JOURNAL OF MORPHOLOGY 221:261-276 (1994)
Com parative Morphology of Caecilian Sperm (Amp h i bi a: Gym nop h ion a)
MAFWALEE H. WAKE Department of Integrative Biology and Museum of Vertebrate Zoology, University of California, Berkeley, California 94720
ABSTRACT The morphology of mature sperm from the testes of 22 genera and 29 species representing all five families of caecilians (Amphibia: Gymnoph- iona) was examined at the light microscope level in order to: (1)determine the effectiveness of silver-staining techniques on long-preserved, rare material, (2) assess the comparative morphology of sperm quantitatively, (3) compare pat- terns of caecilian sperm morphology with that of other amphibians, and (4) determine if sperm morphology presents any characters useful for systematic analysis. Although patterns of sperm morphology are quite consistent intrage- nerically and intrafamilially, there are inconsistencies as well. Two major types of sperm occur among caecilians: those with very long heads and pointed acrosomes, and those with shorter, wider heads and blunt acrosomes. Several taxa have sperm with undulating membranes on the flagella, but limitations of the technique likely prevented full determination of tail morphology among all taxa. Cluster analysis is more appropriate for these data than is phylogenetic analysis. cc: 1994 Wiley-Liss, Inc.
Examination of sperm for purposes of describ- ('70), in a general discussion of aspects of ing comparative sperm morphology within sperm morphology, and especially Fouquette and across lineages and for systematic assess- and Delahoussaye ('77, '921, Morrisett ('741, ment is enjoying a renaissance. The publica- and Wortham et al., ('77, '821, and recently tion of Jamieson's volume ('91) on compara- Lee and Jamieson ('92a,b) and Jamieson et tive morphology and systematics of fish al., ('93) have considered the phylogenetic sperm, with a discussion of techniques and a significance of sperm morphology in amphib- general assessment of the utility of sperm ians, particularly of families of frogs and ultrastructural characters for systematic salamanders. studies of both invertebrates and verte- Descriptions of caecilian sperm morphol- brates, presents an extreme, but useful, eluci- ogy are limited to analyses of patterns of dation of the potential value of sperm charac- spermatogenesis in individual species (de Sa ters to phylogenetic reconstruction (see and Berois, '86; Chthonerpeton indistinc- Discussion). tum; Exbrayat and Sentis, '82; Exbrayat, '86: Among amphibians, sperm have been de- Typhlonectes compressicaudus; Seshachar, scribed for several taxa of frogs and sala- '36, '37a,b: Ichthyophis glutinosus, '39: manders, usually in the context of assessing Uraeotyphlus narayani, '40: several species, patterns of spermatogenesis (e.g., Burgos and '42a,b: Siphonops annulatus and Schistome- Fawcett, '56; Baker, '62, '63, '66; Austin and topum [then Dermophis] gregorii, '43: Ich- Baker, '64; Barker and Biesele, '67; Brandon thyophis glutinosus, '45: Uraeotyphlus naray- et al., '74) or describing sperm at the light ani; Wake, '68; Gymnopis multiplicata, with microscope or ultrastructural levels (e.g., notes on several other species; Van der Horst Noble and Weber, '29; Fawcett and Hilfer, et al., '91, on the ultrastructure of the sperm '61; Picheral, '67; Nicander, '70; Martan and of Typhlonectes natans). Only a few of these Wortham, '72; Reed and Stanley, '72; Furi- reports include comparison of patterns of eri, '75; Wortham et al., '77, '82; Van der development and morphology across species, Horst, '79; Mainoya, '81; Mo, '85; Mizuhara et al., '86; Visser and Van der Horst, '87; Address reprint requests to Marvalrr H Wake, Departmerit or Yoshizaki, '87; Garrido et al., '89). Franzen Integrative Biology, University of Califnrnia. Berkeley, (:A 94720 o 1994 WILEY-LISS,INC. 262 M.H. WAKE and all are at the light microscope level (save were examined without a coverslip in bright- that of Van der Horst et al.). field illumination. I describe the comparative morphology of The maceration, several resuspensions, and mature sperm of a large number of genera especially the rinses in water cause loss of representing all five families of caecilians and numbers of sperm, but other nonsperm cells comment on the range of morphology, bioge- also are removed. Thus searching the field ography, and life history found in the order. I usually produces several sperm that are well examine both quantitative and qualitative stained and isolated from each other. Despite variables across all species and for a large years of preservation and the relative crudity sample within and among individuals of a of the technique, the sperm are quite intact. single population in order to assess patterns Even the acrosome, which I would have ex- of variation. The currently accepted hypoth- pected to be lysed and absent, was present in esis of the phylogenetic relationships of fami- virtually all specimens. However, the fragile lies of caecilians (Duellman and Trueb, '86; undulating membrane of the flagellum was Nussbaum and Wilkinson, '89; modified by present in only a few taxa and best repre- Hedges et al., '93 to include five families, sented in those most recently preserved (al- rather than six, the "Typhlonectidae" now a though it was also present in one long- subfamily of the Caeciliaidae) has been used preserved-70 yr-sample) . I suspect that as a basis for assessing phylogenetic patterns an undulating membrane may be present in presented by features of sperm morphology. more taxa than those that I observed (see MATERIALS AND METHODS Discussion), so I did not include its presence One to three testis lobes were removed or absence in my cluster analysis. The unim- from preserved specimens in various muse- paired morphology of long-preserved sperm ums (see Table 1and Appendix for list of taxa was evident when light microscopy photo- and their sources). Testis