Cryptic diversity and phylogeography of the island‐associated barnacle Chthamalus moro in Asia

Pleistocene glaciations were among the important historic events that shaped the population structures of marine organisms. Genetic studies of different marine fauna and flora have demonstrated the effect of Pleistocene glaciations on taxa that reside in marginal seas. However, how marine island species responded to Pleistocene glaciations remains relatively unstudied, especially in Asia. Genetic analyses based on the island‐associated barnacle Chthamalus moro collected from 14 sites in Asia reveal that C. moro comprises three distinct lineages, with COI divergence ranging from 3.9 to 8.3%. Population genetic analyses on respective lineages reveal signs of demographic expansion within the Pleistocene epoch at different times. The Ogasawara lineage, which has a more oceanic distribution, expanded the earliest, followed by the population expansion of the Ryukyu and Southern lineages that inhabit islands closer to the continent. The data suggest that the inhabitants of outer islands may have been less affected by Pleistocene glaciations than those that reside closer to the continent, as the former were able to maintain a large, stable, effective population size throughout the late Pleistocene.     

West versus East Mediterranean Sea: origin and genetic differentiation of the sea cucumber Holothuria polii

We studied the genetic structure of the sea cucumber Holothuria (Roweothuria) polii (Delle Chiaje 1823) by analysing the mitochondrial DNA variation in two fragments of cytochrome oxidase I (COI) and 16S genes. Individuals were collected in seven locations along the Mediterranean Sea, which cover a wide range of the species distribution. We found high haplotype diversity for COI and moderate diversity for 16S, and low nucleotide diversity for both genes. Our results for the COI gene showed many recent and exclusive haplotypes with few mutational changes, suggesting recent or ongoing population expansion. The Western and Eastern Mediterranean populations exhibited slight but significant genetic differentiation (COI gene) with higher genetic diversity in the East. The most ancient haplotype was not present in the westernmost sampling location (SE Spain). The oldest expansion time was observed in Turkey, corresponding to mid‐Pleistocene. Turkey had also the highest genetic diversity (number of total and exclusive haplotypes, polymorphisms, haplotype and nucleotide diversity). This suggests that this region could be the origin of the subsequent colonizations through the Mediterranean Sea, a hypothesis that should be assessed with nuclear markers in future research.     

Mitochondrial DNA variation reveals distinct lineages in Penaeus semisulcatus (Decapoda,Penaeidae) from the Indo‐West Pacific Ocean

Penaeus semisulcatus, the green tiger prawn, is an ecologically and economically important penaeid shrimp in the Indo‐West Pacific region, especially in rice‐shrimp farming and capture fisheries in Bangladesh and Sri Lanka, respectively. Genetic variation and phylogeography of samples of this species from Bangladesh and Sri Lanka were studied utilizing different mitochondrial DNA markers, i.e. cytochrome oxidase subunit 1 (CO1), control region (CR) and 16S rRNA genes. No evidence of population structure was observed in Bangladesh, but distinct variations were found among the Sri Lankan samples (Φ_ST = 0.04, p = .002; F_ST = 0.07, p = .001), with the western sample differing from the northwestern and southern samples. The Bangladesh population had lower genetic diversity than two of the three Sri Lanka populations. The phylogeography of P. semisulcatus revealed two distinct mitochondrial DNA (mtDNA) lineages, one in the Western Pacific Ocean and second in the Indian Ocean. The Bangladesh samples showed highest levels of similarity with samples from Sri Lanka, India and Malaysia, with the Bangladesh and Sri Lanka populations sharing the most common recent ancestry. Among the Indian Ocean samples, high levels of variation were observed in the samples from Iran, indicating admixture of two distinct mtDNA lineages, one shared by the populations from the Bay of Bengal and the other possibly originating from Eastern Africa. The genetic and phylogeographic information obtained in this study will be useful in appropriate planning for management and conservation of shrimp fisheries in Bangladesh and Sri Lanka, and in the Indo‐West Pacific region.     

