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Comparative analysis of the chorionic patternin Habroleptoides species (Ephemeroptera,Leptophlebiidae)Elda Gaino a , Massimo Mazzini b & Michel Sartori ca Istituto di Zoologia dell'Università , via Balbi 5, 1–16126, Genova, Italyb Dipartimento Scienze Ambientali , Università della Tuscia , via S. Camillo de Lellis,1–01100, Viterbo, Italyc Musée de Zoologie , Palais de Rumine , Place Riponne 6, CH‐1000, Lausanne,SwitzerlandPublished online: 28 Jan 2009.

To cite this article: Elda Gaino , Massimo Mazzini & Michel Sartori (1993) Comparative analysis of the chorionicpattern in Habroleptoides species (Ephemeroptera, Leptophlebiidae), Bolletino di zoologia, 60:2, 155-162, DOI:10.1080/11250009309355805

To link to this article: http://dx.doi.org/10.1080/11250009309355805

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Boll. Zool. 60: 155-162 (1993)

Comparative analysis of the chorionicpattern in Habroleptoides species(Ephemeroptera, Leptophlebiidae)

ELDA GAINOIstituto di Zoologia dell'Università,via Balbi 5, 1-16126 Genova (Italy)

MASSIMO MAZZINIDipartimento Scienze Ambientali, Università della Tuscia,via S. Camillo de Lellis, 1-01100 Viterbo (Italy)

MICHEL SARTORIMusée de Zoologie, Palais de Rumine,Place Riponne 6, CH-1000 Lausanne (Switzerland)

ABSTRACT

The eggs of eleven species belonging to the genus Habroleptoides,namely H. annae, H. assefae, H. auberti, H. berthelemyi, H. budtzi,H. confusa, H. malickyi, H. nervulosa, H. pauliana, H. thomasiand H. umbratilis, were examined by scanning electronmicroscopy. Eggs of H. auberti are readily distinguished by a large-mesh reticulation of the chorion formed by ribs which delimitpolygonal areas. In the other species, egg chorion is covered byraised ribs running along the major axis of the egg to formlongitudinal rows. Ribs are arranged side by side, except in H. annaewhere they are widely separated from each other by a groove. Froman ootaxonomic point of view, the ultrastructural organization of theegg chorion suggests that, except for H. auberti, the species ofHabroleptoides are very closely related. Nevertheless, the wideseparation between ribs occurring in H. annae distinguishes thisspecies from the others in the group characterized by eggs with auniform covering of ribs. Analysis of the metric data, calculated forall the above mentioned species of Habroleptoides, showed acorrelation between egg length and width. Scatter plots of theseparameters yielded separations among species.

KEY WORDS: Egg shell morphology - Ootaxonomy - ScanningElectron Microscopy - Habroleptoides - Mayflies.

ACKNOWLEDGEMENTS

We wish to express our thanks to Dr. Gillies for his valuablesuggestions and criticism in reading the manuscript, and to Dr. B.Burlando for his practical help in statistical calculations of the data.This work was supported by grants from Italian Ministerodell'Università e della Ricerca Scientifica e Tecnológica (MURST 40%and 60% funds).

INTRODUCTION

In a recent revision of the genus HabroleptoidesSchoenemund, 1929, the taxonomic status of the encom-passed species has been settled, taking into accountmainly the morphology of wings and genitalia (Sartori,1986; Sartori & Jacob, 1986; Sartori & Thomas, 1986).The genus now includes fifteen species tentativelyclustered in seven main groups on the basis of the abovementioned characters.

A first approach to the study of egg sculptures in threespecies of Habroleptoides, revealed a spe.cies-specificchorionic pattern (Gaino & Mazzini, 1984; Mazzini &Gaino, 1990), as commonly observed for the eggs ofmany insect groups (review in Hinton, 1981). InEphemeroptera, scanning electron microscope (SEM)techniques have been utilized to enhance specific iden-tification (Alba-Tercedor & Sowa, 1987). In addition, in-vestigation of egg characteristics may contribute totesting the relationships between species (Kopelke &Müller-Liebenau, 1981a, b; 1982; Malzacher, 1982; Gainoet al., 1987, 1989) and to furthering phylogenetic studiesof the order.

The present work has been carried out on the eggs of11 species of Habroleptoides, to describe their chorionicpattern and to gain insight on their relationships.

