Triops Species Research Articles

Raman spectroscopic analysis of the composition of the clam-shrimp carapace (Branchiopoda: Laevicaudata, Spinicaudata, Cyclestherida): a dual calcium phosphate-calcium carbonate composition

Abstract Clam shrimps constitute a paraphyletic group of bivalved branchiopod crustaceans that includes orders Laevicaudata, Spinicaudata, and Cyclestherida. The unique mineral content of the carapace, formed by molt retention in Spinicaudata and Cyclestherida, has been variously ascribed to calcium carbonate or calcium phosphate. We analyzed the composition of modern carapaces from one laevicaudatan species, thirteen spinicaudatan species (including cyzicids, leptestheriids, and limnadiids), one cyclestherid species, and two species of the notostracan TriopsSchrank, 1803 (as an outgroup comparison within Branchiopoda) via Raman spectroscopy. The results were surprisingly variable. The outgroup Triops species varied in either having no mineral content to having a slight amount of calcium phosphate. The laevicaudatans likewise had a minor calcium phosphate peak; the leptestheriid spinicaudatan had strong calcium phosphate and calcium carbonate peaks; the limnadiid spinicaudatans were variable, varying from no mineral content to strong dual calcium carbonate and calcium phosphate peaks; the cyzicid spinicaudatans tended to have strong calcium phosphate peaks with some amount of calcium carbonate; and the cyclestherid had no mineral content. The results support the conclusion that spinicaudatans primitively have a dual mineralization system in their carapace that utilizes both calcium phosphate and calcium carbonate, with the calcium phosphate ability being basal. This suggestion of a dual mineralization system is novel in branchiopods and warrants study from material scientists. Better understanding of the distribution of phosphate versus carbonate among modern spinicaudatans will help us understand the evolution of biomineralization in the group. This, in turn, will shed light on the fossilization potential of different fossil clam-shrimp lineages.

Interspecific competition between two self-fertile and one related sexual species of tadpole shrimp Triops (Branchiopoda, Notostraca) with reproductive interference, Allee effect and environmental fluctuation

Invasive alien species of tadpole shrimp Triops, originated from America, were first introduced into rice fields of Japan in the 1910s and have spread around Japan. In the 1950s, a European species of Triops also seems to have entered Japan. Originally, Japanese paddy fields had Asian Triops species as a prehistoric naturalised animal. A new invasive Triops from Western Australia has been reported in the south part of Japan, occasionally forming mixed populations. The expansion of Triops has resulted in negative ecological and potential economic impact. Longhursts revision did not contain any specimens of the richest collection in the world, i.e., Daday Collection in the Hungarian Natural History Museum. And therefore, Naganawa surveyed at least 20 times as many samples as those by Longhurst, and number the named Triops granarius s.l. species group from Africa-India to Siberia-Asia at eight species, organised in the same genus. As for the establishment or extinction patterns, based on Tilmans competition model, we built a simple mathematical competition model between two self-fertile and one related sexual species of Japan, incorporating reproductive interference. To make the mathematical model more natural, we also assumed environmental fluctuation and Allee effect, which reduces the population growth rate of sexual species with low density. Intense reproductive interference drove the competition to sexual exclusion of self-fertile species. However, even under intense reproductive interference, Allee effect allowed self-fertile species to survive when the competition started with the lower densities; contrary to the survival of sexual species when the competition started with the higher densities.

Molecular and morphological delimitation of Australian Triops species (Crustacea: Branchiopoda: Notostraca)—large diversity and little morphological differentiation

