POPULATION STRUCTURE AND SPECIATION IN TROPICAL SEAS: GLOBAL PHYLOGEOGRAPHY OF THE SEA URCHIN DIADEMA

Abstract.— The causes of speciation in the sea are rarely obvious, because geographical barriers are not conspicuous and dispersal abilities or marine organisms, particularly those of species with planktonic larvae, are hard to determine. The phylogenetic relations of species in cosmopolitan genera can provide information on the likely mode of their formation. We reconstructed the phylogeny of the pantropical and subtropical sea urchin genus Diadema, using sequences of mitochondrial DNA from 482 individuals collected around the world, to determine the efficacy of barriers to gene flow and to ascertain the history of possible dispersal and vicariance events that led to speciation. We also compared 22 isozyme loci between all described species except D. palmeri. The mitochondrial DNA data show that the two deepest lineages are found in the Indian and West Pacific Oceans. (Indo‐Pacific) Diadema setosum diverged first from all other extant Diadema, probably during the initiation of wide fluctuations in global sea levels in the Miocene. The D. setosum clade then split 3‐5 million years ago into two clades, one found around the Arabian Peninsula and the other in the Indo‐West Pacific. On the lineage leading to the other species of Diadema, the deepest branch is composed of D. palmeri, apparently separated when the climate of New Zealand became colder and other tropical echinoids at these islands went extinct. The next lineage to separate is composed of a currently unrecognized species of Diadema that is found at Japan and the Marshall Islands. Diadema mexicanum in the eastern Pacific separated next, whereas D. paucispinum, D. savignyi, and D. antillarum from the western and central Atlantic, and (as a separate clade) D. antillarum from the eastern Atlantic form a shallow polytomy. Apparently, Indo‐Pacific populations of Diadema maintained genetic contact with Atlantic ones around the southern tip of Africa for some time after the Isthmus of Panama was complete. Diadema paucispinum contains two lineages: D. paucispinum sensu stricto is not limited to Hawaii as previously thought, but extends to Easter Island, Pitcairn, and Okinawa; A second mitochondrial clade of D. paucispinum extends from East Africa and Arabia to the Philippines and New Guinea. A more recent separation between West Indian Ocean and West Pacific populations was detected in D. setosum. Presumably, these genetic discontinuities are the result of water flow restrictions in the straits between northern Australia and Southeast Asia during Pleistocene episodes of low sea level. Diadema savignyi is characterized by high rates of gene flow from Kiribati in the central Pacific all the way to the East African Coast. In the Atlantic, there is a biogeographic barrier between the Caribbean and Brazil, possibly caused by fresh water outflow from the Amazon and the Orinoco Rivers. Diadema antillarum populations of the central Atlantic islands of Ascension and St. Helena are genetically isolated and phylogenetically derived from Brazil. Except for its genetic separation by the mid‐Atlantic barrier, Diadema seems to have maintained connections through potential barriers to dispersal (including the Isthmus of Panama) more recently than did Eucidaris or Echinometra, two other genera of sea urchins in which phylogeography has been studied. Nevertheless, the mtDNA phylogeography of Diadema includes all stages expected from models of allopatric differentiation. There are anciently separated clades that now overlap in their geographic distribution, clades isolated in the periphery of the genus range that have remained in the periphery, clades that may have been isolated in the periphery but have since spread towards the center, closely related clades on either side of an existing barrier, and closely related monophyletic entities on either side of an historical barrier that have crossed the former barrier line, but have not attained genetic equilibrium. Except for D. paucispinum and D. savignyi, in which known hybridization may have lodged mtDNA from one species into the genome of the other, closely related clades are always allopatric, and only distantly related ones overlap geographically. Thus, the phylogenetic history and distribution of extant species of Diadema is by and large consistent with allopatric speciation.     