lobes vary in size, graphs of sperm of Typhlonectes natans were shape, and number across taxa (Wake, '68). compared with the ultrastructural detail pre- The lobes were macerated rapidly with dis- sented by Van der Horst et al. ('91).General secting needles in a watch glass to distribute structural fidelity was striking. Further, the sperm into physiological saline solution (0.7% measurements of sperm components ob- NaCl). A modification of the technique of tained are quite similar to those reported by Howell and Butts ('83) for silver-staining Seshachar ('39, '42a, '43, '45) of material sperm extracted from recently dispatched, prepared by sectioning and staining and by unpreserved fishes was used to obtain stained Van der Horst et al. ('911,using transmission sperm. The technique works well for pre- electron microscopy, for the same species. served amphibians, but length of time since The technique allows more details of acro- preservation does affect the quality of the some morphology to be revealed than those sperm produced (see below). reported by Seshachar ('40). Sperm suspensions were centrifuged 5 min Slides were photographed with a Nikon at 300 g; saline was aspirated and the sperm photomicrographic apparatus at 1,000 x with button was resuspended in 4 ml of 10%forma- oil immersion. Photographs of a micrometer lin. This procedure was repeated twice. Then, stage scale were taken periodically to be sure the supernatant was discarded and the sperm that photographs were taken at the same were resuspended in 1 ml of 10% formalin. magnification and that prints were all made Two to five drops of sperm suspension were to the same scale. Measurements were taken placed on a slide, and the suspension was by placing photographs on a digitizing tablet. distributed evenly. Slides were air-dried, The scale was calibrated, and (1) acrosome rinsed in running deionized HzO, and blotted length, (2) acrosome width, (3) head length, dry. Two drops of colloidal developer solution (4) head width anteriorly, (5) head width (2 gm USP gelatin dissolved in 100 ml deion- posteriorly, (6) body (midpiece) length, (7) ized HzO and 1ml formic acid) and 4 drops of body width, (8) flagellum length, (9) flagel- 50%AgNO3 (4 gm AgNO3 dissolved in 8 ml lum width anteriorly (at the proximal attach- deionized HzO) were pipetted to the surface ment to the body, and (10) flagellum width of the slide; the slide was covered with a 24 x posteriorly (at the distal tip) measured. Use 60 mm coverslip and dried on a 70" slide of the digitizer allowed the length of a long warmer for 3 min. The stain and coverslip and multiply curved flagellum to be mea- were then rinsed off in running deionized sured accurately. If it was apparent that a HzO, and the slide was blotted dry. Slides sperm head and body were not flat (i.e., curved COMPARATIVE MORPHOLOGY OF CAECILIAN SPERM 263 out of plane), so that shape was distorted in proportion to length). Long heads are >35 the two-dimensional photograph or the en- pm, moderately long 25-29 p.m, midlength tire flagellum was not in the photograph, the 15- 24 pm, short 8-14 pm. Narrow heads measurements were discarded. The paucity are 1 pm or less wide, medium width 2-3 pm, of sperm on the slides owing to the macera- broad > 4 pm. Midpieces considered long and tion and washing technique, coupled with thin are > 12 pm long and < 4 pm wide; long attempts for accuracy of measurements, re- but thick, > 12 km long but > 4 pm wide, sulted in very small sample sizes for most short and thick, 8 pm or < long and > 4 pm taxa. Meristic data are presented in Table 1. wide; short, slender, 8 or < pm long and < 4 Measurements were subjected to a princi- pm wide; moderate length 8-10 km. Flagella pal components analysis based on covari- are short if
TABLE 1. Sperm meristic data' Families and species NAL AW HL Hw ML MW FL Rhinatrematidae Epicrionops bicolor 3 5 1.5 38-40 1.3-2 10 24.5 105-106 E. petersi 3 3.7-5 .9-1.2 3538 1.0-2.3 8.5-10 2.3-4 52-64 Ichthyophiidae Ichthyophis orthoplicatus 1 4 2 43 2 7 4 200 I. glutinosus 5 4-6 1-3 17-18 3-5 67 3-6 100-120 I. kohtaoensis 3 P6.5 1-3 17-18 3-5 M M 64-117 Caudacaecilia asplenia 4 3 1 18-24 3-4 7-9.6 2-4 37-48 Uraeotyphlidae Uraeotyphlus narayani 4 3 4 18-23 2.5-2.6 6-12 .5-5 48-82 Caeciliaidae Afrocaecilia taitana 1 10 2 13 2 10 5 64 Caecilia occidentalis 2 3-4 1.5-2 10-11 2 7 1-2 60-140 Dermophis mexicanus 16 3-6 2-3 13-19 3-5 54 44.6 105-153 Gegeneophis mmaswamii 1 I 5 20 2.5 12 3 63 Geotrypetes seraphini 2 2 2 8-9 2-3 4-6 3 92-99 Grandisonia alternans 3 2-5 2-3 12-13 2-3 10-13 4-5.5 109-117 Gymnopis multiplicata 3 2-4 2 1417 3-5 7-8 5-8 86-100 Hypogeophis roslratus 2 4 3 15-16 2-3 a9 14 56-94 Idiocranium russelli 2 5 2-3 13 2.5 5-8 2 55-95 Microcaecilia albiceps 1 4 2 35 1.3 11 4 60 Mimosiphonops vermiculatus 1 6 3 15 2.5 9 4.5 82 Minascaecilia sartoria 1 4 3 13 2.5 8 6.5 107 Siphonops annulatus 2 4-8 2 17-18 2.5-3 10 4 61 S.paulensis 3 5-8 1 9-19 3.5 10-13 5-6 81-122 Typhlonectinae C. indistinctum 2 7-15 .5-.1 3135 1.5-2.6 I 6.3-7 86-100 T. natans 2 10-11 1-2 25-28 2 610 24 12&137 Scolecomorphidae Scolecomorwhus ulupuruensis- 2 3 2 8 3-4 7-9 2 105-109 'Legend: N = sample size; AL = acrosome length; AW = acrosome width; HL = head length; HW = head width; M = not measurable; ML = midpiece length; Mw = midpiece width; FL = Aegellar length. All measurementsare in microns.Although measured, head width anterior and head width posterior, and flagellar width anterior and flagellar width posterior, are not listed separately in the table, for there was no variation in these measurementsfor nearly all species. 264 M.H. WAKE
Figs. 1-14. Photomicrographs of sperm of species of Fig. 3. lchthyophis orthoplicatus sperm. Note elon- caecilians. All photographs are at the same scale; hence, gate head and acrosome, very short midpiece, and very scale bars on the separate plates = 25 +m for all figures. long flagellum with an undulating membrane.
