Female genital structures of Metridinidae (Copepoda: Calanoida)

The female genital structures of six calanoid copepod species, belonging to the genera Gaussia, Metridia and Pleuromamma, were studied using light and scanning electron microscopy (SEM). The copulatory pores and seminal receptacles are paired in Gaussia and Metridia, but unpaired in Pleuromamma. A thin epicuticle and a spermatophoral plug are the mechanisms by which the pores are closed before and after copulation. The pores open directly into the receptacles, which are reduced to shallow integumentary depressions in Gaussia. The mode of insemination suggests two matings in females of Gaussia and Metridia, but only one in Pleuromamma. Paired gonopores and egg-laying ducts are present in the three genera, with a characteristic, closed semicircular configuration; these are opened during egg-laying by the action of retractor muscles of the gonoporal plates. The seminal ducts, which open into the gonopores, are long, thin and paired in Gaussia and Metridia, whereas in Pleuromamma they are short, broad and unpaired. The opening of the seminal and egg-laying ducts is synchronized. The shell ducts are paired in Gaussia and Metridia, unpaired in Pleuromamma; these arise from glands situated in the lateral expansions of the last prosomite and lead into the distal part of each egg-laying duct in Gaussia and Metridia and of the egg-laying duct receiving the seminal duct in Pleuromamma. The position and anatomy of these structures are compared to those of other families and genera, and a functional interpretation of their morphology is proposed. The main evolutionary trends of the different structural patterns of female calanoid genitalia are presented in tabular form. Received: 30 December 1996 / Accepted: 11 February 1997     

Distribution,structure and importance of the cephalic dorsal hump,a new sensory organ in calanoid copepods

The occurrence, external morphology and internal ultrastructure of a cephalic integumental organ in calanoid copepods were studied, using the specimens from the Pacific, Indian and Atlantic Oceans. This organ is located on the dorsoanterior surface of the cephalosome, and a name, cephalic dorsal hump (CDH) is proposed. Externally, it usually has two pores, anterior and apical, a dorsal plate, and a thin cuticle along the sides. CDH is found only in the male of Calanidae, Megacalanidae, Mecynoceridae and Paracalanidae, and showed some variation between species or species groups both in size and shape. Transmission electron microscopy (TEM) on the specimens from Sagami Bay, Central Japan, revealed that the CDH of Paracalanus parvus and Calanus sinicus consists of two dermal glands and a receptor, which is assumed to be chemosensory. A comparison of the distributions of CDH and prehensile fifth legs of male calanoid copepods suggests that it plays an important role in mate recognition.     

Recent introduction of an Asian estuarine copepod,Pseudodiaptomus marinus (Copepoda: Calanoida), into southern California embayments

Past studies made between 1938 and the mid-1970's of coastal embayments between northern Baja California and Oregon (USA) established the presence of only one species of the calanoid copepod genus Pseudodiaptomus, P. euryhalinus. This species is endemic to warm temperate latitudes of western North America. We now report the presence of breeding populations of the Asian calanoid copepod P. marinus in two Southern California embayments, Mission Bay and Agua Hedionda Lagoon. The Mission Bay population was monitored monthly from December 1986 to June 1987 and showed evidence of continuous breeding and growth. The regional, endemic species, P. euryhalinus, was not observed in either embayment. Mechanisms that might account for the apparent recent introductions of P. marinus into Southern California include dispersion by ocean circulations and transport by transoceanic ships. In the present case, the explanation is that the introduction is most likely a direct consequence of aquaculture projects. Remaining to be confirmed by systematic sampling is the apparent disappearance of P. euryhalinus from embayments dominated by P. marinus.     

Unexpected new species of deep-water Hydroidomedusae from Korsfjorden,Norway

Two new species of Hydroidomedusae, Parateclaia norvegica sp.nov. and Foersteria quadrata sp.nov., are described from specimens collected in Korsfjorden, western Norway. Neither genus has previously been observed in the North-Atlantic Ocean. The closest congenerics to the new species occur in the Mediterranean, where family Teclaiidae has been considered endemic. P. norvegica and F. quadrata were only collected at sites 500 m or deeper and are possibly deep-water benthopelagic species like their congenerics elsewhere. Sampling took place for the entire year 2003 at 2- to 4-week intervals. F. quadrata, the more numerous of the new species, was absent from the water column only during the spring months, the implications of which regarding its lifecycle are discussed. Hypotheses on speciation in the Foersteria species group and family Teclaiidae are shortly discussed.     