PHYLOGEOGRAPHY OF THE PANTROPICAL SEA URCHIN EUCIDARIS IN RELATION TO LAND BARRIERS AND OCEAN CURRENTS

The pantropical sea urchin genus Eucidaris contains four currently recognized species, all of them allopatric: E. metularia in the Indo-West Pacific, E. thouarsi in the eastern Pacific, E. tribuloides in both the western and eastern Atlantic, and E. clavata at the central Atlantic islands of Ascension and St. Helena. We sequenced a 640-bp region of the cytochrome oxidase I (COI) gene of mitochondrial DNA to determine whether this division of the genus into species was confirmed by molecular markers, to ascertain their phylogenetic relations, and to reconstruct the history of possible dispersal and vicariance events that led to present-day patterns of species distribution. We found that E. metularia split first from the rest of the extant species of the genus. If COI divergence is calibrated by the emergence of the Isthmus of Panama, the estimated date of the separation of the Indo-West Pacific species is 4.7–6.4 million years ago. This date suggests that the last available route of genetic contact between the Indo-Pacific and the rest of the tropics was from west to east through the Eastern Pacific Barrier, rather than through the Tethyan Sea or around the southern tip of Africa. The second cladogenic event was the separation of eastern Pacific and Atlantic populations by the Isthmus of Panama. Eucidaris at the outer eastern Pacific islands (Galapagos, Isla del Coco, Clipperton Atoll) belong to a separate clade, so distinct from mainland E. thouarsi as to suggest that this is a different species, for which the name E. galapagensis is revived from the older taxonomic literature. Complete lack of shared alleles in three allozyme loci between island and mainland populations support their separate specific status. Eucidaris galapagensis and E. thouarsi are estimated from their COI divergence to have split at about the same time that E. thouarsi and E. tribuloides were being separated by the Isthmus of Panama. Even though currents could easily convey larvae between the eastern Pacific islands and the American mainland, the two species do not appear to have invaded each other's ranges. Conversely, the central Atlantic E. clavata at St. Helena and Ascension is genetically similar to E. tribuloides from the American and African coasts. Populations on these islands are either genetically connected to the coasts of the Atlantic or have been colonized by extant mitochondrial DNA lineages of Eucidaris within the last 200,000 years. Although it is hard to explain how larvae can cross the entire width of the Atlantic within their competent lifetimes, COI sequences of Eucidaris from the west coast of Africa are very similar to those of E. tribuloides from the Caribbean. F_ST statistics indicate that gene flow between E. metularia from the Indian Ocean and from the western and central Pacific is restricted. Low gene flow is also evident between populations of E. clavata from Ascension and St. Helena. Rates of intraspecific exchange of genes in E. thouarsi, E. galapagensis, and E. tribuloides, on the other hand, are high. The phylogeny of Eucidaris confirms Ernst Mayr's conclusions that major barriers to the dispersal of tropical echinoids have been the wide stretch of deep water between central and eastern Pacific, the cold water off the southwest coast of Africa, and the Isthmus of Panama. It also suggests that a colonization event in the eastern Pacific has led to speciation between mainland and island populations.     

分支分类学中和谐性概念与和谐性分析方法

和谐性是分支分类学中的一个基本概念。本文给出一个和谐性的数学定义,称为Kexue和谐性。并在Kexue和谐性的基础上开发出一个新的和谐性分析方法。并对该方法在分支分类研究中的应用进行讨论。     

几何形态学方法及其在动物发育与系统进化研究中的应用

简要回顾了几何形态学的发展历史,介绍了30多年来该领域使用的主要方法及其在生物发育与进化研究中的实际应用。除目前应用最为广泛的标点法和轮廓线法外,基于三维数据的表面特征分析和有限元分析也在该领域得到推广。这些方法可运用于分析两性分化、异速生长、种群分化、种上进化以及复杂功能结构的形态集成等科学问题。几何形态学与生物信息学的综合运用还将有利于探讨表型变化与遗传物质变化的相关性。随着生物成像技术和海量数据计算技术的进步,几何形态学将由目前以二维数据为主的研究向三维重建和分析的方向发展。     

Resistance of the heart sea urchin Echinocardium cordatum (Echinoidea: Spatangoida) to extreme environmental changes

The ability to survive under extreme environmental conditions was studied in the adults of the heart sea urchin Echinocardium cordatum (Pennant). At seawater temperatures of 13.3 to 14.8°C and salinity of 33.2–33.4‰, being devoid of the possibility to burrow into the sand or eat, some sea urchins died on day 5 and all individuals had perished by the end of day 8. At a temperature of 19°C, the salinity tolerance range of adults was limited to 33–28‰. Only 30 to 20% of sea urchins transferred to a solid substrate survived for 7 days at a salinity of 33 to 24‰, but all of them perished toward the end of day 8.     

