A non‐destructive tissue sampling technique for holothurians to facilitate extraction of DNA for genetic analysis

Increasing demand and overfishing of high‐value species has promoted interest in both conservation and aquaculture initiatives supporting stock restoration programs for tropical sea cucumbers. Accordingly, there is a need for baseline information on the genetic structures and relatedness of sea cucumber populations to support sustainable implementation of mariculture and conservation programs, which often involve coastal communities in developing countries. Identification of a non‐destructive tissue sampling technique for sea cucumbers that allows extraction of high‐quality genomic DNA in a sustainable and culturally appropriate way is thus required. Six sampling techniques were assessed for their suitability to collect tissue for DNA extraction from sandfish (Holothuria scabra): core needle biopsy, punch biopsy, shave biopsy, buccal swab, anal swab, and evisceration. The quantity, quality, and purity of extracted DNA were compared to assess the relative merit of each sampling method. The swab biopsy method produced the best quality DNA agarose band image, and evisceration resulted in the highest yields of DNA, at an average of 525.9 (±98.0) μg g^?1. However, when considering all criteria assessed, the swab biopsy methods (both buccal and anal) proved superior. Swabbing not only produced the best quality DNA agarose band image, it was also the only technique that produced DNA that amplified 100% of the time across both extraction protocols. We hope that the non‐destructive sampling techniques evaluated in this study provide a foundation for the genetic analysis of sea cucumber stocks to support their conservation and management.     

Genetic and morphological variation in two littorinid gastropods: evidence for recent population expansions along the East African coast

In marine species, population diversity and differentiation is affected by the population history and by the complex interaction between oceanographic dynamics and ecological traits. In the present study, we examined two species of marine gastropods (the mangrove periwinkle Littoraria scabra and the rocky shore Littoraria glabrata) along the East African coast, using both genetic and geometric morphometric methods. We report a greater variation of shell shape in L. scabra compared to the slightly smaller variation in L. glabrata. This variation was probably associated with variation of environmental factors along the coast, such as temperature and hydrodynamics. Despite morphological variation, we found low mitochondrial genetic differentiation among samples from different localities for both species, which is probably a consequence of the ongoing gene flow during the free‐swimming larval stage of these gastropods. Additionally, high levels of haplotype diversity, low nucleotide diversity, and ‘star‐like’ genealogies were found in both species. These observations and the results from mismatch distributions, indicate a possible signature of recent population expansions in both species, which probably started during interglacial periods of the Pleistocene and led to the colonization of the Indian Ocean coast. © 2013 The Linnean Society of London     

A test of Brown's principle in the intertidal limpet Collisella scabra (Gould, 1846)

Aim Brown's principle predicts that a species will peak in density near its range centre, and decline gradually towards the margins of its geographical distribution. The decline is assumed to reflect a decrease in individual performance near range margins. I test this abundance–performance hypothesis by comparing patterns in density and size across the northern half of the geographical distribution of the marine patellogastropod Collisella scabra (Gould, 1846). Location Collisella scabra is a high intertidal patellogastropod species distributed along the Pacific coast of North America from Cape Mendocino (CA, USA) to southern Baja California (Mexico). I surveyed 11 research sites spanning c. 36–44° N. Methods In each of the 11 research sites I surveyed four distinct microhabitats, and compared spatial patterns in density and in the size of solitary limpets. Results Both density and size were highly variable across the species range. Density peaked near the northern range margin, and showed greater variance at small spatial scales (< 10 km) than at large scales (> 100 km). In contrast, large size occurred uniformly across the survey area, and size was strongly associated with microhabitat. Main conclusion Collisella scabra does not show spatial patterns of density or performance that are consistent with Brown's principle. The underlying assumptions of Brown's principle may conflict with specific characteristics of C. scabra's life history and/or patterns of environmental variation across its range. Because such conflicts may be common in a large number of marine and terrestrial species, the generality of Brown's principle is questioned.     

The taxonomy of the endemic golden palm civet of Sri Lanka

Two species of palm civet are currently known from Sri Lanka: the widespread common species, Paradoxurus hermaphroditus (Pallas, 1777), and the endemic golden species, Paradoxurus zeylonensis (Pallas, 1778). The latter has two ‘morphs’, one golden and one dark brown, both of which are recorded from all three major biotic zones in Sri Lanka (wet zone, dry zone, and cloud forest). We have examined specimens of both ‘morphs’ from all zones, and conclude that there are actually several species involved: names are available for two of them, we describe a third as a new species, and we draw attention to a probable fourth species, based on two distinctive specimens, the provenance of which are unfortunately unknown. The name zeylonensis probably does not apply to a golden palm civet at all. © 2009 The Linnean Society of London, Zoological Journal of the Linnean Society, 2009, 155 , 238–251.     