Australia has a very rich and diverse large branchiopod fauna with approximately 140 described or provisionally delimited species, but only one species of Triops, Triops australiensis (Spencer and Hall 1895), is currently recognized. Previous studies identified extensive genetic diversity within T. australiensis that suggested the presence of cryptic species. Herein, we employed an integrative approach to taxonomy to delimit putative species, integrating COI and EF1α sequence data and morphological data. Putative species were initially delimited based on COI by two computational approaches (GMYC and ABGD). The results were interpreted in the light of several species concepts, with particular emphasis on reproductive isolation. Twenty to 27 genetic lineages were delimited. Of these, up to 26 represent species following an evolutionary or phylogenetic species concept. Eighteen are biological species, though reproductive isolation could not be unambiguously established for allopatric species or species without known males. The level of co-occurrences was exceptionally high for Triops, with up to three syntopic and six sympatric species. Species delimitation was impeded by extensive overlap between intraspecific variability and interspecific variation in the genetic as well as morphological datasets. Without prior delimitation of putative species via COI, morphological delimitation would have been impossible. A potential explanation for the morphological variability is the retention of ancestral polymorphisms over long periods of time and across multiple speciation events without subsequent differentiation.

Self-Fertilization and the Role of Males in Populations of Tadpole Shrimp (Branchiopoda: Notostraca: Triops).

Self-fertilization has both negative and positive fitness effects on species evolution. Selfing can increase inbreeding depression, thereby decreasing genetic diversity. In contrast, self-fertilization can preserve beneficial gene combinations and facilitate colonization success. Within the class of crustaceans Branchiopoda, selfing is a primary reproductive mode. Some species of Triops, in the family Notostraca, are a few of the animal species thought to have a mixed mating system between hermaphrodites and males termed androdioecy. The objective of this study is to validate the reproductive mode utilized by Triops newberryi in southern New Mexico by the use of progeny arrays and population simulations. Individuals were reared in the lab from dried soil collected from temporary ponds inhabited by T. newberryi The adults reared and the encysted embryos contained within their brood pouches were genotyped using 7 T. newberryi specific microsatellite markers to determine the relatedness between parent and offspring. Overall microsatellite diversity was low with few heterozygous individuals and limited polymorphisms. Simulated populations and allele segregation analysis suggest hermaphroditism is the primary reproductive mode for T. newberryi In addition, based on the offspring's alleles, there was no direct evidence that a male (ovisacless) T. newberryi outcrossed with a female. Population simulations further suggest that the rate of successful outcrossing events must be low and could explain why outcrossing was not observed in the laboratory rearing trials.

The large branchiopod, Triops cancriformis simplex (Gihi, 1921) (Crustacea: Notostraca) in temporary rain pools, recorded for the first time in Taif-KSA

The present species Triops cancriformis simplex was not recorded in Taif and Kingdom of Saudi Arabia before. The present work is a confirmatory study to indicate the identity of this species in the ephemeral freshwater bodies in Taif, KSA and to ascertain that, if there is any other Triops species in the same area or not. Four sites were investigated in Taif governorate; Wadi Al-Arj, Saysed, Jabajeb pool and Ghadeer Al-Banat. The animal was not recorded in the first two sites all the year. The specimens were collected from Jabajeb pool on Al-Hada road and Ghadeer Al-Banat at Al-Roddaf region. No specimens were found in these sites during winter and autumn but they start in late spring and summer. One hundred and seven specimens were collected from both sites in this period. The animal was morphologically described to be distinguished from the other large branchiopods species. The supra-anal plate is absent, the dorsal organ of the present species is rounded, second maxilla is present, no supernumerary spines on apodous rings, the carina of T. c. simplex is smooth without spines in front of its terminal spine and presence of small spines on the furca.

The complete mitogenome of the Australian tadpole shrimp Triops australiensis (Spencer & Hall, 1895) (Crustacea: Branchiopoda: Notostraca)

The mitochondrial genome sequence of the Australian tadpole shrimp, Triops australiensis is presented (GenBank Accession Number: NC_024439) and compared with other Triops species. Triops australiensis has a mitochondrial genome of 15,125 base pairs consisting of 13 protein-coding genes, 2 ribosomal subunit genes, 22 transfer RNAs, and a non-coding AT-rich region. The T. australiensis mitogenome is composed of 36.4% A, 16.1% C, 12.3% G and 35.1% T. The mitogenome gene order conforms to the primitive arrangement for Branchiopod crustaceans, which is also conserved within the Pancrustacean.