The genetic structure of the exotic ascidian Styela plicata (Tunicata) from Italian ports,with a re‐appraisal of its worldwide genetic pattern

The pleated ascidian Styela plicata (Lesueur, 1823) is a solitary species commonly found in ports and marinas around the world. It has been recorded in the Mediterranean region since the mid‐19th century. In the present work, the species’ genetic diversity was analysed, employing a 613‐bp portion of the mitochondrial cytochrome c oxidase subunit I (COI) gene from 149 individuals collected in 14 ports along Italian coasts at spatial scales ranging from 1 to approximately 2200 km. Haplotype and nucleotide diversity values were h = 0–0.933 (total h = 0.789) and π = 0–0.145 (total π = 0.0094), respectively. A general southward trend of increasing within‐population genetic diversity was observed. Analysis of molecular variance revealed significant genetic structuring but no significant differences were detected among basins, and no isolation by distance was found. Our data were integrated with the COI sequences available from previous studies and re‐analysed in order to investigate the possible routes of introduction of this ascidian into the Mediterranean Sea. The presence of the two COI haplogroups detected in previous molecular investigations on S. plicata at intercontinental spatial scale was confirmed in the Mediterranean Sea. The results revealed multiple introductions of S. plicata, although some locations appear to have experienced rapid expansion from few founding individuals with reduced genetic diversity. However, continuous introductions would confound the pattern deriving from single founder events and make it difficult to estimate the time needed for gene diffusion into established populations. This mixing of effects creates difficulties in understanding the past and current dynamics of this introduction, and managing this alien invasive ascidian whose genetic structure is continuously shuffled by vessel‐mediated transport.     

Hierarchical analysis of the population genetic structure in Concholepas concholepas,a marine mollusk with a long‐lived dispersive larva

For most marine invertebrate species, dispersal is achieved mainly or exclusively by pelagic larvae. When the duration of the pelagic larval stage is long, genetic homogeneity over large geographic scales is expected. However, genetic structure has often been reported over small spatial scales, suggesting that more complex processes occur than a simple positive relationship between pelagic larval duration and gene flow. Concholepas concholepas has a larval stage that can last up to 3 months in the water column with a wide distributional range covering from 6°S to 56°S. We used a hierarchical sampling technique to test if the genetic homogeneity of this highly dispersive species is maintained throughout its total geographic range in spite of environmental heterogeneity. In the three studied regions (Antofagasta Bay, Valdivia and Patagonia), a spatial pattern of isolation by distance in conjunction with a spatial genetic structure was observed. Within each region, different spatial genetic patterns were detected. In Antofagasta Bay and Valdivia there was evidence of substantial gene flow among populations, whereas in Patagonia, populations showed genetic structure and a unique, genetically isolated location was identified. These results revealed the existence of spatial differences in the genetic patterns among regions with different coastal topographies in C. concholepas, and give us new insights into the inter‐relationships of larval dispersal potential, actual larval dispersal and physical processes. Regarding the sustainable management of C. concholepas, two important issues are derived from this study: (i) to highlight the need for a regional context in the management of C. concholepas, (ii) to determine the distinctiveness of the most austral population and to focus on the conservation efforts due to the relevance of this area.     

Significance of biofilm proteins in modulating cyprid metamorphosis of Balanus amphitrite (Cirripedia: Thoracica)

The role of biofilm proteins in modulating cyprid metamorphosis is not well understood. In the present investigation this possibility was explored by disrupting biofilm proteins using commercially available protease. The influence of natural biofilm and artificial biofilms of Aeromonas salmonicida salmonicida and Bacillus brevis and their culture supernatants and exopolysaccharides obtained under different nutritional conditions was evaluated. Natural biofilm facilitated cyprid metamorphosis in Balanus amphitrite, whereas monospecies biofilms did not. The treatment of biofilms with protease resulted in a decline in metamorphosis, except in B. brevis, where although metamorphosis was negated initially, it increased with the protease concentration and observation days. As the response of cyprids to exopolysaccharides produced by A. salmonicida salmonicida was dependent on protein concentration, protease treatment resulted in a decrease in metamorphosis, whereas in case of B. brevis biofilms, protease hydrolysis products could be important in providing settlement cues. Thus, biofilm bacterial components play an important role in the cyprid metamorphosis of Balanus amphitrite.     

