The role of nutrition in regulation of predator-induced crests of Daphnia carinata

1. The effects of food level on the development of crests by Daphnia carinata in the presence of Anisops grains (Notonectidae) were investigated in two laboratory experiments and in the field. 2. In the laboratory experiments D. carinata were grown at different food levels either in the presence of A. gratus or in predator-free water. In the field study the effects of six variables (temperature, food level, density of Daphnia, density of Anisops, brood size and maternal nutrition) on development of crests in the next generation of Daphnia were investigated over a 12-month period at a single field site. 3. In the laboratory food level had a small but significant effect on relative crest height of D. carinata. In the presence of notonectid predators the rate of crest growth relative to body length was higher than for control D. carinata only during embryogenesis and/or the first two neonatal instars. Beyond this age the rate of crest growth was similar in both morphs. 4. In the field study, crest height was negatively correlated with density of Daphnia and positively correlated with density of Anisops and maternal nutrition (maternal nutrition was defined as the somatic weight of a female with a body length of 2.5 mm, calculated from the length-dry weight regression based on a sample of the population). However, these data were biased by a short period when no Anisops were present in the pond. When these data were excluded, the only factor which significantly correlated with crest height was maternal nutrition. This finding indicated that relative crest height of the next generation was determined primarily during embryogenesis on the basis of maternal feeding success and possibly on food availability during the first two instars.     

Reproduction and the energy cost of defense in a Batesian mimicry complex

Summary The amount of energy invested in reproduction and in defense was examined in a Batesian mimicry complex consisting of the modelEleodes obscura and the mimicStenomorpha marginata (both Coleoptera: Tenebrionidae). Models live up to 4 y as adults while mimic adults live only 3 mo. The energy content of the eggs of the model and mimic was determined by microbomb calorimetry. The energy content of the defensive secretions produced by the model was determined by computational chemistry and MNDO computer programming. Contrary to the predictions of some life-history theory, the long-lived model annually produces many small eggs each of low energetic content, while the short-lived mimic annually produces fewer, larger eggs each of high energetic content. However, in terms of total energy, the long-lived model has an annual investment in reproduction equal to that of the short-lived mimic. During the 3 mo of co-ocurrence of models and mimics within a year, an average individual model's cost in using defensive secretions against potential predators is 12% of the amount of energy tied up in the eggs that it produces within the year. The annual cost of defense for the model is 18% of the energy contained in the mean number of eggs produced. When the energy allocated to eggs is added to that allocated to defense, the model has an annual investiment that is greater than the annual investment in reproduction by the mimic. Although the energy invested in defense by the model is small relative to the energy invested in egg production, it buys the model considerable protection from predation. Nevertheless, the cost of defense does not explain the deviations from the predictions of life-history theory.     

Polymorphism and Food Limitation in Three Daphnia carinata Populations

The annual cycles of Daphnia carinata in three fish-free ponds in southern New Zealand (Quarry, Taieri and Raupo Ponds) are described. Although breeding was continuous, the populations were frequently dominated by distinct cohorts and there was a sexual phase in spring. Growth rates, particularly in Quarry Pond, were often slow, and clutch sizes generally small. Egg and neonate sizes changed seasonally, but changes were not closely related to clutch sizes and body lengths. In Taieri Pond and Raupo Pond some D. carinata grew to 6.14 mm and carried clutches of up to 154 eggs. The potentially high fecundity associated with large size makes D. carinata a good coloniser, but our studies showed that it also survives well in poor food conditions by growing slowly and producing only 1–2 eggs per clutch. D. carinata showed marked seasonal polymorphism in tailspine length and development of head crests. In winter, crests were absent and tailspines were short and varied little with body length. Crests were first apparent, and tailspines began to elongate, in early spring, before the first appearance of a notonectid, Anisops wakefieldi. By midsummer, tailspines had increased markedly with body length but the slope of the relationship declined in autumn. Laboratory trials showed that both tailspine length and crest development increased in the presence of Anisops. These increases may be due to water-soluble factor(s) produced by Anisops.     