Geographical range and taxonomic divergence in North Atlantic Calanus (C. helgolandicus,C. finmarchicus and C. glacialis)

The known distribution of Calanus helgolandicus in the North Atlantic Drift is difficult to explain in the absence of a reproductively active population inhabiting continental waters off eastern North America. New evidece indicates that this population, overlooked in the past, does in fact exist. The species has been found in a study of zooplankton samples from a number of MARMAP (Marine Resource Monitoring, Assessment, and Prediction Program) cruises surveying ichthyoplankton between Cape Hatteras and the New York Bight. Sexual activity in these stocks of C. helgolandicus was indicated by the ripeness of ovaries, the frequency of males, the presence of sperm in the females' seminal receptacles and the appearance of a female bearing a Calanus spermatophore. The new records provide a likely origin for the presence of the species in the vicinity of the Labrador Grand Banks as well as further east in the North Atlantic Drift. C. finmarchicus, similarly sexually active, was found to be sympatric with C. helgolandicus in the MARMAP collections which were taken from a region contiguous with the southernmost, known distribution, of C. glacialis. The distribution of integumental organs (i.e., pore signature patterns) was examined in the three species to determine whether they would be taxonomically useful. Strikingly different patterns were found on the female urosome. Pore signature differences between the polar species C. glacialis and the temperate C. helgolandicus proved to be as pronounced as those between the boreal C. finmarchicus and its two neighboring species. The successively overlapping ranges and the distinctive differences in pore signature patterns suggest that divergence from the generic pattern of integumental organ distribution has been a product of selection against hybridizing among the three species. If this is in fact the case, the reproductive range of C. helgolandicus has overlapped with those of C. finmarchicus and C. glacialis for appreciable periods in the history of the three species.     

Population structure of the black tiger prawn, Penaeus monodon, among western Indian Ocean and western Pacific populations

We examined the population structure of the black tiger prawn, Penaeus monodon Fabricius, 1798, in the Indo-West Pacific by analyzing the geographic distribution of elongation factor 1-alpha intron sequences from specimens collected during the winter and spring of 1997. Both the molecular phylogeny of alleles and F-statistics indicated very strong differentiation between populations from the western Indian Ocean and western Pacific. This pattern is concordant with other recent studies of marine species in this region, implying that the Indo-Australian Archipelago represents a biogeographic break between populations in the Indo-West Pacific. F _ST-values among populations in the western Indian Ocean also indicate structure within this region, whereas no structure was found among western Pacific populations. Nucleotide diversity was significantly lower in the western Indian Ocean populations than in the western Pacific, implying that the populations have regional differences in demographic history. Received: 16 November 1998 / Accepted: 26 May 1999     

<Emphasis Type="Italic">Alexandrium catenella</Emphasis> (Dinophyceae), a toxic ribotype expanding in the NW Mediterranean Sea

The presence of the paralytic shellfish poisoning (PSP) dinoflagellate Alexandrium catenella in the north western (NW) Mediterranean Sea has been known since 1983. From this date on, the species has spread along the Spanish and Italian coastlines. Information concerning A. catenella isolates in the NW Mediterranean Sea was gained through phylogenetic studies. Twenty established toxic cultures of A. catenella taken from various NW Mediterranean Sea locations were analysed by nucleotide sequencing of the 5.8S rDNA and internal transcribed spacer regions. These rDNA ribosomal markers resulted useful in delineating the phylogenetic position of this species in the genus Alexandrium as well as in determining relationships between A. catenella isolates from different geographic areas. The phylogenetic position of the Mediterranean A. catenella ribotype, when compared to the “Alexandrium tamarense/catenella/fundyense species complex”, fits this species complex well. All the Mediterranean A. catenella isolates were constituted by only one genetic ribotype. By comparing the isolate sequences with those of other geographic areas, it revealed that the Mediterranean A. catenella ribotype was closely related to the A. catenella from Japan, Western Pacific Ocean. It was also evident that in temperate Japanese waters, a genetic variability was detected within A. catenella isolates; in fact, all strains resulted divergent showing as many as 15 mutational steps. The possibility that A. catenella has been recently introduced into the Mediterranean basin from temperate Asian areas is discussed.     

