Can Aurelia ( Cnidaria , Scyphozoa ) species be differentiated by comparing their scyphistomae and ephyrae ?

Debate exists regarding the number of species of the moon jellyfish (genus Aurelia), a common member of the planktonic community of the coastal shelf seas around the world. Three Aurelia congeners (A. aurita, A. labiata and A. limbata) are currently considered to exist but recent genetic analyses suggested that this is an oversimplification. We analyzed the morphological characteristics of scyphistomae, morphological characteristics of ephyrae and differences in the time span of the strobilation process of Aurelia congeners from 17, 7 and 6 different source populations, respectively, of known species. Morphological characteristics of scyphistomae were similar among the 17 populations but those of ephyrae, such as the shape and form of lappets, were effective discriminators in the 6 cases examined. We recommend identifying species based on differences in 1) the morphological characteristics of scyphistomae and ephyrae (and not only medusae), 2) the genetics of individuals, and 3) the geographical occurrence of the population. This study adds to the growing body of knowledge on scyphozoan scyphistomae and ephyrae, stages of the metagenic life cycle of scyphozoans that have received relatively little study compared to medusae.


Introduction
The perceived increased frequency of blooms of jellyfish in the world's ocean (Mills 2001;Purcell et al. 2007;Purcell 2012;Condon et al. 2012Condon et al. , 2013;;Lee et al. 2013) has intensified research efforts to understand the population dynamics of gelatinous plankton, with the majority of research having been conducted on the conspiculous medusoid life stages (Lucas et al. 2012).Medusae in the genus Aurelia Lamarck, 1816 have been reported to occur in the world's oceans from 70° N to 40° S where they are most commonly found along continental shelves or close to large islands (Mayer 1910;Kramp 1961;Russell 1970;Miyake et al. 2002;Schroth et al. 2002).Despite >100 years of research on Aurelia, the taxonomy of the genus is still unclear.The genus was originally described as 'Aurellia', which was changed to 'Aurelia' by Rees (1957).It had three species: A. aurita (Linné, 1758), the Pacific A. labiata Chamisso & Eysenhardt, 1820 and A. maldivensis Bigelow, 1904 from the Indian Ocean (Linné 1758; Chamisso & Eysenhardt 1820; Bigelow 1904).Debate existed on whether A. labiata and/or A. limbata Brandt, 1838 were valid members of the genus (Brandt 1838).Kramp (1961) described six different species of Aurelia, whereas Russell (1970) declared that only A. aurita and A. limbata were valid species.Gershwin (2001) provided a new description of A. labiata and reported it to be endemic to coastal areas of the Eastern Pacific.Although three species (A. aurita, A. labiata and A. limbata) are currently morphologically considered to comprise the genus (Wrobel & Mills 1998;Gershwin 2001;Miyake et al. 2002;Albert 2005;Widmer 2005), genetic analyses have suggested a more complex situation, including five cryptic species of Aurelia in the North Pacific and up to nine species worldwide (Dawson & Jacobs 2001;Dawson & Martin 2001;Schroth et al. 2002;Dawson 2003;Dawson et al. 2005).
The vast majority of studies using morphology to distinguish species of Aurelia has examined medusae, while only five studies (Uchida & Nagao 1963;Gershwin 2001;Straehler-Pohl 2009;Straehler-Pohl & Jarms 2010;Straehler-Pohl et al. 2011) have considered the morphology of scyphistomae and ephyrae.It is known that scyphistoma populations of scyphozoan jellyfish can be distinguished not only by using morphological features but also by analyzing asexual propagation as well as the timing and intermittency of strobilation (e.g., Condon 2001;Lucas 2001;Ma & Purcell 2005;Willcox et al. 2007;Adler & Jarms 2009;Straehler-Pohl 2009;Holst 2012a).Documenting the morphological characteristics of scyphistomae and ephyrae can not only (potentially) help clarify the taxonomic status of the genus Aurelia, but advancing our knowledge on the strobilation of Aurelia scyphistomae is also an important step towards understanding the bloom dynamics of members of this genus (Lucas 2001;Purcell 2007;Purcell et al. 2012).
We examined aspects of the morphology of scyphistomae and ephyrae and the strobilation dynamics of Aurelia populations collected from 17 locations around the world.Populations were from three species and we tested whether the same species assignments would be made using morphological measurements of scyphistomae and ephyrae.We also measured characteristics of strobilation (duration, number of ephyrae per scyphistoma), contrasting Pacific from Atlantic populations.We performed these comparisons not only to help clarify the taxonomic status of the genus but also to gain knowledge of basic features of scyphistomae and their reproductive dynamics in the laboratory.