SPECIATION AND POPULATION GENETIC STRUCTURE IN TROPICAL PACIFIC SEA URCHINS

Unlike populations of many terrestrial species, marine populations often are not separated by obvious, permanent barriers to gene flow. When species have high dispersal potential and few barriers to gene flow, allopatric divergence is slow. Nevertheless, many marine species are of recent origin, even in taxa with high dispersal potential. To understand the relationship between genetic structure and recent species formation in high dispersal taxa, we examined population genetic structure among four species of sea urchins in the tropical Indo-West Pacific that have speciated within the past one to three million years. Despite high potential for gene flow, mtDNA sequence variation among 200 individuals of four species in the urchin genus Echinometra shows a signal of strong geographic effects. These effects include (1) substantial population heterogeneity; (2) lower genetic variation in peripheral populations; and (3) isolation by distance. These geographic patterns are especially strong across scales of 5000-10,000 km, and are weaker over scales of 2500-5000 km. As a result, strong geographic patterns would not have been readily visible except over the wide expanse of the tropical Pacific. Surface currents in the Pacific do not explain patterns of gene flow any better than do patterns of simple spatial proximity. Finally, populations of each species tend to group into large mtDNA regions with similar mtDNA haplotypes, but these regional boundaries are not concordant in different species. These results show that all four species have accumulated mtDNA differences over similar spatial and temporal scales but that the precise geographic pattern of genetic differentiation varies for each species. These geographic patterns appear much less deterministic than in other well-known coastal marine systems and may be driven by chance and historical accident.     

GEOMETRIC MORPHOMETRICS OF THE SKULL ROOF OF STEREOSPONDYLS (AMPHIBIA: TEMNOSPONDYLI)

Abstract:  Geometric morphometric analysis using relative warps is applied to the skull roof of 62 species of stereospondyls and their closest outgroups (i.e. basal archegosauriforms) from among temnospondyl amphibians. Twenty-one landmarks and five taxonomic groups are used for comparisons. Their skull evolution is quantified in a morphospace defined by two relative warps axes. The majority of groups show poor concordance between morphological and phylogenetic distances. The only exception is represented by Yates and Warren's study of stereospondyl relationships, in which concordance is high. Only basal archegosauriforms and rhinesuchids show significant overlap in morphospace, although this might be due to low sample sizes. Regression of estimated mean disparity against taxon sample size shows that species within both the trematosauroid and the rhytidostean groups are more widely dispersed in morphospace than species belonging to any of the remaining stereospondyl groups. Stereospondyl skull evolution was characterized by divergence between major clades and convergence within those clades. Changes in patterns of morphospace occupation through time agree with the hypothesis of an 'explosive' radiation in the early Early Triassic, after the extinction of basal archegosauriforms at the end of the Permian.     

分支系统学当前的理论和方法概述及华东地区山胡椒属十二个种的分支系统学研究

本文概述了当前分支系统学研究中涉及的主要理论和方法,包括性状的选取、性状状态和极性的确定、数据矩阵的分析计算、结果分支图的处理、分支图可靠性的评价及分支图的应用。本文同时以华东地区樟科山胡椒属Linderal2个种的分支系统学研究为例,讨论了用形态性状进行分支系统学研究中可能遇到的问题,也揭示了一些分支系统学与传统的系统学在应用性状推导进化关系上的不同点。对这12个种的分支系统学研究得出了一些不同于传统系统学方法所推测的山胡椒属内的系统发育关系,如分支系统学研究显示山胡椒组和球果组很近缘。在严格一致性分支图上,杯托组的黑壳楠和江浙山胡椒分别位于最原始和最进化的分支,表明这个组是复系类群。分支图也显示山胡椒组可能是复系类群。     

MITOCHONDRIOGENESIS IN MATURING SEA URCHIN OOCYTES: A COMPUTERIZED RECONSTRUCTION ANALYSIS

The dynamics of chondriome changes in oogenesis of the sea urchinParacentrotus lividuswere studied by electron microscopy. An oocyte-enriched fraction obtained by gonad mechanical dissociation without protease treatment was used. The shape, size and arrangement of mitochondria (Mt) in cells were quantitatively analysed on the basis of data from reconstruction experiments, with serial sections performed using a specific computer program. At all stages of oogenesis, the chondriome was shown to consist of rod-shaped Mt of various lengths and also of small amounts of globular Mt about 0.3 μm in diameter. Chondriome transformation during oogenesis is shown to involve the following processes: (1) a 64-fold increase in number of Mt, with the ratio of cytoplasm to Mt volume quite constant in the course of oogenesis; (2) an increase in length of Mt to a maximum of 1.54 μm in medium oocytes and successive considerable mitochondrial division; (3) changes in Mt ultrastructure; and (4) a clustering of Mt. In a mature egg, the modal value of Mt length was reduced and, unlike the oocytes, was more homogeneous, and the Mt were completely clustered.     

REPRODUCTIVE PERIODICITY OF THE SEA URCHIN,TRIPNEUSTES GRATILLA (L.) IN TAIWAN COMPARED WITH OTHER REGIONS

An annual reproductive periodicity of Tripneustes gratilla in northern Taiwan is revealed by both the gonad development stages and the gonad index. The gonads of T. gratilla recovered from spawning in February—March, propagated gametocytes in April—June, came to prematurity in July—August, and matured in September—November. Spawning occurred in October—December. The breeding pattern of T. gratilla over a broad geographical area appears to be related to seasonal changes of sea temperatures.     