Fig. 1. Epicrionops bicolor sperm. Note elongate head Fig. 4. Uraeotyphlus narayani sperm. The head is and acrosome, and the short midpiece. Abbreviations for medium in length, with a blunt acrosome and a short, all figures: a, acrosome; f, flagellum; h, head; m, mid- thick midpiece. The undulating membrane is not visible piece; u, undulating membrane. in this photomicrograph. Fig. 2. Epicrionops petersi sperm. Note resemblance to sperm of E. bicolor.
Family Ichthyophiidae acrosomes are stubby (4 x 2 pm) and flagella Sperm morphology varies within the genus are long (nearly 200 pm) with undulating Ichthyophis. Ichthyophis orthoplicatus (Fig. membranes (N = 1).Ichthyophis glutinosus 3) has sperm that resemble those of Epicrion- and I. hohtaoensis have relatively short heads ops in havingelongate heads (43 pm), but the (17-18 pm) with acrosomes that are moder- COMPARATIVE MORPHOLOGY OF CAECILIAN SPERM 265 ate in length (4-6 pm) and narrow (1-3 pm), thick at the base and narrowing anteriorly especially anteriorly. Midpieces are short and (7 x 5 pm [basel). The midpiece is long and thick (6-7 x 3-6 pm, respectively). Flagella thin (12 x 3 pm). The flagellum is short (63 are medium in length (100-120 pm) and pm) and has an undulating membrane usually have an undulating membrane (N = 5 (N = 1). and 3, respectively, for the two latter spe- Sperm of Geotrypetes seraphini have short cies). Caudacaecilia asplenia resembles the (8-9 pm), moderately wide (2-3 pm) heads latter species in having a medium-length head with very stubby acrosomes (2 x 2 pm). Mid- (18-24 pm) with a stubby acrosome (3 pm pieces are short (4-6 pm) and slender (3 pm). long, 1 pm wide). Midpieces are modest (7- Flagella are 92-99 pm long (N = 2). 9.6 x 2-4 pm). The flagellum is the shortest Grandisonia alternans sperm (see Fig. 10) observed in all caecilians examined (37-48 have short (12-13 pm) heads of medium pm), but there may be some effect of preser- width (2-3 pm); the acrosomes are short vation (see Materials and Methods and Dis- (2-5 pm) and broad (2-3 pm). The midpieces cussion) (N = 4). are long (10-13 pm) but broad anteriorly Family Uraeotyphlidae (4-5.5 pm), tapering posteriorly to 2 pm. Flagella are 109-117 pm (N = 3). Uraeotyphlus narayani sperm (Fig. 4) have Gymnopis multiplicata sperm have heads long (18-23 pm), medium-width (2.5-2.6 pm) that are 14-17 pm long (N = 3) and thick, 5 heads with stubby acrosomes (3 x 4 pm). pm at the base, 3 pm anteriorly. The acro- They have medium to long (6-12 pm), thin somes are stubby (2-4 x 2 pm). Midpieces (0.5-5 pm) midpieces and rather short fla- are short and thick, 7-8 x 5-8 pm. Flagella gella (48-82 pm) with indications of an undu- are 86-100 pm (N = 3). lating membrane (N = 4). Hypogeophis rostratus sperm (see Fig. 11) Family Caeciliaidae heads are moderate in length and width (15- 16 x 2-3 pm; N = 2). Acrosomes are stubby The sperm of Afrocaecilia taitana have (4 x 3 pm). Midpieces are midlength (8-9 short (13 pm), medium-width (2 pm) heads pm) and slender to broad (1-4 pm); flagella with long, narrow acrosomes (10 x 2 pm). are short (56-94 pm). The midpiece is 10 pm by 5 pm. The flagellum The miniaturized Idiocranium russelli have is short at 64 pm (N = 1). relatively small sperm (Fig. 9); heads are Caecilia occidentalis sperm have short (10-11 pm), medium-width (2 pm) heads short (13 pm) but moderately wide (2.5 pm) with stubby acrosomes (3-4 x 1.5-2.0 pm), and acrosomes are medium length (5 pm) and small midpieces (7 x 1-2 pm). The flagel- and narrow (2-3 pm). Midpieces are short lum varies in length (60-140 pm) but in the (5-8 pm) but very slender (2 pm). Flagella largest specimen is among the longest ob- are not long (55-95 pm) (N = 2). served in caecilians (N = 2). Sperm of Microcaecilia albiceps (see Fig. The large sample of Dermophis mexicanus 14) have an unusual morphology for caecili- measured (N = 16) gives some indication of aids. Heads are very long (35 pm), slender the range of measurements of mature sperm, (1.3 pm), and recurved; acrosomes, however, as well as the