Cultures
We compared the morphology of scyphistomae and ephyrae from populations of Aurelia collected at 17 locations in the North and Baltic Seas as well as the Atlantic and Pacific Oceans (Table 1).A priori species identifications were: populations 1 to 14 = A. aurita, populations 15 and 16 = A. labiata, and culture 17 = A. limbata.Species-level identifications were made a priori based on the morphology of medusae.Scyphistomae were kept in culture for more than 5 years.Scyphistomae and ephyrae were cultured using natural seawater at temperatures (SANYO incubators MIR 553 and MIR 253) and salinities (ATAGO S7Mill refractometer) that represented their local conditions (Table 1).
Scyphistomae were kept in 150-ml glass bowls in darkness and ephyrae were maintained in aerated, 500-ml flasks in daylight with the photoperiods occurring between May 5 th and September 23 rd 2009 in Hamburg, Germany, and room temperature (18 to 21°C).Based on previous work, we tried to induce strobilation by first decreasing the temperature and then raising it again to the initial temperature (Holst 2008, Holst 2012a).In 14 cultures (all except cultures 6 and 14 because strobilation was happening already on a regular basis) temperature was set from 15°C to 10°C and back to 15°C after 7 days.In culture 9 temperature was set from 23°C to 15°C and back to 23°C after 7 days.Three out of the seven cultures in which strobilation actually occurred were additionally treated with potassium iodine (1.5 ml KI in 100 ml seawater; Spangenberg 1967Spangenberg , 1968) because the short-term temperature decrease was not sufficient to induce strobilation (Table 1).Scyphistomae and ephyrae were fed Artemia salina (Linné, 1758) nauplii once a week and every other day, respectively.

Scyphistomae
Scyphistomae were transferred to petri dishes and a 30-min period was allowed for them to relax and fully re-expand.Afterwards, individuals were digitally photographed and morphometric measurements were made using computer image analysis (ColorView, Soft Imaging System GmbH).Various morphological features were documented (colour, shape, number of tentacles) as well as strobilation duration and ephyrae production (Fig. 1).We took morphometric measurements as mentioned above following the method developed by Straehler-Pohl (2009), Straehler-Pohl & Jarms (2010) and Straehler-Pohl et al. (2011).The morphometric measurements of scyphistomae were compared by using the following ratios (expressed in percent; abbreviations are explained in Ephyrae Ephyrae were measured within 24 h after detachment.They were collected from petri dishes using a pipette and transferred with a small amount of water to a glass slide with the manubrium facing up, where they where allowed to relax for 5-10 minutes.They were photographed and measured as described above for scyphistomae, including differences in rhopalial lappet-and gastric canal forms (Fig. 1D).We took morphometric measurements as mentioned above following the method developed by Straehler-Pohl (2009), Straehler-Pohl & Jarms (2010) and Straehler-Pohl et al. (2011).The morphometric measurements of ephyrae were compared by using the following ratios (expressed in percent; abbreviations are given in Table 2): LStL/TMLL; RLL/TMLL; CDD/TBD; ML/TBD; RhTI/TBD; AdD/TBD; CDD/AdD; ML/ AdD; TMLL/TBD; LStL/TBD; RLL/TBD (for original data please see Appendix B).
We explored statistically significant differences between the 17 populations using a post-hoc, stepwise Linear Discriminant Analysis (LDA) of the ratios based on the morphometric measurements (n = 6 individuals per population).Ephyra production and number of tentacles per scyphistoma (both n = 10) were evaluated using a Kruskal-Wallis test, followed by a Tukey's Honestly Significant Difference (HSD) post-hoc test.The significance level was set at alpha < 0.05.Statistical analyses were performed using the software R 2.15.2 (R Core Team 2012).