Phylogeography of related diadromous species in continental and island settings,and a comparison of their potential and realized dispersal patterns

Aim To compare patterns of potential and realized dispersal in ecologically similar and phylogenetically related amphidromous shrimps (Atyidae) in continental and island‐dominated landscapes. Location Eastern Australia and the Caribbean region. Methods Population genetic and phylogeographic analyses of mitochondrial DNA data for Australatya striolata from eastern Australia (a continental landscape) and Atya scabra from the Caribbean (an island‐dominated landscape). Results Australatya striolata contained two highly divergent genetic lineages in eastern Australia, corresponding to the disjunct northern and southern populations, respectively. These lineages probably represent allopatric cryptic species, both of which were found to have genetically homogeneous population structures within their regions of occurrence. Atya scabra was genetically homogeneous throughout the Caribbean. Recent population expansions were detected for Atya scabra in the Caribbean, but not for northern or southern Australatya striolata. Main conclusions The findings of this study are consistent with previously reported patterns of genetic population structure in amphidromous species in both continental and island‐dominated landscapes, suggesting that potential for widespread dispersal is typically matched by realized patterns of panmixia. We therefore raise the hypothesis that landscape setting (i.e. continent or island‐dominated) does not influence dispersal patterns in amphidromous species. Further studies, especially of population genetic patterns of amphidromous species on continents, are needed to test this idea. Interestingly, results of the genetic neutrality tests led us to hypothesize that demographic and drift‐mutation equilibrium is attainable although not always evident for amphidromous species on continents, but is not attainable for those species distributed across island settings.     

Novelties in spermacoceae (Rubiaceae) from Bolivia and Paraguay

One new Bolivian species (Borreria pazensis) and three new varieties (Borreria densiflora var.minima, Richardia, scabra var.chacoensis andStaelia virgata var.killeenii) are described and illustrated. The genusDiodella, with three species (D. apiculata comb. nov., D. radula comb. nov., andD. teres), is reported for the first time.Borreria limae, B. wunschmannii, B. scabiosoides var.scabiosoides, andB. scabiosoides var.anderssonii are also reported as new to Bolivia.     

Behavioral thermoregulation in a tropical gastropod: links to climate change scenarios

Tropical species are vulnerable to global warming because they live at, or near to, their upper thermal threshold limits. Therefore, the predicted increase in the frequency of warming events in the tropics is expected to be critical for the survival of local species. This study explored the major environmental variables which were thought to be correlated with body temperatures (BTs) of the tropical snail Littoraria scabra at the niche level. A correlation between BT and substrate temperature (ST) was detected from field observations which suggests a possible causal relationship between both substrate and BTs. In contrast, there was no correlation between BT and air temperature. Field observations suggest that 33.4 °C may be L. scabra upper limit of substrate surface temperature, although further experiments are needed to assess if the upper limit of physiological tolerance is actually different. As L. scabra individuals were free to choose their substrata, the observed distribution pattern at the niche level is related to L. scabra's behavior. Additionally, substrate surface temperatures were very heterogeneous at centimeter scale (i.e. from 22.5 to 53.1 °C) and L. scabra was shown to select specific STs (i.e. between 22.5 and 33.4 °C) rather than microhabitat type. Therefore, L. scabra did not seem to behaviorally thermoregulate through microhabitat selection nor aggregation. In contrast, behavioral experiments showed that L. scabra has the ability to actively select a thermally favorable site over short temporal scale (i.e. individual average speed of 1.26 cm min^?1) following exposure to high temperatures above 33.4 °C. Hence, this study supports the crucial need to integrate intertidal invertebrate behavioral responses to thermal constraints in climate change studies.     