Comparative life history and parasitism of a new colour morph of the walnut aphid in California

1 The walnut aphid Chromaphis juglandicola is a yellow aphid. In 2003, however, a white colour morph was discovered in the Sacramento Valley of California. The colour dimorphism occurs between clone lines and, when white morphs are present, they occur in mixed colour morph colonies on the underside of walnut leaves. 2 Laboratory experiments were undertaken to evaluate the thermal requirements for development, adult longevity and progeny production of the two colour morphs. Host instar preference of Trioxys pallidus, a parasitoid responsible for the successful biological control of the walnut aphid in California, was examined separately for each colour morph, and host colour preference was investigated for the preferred instar. 3 No differences in thermal requirements for development, adult size or mean longevity were detected between yellow and white colour morphs. A small difference in early reproduction was detected: white colour morphs produced more progeny on each of the two first days of adult reproduction than yellow colour morphs. 4 Trioxys pallidus showed a slight preference for the fourth instar of the yellow morph over the second‐ and third‐, but equal preference for second, third and fourth instars of the white morph. When offered equal numbers of fourth instars of the two colour morphs, T. pallidus did not show any colour preference. 5 The differences in early aphid reproduction and host instar preference by T. pallidus were combined in a stage‐structured matrix model. Model analysis showed a greater potential for population growth of the white morph over the yellow morph, with early reproduction having a greater influence than host instar preference.     

Biology and life tables for the predaceous earwig,Doru taeniatum [Derm.: Forficulidae]

The biology of the earwig,Doru taeniatum (Dohrn), a reported predator ofSpodoptera frugiperda (J. E. Smith), was studied in the laboratory.D. taeniatum under laboratory conditions was easily reared, mated readily and females tended eggs and first instar nymphs.D. taeniatum developed through 4 nymphal instars and reached imago in approximately 30 days. It is an omnivore in the laboratory and capable of developing and reproduction on plant pollen alone, and on Lepidoptera eggs (Sitotroga carealella) (Olivier) alone. Immatures developed most rapidly and females were most fecund when fed either a combination ofS. cereallea eggs and pollen, orS. cerealella eggs alone. Approved as TA 20744 by Director, Texas Agricultural Experiment Station.     

Inducible defences in Daphnia: responses to two closely related predator species

The predator-induced responses of two species of Australian Daphnia, with contrasting distributions and life history patterns, to the kairomones of two species of Anisops predators, were measured. Daphnia longicephala produced a large crest and attained a larger size when exposed to both predators. D. carinata sl matured earlier than D. longicephala and did not produce a crest. Surprisingly, kairomones of both predators inhibited the production of ephippia in D. carinata sl. Anisops stali, the larger of the two predator species, induced a significantly larger crest size in D. longicephala, and larger brood size in both species compared with the smaller A. gratus, indicating a quantitative but not qualitative effect of predator species on inducible defences. Received: 18 August 1999 / Accepted: 14 April 2000     

Characteristics of six cladocerans in relation to ecotoxicity testing

Investigation was made to compare some biological characteristics relevant to ecotoxicity testing among six cladoceran species, including Daphnia magna, Daphnia carinata, Daphnia pulex, Ceriodaphnia quadrangular, Bosmina longirostris, and Simocephalus vetulus. The results show that D. carinata had advantages over other cladoceran species for being used as a test organism, particularly for ecotoxicological assessment of aquatic environments in tropical and subtropical areas. D. carinata had similar body size and total number of offspring per female to D. magna. However, D. carinata was more sensitive to the reference toxicant and had much shorter reproduction cycle than D. magna. D. carinata had similarity to D. pulex, C. quadrangular, S. vetulus and B. longirostris in terms of sensitivity to the reference toxicant and length of reproduction cycle. However, D. carinata was much larger in size and produced much more offspring per female than any of D. pulex, C. quadrangular, S. vetulus and B. longirostris. Among the investigated cladocerans, only the neonates (2- and 4-day-old) of D. carinata exhibited phototaxis that was sufficiently remarkable. The low among-generation variation in phototaxis index (I_p) of D. carinata and the close relationship between I_p and the concentration of the ISO standard toxicant (K₂Cr₂O₇) appears to suggest that I_p can be used as an excellent test endpoint for ecotoxicity testing.     