Comparative observations on the biometry and development of Calanus finmarchicus and C. helgolandicus in Copepodite Stage V,with comments on other Calanidae

A similar, sexual dimorphism in Copepodite Stage V was observed in winter generations of Calanus finmarchicus (Gunnerus) and C. helgolandicus (Claus), co-existing in the Firth of Clyde, Scotland. In both species, the frequency distribution of prosome length in the Copepodite V population was bimodal. Among individual Stage V copepodites kept in the laboratory, those of the larger form alone produced adult males. Indications of morphological distinctions between the forms other than in size, however, were absent. Differences in least-squares regressions of individual cephalosome on metasome lengths, previously attributed to a distinction in prosome shape by Grigg et al. (1985), reflected size alone. These two measurements also were greater in the “large” form, but this independently of each other. Variations in their relative contributions to the prosome length distinguished individuals, and were continuous between the forms. Present and previously published observations on members of the family Calanidae have been compared. The presence, or not, of a sexual distinction in prosome length in the Stage V copepodite is variable among calanids, within and between species. A possible association with the extent of seasonal variations in size and life-cycle characteristics is suggested.     

Winter food utilisation by sympatric mysids in the Baltic Sea,studied by combined gut content and stable isotope analyses

We studied the winter dietary characteristics of two sympatric mysid species, Mysis mixta and M. relicta, which exploit both benthic and pelagic habitats during diel vertical migrations. Samples collected before and after the ice-covered period in the northern Baltic Sea were investigated using both stomach content analyses and stable isotope analyses of carbon and nitrogen. Both of the mysid species were omnivorous during winter and utilised both benthic and pelagic food sources. The main food source before the ice period was calanoid copepods (40 and 36% for M. mixta and M. relicta, respectively), and after ice-out calanoid copepods (23%) and zooplankton resting eggs (23%) for M. mixta and diatoms (44%) and calanoids (25%) for M. relicta. Their patterns of food utilisation broadly followed seasonal fluctuations in the abundance of the main prey groups. Although pelagic food availability is low in winter both mysid species utilised pelagic prey widely. We also show that when combining these different diet analysis methods it is important to take into account the time lag in isotopic signatures, otherwise the obtained results do not correspond but instead show the feeding history at different times.     

Hidden ancient diversification in the circumtropical lancelet Asymmetron lucayanum complex

The tropical lancelet Asymmetron lucayanum (= Epigonichthys lucayanus) is distributed from the western Indian Ocean to the central Pacific Ocean, and the western Atlantic Ocean. Molecular phylogenetic analysis of mitochondrial cytochrome c oxidase subunit I (COI) sequences (1,035 bp) of A. lucayanum (80 specimens from seven localities) showed clearly that this species is genetically distinguished into three major groups of geographical populations based on neighbor-joining tree using maximum likelihood distance (HKY model with invariable sites and gamma correction), suggesting the existence of three cryptic species. Our genetic data show that (1) inter-oceanic divergence time between Clade B (the West-Central Pacific) and Clade C (the Atlantic) (d = 6.6%, ca. 12 million years ago) was smaller than intra-oceanic divergence time between Clade A (the Indo-West Pacific) and Clade B (d=39.5%, ca. 100 million years ago); (2) there are two cryptic species in the West Pacific in sympatry; and (3) high gene flow is implied between the Maldives and the Ryukyus in Clade A (10,000 km distance), the Philippines and Hawaii in Clade B (8,500 km distance), and Barbados and Bermuda in Clade C (2,200 km distance).     

Molecular characterization of the zoanthid genus <Emphasis Type="Italic">Isaurus</Emphasis> (Anthozoa: Hexacorallia) and associated zooxanthellae (<Emphasis Type="Italic">Symbiodinium</Emphasis> spp.) from Japan

