S. divinus asonis: 15 species were leaf and stem feeders, seven (including S. divinus asonis) fed on flower buds, four were flower feeders and one fed on the seeds of So. flavescens. Of these 27 species, four were omnivores. The natural enemies of S. divinus asonis comprised six insect species, 11 spider species and one entomopathogenic fungus species, including six new predator records. The linkage density, total number of trophic links, connectance, average chain length and predator–prey ratio were 1.617, 97, 0.0548, 2.267 and 0.694, respectively. Exclusion of any of the 15 species with four or more trophic links reduced the connectance of the food web. These 15 species included facultative mutualistic attendant ants and predators of S. divinus asonis, herbivores to So. flavescens, an omnivore feeding on S. divinus asonis and So. flavescens, and prey insects. Therefore, future studies should monitor these 15 species.     

Conservation genetics of two island endemic Ribes spp. (Grossulariaceae) of Sardinia: survival or extinction?

Measuring levels of population genetic diversity is an important step for assessing the conservation status of rare or endangered plant species and implementing appropriate conservation strategies. Populations of Ribes multiflorum subsp. sandalioticum and R. sardoum, two endangered endemic species from Sardinia, representing the whole genus on the island, were investigated using ISSR and SSR markers to determine levels and structure of genetic variability in their natural populations. Results indicated medium to low genetic diversity at the population level: Nei's gene diversity for ISSR markers ranged from 0.0840 to 0.1316; the expected heterozygosity (H_E) for SSR ranged from 0.4281 to 0.7012. In addition, only one remnant population of R. sardoum showed a high level of inbreeding, in accordance with its very small size. Regarding the structure of the six R. sandalioticum populations, both principal coordinates analysis (PCoA) and STRUCTURE analysis of ISSR and SSR data highlighted low population structure, although two populations appeared to be clearly distinct from the others. The genetic pattern of the two taxa associated with their different ecological positions indicated resilience of R. sandalioticum populations in fresh and humid habitats and uncertain future resistance for the residual R. sardoum population in xeric calcareous stands. Hence, this study highlights the importance of an integrated conservation approach (genetic plus in situ and ex situ conservation studies/measures) for activating management programmes in these endemic and threatened taxa that can be considered as crop wild relatives of cultivated Ribes species.     

An update on the status of native and exotic freshwater fishes of Italy

An updated, corrected checklist of both native and established alien freshwater fish species in Italy is given based on molecular, morphological and biogeographical data. Some 12 native species, reported as conspecific with transalpine species in official Italian ichthylogical literature, are in fact endemics. Previous taxonomic confusion has resulted in the introduction of several alien species, either with official stockings or mixed in as impurities. Rehabilitated species include the cyprinids Scardinius hesperidicus, Scardinius scardafa and Telestes savigny from northern Italy, as well as Squalius ruffoi and the Telestes comes from southern Italy. Squalius albus is a junior synonym of S. squalus. The endemic gudgeon, previously assigned to the genus Romanogobio, is returned to the genus Gobio (G. benacensis). Phoxinus lumaireul is a junior synonym of P. phoxinus. Among the Salmonidae, Salmo cenerinus is a junior synonym of S. marmoratus, while Salmo farioides represents the trout species of the Adriatic lineage for which a neotype is designated. Thymallus aeliani represents the endemic lineage of grayling of the Adriatic populations. The esocid Esox cisalpinus is an endemic pike species and Esox flaviae is a junior synonym; the extensive exportation as well as the presence of this species throughout Europe is possibly due to humans. Among sculpins, Cottus scaturigo and C. ferrugineus are junior synonyms of C. gobio. The International Union for Conservation of Nature (IUCN) categories for native species of Italy are updated. At present, 52 native freshwater fish species are listed: 2 are extinct (Acipenser sturio and Huso huso), 12 are critically endangered, 7 endangered, 10 vulnerable, 3 near‐threatened, 15 low concern and 3 data‐deficient; 35 species are the result of human transfers. Among the 51 introduced species, 6 are recently established (Leuciscus leuciscus, Oreochromis niloticus, Poecilia reticulata, Xiphophorus helleri, Amatitlania nigrofasciatus, Hemichromis sp.), 37 are already established, 5 are probably established and 3 are non‐established Chinese carp, maintained in the wild by intensive stockings. The family most involved is the Cyprinidae, with 22 alien and 20 native species.     

Isolation and characterization of twelve polymorphic microsatellite markers in the endangered Hopea hainanensis (Dipterocarpaceae)

Microsatellite markers were isolated and characterized for Hopea hainanensis Merrill & Chun, an endangered tree species with scattered distribution in Hainan Island and northern Vietnam. Twenty‐six microsatellite markers were developed based on next‐generation sequencing data and were genotyped by capillary electrophoresis on an ABI 3730xl DNA Analyzer. Twelve markers were found to be polymorphic in H. hainanensis. GENODIVE analyses indicated that the number of alleles ranged from 2 to 6 per locus, and the observed and expected heterozygosity varied from 0 to 0.755 and from 0.259 to 0.779, respectively. Primer transferability was tested with Hopea chinensis Hand.‐Mazz. and Hopea reticulata Tardieu, in which 3 and 7 microsatellite markers were found to be polymorphic, separately. The results showed that H. reticulata and H. hainanensis had similar levels of genetic diversity. A neighbor joining dendrogram clustered all individuals into two major groups, one of which was exclusively constituted by H. hainanensis, while the other consisted of two subgroups, corresponding to H. reticulata and H. chinensis, respectively. The 12 polymorphic microsatellite markers could be applied to study genetic diversity, population differentiation, mating system, and fine‐scale spatial genetic structures of H. hainanensis as well as its close relatives, facilitating the conservation and restoration of these endangered but valuable Hopea species.     