problems in securing accurate are short and stout (4 x 2 pm). The midpiece measurements of these very small struc- is 11 pm long and 4 pm wide. The flagellum tures. Measurements of sperm for which the did not photograph well; its anterior part is head and acrosome were curved out of the 60 pm long, but it clearly is longer than that, horizontal plane and the flagellum was not because there is no taper to the flagellum fully captured were not included. Dermophis (N = 1). (Fig. 5) have short heads of medium width The sperm of Mimosiphonops uermicu- (13-19 pm x 3-5 pm) with moderately long latis (see Fig. 7) has a moderate-size head (3-6 pm), narrow (2-3 pm) acrosomes. Mid- (15 x 2.5 pm) with an acrosome that is mod- pieces are short (5-8 pm) and thick (4-6.6 erately long (6 pm) and that narrows anteri- pm). Flagella are long (105-153 pm), and orly (3-1.5 pm). The midpiece is medium in more than half of those measured (9/16) bear length (9 pm) and width (4.5 pm); the flagel- an undulating membrane (see Discussion for lum is short (82 pm) (N = 1). reasons not all specimens might have the The Minascaecilia sartoria sperm has a undulating membrane present). short, moderately wide head (13 x 2.5 pm) The Gegeneophis ramaswamii sperm has a with a stubby acrosome (4 x 3 pm). The head 20 pm long and 2.5 pm wide, with an midpiece is short and thick (8 x 6.7 pm), and acrosome that is moderate in length, but the flagellum is 107 pm (N = 1). Fig. 5. Dermophis mexicanus sperm. Head and acro- Fig. 7. Mimosiphonops uermiculatus sperm. Note the some are of the stout, blunt morph; the long flagellum resemblanceto sperm of Siphonops annulatus. has an undulating membrane. Fig. 8. Seolecomorphus uluguruensis sperm. Scoleco- Fig. 6. Siphonops annulatus sperm. The head is me- morphid sperm have small, stout heads. dium-size by the criteria used, and the acrosome is large. The midpieee is dispersed. COMPARATIVE MORPHOLOGY OF CAECILIAN SPERM 267 Sperm of Siphonops annulatus (Fig. 6) Midpieces are moderate in length and width have heads of moderate size (17-18 x 2.5-3 (10 x 4 pm), and the flagella are short (61 pm) with acrosomes that are moderate in pm) (N = 2). Siphonops paulensis sperm length (4-8 mm) but slender (2 pm). have short (9-19 pm), wide (3-4.2 pm at base, 3 pm anteriorly) heads with mid-length (5-8 pm) but narrow (1 pm) acrosomes. Mid- pieces are long (10-13 pm) but fairly thick (5-6 pm). Flagella are 81-122 km (N = 3). Based on these small samples, the sperm of the two species of Siphonops are generally similar in shape, but S. paulensis has longer heads and flagella. Subfamily Typhlonectinae Chthonerpeton indistinctum sperm (see Fig. 13) have long (31-35 pm), slender (1.5- 2.6 pm), recurved heads with long, very slen- der acrosomes (7-15 x 0.5-0.7 pm). Mid- pieces, however, are short (7 pm) and thick (6.3-7 pm). Flagella are 86-100 pm, and one of the two examined has an undulating mem- brane (N = 2). The sperm of Typhlonectes natans (Fig. 12) also have long (25-28 pm), slender (2 pm), recurved heads with long, narrow acro- somes (10-11 x 1-2 pm). However, Typhlo- nectes sperm do not achieve the lengths of those of the confamilial Chthonerpeton. Mid- pieces are moderate in size (6-10 x 2-4 pm). Flagella are long (120-137 pm) and an undu- lating membrane is suggested, but not mea- surable, in the photographs (N = 2). Family Scolecomorphidae Sperm of Scolecomorphus uluguruensis (Fig. 8) have short, broad heads (8 x 34 pm) with stubby acrosomes (3 x 2 pm). Mid- pieces are short (7-9 pm) but slender (2 pm); flagella are 105-129 pm long, and one of the two examined has an undulating membrane (N = 2). DISCUSSION Comparative sperm morphology Sperm morphology was generally consis- tent within species and genera. For the gen- era for which two or more species were exam-
Fig. 9. ldiocruniurn russelli sperm. Note small heads with bullet-shape acrosomes. Fig. 10. Grandisonu alternuns sperm. Note bullet- shape acrosomes, short bodies, and proportionately large midbodies. Also, note the faint undulating membrane on the flagellum.