MATERIALS AND METHODS

Source of eggs

Eggs were taken by dissecting mature nymphs and adults collectedin several localities, as follows: Habroleptoides annae Sartori - Picosde Europa, Spain (coll. M. Sartori); Habroleptoides assefae Sartori &Jacob - Haut Atlas, Morocco (coll. M. Sartori); Habroleptoides auber-ti (Biancheri) -Jura, Switzerland (coll. E. Biancheri); Habroleptoidesberthelemyi (Thomas) - Pyrenees, France (coll. A. Thomas);Habroleptoides budtzi (Esben-Petersen) - Corsica (coll. M. Sartori);Habroleptoides confusa Sartori & Jacob - Piedmont, Italy (coll. E.Gaino); Habroleptoides malickyi Gaino & Sowa - Euboea, Greece(coll. M. Sartori); Habroleptoides nervulosa (Eaton) - Sierra Nevada,Spain (coll. M. Sartori); Habroleptoides pauliana (Grandi) - Liguria,Italy (coll. M. Grandi); Habroleptoides thomasi Sartori - Serra Estrela,Portugal (coll. M. Sartori); Habroleptoides umbratilis (Eaton) -Ligurian Alps, Italy (coll. E. Gaino).

Preparation of the eggs

For scanning electron microscope, eggs taken from specimenspreserved in alcohol or formalin were dehydrated in an alcoholseries. Selected material was critical point dried using liquid CO2 ina Bomar apparatus, attached to specimen holders by silver conduc-ting paint, coated with gold in a Balzers Union evaporator and obser-ved with JEOLJSM 5200 and PHILIPS 515 electron microscopes.

Terminology

The basis descriptive terminology already used by Koss &Edmunds (1974) is followed in the present paper.

(Received 20 October 1992 - Accepted 29 December 1992)

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156 E. GAINO, M. MAZZ1NI, M. SARTORI

RESULTS

All the examined species included in this genus presenteggs with oval outline lacking in peculiar differentiatedattachment devices. The chorionic surface is charac-terized by ridges which are arranged to form a) areticulate pattern (in Habroleptoides auberti only) or b) auniform covering of longitudinal rows. Micropyles(usually two at least) of the funnelform type, are locatedin the equatorial area and consist of a sperm guide andmicropylar canal connected by the micropylar opening(Figs IC, 2C, F).

The egg of Habroleptoides auberti differs significantlyfrom the chorionic pattern shown by all other species ofthis genus. Indeed, the surface of the chorion is markedby a bold pattern of polygons outlined by thin ridges(Fig. 1A, B). The reticulate structure is apparent even atlight microscopy (Degrange, I960) and this featureallows a ready identification of this species. Themicropylar apparatus is usually situated at rib join and ap-pears as a chorionic depression that leads to themicropylar canal through the micropylar opening(Fig. 1C).

The remaining species possess eggs with raised ribs run-ning along the major axis of the cell. The interspacesamong ribs are very reduced but in H. annae (Fig. ID, E,F) ribs are separated from each other by grooves (Fig. IE)which contain adhesive material (Fig. IF). In general, norib extends over the whole length of the egg surface, fromone egg pole to the opposite one, but runs for an exten-sion of variable length, connecting with other adjacentribs. This organization results in a uniform egg covering.In the equatorial area, where the micropylar apparati arelocated, the ribs greatly enlarge to include the sperm guide(Figs IC, 2F) or are elevated to form a rim delimiting eachmicropyle (Fig. 2C). The sperm guide appears as a nearly

elliptical opening. Egg chorion may also have adhesivematerial, which forms a thin sheet adhering more or lessto the surface of the ribs (Fig. 2B), whereas in H. annae,this material is included inside the grooves (Fig. IF). Ribsmay be slightly twisted along their longitudinal axis. Athigh magnification this organization is detectable as a thinbanding that forms, oblique to the major axis of the ribs,a pattern particularly evident in H. confusa where the ban-ding is repeated with a period of 0.4 um (Fig. 3F). Such afeature is also present in H. annae, even though the ban-ding periodicity is more irregular and better detectable onthe widest ribs (Fig. IF).