Structure and evolution of the pod in Indigofera (Fabaceae) reveals a trend towards small thin indehiscent pods

Pod morphology and anatomy have been studied in 28 species and four varieties of Indian Indigofera. Pods of Indigofera spp. differ with respect to size, the relative thickness of the fruit wall, the number of sclerenchymatous layers in the endocarp, the presence/absence of hypodermis and trichomes, and the presence of separation tissue. Anatomically, the pericarp is broadly characterized into three types: type I (thin pericarp and three to five sclerenchymatous layers in the endocarp), type II (intermediary pericarp thickness and six to eight sclerenchymatous layers), and type III (thick pericarp and more than eight sclerenchymatous layers). The distribution of these types across the tribe is not congruent with the current phylogenetic analyses. Type III pericarp (present in the early diverging lineages of the tribe) represents the most primitive state, whereas type I and type II pericarps are derived. Fruits of Indigofera generally show normal explosive dehiscence as a means of dispersal of seeds, although some species show adaptations for dispersal by wind. In Indigofera, dehiscence is caused by a separation layer present at the dorsal and ventral sutures except in some species (Indigofera hochstetteri, Indigofera karnatakana, Indigofera glandulosa var. sykesii, and Indigofera trita var. scabra) in which no separation tissue is present; these species show delayed dehiscence or an indehiscent condition. The indehiscent pod type is considered to be apomorphic. The taxonomic, functional, and evolutionary significance of morphological and anatomical features in fruits of the genus Indigofera has been evaluated. © 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 176 , 260–276.     

Cytogenetics of new Guizotia Cass. (Compositae) interspecific hybrids pertaining to genomic and phylogenetic affinities

 The following crosses were made using four recognized species/subspecies and a new population of Guizotia, referred to as Chelelu after the name of the locality in Ethiopia from which it was collected: G. scabra subsp. schimperi × Chelelu, Chelelu × G. scabra subsp. scabra, G. zavattarii × G. arborescens and Chelelu × G. zavattarii (all accessions with 2n=30). Plant morphology as well as mitotic and meiotic chromosome analysis confirmed the hybrid nature of the obtained progeny. At metaphase I of meiosis, the F₁ hybrid plants (2n=30) showed a mean of about 95%, 31%, 63% and 0.50% of the pollen mother cells with 15 bivalents, and a mean of about 14.95, 13.75, 14.40 and 7.86 bivalents per cell, respectively. The respective mean pollen stainability was about 67%, 19%, 31% and 2%. From the results it was concluded that Chelelu is more closely related to G. scabra subsp. schimperi than to G. scabra subsp. scabra but more to the latter than to G. zavattarii. Guizotia zavattarii and G. arborescens are closely related to each other. Based on the cytological observations made, the probable basic chromosome number for the genus is discussed. Received November 5, 1999 Accepted August 21, 2001     

The confusing identity of Pternopetalum molle (Apiaceae)

The misapplication of the name Pternopetalum molle (Franch.) Hand.‐Mazz. has resulted in considerable taxonomic confusion in the genus, involving 11 names belonging to seven taxa (according to a recent treatment). A supplementary taxonomic revision of P. botrychioides (Dunn) Hand.‐Mazz., P. molle and P. vulgare (Dunn) Hand.‐Mazz. is presented, after a detailed examination of the morphological variation. Four new synonyms, P. longicaule var. humile R.H.Shan & F.T.Pu, P. molle var. dissectum R.H.Shan & F.T.Pu, P. radiatum (W.W.Smith) P.K.Mukherjee and P. trifoliatum R.H.Shan & F.T.Pu, are proposed. Pternopetalum delicatulum (H.Wolff) Hand.‐Mazz. should be reduced to P. botrychioides rather than P. radiatum. The previous taxonomic treatments of P. cuneifolium (H. Wolff) Hand.‐Mazz., P. cartilagineum C.Y.Wu ex R.H.Shan & F.T.Pu and P. molle var. crenulatum R.H.Shan & F.T.Pu are reinstated. A key and distribution maps are provided for the accepted species. © 2008 The Linnean Society of London, Botanical Journal of the Linnean Society, 2008, 158 , 274–295.     