Fig. 11. Hypogeophis rostrutus sperm. Note the resem- blance to sperm of Grundisoniu ulternans, but Hypogeo- phis has a more slender and elongate midpiece. Fig. 12. Typhlonectes natans sperm. The head and Fig. 14. Microcuecilia albiceps sperm. This is the only acrosome are elongate, and the midpiece is short. The caeciliaid with an elongate head, and it has a small, pointed undulatingmembrane on the flagellum is barely discernable. acrosome and a rather small midpiece. Fig. 13. Chthonerpeton inclistinctun sperm. Note the elongatehead and acrosome. COMPARATIVE MORPHOLOGY OF CAECILIAN SPERM 269 ined, the morphology was qualitatively phis mexicanus, Minascaecilia sartoria, and similar and the taxa clustered in the same Chthonerpeton indistinctum. All other taxa quadrants of the PCA (Fig. 15). Two main have midpieces that are medium in both qualitative (and quantitative) morphologies length and width, according to the measure- were observed. One group of sperm have ment criteria for long, short, etc. Therefore, long, recurved heads, bodies, and acrosomes, midpiece measurements are not correlated whereas the other group has shorter, wider with either head and acrosome morphology heads and bodies, and rather blunt or bullet- or systematic relationships. shaped acrosomes. Sperm with long, re- Ichthyophis orthoplicatus has the longest curved components are found in most species filament, both absolutely and relatively, fol- examined in the families Rhinatrematidae lowed by Dermophis mexicanus and the typh- (Epicrionops bicolor and E. petersi), Ichthyo- lonectines. Several taxa have proportionally phiidae (Ichthyophisorthoplicatus), but rarely short filaments (Caudacaecilia asplenia, in the Caeciliaidae (Microcaecilia albiceps and Afrocaecilia taitana, Gegeneophis ra- the typhlonectines Chthonerpeton indistinc- maswamii, and Siphonops annulatus),but it tum and Typhlonectes natans). It is notewor- is possible that the end of the tail was out of thy that acrosome morphology varies consid- the plane of the photograph or perhaps lysed erably, because Seshachar (’40)reported that away; thus these measurements may not be the “acrosome is spatulate and though reliable. An undulating membrane was pre- slightly variable in size . . . , is of similar sent on the flagellum in the ichthyophiids appearance’’ in Ichthyophis glutinosus, (Ichthyophiskohtaoensis, I. orthoplicatus, I. Uraeotyphlus narayani, and Siphonops annu- glutinosus, and Caudacaecilia asplenia),the latus. In this study, the acrosomes oflchthyo- rhinatrematid Epicrionops bicolor, both typh- phis and Uraeotyphlus were found to be me- lonectines (T.natans, and C. indistinctum), dium in length, but slender and tapering, and several other caeciliaids (G. alternans, Gege- that of Siphonops to be very blunt. neophis ramaswamii, Schistometopum tho- Most ofthe ichthyophiids (Ichthyophis glu- mensis, and Dermophis mexicanus), the tinosus, I. kohtaoensis, Caudacaecilia asple- uraeotyphlid U. narayani, and the scoleco- nia),the uraeotyphlid examined, the scoleco- morphid Scolecomorphus uluguruensis. How- morphids, and all of the paraphyletic ever, in some species, an undulating mem- caeciliaids except Microcaecilia albiceps have brane was not present on all sperm examined. the shorter, stouter, blunter sperm morphol- This fact, and because of observations that ogy. However, there is considerable variation the membrane was more often present in within the stouter sperm morph. Acrosomes sperm from recently preserved specimens and can be blunt and bullet-shaped (most taxa) or that it is present in some members of some sharply pointed (Siphonopspaulensis, Gym- species in all five families, suggests that the nopis nultiplicata, Caecilia occidentalis). membrane may be present in more taxa than Afrocaecilia taitana has a long, narrow acro- I observed. Further, Seshachar (’39,’40, ’42a, some but a short, moderately wide head. ’43, ’45) reported undulating membranes pre- Conversely, Geotrypetes seraphini and Scole- sent in all species that he examined, but comorphus uluguruensis have stubby, nearly these are taxa, if included in my sample, in square acrosomes atop short, moderately wide which I observed the membrane as well (ex- heads. The head-to-midpiece ratio is highly cept for the equivocal condition in Siphonops variable, although consistent within species annulatus). However, because the sperm in and among species within a genus. The mid- the large Dermophis sample were taken from piece is the width of the body in most species, recently preserved specimens, the apparently but in some it is spherical and considerably fragile undulating membranes should have wider than the head. Species with long, thin been relatively intact. Because some sperm midpieces (>12 pm long, 4 pm or less wide) lack membranes, there are four possible ex- are Uraeotyphlus narayani and Gegeneophis planations, singly or in combination, of their ramaswamii; those with long, thick mid- absence: (1) the membranes are lost very pieces (>12 pm long, > 4 pm wide) are Ich- easily and early, (2) they develop very late in thyophis orthoplicatus, Grandisonia alter- the maturation process, (3) the membranes nuns, and Siphonops paulensis, and those wrap around the flagellum and, therefore, with short, thick midpieces ( < 8 pm long, > 4 are not obviously visual, and (4)some sperm pm wide) include Ichthyophis glutinosus, I. lack the membrane throughout their exis- kohtaoensis, Gymnopis multiplicata, Dermo- tence. This question cannot be resolved by 270 M.H. WAKE
X A X X 0 : xx SMZ x I
0 b A + 0 w on + w
-3 -2 -1 0 1 2 3 PC 1
0 Airocaecilia taitana V lchthyophis glutinosus Caecilia occidentalis lchthyophis orthoplicatus 0 Caudacaecilia asplenia 0 Idiocranium msseli + Chthonerpeton indistincturn 0 Microcaecilia albiceps X Dermophis mexicanus A Mimosiphonops vermiculatus 0 Epicrionops bicolor 0 Minascaecilia sartoria Epicrionops petersi X Scolecomorphus uluguruensis A Gegeneophis ramaswamii Siphonops annulatus Geotrypetes seraphinii Siphonops paulensis 0 Grandisonia allernans A Typhlonectes na tans 0 Gymnopis mulliplicata + Uraeotyphlus narayani rn Hypogeophis rostratus