东方小藤壶Chthamalus challengeri（甲壳动物亚门：无柄目）线粒体基因组：小藤壶科基因排列比较及藤壶亚目系统发育分析

藤壶是潮间带生态、幼体发育以及生物防污损研究中重要的模式生物。目前,线粒体基因组学的发展有助于从线粒体基因组水平上更好地理解系统发育关系。本研究获得东方小藤壶Chthamalus challengeri完整线粒体基因组,大小为15358bp的环状分子。与藤壶亚目其它物种相比,东方小藤壶非编码区的长度较长,而基因区的长度则相近。东方小藤壶线粒体基因组A+T含量为70.5%。现有藤壶物种的线粒体基因组中存在起始和终止密码子的变化。同属的东方小藤壶和触肢小藤壶C. antennus具有共同的基因排列。然而,小藤壶科中不同属之间却具有不同的基因排列,包括两个tRNA基因出现易位,一个基因块出现倒置。值得注意的是,与以往藤壶亚目物种不同,小藤壶属两个物种的srRNA和lrRNA基因都在重链上编码。小藤壶科中进化树的拓扑结构与基因排列证据相互支持,系统发育分析表明小藤壶科是单系群,而藤壶科和古藤壶科则是多系群。     

Temporal variation in cannibalistic infanticide by the shore crab Hemigrapsus oregonensis: implications for reproductive success

Larvae of benthic marine organisms are released amid high densities of suspension feeding and predatory adults and are highly subject to being consumed, even by conspecifics or their own parent. During laboratory feeding trials conducted in June 2006, female shore crabs (Hemigrapsus oregonensis) from Stege Marsh in San Francisco Bay (37°54.530′ N, 122°19.734′ W) that released their larvae during the previous 24 h ate fewer conspecific larvae than females that had not recently released larvae, though the behavior was not repeated during similar trials in 2007. Additionally, the number of larvae eaten increased with increasing starvation time, and hungrier females showed a trend toward eating more larvae from a different species (Carcinus maenas) than larvae of conspecifics. Thus, suppression of suspension feeding may reduce conspecific predation of newly released larvae, but this response partially depends on hunger level. This is the first time crabs have been shown to suppress feeding to reduce cannibalism of larvae, and this behavior could affect reproductive success and population dynamics.     

Life cycle and population dynamics of Idotea balthica basteri (Pallas, 1772), Isopoda Valvifera from the Bizerte lagoon (Southern Mediterranean Sea – Tunisia)

The population dynamics and life cycle of Idotea balthica basteri (Pallas, 1772) were studied at the Menzel Jemil (lagoon of Bizerte, Tunisia) based on monthly samples taken from October 2009 to September 2011. The presence of juveniles and gravid females throughout the sampling period showed that this species exhibits continuous reproduction. Sexual differentiation occurs in approximately 43–59 days. Females took 88–160 days (3–5.5 months) to reach sexual maturation, and males took 102 days (3.5 months). The fecundity and fertility values exhibited a great variation in the number of eggs and embryos or mancae inside the marsupium which was correlated with the length of the gravid females. The sex ratio underwent fluctuations throughout the sampling period but was always in favour of females. Size frequency distributions were analysed recognizing 24 cohorts during the sampling period. Six cohorts were identified in October 2009 and 18 cohorts in November 2009–September 2011. Among these latter cohorts, nine were tracked till they disappeared. The minimum average size of new cohorts ranged from 1.92 ± 0.31 to 2.97 ± 0.26 mm and the maximum values ranged from 12.83 ± 0.96 to 19.23 ± 0.56 mm. Life span was estimated at 10–14 months. I. balthica basteri, in the lagoon of Bizerte, is a semi‐annual species with iteroparous females, and a bivoltine life cycle that produces two generations per year.     