Discriminating between the effects of founding events and reproductive mode on the genetic structure of Triops populations (Branchiopoda: Notostraca).

Crustaceans that initially colonize a freshwater temporary pond can strongly bias the subsequent genetic composition of the population, causing nearby populations to be genetically distinct. In addition, these crustaceans have various reproductive modes that can influence genetic differentiation and diversity within and between populations. We report on two species of tadpole shrimp, Triops newberryi and Triops longicaudatus “short”, with different reproductive modes. Reproduction in the tadpole shrimp can occur clonally (parthenogenesis), with self fertilization (hermaphroditism), or through outcrossing of hermaphrodites with males (androdioecy). For all these reproductive modes, population genetic theory predicts decreased genetic diversity and increased population differentiation. Here we use mitochondrial control region (mtCR) sequences and nuclear microsatellite loci to determine if the difference in reproductive mode affects the high genetic structure typical of persistent founder effects. Previous authors indicated that T. newberryi is androdioecious because populations are composed of hermaphrodites and males, and T. longicaudatus “short” is hermaphroditic or parthenogenetic because males are absent. In our data, T. newberryi and T. longicaudatus “short” populations were highly structured genetically over short geographic distances for mtCR sequences and microsatellite loci (T. newberryi: ΦST = 0.644, F ST = 0.252, respectively; T. l. “short”: invariant mtCR sequences, F ST = 0.600). Differences between the two Triops species in a number of diversity measures were generally consistent with expectations from population genetic theory regarding reproductive mode; however, three of four comparisons were not statistically significant. We conclude the high genetic differentiation between populations is likely due to founder effects and results suggest both species are composed of selfing hermaphrodites with some level of outcrossing; the presence of males in T. newberryi does not appreciably reduce inbreeding. We cannot exclude the possibility that males in T. newberryi are non-reproductive individuals and the two species have the same mating system.

Trehalose and Vitreous States: Desiccation Tolerance of Dormant Stages of the CrustaceansTriopsandDaphnia

Several aquatic organisms are able to withstand extreme desiccation in at least one of their life stages. This is commonly known as "anhydrobiosis." It was often thought that to tolerate such a desiccated state required high amounts of compatible solutes such as the nonreducing disaccharide trehalose, which protects cellular structures by water replacement and glass formation. Trehalose levels of dormant eggs and cysts of five freshwater crustaceans (Daphnia magna, Daphnia pulex, Triops longicaudatus, Triops cancriformis, and Triops australiensis) were observed in different states of hydration and dehydration. Although trehalose was detected in all species, the concentration was under 0.5% of the dry weight (0.05 μg/μg protein), and no change between the different states was observed. Differential scanning calorimetry (DSC) measurements indicated that dried cysts of all Triops species were in a glassy state, supporting the vitrification hypothesis. No indication for a vitreous state was found in dried resting eggs of Daphnia.

Co-occurrence of two tadpole shrimp, Triops cf. australiensis (Branchiopoda:Notostraca), lineages in middle Paroo, north-western New South Wales, with the first record of Triops hermaphrodites for the Australian continent

The only species of the genus Triops in Australia, T. australiensis, is found to reproduce by gonochorism. Morphological and reproductive data and molecular analyses of fragments of mitochondrial genes 12S rRNA (12S) and cytochrome oxidase I (COI) indicate that a Triops population from the middle Paroo in north-western New South Wales is composed of two different entities, Triops cf. australiensis lineage A, and T. cf. australiensis lineage B. Gonad histology in individuals with ovisacs of lineage A revealed no evidence of testicular tissue; however, large testicular lobes were found in individuals with ovisacs of lineage B, indicating that they were anatomically hermaphrodites. This is the first record of Australian hermaphroditic Triops. For each lineage, a single haplotype of each gene was obtained. Molecular genetic distance and phylogenetic analyses confirmed the closer relationship and monophyly of the two lineages with T. australiensis (GenBank) when compared with Triops species from other continents. COI haplotypes of lineage A and lineage B differed by 7.7% from each other and differed by 10.2% and 9.6% from a published T. australiensis sequence, respectively. The 12S haplotypes of lineage A and lineage B differed by 3.3% from each other and differed by 2% and 2.5% from a published T. australiensis sequence, respectively. Our results suggest that the two co-occurring Triops lineages probably represent two species that are distinct from T. australiensis.