Fig. 15. Principal components analysis of sperm measurements of 23 species. Sample sizes vary from 1 to 16 (see text for discussion). From Wake 1'931, in which tables of loadings are given. examination of this sample; electron micro- ther may be lost during long-term holding in scopic examination of a large number of ethanol or may adhere to the flagellum and sperm from a freshly dispatched animal is thus not be obvious in silver-stained prepara- necessary. The membrane is fragile and ei- tions. COMPARATIVE MORPHOLOGY OF CAECILIAN SPERM 271 Quantitative relationships but informative, screening of the data. Larger, Principal components analysis (PCA) was comparable sample sizes are needed to see performed on meristic data because it exam- how discrete species clusters really are. ines size and shape factors. Because it is a The PCA of data for species aggregated by clustering technique, one can examine family (Fig. 16)is also a general, but informa- whether or not groups presumed to be closely tive, screening. Caeciliads are scattered related phylogenetically are associated with throughout; this reflects the extensive varia- each other in PCA space. Other statistical tion in size and shape among members of this paraphyletic taxon. The scolecomorphid and treatments are not possible, because the the uraeotyphlid are in separate quadrants sample sizes range from 1 to 16. Cladistic and do not overlap caeciliaids. The rhina- analyses of these data are equally inappropri- trematids, two of the three ichthyophiids, ate, because of sample size variation and and some caeciliaids, including the typhlonec- other problems. PCA proved to be informa- tines, occupy the same quadrant, reflecting tive. PCA was run to examine all specimens their similarities in size and shape, especially in all species examined (Fig. 151, and then for of head and acrosome morphology. Again, the 20 genera represented (Fig. 16). In both one must not overinterpret these limited data. cases, vector 1, size, explained the most varia- tion (35.5% and 28.0%, respectively) with the Caecilians and other amphibians greatest loads for head length and body width Many features of caecilian sperm are, in (factor loadings are presented in Wake, '93). general, like those of other amphibians. Pro- Vector 2, shape, accounted for 24.7% and portions are similar in frogs and caecilians; 22.2%, respectively, of the variation, with salamander sperm have much longer heads acrosome width and body width loading and flagella (Austin and Baker, '64; Wortham heavily, followed by length of flagellum and et al., '77). The kinds of variation observed in anterior and posterior head width. size and shape of sperm components also In the analysis of all specimens, species obtain in salamanders and frogs. There are within genera clustered fairly closely, al- some differences in morphology, however, though they were not necessarily the closest revealed even by light microscopic examina- neighbor. The two species of Epicrionops clus- tion. The double-tailed morphology of frogs ter in the same quadrant; the two Siphonops of the Hyla rubra group (Fouquetteand Dela- are close together (and Mimosiphonops is houssaye, '77) and of sirenids (Austin and near them). Gymnopis clusters with Dermo- Baker, '64) was not observed in any caecil- phis, and Caecilia and Minascaecilia lie in ians, nor did I observe the cytoplasmic drop- the same quadrant, as do the two genera of let, characteristic of salamander sperm. Acro- typhlonectines. In contrast, the presumably some structure requires more analysis, but closely related Hypogeophis and Grandiso- the caecilian and salamander acrosomes are nia do not cluster, nor do the ichthyophiids. similar in lacking an acrosome cap, whereas Microcaecilia clusters most closely with the in anurans, the acrosome is effectively repre- two species of Epicrionops. Although it is sented only by the cap, without differentia- encouraging that species within genera clus- tion of a true acrosome (Burgos and Fawcett, ter together, and in some cases genera that '56; Morrisett, '74; Van der Horst, '79). Ultra- are presumed closely related on morphologi- structural details provide additional features cal and other grounds also cluster, this analy- that some workers have used to distinguish sis is quite limited by the small sample sizes. the sperm of different taxa (e.g., presence, Further, as shown in the larger Dermophis position, and texture of the cytoplasmic drop- mexicanus sample and the Uraeotyphlus let, nature of the tail membrane, surface sample, there is within-taxon variation in topography, head and midpiece juncture mor- sperm dimensions, but it does not swamp the phology, acrosome morphology: summarized analysis. Uraeotyphlus occupies one quad- in Wortham et al., '82, and Jamieson, '91). rant and is discrete from other species in the Comparisons of sperm morphology at ordi- quadrant. The Dermophis sample occupies nal, familial, generic, specific, and popula- positive PC2 space, although it surrounds tional levels are beginning to provide informa- the median of PC1 space; this is indicative of tion of biological significance, when more extensive size variation than shape comparisons are made consistently and using variation. Because the scale is in microns and similar methods. (It is difficult to compare the sample sizes vary from one to 16, this information obtained from ultrastructural ex- analysis must be treated as a preliminary, amination of fresh material in one group 272 M.H. WAKE
-2.5 -2 -1.5 -1 -.5 0 .5 1 1.5 2 PC 1
0 Caeciliaidae 0 lchthyophiidae A Typhlonectidae 0 Rhmtrematidae + Scdecomorphidae X Uraeotyphliae