EFFECT OF THE IMPase INHIBITOR L690,330 ON SEA URCHIN DEVELOPMENT

A variety of concentrations of the IMPase inhibitor L690,330 were added to sea urchin embryos. Immediate arrest of development was obtained for concentrations from 7.5mm on. Concentrations lower than 3.5mm permitted gastrulation but inhibited skeletogenesis and disturbed elongation along the animal—vegetal axis. The latter results are similar to those obtained by counteracting lithium effect with myoinositol, which are suggested to be due to partial relief of IMPase inhibition.     

ENZYMES OF THE TRICARBOXYLIC ACID CYCLE IN SEA URCHIN EGGS AND EMBRYOS

Changes in the activity of some enzymes of the tricarboxylic acid cycle during development of sea urchins were investigated. Unfertilized eggs showed substantial activity of citrate synthase, aconitase, NAD- and NADP-specific isocitrate dehydrogenases, fumarase and malate dehydrogenase. During development, the activity of citrate synthase, aconitase, NADP-specific isocitrate dehydrogenase and malate dehydrogenase increases gradually, whereas the activity of fumarase remains rather constant. There is no close correlation between changes in the enzyme activity and the increase in oxygen consumption during development. Citrate synthase, aconitase, NADP-specific isocitrate dehydrogenase are mainly localized in the mitochondrial fraction, whereas fumarase and malate dehydrogenase are present in both mitochondrial and cytosol fractions. The intracellular localization of these enzymes does not change during development. A possible mechanism for the regulation of some enzymes of the tricarboxylic acid cycle in sea urchin eggs is discussed.     

FORMATION AND BEHAVIOR OF PINOSOMES IN THE SEA URCHIN OOCYTE DURING OOGENESIS

Formation and behavior of the pinosomes at the surface of the oocyte during oogenesis in the 4 species of sea urchins, Anthocidaris crassispina, Temnopleurus toreumaticus, Mespilia globulus and Pseudocentrotus depressus, were studied. The plasma membrane of the oocyte is almost smooth at the early stage of oogenesis, although a small number of cytoplasmic processes appear on it, facing the germinal epithelium. At the beginning of vitellogenetic stage many processes appear on the whole surface of the oocyte. Near the base of the fully grown process, the pinosome designated as the α-pinosome is formed. The α-pinosome may play a part in maturation of the yolk granule. The processes shorten as a whole at the time of the breakdown of the germinal vesicle. Formation of the pinosome designated as the β-pinosome begins just before vitellogenetic stage and continues during this stage. The β-pinosome may be directly concerned with the formation of cortical granules.     

ANTIGENS IN EGGS AND DEVELOPMENTAL STAGES OF THE SEA URCHIN : II. Localization

Homogenates of fertilized eggs of the sea urchin Paracentrotus lividus were fractionated by differential centrifugation. In addition, whole eggs were fragmented, on a preparative scale, by centrifugation in sea water-sucrose gradients. The fractions and fragments were subsequently assayed for their content of soluble protein antigens described in an earlier publication. Relative concentrations of antigen present in quantitatively isolated cell fractions were estimated by graded antiserum absorption in combination with agar-diffusion technique. Two of six antigens were found to be associated mainly with the low speed sediments. Treatment of the various sediments with hypotonic medium and results obtained with fragmented eggs suggested that these two antigens and possibly a third were probably located in the yolk granules. The other antigens were more evenly distributed among the low speed sediments and the non-sedimented part of the cytoplasm. Only one of the antigens was consistently associated with the microsomal fraction.     