Analysis and comparison of a set of expressed sequence tags of the parthenogenetic water flea <Emphasis Type="Italic">Daphnia carinata</Emphasis>

The water flea Daphnia carinata (D. carinata) reproduces both sexually and parthenogenetically, yet little is known about the genes involved in these processes. To further clarify the reproductive biology of Daphnia and elucidate their unique mechanism of reproductive transformation, we have generated and characterized an expressed sequence tag (EST) data set from D. carinata. A set of 1,495 clusters were generated from sequencing 3,072 randomly chosen clones from a parthenogenetic, juvenile water flea cDNA library. The nucleic acid and deduced amino acid sequences were compared with known GenBank sequences. Functional annotation found that 959 clusters showed significant homology with known genes involved in a broad range of activities, including metabolism, translation, development and reproduction, as well as genes involved in sensing environmental factors. We speculate that genes involved in development and reproduction, along with genes that allow the organism to sense changes in the environment, play important roles in the process of parthenogenetic reproduction and could be markers of the early steps of sexual differentiation. Additionally, 86% of the D. Carinata unique sequences could be stringently mapped to the D. pulex genome, of which 125 mapped to intergenic and intronic regions on the current assembly. Our results provide practical insight into crustacean reproductive biology, in addition to establishing a new animal model for reproductive and developmental biology. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Xiaoqian Xu and Shuhui Song contributed equally to this work. Nucleotide sequence data reported are available in the GenBank databases under the accession numbers: GD269049−GD272045.     

Genetic heterogeneity and adaptive strategies of the ladybird <Emphasis Type="Italic">Harmonia axyridis</Emphasis> Pall. (Coleoptera,Coccinellidae)

Analysis of microstatial variability of the phenetic composition of populations of the ladybird Harmonia axyridis Pall. is performed in Baikal region of the Primorskii krai. It is revealed that the phenetic composition of the Baikal H. axyridis population is significantly more labile than that in Primorie. In individual micropopulations the phenoimage is clarified due to a local increase in frequency of the morph succinea. Individual and family testing of H. axyridis individuals from the Primorie and Baikal populations is performed by the quantitative parameters affecting the adaptability-the larval and pupal survival at starvation, critical weight of pupation, the imago weight and fertility. At starvation of larvae of the IV instars for 3–4 days, only the morph succinea individuals from the Primorie population reached the imago stage. The oviposition value in these imagoes corresponded to the mean for the population (31.2 ± 1.06 eggs). The obtained result confirms the earlier expressed suggestion that the morph succinea is distinguished by a high ecologic plasticity. This morph is able to survive under conditions of the food deficit and is characterized by the intermediate, but stable reproduction.     

Does superparasitism improve host suitability for parasitoid development? A case study in the Microplitis rufiventris-Spodoptera littoralis system

Superparasitism refers to the oviposition behavior of parasitoid females who lay their eggs in an already parasitized host. Recent studies have shown that allocation of additional eggs to an already parasitized host may be beneficial under certain conditions. In the present work, mortality of Microplitis rufiventris wasps was significantly influenced by both host instar of Spodoptera littoralis larvae at parasitism and level of parasitism. In single parasitization, all host instars (first through sixth) were not equally suitable. Percentage of emergence success of wasp larvae was very high in parasitized first through third (highly suitable hosts), fell to 60% in the fourth instar (moderate suitable) and sharply decreased in the penultimate (5th) instars (marginally suitable). Singly parasitized sixth (last) instar hosts produced no wasp larvae (entirely unsuitable), pupated and eclosed to apparently normal adult moths. The scenario was different under superparasitism, whereas supernumerary individuals in the highly suitable hosts were almost always killed as first instars, superparasitization in unsuitable hosts (4th through 6th) had significant increase in number of emergence success of wasp larvae. Also, significantly greater number of parasitoid larvae successfully developed in unsuitable hosts containing three wasp eggs than counterparts containing two wasp eggs. Moreover, the development of surplus wasp larvae was siblicidal in earlier instars and nonsiblicidal gregarious one in the penultimate and last “sixth” instars. It is suggested that the optimal way for M. rufiventris to deal with high quality hosts (early instars) is to lay a single egg, while the optimal way to deal with low quality hosts (late instars) might be to superparasitize these hosts.     