Fig. 16. Principal components analysis of means of sperm measurements for 20 genera (see text for discussion). From Wake (’93). with that of light microscopic analysis of tion of the size of the sperm nucleus (head) preserved material from another.) Work on and the amount of “nuclear material” (based other groups of vertebrates has shown the on chromosome arrangement) in plethodon- utility of this approach (Hirth, ’60; Baccetti, tid salamanders. Therefore, it is possible that ’70; Fawcett, ’70; Nicander, ’70; Linzey and head size could be predictive of genome size Layne, ’74; Jamieson, ’91). Study of sperm in amphibians. My comparison of head length cannot stop with descriptive analysis; charac- in a diversity of plethodontid salamanders teristics of sperm must be included in biologi- (data from Wortham et al., ’77) with their cal analyses, such as those of reproductive genome sizes (C-values from Sessions and mode and life histories of amphibians. Sperm Larson, ’87) suggests that this is indeed likely. morphology can also be used to assess other This opens new questions for which caecil- biological questions of interest. For example, ians are appropriate for investigation, such Macgregor and Walker (’73) noted a correla- as whether both basal rhinatrematids and COMPARATIVE MORPHOLOGY OF CAECILIAN SPERM 273 derived typhlonectines, with their large sperm Jamieson has examined sperm, but I am con- heads, also have large genome sizes, and what cerned that he has not analyzed the degree of the correlation of genome size and phylogeny correlation of his 70 characters. This can be a might be. serious problem in any cladistic analysis when Phylogenetic implications independence of characters is suspect (Wake, of sperm morphology '94). I am also concerned how the treatment of 70 sperm ultrastructural characters incor- Amphibian sperm have been examined for porated into a larger, more diverse data ma- characters of systematic utility for some time trix will affect the analysis (Wake, '94). Fur- (summarized for salamanders by Wortham ther, in their reports on frog sperm and their et al. "821 and for frogs by Fouquette and phylogenetic implications, Jamieson and his Delahoussaye "77, '921 and Lee and Jamie- colIeagues have not stated the characters that son ['92a,bl and Jamieson et al. "933). How- they used in their analysis, how they were ever, sperm have received little attention from polarized and coded, what method of analysis other systematists, and sperm morphology was used to generate their trees, or what the rarely has been used in a phylogenetic con- consistency indices and other statistical mea- text. One problem may be that features of sures are. Further, they extend their discus- sperm morphology are useful at different lev- sions of sperm-based phylogenies to consider els in different groups, e.g., tail morphology such questions as whether internal fertiliza- in frogs at the species-group level (Fouquette tion is plesiomorphic for amphibians. I shall and Delahoussaye, '77), but at the family consider their arguments elsewhere. How- level in salamanders (Martan and Wortham, '72; Brandon et al., '74; Wortham et al., ,821, ever, I do not suggest that the characters and cross section of the axial rod useful at the should not be examined and used. To the interfamilial level in some salamanders but contrary, I simply plea for more rigorous at the intrageneric level in the genus Desmog- analysis and consistency of treatment, in ob- nathus. However, this should not pose a prob- taining the information, its implementation, lem for a careful cladistic analysis if taxa are and its analysis. Characters and character- adequately represented in both characters states must be well understood, both biologi- and sample sizes. Comparability of treat- cally and methodologically, in order to be ment is necessary and may have been the useful. basis for the lack of use of characters already It is clear that sperm morphology does in the literature. provide characters of systematic utility, but Recent innovations in techniques, ranging under certain conditions: (1) investigation from new stains to new uses of scanning and should be focused on sperm morphology of transmission electron microscopy, are being well-defined groups (be they species within applied to examination of sperm morphology genera, or even families), (2) fresh material, with renewed zeal by the above cited authors when available, should be examined by light, and by others. Jamieson ('91) claimed to scanning, and transmission electron micros- have amplified the field of "spermiocladis- copy together to provide detailed information tics" through the use of a host of ultrastruc- and better understanding of both the biology turd characters. He has examined primarily and the independence of the characters eluci- certain invertebrates (sperm morphology hav- dated, (3) it must be recognized that more ing been used in classification of certain inver- techniques are available for examining sperm tebrate groups for some time) and many from preserved specimens (fresh material is groups of fishes, with a survey of a number of simply not obtainable for many taxa), and (4) other chordates. Jamieson found 70 ultra- then, rigorous analysis must be executed in structural characters for fish sperm and has order that the utility of sperm morphology in mapped apomorphies on cladograms gener- phylogenetic analysis may be made clear. As ated by other workers to analyze the relation- we proceed to that point, it is still worthwhile ship of sperm morphology to phylogeny. He to assess sperm morphology by analyzing also has generated cladograms based on how its attributes correlate with phyloge- sperm ultrastructural characters exclusively. netic hypotheses based on other data, i.e., He and his colleagues apparently have not sperm characters should be mapped on phylo- added their data to other morphological and genetic trees to assess: (1)the correlation of molecular data sets and subjected the com- characters with each other, (2) points of ac- bined data to phylogenetic analysis. I ap- quisition of apomorphies, and (3) areas in plaud the consistency and rigor with which which new questions are revealed. For ex- 274 M.H. WAKE ample, although the present survey did not Brandon, R.A., J. Martan, J.W.E. Wortham, and D.C. produce data amenable to cladistic analysis, Englert (1974)The influence of interspecific hybridiza- tion on the morphology of the spermatozoa of Ambys- it has generated a number of questions for toma (Caudata, Ambystomatidae). J. Reprod. Fert. 41: future research. What are the implications of 275-284. the morphological similarities of the head- Burgos, M.H., and D.W. Fawcett (1956) An electron acrosome structure in the basal rhinatrema- microscope study of spermatid differentiation in the tids and the derived typhlonectines? What toad, Bufo arenarum Hensel. Biophys. Biochem. Cytol. 