A biochemical genetic and morphological investigation of the species within the genus Endeis Philippe (Pycnogonida : Endeidae) in Britain

A biochemical genetic and morphological investigation of the species within the genus Endeis Philippe (Pycnogonida : Endeidae) in Britain

J. Exp. Mar. Biol. Ecol., 1986, Vol. 98, pp. 115-128 115 Elsevier JEM 619 A BIOCHEMICAL GENETIC AND MORPHOLOGICAL INVESTIGATION OF THE SPECIES WIT...

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J. Exp. Mar. Biol. Ecol., 1986, Vol. 98, pp. 115-128



JEM 619


P.E. KING Department of Zoology, [email protected] College of Swansea, Singleton Park, Swansea SA2 8PP. U.K.

J. P. THORPE’ Departments of Zoology and Genetics, University College of Swansea. Singleton Park, Swansea SA2 8PP. U.K.


G. P. WALLIS’ Department of Genetics, University College of Swansea, Singleton Park, Swansea SA2 8PP, U.K.

(Received 22 November 1985; revision received 25 February 1986; accepted 26 February 1986) Abstract: Specimens of the pycnogonid genus Endeb from Mumbles Point (southern Wales) were found to differ consistently in certain morphological features when compared with samples from Martin’s Haven (southwestern Wales). The use of biochemical genetic techniques to compare enzyme loci showed the samples from the two localities to have no common allele at 11 of 15 loci examined. Genetic identity and genetic distance between the two samples were 0.241 and 1.42, respectively. Such high levels of genetic differentiation between the two morphs not only indicate that they are not conspecilic but also suggest that they possibly should not be congeneric. It is concluded that there are at least two species of Endeis in Britain and that the valid names for these are E. charybdaea (Dohm) and E. spinosa (Montagu). Key words: Pycnogonida; Endeidae; biochemical genetics; taxonomy

Carpenter (1893), using the characters size, spination and number of cement gland openings, distinguished two British species within the pycnogonid family Endeidae. These he referred to as Endeis spinosa (Montagu, 1808) and E. Zaevis(Grube, 1871). Norman (1908) described both Carpenter’s specimens as E. spinosa, although noting a variation in spination. Bouvier (1923) united the two species under the name Chilophoms spinosu Montagu. Fry & Hedgpeth (1969) also cast doubt on the validity of the criteria originally used to separate Endeis spinosu and a third nominate species, E. chybduea (Dohm, 1881). ’ Present address: Department of Marine Biology, University of Liverpool, Port Erin, Isle of Man, U.K. 2 Present address: Department of Zoology, University of Leicester, Leicester, LEl 7RH, U.K. 0022-0981/86/$03.50 0 1986 Elsevier Science Publishers B.V. (Biomedical Division)




Most British fauna lists place Ended specimens in the single species E. spinosa, although Wyer (1972) considered E. laevis to be a separate species, which he distinguished on the basis of body length, proboscis shape, leg length, claw and accessory claw length, and cement gland pores. Krapp (1975), in a morphological and distributional study, presented further evidence to suggest that there are two distinct species, but considered that these should be referred to as E. charyybdaea(Dohrn) and E. spinosa (Montagu). Thus, considerable confusion exists with regard to both number and status of species present. Since several past workers have considered there to be two species, but have disagreed on nomenclature, a degree of synonymy may be involved. The present study uses morphological and genetic criteria to compare specimens from two localities in the Bristol Channel in an attempt to investigate the status of the various described species.