A study on the effects of food quantity and quality on glutathione S-transferase (GST) activity and growth rate parameters of <Emphasis Type="Italic">Daphnia</Emphasis><Emphasis Type="Italic">carinata</Emphasis> varying in age

The adverse influences of insufficient food and toxins on Daphnia carinata’s body growth, reproduction and tolerance were investigated in the laboratory. Different concentrations of Scendesmus obliquus and a mixture of S. obliquus and microcystin (MC)-containing Microcystis aeruginosa PCC7820 were used to feed D. carinata. Glutathione S-transferase (GST) activity towards five chemical compounds (substrates) was measured and used as an indicator of their tolerance. Body growth rate and clutch size of D. carinata decreased with declined concentration or decreased proportion of S. obliquus in the diet. GST activity decreased with ageing in D. carinata. However, GST activity to several chemical compounds increased when food quantity or food quality decreased. Adult D. carinata had a lower GST activity towards p-nitrophenenzyl chloride (PNBC) than juveniles and exhibited a sharp decline (P < 0.001) in GST activity towards PNBC as the animals aged. It is suggested that the age-specific decrease in GST activity is a possible mechanism for the high morality of adult D. carinata in the summer of eutrophic lakes.     

Reproductive allocation in Daphnia exposed to toxic cyanobacteria

We investigated experimentally how resources were allocated to reproduction in Daphnia pulex and Daphnia longispina when varying levels of toxic Microcystis were added to higher quality food. We used multiple regression models to estimate mean offspring size and clutch size in relation to maternal size and clutch number, and analysed effects of treatments on residuals from the models. We also measured variation in per offspring investment. At a high cyanobacterial level, D.pulex was virtually unable to reproduce. At a lower level, D.pulex produced small clutches. However, the regression model residuals indicated that the presence of cyanobacteria increased the portion of available resources allocated to reproduction. The observed allocation may be a means to maximize reproduction under diminished longevity. Effects on mean offspring size were marginal in D.pulex but variation in per offspring investment sometimes decreased in cyanobacterial exposures. Daphnia longispina was affected by a higher cyanobacterial level only, where offspring sized was reduced. Deviations from the regression model indicated that effects on maternal size alone do not explain this effect. Clutch size residuals and per offspring investment were unaffected by treatments in D.longispina. The observed responses differ from theoretical models on reproductive allocation under food imitation.      

Resource use for reproduction depends on spring arrival time and wintering area in an arctic breeding shorebird

Resources for egg production may come from body reserves stored before breeding (“capital breeders”) or from food acquired at the breeding site (“income breeders”). Arctic migrants were long thought to be capital breeders, because they often arrive at a time when local food availability is still limited. However, later evidence suggested that arctic breeding shorebirds are primarily income breeders, or that they use a mixed strategy depending on laying date. We explored the relationship between laying date and resource use for reproduction in the pectoral sandpiper Calidris melanotos breeding in the Alaskan arctic by contrasting carbon isotope (δ¹³C) values of the local diet and of maternal plasma, cellular blood, feather and claw with those of the eggs produced. Our results revealed that early breeding females utilize resources for egg production that were acquired recently at staging areas, whereas later breeding females mostly relied on nutrients derived from local food sources. These findings suggest that the resource allocation strategy used for reproduction differs among females, and varies depending on the timing of arrival and the start of reproduction. The arrival date at the breeding ground and laying date may critically depend on non‐breeding season events such as winter habitat choice, staging areas or migration routes. By comparing maternal feather δ¹³C, claw δ¹³C and feather δD, we examined whether non‐breeding season events influenced the use of resources for egg production through variation in capture date or clutch initiation date. Female pectoral sandpipers originating from moulting areas characterized by higher (more positive) δD signatures were caught earlier and started laying earlier, and they used stored resources for reproduction. Using regional maps of δD values for precipitation in the wintering sites in South America, we compared the spatial variation in the observed feather δD signatures. This analysis indicated that female pectoral sandpipers with higher δD signatures, presumably coming from more north‐easterly wintering sites in southern America, started laying earlier and used mostly stored resources for egg production, compared to females that wintered (or at least moulted) further south. Our results thus show that winter moulting habitat is linked to breeding resource allocation strategy in this high‐arctic breeding shorebird.     