2223-240. are the implications of the differences of Zch- Christensen, A.K. (1966) Sperm fine structure in the thyophis orthoplicatus sperm morphology California slender salamander. Anat. Rec. 154:499. relative to that of other ichthyophiids and of de Sa, R., and N. Berois (1986) Spermatogenesis and Microcaecilia albiceps to other caeciliaids? histology of the testes of the caecilian, Chthonerpeton indistincturn. J. Herpetol. 20:510-514. Are there correlations of sperm morphology Duellman, W.E., and L. Trueb (1986) Biology of Amphib- with reproductive mode? ians. New York: McGraw Hill. Exbrayat, J.-M. (1986) Le testicule de Typhlonectes com- ACKNOWLEDGMENTS pressicaudust structure, ultrastructure, croissance et cycle de reproduction. Mem. SOC.Zool. France 43:122- I thank Kurt Schwenk, Kipp Baron, and 132. especially Enrique Lessa for executing most Exbrayat, J.-M., and P. Sentis (1982) Homogeneit6 du of the sperm preparations, Enrique Lessa, testicule et cycle annuel chez Typhlonectes compressi- caudus (Dumeril et Bibron, 1841), amphibien apode Kipp Baron, and Jim Hendel for photograph- vivipare. C. R. Acad. Sci. Paris 294:757-762. ing the sperm, and Kipp Baron for running Fawcett, D.W. (1970)A comparative view of sperm ultra- the PCA. I especially appreciate the coopera- structure. Biol. Reprod. Suppl. 2:90-127. tion of several museums and their curators Fawcett, D.W., and S.R. Hilfer (1961) The fine structure (see Appendix 1) for allowing me to remove of the spermatozoa of Triturus uiridescens. Anat. Rec. 193:329. testis lobes from specimens. I thank Barrie Fouquette, M.J., and A.J. Delahoussaye (1977) Sperm Jamieson for sending me manuscripts in press morphology in theHyla rubra group (Amphibia, Anura, and submitted, and I look forward to discus- Hylidae) and its bearing on generic status. J. Herpetol. sions with him. I appreciate the support of 11:387-396. the National Science Foundation for my re- Fouquette, M.J., and A.J. Delahoussaye (1992) System- atic application of spermatology, and the comparative search on the evolutionary morphology of sperm morphology of anuran amphibians. ASIH Meet- caecilians. I am grateful for the support of a ing Abstracts 1992:102. John Simon Guggenheim Memorial Founda- Franzen, A. (1970) Phylogenetic aspects of the morphol- tion Fellowship, under which auspices most ogy of spermatozoa and spermiogenesis. In B. Baccetti (ed): Comparative Spermatology. New York: Academic of the material was collected, and for the Press, pp. 2946. hospitality of the Department of EPO Biol- Furieri, P. (1975)The peculiar morDholopv of the sDerma- ogy, University of Colorado, as the manu- tozoon of Bombina- uariegata (L.1. Mlonit. ZO~.Ital. script was drafted during a sabbatical leave 9: 185-201, there. Garrido, O., E. Pugin, and B. Jorquera (1989) Sperm morphology of Batruchyla (Anura: Leptodactylidae). Amphibia-Reptilia 10: 141-149. LITERATURE CITED Hedges, S.B., R.A. Nussbaum, and L.R. Mason (1993) Austin, C.R., and C.L. Baker (1964) Spermatozoon of Caecilian phyologeny and biogeography inferred from Pseudobranchus striatus axanthus. J. Reprod. 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Tenn. Acad. Sci. 41~2-25. structure of the spermatozoon of the internally fertiliz- Barker, K.R., and J.J. Biesele (1967) Spermateleosis of a ing frog Ascaphus truei (Ascaphidae: Anura: Am- salamander Amphiuma tridactylum Cuvier. A corre- phibia) with phylogenetic considerations.Herpetologica lated light and electron microscope study. La Cellule 49:52-65. 67.9-118. Lee, M.S.Y., and B.G.M. Jamieson (1992a)The ultrastruc- Bernardini, G., R. Stipani, and G. Melone (1986) The ture of the spermatozoa of three species of myobatra- ultrastructure of Xenopus spermatozoon. J. Ultra- chid frogs (Anura,Amphibia) with phylogenetic consid- struct. Mol. Struct. Res. 94~188-194. erations. Acta Zool. (in press). COMPARATIVE MORPHOLOGY OF CAECILIAN SPERM 275
Lee, M.S.Y., and B.G.M. Jamieson (199213) The ultrastruc- Seshachar, B.R. (1939) The spermatogenesis of Uraeo- ture of the spermatozoa of bufonid and hylid frogs typhlus narayani Seshachar. La Cellule 48r63-76. (Anura, Amphibia): implications for phylogeny and Seshachar, B.R. (1940) The apodan sperm. Curr. Sci. fertilization biology. Zool. Scr. (in press). (Bangalore) IOt464465. Linzey, A., and J.N. Layne (1974) Comparative morphol- Seshachar, B.R. (1942a) Stages in the spermatogenesis of ogy of spermatozoa of the rodent genus Peromyscus Siphnops annulatus Mikan and Dermophis gregorii (Muridae).her. Mus. Novit. 2532:l-20. Blgr. (Amphibia:Apoda). Indian Acad. Sci. Proc. 15B: Macgregor, H.C., and M.H. Walker (1973) The arrange- 263-2 77. ment of chromosomes in nuclei of sperm from pleth- Seshachar, B.R. (1942b) The sertoli cells in Apoda. Half- odontid salamanders. Chromosoma (Berlin) 40:243- Yrly. Jour. Mysore N. S.3B:65-71. 262. Seshachar, B.R. 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WAKE APPENDIX Sources of specimens British Museum (Natural History): Sylvacaecilia grandisonae University of California, Berkeley, Museum of Vertebrate Zoology: Afrocaecilia taitana, Boulengerula boulengeri, Caecilia occidentalis, Chthonerpeton indistinctum, Dermophis mexicanus, Epicrionops petersi, Geotrypetes seraphini, Grandisonia alternans, Gymnopis multiplicata, Hypogeophis rostratus, Ichthyophis glutinosus, Ichthyophis kohtaoensis, Ichthyophis orthoplicatus, Oscaecilia bassleri, Oscaecilia ochrocephala, Typhlonectes na- tans, Uraeotyphlus narayani Cambridge University Natural History Museum: Idiocranium russeli Field Museum of Natural History: Gegeneophis ramaswamii Harvard University Museum of Comparative Zoology: Scolecomorphus kirkii, Scolecomor- phus uluguruensis, Scolecomorphus vittatus William Lamar Collection: Siphonops annulatus, Caecilia corpulenta Louisiana State University Museum of Natural History: Epicrionops bicolor, E. petersi University of Kansas Museum of Natural History: Minascaecilia sartoria United States National Museum: Caecilia orientalis, Caudacaecilia asplenia, Microcaecilia albiceps, Mimosiphonops vermiculatus, Schistometopum thomensis, Siphonops paulensis