MATERIALSAND METHODS For the experimental work pycnogonids were collected either on the shore at Mumbles Point, Swansea or subtidally from Martin’s Haven, Pembrokeshire. The latter samples were collected by diving at depths of up to 25 m. The collection of samples from a larger number of sites would have been desirable, but for two reasons this was not possible. First, for electrophoresis, with such small animals, frozen specimens would have been unsuitable and therefore these had to be used live. Since Enderk generally die rapidly in the laboratory samples had to be collected immediately before use. It was logistically difficult to arrange simultaneous collection even from two sites and more would probably have been impossible. The second reason was that these were the only two known sites within reasonable distance of the laboratory where adequate numbers of Endeis could be obtained. Samples from further afield would have been unlikely to have survived the journey back to Swansea. MORPHOLOGY Measurements were made of the lengths of the proboscis, body segments, leg segments, claw, auxiliary claw and the whole body (from the anterior of the cephalon to the end of the fourth body segment). Regression analyses were then applied to determine whether there were any significant differences between the two samples. The cement gland openings on the front femurs were counted and the number recorded for each specimen. GENETICS Enzyme electrophoresis was carried out using the horizontal starch gel technique. The gel baths used were supplied by Shandon (Model U77, SAE 3225) and the constant





voltage for running the gels was from Heathkit constant voltage power supplies (models IP- 17 and SP 17a). Voltages were adjusted to give a current of x 50 mA. Running times were in the region of 3-4 h. The starch used for making the gels was Connaught Hydrolysed (Connaught Diagnostics, 1755 Steeles Avenue West, Willowdale, Ontario, Canada). Four systems of gel and electrode buffers were employed. These were as follows. (1) A discontinuous tris-citrate/citric acid-sodium hydroxide system (pH 8.3) from Poulik (1957). (2) A continuous tris-citrate system (pH 8.0) from Ward & Beardmore (1977). (3) A discontinuous histidine buffer (pH 7.0) modified from Fildes & Harris (1966) by doubling the concentration of the gel buffer. (4) A discontinuous borate/Tris-citrate buffer (pH 8.6), composition: electrode buffer 18.6 g boric acid + 4.0 g sodium hydroxide per litre, gel buffer; 8 g Tris + 1.5 g citric acid per litre. Pycnogonids were prepared for electrophoresis by blotting them to remove excess sea water and placing each animal separately in a 4-mm diameter hole in a Perspex (Plexiglas) block at - 73 “C (Kelvinator ultra deep freeze). After the addition of a few drops of buffer the specimens were homogenized by grinding them in the holes in the Perspex block with the rotating tip of a solid glass rod. Water soluble enzymes were then extracted by absorbing them into small (6 x 4 mm) rectangles of filter paper (Whatman No. 1). These were then placed in line in a slot cut across the starch gel and were arranged in alternating groups of five from each sample area. The total sample size used was 10 animals of each of the two putative species. After electrophoresis, gels were stained for various enzymes using methods given by Shaw & Prasad (1970) and Harris & Hopkinson (1978). Electrophoretic techniques were very similar to those of Wallis & Beardmore (1984) who give further details of the methods. RESULTS


Present morphological observations indicate the probability that there are two distinct species of Endeis present in British waters. Sexually mature individuals collected by diving from Martin’s Haven are twice the size of mature animals collected on the shore at Mumbles Point. The average leg and body lengths of individuals from Martin’s Haven were 12.88 mm and 2.75 mm respectively, whilst those at Mumbles were 7.76 mm and 1.99 mm. The percentage of the total body length occupied by the fourth body segment in specimens collected at Martin’s Haven is significantly different from that of specimens collected at Mumbles Point (t = 3.68, P < 0.001) although the other body segments showed no significant difference. In both putative species the proboscis is long compared with the rest of the body, but whereas specimens from Mumbles Point have few circumoral spines, these are numerous in specimens from Martin’s Haven.



The prupodrrs bears a large claw and a pair of small auxiliary claws. The curvature of the propodus is greater in the individuals collected from Munxbles than in those from Martin’s Haven (Fig. 1) and the ratio of the length of the main claw to the length of the auxiliary claw is significantly Werent in the two goups (t = 2.52; P -c 0.02) (Fig. 2). Mumbles ACCESSORY


(E . charybdaea) Fig. 1, Diagram of propodus with large claw and small auxiliary claws from animals collected in Martin’s Haven and Mumbles.