Resource allocation in a simultaneously hermaphroditic slug with phally polymorphism

The theory of resource allocation assumes that a resource not allocated to one function may be reallocated to another. Thus, in hermaphroditic species, an individual that suppresses the use of one sex function may free resources for the other sex function. We determined the relative importance of male copulatory organs in terms of their fraction of the total dry body weight and tested whether in the pulmonate land slug Deroceras laeve (Müller), individuals that lack the male copulatory organs (aphallics) reallocate this resource towards the female structures and/or towards life-history traits. To this end, we raised 13 families under uniparental reproduction and compared growth, length of the juvenile period, number of eggs produced, percentage of hatched eggs and hatching time among a- and euphallics. We also measured the reproductive and sex allocation of all individuals. Six out of 13 families contained no euphallic individuals. In the other seven families, the proportion of euphallic individuals ranged from 0.13 to 0.43. There was an enormous variation in life-history traits and reproductive and sex allocation among individuals, even among individuals of the same family. Allocation to the male function was very low in euphallic slugs (i.e. 1.35% of the total body dry mass and 12.33% of the total reproductive dry mass). Our results did not reveal a reallocation from the lost male function towards the female function, nor towards one of the life-history traits. Finally, we propose a scenario that could explain the maintenance of phally polymorphism in D. laeve.     

Diet, fecundity and egg size in some polyphagous predatory carabid beetles

Egg size was measured at different rates of egg laying in three polyphagous carabid species, known to be useful predators of cereal aphids; the small Bembidion lampros Herbst and the medium-sized Pterostichus cupreus L. and P. melanarius Illiger. Variations in fecundity, as well as the ability of the medium-sized species to also build up fat reserves, were obtained when beetles were subjected to different dietary regimes consisting of aphids, or foods with a lower or higher protein content. Egg size was found to be dependent on the rate of egg laying within a species. A diet of cereal aphids appeared to be adequate for egg production in these polyphagous carabids, but female P. cupreus were unable to build up fat reserves when they ingested aphids contaminated with the aphicide pirimicarb. Beetles were able to devote resources to more and larger eggs (B. lampros), or to larger eggs and/or fat reserves (P. melanarius/P. cupreus) when given access to a carbohydrate-rich food with low protein content. The highest rate of egg laying was obtained when female P. cupreus and P. melanarius were given a more varied diet at frequent intervals; including regular shifts between unsprayed aphids, carbohydrate-rich food and protein-rich maggots. Within the varied diet treatment a negative relationship was obtained between egg size and egg number among similar-sized individuals of P. cupreus and P. melanarius; females producing the largest number also laid the smallest eggs. Egg size affected larval survival, since first instars hatching from large eggs were found to survive longer than those hatching from small eggs. The influence of differences in food intake on reproduction, maintenance metabolism, and survival of fieldinhabiting carabids is discussed.     

The demographic costs of nectar production in the desert perennial Prosopis glandulosa (Mimosoideae): a modular approach

Nectar production in angiosperms is considered to represent a reproductive cost, and has been associated with a decrease in fruit set or an overall decrease in the energetic budget of the plant. Populations of Prosopis glandulosa var. torreyana (honey mesquite) are a suitable system to evaluate the demographic costs of nectar production, as populations are composed of a 1:1 proportion of nectarful to nectarless individuals. The study was carried out in a population of 404 individuals of Prosopis g1andulosa var. torreyana found in an area with differing water availability in the Southern Chihuahuan Desert. The possible costs of nectar production were assessed on 1212 shoots of the honey mesquite that were tagged in 1994 and followed until 1998. We used two methods of analysis to describe the effect of nectar production on modular population dynamics: matrix analysis and log-xlinear models. Water availability and the varying environmental conditions affected plant growth, but nectar production did not have an effect on the demographic parameters we measured. The values of λ did not differ between nectar morphs and the only important effects we detected were the year to year variation in precipitation and microclimate differences at each site. Furthermore, the elasticity of each demographic process (growth, fecundity, retrogression and stasis) between nectar morphs did not differ. The log-linear models suggested a similar pattern but could discriminate the importance of each factor (nectar morph, year and site) on module fate. We were not able to detect a demographic cost of nectar production in the honey mesquite. The absence of a demographic response could be due to the negligible cost of producing nectar for this species or that the resources allocated for growth are different from those allocated for reproduction. Our results suggest that the modular fates of mesquites are mainly determined by environmental factors. This revised version was published online in August 2006 with corrections to the Cover Date.     