Genetic connectivity of the coral‐eating sea star Acanthaster planci during the severe outbreak of 2006–2009 in the Society Islands,French Polynesia

Occasional population outbreaks of the crown‐of‐thorns sea star, Acanthaster planci, are a major threat to coral reefs across the Indo‐Pacific. The presumed association between the serial nature of these outbreaks and the long larval dispersal phase makes it important to estimate larval dispersal; many studies have examined the population genetic structure of A. planci for this purpose using different genetic markers. However, only a few have focused on reef‐scale as well as archipelago‐scale genetic structure and none has used a combination of different genetic markers with different effective population sizes. In our study, we used both mtDNA and microsatellite loci to examine A. planci population genetic structure at multiple spatial scales (from <2 km to almost 300 km) within and among four islands of the Society Archipelago, French Polynesia. Our analysis detected no significant genetic structure based on mtDNA (global F_ST = ?0.007, P = 0.997) and low levels of genetic structure using microsatellite loci (global F_ST = 0.006, P = 0.005). We found no significant isolation by distance patterns within the study area for either genetic marker. The overall genetically homogenized pattern found in both the mitochondrial and nuclear loci of A. planci in the Society Archipelago underscores the significant role of larval dispersal that may cause secondary outbreaks, as well as possible recent colonization in this area.     

Genetic variability of the Lessepsian migrant mussel Brachidontes pharaonis (Bivalvia: Mytilidae) in Tunisia

The present study used two mitochondrial markers (16S rRNA and COI) to assess the genetic diversity of a newly founded Lessepsian migrant mussel, Brachidontes pharaonis, in Tunisian waters. The species appears to be restricted to only one population in Rades Harbour, in the northern part of the country. Phylogenetic analyses revealed the monophyly of B. pharaonis in Tunisia. Both molecular markers revealed high genetic variability of the B. pharaonis population. Haplotype networks and demographic analyses confirmed the recent expansion events within this population. Multiple human-mediated introduction events involving several founder populations and intensive population growth rates are probably the main causes of the high polymorphism observed within this invasive mollusc.     

Breeding cycle of the red squat lobster Pleuroncodes monodon H. Milne Edwards, 1837 (Decapoda,Muninidae) from deepwater Pacific of Costa Rica

Reproduction and larval development of the red squat lobster Pleuroncodes monodon is strongly linked to temperature changes provoked by upwelling along the Chilean coast. Here, we propose the hypothesis that both the breeding cycle and the spatial distribution of egg‐bearing females of P. monodon in Costa Rica are related to decreasing water temperatures during seasonal coastal upwelling. To describe the breeding cycle, squat lobsters were collected between February 2007 and January 2008 from the Central Pacific coast of Costa Rica. The presence of egg‐bearing females in other areas of the Costa Rican coast was studied from samples collected during two latitudinal research cruises (August 2008: rainy season; May 2009: dry season). Our results revealed that P. monodon has a marked seasonal breeding period (from November to March), which is associated with decreasing water surface temperatures registered during coastal upwelling events. All females with embryos close to hatching were found in areas surrounding the Gulf of Nicoya, when upwelling events have been reported. The near absence of egg‐bearing females in zones where upwelling does not occur suggests the existence of a strong correlation between upwelling events and the breeding cycle of P. monodon. Our information should be considered when developing management measures for the sustainable use of this potential fishery resource in the Pacific coast of Central America.     

Dietary analysis of harbour seals (Phoca vitulina) from faecal samples and overlap with fisheries in Erimo,Japan

The number of harbour seals in Japan has been rebounding since protection began in the mid‐1980s. With the increase in seal numbers, increased conflict with fisheries has occurred through depredation and the belief that seals compete with fisheries for prey. However, competition can only be determined if the prey species and quantities seals consume over time are known. We studied the diet of harbour seals in Erimo, site of the largest population of harbour seals in Japan, from 2011 to 2012 and assessed the degree of prey overlap with local fisheries. We used both hard parts and DNA techniques to identify prey items in seal scats, and compared these results to local fisheries data. A total of 46 prey occurrences was detected by both methods, of which 17 matched between techniques at least to the family level. Hard parts methods identified five incidences of prey undetected by DNA methods in five scats (one incidence per scat). DNA methods identified 24 additional prey occurrences in 13 scats, for which no hard part evidence for that prey had been found. This more than doubled the total number of prey occurrences across the 15 scats compared. Overall, the most frequently occurring harbour seal prey were walleye pollock (Theragra chalcogramma), sculpins and snailfishes. In contrast, the top three groups targeted by fisheries were codfishes, salmon and invertebrates. Many species common in the harbour seal's diet such as snailfishes and blennies were not targeted by fisheries. Fishes such as greenlings, sculpins, rockfishes and Japanese anchovy (Engraulis japonicus) were common in the diet of harbour seals, but made up a very small proportion of fisheries catches in Erimo. The importance of other prey species varied between seasons. Sculpins, greenlings and forage fishes were the top three prey groups for harbour seals in spring by percent modified frequency of occurrence, whereas the most important target groups by percentage mass caught by fisheries were codfishes, cephalopods and other invertebrates. Sculpins and rockfishes remained as important prey items for harbour seals in summer and autumn, in addition to codfishes, snailfishes and blennies. In contrast, the main groups caught by fisheries in summer were other fishes, particularly Japanese amberjack (Seriola quinqueradiata) and various species of sharks and eels, and invertebrates. By autumn, Erimo fisheries had focussed on catching salmon. Salmon were taken by harbour seals as well during this period, but at a relatively lower frequency compared to other prey groups. The results of our study show that although harbour seals consumed several of the prey species targeted by fisheries, the relative importance of these species to seals and fisheries and the seasons in which they were targeted were different.     