The zoanthid genus Isaurus (Anthozoa: Hexacorallia) is known from both the Indo-Pacific and Atlantic Oceans, but phylogenetic studies examining Isaurus using molecular markers have not yet been conducted. Here, two genes of markers [mitochondrial cytochrome oxidase subunit I (COI) and mitochondrial 16S ribosomal DNA (mt 16S rDNA)] from Isaurus specimens collected from southern Japan (n = 19) and western Australia (n = 3) were sequenced in order to investigate the molecular phylogenetic position of Isaurus within the order Zoantharia and the family Zoanthidae. Additionally, obtained sequences and morphological data (polyp size, mesentery numbers, mesogleal thickness) were utilized to examine Isaurus species diversity and morphological variation. By comparing our obtained sequences with the few previously acquired sequences of genera Isaurus as well as with Zoanthus, Acrozoanthus (both family Zoanthidae), and Palythoa spp. (family Spenophidae) sequences, the phylogenetic position of Isaurus as sister to Zoanthus within the Family Zoanthidae was suggested. Based on genetic data, Isaurus is most closely related to the genus Zoanthus. Despite considerable morphological variation (in particular, polyp length, mesentery numbers, external coloration) between collected Isaurus specimens, all specimens examined are apparently conspecific or very closely related based on molecular data and observed morphological variation within colonies. Additionally, obtained internal transcribed spacer of ribosomal DNA (ITS-rDNA) sequences from symbiotic zooxanthellae (Symbiodinium spp.) from all Isaurus specimens were shown to be subclade C1-related Symbiodinium. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Hidden genetic diversity in a key model species of coral

A total of 43 colonies of the scleractinian coral Pocillopora damicornis from lagoonal and reef slope sites in the western Indian Ocean (WIO) region were genetically characterised at one nuclear and two mitochondrial sequence markers and six microsatellite loci. Both mitochondrial and microsatellite data support the existence of two reciprocally monophyletic clusters (F- and NF-types) and provide evidence of the existence of two cryptic species of P. damicornis on reefs in WIO region and put current morphological delineation and geographical boundaries of P. damicornis and Pocillopora molokensis into question. The results add to ongoing studies on the phylogeny and phylogeography within the genus Pocillopora, which all point towards a range of unresolved morphological and molecular species boundaries. Nuclear phylogenies derived from the present and previously published sequences show evidence for incomplete lineage sorting and/or introgressive hybridisation between Pocillopora morphospecies. However, the two WIO types largely remain in separate clusters, further supporting the theory that these represent two different species.     

Using fatty acid analysis to elucidate the feeding habits of Southern Ocean mesopelagic fish

Fatty acid biomarkers were used to investigate the feeding ecology of 17 mesopelagic fish species occurring in the Southern Ocean. Fatty acid signatures of species where little or no dietary information exists were compared to fatty acid signatures of species of known diets in order to elucidate their trophic position. Principal component analysis grouped species of known diets into two clusters with amphipods and copepods comprising the main prey species, respectively. Although the majority of species of unknown diet were grouped with either of these feeding guilds, a third cluster comprising only Gymnoscopelus bolini was identified suggesting a significantly different diet for this species. Electrona antarctica also exhibited significant changes in fatty acid signatures with size. Furthermore, discriminant analysis of the four most abundant species classified species with a 90% success rate thus validating the usefulness of fatty acid signatures when trying to resolve the trophic position of species where no or little dietary information exists.     

Seasonal variation in some population parameters of Euchaeta species (copepoda: Calanoida)

Studies of the biology of oceanic copepods are few relative to those of coastal species. Females of the genus Euchaeta have spermatophores attached to the genital somite by the male and carry their broods of eggs attached to this somite, so defining the breeding season. Populations of E. norvegica in the fjordic environment of Loch Etive (collected between 1971 and 1974 and from 1978 to 1979), Scotland and in the marginal oceanic region of the Rockall Trough, northeastern Atlantic Ocean (collected between 1973 and 1976) and of a further eight species of Euchaeta in the Trough are examined. Seasonal changes in the incidence of egg masses and spermatophores attached to the females, sex ratio and population numbers are determined. Sampling errors in the estimation of these parameters are larger in the oceanic Rockall Trough. The Loch Etive populations of E. norvegica produce two generations per year with a proportion, variable between years, of the population producing a third generation. This species produces a single annual generation in the Rockall Trough as do E. acuta and E. pseudotonsa. Two generations per year are probably passed through by E. gracilis in the Trough while the large bathypelagic E. sarsi may produce a single generation in the autumn of every second year. The other bathypelagic species, E. barbata, E. scotti, E. abbreviata and E. longissima, breed continuously throughout the year and no estimate of their generation times is possible. Consequently, this approach to population analysis is useful where breeding is seasonal, but resolving generation time in bathypelagic crustaceans remains a problem.     