A NEW LOCATION OF TADPOLE SHRIMP (TRIOPS CANCRIFORMIS L.) IN LITHUANIA

Tadpole shrimps (Triops cancriformis L.) were registered on 10 August 2007 and during the repeated observation on 8 September 2007 (Fig. 1). Only two habitats of this species of triops are known to exist in Lithuania (Raðomavieius 2007). To date no records of T. cancriformis have been reported from the northern part of Lithuania (Raðomavieius 2007). The habitat of these rare crustaceans (Red Data Book of Lithuania category 2 (V), MoE 2007) has formed in quite an unexpected place – at the sodden edge of a rape field situated at a distance of 5.5 m from the carriageway side (Fig. 2). From the viewpoint of administrative division, the habitat of Triops cancriformis L. is in Pasvalys region, on the left side of the No. 150 motorway (Ðiauliai–Pakruojis–Pasvalys), 59.7 km from Ðiauliai. The nearest settlement Meðkalaukis lies 3.70 km south-west of the location. Coordinates of the location are 24°16'11.2'' east longitude and 56°01'53.0'' north latitude (error – 6 m). In the north of the habitat there is a 150 m wide tract of a rape field with a mediumaged mixed forest behind it. Approximately 50 m east of the habitat, there is a mature deciduous forest and a wheat field (Triticum sp. L.) to the south of it (on the other side of the above mentioned motorway). Suitable conditions for the development of T. cancriformis in the rape field formed for several reasons, water accumulation being of the primary importance. The location is situated in the lowland of central Lithuania, in a moderate descent of the flat landscape relief, just 41 m above sea level. However, the presumption that the habitat is supplied with groundwater has to be ruled out, for 45 m north-east of the site there is a ~10 m wide and ~5 m deep drain ditch which drains underground water. The only source of moisture for this temporary water body is precipitation. Thus, the second prerequisite for the formation of the T. cancriformis habitat comes out. It is specificity of soil. According to Miliauskas and Nedzinskas (2000), soils in these areas are periodically dry. Hence, soil moisture regime is mainly preconditioned by the amount of precipitation. Surface layers of carbonate-rich morenic clay loam and clay have formed a natural hydrological barrier (Galvydyte 2000). In 2007 the amount of precipitation was sufficient for the formation of a perennial puddle and develoment of T. cancriformis eggs. The fact that a number of water plants, plants of damp sites and algae managed to root and even to flourish there – some of them reaching generative and senile stages, proves that the terrain has been sodden for a long period of time (Vilkonis 2001). Among such plants the following should be mentioned: great reedmace (Typha latifolia L.), common reed-grass (Phragmites australis (Cav.) Trin. Ex Steud.), marsh woundwort (Stachys palustris L.), murr marigold (Bidens tripartita L.), marsh cranesbill (Geranium palustre L.), common water-plantain (Alisma plantagoaquatica L.), common mudwort (Limosella aquatica L.), muskgrasses (Chara sp.) (Gudþinskas 1999; Vilkonis 2001). The following water animals managed to inhabit the temporary water reservoir: pond scatter (Gerris sp.), great diving beetle (Dytiscus marginalis L.), and a lot of fire-bellied toads (Bombina bombina L.).