Scyphistomae
Scyphistomae of the 17 Aurelia populations were morphologically variable with respect to colour and shape of the calyx, the shape of the hypostome, and the number of tentacles (Table 3).We were able to distinguish four different calyx shapes (barrel, square, chalice, and cone) and four different hypostome shapes (convex, cap, cylindrical, and cone).The number of tentacles varied significantly among the populations (Kruskal-Wallis, H 16 = 67.3,p > 0.00001, n = 10).Pairwise comparison revealed that population 12 (West Atlantic) was significantly different from all others (HSD, p ≤ 0.05).In 10 of the 17 populations there was a consistent ranking of body proportion (CL > HL > StL) and stalks in populations 4-6 (Baltic Sea and East Atlantic), 9 (Mediterranean Sea), 13 (East Pacific), 15 (East Pacific) and 16 (East Pacific) were longer than the hypostome (CL > StL > HL; Fig. 2).There were significant differences between ratios of hypostome length, calyx length and stem length in relation to   Abbreviations: see Table 2.

Strobilation
Strobilation occurred in 7 of the 17 populations (Tables 1, 5).In cultures 1 (North Sea) and 16 (East Pacific), scyphistomae strobilated after a decrease in temperature.In cultures 2 (North Sea), 8 (North Sea / Skagerrak) and 9 (Mediterranean Sea), strobilation commenced after exposure to KI and a decrease in temperature.Cultures 6 (East Atlantic) and 14 (Pacific) were maintained at 15°C and strobilated several times.The strobilation process from the development of the first constriction until detachment of the last ephyra lasted 17 to 27 days.In six of seven cultures, at least ten scyphistomae strobilated.Only 1 scyphistoma strobilated in culture 1 (North Sea) and this culture was not included in further analyses concerning the strobilation process.Cultures from the Pacific Ocean (cultures 14 and 16) had much shorter strobilation times (17 to 18 days) compared to the four cultures from the East Atlantic Ocean / North Sea (cultures 1, 2, 6 and 8) and the Mediterranean (culture 9).In these six cultures (i.e., excluding culture 1), the mean (± standard deviation) number of ephyrae per strobila varied from 6.0 ± 0.9 to 21.1 ± 3.0 and was significantly different among cultures (Kruskal-Wallis H5 = 47.4,p < 0.001, n = 10).The number of ephyrae per strobila in the Pacific cultures was significantly lower than that of the remaining experimental cultures (HSD, p ≤ 0.05).

Ephyrae
The colour of the ephyrae ranged from translucent white to translucent pink to reddish brown (Table 5).The shape of the rhopalial lappets as well as the shapes of the rhopalial and velar canals differed between cultures (Table 5).Lappet proportions of the ephyrae exhibited differences.Culture 6 (East Atlantic) produced ephyrae showing ratios of rhopalial lappet (RLL) to lappet stem length (LStL) of almost 60% whereas others were about 50% (Fig. 3).A factorial ANOVA revealed significant differences of body proportions between all species (p < 0.001, Fig. 3, Table 4).2.

Linear Discriminant Analysis (LDA) Scyphistomae
The LDA based on ratios of morphometric measurements for scyphistomae did not identify any kind of separation among the 17 populations.All populations overlapped to varying degrees except for populations 2 (North Sea) and 16 (East Pacific; Fig. 4A).

Ephyrae
The LDA based on ratios of morphometric measurements for ephyrae suggested distinct classifications (Fig. 4B) with overlaps between populations 9 (Mediterranean Sea) and 14 (Pacific), 1 and 2 (North Sea), and a similar placement of populations 16 (East Pacific) and 9 (Mediterranean Sea).Ephyrae from populations 6 (East Atlantic) and 8 (North Sea / Skagerrak) were completely isolated from each other and from the other populations.