Mating behavior of Labidocera aestiva (Copepoda: Calanoida)

The calanoid copepod Labidocera aestiva Wheeler exhibits a ritualized mating behavior. Sensory hairs and spines and integumental pores on the male and female are believed to play a regulatory role in the mating encounter. Scanning electron microscopy (SEM) revealed approximately 20 unusual pore structures (pit-pores) on the right ventrolateral surface of the female's abdominal segment, the area where the spermatophore coupling device is attached. Prior to spermatophore transfer, the male vigorously strokes this area with the modified endopod of his left fifth leg to prepare the pit-pore region for attachment of a new spermatophore. Transmission electron microscopy (TEM) indicated that the cells underlying each pit-pore may have a secretory function. We hypothesize that the pit-pores secrete a substance which dissolves the cement binding the coupling device to the abdomen and aids the female to remove a discharged spermatophore from her urosome.Contribution No. 116, Harbor Branch Foundation, Inc.     

Spatio-temporal changes in diversity and community structure of planktonic copepods in Sagami Bay, Japan

Seasonal changes in diversity and community structure of planktonic copepods at a shelf site in Sagami Bay, Japan was studied in relation to cross-shelf interaction of species components. Seasonal mesozooplankton samples were collected from the shelf station (St. M) of the north-west part of Sagami Bay from 1995 to 1997. Vertical multi-layered samples were collected near the center of Sagami Bay (St. P) in June 1996. A total 185 copepod species were identified from the two stations. We observed a clear seasonal succession in calanoid diversity and community structure at St. M from a simple shelf water community (>11 species) during spring blooming periods to highly diverse and mixed communities (ca 20–30 species) of shelf water species coupled with various Kuroshio Current species during late summer to autumn. Cluster and non-metric multidimensional scaling ordination analyses showed two distinct calanoid community groups. One group, which included samples of St. M and the surface layer of St. P, consisted of shelf water species, such as Calanus sinicus, Ctenocalanus vanus, Paracalanus spp., and Kuroshio species, such as, Canthocalanus pauper, Scolecithrix danae, etc. The other cluster was restricted to the samples collected from mid and deep layers at St. P, which consisted of meso- and bathypelagic species and Oyashio species (cold-current species, such as Neocalanus cristatus, Pseudocalanus spp., Eucalanus bungii and Metridia pacifica). In the mid and deep layers at St. P, the population of dormant copepodid stage V (CV) of Eucalanus californicus and C. sinicus were dominant. The deep CV population of C. sinicus might be ecologically discriminated from the surface and shelf water population due to their larger body length and dormant life cycle. E. californicus was also collected at the shelf site during each spring bloom period, whereas the population might descend into the mid- and deep-layers of the central bay before summer. Our results suggest that the seasonal fluctuation of community structure in the shelf water was controlled by both physical (Kuroshio Current) and biological factors, i.e., spring bloom and ontogenetic vertical migration of E. californicus. In particular, transport and diffusion processes of Kuroshio Current in Sagami Bay played a key role in controlling the shelf water calanoid community.     

Symbiont diversity within the widespread scleractinian coral<Emphasis Type="Italic"> Plesiastrea versipora</Emphasis>, across the northwestern Pacific

The molecular diversity of symbiotic dinoflagellates associated with the widespread western Pacific coral Plesiastrea versipora was explored in order to examine if associations between reef-building corals and symbiotic dinoflagellates change with environment. Several ribosomal DNA genes with different evolutionary rates were used, including the large subunit (28S), the 5.8S region and the internal transcribed spacers (ITS). The phylogenetic analysis of the 28S and 5.8S rDNA regions indicated that a single endosymbiont species, highly related to one of the species of Symbiodinium in clade C (= Symbiodinium goreaui, Trench et Blank), associates with P. versipora along the Ryukyu Archipelago. The persistence of the same endosymbiont within P. versipora across this wide array of latitudes may be a result of such features as the Kuroshio Current, which brings tropical temperatures as far north as Honshu, Japan. Analysis of the faster evolving ITS rDNA region revealed significant genetic variability within endosymbionts from different populations. This variation was due to a high degree of interpopulation variability, based on the proportion of pairwise variation detected among the populations (0.95% approximately). By comparison with other studies, the results also indicate that some ITS1 haplotypes from P. versipora endosymbionts seem to be widely distributed within the western Pacific Ocean, ranging from the Great Barrier Reef to the northeast of the China Sea.     