Molecular characterization of the tadpole shrimp Triops (Branchiopoda: Notostraca) from the Baja California Peninsula, México: New insights on species diversity and phylogeny of the genus

Using sequence analyses of fragments of the small and large subunits of mitochondrial genes 12S and 16S rRNA, we studied the molecular identity of five Triops populations from the Baja California Peninsula, Mexico. Additionally, we explored the phylogeny of the genus by comparing with sequence data from gonochoric T. longicaudatus (Zacatecas, Mexico), commercial Triops kit (U.S.A.), T. ‘granarius’ (Japan), T. cancriformis (Austria), T. australiensis (Australia) and Lepidurus lemmoni (U.S.A.). The 16S fragment was not useful to discriminate the American Triops forms because their sequences were more than 99% similar. Molecular and phylogenetic analyses using the 12S gene fragments, in agreement with previous allozyme studies, indicate that the nominal (morphological) species T. longicaudatus is a mixture of several species such that, of the seven Triops American populations studied, six phylogenetic species can be identified and two morphologically and reproductively highly divergent forms can be grouped into a single monophyletic clade. The molecular data, rather than supporting our previous proposal that the phylogenetic relationships of Triops species could be deduced by similarities in the number of total and legless rings, suggest that T. cancriformis may represent an independent group from the rest of the species in that genus. In spite of detectable differences among American populations, our analyses indicate these represent a single monophyletic group when compared to Triops from outside of the New World.

Ultrastructure of the Tertiary Envelope of the Cyst of the Tadpole Shrimp Triops longicaudatus (Branchiopoda; Notostraca)

Abstract Tadpole shrimp inhabit temporary freshwater pools and ponds where their occurrence is largely regulated by rainfall events and water temperature. When dry basins are flooded, cysts of Triops imbibe water and hatch to produce rapidly growing, carapaced larvae. While previous studies show anostracan (fairy shrimp) cyst-surface morphology often species specific, few studies illustrate shell ultrastructure of Triops and none has considered T. longicaudatus. Here we examine the shell of T. longicaudatus (Notostraca) and compare its fine structure to other species of Triops and to that of Artemiafranciscana(Anostraca), which we previously studied. Cysts, produced in culture from Utah broodstock, were purchased from Triops, Inc., 1924 Creighton Rd., Pensacola, FL 32504. Thin sections of cysts were prepared for transmission electron microscopy (TEM) as previously described (Fig. 1). Cysts were also examined with scanning electron microscopy (SEM), dry, whole or fractured (Figs. 2,3), or after imbibition and/or hatching in oxygen saturated, double-distilled water, at 25 ° C.

Morphological stasis and phylogenetic relationships in Tadpole shrimps,Triops(Crustacea: Notostraca)

The tadpole shrimp (Triops) is a well-known ‘living fossil’ whose fundamental morphology has been unchanged for over 170 million years. Thus, tadpole shrimps are suitable subjects for the study of morphological stasis. We were able to obtain samples of three species ofTriops(T. granarius, T. longicaudatusandT. cancriformis) from four regions in Japan. Taxonomic species were identified by diagnostic morphology. We inferred phylogenetic relationships between individual samples using mitochondrial 16S rRNA. Carapace shapes were compared among populations using shape coordinate methods. The phylogeny inferred from mtDNA shows thatT. granariusis phylogenetically more similar toT. longicaudatusthan toT. cancriformis. mtDNA sequences did not differ among the populations ofT. granarius. However, there were two distinct phylogenetic species withinT. longicaudatus. In spite of the similarity in fundamental morphological characteristics amongTriopsspecies, mtDNA sequences ofTriopsshowed marked differences among the four phylogenetic species. Among the populations ofT. granarius, the carapace shape of the Fukuoka population was significantly different from those of other populations ofT. granarius. The carapace shape of the Kagawa population ofT. longicaudatuswas more similar to those of the Shizuoka and Kagawa populations ofT. granarius.The shape of the carapace ofT. cancriformiswas significantly different from those ofT. granariusandT. longicaudatus.Thus, taxonomic species, phylogenetic species and populations with similar carapace morphology did not correspond with each other. The present results indicate that most of the morphological change did not occur at the time of speciation (lineage separation) and that morphological stases are important evolutionary patterns, but they are not species-level properties.