(E. charybdaea


l-7 main

1.8 l-9 cfaw length /


2-O 2.1 act. claw length



Fig. 2. Histogram comparing the ratio of the length of the main claw to that of the auxiliary claws in animals

from Martin’s Haven and Mumbles.




Individuals collected from Martin’s Haven had 23-25 cement gland pores on each femur whereas those from Mumbles had 17-19 (t = 11.14; P < 0.001) (Fig. 3).



Fig. 3. Diagram showing the position of the cement gland openings in the kont femurs in animals from Martin’s Haven and Mumbles.

The ratios of total leg length to that of tibia I (d = 23.11; P < 0.001) and tibia II (t = 4.13; P < O.OOl},proboscis to body length (f = 5.29; P < O.OOl),and proboscis to leg length (t = 6.96; P < 0.001) are significantly different in the two groups. GENETICS All specimens of both samples were successftiy typed for a total of 15 putative genetic loci coding for nine different enzymes. Details of estimated allele frequencies at these loci are given in Table I. Both alleles and loci for any given enzyme are numbered successively from the point of origin; allele 1 in each case therefore is that of the lowest electrophoretic mobility. Only three loci, Mdh-2 (malate dehy~og~ase, EC, Me- 1 and Me-2 (ma&c enzyme, EC showed no detectable variation between the two samples. At all three of these loci both putative species were entirely monomorphic (i.e. there was no intraspecific genetic variation between any of the specimens which were examined). At the Mpi (mannose phosphate isomerase, EC locus there was intraspecific variation with the same two alleles present in both species (Fig. 4, Table I) but with considerable variation in the estimated allele frequencies. On the basis of the fairly small sample sizes used in the present work the differences at this locus alone are not statistically significant as between allopatric populations some variation in allele frequency may be expected. At a further 11 loci the two species were, however, totally diRerent with no common alleles. Nine of these loci appeared to be fmed for different alleles in the two species but showed no intraspecific genetic variation. These loci are Pep-l (peptidase, EC 3.4.11-13.-), Mdh-1, Pgm (phosphoglucomutase, EC, Pgi-1, Pgi-2 (phos-



phoglucose isomerase, EC ( = glucosephosphate isomerase, Gpi), Idh (isocitrate dehydrogenase, EC, Xod- 1, Xod-2 (xanthine oxidase, EC, and Sod-l (superoxide dismutase, EC (Fig. 4). At the Pep-2 locus both species showed some intraspecific variation (Fig. 4) whilst at Pep-3 the Martin’s Haven samples were highly polymorphic but those from Mumbles were monomorphic. There were no common alleles between the species at either of these loci. TABLE I Allele frequencies at 15 enzyme loci in samples ofEnd& from Martin’s Haven and Mumbles Point: number of alleles sampled per locus = 20. Locus Pep- 1 Pep-2


Mdh-1 Mdh-2 Pgm Pgi-1 Pgi-2 Mpi


Martin’s Haven

Mumbles Point




1.00 0.95 0.05 0.00 0.00 035 0.65 0.00 0.00 1.00 1.00 0.00 1.00 1.00 0.00 1.00 0.00 0.05 0.95 1.00 0.00 1.00 1.00 0.00 1.00 0.00 1.00 1.00 0.00

1.00 0.00 0.00 0.00 0.90 0.10 0.00 0.00 1.00 1.00 0.00 1.00 1.00 0.00 0.00 1.00 0.00 1.00 0.50 0.50 0.00 1.00


2 1 2 3 4 1 2 3 1 2 1


2 2 4

1 2 1 2 1 2 1 2



Me- 1 Me-2 Xod- 1

2 1 1 1 2


1 2 1 2

1.00 1.00 0.00 1.00 0.00 0.00 1.00



2 2 2 2 2 2 2

4 2

Genetic interpretations of the banding patterns observed for some of the enzymes probably require further explanation. There are clearly at least three Pep loci (Fig, 4). The fainter bands of slightly greater mobility near the Pep-l locus have been interpreted as being satellite bands of this locus although it is possible that they may represent products of a separate (fourth) locus. At the Pep-2 locus the two-banded samples are






interpreted as heterozygotes, the two bands showing the enzyme to be (at this locus) a functional monomer. The three-banded heterozygotes at the Pep-3 locus show this form of the enzyme to be dimeric. (For further explanation of banding patterns see Harris & Hopkinson, 1978; Ferguson, 1980.)