High molecular and phenotypic diversity in the Merodon avidus complex (Diptera,Syrphidae): cryptic speciation in a diverse insect taxon

This paper examines molecular and phenotypic variability in the widely spread European hoverfly species complex Merodon avidus. Herein, based on the mitochondrial DNA (mtDNA) sequences of the cytochrome c oxidase subunit I (COI) and morphometric wing parameters, M. avidus is shown to comprise a complex of cryptic species, and one variety is redefined as a valid species: M. bicolor Gil Collado, 1930 (as var. of spinipes). The species M. bicolor, M. avidus A, and M. avidus B were clearly delimited based on their wing size. A total of 29 M. avidus and M. bicolor individuals presented 20 mtDNA haplotypes, four of which were shared by M. avidus A and M. avidus B, three were confined to M. bicolor, seven to M. avidus A, and six to M. avidus B. Sequence divergences between lineages occurring in the Balkan and in Spain ranged from 4.93 to 6.0 (uncorrected p in %) whereas divergences between M. avidus A and M. avidus B were 0.26 to 1.56. Divergence among morphologically identified individuals of M. avidus A and M. avidus B species ranged from 0.13 to 1.58, and from 0.13 to 0.52, respectively. The phenotypic substructuring and observed genetic uniqueness of populations in spatially and temporally fragmented M. avidus taxa were used to identify genetic units. The early split of two allopatric lineages, Spanish M. bicolor and Balkan M. avidus, was followed by diversification in each lineage. Present‐day morphological uniformity masks much of the genetic complexity of lineages within the M. avidus complex. © 2009 The Linnean Society of London, Zoological Journal of the Linnean Society, 2009, 155 , 819–833.     

Biosystematic studies on the genus Heloniopsis (Melanthiaceae) I. Phylogeny inferred from plastid DNA sequences and taxonomic implications

Based on a combined dataset of plastid DNA sequences (atpB‐rbcL, trnG, trnL‐trnL‐trnF, trnK 5' intron and matK) from 60 individuals, we conducted parsimony and likelihood analyses to clarify the phylogenetic relationships among the six species and three varieties that are commonly recognised in Heloniopsis, in addition to the related genera Ypsilandra and Helonias, using Chamaelirium and Chionographis as an outgroup. According to the single most parsimonious tree, which was identical to the maximum‐likelihood tree in topology, Helonias, Ypsilandra and Heloniopsis are all monophyletic with 100% bootstrap support (BS). In Heloniopsis, there are two highly supported clades (BS 94–97%): a clade of Korean species and a clade of Japanese and Taiwanese species. The latter clade comprised the following four subclades (BS 99–100%): 1) H. orientalis var. orientalis, 2) H. orientalis var. breviscapa and var. flavida, 3) H. kawanoi and 4) H. leucantha and H. umbellata. Because subclades 1 and 2 did not form a monophyletic group, and do show clear morphological differences – including nectary position, nectary‐sac structure and leaf margin undulation – they should be distinguished at the species level: H. orientalis for subclade 1 and H. breviscapa for subclade 2. In subclade 2, neither var. breviscapa nor var. flavida was monophyletic; instead, var. breviscapa plus var. flavida (thick‐leaved entity) was monophyletic (BS 62–63%) and var. flavida (thin‐leaved entity) was monophyletic (BS 86–87%). As var. breviscapa and var. flavida (thick‐leaved entity) share basally ± pinkish wide tepals and dark‐coloured thick leaves, in contrast to var. flavida (thin‐leaved entity), which has completely white narrow tepals and light‐coloured thin leaves, the two varieties should may be kept distinct after the merge of var. flavida (thick‐leaved entity) with var. breviscapa.     

Neurocranial osteology and systematic relationships of Varanus (Megalania) prisca Owen, 1859 (Squamata: Varanidae)

The neurocranial osteology of the giant monitor lizard Varanus (Megalania) prisca Owen, 1859 is described in detail for the first time. Optimization of neurocranial characters onto phylogenetic topologies for varanoids, including Lanthanotus, Heloderma, and Varanus species nests V. prisca within an Indo‐Australian clade of Varanus on the basis of characters of the otic capsule. A sister‐taxon relationship between V. prisca and Varanus komodoensis Ouwens, 1912 is proposed based on apomorphies of the crista prootica, fenestra vestibuli, occipital recess, and supraoccipital. These results support a monophyletic clade of giant monitors among Indo‐Australian species, and unambiguously synonymize Megalania with Varanus at both generic and subgeneric levels. © 2009 The Linnean Society of London, Zoological Journal of the Linnean Society, 2009, 155 , 445–457.     