Apart from this peculiarity, the chorionic pattern of thespecies of Habroleptoides, except that of//, annae and H.auberti, is rather uniform, as is seen in Figures 2, 3 and 4,which show both egg and rib shape in each species.Nevertheless, the results of the measurements of egg sizesallow some differences among species to be pointed out,as summarized in Table I. For this comparative metricanalysis, egg length, egg width, and the width of chorionicribs have been measured in different eggs of each species(Table I). For each of these variables, Duncan's multiplerange test shows significant differences among singlespecies and groups (Table I). H. berthelemyi and H.assefae rank among the highest values for all variables,whereas H. annae has relatively large eggs but thin ribs.Egg length and width are significantly correlated if thedata from all species are taken together. However, if acorrelation is estimated from the data of a single species,non-significant values are found in some cases (Fig. 5).

DISCUSSION

The diagnostic relevance of ootaxonomy has beendemonstrated for several groups of insects and this has

TABLE I - Summary statistics and Duncan grouping (a = 0.05) of egg variables (expressed in \im).

SP

Egg length

SE SP

Egg width

SE SP

Rib width

SE

anbeasbuCO

math

umaupane

55565656546

221220217206192183181180178176170

5.24.02.01.02.32.42.72.21.41.20.7

beanaspamaurnnethaubuCO

55546665565

1201051029990858482827977

2.92.80.81.01.20.74.61.61.80.90.9

beasbuumCO

manethpaanau

556656687

115

2.72.22.11.91.91.81.81.81.41.31.0

0.090.090.100.030.020.080.030.030.060.080.09

SP, species symbol: an, annae; as, assefae; au, auberti; be, berthelemyi; bu, budtzi; co, confusa; ma, malickyi; ne, nervulosa; pa, pauliana,urn, umbratilis, th, thomasi. SE, standard error.

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EGGS OF ELEVEN HABROLEPTOIDES SPECIES 157

^VV/^\^!P" :''.•'-."

Fig. 1 - Habroleptoides auberti, SEM view of the egg (A-C). A, outline of the egg shape; B, detail of the large-mesh reticulation of the chorionicsurface; C, micropyle showing the sperm guide (sg) and the micropylar opening (arrow) leading to the micropylar canal. Habroleptoides an-nae, SEM view of the egg (D-F); D, outline of the egg shape; E, chorionic surface with spaced ribs; F, detail of the ribs with an irregular thinbanding and with adhesive material (am) interposed between them. Scale bars: A, 25um; B, 5(im; C, 2¡im; D, 25|Jm; E, lO^m; F, 2(im.

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158 E. GAINO, M. MAZZINI, M. SARTORI

Fig. 2 - Habroleptoides pauliana, SEM view of the egg (A-C). A, outline of the egg shape; B, detail of the chorionic surface showing closelyadhering ribs covered by an adhesive sheet (arrows); C, micropyle delimited by raised ribs. Habroleptoides budtzi, SEM view of the egg (D-F).D, outline of the egg shape; E, detail of the chorionic ribs; F, micropyle partially delimited by a greatly enlarged rib. Scale bars: A, 25|im; B,

; C, 3nm; D, 25nm; E, 4|xm; F,

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EGGS OF ELEVEN HABROLEPTOIDES SPECIES 159

Fig. 3 - SEM view of egg and chorionic ribs, respectively, in Habroleptoides berthelemyi (A, B), //. umbratilis (C, D) and //. confusa (E, F). Notein F, the banding on the rib surface of//, confusa. Scale bars: A, 25(.im; B, 4\im; C, 25nm; D, 3(.im; E, 25nni; F, 3(wn.

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160 E. GAINO, M. MAZZINI, M. SARTORI

Fig. 4 - SEM view of egg and chorionic ribs, respectively, in Habroleptoides assefae (A, B), H. malickyi (C, D), H. nervulosa (E, F), and H.thomasi (G, H). Scale bars: A, C, E, G, 50nm; B, D, F, H, 5¡*m.