mmmam omaumm




_____ mmmmm








Fig. 4. Diagrams of eight gels stained for different enzymes: in each case (numbering from left) samples l-5 and 11-15 are from animals from Martin’s Haven and samples 6-10 and 16-20 are from animals from Mumbles Point; in each diagram the base line indicates the point of origin; the enzymes are A, MPI; B, PEP; C, MDH; D, PGM; E, PGI; F, IDH; G, XOD; H, SOD.



The three-banded pattern of each sample for MDH (Fig. 4) was considered to show the existence of two loci coding for this enzyme with the middle band in each case being a hybrid zone between these two loci. Such hybridization would indicate a dimeric structure for MDH which is as expected (see Harris & Hopkinson, 1978; Ward, 1978). The three PGI bands in each sample were also interpreted as showing two loci with an intermediate hybrid zone of activity. Again the enzyme is dimeric in other animals (Harris & Hopkinson, 1978; Ward, 1978) and so a hybrid zone is to be expected. MPI is expected to be monomeric and this is in agreement with the interpretation of two-banded samples as heterozygotes (Fig. 4). A further six putative enzyme loci were typed in all specimens from Martin’s Haven but did not give satisfactory results in Mumbles specimens. These loci were Ada (adenosine deaminase, EC, Gld (glutamate dehydrogenase, EC, Gpd-1, Gpd-2, Gpd-3 (glycerophosphate dehydrogenase, EC ( = glycerol 3 phosphate dehydrogenase), and Pgd (6-phosphogluconate dehydrogenase, EC All were monomorphic except for Pgd, which was highly polymorphic and appeared to show at least four alleles. The reasons these loci did not stain for the other species is probably that specimens from Martin’s Haven were considerably larger and therefore more enzyme was likely to be present in each sample. Four enzymes showed only very weak or zero activity, and gave no useful results. These were CK (creatine kinase, EC LAP (leucine aminopeptidase, EC 3.4. Il. l), GOT (glutamate-oxaloacetate transaminase, EC 2.6.1) ( = aspartate aminotransferase, AAT) and EST (esterase EC


The two samples studied in the present work may be distinguished morphologically on the basis of size, the ratio of main claw length to that of the auxiliary claw, and the number of cement gland openings on the male femurs (Figs. 1,2, and 3). The results of the study using enzyme electrophoresis show a remarkably high level of genetic differentiation between samples from Mumbles and Martin’s Haven. The two populations were identical at only three of 15 gene loci which were examined. Such a result must be considered to provide strong evidence that they cannot be conspecific. The biochemical technique of enzyme electrophoresis has, for several years, been extensively used as a tool in population genetics. The basis of the technique and its genetic uses have been described in detail in many reviews (e.g. Gordon, 1975; Sargent & George, 1975; Harris & Hopkinson, 1978; Ferguson, 1980) and it has considerable potential for the taxonomist (see Avise, 1974; Thorpe, 1979,1982; Oxford & Rollinson, 1983). Individuals not belonging to at least potentially interbreeding gene pools cannot, by definition, be of the same species, whilst conversely those showing little genetic differentiation are unlikely to be of different species. There is now available a considerable body of published data from a wide variety of animals and plants which shows that,