Morphological stasis and phylogenetic relationships in Tadpole shrimps, Triops (Crustacea: Notostraca)

The tadpole shrimp (Triops) is a well-known ‘living fossil’ whose fundamental morphology has been unchanged for over 170 million years. Thus, tadpole shrimps are suitable subjects for the study of morphological stasis. We were able to obtain samples of three species ofTriops(T. granarius, T. longicaudatusandT. cancriformis) from four regions in Japan. Taxonomic species were identified by diagnostic morphology. We inferred phylogenetic relationships between individual samples using mitochondrial 16S rRNA. Carapace shapes were compared among populations using shape coordinate methods. The phylogeny inferred from mtDNA shows thatT. granariusis phylogenetically more similar toT. longicaudatusthan toT. cancriformis. mtDNA sequences did not differ among the populations ofT. granarius. However, there were two distinct phylogenetic species withinT. longicaudatus. In spite of the similarity in fundamental morphological characteristics amongTriopsspecies, mtDNA sequences ofTriopsshowed marked differences among the four phylogenetic species. Among the populations ofT. granarius, the carapace shape of the Fukuoka population was significantly different from those of other populations ofT. granarius. The carapace shape of the Kagawa population ofT. longicaudatuswas more similar to those of the Shizuoka and Kagawa populations ofT. granarius.The shape of the carapace ofT. cancriformiswas significantly different from those ofT. granariusandT. longicaudatus.Thus, taxonomic species, phylogenetic species and populations with similar carapace morphology did not correspond with each other. The present results indicate that most of the morphological change did not occur at the time of speciation (lineage separation) and that morphological stases are important evolutionary patterns, but they are not species-level properties.

Comparison of the hatching process of the tadpole shrimps Triops cancriformis and Lepidurus apus lubbocki (Notostraca) and its relation to their distribution in rain-pools in Israel

Rain-pool habitats are gradually disappearing in Israel as a result of agricultural and urban development. Present and past records of notostracan distribution here reveal a difference in the occurrence of Triops cancriformis and Lepidurus apus lubbocki, the former rather rare, and support the suggestion that species of Triops are more thermophilic than Lepidurus, with optimal hatching temperature 8–12°C higher. The limited distribution of T. cancriformis in Israel may be partly attributed to sub-optimal temperatures (<20°C) in early winter.

Effect of Light on the Hatching of Eggs in Triops granarius (Notostraca: Triopsidae)

A tadpole shrimp, Triops granarius appears in paddy fields a few days after the water submergence and ploughing. The Triops species have many similarities in their ecological characteristics with those of the annual plant weeds in paddy field, such as the life cycle in an annually disturbed habitat, the wide range of food or nutritional requirement, the high persistency of eggs or seeds in drought, the rapid development and maturation, the great overlapping of growing and reproductive stages, the high reproductive power, and the self compatibility in reproduction. As some annual weed species in paddy fields are light-sensitive in their seed germination, T. granarius eggs were also examined in this respect and it was found that the light condition has significant effect on their egg hatching.The eggs, collected from a paddy field with soils at Ibaraki City, Osaka (Experiments 1 and 2), and oviposited in the laboratory (Experiment 3), were submerged in water and the rate of egg hatching was examined in the darkness and under the various day lengths of illumination by the fluorescent lamp (Toshiba white 20W NL, 40 cm apart) at 20°C. A high percentage of eggs hatched rapidly under the light condition between 8 and 24 hrL. The egg hatching was significantly prevented under the shorter day length of less than 4 hrL as well as in the darkness.

Some aspects of ionic regulation in the tadpole shrimp Triops longicaudatus

1. 1. In deionized water and in the presence of a natural food source Trips longicaudatus rapidly lost hemolymph sodium and chloride, whereas calcium and magnesium remained little changed. Mean and maximum survival times were 10·2 adn 15 hr, respectively. In natural pond water hemolymph sodium and chloride were maintained agaist a high concentration gradient. 2. 2. These results on the American species, T. longicaudatus, are distinctly dissimilar to those obtained by Parry (1961) on the Eurasian species, T. cancriformis. Apparently these two species of Triops depend on different ionic regulatory mechanisms to maintain their hyperosmoticity.