Reproductive biology of five native plant species from the Monte Desert of Argentina

Research into plant breeding systems enables the evaluation of whether seed production depends on pollination agents and gene flow mechanisms within and among populations. This aids, in turn, the estimation of the appropriate population sizes needed to maintain both genetic and species' diversity. Little is known about plant reproductive biology in the Monte Desert (Patagonia, Argentina), a habitat threatened by desertification as a result of human impact. The mating systems, flowering phenologies and pollinator networks were studied in five representative plant species of the Monte Desert. The mating systems studied ranged from anemophilous pollen dispersion in the dioecious Atriplex lampa, to a gradient of dependence on pollinators, from the less dependent (facultative self‐compatible) Gutierrezia solbrigii and two Larrea spp. (L. divaricata and L. cuneifolia) to the most dependent Grindelia chiloensis (self‐incompatible). Flowering phenology was restricted to spring and coincided with pollinator abundance. Solitary bees were the main pollinator group, but beetles, flies and butterflies were also important. The four insect‐pollinated species were moderately generalist, but they maintained their own pollinator assemblage. Coleopterans depended more on Grindelia chiloensis and dipterans on Gutierrezia solbrigii. Lepidopterans frequently visited Gutierrezia solbrigii and Larrea divaricata, whereas hymenopterans visited both Larrea spp. more frequently. The studied plant species are ecologically important, not only as resources for a wide range of pollinator species but also for other insects. The Monte Desert is a very disturbance‐sensitive environment and, as these plant species hardly show vegetative regeneration, sexual reproduction is essential for their survival. © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 161 , 190–201.     

Character analysis and taxonomic revision of the Microtoena insuavis complex (Lamiaceae)

The taxonomy of the Microtoena insuavis complex (Lamiaceae) is controversial. No agreement on the taxonomic treatments of M. esquirolii, M. insuavis, M. mollis and M. patchoulii has been reached. In addition, some species and varieties described on the basis of a single specimen or a limited number of specimens are doubtful. Based on the examination of a large number of herbarium specimens, extensive field observations, population sampling, scanning electron microscopy observation and statistical analysis of some characters, M. esquirolii is separated from the M. insuavis complex, and M. subspicata and M. subspicata var. intermedia are treated as synonyms of M. esquirolii, M. pauciflora is treated as a synonym of M. patchouli, and M. siamica is treated as a synonym of M. insuavis. Only three species (M. insuavis, M. mollis and M. patchouli) are recognized in this complex. © 2011 The Linnean Society of London, Botanical Journal of the Linnean Society, 2011, 165 , 315–327.     

A new species of Paraboea (Gesneriaceae) from a karst cave in Guangxi,China, and observations on variations in flower and inflorescence architecture

A new species of Gesneriaceae, Paraboea trisepala W.H.Chen & Y.M.Shui, from a karst cave in Guangxi, China is described and illustrated. The new species differs from other species of Paraboea by its three‐lobed calyx. Variation in flower and inflorescence architecture was observed under cultivation. © 2008 The Linnean Society of London, Botanical Journal of the Linnean Society, 2008, 158 , 681–688.     

Efficacy of chemical markers and physical tags for large-scale release of an exploited holothurian

Hatchery-produced juveniles need to be distinguished from wild conspecifics in order to evaluate the success of restocking experiments and stock enhancement. The commercially valuable sea cucumber Holothuria scabra, or ‘sandfish’, has potential for stock enhancement but cheap, long-lasting tags have not yet been developed. We evaluated five non-genetic tags against several criteria: cost, ease of application, retention rate, and ease of detection. In a 1-month trial, T-bar tags and visible elastomer implants proved unsuitable on hatchery-produced sandfish juveniles. Coded-wire tags and two fluorochromes, tetracycline and calcein, showed more promise, so retention rates of these tags were tested on juveniles held in an earthen seawater pond for 1 year. Sixty percent of juveniles retained at least one of two coded-wire tags for 1 year. Under UV illumination, spicules from the body wall of sandfish immersed in solutions of tetracycline are yellow, whereas calcein-stained spicules are green. From months 1 to 12 after immersion, the average percentage of stained spicules declined from 40% to 8% with tetracycline and 54% to 5% with calcein. After 1 year stained spicules were detected in all immersed animals, i.e. mark retention was 100%. Stained spicules from the ventral body wall of juveniles were generally in greater proportions, and fluoresced brighter, than those from the dorsal body wall. Spicule density exceeded 7000 spicules per mm² of body wall in juveniles of 2-13 g, and decreased with sandfish weight. Fluorescence provides a simple diagnostic tool for identifying marked individuals and the sampling is non-destructive. Several mm² of outer body wall is ample for tag identification. Immersion-staining with fluorochromes is preferred to coded-wire tags and provided a cheap and reliable batch-mark for distinguishing released and wild sandfish.