Mating behavior in three species of Pseudodiaptomus (Copepoda: Calanoida)

Quantitative studies of mating behavior between conspecific and heterospecific pairs of three closely related species in the genus Pseudodiaptomus reveal differences in the timing of life-history events, species-specific attraction of mates, distinctive tactile cues, multiple spermatophore attachments, and gametic incomptibility or hybrid inviability. These differences may function as reproductive isolating mechanisms among calanoid copepods. Differences in breeding behaviors and associated morphological features are greater between sympatric than between allopatric species, indicating that selection reinforces both behavioral and morphological divergences.     

Phylogenetic relationships of coral-associated gobies (Teleostei,Gobiidae) from the Red Sea based on mitochondrial DNA data

Bryaninops, Gobiodon, Paragobiodon and Pleurosicya are the most abundant genera of coral-associated gobies. These genera are adapted to live among coral, while other small reef gobies (e.g., the genus Eviota) show no obligate association with this living substrate. Thirteen coral-associated species and two Eviota species were sampled from different regions of the Red Sea, along with four populations/species of Gobiodon from the Indian and western Pacific Oceans. A molecular phylogenetic analysis was performed using partial sequences of 12S rRNA, 16S rRNA and cytochrome b mitochondrial genes, 1,199 base pairs in total. Several clades were consistently resolved in neighbor joining-, maximum parsimony-, maximum likelihood and Bayesian analyses. While each of the four genera Gobiodon, Paragobiodon, Bryaninops and Pleurosicya proved to be monophyletic, their relative position in the phylogeny did not support an emergence of coral-associated gobiids as a monophyletic assemblage. Instead, two separate monophyletic sub-groups were discovered, the first comprising Gobiodon and Paragobiodon, and the second Bryaninops and Pleurosicya. Our molecular phylogenetic examinations also revealed one unassigned species of Gobiodon from the Maldives as a distinct species and confirmed three putative and yet unassigned species from the Red Sea. Moreover, the uniformly black colored species of Gobiodon are not monophyletic but have evolved independently within two distinct species groups. Genetic distances were large in particular within Pleurosicya and Eviota. Estimated divergence times suggest that coral-associated gobies have diversified in parallel to their preferred host corals. In particular, divergence times of Gobiodon species closely match those estimated for their typical host coral genus Acropora.     

Deconstructing an assemblage of “turtle” barnacles: species assignments and fickle fidelity in Chelonibia

Barnacles in the genus Chelonibia are commensal with a variety of motile marine animals including sea turtles, crustaceans, and sirenians. We conducted a worldwide molecular phylogenetic survey of Chelonibia collected from nearly all known hosts to assess species relationships, host-fidelity, and phylogeographic structure. Using DNA sequences from a protein-coding mitochondrial gene (COI), a mitochondrial rRNA gene (12S), and one nuclear rRNA gene (28S), we found that of four species, three (C. testudinaria, C. patula, and C. manati) are genetically indistinguishable. In addition, we show each utilizes a rare androdioecious mode of reproduction involving complemental males. In contrast, the fourth species (C. caretta), which is hermaphroditic and specializes on turtles, is genetically distinct—leading to the conclusion that the three former taxa are morphotypes of the same species and should be synonymized under C. testudinaria. Phylogenetic analysis resulted in three geographic clades (Atlantic, Indian Ocean/western Pacific, and eastern Pacific) with haplotype parsimony networks revealing no shared haplotypes among geographic regions. Analysis of molecular variance detected significant differences among sequences by region (p < 0.005); conversely, there were no significant differences among sequences when grouped by host or taxonomic designation. Average pairwise genetic distances were lower between the eastern Pacific and Atlantic clades (0.053 ± 0.006) than between the eastern Pacific and Indian Ocean/western Pacific clades (0.073 ± 0.008), suggesting Atlantic and eastern Pacific populations were connected more recently, perhaps until the rise of the Isthmus of Panama. Host use by Chelonibia morphotypes is discussed along with speculation on possible ancestral hosts and support for a “turtle-first” hypothesis.