Size‐frequency distributions of Joania cordata and Argyrotheca cuneata (Brachiopoda: Megathyrididae) from the Central Tyrrhenian Sea

Size‐frequency distributions can support reliable inferences concerning population dynamics of brachiopods, but only a few data are available so far. In this study, length and width frequency distributions of dead specimens of the Recent brachiopods Joania cordata and Argyrotheca cuneata from the Marine Protected Area ‘Secche di Tor Paterno’, Central Tyrrhenian Sea, Italy (41°35′ N, 12°20′ E), are reported in order to add new data about size‐frequency distributions of brachiopods. The studied specimens came from death assemblages in the coralligenous substrate, in the Posidonia oceanica meadows, and in the sand channels. The observed patterns vary from left‐skewed (J. cordata) to right‐skewed (A. cuneata), indicating respectively a low and high mortality of smaller individuals. Significant differences between the coralligenous substrate and the P. oceanica meadow were observed for both species, revealing a variation among different habitats. All length and width distributions are clearly polymodal, but the biological meaning of the peaks is difficult to interpret, as the two species seem to have a 2‐year life span. A biometric analysis of shell sizes revealed that length and width are the most variable parameters during the growth of the animal.     

PCR‐based techniques to determine diet of the Australian sea lion (Neophoca cinerea): a comparison with morphological analysis

Comprehensive dietary information for the endangered Australian sea lion (Neophoca cinerea) is currently limited by the deficiency and poor quality of identifiable prey remains recovered from regurgitate and faeces and the difficulty of observing feeding in the wild. In this study, we investigated DNA‐based prey detection methods using conventional (end‐point) and quantitative real‐time PCR (qPCR) on faeces collected from two captive Australian sea lions fed experimental diets of whole teleost fish, squid and shark tissue. PCR prey detection methods using the mitochondrial cytochrome oxidase subunit I (COI) and 16S genes combined with clone sequencing were compared with prey identified using traditional hard part analysis. The molecular results indicated that prey DNA was degraded. However, prey amplification was successful by targeting short (71 bp) DNA fragments. Both conventional PCR and qPCR techniques significantly increased prey detection compared with analysis of hard parts. For both sea lions, the hard part analysis was constrained by sporadic and extremely low recovery of fish otoliths (<2%), and cephalopod beaks were not recovered from the 116 squid fed. Comparisons between PCR techniques indicated comparable prey detection frequencies for all species tested; however, the sensitivity and greater resolution of qPCR improved prey detection by ~25% in one sea lion fed the experimental squid and perch. The detection of squid DNA ≤ 6 day post‐ingestion by qPCR further exhibits the ability and potential of this method to detect low concentrations of infrequent or pulse prey. This study highlights the use of DNA‐based analysis to detect prey taxa in the absence of identifiable hard prey remains.     