Effect of the epizoic rotifer Brachionus rubens on the population growth of three cladoceran species

Using population densities and growth rates as criteria, we studied interactions between the epizoic rotifer Brachionus rubens and each of three cladoceran species differing in size and reproductive rates — Daphnia carinata, Moina macrocopa and Ceriodaphnia rigaudi. In all mixed — species experiments, B. rubens existed in both the epizoic mode, attached to the cladoceran host, and in the free-swimming mode. Rotifer population growth rates were significantly depressed in the presence of M. macrocopa, presumably as a consequence of exploitative and interference competition. The largest cladoceran, D. carinata probably did not suppress B. rubens, because the epizoic component of the rotifer population escaped from the deleterious effects of mechanical interference. Peak population numbers and initial population growth rates reached by all three cladocerans were lower in the presence of B. rubens, probably because of the adverse effects of the epizoic infestation, which was maximal on D. carinata and least on C. rigaudi. In mixed-species cultures of D. carinata and M. macrocopa, the presence of B. rubens helped D. carinata coexist with M. macrocopa, which otherwise would have suppressed the Daphnia.     

Impact of microphallid trematodes on the survivorship, growth, and reproduction of an isopod (Cyathura carinata)

Crustaceans are second intermediate hosts to several microphallid species (Trematoda). Some of these parasites are potentially pathogenic or manipulative. A laboratory experiment was performed to assess the impact of microphallids on the survival, growth and fecundity of Cyathura carinata, a protogynous hermaphroditic isopod, widespread within European estuaries. For nearly 12 weeks, experimental populations of infected and non-infected isopods were kept at 25 °C. C. carinata carrying microphallid cysts showed higher mortality rates than non-infected specimens and were not able to produce embryos. The reduced fecundity of infected isopods could be caused by parasite-induced castration and/or by mating failure due to behavioural modifications in one of the sexes. It might also be associated with lower growth rates and lower moulting frequencies, since infected C. carinata were significantly smaller than the non-infected after 9 weeks. This may imply a setback for the isopods to achieve sexual maturity (which may also affect the population sex ratio) and for females to lay their eggs in the marsupia. Regardless of the mechanisms involved, microphallids may have severe consequences for their host populations, through negative effects on survival, growth and fecundity. For species with direct development, such as C. carinata, parasite-induced reproduction failure may contribute to temporal fluctuations of abundance. Based on the present results, it is recommended to include parasites as an important factor influencing host populations from shallow-water ecosystems.     

The brine shrimp Artemia sp. as alternative prey for rearing the predatory bug Macrolophus caliginosus

The predatory bug Macrolophus caliginosus, which is widely used in greenhouse crops, is limited in its application by its high price. An important factor in the cost is the high price of Ephestia kuehniella eggs, the prey used in their mass rearing. In order to reduce their price, alternatives to moth eggs are currently being investigated. The brine shrimp Artemia sp. is produced in large quantities in saline lakes and is fed as live food source to the larvae of a variety of marine and freshwater organisms. In this study, we tested Artemia sp. as prey for rearing M. caliginosus from two strains. We evaluated developmental and reproduction parameters of the predator when fed nauplii, enriched nauplii with a fatty acid, dry cysts and hydrated cysts, and were compared with those obtained when the predator was fed with E. kuehniella eggs. Nauplii had a significant reduction in survivorship, a delay in development of nymphs and a low reproduction of adults. Nauplii enriched with docosahexaenoic acid (DHA, 22:6n − 3), a common practice for larviculture of some marine fish species, resulted toxic to M. caliginosus nymphs and survival was quite low. On the contrary, either dry or hydrated cysts from the two strains tested of the brine shrimp produced the same nymphal survivorship, nymphal development time and weight and fecundity of adults as those obtained with E. kuehniella eggs. Demographic parameters of the eighth generation of the predator reared with cysts of the two strains, either dry or hydrated, were as good as those of moth eggs. We concluded that Artemia sp. cysts were a good substitution prey for the mass rearing of M. caliginosus.     

The systematics of Australian Daphnia (Cladocera: Daphniidae). Electrophoretic analyses of the Daphnia carinata complex

Genetic variation at eleven allozyme loci was scored in approximately 1600 individual Daphnia. The samples included representatives of all nine taxa described from the D. carinata complex in Australia. Principal coordinates and cluster analyses revealed only three groups of genotypes within the complex, corresponding to the taxa D. cephalata, D. nivalis, and a conglomerate of all other taxa described from the complex, D. carinata (sensu lato). These results are consistent with recent multivariate morphological analyses of the complex.