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EGGS OF ELEVEN HABROLEPTOIDES SPECIES 161

taofTOTAL r > 0.60 i

(an)nae r * NS

( i s ) ie lae r = NS

(au)berti i • NS

(be)rthalerayi r - 0.9S

be

; (bu)dt i i r = NS

neau'

CO

(co)n iusa r = 0.99¡

! (m a ) 1 i c k y i r • O.S0

! (ne)rvulosa i = NS

(pa)uliana r = 0.99

{lli)omisj f = NS

i (um)brali l is r = 0.86

length

220

Fig. 5 - Scatter plots of the average values of egg length and width(expressed in um) in each species. Inserts show Pearson correlationcoefficients estimated for all data and for each species. NS, non-significant correlation (P > O.O5).

helped in the task of defining relationships betweenspecies (review in Hinton, 1981). With regard to theEphemeroptera, it has been shown that the chorionicpattern provides a valuable character set in the study oftaxonomic problems (Koss & Edmunds, 1974). Thus,Alba-Tercedor & Sowa (1987) and Gaino et al. (1989)were able to use SEM studies to distinguish closelyrelated species of Rhithrogena, which are included in thediaphana and alpestris groups, respectively. In addition,the richness in chorionic sculptures and associated eggshell architecture favour the recognition of species(Malzacher, 1982; Gaino et al., 1987; Gaino &Bongiovanni, 1992a, b).

The ultrastructural analysis carried out on the eggs ofthe species belonging to the genus Habroleptoidesallows two main models related to the organization ofridges running on the egg surface to be proposed: a) alarge-mesh reticulation, limited to H. auberti, and b) auniform covering by ribs. The latter represents the mosttypical and widespread character of the genus. Indeed,apart from H. annae in which wide separation betweenadjacent ribs allows easy identification, metric datarepresent the only parameters supporting a separationamong species with the same chorionic pattern. Theuniform egg shell morphology suggests that thesespecies are closely related. By contrast, the peculiar eggsculpturing of H. auberti, beside being very diagno-stic, stresses the taxonomic position of this speciesamong Habroleptoides. This feature has been previouslypointed out in a first approach to the study of the eggs ofthis genus (Mazzini & Gaino, 1990), and is confirmed

here by a comparative analysis on a greater number ofspecies.

It seems acceptable to envisage two divergent lines inthe genus Habroleptoides: the first, characterized by auniform cover of ribs, includes a wide number ofrepresentatives, while the second, with a reticulate pat-tern, is limited to H. auberti. We emphasize that theplesiomorphic character state is represented by ribsevenly covering the egg surface. This conclusion is con-firmed by the fact that the same chorionic pattern ispresent even in a species of Calliarcys (C. humilis, un-published data), a genus closely related to Habrolep-toides. The peculiar egg shell organization of H. aubertiseems to be highly apomorphic because, as far as weknow, no species of Habroleptoides presents a chorionicpattern like that of//, auberti. The habitat of this speciestends to be restricted to a few rocky mountain localities(Gaino etal., 1984; Sartori, 1986).

Among the species with a chorionic pattern of ribs, H.annae deserves particular attention. Indeed, the reducedwidth of the ribs and the presence of grooves interposedbetween them are peculiar to this species, which isknown mainly on the basis of male characters. Thechorionic pattern could be useful in identifying nymphalstages also, and this stresses the main role of egg sculp-turing in taxonomic applications. In addition, theadhesive material accumulating inside the grooves couldrepresent an important device in improving egg adhesionto the substratum after deposition in water. It is wellknown that mayflies have evolved different stratagems toassure egg anchoring (Koss & Edmunds, 1974; Gaino &Mazzini, 1987; Gaino & Bongiovanni, 1992a, b). Amongthem adhesive layers perform an important functionowing to their ability to swell in water, thus preventingeggs from being dragged away (Mazzini & Gaino, 1985).

Species with a chorion characterized by a uniform coverof ribs have different geographical distribution: H. confusais the most widespread species, whereas a large number ofthem are endemic representatives (Sartori, 1986).

From a metric point of view, the correlation betweenegg width and length emphasizes the fact that H. annae,H. berthelemyi and H. budtzi possess eggs that reach thehighest values among the examined species of Habrolep-toides; eggs of H. berthelemyi are distinguished by theirwide ribs. Nevertheless, metric data are insufficient topermit one species to be discriminated from another,thus stressing the main role of the chorionic pattern inootaxonomy. This is particularly evident when com-paring H. confusa and H. annae whose affinity, alreadypointed out by Sartori (1986), is confirmed by theirtwisted ribs, a condition not detectable in any otherspecies of Habroleptoides.

In conclusion, even though egg chorionic pattern doesnot constitute the only source of data for ascertaining therelationships among species, egg shape and organizationmay be used, in addition to other morphological details,to outline the affinities among species in an attempt toreconstruct the way the taxa have spread.

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