Discussion
Some morphological features of scyphistomae and ephyrae can be used to distinguish congeners while others cannot.Straehler-Pohl & Jarms (2010) reported that the relation between rhoparlar lappet length (RLL) and lappet stem length (LStL) and the development of the gastric system could be important distinguishing features of genera but not species.

A B
higher latitude scyphistomae produce white and translucent ephyrae and the degree of pigmentation increased with decreasing latitude.Moreover, ephyrae produced by Pacific scyphistomae differed in colour from those produced by scyphistomae collected from the Atlantic Ocean and Mediterranean Sea.Therefore, colour can be a useful trait if compared among individuals maintained using the same culture/feeding conditions.
The LDA we conducted using the morphometric data of ephyrae reveal a clear separation of the different populations (Fig. 4b), which again supports the genetically indicated existence of more than three species.The obvious partitioning clearly allows classifications to be made and these are discussed (below) in light of known differences among the 17 populations.
In scyphistomae the colour of the calyx was not a reliable characteristic to differentiate among species, since it was more or less the same in all scyphistomae.We expected differences similar to the ones found in ephyrae, due to the same feeding conditions (Straehler-Pohl 2009;Straehler-Pohl & Jarms 2010;Holst 2012b).The shape of the calyx was a suitable character for separating species (e.g., a cup-like calyx is characteristic of A. limbata [Straehler-Pohl 2009; this study]), although the morphology of the calyx and hypostome can vary and can be misinterpreted (Berrill 1949;Straehler-Pohl 2009;Straehler-Pohl et al. 2011).Moreover, biotic and abiotic factors such as food intake, light intensity, salinity and temperature may affect the shape and colour of these features (Spangenberg 1964;Willcox et al. 2007).The number of tentacles is an obvious morphological feature of scyphistomae which also displays interspecific variability, with A. aurita and A. labiata having 14 to 28 (Holst 2008;Straehler-Pohl 2009; this study) and 14 to 21 (Gershwin 2001;Widmer 2006; this study), respectively.Scyphistomae of population 12 had between 27 and 33 tentacles and, based on this morphology, may not belong to Aurelia.Finally, the length of the hypostome in relation to the stem has been reported to distinguish members of Scyphozoa (Semaeostomeae: CL > HL > StL, Cepheida: StL > CL > HL, Rhizostomida: CL > StL > HL; Straehler-Pohl 2009;Straehler-Pohl et al. 2011).In this study, StL was > HL in only 40% of the specimens; this relationship is not suitable for distinguishing Aurelia congeners.Furthermore, we demonstrate that morphometric measurements of scyphistomae cannot be used to distinguish among species within this genus (Fig. 4A).Lucas et al. (2012) provide a review of studies conducted on scyphistoma populations, including the species-specific triggers for strobilation.We were able to induce strobilation in 7 of the 17 populations using cues known to be effective for Aurelia species, including a decrease in temperature and, in some cases, the addition of KI (Berrill 1949;Spangenberg 1967;see Lucas et al. 2012).Spangenberg (1964) discovered that scyphistomae with large calices produce more ephyrae than small scyphistomae and still have enough energy to regenerate the residuum (Straehler-Pohl & Jarms 2005).Scyphistomae are often larger when grown at low salinities and low temperatures (Schroth et al. 2002;Willcox et al. 2007) and could therefore produce more ephyrae than smaller scyphistomae in warmer waters, possibly due to higher availability of tissue.Our results suggested longer durations of strobilation and greater numbers of ephyrae produced by scyphistomae collected from higher (colder) versus lower (warmer) latitudes despite scyphistomae being maintained at the same temperature in the laboratory, even though some populations needed the temperature drop and the consequent rise to start the strobilation process.
Naturally, the number of ephyrae produced by scyphistomae depends upon both endogenous and exogenous factors (Lucas et al. 2012) and the effect of the latter make it difficult to use this trait to differentiate species within one genus.