PHYLOGENY AND FORELIMB DISPARITY IN WATERBIRDS

Previous work has shown that the relative proportions of wing components (i.e., humerus, ulna, carpometacarpus) in birds are related to function and ecology, but these have rarely been investigated in a phylogenetic context. Waterbirds including “Pelecaniformes,” Ciconiiformes, Procellariiformes, Sphenisciformes, and Gaviiformes form a highly supported clade and developed a great diversity of wing forms and foraging ecologies. In this study, forelimb disparity in the waterbird clade was assessed in a phylogenetic context. Phylogenetic signal was assessed via Pagel's lambda, Blomberg's K, and permutation tests. We find that different waterbird clades are clearly separated based on forelimb component proportions, which are significantly correlated with phylogeny but not with flight style. Most of the traditional contents of “Pelecaniformes” (e.g., pelicans, cormorants, and boobies) cluster with Ciconiiformes (herons and storks) and occupy a reduced morphospace. These taxa are closely related phylogenetically but exhibit a wide range of ecologies and flight styles. Procellariiformes (e.g., petrels, albatross, and shearwaters) occupy a wide range of morphospace, characterized primarily by variation in the relative length of carpometacarpus and ulna. Gaviiformes (loons) surprisingly occupy a wing morphospace closest to diving petrels and penguins. Whether this result may reflect wing proportions plesiomorphic for the waterbird clade or a functional signal is unclear. A Bayesian approach detecting significant rate shifts across phylogeny recovered two such shifts. At the base of the two sister clades Sphenisciformes + Procellariiformes, a shift to an increase evolutionary rate of change is inferred for the ulna and carpometacarpus. Thus, changes in wing shape begin prior to the loss of flight in the wing‐propelled diving clade. Several shifts to slower rate of change are recovered within stem penguins.     

有关节肢动物系统发育若干问题的研究进展

近年来对节肢动物系统发育问题的认识有了相当大的变化,尤其是支序分析研究方法的使用,以及来自分子系统学和分子发育生物学的证据,成为继比较形态学之后有力的研究手段,近年来的研究显示线虫,缓步类和有爪类是节肢动物的近缘类群,而非以前绝大多数学者所认为的软体动物的环节动物,对于节肢动物门的单系性及其内部类群间关系的问题,目前倾向于认为（1）节肢动物为单系群;（2）由甲壳类,六足类和多足类联合形成单系群－具颚类;（3）与六足类分子系统学两方面研究的支持;对第3点的争议虽较大,但就论据而言确为优势观点。     

SOME STRUCTURAL AND FUNCTIONAL ASPECTS OF THE MITOTIC APPARATUS IN SEA URCHIN EMBRYOS

The mitotic figures in dividing cells of sea urchin embryos, from first division to the onset of cilia formation, were studied with regard to the filament system and its relation to kinetochores, chromosomes, and poles, as well as to fixation conditions which would best preserve these structures. With regard to fixation, variations in the salt concentration and pH of the fixative indicated that an extraction effect on the chromosomes noted in earlier work was probably due to a combination of neutral pH and salt concentration equivalent to sea water. The presence of the 15 mµ filaments depended on the presence of either of two stabilizing conditions: pH 6.1 or presence of the salts of sea water, presumably the divalent cations of Ca and Mg. Kinetochores and centrioles were unaffected by the fixative variations. The 15 mµ filaments, reported earlier in the central spindle, are also found in great numbers in the asters of early cleavage divisions. However, with successive divisions and reduction in cell size, the aster disappears at about the 32 to 64 cell stage, and the 15 mµ filaments are entirely associated with the central spindle. This disappearance of the aster suggests that it may be, in fact, merely a specialization of large cells for cytokinesis.     

THE FINE STRUCTURE OF PRONUCLEAR DEVELOPMENT AND FUSION IN THE SEA URCHIN, ARBACIA PUNCTULATA

Fertilization events following coalescence of the gamete plasma membranes and culminating in the formation of the zygote nucleus were investigated by light and electron microscopy in the sea urchin, Arbacia punctulata. Shortly after the spermatozoon passes through the fertilization cone, it rotates approximately 180° and comes to rest lateral to its point of entrance. Concomitantly, the nonperforated nuclear envelope of the sperm nucleus undergoes degeneration followed by dispersal of the sperm chromatin and development of the pronuclear envelope. During this reorganization of the sperm nucleus, the sperm aster is formed. The latter is composed of ooplasmic lamellar structures and fasciles of microtubules. The male pronucleus, sperm mitochondrion, and flagellum accompany the sperm aster during its migration. As the pronuclei encounter one another, the surface of the female pronucleus proximal to the advancing male pronucleus becomes highly convoluted. Subsequently, the formation of the zygote nucleus commences with the fusion of the outer and the inner membranes of the pronuclear envelopes, thereby producing a small internuclear bridge and one continuous, perforated zygote nuclear envelope.     

雀形目高级阶元分类与起源研究概况

对雀肜目鸟类高级阶元的分类和系统发育的研究进行了简要概述.经典分类与应用分子生物学方法建立的分类系统在高级阶元(总科)有较大差异,但科级的分类基本一致.在雀形目鸟类起源研究方面,古生物学研究结果认为其起源于劳亚古陆;而分子生物学的证据则认为其起源于冈瓦纳古陆.由于化石的证据与"分子钟"的推测年代相差较大,因此对雀形目鸟类的起源还存在争议,但是目前的研究更倾向于"冈瓦纳起源"假说.