Low genetic diversity in the endangered great Indian bustard (Ardeotis nigriceps) across India and implications for conservation

The great Indian bustard (Ardeotis nigriceps) is an endemic endangered bird of the Indian subcontinent with a declining population, as a result of hunting and continuing habitat loss. In this first genetic study of this little-known species, we investigate the diversity of the mitochondrial DNA (hypervariable control region II and cytochrome b gene) among samples (n = 63) from five states within the current distribution range of great Indian bustards in India. We find just three haplotypes defined by three variable sites, a comparatively low genetic diversity of π = 0.0021 ± 0.0012 for cytochrome b, 0.0008 ± 0.0007 for the control region (CR), and 0.0017 ± 0.0069 for combined regions and no phylogeographic structure between populations. We provide evidence for a bottleneck event, estimate an effective population size (Ne) that is roughly concordant with recent population size estimates based on field surveys (~200 to 400), but extremely low for a widely distributed species. We also discuss the conservation implications. Based on our findings, we strongly recommend upgrading the IUCN threat status from Endangered to Critically Endangered.     

A review of cross‐backed grasshoppers of the genus Dociostaurus Fieber (Orthoptera: Acrididae) from the western Mediterranean: insights from phylogenetic analyses and DNA‐based species delimitation

Phylogenetic analyses and species delimitation methods are powerful tools for understanding patterns of species diversity. Given the current biodiversity crisis, such approaches are invaluable for urgent assessment and delimitation of truthful species, particularly of endangered and morphologically cryptic taxa from vulnerable areas submitted to strong climate change and progressive human intervention such as the M editerranean region. In this study, we applied two DNA ‐based species delimitation methods and performed a B ayesian phylogenetic reconstruction using three mitochondrial gene fragments (12S , 16S and COI) to solve several taxonomic uncertainties among species of cross‐backed grasshoppers (genus Dociostaurus F ieber) from the western M editerranean. P hylogenetic analyses demonstrate the polyphyletic character of subgenera Dociostaurus , Kazakia B ey‐B ienko and Stauronotulus T arbinsky and, thus, the necessity of revising the currently accepted taxonomic subgenera within the genus Dociostaurus . We propose the split of closely related taxa with allopatric distributions such as D. (S.) kraussi and D. (S.) crassiusculus , considering the later a distinct species limited to the I berian P eninsula and excluding the name crassiusculus from other forms of D. (S.) kraussi from E ast E urope and A sia. Estimates of divergence times indicate that diversification of Dociostaurus probably happened during the M iocene–P liocene (3–7 Ma), and the split of the studied pairs of sister taxa took place during the middle and late P leistocene (1–2 Ma). This study highlights the need for more molecular studies on the genus and their different species for a better understanding of their evolution, genetic variation and population dynamics in order to prioritize strategies for their adequate conservation and management.     

Conservation implications of high genetic variation in two closely related and highly threatened species of Crambe (Brassicaceae) endemic to the island of Gran Canaria: C. tamadabensis and C. pritzelii

We used data from 12 allozyme loci for two endemic Brassicaceae from Gran Canaria (the endangered narrow endemic Crambe tamadabensis and its more widespread congener C. pritzelii) to assess whether their genetic diversity patterns reflect their phylogenetic closeness and contrasting population sizes and distribution areas, and to derive conservation implications. Genetic diversity values are high for both species and slightly higher in C. tamadabensis, despite its narrow distribution in north‐western Gran Canaria. At odds with the generally high interpopulation diversity levels reported in Canarian endemics, values of G_ST in C. tamadabensis and C. pritzelii are rather low (0.067 and 0.126, respectively). We construe that the higher genetic structure detected in C. pritzelii is mainly a result of unbalanced allele frequencies and low population sizes at the edges of its distribution. The overall high allozyme variation detected in C. tamadabensis and C. pritzelii is nevertheless compatible with an incipient but consistent genetic differentiation between the two species, modulated by recurrent bottlenecks caused by grazing and drift. Our data suggest that conservation efforts aimed at maintaining the existing genetic connectivity in each species and ex situ conservation of seeds are the best strategies to conserve their genetic diversity.     

Multiple lines of evidence for an Australasian geographic boundary in the Indo-Pacific humpback dolphin (<Emphasis Type="Italic">Sousa chinensis</Emphasis>): population or species divergence?

The taxonomic status of humpback dolphins (genus Sousa, sub-family Delphininae) is unresolved. While the classification of this genus ranges from a single to three nominal species, the International Union for Conservation of Nature and the International Whaling Commission only recognise a ‘two-species’ taxonomy (S. teuszii in west Africa, and S. chinensis in the Indo-Pacific). Under the IUCN (2008), S. chinensis is listed as ‘near threatened’, but is only considered as a ‘migratory’ species in Australia. Taxonomic resolution of the genus Sousa is needed to define particular conservation status and develop appropriate management actions. Using phylogenetic analyses of 1,082 bp of mitochondrial and 1,916 bp of nuclear DNA, we provide multiple lines of genetic evidence for the genetic distinction of S. chinensis in China and Indonesia from S. chinensis in Australia. The separation of Australian Sousa from Sousa of Southeast Asia requires a review of their current conservation status and respective management actions.     

<Emphasis Type="Italic">Stemonurus corrugatus</Emphasis> (Stemonuraceae,Icacinaceae s.l.) a new species from Sarawak

Summary   Stemonurus corrugatus Utteridge & Schori is described and illustrated as a new species from Sarawak, Malaysia. The new species is similar to S. grandifolius, S. scorpioides, and S. umbellatus, and morphological differences among the taxa are discussed. A distribution map and notes on the new species’ conservation status are provided.