Classification
Historical reports and descriptions of species within Aurelia often used geographical distribution as a distinguishing characteristic.However, geographical distributions of many planktonic organisms have changed via transport in ballast waters and jellyfish introductions are commonly reported (Greenberg et al. 1996;Purcell et al. 2007).The potential for mixing of different (potentially cryptic) species within the same area demands that methods be found to reliably identify species.In the following, we discuss the 17 populations in terms of the results of our morphological observations of scyphistomae and ephyrae, morphometric measurements of ephyrae, recent genetic analyses as well as information on geographical distribution.
Group 1 Populations 1-5, 7, 8: Agreement -A.aurita.All of these scyphistomae were collected from eastern parts of the Atlantic Ocean and the Baltic Sea.Molecular (Dawson & Jacobs 2001;Schroth et al. 2002;Dawson et al. 2005), distributional (Mayer 1910(Mayer , 1917;;Kramp 1961;Russell 1970) and our data as described in Table 3 support this species identification for populations 1-5, 7 and 8.The effects of changes in salinity on the morphological characteristics of scyphistomae and ephyrae are not known and could influence classification based merely on morphology.However, morphometric measurements of ephyrae (see Table 5) suggested that population 8 was morphologically distinct from populations 1 and 2. We speculate that populations 1 and 8 may be boreal species as suggested by Schroth et al. (2002).The results of the LDA suggest that population 8 may be transitional between North Sea and Atlantic populations, which is supported by the findings of Dawson et al. (2005).Group 1 is considered to be a member to the initially described species, Aurelia aurita (Table 6).

Group 2
Population 6: Disagreement -Aurelia aurita which is likely another species.Scyphistomae in populations 6, 9 and 10 originated from geographically separate areas (North Sea, the Atlantic Ocean and Red Sea, respectively); nevertheless their scyphistomae and ephyrae share some common morphological characteristics (see Tables 3, 5).Unlike the genetic analyses reported by Schroth et al. (2002), our morphometric measurements distinguish population 6 from all the other groups.Dawson & Jacobs (2001) as well as Schroth et al. (2002) propose populations 6 and 14 to be the same species, and geographic differences may not be valid given ballast water transport of conspecifics.Still, based on the literature (Mayer 1910(Mayer , 1917;;Kramp 1961;Russell 1970) and our measurements, we suggest that population 6 represents the only member of group 2 and a new species, Aurelia sp. 1 (Table 6).

Group 3
Population 9: Disagreement -A.aurita which is likely another species.Animals from culture 9 were collected in the Mediterranean Sea and the separation into a new species is supported by genetic uniqueness (Dawson & Jacobs 2001;Dawson & Martin 2001;Schroth et al. 2002, Dawson et al. 2005).Also, the morphology of the ephyrae shows unique features, e.g., the yellowish colour in combination with the shape of the lappets and the shapes of rhopalial-and velar canals.Nonetheless, population 9 displays similar morphological features to A. aurita (Kramp 1961).Schroth et al. (2002) suggested a transitional habitat called "Tethys," which includes populations in the Mediterranean and Red Seas.We therefore provisionally place culture 9 within the taxon Aurelia cruciata Haeckel, 1880 (Mayer 1910;Kramp 1961) as it is located within the "Tethys" habitat.

Group 4
Population 10: Disagreement -A.aurita which is likely another species.Animals from population 10 were collected from the Gulf of Aqaba, also within this "Tethys" region defined by Schroth et al. (2002) and Dawson et al. (2005).Individuals display a similar morphology to A. aurita (Kramp 1961).We suggest placing population 10 within the taxon Aurelia maldivensis Bigelow, 1904(Stiansy 1938;Kramp 1961) considering its range of distribution within the "Tethys" habitat (see Table 6).

Group 5
Population 11: Disagreement -Aurelia aurita which is likely another, transitional species.Based on known distributions of adult medusae (given the caveats of potential mixing) and morphological features (Table 5), population 11 can be separated from the other Atlantic populations of A. aurita.This population is possibly a member of the taxon Aurelia flavidula Péron & Lesueur, 1809(Péron & Lesueur 1809;Mayer 1910;Kramp 1961) or Aurelia marginalis Agassiz, 1862 (Mayer 1910).Classification into a different taxon seems reasonable at this point, even though these findings are not yet supported by molecular data.