Multiple paternity in the common long‐armed octopus Octopus minor (Sasaki, 1920) (Cephalopoda: Octopoda) as revealed by microsatellite DNA analysis

Multiple paternity was assessed in Octopus minor using microsatellite DNA markers. Octopus minor adults were captured in traps and kept in indoor cement ponds. The females spawned after several days. Ten broods (B1–B10), each containing 15 embryos and the maternal octopus, were maintained until the embryos reached the stage at which DNA could be extracted and used for genotyping. Multiple paternity was tested using five microsatellite DNA markers and the results proved the hypothesis that multiple paternity occurs in O. minor (observed in six out of the 10 broods). Analysis with GERUD software gave the minimum number of fathers and showed that four broods were sired by a single male, three were sired by two different males and three were sired by three different males. In brood B10, the number of fathers identified by the single‐locus method was one fewer than that by the GERUD method. Of the six multiple paternity broods, four (66.7%) showed significant skew from equal paternal contributions, which indicated that sperm competition and/or cryptic female choice may be important for post‐copulatory paternity bias in this species.     

Symbiodinium (internal transcribed spacer 2) diversity in the coral host Agaricia lamarcki (Cnidaria: Scleractinia) between shallow and mesophotic reefs in the Northern Caribbean (20–70 m)

This study investigated differences in Symbiodinium diversity in the scleractinian coral species Agaricia lamarcki between shallow (20–25 m) and mesophotic (50–70 m) depths in the Northern Caribbean. Corals were sampled in each of four shallow sites (20–25 m; n = 18) and three mesophotic sites (50–70 m; n = 18) from Mona Island (Puerto Rico) and the US Virgin Islands during a mesophotic exploratory cruise and from the La Parguera shelf edge, off Southwestern Puerto Rico. Symbiodinium diversity was assessed using internal transcribed spacer 2 sequences clustered into operational taxonomic units (OTUs). Clustering resulted in eight clade C OTUs and one clade D OTU. Of these, there were three common Symbiodinium OTUs consisting of C3 and D1a.N14 in shallow reefs and C11.N4 in mesophotic reefs. Statistical tests (permutational multivariate analysis of variance and analysis of similarity) showed significant differences between clade C Symbiodinium OTUs in A. lamarcki colonies located at shallow and mesophotic depths, indicating symbiont zonation. Symbiodinium diversity in A. lamarcki from the Northern Caribbean is comparable to previous reports in the Southern Caribbean for this species. This is the first report of the thermal tolerant species Symbiodinium trenchii (D1a) in A. lamarcki.     

Growth and population dynamics of the non‐indigenous species Branchiomma luctuosum Grube (Annelida,Sabellidae) in the Ionian Sea (Mediterranean Sea)

The population dynamics and gametogenesis of the non‐indigenous polychaete species Branchiomma luctuosum Grube, 1869 (Annelida, Sabellidae) has been investigated at three sites in the Taranto Seas (Ionian Sea, Mediterranean Sea). The species, probably introduced from the Red Sea, has been reported in the Mediterranean Sea since 1983. The species is hermaphrodite, and the reproductive season is between June and October when the largest mean size of oocytes was recorded together with the presence of mature spermatozoa. Small oocytes are present in specimens reaching about 20–25 mm in length. Therefore oogenesis seems to begin early during the first year of life, but the first reproduction can occur when the worms attain a larger size and are at least 6 months of age. Although most of the individuals reproduce seasonally within a discrete period, some individuals can reproduce in different periods during the year, the oogenesis of individuals not being synchronous. A life span of at least 2 years is highlighted, with a faster growth rate during the first months (about 20 mm per month) decreasing to about 10 mm from the 3rd to the 8th months and slowing down again after the worm reaches 100 mm in size. Some differences in growth performance are discussed, enhanced by comparing the sites located at greater depth (5–7 m) and those located at 0.5 m depth, together with the possible interactions of this alien species with the autochthonous sabellid Sabella spallanzanii.     

鳞侧石鳖属(Leptochiton)一物种在西太平洋雅普海沟分布的新记录

蛟龙号载人潜水器在位于雅普海沟北部6 745 m深处的底栖沉积环境中采集到两只石鳖样本,这是石鳖物种分布于此的新记录。形态学和DNA分子标记均支持两只石鳖为鳞侧石鳖属（Leptochiton）同一物种。该种在形态上与L.vanbellei和L.deforgesi接近;在系统发生方面,与L.vanbellei,L.deforgesi和L.boucheti的亲缘关系密切。这是截至目前世界石鳖分布报道的第三深度记录。