on average, individual organisms from allopatric populations of a single species show genetic differences at only a small proportion (generally < 10%) of gene loci. Interspecific comparisons usually show far greater levels of genetic differentiation (for congeneric species typically =50x) (see Thorpe, 1982, 1983). Genetic differences (as measured by enzyme electrophoresis) between populations or species may be quantified and reduced to a single figure by a variety of statistical methods. Two such methods are the genetic identity measure (Z) of Nei (1972) and genetic similarity (S) of Thorpe (1979). Both are measures of similarity on scales of zero to one, are very closely correlated, and give very similar results (Thorpe, 1979). Their converse measures are the genetic distance (0) of Nei (1972) and the genetic distance (also called D) of Thorpe (1979). These are measures of dissimilarity; Thorpe’s D is on a linear scale of zero to one whilst Nei’s D is on a log scale from zero to infinity. A great deal of work has been carried out on a wide range of organisms to examine levels of genetic variation between related populations of various degrees of taxonomic afIinity. Between allopatric conspecific populations values of Z or S are rarely to.90 and usually > 0.95 whilst between congeneric species these values are normally within the range 0.25-0.85 ( see Avise, 1974; Ayala et al., 1974; Ayala, 1975; Thorpe, 1979, 1982, 1983). Between species of related genera Z or S values are usually below x 0.35. These levels of genetic differentiation show general similarity over a great variety of species of vertebrates, invertebrates and plants (see Thorpe, 1979, 1982, 1983). In the present study the results give a value of S (Thorpe, 1979) between Mumbles and Martin’s Haven specimens of 0.237 and Z (Nei, 1972) of 0.241. The corresponding distance values are D (Thorpe, 1979) = 0.763 and D (Nei, 1972) = 1.42. It should be noted that the fairly small sample sizes used to estimate Z and S (10 animals of each species) only contribute to the intralocus sampling errors. These are negligible when compared with the far larger interlocus errors (the latter are effectively a function of the number of loci examined) (see Nei & Roychoudhury, 1974; Thorpe, 1979, 1982). Even samples as small as one individual per species are unlikely to affect seriously the reliability of genetic identity estimates (Nei, 1978; Gorman & Renzi, 1979). These similarity values between the two species are so low as to suggest that not only are they clearly not conspecific but that they might be more correctly placed in separate genera. On the present data such fundamental taxonomic separation, however, cannot be justified since the errors of Z and S are necessarily substantial (Nei & Roychoudhury, 1974; Li & Nei, 1975; Nei, 1978; Thorpe, 1979) with the result that present estimates of Z and S are not significantly different from those expected for the lower end of the range of congeneric species. By the standards of other animal groups these two pycnogonids are not even very closely related and are far from being sibling species. As Endeis spp., like all known British pycnogonids, are never hermaphroditic, genetic differentiation between populations cannot be a result of inbreeding through self-fertilization. In any case the differences found in the present work are far too large to have occurred by such means. Suggestions that the two samples may represent ends of a cline can also be precluded for similar reasons.