Group 6
Population 12: Disagreement -A.aurita which is likely another species.Collected from Brazilian waters, the morphology of scyphistomae (27-33 tentacles) of population 12 differs from all other populations.The shape of the calyx in combination with the cap-like hypostome, as well as the very small size of the scyphistomae, suggest differences from the other populations.Dawson & Jacobs (2001) found an Aurelia species on the east coast of South America that also has a unique morphology.Mayer (1910) described individuals off the Brazilian coast as A. aurita.Either there have been some misclassifications or both species coexist in this area.Based on the results of this study and the currently available literature, we suggest this population to be a separate species, which we call Aurelia sp. 2.
Group 7 Populations 13-16: Agreement -A.labiata.Both genetic analyses and morphological observations of scyphistomae, strobilae and ephyrae and morphometric measurements in ephyrae correctly distinguished populations 13-16 as A. labiata (Gershwin 2001;Dawson & Jacobs 2001;Dawson et al. 2005;Widmer 2005).The genetic relationship between populations 16 and 6 as reported by Schroth et al. (2002) cannot be substantiated.Genetic analyses classify US west coast populations of Aurelia as A. labiata (Dawson & Jacobs 2001;Dawson & Martin 2001;Schroth et al. 2002) and the morphology of both ephyrae and adult medusae (Mayer 1910;Gershwin 2001;Dawson 2003;Widmer 2005) support this classification.Nevertheless, differences in morphology and life cycle suggest that southern, central and northern varieties exist (Gershwin 2001) or that these may represent different species (Dawson 2003).

Conclusion
We provide a detailed morphological dataset for the scyphistomae of 17 populations of Aurelia congeners.Morphometric characteristics of ephyrae from 7 populations were good indicators of different species/ groups; still, morphometric data of scyphistomae collected and processed by the method of Straehler-Pohl (2009), Straehler-Pohl & Jarms (2010) and Straehler-Pohl et al. (2011) where not separative in order to distinguish among Aurelia species.Based on morphological differences (this study), genetic differences (Dawson & Jacobs 2001;Dawson & Martin 2001;Schroth et al. 2002;Dawson 2003;Dawson et al. 2005) and differences in geographical distribution (Mayer 1910;Kramp 1961;Russell 1970;Gershwin 2001;Widmer 2005), we suggest a separation of these 17 populations, which would include 8 groups: A. aurita, A. labiata, A. limbata, two new Aurelia spp., and three formerly recognized species.Our assessments agree with the a priori species assignments for 10 of the 17 populations.This is another step towards understanding the complexity of the genus Aurelia and it reveals the importance of considering the whole cnidarian life cycle, particularly differences in asexual propagation and morphological characteristics of ephyrae, when attempting to distinguish species.
Fig. 4. Linear Discriminant Analysis based on the morphology.A. Scyphistomae (17 cultures) of Aurelia congeners.B. Ephyrae (7 of the 17 cultures) of Aurelia congeners.In each case, cultures can be distinguished from one another by different symbols and corresponding numbers (see legend).

Table 1 .
Origin, salinity and incubation temperature of 17 different cultures of Aurelia sp.Strobilation was induced by temperature drop and, if required, additional potassium iodine (KI).

Table 3 .
Collected morphological features of the scyphistomae of Aurelia sp.; tentacles were counted on 10 individuals

Table 4 .
Analysis of Variances (ANOVA) for morphometric indices of scyphistomae and ephyrae.Abbreviations: df = degrees of freedom; F = F-Ratio; p = p-Value.Other abbreviations: see Table2

Table 5 .
Collected morphological features of the ephyrae of Aurelia sp.

Table 6 .
Classification of the different populations (cultures 1-17) into eight different groups/species based on our results (morphological and statistical: Linear Discriminant Analysis [LDA]) under consideration of the literature (genetic and distributional data).
Appendix B. Morphological measurements of ephyrae (mean ± sd) in mm.