If the “molecular clock” hypothesis (reviews by Wilson et al., 1977; Thorpe, 1982) is accepted it follows that Nei’s (1972) measure of genetic distance (0) is likely to be a function of evolutionary time. Various attempts at calibration have been made and rates of molecular evolution may or may not vary significantly between taxa (Avise & Aquadro, 1982; Thorpe, 1982, 1983). Using the approximate general calibration that 1 D unit may be equivalent to z 18 million years of evolutionary time (see Yang et al., 1974; Sarich, 1977; Wilson et al., 1977; Thorpe, 1982), divergence time between the two pycnogonid species in the present work may, however, be estimated at x 26 million years. The results above are in agreement with those of Carpenter (1893) and Wyer (1972) both of whom concluded that there are two distinct species of Endeis in British waters. The number of cement gland openings and the measurements of leg length, proboscis length and main claw and auxiliary claw lengths given for E. charybdaea (Dohrn, 188 1) by Krapp (1975), however, agree with those of E. “spinosu”given by Wyer (1972) and the Endeis sp. from Martin’s Haven in the present study (Table II), indicating that all are probably conspecific. There are in fact some differences in size between E. chqbduea described by Krapp (1975) and animals from Martin’s Haven, but Krapp (1975) suggested that a size reduction in E. charybdaea occurs from south to north over the range of their distribution (from the Mediterranean to southwestern Norway). Wyer (1972) revived the name E. laevis (Grube, 1871) to account for differences in the lengths of body, leg, main claw and auxiliary claw between the two species. This species is clearly distinct from E. chmybduea, but the dimensions agree closely with those for E. spinosa as described by Krapp (1975) and some doubt has been raised (F. Krapp, pers. comm., 1978) regarding the validity of Grube’s (1871) type material for E. laevis. Therefore, it is suggested that E. spinosa be used in preference to E. laevis. Consequently, specimens found offshore at Martin’s Haven in the present study should be referred to as E. charybduea (Dohrn, 1881), whereas those collected from the littoral and immediate sublittoral at Mumbles Point should be referred to as E. spinosu (Montagu, 1808). The distribution of E. charybdaea is incompletely known and in view of the taxonomic problems records must, of course, be regarded with caution. The species has, however, been described as being found in the Mediterranean (Dohrn, 1881; Stock & Soyer, 1965; Soyer, 1966; Stock, 1966, 1970) and in the Atlantic, off St. Vincent (Cape Verde Islands) (Bouvier, 1937), from two stations off the Moroccan coast (Stock, 1970), off the French coast (Grube, 1871), and on the shelf of the United Kingdom and southwestern Norway. While E. chmybduea is found generally at depths > 12 m, E. spinosa is considered a shallow-water species, occurring only down to 15 m. In the British Isles, E. spinosa has been recorded from North and South Wales, southwestern England, Yorkshire, the southern and western coasts of Ireland, and the Isle of Man, and from the shores of Brittany, Belgium and Norway (Wyer, 1972; King, 1974). The known distribution of the two species in the Bristol Channel is shown in Fig. 5 and also confirms the differences in depth preferences.

Wyer (1972)

Krapp (1975)

Present work

Present work

E. spinosa

E. charybdaea

E. sp. Martin’s Haven

E. sp. Mumbles Point


Wyer (1972)


E. laevis



(:Iimm) I:.5 (3 : I5 mm) 1:4 (2.5 : 10 mm) 1:45 (2.76 : 12.88 mm) 1 : 3.9 (2 : 7.77 mm)

Ratio body Iength: leg length


? Less curved Less curved


Shape of propodus

Some characters used to distinguish between End& species.

>20 (23-25) <20 (17-19)

(I92& >20 (25-26) 26

Cement gland pores on femur




Relatively longer

Relatively shorter

% length of auxiliary: main claw


g hb 2


I?! 3 =! 8

E E z $



In view of the total absence of any published data on the population genetics of pycnogonids, estimates of observed and expected mean heterozygosity per locus and proportion of loci polymorphic in E. spinosu and E. charybdaea are given in Table III. This shows that levels of genetic variability in these populations are possibly a little below average but values are well within the range commonly found in other animal species (reviews by Selander, 1977; Nevo, 1978). No locus in either species showed gene frequencies differing significantly from


E. charybdaea

Fig. 5. Recorded distribution of E. spinosa and E. charybdueu in the Bristol Channel: -, contour; -----------, 1004 contour.



observed (I?(obs.)) and expected (R(exp.)) heterozygosities per locus and proportion of loci polymorphic (P) for EndeB charybdaea from Martin’s Haven and E. spinosa from Mumbles Point: data are for the 15 loci shown in Table I.

E. charybdaea E. spinosa




0.046 0.053

0.043 0.045

0.20 0.13



Hardy-Weinberg expectations, although this is not surprising given the relative weakness of such tests on other than large sample sizes (see Lewontin, 1958; Fairbairn & Roth, 1980). In both samples the value for observed mean heterozygosity per locus (Table III) is close to the expected value so it is probable that both species are routinely outbreeding as is to be expected in dioecious populations. ACKNOWLEDGEMENTS

We would like to thank Professors E. W. Knight-Jones, J. A. Beardmore, and T. A. Norton for the provision of facilities.

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